Deciphering the Role of the Coagulation Cascade and Autophagy in Cancer-Related Thrombosis and Metastasis

Abstract

Thrombotic complications are the second leading cause of death among oncology patients worldwide. Enhanced thrombogenesis has multiple origins and may result from a deregulation of megakaryocyte platelet production in the bone marrow, the synthesis of coagulation factors in the liver, and coagulation factor signaling upon cancer and the tumor microenvironment (TME). While a hypercoagulable state has been attributed to factors such as thrombocytosis, enhanced platelet aggregation and Tissue Factor (TF) expression on cancer cells, further reports have suggested that coagulation factors can enhance metastasis through increased endothelial-cancer cell adhesion and enhanced endothelial cell activation. Autophagy is highly associated with cancer survival as a double-edged sword, as can both inhibit and promote cancer progression. In this review, we shall dissect the crosstalk between the coagulation cascade and autophagic pathway and its possible role in metastasis and cancer-associated thrombosis formation. The signaling of the coagulation cascade through the autophagic pathway within the hematopoietic stem cells, the endothelial cell and the cancer cell are discussed. Relevant to the coagulation cascade, we also examine the role of autophagy-related pathways in cancer treatment. In this review, we aim to bring to light possible new areas of cancer investigation and elucidate strategies for future therapeutic intervention.

Introduction

While the precise role of the extrinsic coagulation cascade in the pathophysiology of cancer progression is still largely unknown, a hypercoagulable state has been intimately linked to cancer progression for more than a century (1). Cancer-associated thrombosis (CAT), or Trousseau’s syndrome, is attributed as the second leading cause of cancer patient death after organ failure upon metastatic disease (2, 3). High D-dimer levels, a product of coagulation cascade activation, are associated with advance cancer stage and accordingly there is a high prevalence of venous thromboembolism (VTE) in stage IV cancer patients (4–6). Given the risk of VTE being 4- to 7-fold higher in the cancer patient, modulation of the coagulation system is currently an essential aspect of cancer treatment (7), with the potential to improve in the coming years. A chronic hypercoagulable state in oncologic patients may have a multifaceted role in conferring both a survival advance and dissemination potential to a bourgeoning tumor. This hypercoagulable state can be explained by several factors, including increased megakaryocyte (MK) and platelet production (Figure 1A), increased platelet activation and deregulation of other cells intertwined with coagulation systems such as endothelial cells (ECs) and neutrophils (Figure 1B).

Figure 1

Different cell types express Tissue Factor (TF), including cancer cells, platelets, and activated endothelial and immune cells. The antibody blocking of TF has been shown to delay the initiation of tumor formation, growth and vascularization in immunodeficient mice (8). Collectively, data suggest that oncogenic and differentiation pathways regulate TF and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. At the molecular level, TF, a transmembrane receptor belonging to cytokine receptor family, initiates the extrinsic blood coagulation pathway once bound in a quaternary complex with activated factor VII (FVIIa) and coagulation factor X (FX) (Figure 1C) [reviewed in (9)]. The cleavage of FX by FVIIa gives rise to the active protease (FXa), which in turn cleaves prothrombin to thrombin. This coagulation initiation complex can also directly activate cofactor VIII and coagulation factor IX, leading to thrombin generation (9).

The principal mediators of FXa and Thrombin protease signal transduction are the protease-activated receptors (PARs). These receptors belong to the G protein-coupled receptor (GPCR) family and become activated upon proteolytic cleavage of their N-terminal domain by extracellular proteases. PAR signaling has been implicated in several inflammatory diseases, including cancer (10, 11). Mammalian genomes contain four PARs that are ubiquitous within the body. PAR1, PAR3, and PAR4 are thrombin receptors, unlike PAR2. Classically, soluble proteases that are active during vascular injury, coagulation, and inflammation are responsible for PAR activation (12). Among these proteases are plasmin, matrix metalloproteinases (MMPs), activated Protein C (aPC), FVIIa, and FXa. However, the mechanisms remain elusive (13). The proteolytic cleavage of PARs activates numerous downstream signaling pathways, including intracellular Ca²⁺ mobilization, ERK1/2, NFκB signaling pathways and the induction of cytokines such as Interleukin-8 (IL-8) and IL-6 (14), allowing the PARs to mediate several processes in the coagulation-inflammation interplay, including those implicated in cancer progression (15, 16). Thus, PARs regulate platelet activation, assist in maintaining vascular barrier function through adhesion molecules (17, 18), and are associated with immune cell activation and migration (19–21). Upon activation, PARs are rapidly uncoupled from heterotrimeric G proteins through internalization to endosomes and then sorted to lysosomes and degraded. However, recent studies indicate that activated internalized PARs signal from endosomes through the recruitment of β-arrestins and potentially other pathways [reviewed in (10, 22)]. Interestingly, crosstalk between PAR signaling and autophagy has been described in different cell types (23–25), the physiological and pathophysiological role of this crosstalk still is an open field for investigation and discussion.

Autophagy, which comes from the Greek and means “self-eating”, is a highly regulated catabolic pathway by which cytoplasmic cargo is delivered to lysosomes for degradation and recycling, in order to preserve cellular homeostasis (26–29). In cancer, autophagy is broadly recognized as a “double-edged sword”, participating in both cancer suppression and promotion depending on tumor type, stage and microenvironment (30–32). Three principal types of autophagy have been identified: macroautophagy, microautophagy and chaperone-mediated autophagy (33, 34). Macroautophagy hereafter referred to as autophagy, is the most extensively characterized in cancer context and will be the focus of discussion within this review (35–37).

Autophagy involves the sequestration of cytoplasmic material, such as damaged organelles (i.e. mitochondria, endoplasmic reticulum) and protein aggregates, in a double membrane organelle called the autophagosome, which subsequently fuses to lysosomes, forming a new vesicle known as the autolysosome. Lysosomal enzymes within the autolysosome initiate the hydrolytic breakdown of their cargo (30). The resulting degradation products (i.e., sugars, amino acids, fatty acids, nucleosides/nucleotides) are transferred back to the cytoplasm for macromolecule synthesis and energy production (Figure 2) (38). Therefore, autophagy sustains cellular homeostasis by regulating the quality of cytoplasmic organelles. Moreover, this is also an adaptive mechanism that promotes cell survival in response to stress conditions such as nutrient deprivation, hypoxia, reactive oxygen species (ROS), DNA damage, intracellular pathogens or an increase in misfolded proteins (39, 40). Cancer cells can exploit this mechanism when exposed to metabolic, oxidative and inflammatory stress (41–43). Different “autophagy-related proteins” (ATGs) participate in the autophagic process (44), which can be divided into different stages: initiation, nucleation, elongation, autophagosome maturation and fusion with the lysosome, and finally cargo degradation followed by the release of breakdown products into the cytosol.

Figure 2

As schematically represented in Figure 2, the “initiation stage” involves the activation of the ULK1 complex, which is composed of the Unc-51-Like Kinase 1 (ULK1), the Focal adhesion-kinase family Interacting Protein of 200 kDa (FIP200), ATG101, and ATG13. The ULK1 complex integrates nutrient and energy stress signals through the activity of the Serine/Threonine kinase Mammalian Target of Rapamycin Complex 1 (mTORC1), which is known as the master negative regulator of autophagy (29). In parallel, an isolation membrane called phagophore is formed by membrane contributions from various organelles, including the endoplasmic reticulum (ER), Golgi apparatus, and mitochondria (45). Once activated, the ULK1 complex translocates to a membranous site where the phagophore is formed, where it serves as a scaffold for the recruitment of ATG proteins to the isolation membrane and thereby initiating autophagosome biogenesis (46, 47).

In the following step, referred to as the “nucleation stage” (Figure 2), the ULK1 complex phosphorylates the class III Phosphatidylinositol 3-Kinase (PI3K) complex, composed of Vacuolar Sorting Protein 34 (VPS34), Beclin-1 (BECN1), VPS15, and Autophagy Related 14-Like protein (ATG14L). The class III PI3K complex promotes local production of phosphatidylinositol 3-phosphate (PI3P) at the phagophore and initiates the recruitment of effector proteins to assist autophagosome formation (48, 49). ATG9, the only transmembrane protein that is part of the ATG machinery, cycles between the phagophore and the Golgi/endosomes, contributing to the recruitment of membranes for the formation and subsequent expansion of the phagophore (47, 50, 51). The sorting of ATG9 and its following transport to phagophore membranes occurs at RAB11-positive recycling endosomes (52, 53). Besides, ATG2 participates in the transfer of lipids from the ER to the phagophore leading to its expansion (54, 55).

The subsequent step is the “elongation stage” and involves the extension of the phagophore membrane (Figure 2). This process requires two ubiquitin-like systems: the ATG5–ATG12 system and the Microtubule-Associated Protein 1A/1B-Light Chain 3 (MAP1LC3A/B, also known as LC3)-phosphatidylethanolamine (PE) system (56). First, ATG12 is conjugated to ATG5 by the sequential activity of ATG7 and ATG10. The resulting ATG5-ATG12 complex interacts with ATG16L, leading to the formation of the multimeric complex ATG5-ATG12-ATG16L which is fundamental for LC3 lipidation (57, 58). Second, pro-LC3 is cleaved by ATG4 to form the cytosolic soluble LC3-I. Subsequently, ATG7 and ATG3 activity enable LC3-I conjugation to the lipid PE to form LC3-II, which is then recruited to the inner and outer surface of the autophagosome in an ATG5-ATG12 dependent manner (59). This process elongates and seals the phagophore leading to the subsequent formation of the autophagosome. Importantly, under basal conditions, LC3-I is uniformly distributed across the cytoplasm; however, upon autophagy induction, the lipidated form of LC3 (LC3-II) accumulates on nascent autophagosomes, and thus, represents a marker to monitor the formation of autophagosomes and autophagy-related structures (60–62). Autophagic cargo selection occurs in parallel to the processes of sensing, initiation and elongation. Cargo adaptors such as the receptor protein Sequestosome 1/p62 (SQSTM1/p62) can interact with both ubiquitin chains and LC3, and thereby promote ubiquitinated cargo recruitment to autophagosomes for selective degradation (63). As p62 becomes incorporated within the autophagosomes and are degraded in autolysosomes, thus serving as an index of autophagic degradation (60). Upon closure, the autophagosome dissociates from the assembly site and undergoes “maturation” via fusion with endosomes and subsequently with lysosomes to form a degradative autolysosome (64, 65). Maturation and autophagosome-lysosome fusion requires several proteins including Rab GTPases, membrane-tethering complexes and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) (66–68).

Finally, the lysosomal hydrolases degrade the autophagic cargo, and the resulting metabolites get recycled and returned to the cytosol through autolysosome efflux transporters, and thus cellular homeostasis is maintained (34, 69, 70).

Autophagy is highly regulated by different signaling pathways implicated in cancer (36, 71, 72). Nutrient starvation is the best-characterized autophagy inductor, where the serine/threonine protein kinase mTOR plays a critical role as an energy sensor (73). Within the human cell, mTOR can be found in at least two distinct multiprotein complexes, referred to as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (74). The mTORC1 complex is considered the primary negative regulator of autophagy (75, 76). Under nutrient-rich conditions, class I PI3K and AKT/PKB activate mTORC1 complex which by phosphorylating ULK1 and ATG13, prevents the induction of autophagy as shown in Figure 2 (77–80). A sensor of available energy is the AMP-activated protein kinase (AMPK), which is directly activated by a low ATP:ADP ratio (81, 82). Under starving, AMPK directly phosphorylates and inactivates mTORC1 (83). Through AMPK regulation, the inhibition of mTORC1 and the activation of the ULK complex can initiate the autophagy process (Figure 2) (46, 77).

Numerous factors that regulate autophagy are also classified as either oncoproteins or products of tumor suppressor genes [reviewed in (36, 71, 84)]. Thus, autophagy-signaling pathways are caught up in cancer regulation and control (Figure 2). Oncoproteins, including the small GTPase RAS, RHEB, and Nuclear Factor-κB (NF-κB), can activate mTORC1 and in consequence inhibit autophagy (85). NF-κB activates autophagy by inducing the expression of proteins involved in autophagosome formation, including BECN1, ATG5, and LC3. Conversely, NF-κB can also inhibit the autophagic process by increasing the expression of autophagy repressors, such like B cell lymphoma 2 (Bcl-2) family members (86). The anti-apoptotic members of the B Cell Lymphoma 2 (Bcl-2)-family bind and sequester BECN1 to prevent autophagy induction (87). On the contrary, tumor suppressors such as the transcription factor Forkhead box O1 (FOXO1) and nuclear p53 are known to induce autophagy (88). Interestingly, ROS production, a hallmark of cancer, and the subsequent activation of the oncogene c-Jun N-terminal kinase1 (JNK1) (89) can lead to the phosphorylation of Bcl-2; this prevents the interaction of this latter protein with BECN1 and thereby induces autophagy (88).

Pharmacological agents are frequently used to either enhance or suppress autophagy (Figure 2) (90). A frequent used approach for autophagy induction is mTOR inhibition by rapamycin (91). Conversely, 3-methyladenine (3-MA) can suppress the nucleation stage by inhibiting the PI3K complex, thereby inhibiting autophagosome formation (92). Autophagy can be blocked at later stages resulting in the inhibition of autophagic flux. This refers to the entire process from autophagosome synthesis to lysosomal degradation (93). Bafilomycin A1 (BafA1) is a potent V-ATPase inhibitor that impairs lysosomal acidification and thus the degradation of autophagic cargo (94). By a similar approach, chloroquine (CQ) can inhibit autophagy by increasing the lysosomal pH and therefore reducing the activity of degradative enzymes (95). Accordingly, BafA1 and CQ are commonly used to decrease the autophagic flux.

Although canonically characterized as a degradation mechanism, recent evidence has demonstrated a role for the autophagic machinery in extracellular secretion, a process termed as “secretory autophagy” or more linguistically precise “ATG gene-dependent secretion” (96–98). Accordingly, canonical autophagy involves the fusion of the autophagosomes with lysosomes for cargo degradation, whereas the secretory pathway bypasses this degradative process to allow unconventional extracellular delivery of cytosolic proteins via LC3-positive vesicles (Figure 2) (99, 100). Even though the molecular pathways in secretory autophagy are not entirely deciphered, the molecular machinery of the degradative processes is required (99). ATG5 and BCN1, together with other factors participating in canonical autophagy, are also activated as part of the secretory pathway (98, 101). The secretory autophagy pathway plays a key role in the progression of several diseases, including cancer (102, 103). It is involved in the secretion of cytokines such as IL-6, IL-8, and IL-1β, damage response mediators such as High mobility group box 1 protein (HMGB1) or ATP, and granule content such as von Willebrand factor (vWF) (104–107). Particularly, autophagy-dependent secretion of IL-6 has been implicated in tumor cachexia (103) and the metastatic potential of Ras-transformed cancer cells (108). The secretory autophagy pathway is reported to mediate cytokine release from cancer-associated fibroblasts, contributing to the development of head and neck squamous cell carcinoma (102). Thus, within the TME, both the cancer cells and supporting stromal cells rely on autophagy-dependent secretion for malignant progression (98).

Interestingly, it has been observed that decreased autophagosome degradation is associated with increased release of extracellular vesicles (EVs) in human malignant cervical and breast cancer cell lines (109). Extracellular secretion may provide a supplementary pathway to maintain cellular homeostasis when the degradative autophagy pathway is blocked (109). These suggest possible crosstalk between degradative and secretory autophagy to maintain cellular homeostasis and tumor cell survival. Furthermore, following pathological stress, there is a reported release of cytosolic proteins, molecular chaperones, harmful nucleic acids and misfolded proteins into the extracellular space through EVs, and this may contribute to tumor progression and metastasis (110, 111).

There is a complex association between cancer and autophagy. While in late-stage disease autophagy promotes tumor progression by providing nutrients to a rapidly dividing yet under vascularized and undernourished tumor, at early stages autophagy may suppress the bourgeoning tumor by suppressing reactive oxygen species and thus limiting genomic instability and, furthermore, promoting an anti-inflammatory microenvironment (36, 112–115).

Noteworthy is that the participation of the coagulation cascade in cancer progression is not limited only to thrombogenesis and thrombocytosis. The hemostatic system is known to promote tumor growth and metastasis, with thrombin increasing proliferation, migration and angiogenesis in preclinical models (116, 117). Circulating Tumor Cells (CTCs) are a marker of poor prognosis and are associated with increased risk of VTE in cancer patients (6, 118). TF has been identified to promote and be present on cancer stem cells (119, 120). Moreover, cancer and adjacent cell expression of PAR1 was recently postulated as a predictive marker of metastasis (121). The plasmatic concentration of Thrombin–Anti-Thrombin III (TAT) complex, a surrogate marker for activated thrombin used to assess coagulation state, inversely correlates with cancer patient survival (6). Independently of a direct role in the clotting process, the TF-FVIIa-PAR2 signaling was also linked to breast and liver cancer progression (23, 122).

Mouse models of cancer have demonstrated that FXa can increase tumor growth (15, 19, 20). Moreover, FXa increased lung, lymph node, liver, kidney metastasis in a syngeneic melanoma model, while promoting vascular permeability and increased infiltration (15). How PAR activation impinges on the autophagic pathway and how this regulates both physiological and pathophysiological processes is still an area requiring further study. We discuss herein that the coagulation system is not merely a bystander in cancer metastasis, but instead an integral part of the multifaceted approach taken by the malignant cell to survive, propagate, and ultimately exhaust the body’s capacity to function. We speculate that the malignant cell may use common mechanisms to hijack immune cells, ECs, hematopoietic cells, and platelets to integrate with the coagulation system for its own end. In this review, we postulate that a common mechanism present in each of these components may be the hijacking of the autophagy pathway, given the reported correlation between CTCs, hypercoagulable state and reduced survival in cancer. We examine the evidence for shared mechanisms and pathway integration to better understand cancer pathophysiology and to uncover novel druggable targets for the oncology clinic.

Autophagy Regulates Thrombus Formation Through Megakaryocyte Differentiation, Platelet Production, and Platelet Activation

Autophagy Is Implicated in Megakaryopoiesis and Thrombopoiesis

The body produces 1 × 10¹¹ platelets per day to maintain platelet count through the processes of megakaryopoiesis and thrombopoiesis (123). As schematically represented in Figure 3, within the bone marrow, the process of megakaryopoiesis gives origin to MKs from Hematopoietic Stem Cells (HSCs) through successive lineage commitment steps, followed by a maturation process. HSC differentiate through sequential steps into multipotent progenitors (MPPs), common myeloid progenitors (CMPs), bipotential MK-Erythroid progenitors (MEPs), and unipotent MK progenitors (MKPs), which then mature into MKs (124–127). The principal growth factor regulating steady-state megakaryopoiesis and thrombopoiesis is Thrombopoietin (TPO), which influences almost every step of the differentiation and maturation process. TPO is involved in HSC self-renewal, the proliferation of MKPs, MK maturation, and platelet production. TPO binds to the Thrombopoietin receptor (Mpl) activating JAK2 signaling, STAT3/5 and MAPK pathways and in this way positively regulates MK and platelet production (128).

Figure 3

Autophagy and megakaryocytic differentiation are overlapping processes. An up-regulation of BECN1 and LC3II precedes megakaryocytic differentiation. Moreover, autophagy is necessary for MK differentiation in vitro (129). Human CD34+ hematopoietic progenitor cell differentiation into MKs is slowed down by the autophagy inductor rapamycin, resulting in a lower percentage of and smaller MKs (130). It was further shown that ATG2B and the GSK3B-interacting protein (GSKIP) enhanced the differentiation of CD34+ progenitors and MKPs into MKs by increasing progenitor TPO sensitivity (131).

Moreover, it seems that autophagic flux regulation and its counterbalance of the apoptosis pathway is a crucial aspect during megakaryopoiesis (132). Interestingly, TPO stimulation results in TPO-Receptor internalization and targeting to the autolysosome for degradation (133, 134). Besides the canonical ER-Golgi route, the receptor traffics to the membrane through secretory autophagy (133, 134). Based on these studies, steady-state megakaryopoiesis and thrombopoiesis may be maintained by MAPK activation, which in turns targets the TPO receptor to autolysosomes and thus decreases TPO sensitivity in MPPs. However, an increase in p62 and thus disruption of the autophagic flux is observed (129), indicating that TPO could increase autolysosome formation but not necessarily degradation, thus favoring secretory autophagy. TPO activation of MAPK signaling may be promoting autophagy and therefore reducing TPO sensitivity; however, sensitivity could increase through secretory autophagy mediated by BECN1, ATG2B, and GSKIP (Figure 3). Therefore, the balance between degradative and secretory autophagy may play a key role in TPO sensitivity and steady-state megakaryopoiesis.

Further demonstrating the dependence of the autophagic pathway in steady-state megakaryopoiesis, VPS34, a protein required at the “nucleation stage”, is implicated in MK migration, Demarcation Membrane System (DMS) development, pro-platelet formation and platelet release (135, 134). Although rapamycin strongly inhibits polyploidization and pro-platelet formation (130, 137), hematopoietic lineage-specific Atg7 deletion demonstrated that the autophagy machinery is necessary for thrombopoiesis (138). Atg7 deletion not only impaired autophagy but also megakaryopoiesis, MK differentiation and thrombopoiesis. In particular, Atg7 knockout (KO) mice presented fewer platelets and failed to maintain hemostasis (138). In contrast, a separate study using Atg7^f/f; PF4-Cre mice failed to demonstrate a role for Atg7 in thrombopoiesis (139). Differing results may be explained by HSC relying exclusively on ATG7-dependent autophagy; however, differentiated cells may trigger compensatory non-canonical signaling pathways (140). Given the role of Atg7 in the initiation of canonical autophagy, this process appears to be crucial for both MK and platelet production and function.

Under stress conditions such as injury and infections, platelets are rapidly consumed, representing a high risk for health. Inflammatory signaling leads to a 10-fold increase in platelet production and platelet size (141). As shown in Figure 3, the regulatory mechanism of thrombopoiesis is mainly attributed to IL-6 promoting TPO secretion from the liver (142). However, inflammatory signaling could have a more direct effect as STAT3 activation is required for MKP expansion, MK maturation, and platelet production in a TPO independent fashion (143). Further mechanisms may involve the potentiation of megakaryopoiesis through TLR-mediated activation of quiescent stem-like MK-committed progenitors and IL1a driven MK rapid cytoplasmic fragmentation (144, 145). While autophagy regulation by inflammatory stimuli is well studied (42, 97), to our knowledge, there are no reports to date implicating autophagy in inflammation-mediated thrombopoiesis.

Taken together, published data strongly suggest the relevance of the autophagic process during steady-state megakaryopoiesis. Interestingly, autophagic activity may vary between stages of commitment and differentiation and may be involved in TPO sensitivity and pro-platelet production in steady-state thrombopoiesis. The relationship between autophagy and inflammatory signaling, as well as the existence of inflammatory induced thrombopoiesis, raises the question if these processes are connected. Further studies are required to clarify the role of autophagy during megakaryopoiesis and thrombopoiesis.

Crosstalk Between Coagulation Cascade and Autophagy During Platelet Activation

Despite being a developing field of research, the machinery and cellular structures of autophagy have been identified to be present and operative in platelets, indicating that platelets undergo basal autophagy much like nucleated cells. Autophagosome-like structures in platelets were first described during the 70s (146) and recently confirmed by super-resolution microscopy (147). Furthermore, the presence of autophagy-related structures and proteins are also evidenced in resting platelets (139, 147–150). Starvation or rapamycin treatment increases the autophagy in platelets in an ATG5-dependent fashion, which is reversed by 3-MA (151, 152). Although still under study, the physiological relevance of autophagy in platelets appears to be related to their activation. Firstly, autophagy can be induced during platelet activation by hemostatic agonists (139, 151). Secondly, defective platelet activation has been reported in Atg7-, Atg5-, Becn1-, Vps34- knockout mice (135, 139, 153).

Platelet activation occurs upon collagen binding to platelet glycoproteins (such as GPVI), thrombin engagement of PARs, and ADP activation of P2X receptors [reviewed in (154)]. Activation of these pathways triggers positive feedback, with platelets releasing their granules containing more agonists such as prothrombin, ADP and among other growth factors (Figure 4). In parallel, integrins are upregulated on platelet membranes, allowing platelet aggregation and ultimately clot formation (155). Noteworthy, is that Becn1 KO and platelet specific Vps34 KO mice display defective collagen-induced platelet aggregation, adhesion and thrombus formation (136, 152, 153). This association still needs further investigation as Becn1 is also dampened in ECs, among other cells relevant for hemostasis.

Figure 4

Interestingly, platelet specific Vps34 KO showed impaired thrombus formation in two independent studies, indicating that platelet VPS34 is necessary for the clotting process (133153). Accordingly, thrombin or C Reactive Protein (CRP) treatment significantly increases VPS34 dependent PI3P production in platelets while in Vps34^−/− platelets and human platelets treated with 3-MA displayed impaired responses to thrombin, collagen and ADP (133). Furthermore, Vps34^−/− platelets displayed impaired aggregation, dense granule secretion and decreased levels of Syk and PLCγ2 phosphorylation (153). In line with these findings, 3-MA impaired human platelet activation in response to Convulxin (a GPVI agonist) and thrombin, suggesting involvement of VPS34 in platelet activation downstream of PARs and GPVI (153). Although Vps34^−/− platelets showed reduced mTOR signaling and increased LC3-II levels, the authors of this study did not demonstrate an association between autophagy and platelet activation (153). Alternatively, they proposed that VPS34 promotes PI3P–guided NADPH oxidase assembly and subsequent ROS generation, supporting PAR- and GPVI-mediated platelet activation (153). However, a separate study showed an increased rate of secretion in response to platelet agonists in VPS34-deficient platelets ex vivo and underflow conditions (133). Moreover, under arterial flow, VPS34-deficient platelets display an inefficiency in recruiting circulating WT platelets to the growing thrombus (133). These authors proposed that VPS34 production of PI3P contributes to the spatiotemporal regulation of granule secretion, possibly by recruitment of intracellular proteins that regulate granule fusion and secretion (133). Interestingly, ROS production in platelets suppresses the downstream activity of the PI3K/AKT/mTOR signaling pathway promoting autophagy and consequently exacerbating platelet aggregation (156). These observations raise the possibility that VPS34 mediation of ROS production may promote autophagy and subsequently platelet aggregation; however, this hypothesis still needs to be tested.

Interestingly, 65% of mice with a platelet specific deletion of Atg7 (Atg7^f/f;PF4-Cre/⁺) were unable to arrest bleeding 10 min after tail transection despite no significant effects on platelet counts or volumes (139), further demonstrating that selective autophagy is an aspect for platelet activation. In the same line, it has been proposed that Atg5 enables mitophagy in platelets, which is a requirement for correct platelet activation (157).

The concept that autophagy could be promoted downstream of platelet agonist signaling, and that in turns regulates platelet activation, is supported by the observation that autophagy inhibitors block platelet aggregation and adhesion. Pre-incubation of platelets with 3-MA, BafA1 or CQ is shown to inhibit Collagen I (COL1)- and thrombin-stimulated aggregation (151). Interestingly, thrombin reduces LC3II levels, indicating an increase of the autophagic flux upon platelet activation (139). Similarly, a PAR1 peptide and ADP both mediated a reduction in LC3II (139). LC3II reduction requires degradation through canonical autophagy which is mediated by key downstream elements of platelet activation signaling cascade, including phospholipase C, protein kinase C, Ca²⁺, and Src-family kinases, as shown in Figure 4 (139) Although Src-family kinases seem to be upstream of LC3II increase in platelets, the specific mechanism has yet to be elucidated. On the other hand, extrapolating mechanisms reported in other cell types, Ca²⁺ may promote autophagy through CAMKKβ/AMPK/mTOR signaling (158). Hence, GPCR mediated platelet activation and subsequent increase in [Ca²⁺]_i could promote autophagy through this pathway (Figure 4). However, AMPK-independent Ca²⁺ regulation of autophagy has also been described (159). Thus, the possibility exists that Ca²⁺ promotes autophagy through a different mechanism.

The evidence of autophagy in resting platelets and, more importantly, of an increased autophagic flux regulation during platelet activation, clearly points to crosstalk between platelet agonists and autophagy and strongly suggests that autophagy is essential for coagulation. Furthermore, deficiency in key mediators of autophagosome formation such as VPS34 and ATG7 are associated with secretion and packaging of platelet granules, a fundamental aspect in thrombus formation. Taken together, platelet agonists signaling through PARs and GPVI could activate the autophagy signaling machinery, potentially mediated by Ca²⁺ and Src signaling, to promote autophagosome formation, which in turn promotes efficient granule packing and secretion (Figure 4).

Autophagy Implication in Cancer-Associated Thrombocytosis Through the Regulation of Platelet Production and Activation

It is established that thrombocytosis at diagnosis correlates with enhanced tumor invasion, metastasis, and poor prognosis in several solid cancers. Mean platelet volume was recently proposed as a diagnostic biomarker for lung cancer (160), while platelet counts have been correlated with stage and survival in melanoma patients, were thrombocytosis at diagnosis was significantly associated with distant metastasis (161). The current line of thought is that tumors benefit from thrombocytosis through platelet interaction with the CTCs, the latter encapsulating the tumor cells protecting them from NK cells and promoting the maintenance of an epithelial to mesenchymal transition (EMT) state (119, 162–165).

Altered platelet production in cancer could be explained by inflammation-mediated thrombopoiesis. A hypothesis is that tumor production of IL-6 promotes megakaryopoiesis via hepatic TPO, leading to thrombocytosis (142, 166). However, other plasmatic thrombopoietic cytokines such as Stem Cell Factor (SCF), Interleukin 1 alpha (IL-1α), Tumor Necrosis Factor alpha (TNF-α), stromal cell-derived factor 1 (SDF1), and Vascular Endothelial Growth Factor A (VEGF-A) are increased in cancer patients, providing alternative pathways for thrombopoiesis that could be independent or complementary to the IL-6/TPO axis. The SDF1a-CXCR4 axis is independent of the TPO/Mpl axis in murine models (167). Moreover, VEGFR1 promotes MKP maturation, possibly through CCXR4 up-regulation, leading to increased platelet counts in vivo (168, 169). Likewise, VEGFR2 activation increases MK proliferation, survival and differentiation (170).

Moreover, it has been reported that quiescent MKPs can rapidly differentiate into mature MKs and replenish platelet counts in response to inflammatory stimuli (144). This may be triggered by malignant signaling, as Toll-Like Receptor (TLR) activation is implicated in both hematological and solid tumors (171). As previously discussed, autophagy inhibition in early megakaryopoiesis appears to impair MK maturation, reduce platelet formation, and affect platelet function. Although many of the thrombopoietic cytokines such as TPO, IL-6, TNF-α, and VEGF have been associated with autophagy in MK or other cell types (131, 172) it remains unknown if, through deregulation in cancer, autophagy is central to promoting platelet production and thus sustaining a vicious cycle between malignancy and thrombopoiesis.

Suggesting that platelet autophagy is somehow deregulated in cancer, Lewis et al. described an increase in autophagosome-like structures in platelets from cancer patients (146). In malignancy, an obvious path for platelet autophagy regulation and platelet activation is the deregulation of the coagulation-signaling cascade. As previously discussed, platelet agonists such as thrombin, ADP and collagen can increase autophagic flux which is necessary for efficient platelet activation. Pro-inflammatory cytokines such as TNFα, IL-6, and IL-8, which are frequently elevated in cancer patient serum samples (173), are also involved in both platelet activation and autophagy. TNFα was shown to promote the platelet activation independently of platelet agonists, increasing TF expression, thrombin generation and subsequent clot formation (174). Other pro-inflammatory cytokines such as IL-6 and IL-8 and NF-κ B activation can also promote platelet activation (175, 176). TLR, TNFα, NF-κB, and JAK/STAT signaling pathways have been widely reported to regulate autophagy (42, 172, 177); however, this has yet to be evaluated in platelets.

ROS regulates autophagy in various cells by modulating the PI3K/AKT/mTOR pathway, and recent studies show that this is also true in platelets (156). High blood levels of oxidized LDL (oxLDL) have been reported in cancer patients and associated with metastasis in breast, ovarian, gastric and prostate cancer (178–180). Noteworthy, these cancers are also associated with a higher risk for thrombosis (181). ox-LDL can increase platelet activation in a VPS34 dependent manner, suggesting the involvement of the autophagy process (156). In line with these findings, the Oxidized low-density lipoprotein receptor 1 (LOX-1), recognizes and binds activated platelets assisting in the formation of a thrombus (182).

Furthermore, a separate study revealed that autophagy was activated in platelets through an oxidative stress-induced JNK pathway, which was evidenced by increased co-localization of LC3II with the Lysosomal Associated Membrane Protein 1 (LAMP1), suggesting enhanced autolysosome formation, after hydrogen peroxide treatment (152). It was also observed that mitophagy reduced phosphorylated p53, thus preventing apoptosis, and conversely the absence of mitophagy resulted in increased thrombosis (152). In this way, tumor micro-environmental cues such as ROS could alter platelet selective autophagy of mitochondria and thereby regulate platelet activation and thrombus formation.

Taken together, tumor-derived soluble factors could promote thrombopoiesis and potentially deregulate autophagy in hematopoietic progenitors and MKs. TME factors may regulate autophagy and subsequent platelet activation, furthermore, a high coagulative state and platelet-derived factors may promote tumor growth, metastasis and chemotherapy resistance.

Coagulation and Autophagy Cascade in Cancer Cells

TF and its protease products, such as Thrombin and FXa, are associated with cancer progression and cancer-related thrombosis (15, 20, 119, 183). Furthermore, this association may not be solely related to TF expression in the primary tumors as results have now shown that circulating microparticles carrying TF are 16- to 26-fold higher in pancreatic cancer patients with thrombosis when compared to healthy controls (183). There is emerging evidence that there is an involvement of the autophagy cascade in the mechanism controlling and promoting cancer progression and cancer-related thrombosis (23, 184). Proteins of the autophagy pathway are associated with cancer cell chemotherapy and radiation sensitivity through the alleviation of cellular stress and the dampening of apoptosis. Somewhat paradoxically, BECN1 has been postulated as a tumor suppressor due to its role in regulating p53 stability (185). As already mentioned, mTORC1 is the master negative regulator of the autophagic pathway and plays a critical role in cancer cell growth and progression (186, 187). Due to insufficient vascularization, tumors experience nutrient deprivation and hypoxia (188–190). In this scenario, mTORC1 may act as a restriction point between proliferation and differentiation (191). A hypoxic microenvironment also leads to an increase in both TF and FVII, correlating with tumor progression, local invasion, distant metastasis and therapeutic resistance (192).

As shown in Figure 5, thrombin mediated-PAR1 activation signals through PI3K/AKT/mTORC1 and activates Hypoxia-Inducible Factor-1 alpha (HIF-1α) (193), a well-known mediator of tumor survival, EMT, angiogenesis, and metastasis (193–195). Activation of PAR2 also leads to the downstream activation of the PI3K/AKT signaling pathway, promoting cell migration and invasion in both oral squamous and renal cell carcinoma (196, 197). In accordance, activation of both PI3K-AKT and mTORC1 signaling pathway by TF-FVIIa-Xa complex mediated by PAR1 and PAR2 has been associated with enhanced cell migration in a human breast cancer cell line (198, 199). Furthermore, recombinant TF, recombinant FVII or a PAR2 agonist upregulated mTORC1 signaling pathway in a hepatocellular carcinoma cell line (23). Moreover, levels of the autophagic marker LC3-II and the coagulation proteins TF, FVII and PAR2 were inversely correlated in human hepatocellular carcinoma tissues (23), suggesting a possible role of the coagulation pathway on autophagy suppression in cancer. In further accordance with an interaction between PAR signaling and autophagy, it was reported in human kidney tubular epithelial cells that PAR2 acting through the PI3K/AKT/mTOR pathway suppressed the process of autophagy, by affecting ATG5 and ATG12, and thus decreasing autophagosome formation (24). Additionally, downregulation of autophagy is associated with enhanced secretion of inflammatory mediators, such as IL-1β, TNF-α, and Monocyte Chemoattractant Protein-1 (MCP-1) (24). The aforementioned data suggest that activation of PAR2 by TF-FVII-Xa suppresses the autophagic pathway in a PI3K/AKT/mTOR-dependent manner (184), contributing to the generation of an inflammatory microenvironment that may lead to increased cancer cell migration and invasiveness. In this way, PAR signaling could directly promote tumor growth.

Figure 5

Interestingly, allograft models indicate that PAR1 promotes tumor growth by mediating immune escape. Tumor depletion of TF or PAR1 in allograft studies showed that CD8⁺ T cells effectively eliminated Par1 KO cancer cells in immune-competent mice (21). Similarly, in a separate study, cancer cell derived-extracellular vesicles and tumor-released autophagosomes (TRAPs) mediated immune escape by T cell suppression. In particular, these vesicles induced autophagy and activation and polarization of neutrophils and macrophages into an anti-inflammatory phenotype that promoted tumor growth and immune escape in a Programmed Death-ligand 1 (PDL-1) dependent fashion (200, 201). Furthermore, blockade of autophagy in tumor cells promoted the switch of macrophages into the anti-tumor M1-like phenotype and restored immune function of tumor-infiltrating lymphocytes (TILs) (201). A tempting hypothesis would be that PAR signaling mediates the secretion of TRAPs through autophagy regulation, thus promoting secretory autophagy while decreasing degradative autophagy, which may promote both cancer cell survival and immune escape.

While extracellular vesicles may allow intercellular communications throughout the body, direct communication between tumor cells and ECs mediated by connexins may also be critical to tumor cell extravasation at a potential metastatic site (202). Connexin 43 (Cx-43) has been reported as an obstacle and a promoter of cancer progression. As examples, Cx-43 may obtain tumor suppressor gene status as its loss contributes to metastasis (203–205). Conversely, expression of Cx-43 has also been shown to enhance tumor metastasis through increased attachment and communication with the vascular endothelium (Figure 5) (206–209). In metastatic melanoma cell lines, PAR1 silencing decreased Cx-43 expression as well as cancer cell attachment to ECs and extravasation, suggesting that PAR1 contributes to invasion and metastasis via regulation of Cx-43 (210). However, it should be noted that most connexins are characterized by a rapid turn-over mediated by different degradation pathways, including autophagy, where p62 served as a cargo-recognition factor, forming a bridge between ubiquitinated Cx-43 and LC3, thereby leading it’s to degradation (211–213). In turn, connexins might negatively regulate the autophagic process at initial stages (214). As represented schematically in Figure 5, Cx-43 may recruit ATG16 and the PI3K-complex to the plasma membrane, limiting their availability and capacity for regulating autophagy (212). Thus, Cx-43 up-regulation induced by PAR1 could impair autophagy leading to enhanced migration and invasion in cancer.

Furthermore, PAR4 is also associated with cancer development (215), and its activation in esophageal squamous cell carcinoma (ESCC) leads to the downregulation of proteins associated with chaperone-mediated autophagy, such as the Heat shock protein family A (Hsp70) (216). This evidence strongly suggests that PARs activation mediates cancer progression by regulating signaling pathways associated with the autophagy process (Figure 5).

MicroRNAs (miRNAs) are now widely reported to play critical roles in the modulation of autophagy in cancer cells and as potential markers for cancer detection (217, 218). It is thus unsurprising that an association exists between coagulation-mediated autophagic suppression and tumor malignancies involving miRNAs participation (25). Huang and collaborators observed in hepatocellular carcinoma tissues that elevated levels of both miR-135a and FVII were associated with tumor stage, recurrence, microvascular invasion, and decreased disease-free survival (Figure 5) (25). Moreover, in Hep3B cells treated with recombinant TF, FVIIa, or a PAR2 peptide agonist, the expression levels of miR135a were increased. They further demonstrated that expression of this miRNA was dependent on mTOR levels and that miR-135a acts as a downstream effector of PAR2 activation, abrogating the autophagic process in an mTOR dependent manner (25). In the same line, in breast cancer cells, miR-142-3p inhibits autophagy by targeting HMGB1 (219), which can be delivered by platelets and is associated to autophagy in other cell types (220). Platelet-derived exosomes contain miR-126 (221), which inhibition enhances autophagy as determined by LC3-II increased and p62 decreased protein levels in ESCC (222). These few examples demonstrate that miRNAs secreted by the coagulation through the release of platelet exosomes may impinge on autophagy-related pathways and mediate cancer cell progression. Thus, future investigation should seek to evaluate the interplay between coagulation, autophagy and microRNA in the same biological models.

Coagulation Cascade and Autophagy in the Tumor Microenvironment

The tumor microenvironment (TME) plays a principal role during tumor growth and metastasis. Within the tumor, ECs, immune cells and stromal cells alter their phenotype into a tumor-promoting state through secretion of growth factors and cytokines that sustain cancer cell survival and proliferation, while dampening the immune response. Interestingly, all these cells are subjected to stressors generated by cancer cells, such as glucose deprivation, hypoxia and inflammatory signaling; thus, autophagy plays a central role in the regulation of the TME. The TME is also a source of pro-thrombotic proteins, such as TF and FX, and thus in the subsequent section we will discuss how the interplay between the coagulation and autophagy cascades could maintain and promote tumor growth.

Coagulation Cascade and Autophagy in the Tumor Endothelium

During metastasis, cancer cells acquire an invasive phenotype and detach from the primary tumor and enter the bloodstream in a process called intravasation. Subsequently, cancer cells migrate out of the bloodstream by the process of extravasation and establish metastatic foci at distant organs (223). Fundamental to both these processes is endothelial permeability, which is promoted by the coagulation factors thrombin and FXa (15, 224). Interestingly, recent studies have shown that autophagy is involved in thrombin-induced endothelial dysfunction. Thrombin promotes VE-cadherin disassembly and degradation, allowing endothelial hyper-permeability in through BECN1 (225). Likewise, in vitro knockdown of Atg5 inhibited thrombin-induced actin stress fiber formation and VE-cadherin loss at the cell surface, thus preventing endothelial barrier dysfunction (226). Furthermore, pharmacological inhibition of autophagy with 3-MA, BafA1, or CQ can abrogate thrombin-induced hyperpermeability (227). Thus, in response to thrombin, VE-cadherin degradation through autophagy may lead to vessel hyperpermeability, as shown in Figure 6.

Figure 6

Poor vascular integrity contributes to the TME. Thrombin induces macrophage migration Inhibitory Factor (MIF) secretion from ECs, and the process of autophagy is involved in MIF-mediated endothelial hyperpermeability. Chao and colleagues showed that blocking autophagy attenuated thrombin-induced hyperpermeability in EC lines. Furthermore, blocking of autophagy or MIF effectively alleviated vascular leakage (227). These data suggest that endothelial permeability modulated by coagulation factors is dependent on autophagy (Figure 6), although further studies are required to evaluate these processes in the context of cancer.

How coagulation factor signaling pathways interact with autophagy is still an open question. However, upon ECs we know that thrombin binding to PAR1 initially induces Ca⁺² mobilization through activation of the Gq/11-phospholipase C pathway and members of the transient receptor potential canonical (TRPC) family of channels (228, 229). This raise in [Ca²⁺]i activates CaMKKβ, which in turn, activates AMPK (228, 230, 231). Hence, it is tentative to suggest that activation of PARs by coagulation factors induce AMPK activation and thus initiate autophagy (Figure 6).

Within ECs, the autophagy machinery is associated with vWF and P-selectin secretion in autophagic vacuoles (106, 232, 232). The primary function of vWF is to create a cell-surface adhesion site for coagulation factor VIII or platelet adhesion (among other proteins) at the endothelium membrane. Upon vascular damage, secretory granules called Weibel-Palade Bodies (WPBs), which contain thrombosis promoting factors, are assembled into chains that bind to adjacent connective tissue and in turn trap circulating platelets (234). Notably, Torisu et al. showed that autophagosomes contain vWF and that pharmacological inhibition of the autophagy or knockdown of Atg5 or Atg7 inhibits vWF secretion (106). Accordingly, EC-specific KO of Atg5 or Atg7 increased mice bleeding time (106). However, the size and composition of thrombus did not vary in EC specific Atg5-KO mice (235). Moreover, the secretion of vWF and P-selectin in response to the ox-LDL pro-thrombotic stimulus is associated with a decrease in Sirt1/FoxO1 signaling, and therefore autophagic flux. Moreover, the increased release of vWF and P-selectin is mediated by the inhibition of the Sirt1/FoxO1 pathway that depresses the fusion of the autophagosome with the lysosome, thus favoring the secretion of these factors (Figure 6) (232).

Interestingly, it has been observed in cervical and breast cancer cell lines that decreased autophagosome-lysosome fusion is associated with increased release of extracellular nanovesicles, suggesting that extracellular secretion might provide a supplementary pathway to maintain cellular homeostasis when the autophagy degradative pathway is damaged (109). Moreover, extracellular vesicle secretion may contribute to tumor progression and metastasis (110, 111). Indeed, exosome secretion is shown to be essential for directional and efficient migration of HT1080 fibrosarcoma cells (236). Thus, these examples suggest a possible crosstalk between degradative and secretory autophagy pathways to maintain cellular homeostasis and tumor cell survival (99).

Within the TME, endothelial autophagy regulation of coagulative factors may contribute to cancer progression and cancer-related thrombosis. Cancer cells promote the secretion of WPBs and vWF from ECs (237, 238) and elevated plasmatic vWF correlate with tumor grade and metastasis (239). Moreover, vWF secretion has been reported to contribute to the process of EMT (240), the secretion of pro-inflammatory cytokines, and vascular permeability (234). Several studies show vWF-dependent cancer cell adhesion to the endothelium is mediated by integrin receptors and facilitates extravasation during metastasis (239, 241–243). It is tempting to speculate that autophagy plays a central role in mediating cancer-related thrombosis and metastasis by regulating vWF release from the activated endothelium.

Autophagy also possesses a role in the constitutive recycling of PAR1, a pivotal process to maintain the receptor pool and enable re-sensitization to its potential coagulation cascade agonists. Rab11A and Rab11B are involved in autophagosome formation by regulating membrane transport from recycling endosomes (52). Grimsey and collaborators found that under basal conditions PAR1 is constitutively internalized and recycled back to the cell surface by a Rab11B-dependent pathway, whereas Rab11A regulates PAR1 basal lysosomal degradation (244). Interestingly, when recycling is disrupted in Rab11B-deficient cells, PAR1 is sorted from endosomes to autophagosomes and subsequently degraded in autolysosomes, in a Rab11A and ATG5 dependent manner (244). These results support a role for Rab11A in PAR1 basal autophagosomal-lysosomal sorting. Consistently, in EC cell lines, Rab11-B depleted cells showed decreased expression of PAR1, as a sign of its increased degradation. Conversely, in Rab11A deficient ECs, PAR1 protein expression was elevated. Thus, Rab11B and Rab11A serve distinct functions and regulate PAR1 recycling or basal autophagic/lysosomal degradation, respectively (244).

Autophagy may regulate the signaling pathways of different coagulation factors and their appropriate cellular responses in ECs by altering the recycling and endosomal sorting of PAR1 as well as in pro-thrombotic factors release. Furthermore, the pro-inflammatory, hypoxic and nutrient-starved state of the TME may deregulate EC autophagy, promoting coagulation and in turn paving the way for metastasis. Further experimental approaches should be applied to confirm this hypothesis.

Coagulation Cascade and Autophagy Regulate Myeloid Cell Polarization Within the Tumor Microenvironment

Autophagy and inflammation work synergistically in the TME to facilitate tumor growth and metastasis (245). Within the TME, monocytes and macrophages are essential sources of extravascular FX and TF. Notably, the synthesis of FX myeloid cells determines the Tumor-Associated Macrophage (TAM) phenotype (20). Inhibition of FXa-PAR2 signaling causes reprogramming of TAMs and attenuates the recruitment of immunosuppressive neutrophils and regulatory T cells promoting anti-tumor immunity (20). Coagulation factors could facilitate invasion and metastasis by transforming monocytes and macrophages into TAM-like cells. In the same line, monocytes and macrophages treated with FXII exhibited polarized M2 phenotypes with up-regulation of CD163, IL-10, IL-8, CCL18, CCR2, and CXCR2 (246). It has been reported that FXII and FXIIa upregulate neutrophil functions, contributing to macrophage polarization and T-cell differentiation that may contribute to cancer progression [Reviewed in (247)]. Furthermore, epithelial ovarian cancer cells exposed to conditioned medium from FXII-stimulated monocytes/macrophages showed increased invasive potential (246). In the same way, TF-FVIIa complex produced CD14 and CD163 up-regulation in monocytes in addition to an increase in the expression of IL-10, IL-8, TNF-α, CXCR2, and CCR2 (248). Moreover, co-cultures of epithelial ovarian cancer cells with TF-FVIIa stimulated monocytes increased the invasive potential (248). Additionally, THP-1 human monocytic cell line stimulated with TF or FVIIa displayed an M2-like phenotype with high levels of IL-4, IL-10, TGF-β, and TNF-α. Gastric cancer cells co-cultured with TF-stimulated TAMs also showed increased migration and invasion (249). These TF-stimulated TAMs induced VEGF and MMP-9 expression, which could promote the invasive potential and angiogenesis (249). Furthermore, co-cultivation of TF-expressing cancer cell lines with human monocytes stimulated invasive capacity, an effect inhibited by a TF neutralizing antibody (250). In non-small cell lung cancer patients with lymph node metastasis, there are reported higher levels of monocyte TF mRNA, which correlate with overall survival (251).

Interestingly, Graf et al. showed that PAR2 signaling directly regulates TAM mediated immune-evasion (19). Given that macrophage-specific deletion of FX prevented in vitro macrophage polarization, these observations suggest that coagulation factors contribute to cancer progression by promoting the formation of TAM-like cells. Furthermore, TAM phenotypic changes were similar in both macrophage FX-deficient mice and PAR2 mutant mice and accompanied by increased T cell infiltration, suggesting that PAR2 activation by FXa impairs anti-tumor immunity (19, 20). FXa production through TF-FVIIa leading to PAR2 activation and the formation of the TF-FVIIa-FXa-Endothelial protein C receptor (EPCR) complex is essential in the TLR4-mediated innate immune responses [reviewed in (9)]. Moreover, the TF-FVIIa-FXa-EPCR complex selectively induces expression of the TLR3/4 signaling adaptor protein Pellino-1, the transcription factor interferon regulatory factor 8 (IRF8) and a set of interferon (IFN)-regulated genes (252).

Autophagy is also associated with macrophage phenotype and activation as the AKT pathway converges both inflammatory and metabolic signals (253). The activation of mTORC1 promotes M1 polarization (254) and in accordance autophagy modulates the activity of macrophages and their responses (255–257). In general, the induction of autophagy downstream of TLR/NF-κB promotes monocyte differentiation and M2 polarization [reviewed in (258)]. Since the coagulation cascade also interacts with TLR-NF-κB signaling promoting polarization toward an M2 phenotype, it would be interesting to evaluate if there is an interaction between autophagy and the coagulation cascade in terms of innate immune signaling within the TME.

Coagulation Cascade, Autophagy, and Neutrophil Extracellular Traps in the Tumor Microenvironment and Cancer-Associated Thrombosis

Neutrophils and platelets cooperate to enhance coagulation (259). The release of intracellular components by neutrophils promotes coagulation and this has been associated with hypercoagulability and cancer-related thrombosis (260). As clinically observed in patients with sepsis and deep vein thrombosis, the activation of platelets can induce neutrophil extracellular traps (NETs), which in turn potentiate platelet aggregation (261–263). Once again demonstrating a connection to thrombosis and cancer, NETs can also formed under conditions of inflammation (264–267). As schematized in Figure 7, neutrophils can translocate TF to the generated NETs, where neutrophil elastase enhances TF pro-thrombotic activity through the degradation of TFPI (9). Elastase is capable of activating PAR1 and PAR2 receptors (268, 269), which may contribute to thrombus formation by promoting platelet and EC adhesion and activation.

Figure 7

Interestingly, the autophagy pathway is involved in mediating the delivery of thrombogenic TF to NETs, thus promoting thrombin generation and subsequent PAR-1 signaling (270). Moreover, in neutrophils, the inclusion of TF in autophagosomes is associated with its extracellular delivery. Disruption of autophagy by addition of 3-MA or BafA1 can abrogate NET release and TF trafficking, respectively (270). Notably, autophagy-mediated NET formation has been associated with CAT (271, 272), suggesting a potential interaction between autophagy, neutrophils and platelets within TME.

In line with this hypothesis, tumor-bearing mice incapable of forming NETs display decreased platelet aggregation and decreased circulating TF (271). Moreover, under in both in vitro and ex vivo experimental conditions, the pre-treatment of neutrophils with CQ inhibited NET formation (273, 274). TF, located in acidified autophagosomes, is released into NETs upon neutrophils exposure to inflammatory stimuli. Furthermore, TF from NETs can induce both thrombin generation and platelet activation mediated by PAR1 signaling (270). These data suggest that the release of TF through NETs could cause localized activation of the coagulation cascade and subsequent PAR dependent activation of platelets and ECs.

Interestingly, neutrophils from tumor-bearing orthotropic mice have increased LC3-II expression (274). Inhibition of autophagy with CQ or genetic ablation of the Receptor for Advanced Glycation End-products (RAGE), a Class III MHC protein receptor that mediates autophagy, resulted in reduced NET formation frequency within the TME (274). Based on these findings, Boone et al., proposed that NETs are upregulated in pancreatic cancer through RAGE (274). In the same line of thinking, a separate study found that, in response to platelet-derived microparticles, neutrophils can increase autophagy, mobilization of the granule content, enhanced proteolytic activity, prolonged survival, and generation of NETs (275). Neutrophil autophagy and the generation of NETs were also blunted in the presence of a competitive inhibitor of the cytokine mediator of inflammation HMGB1 (275). These findings are in line with other studies showing that autophagy in the neutrophil is essential for the NET formation, and this process is deregulated in cancer.

Targeting the Coagulation Cascade and Autophagy for Cancer Treatment

How can the knowledge of the interplay between coagulation and autophagy be put in practice in the oncology clinic? In truth, the axis of TF-FXa/thrombin-PAR-AKT- mTOR is already exploited in numerous treatments such as the rapamycin family of drugs, anticoagulants, metformin and certain targeted therapies. VTE is a significant cause of cancer-related death (276) and its prophylactic treatment reduces mortality, with low molecular weight heparin (an anticoagulant) often employed as the first-line treatment (7). As discussed previously, anticoagulants such as low molecular weight heparin, Rivaroxaban and Dalteparin have also demonstrated direct effects on tumor growth, immune-evasion and metastasis. It has already been speculated that the specific targeting of FXa could reduce metastasis and promote anti-tumor immunity (15, 20). It was previously reported that exogenous FXa increases melanoma metastasis to the lung, spleen and lymph nodes, together with an accumulation of intra-peritoneal fluid in a syngeneic mice model (15). Furthermore, the co-administration of the anticoagulant Dalteparin reduced the FXa- increased lung metastasis, while no metastasis was observed in other organs (15). A separate study demonstrated that the targeted deletion of this coagulation factor in myeloid cells reduced tumor progression in animal models, adding justification for pharmaceutical intervention in this pathway.

Furthermore, the anticoagulant Rivaroxaban has been demonstrated to give similar results to that of anti–PD-L1 therapy, and these two treatments were shown to synergize to improve anti-cancer immunity. As a mechanism, the authors propose that FXa signals through PAR2 to promote immune evasion, an effect attenuated by Rivaroxaban through the reprogramming of TAMs (20). Furthermore, co-treatment with a thrombin inhibitor, Dabigatran, and cisplatin in a model of ovarian cancer reduced tumor growth and levels of circulating activated platelets compared to Dabigatran or cisplatin alone. Interestingly, these authors demonstrated that this co-treatment with Dabigatran promoted anti-tumor activity of cisplatin by alleviating the immunosuppressive microenvironment. Co-treatment significantly decreased the number of Myeloid-Derived Suppressor Cells (MDSCs) and dendritic cells while increasing IFN-γ production by CD8⁺ effector T cells in ascites (277).

Similarly, in a melanoma metastasis model FXa increases endothelial permeability and promotes immune infiltration into mouse lungs, and this accumulation was reduced by the presence of the anticoagulant Daltepatin (15). These results further support the use of direct oral anticoagulants to reduce metastasis and favor an immune response against the tumor. Of note is that the inhibition of FXa on metastasis may be distinct to that of thrombin, shedding further light on the non-coagulation related roles of these coagulation factors. These observations also highlight the possibility that compounds may be developed that differentially inhibit either the coagulation or the non-coagulation actions of coagulation factors.

PARs, central proteins in the pathway connecting coagulation and in malignancy, are already a recognized cancer target (278). TF-mediated signaling via PAR2 has been associated with proliferation, migration and invasion of the cancer cell, and accordingly, the use of an anti-TF antibody has been shown to block PAR2 activation and suppresses tumor growth while demonstrating minimal effects on the coagulation process (279, 280). PAR inhibitors like vorapaxar, atopaxar, and PZ-128 have undergone clinical evaluation in the oncology setting. Evidence from metastatic breast cancer suggests that PAR1 blockade with PZ-128 in combination with Taxotere could be beneficial. Furthermore, a benefit may be present in dual-standard chemotherapy regimens and PZ-128 treatment in breast and ovarian cancers (278).

A further potential therapeutic target is oxidoreductase-protein disulfide isomerase (PDI) that catalyzes a thiol-disulfide exchange. In monocytes and macrophages, the activation of the TF cascade is reported to require a thiol-disulfide exchange and PDI (9). Liberated from both the EC and platelets upon agonist vascular injury, the subsequent use of PDI inhibitors could potentially attenuate platelet thrombus formation and fibrin deposition (9). Inhibition of PDI, by either antibodies or a non-specific thiol inhibitor decrease thrombus formation and fibrin generation in a mouse model of thrombosis and thus it will be interesting to examine if this could give potential benefit in the cancer setting (281).

As a central pathway for cellular function, the autophagy pathway is also a potential target for therapeutic intervention in cancer. To this end, the understanding of the action of rapamycin, discovered from a plant on Easter Island, has led to a series of “rapalogs” entering clinical oncology practice. Rapamycin, a specific mTOR inhibitor, can decrease cell migration promoted by the formation of TF-FVIIa-FXa complex in breast cancer cell model (199). Rapamycin, in combination with doxorubicin, can bring about remission in an AKT-positive lymphoma mouse model by blocking AKT signaling and overcoming chemotherapy resistance (282). Furthermore, in a vascular malformational model, the levels of D-dimer, a direct indicator of coagulation, were significantly decreased following treatment with an mTOR inhibitor (283).

Interestingly, long non-coding RNAs have been suggested to be a mechanistic target of mTOR signaling, ubiquitin-mediated proteolysis, and the coagulation cascade, opening the door to future intervention with RNA targeted therapy in cancer (284). However, as with any distribution of a ubiquitous process in cellular machinery, the use of mTOR inhibition may bring caveats. In an EC model, rapamycin strongly enhances the VEGF-induced expression of TF, possibly due to the interference in the negative feedback mechanism controlling this cycle. As VEGF is upregulated in tumors, this may explain the tumor vessel thrombosis observed in patients undergoing rapamycin therapy (285). Understanding the signaling of the coagulation system and its interaction upon cancer cell progression will be necessary for the targeted selection by the plethora of anticoagulants currently on the market to reduce tumor burden without perturbing essential hemostatic signaling.

Precision medicine based on Tyrosine Kinase Inhibitors (TKIs) has been reported to induce autophagy in many types of cancer cells (286). Noteworthy, the combination of certain TKIs with azithromycin (which dampens autophagy), enhanced cytotoxicity (286, 287). In the same line, an antibody-modified nanoparticle containing a combination of gefitinib and CQ was shown to have potential benefit in overcoming acquired EGFR-TKI resistance (288). Similarly, a combination of BafA1 with Gefitinib improved anti-tumor activity in a mouse model of triple-negative breast cancer (289). This burgeoning field may show the potential of multi-targeting to achieve both tumor-targeting selectivity and autophagy inhibition.

As previously discussed, recent studies have brought to light the immune regulatory role of coagulation components in the TME. FXa promotes immune evasion by signaling through PAR2, and the consequent addition of Rivaroxaban reprograms TAMs, supporting the translational potential of direct oral anticoagulants to overcome resistance to immunotherapy. A recent publication reported a before unidentified role of the immune checkpoint Programmed cell death protein 1 (PD-1) in regulating both lineage commitment and cell metabolism in cancer-associated myelopoiesis (290). These authors demonstrated that myeloid progenitors deficient for PD-1 manifested enhanced activation of mTORC1 in response to Granulocyte-Colony Stimulating Factor (G-CSF). This myeloid cell-specific ablation of PD-1 increased T memory cell function and anti-tumor activity. This evidence opens the door to the potential manipulation of the autophagy pathway during cancer immunotherapy.

Conclusion

The coagulation cascade and specifically its primary initiator TF possess effects that extend well beyond hemostasis and into the poorly characterized cauldron of cancer progression. Furthermore, the PARs, once believed to be exclusive mediators of the thrombin activation of platelets, have now manifested their versatility in regulating the intracellular pathways of almost every cell type examined. At the center of every cell lies the machinery for the process of autophagy, another black hole of information, yet this cascade is known to impinge on every biological process imaginable to the minds of cell biologists and physiologists. A NCBI Pubmed search in late 2020 with the keywords coagulation, PAR, autophagy and cancer will bring back only a handful of publications. However, each word alone will offer up more references than the most diligent biomedical investigator could read in their lifetime. Therefore, it is evident that we are extensively researching each integral pathway that classically appears in our physiology textbooks. However, there is little integration between the disciplines. In this review, we made a first attempt to examine coagulation and cancer signaling pathways to try and find common ground between each cascade and in particular, identify if autophagy is at the center of the intersections. Herein, we illustrated that each pathway has components in common.

Although the functional relevance of autophagy in cancer requires further study, it is proposed that autophagy acts as a protective mechanism during cancer initiation, yet promotes later stage tumor growth and metastasis. Evidence that autophagy-associated cell death acts as an initial tumor suppressor comes from the observation that many tumors present deletions in autophagy-related genes; moreover, loss of autophagy can induce genomic instability. Conversely, autophagy could promote cancer cell survival under metabolic stress, thereby facilitating metastasis by promoting cancer cell survival. The later hypoxic, nutrient-starved and pro-inflammatory TME may deregulate local and distant autophagic pathways in ECs, platelets, immune cells, and HSCs promoting coagulation and paving the way for metastasis and thrombosis. Moreover, the knowledge that components of the autophagy machinery are required for non-conventional protein secretion of pro-inflammatory and thrombotic mediators into the TME suggests that not only conventional autophagy but also secretory pathway may have a role in CAT and metastasis. Hopefully, this first in-depth analysis of the crossovers in these differing pathways will serve to bring to light possible new areas of investigation and elucidate strategies for future therapeutic intervention.

Funding

This study was funded by the grants issued by the Government of Chile: CONICYT FONDAP-15130011 (GO), Millennium Institute on Immunology & Immunotherapy IMII P09/016-F (GO), FONDECYT 1180241 (GO). The first author (CH) is a doctoral scholarship recipient of the Vice-rectory of Investigation of the Pontifical Universidad Católica de Chile. The authors (MH-C) and (CA) are recipients of CONICYT PhD fellowships (N°21151609 and 21201596, respectively). All figures were created with BioRender.com.

References

• 1

  TrousseauA. Phlegmasia Alba Dolens. In: . On Clinical Medicine, Vol II. (1873).

  • Google Scholar

• 2

  ElyamanyGAlzahraniAMBukharyE. Cancer-associated thrombosis: An overview. Clin Med Insights Oncol (2014) 8:129–37. doi: 10.4137/CMO.S18991

  • CrossRef
  • Google Scholar

• 3

  FalangaARussoLVerzeroliC. Mechanisms of thrombosis in cancer. Thromb Res (2013) 131:S59–62. doi: 10.1016/S0049-3848(13)70024-0

  • CrossRef
  • Google Scholar

• 4

  GoldhaberSZAgenoWCasellaIBCheeKHSchellongSSingerDEet al. Profile of Patients Diagnosed With Acute Venous Thromboembolism in Routine Clinical Practice: The RE-COVERY DVT/PE^TM Study. Am J Med (2020) 133:936–45. doi: 10.1016/j.amjmed.2020.03.036

  • CrossRef
  • Google Scholar

• 5

  SparsaADuroxHDoffoel-HantzVMunyangangoEMBédaneCCendrasJet al. High prevalence and risk factors of thromboembolism in stage IV melanoma. J Eur Acad Dermatol Venereol (2011) 25:340–4. doi: 10.1111/j.1468-3083.2010.03795.x

  • CrossRef
  • Google Scholar

• 6

  KirwanCCDescampsTCastleJ. Circulating tumour cells and hypercoagulability: a lethal relationship in metastatic breast cancer. Clin Transl Oncol (2020) 22:870–7. doi: 10.1007/s12094-019-02197-6

  • CrossRef
  • Google Scholar

• 7

  IkushimaSOnoRFukudaKSakayoriMAwanoNKondoK. Trousseau’s syndrome: Cancer-associated thrombosis. Jpn J Clin Oncol (2016) 46:204–8. doi: 10.1093/jjco/hyv165

  • CrossRef
  • Google Scholar

• 8

  MilsomCRakJ. Tissue factor and cancer. Pathophysiol Haemost Thromb (2008) 36:160–76. doi: 10.1159/000175154

  • CrossRef
  • Google Scholar

• 9

  ZelayaHRothmeierASRufW. Tissue factor at the crossroad of coagulation and cell signaling. J Thromb Haemost (2018) 16:1941–52. doi: 10.1111/jth.14246

  • CrossRef
  • Google Scholar

• 10

  HeubergerDMSchuepbachRA. Correction to: Protease-activated receptors (PARs): Mechanisms of action and potential therapeutic modulators in PAR-driven inflammatory diseases (Thrombosis Journal (2019) 17:4. doi: 10.1186/s12959-019-0194-8). Thromb J (2019) 17:1–24. doi: 10.1186/s12959-019-0212-x

  • CrossRef
  • Google Scholar

• 11

  SébertMSola-TapiasNMasEBarreauFFerrandA. Protease-Activated Receptors in the Intestine: Focus on Inflammation and Cancer. Front Endocrinol (Lausanne) (2019) 10:717. doi: 10.3389/fendo.2019.00717

  • CrossRef
  • Google Scholar

• 12

  DriesbaughKHBuzzaMSMartinEWConwayGDKaoJPYAntalisTM. Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin. J Biol Chem (2015) 290:3529–41. doi: 10.1074/jbc.M114.628560

  • CrossRef
  • Google Scholar

• 13

  Willis FoxOPrestonRJS. Molecular basis of protease-activated receptor 1 signaling diversity. J Thromb Haemost (2020) 18:6–16. doi: 10.1111/jth.14643

  • CrossRef
  • Google Scholar

• 14

  SohUJDoresMRChenBTrejoJ. Signal transduction by protease-activated receptors. Br J Pharmacol (2010) 160:191–203. doi: 10.1111/j.1476-5381.2010.00705.x

  • CrossRef
  • Google Scholar

• 15

  ArceMPintoMPGalleguillosMMuñozCLangeSRamirezCet al. Coagulation factor Xa promotes solid tumor growth, experimental metastasis and endothelial cell activation. Cancers (Basel) (2019) 11:1103. doi: 10.3390/cancers11081103

  • CrossRef
  • Google Scholar

• 16

  LangeSGonzalezIPintoMPArceMValenzuelaRArandaEet al. Independent Anti-Angiogenic Capacities of Coagulation Factors X and Xa. J Cell Physiol (2014) 229:1673–80. doi: 10.1002/jcp.24612

  • CrossRef
  • Google Scholar

• 17

  BaeJ-SRezaieA. Thrombin Inhibits Nuclear Factor kB and RhoA Pathways in Cytokine-stimulated Vascular Endotheliar Cells when EPCR is Occupied by Protein C. Thromb Haemost (2009) 101:513–20. doi: 10.1038/jid.2014.371

  • CrossRef
  • Google Scholar

• 18

  PontarolloGMannABrandãoIMalinarichFSchöpfMReinhardtC. Protease-activated receptor signaling in intestinal permeability regulation. FEBS J (2020) 287:645–58. doi: 10.1111/febs.15055

  • CrossRef
  • Google Scholar

• 19

  GrafCWilgenbusPKitanoMMacher-GöppingerSRufW. Macrophage Factor Xa Signaling Promotes Cancer Immune Evasion. Blood (2018) 132:135. doi: 10.1182/blood-2018-99-119167

  • CrossRef
  • Google Scholar

• 20

  GrafCWilgenbusPPagelSPottJMariniFReydaSet al. Myeloid cell-synthesized coagulation factor X dampens antitumor immunity. Sci Immunol (2019) 4:eaaw8405. doi: 10.1126/sciimmunol.aaw8405

  • CrossRef
  • Google Scholar

• 21

  YangYStangASchweickertPGLanmanNAPaulENMoniaBPet al. Thrombin Signaling Promotes Pancreatic Adenocarcinoma through PAR-1-Dependent Immune Evasion. Cancer Res (2019) 79(13):3417–30. doi: 10.1158/0008-5472.CAN-18-3206

  • CrossRef
  • Google Scholar

• 22

  GrimseyNLinHTrejoJ. Endosomal signaling by protease-activated receptors. Methods Enzymol (2014) 535:389–401. doi: 10.1016/B978-0-12-397925-4.00022-5

  • CrossRef
  • Google Scholar

• 23

  ChenKDWangCCTsaiMCWuCHYangHJChenLYet al. Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma. Cell Death Dis (2014) 5:e1244. doi: 10.1038/cddis.2014.212

  • CrossRef
  • Google Scholar

• 24

  DuCZhangTXiaoXShiYDuanHRenY. Protease-activated receptor-2 promotes kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway. Biochem J (2017) 474:2733–47. doi: 10.1042/BCJ20170272

  • CrossRef
  • Google Scholar

• 25

  HuangKTKuoIYTsaiMCWuCHHsuLWChenLYet al. Factor VII-Induced MicroRNA-135a Inhibits Autophagy and Is Associated with Poor Prognosis in Hepatocellular Carcinoma. Mol Ther Nucleic Acids (2017) 9:274–83. doi: 10.1016/j.omtn.2017.10.002

  • CrossRef
  • Google Scholar

• 26

  BabaMTakeshigeKBabaNOhsumiY. Ultrastructural analysis of the autophagic process in yeast: Detection of autophagosomes and their characterization. J Cell Biol (1994) 124:903–13. doi: 10.1083/jcb.124.6.903

  • CrossRef
  • Google Scholar

• 27

  BabaMOhsumiYOsumiM. Analysis of the Membrane Structures Involved in Autophagy in Yeast by Freeze-Replica Method. Cell Struct Funct (1995) 20:465–71. doi: 10.1247/csf.20.465

  • CrossRef
  • Google Scholar

• 28

  NakamuraNMatsuuraAWadaYOhsumiY. Acidification of vacuoles is required for autophagic degradation in the yeast, Saccharomyces cerevisiae. J Biochem (1997) 121:338–44. doi: 10.1093/oxfordjournals.jbchem.a021592

  • CrossRef
  • Google Scholar

• 29

  YangZKlionskyDJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol (2010) 22:124–31. doi: 10.1016/j.ceb.2009.11.014

  • CrossRef
  • Google Scholar

• 30

  EskelinenEL. Autophagy: Supporting cellular and organismal homeostasis by self-eating. Int J Biochem Cell Biol (2019) 111:1–10. doi: 10.1016/j.biocel.2019.03.010

  • CrossRef
  • Google Scholar

• 31

  MorselliEGalluzziLKeppOVicencioJMCriolloAMaiuriMCet al. Anti- and pro-tumor functions of autophagy. Biochim Biophys Acta (2009) 1793:1524–32. doi: 10.1016/j.bbamcr.2009.01.006

  • CrossRef
  • Google Scholar

• 32

  WhiteEDiPaolaRS. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res (2009) 15:5308–16. doi: 10.1158/1078-0432.CCR-07-5023

  • CrossRef
  • Google Scholar

• 33

  ParzychKRKlionskyDJ. An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal (2014) 20:460–73. doi: 10.1089/ars.2013.5371

  • CrossRef
  • Google Scholar

• 34

  YangYKlionskyDJ. Autophagy and disease: unanswered questions. Cell Death Differ (2020) 27:858–71. doi: 10.1038/s41418-019-0480-9

  • CrossRef
  • Google Scholar

• 35

  DowerCMWillsCAFrischSMWangHG. Mechanisms and context underlying the role of autophagy in cancer metastasis. Autophagy (2018) 14:1110–28. doi: 10.1080/15548627.2018.1450020

  • CrossRef
  • Google Scholar

• 36

  GalluzziLPietrocolaFBravo-San PedroJMAmaravadiRKBaehreckeEHCecconiFet al. Autophagy in malignant transformation and cancer progression. EMBO J (2015) 34:856–80. doi: 10.15252/embj.201490784

  • CrossRef
  • Google Scholar

• 37

  HansenMRubinszteinDCWalkerDW. Autophagy as a promoter of longevity: insights from model organisms. Nat Rev Mol Cell Biol (2018) 19:579–93. doi: 10.1038/s41580-018-0033-y

  • CrossRef
  • Google Scholar

• 38

  KaurJDebnathJ. Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol (2015) 16:461–72. doi: 10.1038/nrm4024

  • CrossRef
  • Google Scholar

• 39

  EskelinenEL. New insights into the mechanisms of macroautophagy in mammalian cells. Int Rev Cell Mol Biol (2008) 266:207–47. doi: 10.1016/S1937-6448(07)66005-5

  • CrossRef
  • Google Scholar

• 40

  KroemerGMarinoGLevineB. Autophagy and the integrated stress response. Mol Cell (2010) 40:280–93. doi: 10.1016/j.molcel.2010.09.023

  • CrossRef
  • Google Scholar

• 41

  KimmelmanACWhiteE. Autophagy and Tumor Metabolism. Cell Metab (2017) 25:1037–43. doi: 10.1016/j.cmet.2017.04.004

  • CrossRef
  • Google Scholar

• 42

  MonkkonenTDebnathJ. Inflammatory signaling cascades and autophagy in cancer. Autophagy (2018) 14:190–8. doi: 10.1080/15548627.2017.1345412

  • CrossRef
  • Google Scholar

• 43

  RavananPSrikumarIFTalwarP. Autophagy: The spotlight for cellular stress responses. Life Sci (2017) 188:53–67. doi: 10.1016/j.lfs.2017.08.029

  • CrossRef
  • Google Scholar

• 44

  KlionskyDJCreggJMDunnWAJr.EmrSDSakaiYSandovalIVet al. A unified nomenclature for yeast autophagy-related genes. Dev Cell (2003) 5:539–45. doi: 10.1016/s1534-5807(03)00296-x

  • CrossRef
  • Google Scholar

• 45

  ToozeSAYoshimoriT. The origin of the autophagosomal membrane. Nat Cell Biol (2010) 12:831–5. doi: 10.1038/ncb0910-831

  • CrossRef
  • Google Scholar

• 46

  MizushimaNKomatsuM. Autophagy: renovation of cells and tissues. Cell (2011) 147:728–41. doi: 10.1016/j.cell.2011.10.026

  • CrossRef
  • Google Scholar

• 47

  ReggioriFUngermannC. Autophagosome Maturation and Fusion. J Mol Biol (2017) 429:486–96. doi: 10.1016/j.jmb.2017.01.002

  • CrossRef
  • Google Scholar

• 48

  AxeELWalkerSAManifavaMChandraPRoderickHLHabermannAet al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol (2008) 182:685–701. doi: 10.1083/jcb.200803137

  • CrossRef
  • Google Scholar

• 49

  PolsonHEde LartigueJRigdenDJReedijkMUrbeSClagueMJet al. Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation. Autophagy (2010) 6:506–22. doi: 10.4161/auto.6.4.11863

  • CrossRef
  • Google Scholar

• 50

  ReggioriFTuckerKAStromhaugPEKlionskyDJ. The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure. Dev Cell (2004) 6:79–90. doi: 10.1016/s1534-5807(03)00402-7

  • CrossRef
  • Google Scholar

• 51

  YamamotoHKakutaSWatanabeTMKitamuraASekitoTKondo-KakutaCet al. Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol (2012) 198:219–33. doi: 10.1083/jcb.201202061

  • CrossRef
  • Google Scholar

• 52

  LongattiALambCARaziMYoshimuraSBarrFAToozeSA. TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes. J Cell Biol (2012) 197:659–75. doi: 10.1083/jcb.201111079

  • CrossRef
  • Google Scholar

• 53

  NakatogawaH. Mechanisms governing autophagosome biogenesis. Nat Rev Mol Cell Biol (2020) 21:439–58. doi: 10.1038/s41580-020-0241-0

  • CrossRef
  • Google Scholar

• 54

  ChowdhurySOtomoCLeitnerAOhashiKAebersoldRLanderGCet al. Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex. Proc Natl Acad Sci U S A (2018) 115:E9792–801. doi: 10.1073/pnas.1811874115

  • CrossRef
  • Google Scholar

• 55

  MaedaSOtomoCOtomoT. The autophagic membrane tether ATG2A transfers lipids between membranes. Elife (2019) 8:e45777. doi: 10.7554/eLife.45777

  • CrossRef
  • Google Scholar

• 56

  OhsumiYMizushimaN. Two ubiquitin-like conjugation systems essential for autophagy. Semin Cell Dev Biol (2004) 15(2):231–6. doi: 10.1016/j.semcdb.2003.12.004

  • CrossRef
  • Google Scholar

• 57

  KlionskyDJSchulmanBA. Dynamic regulation of macroautophagy by distinctive ubiquitin-like proteins. Nat Struct Mol Biol (2014) 21:336–45. doi: 10.1038/nsmb.2787

  • CrossRef
  • Google Scholar

• 58

  OtomoCMetlagelZTakaesuGOtomoT. Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy. Nat Struct Mol Biol (2013) 20:59–66. doi: 10.1038/nsmb.2431

  • CrossRef
  • Google Scholar

• 59

  HanadaTNodaNNSatomiYIchimuraYFujiokaYTakaoTet al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem (2007) 282:37298–302. doi: 10.1074/jbc.C700195200

  • CrossRef
  • Google Scholar

• 60

  KlionskyDJAbdelmohsenKAbeAAbedinMJAbeliovichHAcevedo ArozenaAet al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy (2016) 12:1–222. doi: 10.1080/15548627.2015.1100356

  • CrossRef
  • Google Scholar

• 61

  TanidaIUenoTKominamiE. Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes. J Biol Chem (2004) 279:47704–10. doi: 10.1074/jbc.M407016200

  • CrossRef
  • Google Scholar

• 62

  YoshiiSRMizushimaN. Monitoring and Measuring Autophagy. Int J Mol Sci (2017) 18:865. doi: 10.3390/ijms18091865

  • CrossRef
  • Google Scholar

• 63

  PengHYangJLiGYouQHanWLiTet al. Ubiquitylation of p62/sequestosome1 activates its autophagy receptor function and controls selective autophagy upon ubiquitin stress. Cell Res (2017) 27:657–74. doi: 10.1038/cr.2017.40

  • CrossRef
  • Google Scholar

• 64

  EskelinenEL. Maturation of autophagic vacuoles in Mammalian cells. Autophagy (2005) 1:1–10. doi: 10.4161/auto.1.1.1270

  • CrossRef
  • Google Scholar

• 65

  ZhaoYGZhangH. Autophagosome maturation: An epic journey from the ER to lysosomes. J Cell Biol (2019) 218:757–70. doi: 10.1083/jcb.201810099

  • CrossRef
  • Google Scholar

• 66

  NakamuraSYoshimoriT. New insights into autophagosome-lysosome fusion. J Cell Sci (2017) 130:1209–16. doi: 10.1242/jcs.196352

  • CrossRef
  • Google Scholar

• 67

  JiaRGuardiaCMPuJChenYBonifacinoJS. BORC coordinates encounter and fusion of lysosomes with autophagosomes. Autophagy (2017) 13:1648–63. doi: 10.1080/15548627.2017.1343768

  • CrossRef
  • Google Scholar

• 68

  JiangPNishimuraTSakamakiYItakuraEHattaTNatsumeTet al. The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol Biol Cell (2014) 25:1327–37. doi: 10.1091/mbc.E13-08-0447

  • CrossRef
  • Google Scholar

• 69

  RongYMcPheeCKDengSHuangLChenLLiuMet al. Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation. Proc Natl Acad Sci U S A (2011) 108:7826–31. doi: 10.1073/pnas.1013800108

  • CrossRef
  • Google Scholar

• 70

  YuLMcPheeCKZhengLMardonesGARongYPengJet al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature (2010) 465:942–6. doi: 10.1038/nature09076

  • CrossRef
  • Google Scholar

• 71

  AvalosYCanalesJBravo-SaguaRCriolloALavanderoSQuestAF. Tumor suppression and promotion by autophagy. BioMed Res Int (2014) 2014:603980. doi: 10.1155/2014/603980

  • CrossRef
  • Google Scholar

• 72

  LiXHeSMaB. Autophagy and autophagy-related proteins in cancer. Mol Cancer (2020) 19:12. doi: 10.1186/s12943-020-1138-4

  • CrossRef
  • Google Scholar

• 73

  EfeyanAZoncuRSabatiniDM. Amino acids and mTORC1: from lysosomes to disease. Trends Mol Med (2012) 18:524–33. doi: 10.1016/j.molmed.2012.05.007

  • CrossRef
  • Google Scholar

• 74

  ZoncuREfeyanASabatiniDM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol (2011) 12:21–35. doi: 10.1038/nrm3025

  • CrossRef
  • Google Scholar

• 75

  EfeyanAZoncuRChangSGumperISnitkinHWolfsonRLet al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature (2013) 493:679–83. doi: 10.1038/nature11745

  • CrossRef
  • Google Scholar

• 76

  JungCHRoSHCaoJOttoNMKimDH. mTOR regulation of autophagy. FEBS Lett (2010) 584:1287–95. doi: 10.1016/j.febslet.2010.01.017

  • CrossRef
  • Google Scholar

• 77

  AlersSLofflerASWesselborgSStorkB. Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol (2012) 32:2–11. doi: 10.1128/MCB.06159-11

  • CrossRef
  • Google Scholar

• 78

  ChanEY. mTORC1 phosphorylates the ULK1-mAtg13-FIP200 autophagy regulatory complex. Sci Signal (2009) 2:pe51. doi: 10.1126/scisignal.284pe51

  • CrossRef
  • Google Scholar

• 79

  HosokawaNHaraTKaizukaTKishiCTakamuraAMiuraYet al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell (2009) 20:1981–91. doi: 10.1091/mbc.E08-12-1248

  • CrossRef
  • Google Scholar

• 80

  KimJKunduMViolletBGuanKL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol (2011) 13:132–41. doi: 10.1038/ncb2152

  • CrossRef
  • Google Scholar

• 81

  HaJGuanKLKimJ. AMPK and autophagy in glucose/glycogen metabolism. Mol Aspects Med (2015) 46:46–62. doi: 10.1016/j.mam.2015.08.002

  • CrossRef
  • Google Scholar

• 82

  HardieDG. AMPK–sensing energy while talking to other signaling pathways. Cell Metab (2014) 20:939–52. doi: 10.1016/j.cmet.2014.09.013

  • CrossRef
  • Google Scholar

• 83

  DunlopEATeeAR. The kinase triad, AMPK, mTORC1 and ULK1, maintains energy and nutrient homoeostasis. Biochem Soc Trans (2013) 41:939–43. doi: 10.1042/BST20130030

  • CrossRef
  • Google Scholar

• 84

  Chavez-DominguezRPerez-MedinaMLopez-GonzalezJSGalicia-VelascoMAguilar-CazaresD. The Double-Edge Sword of Autophagy in Cancer: From Tumor Suppression to Pro-tumor Activity. Front Oncol (2020) 10:578418. doi: 10.3389/fonc.2020.578418

  • CrossRef
  • Google Scholar

• 85

  ChoiAMRyterSWLevineB. Autophagy in human health and disease. N Engl J Med (2013) 368:1845–6. doi: 10.1056/NEJMc1303158

  • CrossRef
  • Google Scholar

• 86

  VerzellaDPescatoreACapeceDVecchiottiDUrsiniMVFranzosoGet al. Life, death, and autophagy in cancer: NF-κB turns up everywhere. Cell Death Dis (2020) 11:210. doi: 10.1038/s41419-020-2399-y

  • CrossRef
  • Google Scholar

• 87

  PattingreSTassaAQuXGarutiRLiangXHMizushimaNet al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell (2005) 122:927–39. doi: 10.1016/j.cell.2005.07.002

  • CrossRef
  • Google Scholar

• 88

  SinghSSVatsSChiaAYTanTZDengSOngMSet al. Dual role of autophagy in hallmarks of cancer. Oncogene (2018) 37:1142–58. doi: 10.1038/s41388-017-0046-6

  • CrossRef
  • Google Scholar

• 89

  SonYCheongYKKimNHChungHTKangDGPaeHO. Mitogen-Activated Protein Kinases and Reactive Oxygen Species: How Can ROS Activate MAPK Pathways? J Signal Transduct (2011) 2011:792639. doi: 10.1155/2011/792639

  • CrossRef
  • Google Scholar

• 90

  GalluzziLBravo-San PedroJMLevineBGreenDRKroemerG. Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles. Nat Rev Drug Discov (2017) 16:487–511. doi: 10.1038/nrd.2017.22

  • CrossRef
  • Google Scholar

• 91

  PanTRawalPWuYXieWJankovicJLeW. Rapamycin protects against rotenone-induced apoptosis through autophagy induction. Neuroscience (2009) 164:541–51. doi: 10.1016/j.neuroscience.2009.08.014

  • CrossRef
  • Google Scholar

• 92

  PetiotAOgier-DenisEBlommaartEFMeijerAJCodognoP. Distinct classes of phosphatidylinositol 3’-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem (2000) 275:992–8. doi: 10.1074/jbc.275.2.992

  • CrossRef
  • Google Scholar

• 93

  GalluzziLBaehreckeEHBallabioABoyaPBravo-San PedroJMCecconiFet al. Molecular definitions of autophagy and related processes. EMBO J (2017) 36:1811–36. doi: 10.15252/embj.201796697

  • CrossRef
  • Google Scholar

• 94

  MauvezinCNeufeldTP. Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. Autophagy (2015) 11:1437–8. doi: 10.1080/15548627.2015.1066957

  • CrossRef
  • Google Scholar

• 95

  PasquierB. Autophagy inhibitors. Cell Mol Life Sci (2016) 73:985–1001. doi: 10.1007/s00018-015-2104-y

  • CrossRef
  • Google Scholar

• 96

  CadwellKDebnathJ. Beyond self-eating: The control of nonautophagic functions and signaling pathways by autophagy-related proteins. J Cell Biol (2018) 217:813–22. doi: 10.1083/jcb.201706157

  • CrossRef
  • Google Scholar

• 97

  DereticVLevineB. Autophagy balances inflammation in innate immunity. Autophagy (2018) 14:243–51. doi: 10.1080/15548627.2017.1402992

  • CrossRef
  • Google Scholar

• 98

  NewJThomasSM. Autophagy-dependent secretion: mechanism, factors secreted, and disease implications. Autophagy (2019) 15:1682–93. doi: 10.1080/15548627.2019.1596479

  • CrossRef
  • Google Scholar

• 99

  GonzalezCDResnikRVaccaroMI. Secretory Autophagy and Its Relevance in Metabolic and Degenerative Disease. Front Endocrinol (2020) 11:266. doi: 10.3389/fendo.2020.00266

  • CrossRef
  • Google Scholar

• 100

  JiangSDupontNCastilloEFDereticV. Secretory versus degradative autophagy: unconventional secretion of inflammatory mediators. J Innate Immun (2013) 5:471–9. doi: 10.1159/000346707

  • CrossRef
  • Google Scholar

• 101

  GalluzziLGreenDR. Autophagy-Independent Functions of the Autophagy Machinery. Cell (2019) 177:1682–99. doi: 10.1016/j.cell.2019.05.026

  • CrossRef
  • Google Scholar

• 102

  NewJArnoldLAnanthMAlviSThorntonMWernerLet al. Secretory Autophagy in Cancer-Associated Fibroblasts Promotes Head and Neck Cancer Progression and Offers a Novel Therapeutic Target. Cancer Res (2017) 77:6679–91. doi: 10.1158/0008-5472.CAN-17-1077

  • CrossRef
  • Google Scholar

• 103

  PettersenKAndersenSDegenSTadiniVGrosjeanJHatakeyamaSet al. Cancer cachexia associates with a systemic autophagy-inducing activity mimicked by cancer cell-derived IL-6 trans-signaling. Sci Rep (2017) 7:2046. doi: 10.1038/s41598-017-02088-2

  • CrossRef
  • Google Scholar

• 104

  DupontNJiangSPilliMOrnatowskiWBhattacharyaDDereticV. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1beta. EMBO J (2011) 30:4701–11. doi: 10.1038/emboj.2011.398

  • CrossRef
  • Google Scholar

• 105

  ThorburnJHoritaHRedzicJHansenKFrankelAEThorburnA. Autophagy regulates selective HMGB1 release in tumor cells that are destined to die. Cell Death Differ (2009) 16:175–83. doi: 10.1038/cdd.2008.143

  • CrossRef
  • Google Scholar

• 106

  TorisuTTorisuKLeeIHLiuJMalideDCombsCAet al. Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor. Nat Med (2013) 19:1281–7. doi: 10.1038/nm.3288

  • CrossRef
  • Google Scholar

• 107

  ZahoorMFarhanH. Crosstalk of Autophagy and the Secretory Pathway and Its Role in Diseases. Int Rev Cell Mol Biol (2018) 337:153–84. doi: 10.1016/bs.ircmb.2017.12.004

  • CrossRef
  • Google Scholar

• 108

  LockRKenificCMLeidalAMSalasEDebnathJ. Autophagy-dependent production of secreted factors facilitates oncogenic RAS-driven invasion. Cancer Discov (2014) 4:466–79. doi: 10.1158/2159-8290.CD-13-0841

  • CrossRef
  • Google Scholar

• 109

  WangKWeiYLiuWLiuLGuoZFanCet al. Mechanical Stress-Dependent Autophagy Component Release via Extracellular Nanovesicles in Tumor Cells. ACS Nano (2019) 13:4589–602. doi: 10.1021/acsnano.9b00587

  • CrossRef
  • Google Scholar

• 110

  ZhangLZhangSYaoJLoweryFJZhangQHuangWCet al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature (2015) 527:100–4. doi: 10.1038/nature15376

  • CrossRef
  • Google Scholar

• 111

  ZomerAMaynardCVerweijFJKamermansASchaferRBeerlingEet al. In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell (2015) 161:1046–57. doi: 10.1016/j.cell.2015.04.042

  • CrossRef
  • Google Scholar

• 112

  DegenhardtKMathewRBeaudoinBBrayKAndersonDChenGet al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell (2006) 10:51–64. doi: 10.1016/j.ccr.2006.06.001

  • CrossRef
  • Google Scholar

• 113

  MathewRKarpCMBeaudoinBVuongNChenGChenHYet al. Autophagy Suppresses Tumorigenesis through Elimination of p62. Cell (2009) 137:1062–75. doi: 10.1016/j.cell.2009.03.048

  • CrossRef
  • Google Scholar

• 114

  MathewRKarantza-WadsworthVWhiteE. Role of autophagy in cancer. Nat Rev Cancer (2007) 7:961–7. doi: 10.1038/nrc2254

  • CrossRef
  • Google Scholar

• 115

  LorinSHamaïAMehrpourMCodognoP. Autophagy regulation and its role in cancer. Semin Cancer Biol (2013) 23:361–79. doi: 10.1016/j.semcancer.2013.06.007

  • CrossRef
  • Google Scholar

• 116

  RemikerASPalumboJS. Mechanisms coupling thrombin to metastasis and tumorigenesis. Thromb Res (2018) 164:S29–33. doi: 10.1016/j.thromres.2017.12.020

  • CrossRef
  • Google Scholar

• 117

  HenriksonKPSalazarSLFentonJWPentecostBT. Role of thrombin receptor in breast cancer invasiveness. Br J Cancer (1999) 79:401–6. doi: 10.1038/sj.bjc.6690063

  • CrossRef
  • Google Scholar

• 118

  DawoodSAustinLCristofanilliM. Cancer stem cells: implications for cancer therapy. Oncol (Williston Park) (2014) 28:1101–7, 1110.

  • Google Scholar

• 119

  OrellanaRKatoSEricesRBravoMLGonzalezPOlivaBet al. Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells. BMC Cancer (2015) 15:290. doi: 10.1186/s12885-015-1304-z

  • CrossRef
  • Google Scholar

• 120

  ShakerHHarrisonHClarkeRLandbergGBundredNJVersteegHHet al. Tissue Factor promotes breast cancer stem cell activity in vitro. Oncotarget (2017) 8:25915–27. doi: 10.18632/oncotarget.13928

  • CrossRef
  • Google Scholar

• 121

  HattoriTKawanoSTanakaSMatsubaraRSakamotoTHashiguchiYet al. Elevated expression of protease-activated receptor 1 via DNp63 downregulation contributes to nodal metastasis in oral squamous cell carcinoma. Int J Oral Maxillofac Surg (2020) S0901–5027(20):30197–1. doi: 10.1016/j.ijom.2020.04.021

  • CrossRef
  • Google Scholar

• 122

  RydénLGrabauDSchaffnerFJönssonPERufWBeltingM. Evidence for tissue factor phosphorylation and its correlation with protease-activated receptor expression and the prognosis of primary breast cancer. Int J Cancer (2010) 126:2330–40. doi: 10.1002/ijc.24921

  • CrossRef
  • Google Scholar

• 123

  GrozovskyRGianniniSFaletHHoffmeisterKM. Regulating billions of blood platelets: Glycans and beyond. Blood (2015) 126:1877–84. doi: 10.1182/blood-2015-01-569129

  • CrossRef
  • Google Scholar

• 124

  BehrensKAlexanderWS. Cytokine control of megakaryopoiesis. Growth Factors (2018) 36:89–103. doi: 10.1080/08977194.2018.1498487

  • CrossRef
  • Google Scholar

• 125

  GeddisAE. Megakayopoiesis. Semin Hematol (2010) 47:212–9. doi: 10.1053/j.seminhematol.2010.03.001.Megakaryopoiesis

  • CrossRef
  • Google Scholar

• 126

  GeddisAEFoxNEKaushanskyK. Phosphatidylinositol 3-Kinase Is Necessary but Not Sufficient for Thrombopoietin-induced Proliferation in Engineered Mpl-bearing Cell Lines as Well as in Primary Megakaryocytic Progenitors. J Biol Chem (2001) 276:34473–9. doi: 10.1074/jbc.M105178200

  • CrossRef
  • Google Scholar

• 127

  GuoTWangXQuYYinYJingTZhangQ. Megakaryopoiesis and platelet production: insight into hematopoietic stem cell proliferation and differentiation. Stem Cell Invest (2015) 2:3. doi: 10.3978/j.issn.2306-9759.2015.02.01

  • CrossRef
  • Google Scholar

• 128

  MatsumuraINakajimaKWakaoHHattoriSHashimotoKSugaharaHet al. Involvement of Prolonged Ras Activation in Thrombopoietin-Induced Megakaryocytic Differentiation of a Human Factor-Dependent Hematopoietic Cell Line. Mol Cell Biol (1998) 18:4282–90. doi: 10.1128/mcb.18.7.4282

  • CrossRef
  • Google Scholar

• 129

  ColosettiPPuissantARobertGLucianoFJacquelAGounonPet al. Autophagy is an important event for megakaryocytic differentiation of the chronic myelogenous leukemia K562 cell line. Autophagy (2009) 5:1092–8. doi: 10.4161/auto.5.8.9889

  • CrossRef
  • Google Scholar

• 130

  RaslovaHBacciniVLoussaiefLCombaBLargheroJDebiliNet al. Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation. Blood (2006) 107:2303–10. doi: 10.1182/blood-2005-07-3005

  • CrossRef
  • Google Scholar

• 131

  SalibaJSaint-MartinCDi StefanoALengletGMartyCKerenBet al. Germline duplication of ATG2B and GSKIP predisposes to familial myeloid malignancies. Nat Genet (2015) 47:1131–40. doi: 10.1038/ng.3380

  • CrossRef
  • Google Scholar

• 132

  KozumaYYukiSNinomiyaHNagasawaTKojimaH. Caspase activation is involved in early megakaryocyte differentiation but not in platelet production from megakaryocytes. Leukemia (2009) 23:1080–6. doi: 10.1038/leu.2009.7

  • CrossRef
  • Google Scholar

• 133

  CleyratCDarehshouriASteinkampMVilaineMBoassaBEllismanMet al. Mpl traffics to the cell surface through conventional and unconventional routes. Traffic (2014) 15:961–82. doi: 10.1161/CIRCULATIONAHA.110.956839

  • CrossRef
  • Google Scholar

• 134

  HitchcockISChenMMKingJRKaushanskyK. YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation. Blood (2008) 112:2222–31. doi: 10.1182/blood-2008-01-134049

  • CrossRef
  • Google Scholar

• 135

  ValetCLevadeMChicanneGBilangesBCabouCViaudJet al. A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth. Blood (2017) 130:2032–42. doi: 10.1182/blood-2017-04-781641

  • CrossRef
  • Google Scholar

• 136

  BertovićIKurelićRMiloševićIBenderMKraussMHauckeVet al. Vps34 derived phosphatidylinositol 3-monophosphate modulates megakaryocyte maturation and proplatelet production through late endosomes/lysosomes. J Thromb Haemost (2020), 18:1–17. doi: 10.1111/jth.14764

  • CrossRef
  • Google Scholar

• 137

  GuerrieroRParoliniITestaUSamoggiaPPetrucciESargiacomoMet al. Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes. J Cell Sci (2006) 119:744–52. doi: 10.1242/jcs.02784

  • CrossRef
  • Google Scholar

• 138

  CaoYCaiJZhangSYuanNLiXFangYet al. Loss of autophagy leads to failure in megakaryopoiesis, megakaryocyte differentiation, and thrombopoiesis in mice. Exp Hematol (2015) 43:488–94. doi: 10.1016/j.exphem.2015.01.001

  • CrossRef
  • Google Scholar

• 139

  OusephMMHuangYBanerjeeMJoshiSMacDonaldLZhongYet al. Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. Blood (2015) 126:1224–33. doi: 10.1182/blood-2014-09-598722

  • CrossRef
  • Google Scholar

• 140

  CaoYZhangSYuanNWangJLiXXuFet al. Hierarchal autophagic divergence of hematopoietic system. J Biol Chem (2015) 290:23050–63. doi: 10.1074/jbc.M115.650028

  • CrossRef
  • Google Scholar

• 141

  ThompsonCBJakubowskiJA. The pathophysiology and clinical relevance of platelet heterogeneity. Blood (1988) 72:1–8. doi: 10.1182/blood.v72.1.1.bloodjournal7211

  • CrossRef
  • Google Scholar

• 142

  KaserABrandacherGSteurerWKaserSOffnerFAZollerHet al. Interleukin-6 stimulates thrombopoiesis through thrombopoietin: Role in inflammatory thrombocytosis. Blood (2001) 98:2720–5. doi: 10.1182/blood.V98.9.2720

  • CrossRef
  • Google Scholar

• 143

  KiritoKOsawaMMoritaHShimizuRYamamotoMOdaAet al. A functional role of Stat3 in in vivo megakaryopoiesis. Blood (2002) 99:3220–7. doi: 10.1182/blood.V99.9.3220

  • CrossRef
  • Google Scholar

• 144

  HaasSHanssonJKlimmeckDLoefflerDVeltenLUckelmannHet al. Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors. Cell Stem Cell (2015) 17:422–34. doi: 10.1016/j.stem.2015.07.007

  • CrossRef
  • Google Scholar

• 145

  NishimuraSNagasakiMKunishimaSSawaguchiASakataASakaguchiHet al. IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs. J Cell Biol (2015) 209:453–66. doi: 10.1083/jcb.201410052

  • CrossRef
  • Google Scholar

• 146

  LewisJCMaldonadoJEMannKGMoertelCG. Ultrastructural cytochemistry of platelets and megakaryocytes in the carcinoid syndrome. Mayo Clin Proc (1976) 51:585—593.

  • Google Scholar

• 147

  BanerjeeMHuangYOusephMMJoshiSPokrovskayaIStorrieBet al. Autophagy in Platelets. Methods Mol Biol (2019) 1880:511–28. doi: 10.1007/978-1-4939-8873-0_32

  • CrossRef
  • Google Scholar

• 148

  KumaAMizushimaN. Chromosomal mapping of the GFP-LC3 transgene in GFP-LC3 mice. Autophagy (2008) 4:61–2. doi: 10.4161/auto.4846

  • CrossRef
  • Google Scholar

• 149

  ArsovILiXMatthewsGCoradinJHartmannBSimonAKet al. BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development. Cell Death Differ (2008) 15:1385–95. doi: 10.1038/cdd.2008.59

  • CrossRef
  • Google Scholar

• 150

  RowleyJWOlerAJTolleyNDO. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes (Blood (2011) 118, 14 (e101-e111)). Blood (2014) 123:3843. doi: 10.1182/blood-2011-03-339705.An

  • CrossRef
  • Google Scholar

• 151

  FengWChangCLuoDSuHYuSHuaWet al. Dissection of autophagy in human platelets. Autophagy (2014) 10:642–51. doi: 10.4161/auto.27832

  • CrossRef
  • Google Scholar

• 152

  LeeSHDuJStithamJAtteyaGLeeSXiangYet al. Inducing mitophagy in diabetic platelets protects against severe oxidative stress. EMBO Mol Med (2016) 8:779–95. doi: 10.15252/emmm.201506046

  • CrossRef
  • Google Scholar

• 153

  LiuYHuMLuoDYueMWangSChenXet al. Class III PI3K positively regulates platelet activation and thrombosis via PI(3)P-directed function of NADPH oxidase. Arterioscler Thromb Vasc Biol (2017) 37:2075–86. doi: 10.1161/ATVBAHA.117.309751

  • CrossRef
  • Google Scholar

• 154

  MenterDGKopetzSHawkESoodAKLoreeJMGreselePet al. Platelet “first responders” in wound response, cancer, and metastasis. Cancer Metastasis Rev (2017) 36:199–213. doi: 10.1007/s10555-017-9682-0

  • CrossRef
  • Google Scholar

• 155

  NieswandtBVarga-SzaboDElversM. Integrins in platelet activation. J Thromb Haemost (2009) 206–9. doi: 10.1111/j.1538-7836.2009.03370.x

  • CrossRef
  • Google Scholar

• 156

  WangXFuYFLiuXFengGXiongDMuGFet al. ROS Promote ox-LDL-induced platelet activation by up-regulating autophagy through the inhibition of the PI3K/AKT/mTOR pathway. Cell Physiol Biochem (2018) 50:1779–93. doi: 10.1159/000494795

  • CrossRef
  • Google Scholar

• 157

  ZhangWRenHXuCZhuCWuHLiuDet al. Hypoxic mitophagy regulates mitochondrial quality and platelet activation and determines severity of I/R heart injury. Elife (2016) 5:1–20. doi: 10.7554/eLife.21407

  • CrossRef
  • Google Scholar

• 158

  KaniaEPajakBOrzechowskiA. Calcium homeostasis and ER stress in control of autophagy in cancer cells. BioMed Res Int (2015) 2015:352794. doi: 10.1155/2015/352794

  • CrossRef
  • Google Scholar

• 159

  GrotemeierAAlersSPfistererSGPaaschFDaubrawaMDieterleAet al. AMPK-independent induction of autophagy by cytosolic Ca2+ increase. Cell Signal (2010) 22:914–25. doi: 10.1016/j.cellsig.2010.01.015

  • CrossRef
  • Google Scholar

• 160

  ZhuXChenYCuiY. Absolute Neutrophil Count and Mean Platelet Volume in the Blood as Biomarkers to Detect Lung Cancer. Dis Markers (2020) 2020):1371964. doi: 10.1155/2020/1371964

  • CrossRef
  • Google Scholar

• 161

  RachidiSKaurMLautenschlaegerTLiZ. Platelet count correlates with stage and predicts survival in melanoma. Platelets (2019) 30:1042–6. doi: 10.1080/09537104.2019.1572879

  • CrossRef
  • Google Scholar

• 162

  St-OngeMLagardeSLaflammeCRollet-LabelleEMaroisLNaccachePHet al. Proteinase-activated receptor-2 up-regulation by Fcγ-receptor activation in human neutrophils. FASEB J (2010) 24:2116–25. doi: 10.1096/fj.09-146167

  • CrossRef
  • Google Scholar

• 163

  LabelleMBegumSHynesRO. Direct Signaling between Platelets and Cancer Cells Induces an Epithelial-Mesenchymal-Like Transition and Promotes Metastasis. Cancer Cell (2011) 20:576–90. doi: 10.1016/j.ccr.2011.09.009

  • CrossRef
  • Google Scholar

• 164

  LabelleMBegumSHynesRO. Platelets guide the formation of early metastatic niches. Proc Natl Acad Sci U S A (2014) 111:E3053–61. doi: 10.1073/pnas.1411082111

  • CrossRef
  • Google Scholar

• 165

  EricesRCubillosSAravenaRSantoroFMarquezMOrellanaRet al. Diabetic concentrations of metformin inhibit platelet-mediated ovarian cancer cell progression. Oncotarget (2017) 8:20865–80. doi: 10.18632/oncotarget.15348

  • CrossRef
  • Google Scholar

• 166

  JosaVFerencziSSzalaiRFuderEKutiDHorvathKet al. Thrombocytosis and E ff ects of IL-6 Knock-Out in a Colitis-Associated Cancer Model. Int J Mol Sci (2020) 21(17):6218. doi: 10.3390/ijms21176218

  • CrossRef
  • Google Scholar

• 167

  AvecillaSTHattoriKHeissigBTejadaRLiaoFShidoKet al. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med (2004) 10:64–71. doi: 10.1038/nm973

  • CrossRef
  • Google Scholar

• 168

  CasellaIFecciaTChelucciCSamoggiaPCastelliGGuerrieroRet al. Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor. Blood (2003) 101:1316–23. doi: 10.1182/blood-2002-07-2184

  • CrossRef
  • Google Scholar

• 169

  PitchfordSCLodieTRankinSM. VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice. Blood (2012) 120:2787–95. doi: 10.1182/blood-2011-09-378174

  • CrossRef
  • Google Scholar

• 170

  CoppolaSNarcisoLFecciaTBonciDCalabròLMorsilliOet al. Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line. Cell Death Differ (2006) 13:61–74. doi: 10.1038/sj.cdd.4401698

  • CrossRef
  • Google Scholar

• 171

  MonlishDABhattSTSchuettpelzLG. The role of toll-like receptors in hematopoietic malignancies. Front Immunol (2016) 7:390. doi: 10.3389/fimmu.2016.00390

  • CrossRef
  • Google Scholar

• 172

  GeYHuangMYaoYM. ming. Autophagy and proinflammatory cytokines: Interactions and clinical implications. Cytokine Growth Factor Rev (2018) 43:38–46. doi: 10.1016/j.cytogfr.2018.07.001

  • CrossRef
  • Google Scholar

• 173

  LippitzBE. Cytokine patterns in patients with cancer: A systematic review. Lancet Oncol (2013) 14:e218–28. doi: 10.1016/S1470-2045(12)70582-X

  • CrossRef
  • Google Scholar

• 174

  ManfrediAABaldiniMCameraMBaldisseraEBrambillaMPerettiGet al. Anti-TNFα agents curb platelet activation in patients with rheumatoid arthritis. Ann Rheum Dis (2016) 75:1511–20. doi: 10.1136/annrheumdis-2015-208442

  • CrossRef
  • Google Scholar

• 175

  KojokKEl AkoumSMohsenMMouradWMerhiY. CD40L priming of platelets via NF-κB activation is CD40-and TAK1-dependent. J Am Heart Assoc (2018) 7:eo3677. doi: 10.1161/JAHA.118.009636

  • CrossRef
  • Google Scholar

• 176

  LumadueJALanzkronSMKennedySDKuhlDT. K.T.S. Cytokine Induction of Platelet Activation. Am J Clin Pathol (1996) 106:795–8. doi: 10.1017/CBO9781107415324.004

  • CrossRef
  • Google Scholar

• 177

  YouLWangZLiHShouJJingZXieJet al. The role of STAT3 in autophagy. Autophagy (2015) 11:729–39. doi: 10.1080/15548627.2015.1017192

  • CrossRef
  • Google Scholar

• 178

  CaiqiMAJinyeXIEChuanghuaLUOHaofanYINRuopuLIWangXet al. OxLDL promotes lymphangiogenesis and lymphatic metastasis in gastric cancer by upregulating VEGF-C expression and secretion. Int J Oncol (2019) 54:572–84. doi: 10.3892/ijo.2018.4648

  • CrossRef
  • Google Scholar

• 179

  WanFQinXZhangGLuXZhuYZhangHet al. Oxidized low-density lipoprotein is associated with advanced-stage prostate cancer. Tumour Biol (2015) 36:3573–82. doi: 10.1007/s13277-014-2994-6

  • CrossRef
  • Google Scholar

• 180

  DelimarisIFaviouEAntonakosGStathopoulouEZachariADionyssiou-AsteriouAet al. serum oxidizability and serum lipid levels in patients with breast or ovarian cancer. Clin Biochem (2007) 40:1129–34. doi: 10.1016/j.clinbiochem.2007.06.007

  • CrossRef
  • Google Scholar

• 181

  TimpJFBraekkanSKVersteegHHCannegieterSC. Epidemiology of cancer-associated venous thrombosis. Blood (2013) 122:1712–23. doi: 10.1182/blood-2013-04-460121

  • CrossRef
  • Google Scholar

• 182

  ChenMKakutaniMMasakiTSawamuraTChenMNarumiyaSet al. Activation-dependent surface expression of LOX-1 in human platelets. Biochem Biophys Res Commun (2001) 282:153–8. doi: 10.1006/bbrc.2001.4516

  • CrossRef
  • Google Scholar

• 183

  GeddingsJEHisadaYBoulaftaliYGetzTMWhelihanMFuentesRet al. Tissue factor-positive tumor microvesicles activate platelets and enhance thrombosis in mice. J Thromb Haemost (2016) 14:153–66. doi: 10.1111/jth.13181

  • CrossRef
  • Google Scholar

• 184

  ChenKDLinCCTsaiMCHuangKTChiuKW. Tumor microenvironment mediated by suppression of autophagic flux drives liver malignancy. BioMed J (2018) 41:163–8. doi: 10.1016/j.bj.2018.03.002

  • CrossRef
  • Google Scholar

• 185

  LiuJXiaHKimMXuLLiYZhangLet al. Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell (2011) 147:223–34. doi: 10.1016/j.cell.2011.08.037

  • CrossRef
  • Google Scholar

• 186

  BeauchampEMPlataniasLC. The evolution of the TOR pathway and its role in cancer. Oncogene (2013) 32:3923–32. doi: 10.1038/onc.2012.567

  • CrossRef
  • Google Scholar

• 187

  DunlopEATeeAR. mTOR and autophagy: a dynamic relationship governed by nutrients and energy. Semin Cell Dev Biol (2014) 36:121–9. doi: 10.1016/j.semcdb.2014.08.006

  • CrossRef
  • Google Scholar

• 188

  DangCV. Links between metabolism and cancer. Genes Dev (2012) 26:877–90. doi: 10.1101/gad.189365.112

  • CrossRef
  • Google Scholar

• 189

  HowellJJManningBD. mTOR couples cellular nutrient sensing to organismal metabolic homeostasis. Trends Endocrinol Metab (2011) 22:94–102. doi: 10.1016/j.tem.2010.12.003

  • CrossRef
  • Google Scholar

• 190

  Vander HeidenMGCantleyLCThompsonCB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Sci (80-) (2009) 324:1029–33. doi: 10.1126/science.1160809

  • CrossRef
  • Google Scholar

• 191

  LoveNKKeshavanNLewisRHarrisWAAgathocleousMA. nutrient-sensitive restriction point is active during retinal progenitor cell differentiation. Development (2014) 141:697–706. doi: 10.1242/dev.103978

  • CrossRef
  • Google Scholar

• 192

  ChangQJurisicaIDoTHedleyDW. Hypoxia predicts aggressive growth and spontaneous metastasis formation from orthotopically grown primary xenografts of human pancreatic cancer. Cancer Res (2011) 71:3110–20. doi: 10.1158/0008-5472.CAN-10-4049

  • CrossRef
  • Google Scholar

• 193

  ChangLHChenCHHuangDYPaiHCPanSLTengCM. Thrombin induces expression of twist and cell motility via the hypoxia-inducible factor-1alpha translational pathway in colorectal cancer cells. J Cell Physiol (2011) 226:1060–8. doi: 10.1002/jcp.22428

  • CrossRef
  • Google Scholar

• 194

  RankinEBGiacciaAJ. The role of hypoxia-inducible factors in tumorigenesis. Cell Death Differ (2008) 15:678–85. doi: 10.1038/cdd.2008.21

  • CrossRef
  • Google Scholar

• 195

  SemenzaGL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer (2003) 3:721–32. doi: 10.1038/nrc1187

  • CrossRef
  • Google Scholar

• 196

  SunZCaoBWuJ. Protease-activated receptor 2 enhances renal cell carcinoma cell invasion and migration via PI3K/AKT signaling pathway. Exp Mol Pathol (2015) 98:382–9. doi: 10.1016/j.yexmp.2015.03.018

  • CrossRef
  • Google Scholar

• 197

  TangKLTangHYDuYTianTXiongSJ. PAR-2 promotes cell proliferation, migration, and invasion through activating PI3K/AKT signaling pathway in oral squamous cell carcinoma. Biosci Rep (2019) 39:721–32. doi: 10.1042/BSR20182476

  • CrossRef
  • Google Scholar

• 198

  JiangXGuoYLBrombergME. Formation of tissue factor-factor VIIa-factor Xa complex prevents apoptosis in human breast cancer cells. Thromb Haemost (2006) 96:196–201. doi: 10.1160/TH06-01-0057

  • CrossRef
  • Google Scholar

• 199

  JiangXZhuSPanettiTSBrombergME. Formation of tissue factor-factor VIIa-factor Xa complex induces activation of the mTOR pathway which regulates migration of human breast cancer cells. Thromb Haemost (2008) 100:127–33. doi: 10.1160/TH07-12-0722

  • CrossRef
  • Google Scholar

• 200

  ZhangXShiHYuanXJiangPQianHXuW. Tumor-derived exosomes induce N2 polarization of neutrophils to promote gastric cancer cell migration. Mol Cancer (2018) 17:1–16. doi: 10.1186/s12943-018-0898-6

  • CrossRef
  • Google Scholar

• 201

  WenZFLiuHGaoRZhouMMaJZhangYet al. Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1. J Immunother Cancer (2018) 6:1–16. doi: 10.1186/s40425-018-0452-5

  • CrossRef
  • Google Scholar

• 202

  AasenTLeitheEGrahamSVKameritschPMayanMDMesnilMet al. Connexins in cancer: bridging the gap to the clinic. Oncogene (2019) 38:4429–51. doi: 10.1038/s41388-019-0741-6

  • CrossRef
  • Google Scholar

• 203

  CzyzJ. The stage-specific function of gap junctions during tumourigenesis. Cell Mol Biol Lett (2008) 13:92–102. doi: 10.2478/s11658-007-0039-5

  • CrossRef
  • Google Scholar

• 204

  GershonEPlaksVDekelN. Gap junctions in the ovary: expression, localization and function. Mol Cell Endocrinol (2008) 282:18–25. doi: 10.1016/j.mce.2007.11.001

  • CrossRef
  • Google Scholar

• 205

  MitchellJALyeSJ. Differential activation of the connexin 43 promoter by dimers of activator protein-1 transcription factors in myometrial cells. Endocrinology (2005) 146:2048–54. doi: 10.1210/en.2004-1066

  • CrossRef
  • Google Scholar

• 206

  BatesDCSinWCAftabQNausCC. Connexin43 enhances glioma invasion by a mechanism involving the carboxy terminus. Glia (2007) 55:1554–64. doi: 10.1002/glia.20569

  • CrossRef
  • Google Scholar

• 207

  ElzarradMKHaroonAWilleckeKDobrowolskiRGillespieMNAl-MehdiAB. Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium. BMC Med (2008) 6:20. doi: 10.1186/1741-7015-6-20

  • CrossRef
  • Google Scholar

• 208

  Kanczuga-KodaLSulkowskiSLenczewskiAKodaMWincewiczABaltaziakMet al. Increased expression of connexins 26 and 43 in lymph node metastases of breast cancer. J Clin Pathol (2006) 59:429–33. doi: 10.1136/jcp.2005.029272

  • CrossRef
  • Google Scholar

• 209

  PollmannMAShaoQLairdDWSandigM. Connexin 43 mediated gap junctional communication enhances breast tumor cell diapedesis in culture. Breast Cancer Res (2005) 7:R522–34. doi: 10.1186/bcr1042

  • CrossRef
  • Google Scholar

• 210

  VillaresGJDobroffASWangHZiglerMMelnikovaVOHuangLet al. Overexpression of protease-activated receptor-1 contributes to melanoma metastasis via regulation of connexin 43. Cancer Res (2009) 69:6730–7. doi: 10.1158/0008-5472.CAN-09-0300

  • CrossRef
  • Google Scholar

• 211

  BejaranoEGiraoHYusteAPatelBMarquesCSprayDCet al. Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner. Mol Biol Cell (2012) 23:2156–69. doi: 10.1091/mbc.E11-10-0844

  • CrossRef
  • Google Scholar

• 212

  BejaranoEYusteAPatelBStoutRFJr.SprayDCCuervoAM. Connexins modulate autophagosome biogenesis. Nat Cell Biol (2014) 16:401–14. doi: 10.1038/ncb2934

  • CrossRef
  • Google Scholar

• 213

  LichtensteinAMinoguePJBeyerECBerthoudVM. Autophagy: a pathway that contributes to connexin degradation. J Cell Sci (2011) 124:910–20. doi: 10.1242/jcs.073072

  • CrossRef
  • Google Scholar

• 214

  IyyathuraiJDecuypereJPLeybaertLD’HondtCBultynckG. Connexins: substrates and regulators of autophagy. BMC Cell Biol (2016) 17 Suppl 1:20. doi: 10.1186/s12860-016-0093-9

  • CrossRef
  • Google Scholar

• 215

  ElsteAPPetersenI. Expression of proteinase-activated receptor 1-4 (PAR 1-4) in human cancer. J Mol Histol (2010) 41:89–99. doi: 10.1007/s10735-010-9274-6

  • CrossRef
  • Google Scholar

• 216

  WangMAnSWangDJiHGengMGuoXet al. Quantitative Proteomics Identify the Possible Tumor Suppressive Role of Protease-Activated Receptor-4 in Esophageal Squamous Cell Carcinoma Cells. Pathol Oncol Res (2019) 25:937–43. doi: 10.1007/s12253-018-0395-7

  • CrossRef
  • Google Scholar

• 217

  MitchellPSParkinRKKrohEMFritzBRWymanSKPogosova-AgadjanyanELet al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A (2008) 105:10513–8. doi: 10.1073/pnas.0804549105

  • CrossRef
  • Google Scholar

• 218

  ChenXBaYMaLCaiXYinYWangKet al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res (2008) 18:997–1006. doi: 10.1038/cr.2008.282

  • CrossRef
  • Google Scholar

• 219

  LiangLFuJWangSCenHZhangLMandukhailSRet al. MiR-142-3p enhances chemosensitivity of breast cancer cells and inhibits autophagy by targeting HMGB1. Acta Pharm Sin B (2020) 10:1036–46. doi: 10.1016/j.apsb.2019.11.009

  • CrossRef
  • Google Scholar

• 220

  ZhouDMRanFNiHZSunLLXiaoLLiXQet al. Metformin inhibits high glucose-induced smooth muscle cell proliferation and migration. Aging (Albany NY) (2020) 12:5352–61. doi: 10.18632/aging.102955

  • CrossRef
  • Google Scholar

• 221

  SunYLiuXZhangDLiuFChengYMaYet al. Platelet-Derived Exosomes Affect the Proliferation and Migration of Human Umbilical Vein Endothelial Cells Via miR-126. Curr Vasc Pharmacol (2018) 17:379–87. doi: 10.2174/1570161116666180313142139

  • CrossRef
  • Google Scholar

• 222

  LiMMengXLiM. MiR-126 promotes esophageal squamous cell carcinoma via inhibition of apoptosis and autophagy. Aging (Albany NY) (2020) 12:12107–18. doi: 10.18632/aging.103379

  • CrossRef
  • Google Scholar

• 223

  NguyenDXBosPDMassaguéJ. Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer (2009) 9:274–84. doi: 10.1038/nrc2622

  • CrossRef
  • Google Scholar

• 224

  van Nieuw AmerongenGPvan DelftSVermeerMACollardJGvan HinsberghVW. Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases. Circ Res (2000) 87:335–40. doi: 10.1161/01.res.87.4.335

  • CrossRef
  • Google Scholar

• 225

  LeonardAMillarMWSlavinSABijliKMDionisio SantosDADeanDAet al. Critical role of autophagy regulator Beclin1 in endothelial cell inflammation and barrier disruption. Cell Signal (2019) 61:120–9. doi: 10.1016/j.cellsig.2019.04.013

  • CrossRef
  • Google Scholar

• 226

  SlavinSALeonardAGroseVFazalFRahmanA. Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury. Am J Physiol Lung Cell Mol Physiol (2018) 314:L388–96. doi: 10.1152/ajplung.00555.2016

  • CrossRef
  • Google Scholar

• 227

  ChaoCHChenHRChuangYCYehTM. Macrophage Migration Inhibitory Factor-Induced Autophagy Contributes to Thrombin-Triggered Endothelial Hyperpermeability in Sepsis. Shock (2018) 50:103–11. doi: 10.1097/SHK.0000000000000976

  • CrossRef
  • Google Scholar

• 228

  BairAMThippegowdaPBFreichelMChengNYeRDVogelSMet al. Ca 2+ entry via TRPC channels is necessary for Thrombin-induced NF-kB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cδ. J Biol Chem (2009) 284:563–74. doi: 10.1074/jbc.M803984200

  • CrossRef
  • Google Scholar

• 229

  TiruppathiCNaqviTSandovalRMehtaDMalikAB. Synergistic effects of tumor necrosis factor-alpha and thrombin in increasing endothelial permeability. Am J Physiol Lung Cell Mol Physiol (2001) 281:L958–68. doi: 10.1152/ajplung.2001.281.4.L958

  • CrossRef
  • Google Scholar

• 230

  StahmannNWoodsACarlingDHellerR. Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca2+/Calmodulin-Dependent Protein Kinase Kinase β. Mol Cell Biol (2006) 26:5933–45. doi: 10.1128/mcb.00383-06

  • CrossRef
  • Google Scholar

• 231

  ThorsBHalldórssonHThorgeirssonG. ENOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1. Biochim Biophys Acta - Mol Cell Res (2011) 1813:322–31. doi: 10.1016/j.bbamcr.2010.12.001

  • CrossRef
  • Google Scholar

• 232

  WuQHuYJiangMWangFGongG. Effect of autophagy regulated by sirt1/FoxO1 pathway on the release of factors promoting thrombosis from vascular endothelial cells. Int J Mol Sci (2019) 20:1–14. doi: 10.3390/ijms20174132

  • CrossRef
  • Google Scholar

• 233

  NaritaMYoungARJArakawaSSamarajiwaSANakashimaTYoshidaSet al. Spatial coupling of mTOR and autophagy augments secretory phenotypes. Science (2011) 332:966–70. doi: 10.1126/science.1205407

  • CrossRef
  • Google Scholar

• 234

  KaweckiCLentingPJDenisCV. von Willebrand factor and inflammation. J Thromb Haemost (2017) 15:1285–94. doi: 10.1111/jth.13696

  • CrossRef
  • Google Scholar

• 235

  RautouPEBusseJKheloufiMVionACBoulaftaliYStarkKet al. Endothelial Autophagy Does Not Influence Venous Thrombosis in Mice. Thromb Haemost (2018) 118:1113–5. doi: 10.1055/s-0038-1641753

  • CrossRef
  • Google Scholar

• 236

  SungBHKetovaTHoshinoDZijlstraAWeaverAM. Directional cell movement through tissues is controlled by exosome secretion. Nat Commun (2015) 6:7164. doi: 10.1038/ncomms8164

  • CrossRef
  • Google Scholar

• 237

  KerkNStrozykEAPöppelmannBSchneiderSW. The mechanism of melanoma-associated thrombin activity and von Willebrand factor release from endothelial cells. J Invest Dermatol (2010) 130:2259–68. doi: 10.1038/jid.2010.136

  • CrossRef
  • Google Scholar

• 238

  XuYPanSLiuJDongFChengZZhangJet al. GATA3-induced vWF upregulation in the lung adenocarcinoma vasculature. Oncotarget (2017) 8:110517–29. doi: 10.18632/oncotarget.22806

  • CrossRef
  • Google Scholar

• 239

  PatmoreSDhamiSPSO’SullivanJM. Von Willebrand Factor and Cancer; metastasis and coagulopathies. J Thromb Haemost. (2020). doi: 10.1111/jth.14976.

  • CrossRef
  • Google Scholar

• 240

  LingJSunYPanJWangHMaZYinJet al. Feedback modulation of endothelial cells promotes epithelial-mesenchymal transition and metastasis of osteosarcoma cells by Von Willebrand Factor release. J Cell Biochem (2019) 120:15971–9. doi: 10.1002/jcb.28875

  • CrossRef
  • Google Scholar

• 241

  GomesNLegrandCFauvel-LafèveF. Shear stress induced release of von Willebrand factor and thrombospondin-1 in HUVEC extracellular matrix enhances breast tumour cell adhesion. Clin Exp Metastasis (2005) 22:215–23. doi: 10.1007/s10585-005-7359-5

  • CrossRef
  • Google Scholar

• 242

  MorgantiMCarpiAAmo-TakyiBSagripantiANicoliniAGiardinoRet al. Von Willebrand’s factor mediates the adherence of human tumoral cells to human endothelial cells and ticlopidine interferes with this effect. BioMed Pharmacother (2000) 54:431–6. doi: 10.1016/s0753-3322(00)00006-8

  • CrossRef
  • Google Scholar

• 243

  O’SullivanJMPrestonRJSRobsonTO’DonnellJS. Emerging Roles for von Willebrand Factor in Cancer Cell Biology. Semin Thromb Hemost (2018) 44:159–66. doi: 10.1055/s-0037-1607352

  • CrossRef
  • Google Scholar

• 244

  GrimseyNJCoronelLJCordovaICTrejoJ. Recycling and endosomal sorting of protease-activated receptor-1 is distinctly regulated by Rab11A and Rab11B proteins. J Biol Chem (2016) 291:2223–36. doi: 10.1074/jbc.M115.702993

  • CrossRef
  • Google Scholar

• 245

  Martinez-OutschoornUEWhitaker-MenezesDLinZFlomenbergNHowellAPestellRGet al. Cytokine production and inflammation drive autophagy in the tumor microenvironment: Role of stromal caveolin-1 as a key regulator. Cell Cycle (2011) 10:1784–93. doi: 10.4161/cc.10.11.15674

  • CrossRef
  • Google Scholar

• 246

  WangRZhangTMAZWangYChengZXuHet al. The interaction of coagulation factor XII and monocyte/macrophages mediating peritoneal metastasis of epithelial ovarian cancer. Gynecol Oncol (2010) 117:460–6. doi: 10.1016/j.ygyno.2010.02.015

  • CrossRef
  • Google Scholar

• 247

  RennéTStavrouEX. Roles of Factor XII in Innate Immunity. Front Immunol (2019) 10:2011. doi: 10.3389/fimmu.2019.02011

  • CrossRef
  • Google Scholar

• 248

  MaZZhangTWangRChengZXuHLiWet al. Tissue factor-factor VIIa complex induces epithelial ovarian cancer cell invasion and metastasis through a monocytes-dependent mechanism. Int J Gynecol Cancer (2011) 21:616–24. doi: 10.1097/IGC.0b013e3182150e98

  • CrossRef
  • Google Scholar

• 249

  MaYYHeXJWangHJXiaYJWangSLYeZYet al. Interaction of coagulation factors and tumor-associated macrophages mediates migration and invasion of gastric cancer. Cancer Sci (2011) 102:336–42. doi: 10.1111/j.1349-7006.2010.01795.x

  • CrossRef
  • Google Scholar

• 250

  LindholmPFLuYAdleyBPVladislavTJovanovicBSivapurapuNet al. Role of monocyte-lineage cells in prostate cancer cell invasion and tissue factor expression. Prostate (2010) 70:1672–82. doi: 10.1002/pros.21202

  • CrossRef
  • Google Scholar

• 251

  DengCWuSZhangLYangMLinQXieQet al. Role of monocyte tissue factor on patients with non-small cell lung cancer. Clin Respir J (2018) 12(3):1125–33. doi: 10.1111/crj.12640

  • CrossRef
  • Google Scholar

• 252

  AntoniakSMacKmanN. Multiple roles of the coagulation protease cascade during virus infection. Blood (2014) 123:2605–13. doi: 10.1182/blood-2013-09-526277

  • CrossRef
  • Google Scholar

• 253

  VergadiEIeronymakiELyroniKVaporidiKTsatsanisC. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J Immunol (2017) 198:1006–14. doi: 10.4049/jimmunol.1601515

  • CrossRef
  • Google Scholar

• 254

  HaloulMOliveiraERAKaderMWellsJZTominelloTREl AndaloussiAet al. mTORC1-mediated polarization of M1 macrophages and their accumulation in the liver correlate with immunopathology in fatal ehrlichiosis. Sci Rep (2019) 9:14050. doi: 10.1038/s41598-019-50320-y

  • CrossRef
  • Google Scholar

• 255

  XuYLiuXGongXEissaNT. Signaling pathway of autophagy associated with innate immunity. Autophagy (2008) 4:1:110–2. doi: 10.4161/auto.5225

  • CrossRef
  • Google Scholar

• 256

  LiaoXSluimerJCWangYSubramanianMBrownKPattisonJSet al. Macrophage autophagy plays a protective role in advanced atherosclerosis. Cell Metab (2012) 15(4):545–553. doi: 10.1016/j.cmet.2012.01.022

  • CrossRef
  • Google Scholar

• 257

  MihalacheCCSimonHU. Autophagy regulation in macrophages and neutrophils. Exp Cell Res (2012) 318(11):1187–92. doi: 10.1016/j.yexcr.2011.12.021

  • CrossRef
  • Google Scholar

• 258

  WuM-YLuJ-H. Autophagy and Macrophage Functions: Inflammatory Response and Phagocytosis. Cells (2019) 9(1):70. doi: 10.3390/cells9010070

  • CrossRef
  • Google Scholar

• 259

  MassbergSGrahlLvon BruehlMLManukyanDPfeilerSGoosmannC. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med16(8):887–96. doi: 10.1038/nm.2184

  • CrossRef
  • Google Scholar

• 260

  JungHSGuJKimJENamYSongJWKimHKCancer cell-induced neutrophil extracellular traps promote both hypercoagulability and cancer progression. PLoS One (2019) 14(4):e0216055. doi: 10.1371/journal.pone.0216055

  • CrossRef
  • Google Scholar

• 261

  FuchsTABrillADuerschmiedDSchatzbergDMonestierMMyersJDDet al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A (2010) 107:15880–5. doi: 10.1073/pnas.1005743107

  • CrossRef
  • Google Scholar

• 262

  MaugeriNCampanaLGavinaMCovinoCDe MetrioMPanciroliCet al. Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. J Thromb Haemost (2014) 12(12):2074–88. doi: 10.1111/jth.12710

  • CrossRef
  • Google Scholar

• 263

  McDonaldBDavisRPKimSJTseMEsmonCTKolaczkowskaEet al. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood (2017) 129:1357–67. doi: 10.1182/blood-2016-09-741298

  • CrossRef
  • Google Scholar

• 264

  CedervallJZhangYOlssonAK. Tumor-Induced NETosis as a Risk Factor for Metastasis and Organ Failure. Cancer Res (2016) 76:4311–5. doi: 10.1158/0008-5472.CAN-15-3051

  • CrossRef
  • Google Scholar

• 265

  JorchSKKubesP. An emerging role for neutrophil extracellular traps in noninfectious disease. Nat Med (2017) 23:279–87. doi: 10.1038/nm.4294

  • CrossRef
  • Google Scholar

• 266

  PapayannopoulosV. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol (2018) 18:134–47. doi: 10.1038/nri.2017.105

  • CrossRef
  • Google Scholar

• 267

  OlssonAKCedervallJ. NETosis in Cancer - Platelet-Neutrophil Crosstalk Promotes Tumor-Associated Pathology. Front Immunol (2016) 7:373. doi: 10.3389/fimmu.2016.00373

  • CrossRef
  • Google Scholar

• 268

  LiuKZhaoEIlyasGLalazarGLinYHaseebMet al. Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization. Autophagy (2015) 11:271–84. doi: 10.1080/15548627.2015.1009787

  • CrossRef
  • Google Scholar

• 269

  MiharaKRamachandranRRenauxBSaifeddineMHollenbergMD. Neutrophil elastase and proteinase-3 trigger G proteinbiased signaling through proteinase-activated receptor-1 (PAR1). J Biol Chem (2013) 288:32979–90. doi: 10.1074/jbc.M113.483123

  • CrossRef
  • Google Scholar

• 270

  KambasKMitroulisIApostolidouEGirodAChrysanthopoulouAPneumatikosIet al. Autophagy Mediates the Delivery of Thrombogenic Tissue Factor to Neutrophil Extracellular Traps in Human Sepsis. PloS One (2012) 7:1–14. doi: 10.1371/journal.pone.0045427

  • CrossRef
  • Google Scholar

• 271

  BooneBAMurthyPMiller-OcuinJDoerflerWREllisJTLiangXet al. Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps. BMC Cancer (2018) 18:1–12. doi: 10.1186/s12885-018-4584-2

  • CrossRef
  • Google Scholar

• 272

  DemersMKrauseDSSchatzbergDMartinodKVoorheesJRFuchsTAet al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A (2012) 109(32):13076–81. doi: 10.1073/pnas.1200419109

  • CrossRef
  • Google Scholar

• 273

  BooneBASchapiroNENealMDEllisJTLotzeMTZehHJ. Neutrophil Extracellular Traps (NETs) are Upregulated in Pancreatic Cancer as a Result of Autophagy and Promote Hypercoagulability. J Surg Res (2014) 186(2):638. doi: 10.1016/j.jss.2013.11.657

  • CrossRef
  • Google Scholar

• 274

  BooneBAOrlichenkoLSchapiroNELoughranPGianfrateGCEllisJTet al. The receptor for advanced glycation end products (RAGE) enhances autophagy and neutrophil extracellular traps in pancreatic cancer. Cancer Gene Ther (2015) 22:326–34. doi: 10.1038/cgt.2015.21

  • CrossRef
  • Google Scholar

• 275

  MaugeriNCapobiancoARovere-QueriniPRamirezGATombettiEDella VallePet al. Platelet microparticles sustain autophagy-associated activation of neutrophils in systemic sclerosis. Sci Transl Med (2018) 10:eaao3089. doi: 10.1126/scitranslmed.aao3089

  • CrossRef
  • Google Scholar

• 276

  YoungAChapmanOConnorCPooleCRosePKakkarAK. Thrombosis and cancer. Nat Rev Clin Oncol (2012) 9(8):437–49. doi: 10.1038/nrclinonc.2012.106

  • CrossRef
  • Google Scholar

• 277

  AlexanderETMintonARPetersMCvan RynJGilmourSK. Thrombin inhibition and cisplatin block tumor progression in ovarian cancer by alleviating the immunosuppressive microenvironment. Oncotarget (2016) 7(51):85291–305. doi: 10.18632/oncotarget.13300

  • CrossRef
  • Google Scholar

• 278

  CovicLKuliopulosA. Protease-activated receptor 1 as therapeutic target in breast, lung, and ovarian cancer: Pepducin approach. Int J Mol Sci (2018) 19(8):2237. doi: 10.3390/ijms19082237

  • CrossRef
  • Google Scholar

• 279

  VersteegHHSchaffnerFKerverMPetersenHHAhamedJFelding-HabermannBet al. Inhibition of tissue factor signaling suppresses tumor growth. Blood (2008) 111:190–9. doi: 10.1182/blood-2007-07-101048

  • CrossRef
  • Google Scholar

• 280

  VersteegHHRufW. Emerging insights in tissue factor-dependent signaling events. Semin Thromb Hemost (2006) 32:24–32. doi: 10.1055/s-2006-933337

  • CrossRef
  • Google Scholar

• 281

  ChoJFurieBCCoughlinSRFurieB. A critical role for extracellular protein disulfide isomerase during thrombus formation in mice. J Clin Invest (2008) 118(3):1123–31. doi: 10.1172/JCI34134

  • CrossRef
  • Google Scholar

• 282

  WendelHGDe StanchinaEFridmanJSMalinaARaySKoganSet al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature (2004) 428(6980):332–7. doi: 10.1038/nature02369

  • CrossRef
  • Google Scholar

• 283

  MackJMVerkampBRichterGTNicholasRStewartKCrarySE. Effect of sirolimus on coagulopathy of slow-flow vascular malformations. Pediatr Blood Cancer (2019) 66(10):e27896. doi: 10.1002/pbc.27896

  • CrossRef
  • Google Scholar

• 284

  LiuZYanYCaoSChenY. Long non-coding RNA SNHG14 contributes to gastric cancer development through targeting miR-145/SOX9 axis. J Cell Biochem (2018) 119:6905–13. doi: 10.1002/jcb.26889

  • CrossRef
  • Google Scholar

• 285

  GubaMYezhelyevMEichhornMESchmidGIschenkoIPapyanAet al. Rapamycin induces tumor-specific thrombosis via tissue factor in the presence of VEGF. Blood (2005) 105(11):4463–9. doi: 10.1182/blood-2004-09-3540

  • CrossRef
  • Google Scholar

• 286

  TanakaHHinoHMoriyaSKazamaHMiyazakiMTakanoNet al. Comparison of autophagy inducibility in various tyrosine kinase inhibitors and their enhanced cytotoxicity via inhibition of autophagy in cancer cells in combined treatment with azithromycin. Biochem Biophys Rep (2020) 22:100750. doi: 10.1016/j.bbrep.2020.100750

  • CrossRef
  • Google Scholar

• 287

  MukaiSMoriyaSHiramotoMKazamaHKokubaHCheXFet al. Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines. Int J Oncol (2016). doi: 10.3892/ijo.2015.3237

  • CrossRef
  • Google Scholar

• 288

  ZhaoLYangGShiYSuCChangJ. Co-delivery of Gefitinib and chloroquine by chitosan nanoparticles for overcoming the drug acquired resistance. J Nanobiotechnology (2015) 13:57. doi: 10.1186/s12951-015-0121-5

  • CrossRef
  • Google Scholar

• 289

  LiuZHeKMaQYuQLiuCNdegeIet al. Autophagy inhibitor facilitates gefitinib sensitivity in vitro and in vivo by activating mitochondrial apoptosis in triple negative breast cancer. PloS One (2017) 12(5):e0177694. doi: 10.1371/journal.pone.0177694

  • CrossRef
  • Google Scholar

• 290

  StraussLMahmoudMAAWeaverJDTijaro-OvalleNMChristofidesAWangQet al. Targeted deletion of PD-1 in myeloid cells induces antitumor immunity. Sci Immunol (2020) 5(43):eaay1863. doi: 10.1126/sciimmunol.aay1863

  • CrossRef
  • Google Scholar