Regulation of Heparanase in Diabetes-Associated Pancreatic Carcinoma

While at least six types of cancer have been associated with diabetes, pancreatic ductal adenocarcinoma (PDAC) and diabetes exhibit a unique bidirectional relationship. Recent reports indicate that majority of PDAC patients display hyperglycemia, and ~50% have concurrent diabetes. In turn, hyperglycemic/diabetic state in PDAC patients fosters enhanced growth and dissemination of the tumor. Heparanase enzyme (the sole mammalian endoglycosidase degrading glycosaminoglycan heparan sulfate) is tightly implicated in PDAC progression, aggressiveness, and therapy resistance. Overexpression of heparanase is a characteristic feature of PDAC, correlating with poor prognosis. However, given the lack of heparanase expression in normal pancreatic tissue, the regulatory mechanisms responsible for induction of the enzyme in PDAC have remained largely unknown. Previously reported inducibility of heparanase gene by diabetic milieu components in several non-cancerous cell types prompted us to hypothesize that in the setting of diabetes-associated PDAC, hyperglycemic state may induce heparanase overexpression. Here, utilizing a mouse model of diet-induced metabolic syndrome/diabetes, we found accelerated PDAC progression in hyperglycemic mice, occurring along with induction of heparanase in PDAC. In vitro, we demonstrated that advanced glycation end-products (AGE), which are largely thought as oxidative derivatives resulting from chronic hyperglycemia, and the receptor for AGE (RAGE) are responsible for heparanase induction in PDAC cells. These findings underscore the new mechanism underlying preferential expression of heparanase in pancreatic cancer. Moreover, taken together with the well-established causal role of the enzyme in PDAC progression, our findings indicate that heparanase may sustain (at least in part) reciprocal causality between diabetes and pancreatic tumorigenesis.


INTRODUCTION
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of malignancy and expected to become the second-leading cause of cancer-related death in the United States by 2030 (1). Dysregulation of glucose metabolism occurs in majority of PDAC patients: at PDAC diagnosis up to 85% of subjects have hyperglycemia and ∼50% have diabetes (2)(3)(4). In a subset of PDAC patients diabetes occurs as early as 1-3 years before a detection of PDAC and is regarded as "new onset diabetes" (2)(3)(4)(5). Long-standing type 2 diabetes also acts as a risk factor for pancreatic cancer (6,7). Thus, elevation of glucose is a common phenomenon in PDAC (2)(3)(4). Additionally, positive association was reported between PDAC and insulin resistance/hyperinsulinemia (3,8,9). Conversely, recent reports suggest that diabetic state promotes PDAC and renders it highly aggressive, resistant to the existing therapies, and is associated with extremely poor prognosis (3,4,(10)(11)(12). Hence, PDAC and diabetes exhibit a unique bidirectional relationship, with diabetes being both an effect and etiological factor of the pancreatic cancer (3,4,11).
Here, applying in vivo model of diet-induced metabolic syndrome [a cluster of conditions that includes hyperglycemia, insulin resistance, hyperinsulinemia, diabetes, and obesity (37)], we found that accelerated PDAC progression in mice with impaired glucose metabolism coincided with induction of heparanase in pancreatic tumors. In vitro, we demonstrated that advanced glycation end-products [AGE, oxidative derivatives resulting from hyperglycemia, whose levels are increased in clinical/experimental diabetes (38)(39)(40)(41)(42)] and its receptor (RAGE) are responsible for upregulation of heparanase in PDAC cells. AGEs form at a constant but slow rate in the normal body, however, their formation is markedly accelerated in diabetes because of the increased availability of glucose. Given deterioration in glycemic control in a majority of PDAC patients (2)(3)(4), these findings provide molecular explanation for induction of heparanase in pancreatic carcinoma. Moreover, taken together with the previously demonstrated causal role of the enzyme in PDAC progression (13,26), our observations indicate that heparanase may be a part of the bi-directional link between diabetes and pancreatic tumorigenesis.

Cell Culture
The mouse pancreatic carcinoma cell line Panc02 [(43), a generous gift from M. Dauer (Munich, Germany)], and human pancreatic carcinoma cell lines MIA PaCa2 and PANC-1 (authenticated by STR profiling at the Genomics Center of the Biomedical Core Facility, Technion University, Israel), was grown in DMEM supplemented with 1 mM glutamine, 50 µg/ml streptomycin, 50 U/ml penicillin and 10% FCS (Biological Industries) at 37 • C and 8% CO 2 . At 60-80% confluence, cells were maintained for 24 h in serum-free DMEM, and either remained untreated or were incubated with AGE (AGE-BSA, catalog #JM-2221-10; MBL International Corporation), or BSA (Sigma-Aldrich).
In some experiments Panc02 cells were pretreated with RAGE neutralizing antibody (AF1179, R&D Systems) or TAK-242, TLR4 inhibitor (InvivoGen). The final endotoxin levels in experimental media containing AGE/BSA were 0.024-8 pg/mL, which were significantly lower than the concentrations typically found in diabetic patients (44), or than those required to activate Toll-like receptor (TLR) 4 or the classic NFκB pathway (45,46). Cells were lysed and processed for RNA isolation. In some experiments, cells were cultured on glass coverslips (12 mm; Carolina Biological Supply Company), fixed with 100% ice-cold methanol and processed for imunofluorescent staining.  (49). At week 12, when experimental mice developed metabolic syndrome and became hyperglycemic, Panc02 pancreatic carcinoma cells were injected subcutaneously (10 6 cells per mouse). Volume of tumors was monitored for 2 weeks following injection, then animals were sacrificed and tumors were snapfrozen for protein extraction. Part of the tumor tissue was processed for histology. Mice were kept under pathogen-free conditions; all experiments were performed in accordance with the Hebrew University Institutional Animal Care and Use Committee.

Immunoblotting
Tumor tissue samples were homogenized in lysis buffer containing 0.6 % SDS, 10 mM Tris-HCl, pH 7.5, supplemented with a mixture of protease inhibitors (Roche), and phosphatase inhibitors (Thermo Scientific). Equal protein aliquots were subjected to SDS-PAGE (10% acrylamide) under reducing conditions, and proteins were transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 3% BSA for 1 h at room temperature and probed with the appropriate antibody, followed by horseradish peroxidase-conjugated secondary antibody (KPL) and a chemiluminescent substrate (Biological Industries). Band intensity was quantified by densitometry analysis using Scion Image software.

Immunohistochemistry
Paraffin-embedded slides were deparaffinized and incubated in 3% H 2 O 2 . Antigen unmasking was carried out by heating (20 min) in a microwave oven in 10 mmol/L Tris buffer The band intensity was quantified using ImageJ software; intensity ratio for pAKT/total AKT is shown, error bars represent ±SE. Two-sided Student's t-test *p = 0.048; n ≥ 3 mice per condition. containing 1 mmol/L EDTA. Slides were incubated with primary antibodies diluted in CAS-Block (Invitrogen) or with CAS-Block alone, as a control. Appropriate secondary antibodies (Nichirei) were then added, and slides were incubated at room temperature for 30 min. Mouse stain kit (Nichirei) was used when primary mouse antibodies were applied to stain mouse tissues. Color was developed using the DAB Substrate Kit (Thermo Scientific) or Zymed AEC Substrate Kit (Zymed Laboratories), followed by counterstaining with Mayer's Hematoxylin. Controls without addition of primary antibody showed low or no background staining in all cases. Immunohistochemistry was scored based on staining intensity, as described in figure legends.

Analysis of Gene Expression by Quantitative Real Time PCR (qRT-PCR)
Total RNA was isolated from 3 x 10 6 cells using TRIzol (Invitrogen), according to the manufacturer's instructions, and quantified by spectrophotometry. After oligo (dT)-primed reverse transcription of 1 µg of total RNA, the resulting cDNA was amplified using the primers listed below. Realtime quantitative PCR (qRT-PCR) analysis was performed with an automated rotor gene system RG-3000A (Corbett Research). The PCR reaction mix (20 µl) was composed of 10 µl QPCR sybr master mix (Finnzymes), 5 µl of diluted cDNA (each sample in triplicate) and a final concentration of 0.3 µM of each primer. Hypoxanthine guanine phosphoribosyl transferase (HPRT) primers were used as an internal standard. The following primers were utilized: human HPRT sense:

Statistical Analysis
The results are presented as the mean ±SD or ±SE. P ≤ 0.05 were considered statistically significant. Statistical analysis was performed using unpaired Student's t-test. All statistical tests were two-sided.

Dysregulation of Glucose Metabolism Accelerates PDAC Progression and Induces Heparanase Expression
Deterioration in glycemic control is characteristic of PDAC: hyperglycemia has been repeatedly observed in majority (according to some reports-up to 85%) of PDAC patients (2)(3)(4); and epidemiologic studies report increased incidence of pancreatic carcinoma in diabetic populations (6,7). Thus, to investigate heparanase regulation in PDAC under dysregulated glucose metabolism, we utilized a murine experimental system, based on Panc02 mouse pancreatic carcinoma cells growing in C57BL/6J mice with the diet-induced metabolic syndrome, as described in Methods. Diet-induced metabolic syndrome in male C57BL/6J mice represents a reliable model, which closely parallels metabolic abnormalities in diabetic patients, such as increased circulating concentrations of glucose, hyperinsulinemia and impairment of glucose tolerance (47)(48)(49). Following 12 weeks of the diet intake, metabolic syndrome/impaired glucose metabolism was documented in experimental mice fed with high fat diet (but not in the control mice fed with the regular diet), as manifested by hyperglycemia, significantly increased glycated hemoglobin A1c (HbA1c) blood levels and glucose intolerance (Figures 1A-C), along with hyperinsulinemia ( Supplementary Figure 1) and increased body weight. Then, both control (normoglycemic) and experimental (hyperglycemic) mice were injected subcutaneously with Panc02 cells, as described in Methods. In agreement with previous reports (51,52), growth of Panc02 pancreatic carcinoma in vivo was markedly accelerated in hyperglycemic mice ( Figure 1D). Additionally, tumors grown in hyperglycemic mice expressed markedly elevated levels of phospho-AKT [pAKT] (Figure 1E), one of the hallmarks of the PDAC tumorigenesis (53,54).
We next compared heparanase expression in Panc02 tumors grown in experimental vs. control groups, applying immunoblotting. As shown in Figures 2A,B, markedly increased levels of heparanase protein were detected in Panc02 tumors growing in hyperglycemic, as compared to control (normoglycemic) mice. In agreement, quantitative RT-PCR analysis revealed ∼2-fold increase in heparanase mRNA levels in hyperglycemic vs. control mice. Additionally, immunostaining of the mouse tumor tissues with heparanase antibody revealed that Panc02 carcinoma cells, rather than host-derived stromal cells, represent the main source of the enzyme in tumors growing in hyperglycemic mice (Figures 2C,D).

AGE Induces Heparanase Expression in PDAC Cells in vitro
It was previously shown that various components of the diabetic milieu, including high glucose, free fatty acids, AGE, inflammatory cytokines (IL-6, TNF-α), upregulate heparanase in cells of non-pancreatic origin (29-31, 33-35, 50, 55). Importantly, increased levels of the aforementioned diabetic milieu constituents are present in the mouse model of metabolic syndrome/diabetes, used in our study (42,47,(56)(57)(58). We therefore tested effects of various diabetic milieu components on heparanase expression in Panc02 cells in vitro. While treatment with either high glucose, fatty acids, insulin, or IL-6 failed to induce the enzyme expression (Supplementary Figure 2), treatment with AGE significantly increased expression of heparanase mRNA in Panc02 cells ( Figure 3A). Immunofluorescent staining analysis also demonstrated increased heparanase protein levels in Panc02 cells following AGE treatment in vitro (Figures 3B,C), echoing in vivo observations (Figures 2C,D). Similar increase in heparanase expression in the presence of AGE was revealed in human PDAC cell lines MIA PaCa-2 and PANC-1 (Supplementary Figure 3).
AGE, whose formation is particularly augmented in diabetes due to combined effects of hyperglycemia and oxidative stress (39,40), interact with the receptor for advanced glycation end products [RAGE], a multiligand receptor, expressed by numerous cell types, including PDAC cells (59,60). Additionally, AGE are among the endogenous ligands known to activate toll-like receptor 4 (TLR4) (61)(62)(63), which is also expressed by PDAC cells (64,65). As reported in Vaz and Andersson (64) and Kang et al. (65) and confirmed by qRT-PCR, Panc02 cells express both RAGE and TLR4. Responsiveness of Panc02 cells to AGE stimulation was further supported by upregulation of IL-6 and COX-2 following AGE treatment (Supplementary Figures 4A,B), as well as enhanced NFκB signaling, evidenced by increased levels/nuclear localization of phospho-p65 in AGE-treated Panc02 cells (Supplementary Figure 4C). Notably, it was previously shown that NFκB is involved in heparanase upregulation in PDAC cells (66). Since NFκB activation is a known consequence of either TLR (67) or RAGE (68,69) signaling, we next applied inhibitory approach to distinguish between these two pathways. While presence of TLR4-specific inhibitor TAK242 (70) did not affect heparanase induction by AGE in our system (Supplementary Figure 5), presence of RAGE-neutralizing antibody significantly decreased AGEmediated heparanase induction, both at the mRNA and protein level (Figures 3A-C).

DISCUSSION
Among six cancer types attributable to diabetes (71), PDAC and diabetes display a unique reciprocal connection: PDAC is a presumed cause of derangement in glucose metabolism in a large number of cases, while diabetic state is known to promote pancreatic tumor progression (3,4,11,71). Diabetes and PDAC are two heterogeneous diseases with a tremendous impact on health: PDAC has the lowest 5-year relative survival rate compared with all other solid tumor malignancies (1) and diabetes has become a pandemic (72). Thus, identification of pathways linking PDAC and impaired glucose metabolism is of high importance.
Here, applying mouse model of metabolic syndrome/diabetes and concurrent pancreatic carcinoma, we show that diabetic state leads to induction of heparanase expression in PDAC (Figures 1,  2). This induction appears to be driven by AGE (Figure 3), a well-characterized member of the diabetic milieu. It should be noted that the limitation of the present study is that the single model was used for in vivo confirmation-due to enormous complexity of both diseases (PDAC and diabetes) it remains extremely challenging to establish additional mouse models faithfully reflecting concurrent pancreatic tumor progression and diabetes.
Given abundant evidence implicating heparanase in PDAC pathogenesis/aggressiveness/therapy resistance (13)(14)(15)(16)(17)25), our finding may provide a partial explanation for the mechanism through which diabetic state contributes to pancreatic carcinoma progression. Indeed, elevated levels of the enzyme have been found in PDAC tissue samples (13) and in body fluids of patients with active pancreatic cancer disease as compared to healthy donors (16). Pancreatic cancer patients whose tumors exhibit high levels of heparanase mRNA had a significantly shorter postoperative survival time than patients whose tumors contained relatively low levels (13,15,73). Heparanase is a highly significant independent variable for lymph node metastasis in pancreatic cancer patients, further supporting crucial involvement of the enzyme in PDAC progression (14). Importantly, the aforementioned epidemiological observations are backed by the experimental data demonstrating accelerated tumor growth/increased invasiveness in PDAC cells engineered to over-express heparanase (13,15,26), as well as a reduction of primary tumor progression/metastasis in murine models of PDAC following administration of heparanase-inhibiting compounds (25,74). Although several mechanisms controlling expression of the enzyme in various tissues have been described (30,31,55,75), regulation of heparanase induction in PDAC remained under investigated.
To promote PDAC development heparanase acts through augmented release of HS-bound growth factors, removal of extracellular barriers for invasion (13)(14)(15)(16)25) and creation of tumor-stimulating "aseptic" inflammatory conditions, i.e., increased production of IL-6 (a key cytokine driving pancreatic tumorigenesis) by heparanase-stimulated tumor associated macrophages (TAM) (26). In agreement with this mode of action, we found significantly increased levels of IL-6 (and in accordance-increased TAM infiltration) in heparanaseoverexpressing Panc02 tumors derived from hyperglycemic mice (Supplementary Figure 6). Additionally, ability of the enzyme to augment insulin/insulin-like growth factor 1 receptor signaling (76,77), along with the well-documented hyperinsulinemia in PDAC patients [either in the setting of new inset or longstanding diabetes (3,8,9)], suggests that in heparanase-rich PDAC microenvironment insulin is expected to induce stronger pro-tumorigenic response. Thus, heparanase induction appears to be a part of the mechanism(s) through which diabetic state promotes PDAC and renders it highly aggressive, therapyresistant and associated with particularly poor prognosis (3,4,(10)(11)(12).
On the other hand, emerging involvement of heparanase in diabetes, including its role in the islet/beta cell damage (32,(78)(79)(80), taken together with augmented production of the enzyme by pancreatic carcinoma cells under hyperglycemic conditions (this study), implies that the enzyme may exacerbate PDAC-associated diabetes. Indeed, pioneering studies by C R. Parish and his group identified multiple  (13,15,25,26,74). (D) In parallel, contribution of heparanase to the islet damage [originally described in the setting of type 1 diabetes (32, 78-80), but highly relevant to the pathogenesis of PDAC-associated diabetes as well] may impair beta cell function (E), exacerbating diabetic state. (F) Aggravation of diabetes further escalates AGE production, advancing PDAC heparanase expression and its protumorigenic action. roles for heparanase in islet damage (originally-in the setting of type 1 diabetes) (32,(78)(79)(80). The islet-damaging heparanase actions include promotion of the leukocyte migration from pancreatic blood vessels and their passage across the islet basement membrane, as well as depletion of heparan sulfate which is required for beta cell survival (32,(78)(79)(80).
Importantly, beta cell damage, islet inflammation and isletinfiltrating leukocytes (particularly, macrophages) appear to promote type 2 diabetes (T2D) as well (81). Along with insulin resistance, beta cell dysfunction is a major component of T2D pathology, and clinical onset of T2D does not occur until beta cells fail to secrete sufficient insulin to maintain normoglycemia in the face of insulin resistance (81)(82)(83)(84)(85). Macrophages infiltrate islets in clinical and experimental T2D (86,87) and are causally involved in beta cell dysfunction (81,85,88). Of note, patients with PDAC-associated diabetes often have high insulin levels and marked peripheral insulin resistance, similar to T2D [reviewed in (3)]. PDAC-associated diabetes also shares with T2D temporal relationship between insulin resistance, beta-cell dysfunction and development of impaired glucose tolerance (89): at earlier stages beta cells compensate for insulin resistance by increased insulin secretion, but progressive damage to beta cells leads to their dysfunction, deterioration in glycemic control, and at the later stage eventually leading to diabetes.
Given the secreted nature of the enzyme and its involvement in beta cell injury [via depletion of heparan sulfate (32,(78)(79)(80) and through tissue-damaging effects of the adverselyactivated islet-infiltrating macrophages, similarly to those demonstrated in other pathologies (17,26,28,50,90)], it is conceivable that in hyperglycemic patients elevated levels of heparanase, originating from the tumor of exocrine pancreas (i.e., PDAC), can exert pathogenic effects within the endocrine compartment (i.e., islets), further impairing glucose metabolism and leading to the onset/aggravation of diabetes.
Thus, our study not only reveals the mechanism of heparanase upregulation in PDAC, but also implies that the enzyme may contribute to a self-reinforcing sequence of events underlying bidirectional association between diabetes and PDAC (Figure 4): hyperglycemic state, that occurs in the majority of PDAC patients (Figure 4A), leads to heparanase overexpression in carcinoma cells via AGE-dependent mechanism ( Figure 4B); increased levels of heparanase, in turn, promote PDAC progression ( Figure 4C) through several previously-described mechanisms (13,15,25,26,74). In parallel, heparanase is capable of facilitating islet damage (Figure 4D), thus leading to beta cell dysfunction (Figure 4E), aggravating diabetic state and escalating AGE production, which further enhances PDAC heparanase expression and its protumorigenic action ( Figure 4F).
Reciprocal relationships between PDAC and diabetes are certainly multifactorial in origin, and an array of molecular/cellular events underlying these relationships is far from being fully elucidated. Yet, our findings help to recognize the multilevel control that heparanase provides to heterotypic interactions among exocrine, endocrine and immune compartments of the pancreas in PDAC-diabetes link, suggesting that disruption of reciprocal causality between diabetes and PDAC through heparanase-targeting approaches may be of clinical benefit.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by The Hebrew University Institutional Animal Care and Use Committee, Hebrew University of Jerusalem, Israel.

AUTHOR CONTRIBUTIONS
RG, EH, AR, AA, and DN conducted the experiments. AM and AG acquired the data. RG, AM, EH, and ME analyzed the data. TP and ME designed the studies. AM, AG, and TP reviewed the manuscript. ME was responsible for conceptualization, research design, supervised the study, and wrote the manuscript.