Exosomal miRNAs and lncRNAs: The Modulator Keys of Cancer-Associated Fibroblasts in the Genesis and Progression of Malignant Neoplasms

The tumor microenvironment is made up of a universe of molecular and cellular components that promote or inhibit the development of neoplasms. Among the molecular elements are cytokines, metalloproteinases, proteins, mitochondrial DNA, and nucleic acids, within which the ncRNAs: miRNAs and lncRNAs stand out due to their direct modulating effects on the genesis and progression of various cancers. Regarding cellular elements, the solid tumor microenvironment is made up of tumor cells, healthy adjacent epithelial cells, immune system cells, endothelial cells, and stromal cells, such as cancer-associated fibroblasts, which are capable of generating a modulating communication network with the other components of the tumor microenvironment through, among other mechanisms, the secretion of exosomal vesicles loaded with miRNAs and lncRNAs. These ncRNAs are key pieces in developing neoplasms since they have diverse effects on cancer cells and healthy cells, favoring or negatively regulating protumoral cellular events, such as migration, invasion, proliferation, metastasis, epithelial-mesenchymal transition, and resistance to treatment. Due to the growing number of relevant evidence in recent years, this work focused on reviewing, analyzing, highlighting, and showing the current state of research on exosomal ncRNAs derived from cancer-associated fibroblasts and their effects on different neoplasms. A future perspective on using these ncRNAs as real therapeutic tools in the treatment of cancer patients is also proposed.

For everything mentioned above, the present work is based on the recent findings on the protumoral and antitumoral effects of CAFsderived exosomal miRNAs and lncRNAs; it focuses on analyzing and highlighting the importance of these ncRNAs in the genesis and progression of different cancers. At the same time, it shows the current situation of this new growing research area, and a future perspective on the use of these ncRNAs as real therapeutic tools in the treatment of cancer patients is also proposed.

EXOSOMES: INDUCING FACTOR IN THE FORMATION OF CAFS AND TUMOR MICROENVIRONMENT MODULATOR
Exosomes are among the most recognized inducers for generating CAFs (Li K. et al., 2020). CAFs are known as cells capable of secreting exosomes (You et al., 2019), and in turn, exosomes derived from cancer cells containing TGF-β can induce the generation of CAFs from stromal cells (Goulet et al., 2019). It has also been observed that exosomes derived from CAFs containing TGF-β activate the SMAD signaling pathway in cancer cells through a particular type of epithelial-mesenchymal transition (EMT), which increases their malignant behavior , thus generating a loop between CAFs and exosomes. It has been shown that chronic lymphocytic leukemia cells-derived exosomes can transfer miR-146a to bone marrow-derived mesenchymal stem cells to generate CAFs; this is done by promoting EMT by targeting ubiquitin specific peptidase 16 (USP16) (Yang et al., 2020). Another cellular transition process in which exosomes are involved to originate CAFs is the endothelial-mesenchymal transition; this event was demonstrated by observing that melanoma-derived exosomes loaded with TGF-β induced the transition from human umbilical vein endothelial cells (HUVECs) to differentiated CAFs (Yeon et al., 2018). For their part, Baroni et al. demonstrated that triple-negative breast CAFs-derived exosomal miR-9 could generate a cell type with CAF-like properties in human breast fibroblasts, in which this miRNA enhanced the capacity for migration and invasion, as in breast cancer cell lines. Additionally, overexpression of this ncRNA in normal fibroblasts was able to promote tumor growth in a murine orthotopic xenograft model (Baroni et al., 2016).
It has also been observed that the exosomes produced by CAFs can modulate TME. For example, miR-21, which is packed in CAFderived exosomes, induces the generation of monocytic myeloidderived suppressor cells by activating STAT3 . On a breast cancer model, exosomes produced by CAFs can also interfere with immunologic processes. In breast cancer cells treated with exosomes derived from CAFs, an increase of miR-92 was observed, which was essential for migration and invasion and correlated with the suppression of the immune cell function and the promotion of PD-L1 expression in these cells (Dou et al., 2020). Also, in the metastatic lung niche, a higher capacity to induce the transformation fibroblast into CAFs has been observed due to exosomal transport of miR-1247-3p from high-metastatic hepatocellular carcinoma cells; leading to a pro-inflammatory microenvironment promoted by CAFs, which secret IL-6 and IL8, among other cytokines (Fang et al., 2018).
Considering this evidence, the important reciprocal relationship between exosomes and CAFs in the latter's self-generation and consequently in neoplasms' development is manifest. This relationship could be explored in greater depth and considered a previous or potentiating stage of carcinogenesis to inhibit the exosomal release and block exosomal molecules essential for the initiation, establishment, and progression of different cancers.

Migration and Invasion, Processes Upregulated by CAFs-Derived Exosomal ncRNAs
Migration and invasion of cancer cells are essential for the establishment of the malignant neoplasm. Sun et al. isolated CAFs from patients with oral squamous cell carcinoma (OSCC) and showed that in tongue squamous cell carcinoma CAL-27 cells, CAFs could promote these two events through the transfer of exosomal miR-382-5p, which facilitated the OSCC progression. Despite not having biologically determined the interaction, an analysis in silico predicted PTEN, YBX1, RUNX1, STC1, JAM2, and MMP16 as candidate target genes for this miRNA . Another exosomal miRNA that has been shown to promote cell migration and invasion is miR-1228; this small non-coding regulatory RNA was enriched in exosomes secreted by CAFs and could downregulate endogenous SCAI mRNA and protein level in osteosarcoma, contributing to carcinogenesis of this neoplasm (Wang J.-W. et al., 2019).
CAFs-derived exosomes can also carry lncRNAs; LINC00659 is an example of this; it was found enriched in CAFs-derived exosomes and was shown to have multiple effects on human colorectal cancer (CRC) cells, it was able to induce cell proliferation, migration, invasion, and EMT in vitro. Furthermore, it was determined that these events were promoted by directly interacting with the tumor suppressor miR-342-3p to increase ANXA2 expression in CRC cells .
The protumoral effects exerted by various CAFs-derived exosomal miRNAs and lncRNAs enhance the migration and invasion of cancer cells and convert them into key pieces within the universe of participants in developing neoplasms.

CAFs-Derived Exosomal ncRNAs Promote Proliferation and Metastasis, Essential Malignant Characteristics for Tumor Progression
Among the multiple protumoral effects that CAFs-derived exosomal ncRNAs exert on cancer cells are the promotion of cell proliferation and metastasis. Chen et al. determined that CAFs isolated from samples of patients with invasive ductal carcinoma were capable of transferring exosomal miR-500a-5p to breast cancer cell lines, this miRNA bound to ubiquitin-specific peptidase 28 (USP28) promoting cell proliferation and metastasis in a nude mouse xenograft model . It has also been shown that miR-181d-5p can promote various protumoral cellular events in breast cancer cells, such as proliferation, invasion, and migration. If not enough, it can also induce EMT, antagonize apoptosis in vitro of breast cancer cells and promote tumor growth in nude mice xenografted with MCF-7 cells via downregulation of the transcription factors CDX2 and HOXA5 .
Another important effect carried out by CAFs exosomal ncRNAs is the reprogramming of metabolic pathways. Although to date, this aspect has not yet been studied in-depth, there is a report where a protumoral role is proposed for lncRNA SNHG3, which positively regulated pyruvate kinase isozymes M1/M2 (PKM) expression, inhibited mitochondrial oxidative phosphorylation, increased glycolysis, and proliferation of breast tumor cells through a mechanism similar to a molecular sponge for antitumoral miR-330-5p ; this highlights the multifunctionality that CAFs-derived exosomal ncRNAs can exert on various processes related to carcinogenesis and tumor progression.
Considering the evidence reported, CAFs-derived exosomal miRNAs and lncRNAs can participate directly in tumor growth and the dissemination of cancer cells to other anatomical sites adjacent or far from the primary site, which could cause functional compromise of multiple organs and consequent serious complications in cancer patients.

Treatment Resistance Generated by CAFs-Derived Exosomal miRNAs and lncRNAs
CAFs are an important factor in generating tumor resistance to treatment (Fiori et al., 2019); this protumoral feature is associated with worsening cancer patients' prognosis (Domvri et al., 2020;Huang et al., 2020). CAFs possess chemoresistance by innate nature and, it has been observed that in the presence of gemcitabine, a cytotoxic anticancer chemotherapy drug, they can transfer this characteristic to cancer cells. CAFs exposed to this antineoplastic drug could increase the release of miR-146aloaded exosomes; in in vitro assays, this miRNA transmitted gemcitabine resistance to pancreatic cancer epithelial cell lines, promoting cell proliferation and survival. In the same work, it was shown that miR-146a is directly regulated by the promoter binding transcription factor, Snail, which acts as a chemoresistance-inducing factor, and that it was also found to be overexpressed in human pancreatic CAFs-derived exosomes (Richards et al., 2017). Recently, Gao et al. identified and characterized a subtype of CAFs which particularly has the presence of the cell surface protein CD63; these CD63 + CAFs are capable of secreting exosomes enriched in miR-22, which can bind to their targets, ERα and PTEN, and deregulate their expression, thus conferring tamoxifen resistance to breast cancer cells (Gao et al., 2020).
Gemcitabine and tamoxifen are not the only drugs for which CAFs-derived exosomal miRNAs generate resistance in cancer cells; in fact, a considerable number of reports show that the cytotoxic effect caused by cisplatin in cancer cells can be diminished or eliminated by the action of CAFs-derived exosomal ncRNAs. It has been shown that, in cisplatin-sensitive ovarian cancer cells, CAFs-derived exosomal miR-98-5p was capable of binding to cyclin-dependent kinase inhibitor 1A (CDKN1A) to inhibit its expression, which increased ovarian cancer cell proliferation and cell cycle entry, suppressed cell apoptosis, and promoted cisplatin resistance in vitro and in a xenotransplanted nude mouse model . The intrinsic resistance that CAFs have towards cisplatin also favors the progression of head and neck cancer (HNC). It has been observed that CAFs can transfer exosomal miR-196a to HNC cells, which generated cell survival, proliferation, and inhibition of apoptosis, also conferred cisplatin resistance by targeting CDKN1B and ING5, cell cycle inhibitor and tumor suppressor molecules, respectively. Additionally, high levels of exosomal miR-196a in plasma were clinically correlated with poor overall survival and chemoresistance in patients with HNC (Qin et al., 2019).
There is a great diversity between the protumoral effects generated by CAFs-derived exosomal miRNAs and lncRNAs; it has been shown that miR-522 is capable of inhibiting ferroptosis, a novel mode of non-apoptotic cell death induced by a build-up of toxic lipid peroxides (lipid-ROS) in an irondependent manner, in gastric cancer cells by targeting ALOX15 and blocking lipid-ROS accumulation. Additionally, assays performed in an orthotopic implantation model in nude mice to evaluate gastric tumor growth and chemosensitivity led the authors to suggest that CAFs-derived exosomes containing miR-522 promote a new mechanism of acquired chemoresistance to cisplatin through an intercellular pathway, comprising USP7, hnRNPA1, miR-522, and ALOX15 (Zhang H. et al., 2020).
On the other hand, it has been observed that miR-423-5ploaded exosomes derived from CAFs can decrease the chemosensitivity of prostate cancer cells and increase the resistance of cells resistant to taxanes; in addition, it was shown that the inhibition of this miRNA enhanced the drug sensitivity of prostate cancer cells in a tumor xenograft model in nude mice. These protumoral events were favored due to the inhibitory effect of miR-423-5p on GREM2 and the impact exerted on the TGF-β pathway (Shan et al., 2020). In another type of neoplasm, in bladder cancer, it was found that miR-148b-3p can also induce resistance to treatment. Shan et al. validated PTEN as a target of miR-148b-3p; the negative dysregulation of PTEN promoted metastasis, EMT, and resistance to doxorubicin and paclitaxel both in in vitro assays using bladder cancer cells and in in vivo assays using a xenograft mouse model. These events were generated due to the tumor-promoting effects of miR-148b-3p via the Wnt/β-catenin pathway (Shan et al., 2021).
CAFs-derived exosomal miRNAs have also shown an inhibitory effect on the cytotoxic action that methotrexate exerts on the metabolism of colon cancer cells. In an in vivo model of colon cancer, it was shown that miR-24-3p induced resistance to this drug, favoring tumor growth under treatment of methotrexate by down-regulating the CDX2/HEPH axis .
As mentioned previously, CAFs are also capable of secreting exosomal lncRNAs that promote tumor progression. Such is the case of exosomal lncUCA1, which conferred resistance to cisplatin in vulvar squamous cell carcinoma cells. Mechanistically, lncUCA1 functioned as a sponge for miR-103a, a miRNA with antitumoral function in various human cancers, this sequestration of miR-103a by lncUCA1 promoted the expression of WEE1 G2 checkpoint kinase (WEE1), a direct target of miR-103a, enhancing tumor growth and cisplatin resistance in a BALB/c nude xenograft model (Gao et al., 2021). Another CAFs-derived exosomal lncRNA with protumoral effect is H19, highly expressed in tumors generated in an azoxymethane (AOM)/dextran sodium sulfate (DSS) model of colitis-associated cancer, as well as in CRC samples from patients in different tumor-node-metastasis stages. Additionally, this lncRNA promoted the stemness of CRC stem cells, increased the frequency of tumor-initiating cells, and promoted the resistance of CRC cells to oxaliplatin both in vitro and in vivo. Mechanistically, H19 activated the β-catenin pathway by acting as an endogenous competitor for miR-141, a miRNA with antitumoral effect, in CRC cells .
These findings propose miRNAs and lncRNAs as important obstacles to achieving a successful chemotherapeutic treatment; Frontiers in Cell and Developmental Biology | www.frontiersin.org November 2021 | Volume 9 | Article 717478 subject to further studies confirming this evidence, the elimination or reduction of CAFs-derived exosomal ncRNAs that generate chemoresistance should be considered as a new oncological therapeutic strategy.

Exosomal miRNAs With Antitumoral Effects
miRNAs have generally been reported as protumoral molecules; however, some reports suggest that their silencing or elimination could favor tumor development. A feature shared between the antitumoral miRNAs is that they are significantly reduced in CAFs-derived exosomes. In vitro and in vivo studies carried out by Li et al. revealed that miR-148b could function as a tumor suppressor, the downregulation of this miRNA induced EMT, migration, invasion, and increased MMP-9 activity in endometrial cancer cell lines, as well as metastasis in a nude mouse model, additionally, DNA methyltransferase 1 (DNMT1) was determined as the target gene of miR-148b . This enzyme has also been associated with tumorigenesis in breast cancer due to its indispensable role in the maintenance of mammary stem/progenitor cell and cancer stem cell maintenance (Pathania et al., 2015), and chemoresistance to cisplatin in human non-small cell lung cancer cell lines (Sui et al., 2015). Another study also carried out in endometrial cancer showed that the CAFs-derived exosomal miR-320a inhibited the proliferation of cell lines by direct targeting and downregulation of hypoxia-inducible factor 1-α (HIFα) (Zhang N. et al., 2020). This transcription factor has been strongly related to metastasis, angiogenesis, poor patient prognosis, and tumor resistance therapy (Masoud and Li, 2015). In another neoplasm typical of women, the CAFs-derived exosomal miR-4516 was isolated from tumor tissue obtained from a patient with invasive breast ductal carcinoma; this miRNA suppressed the proliferation of breast cancer cell lines by targeting FOSL1 (Kim et al., 2020). The antitumoral effects that some miRNAs possess have not only been reported in gynecological cancers. In vitro and in vivo studies carried out in human hepatocellular carcinoma cell lines and in a nude mouse model, respectively, showed that miR-320a could function as an antitumoral miRNA, whose binding to PBX3 affected the protein expression of cyclin-dependent kinase 2 (CDK2) and MMP-2 due to the reduction of the phosphorylation of ERK1/2. In this way, the miR-320a/PBX3 axis suppressed essential events for cancer progression such as EMT, cell proliferation, migration, invasion, tumorigenesis, and metastasis . miR-3188 is another miRNA that has an antitumoral effect; its loss of CAFs-derived exosomes contributed to the malignancy of HNC cells, increasing cell proliferation, migration, and invasion, and inhibited apoptosis by derepressing its target B-cell lymphoma 2 (BCL2) mRNA, this miRNA was also able to inhibit tumor growth in a BALB/c nude mice model (Wang X. et al., 2019).
Another of the few reports in which evidence is presented about the antitumoral effect of miRNAs is made by Li et al. It was observed that the overexpression of CAFs-derived exosomal miR-34a-5p suppressed the tumorigenesis of OSCC cells in an immunodeficient BALB/c mice subcutaneous tumor model. Additionally, in OSCC cell lines, it was shown that this miRNA is capable of binding directly to AXL, thus modulating the AKT/GSK-3β/β-catenin signaling pathway; this event reduced proliferation, migration, and invasion, and decreased nuclear translocation of β-catenin, which led to decreased expression of Snail, a transcription factor of MMP-2 and MMP-9, important for EMT .
Considering the reported evidence of some miRNAs' antitumoral effects in gynecological and non-gynecological cancers, these ncRNAs could become valuable tools to inhibit essential points in carcinogenesis and disease progression.

CAFS-DERIVED EXOSOMAL MIRNAS AND LNCRNAS AS CANDIDATES FOR THERAPEUTIC TOOLS AND BIOMARKERS
Given the importance of protumoral and antitumoral miRNAs and lncRNAs in processes such as cell proliferation, migration, invasion, EMT, metastasis, metabolism, resistance to treatment (Table 1), these exosomal ncRNAs derived from CAFs have been suggested as strong candidates that could be used as new targets or therapeutic tools in breast, ovarian, vulvar squamous cell, endometrial, head and neck, pancreas, oral squamous cell, gastric, bladder, colon, liver, prostate cancer, as well as biomarkers of clinical relevance; CAFs-derived exosomal ncRNAs can be considered cornerstones in neoplastic processes.

CONCLUSION AND PERSPECTIVES
Exosomes loaded with miRNAs and lncRNAs are a key communication pathway between CAFs and the different elements of the TME. Its relevance lies mainly in the promoter effect of protumoral and antitumoral events that modulate the genesis and the progression of various neoplasms. In the present work, the conventional effect of two types of exosomal ncRNAs derived from CAFs: miRNAs and lncRNAs, has been reviewed; this effect inhibits the translation by binding to its target mRNAs (Dragomir et al., 2018). Considering that cancer cells need different stimuli to form tumors and that CAFs, through the secretion of exosomal ncRNAs, among other mechanisms, can contribute significantly by favoring protumoral events, this type of extracellular vesicles derived from CAFs loaded with miRNAs and lncRNAs can be considered as a potential therapeutic target to prevent the development of cancer. Nevertheless, despite the increase in the last 5 years of strong evidence suggesting CAFs-derived exosomal ncRNAs as therapeutic targets, more studies are needed to confirm the protumoral or antitumoral effects exerted by the various miRNAs and lncRNAs. Probably like miRNAs, lncRNAs have unconventional effects on gene expression that are still awaiting to be studied.
Regardless of future studies, the translational way from the laboratory to the clinic has begun. At present, an extensive database of ncRNAs in extracellular vesicles called EVAtlas, a product of enormous work, is available . In the site http://bioinfo.life.hust.edu.cn/EVAtlas, different characteristics such as extracellular vesicle types and isolation methods, expression level, functions, related drugs, and target genes of seven types of ncRNA of human extracellular vesicles can be consulted, as well as the pathological or physiological condition, tissues, cells, and biological fluids from which they have been isolated. This great bioinformatic tool could be part of the foundations to transfer the findings found in vitro, ex vivo and, in silico on the modulating effects of exosomal ncRNAs, derived and not derived from CAFs in the development of neoplasms, towards therapeutic regimens that improve the prognosis of cancer patients.
Although it is true that it will not be an easy task to incorporate miRNAs and lncRNAs as targets or tools in cancer treatment schemes, their relevance should not be underestimated. In the analysis carried out by GLOBOCAN (Ferlay et al., 2019;Ferlay et al., 2020), the global incidence and mortality rates of different cancers show an increase as the years go by, highlighting the need for additional options than those currently available to help in the fight against neoplasms. Designing and performing safe clinical trials, in which miRNAs and lncRNAs are used as therapeutic tools, is a great challenge and it will be essential to consider them a new real therapeutic alternative that benefits oncological patients.

AUTHOR CONTRIBUTIONS
JV-P conceptualized and drafted the manuscript. ML-T designed the table and the figure. AR-d-A and AP-S critically reviewed the manuscript. AP-S led the activities. All authors approved the submitted version of the manuscript. Frontiers in Cell and Developmental Biology | www.frontiersin.org November 2021 | Volume 9 | Article 717478