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Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors that support tumor growth. Heparanase expression is upregulated in human carcinomas, sarcomas, and hematological malignancies, correlating with increased tumor metastasis, vascular density, and shorter postoperative survival of cancer patients, and encouraging the development of heparanase inhibitors as anti-cancer drugs. Among these are heparin/HS mimetics, the only heparanase-inhibiting compounds that are being evaluated in clinical trials. We have synthesized dicarboxylated oxy-heparins (DCoxHs) containing three carboxylate groups per split residue (DC-Hep). The resulting lead compound (termed XII) was upscaled, characterized, and examined for its effectiveness in tumor models. Potent anti-tumorigenic effects were obtained in models of pancreatic carcinoma, breast cancer, mesothelioma, and myeloma, yielding tumor growth inhibition (TGI) values ranging from 21 to 70% and extending the survival time of the mice. Of particular significance was the inhibition of spontaneous metastasis in an orthotopic model of breast carcinoma following resection of the primary tumor. It appears that apart from inhibition of heparanase enzymatic activity, compound XII reduces the levels of heparanase protein and inhibits its cellular uptake and activation. Heparanase-dependent and -independent effects of XII are being investigated. Collectively, our pre-clinical studies with compound XII strongly justify its examination in cancer patients. [ABSTRACT FROM AUTHOR]

Background: Mammalian cells express a single functional heparanase, an endoglycosidase that cleaves heparan sulfate and thereby promotes tumor metastasis, angiogenesis, and inflammation. Malignant mesothelioma is highly aggressive and has a poor prognosis because of the lack of markers for early diagnosis and resistance to conventional therapies. The purpose of this study was to elucidate the mode of action and biological significance of heparanase in mesothelioma and test the efficacy of heparanase inhibitors in the treatment of this malignancy.Methods: The involvement of heparanase in mesothelioma was investigated by applying mouse models of mesothelioma and testing the effect of heparanase gene silencing (n = 18 mice per experiment; two different models) and heparanase inhibitors (ie, PG545, defibrotide; n = 18 per experiment; six different models). Synchronous pleural effusion and plasma samples from patients with mesothelioma (n = 35), other malignancies (12 non-small cell lung cancer, two small cell lung carcinoma, four breast cancer, three gastrointestinal cancers, two lymphomas), and benign effusions (five patients) were collected and analyzed for heparanase content (enzyme-linked immunosorbent assay). Eighty-one mesothelioma biopsies were analyzed by H-Score for the prognostic impact of heparanase using immunohistochemistry. All statistical tests were two-sided.Results: Mesothelioma tumor growth, measured by bioluminescence or tumor weight at termination, was markedly attenuated by heparanase gene silencing (P = .02) and by heparanase inhibitors (PG545 and defibrotide; P < .001 and P = .01, respectively). A marked increase in survival of the mesothelioma-bearing mice (P < .001) was recorded. Heparanase inhibitors were more potent in vivo than conventional chemotherapy. Clinically, heparanase levels in patients' pleural effusions could distinguish between malignant and benign effusions, and a heparanase H-score above 90 was associated with reduced patient survival (hazard ratio = 1.89, 95% confidence interval = 1.09 to 3.27, P = .03).Conclusions: Our results imply that heparanase is clinically relevant in mesothelioma development. Given these preclinical and clinical data, heparanase appears to be an important mediator of mesothelioma, and heparanase inhibitors are worthy of investigation as a new therapeutic modality in mesothelioma clinical trials. [ABSTRACT FROM AUTHOR]

Heparanase, the sole heparan sulfate (HS)-degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, metastasis, angiogenesis, and inflammation. Heparanase accomplishes this by degrading HS and thereby regulating the bioavailability of heparin-binding proteins; priming the tumor microenvironment; mediating tumor–host crosstalk; and inducing gene transcription, signaling pathways, exosome formation, and autophagy that together promote tumor cell performance and chemoresistance. By contrast, heparanase-2, a close homolog of heparanase, lacks enzymatic activity, inhibits heparanase activity, and regulates selected genes that promote normal differentiation, endoplasmic reticulum stress, tumor fibrosis, and apoptosis, together resulting in tumor suppression. The emerging premise is that heparanase is a master regulator of the aggressive phenotype of cancer, while heparanase-2 functions as a tumor suppressor. [ABSTRACT FROM AUTHOR]

Syndecan-1 (Sdc1) is an important member of the cell surface heparan sulfate proteoglycan family, highly expressed by epithelial cells in adult organisms. Sdc1 is involved in the regulation of cell migration, cell-cell and cell-matrix interactions, growth-factor, chemokine and integrin activity, and implicated in inflammatory responses and tumorigenesis. Gastrointestinal tract represents an important anatomic site where loss of Sdc1 expression was reported both in inflammation and malignancy. However, the biological significance of Sdc1 in chronic colitis-associated tumorigenesis has not been elucidated. To the best of our knowledge, this study is the first to test the effects of Sdc1 loss on colorectal tumor development in inflammation-driven colon tumorigenesis. Utilizing a mouse model of colitis-related colon carcinoma induced by the carcinogen azoxymethane (AOM), followed by the inflammatory agent dextran sodium sulfate (DSS), we found that Sdc1 deficiency results in increased susceptibility to colitis-associated tumorigenesis. Importantly, colitis-associated tumors developed in Sdc1-defficient mice were characterized by increased local production of IL-6, activation of STAT3, as well as induction of several STAT3 target genes that act as important effectors of colonic tumorigenesis. Altogether, our results highlight a previously unknown effect of Sdc1 loss in progression of inflammation-associated cancer and suggest that decreased levels of Sdc1 may serve as an indicator of colon carcinoma progression in the setting of chronic inflammation. [ABSTRACT FROM AUTHOR]

High heparanase expression is associated with enhanced tumor growth, angiogenesis, and metastasis in many types of cancer. However, the mechanisms driving high heparanase expression are not fully understood. In the present study, we discovered that drugs used in the treatment of myeloma upregulate heparanase expression. Frontline anti-myeloma drugs, bortezomib and carfilzomib activate the nuclear factor-kappa B (NF-κB) pathway to trigger heparanase expression in tumor cells. Blocking the NF-κB pathway diminished this chemotherapy-induced upregulation of heparanase expression. Activated NF-κB signaling was also found to drive high heparanase expression in drug resistant myeloma cell lines. In addition to enhancing heparanase expression, chemotherapy also caused release of heparanase by tumor cells into the conditioned medium. This soluble heparanase was taken up by macrophages and triggered an increase in TNF-α production. Heparanase is also taken up by tumor cells where it induced expression of HGF, VEGF and MMP-9 and activated ERK and Akt signaling pathways. These changes induced by heparanase are known to be associated with the promotion of an aggressive tumor phenotype. Importantly, the heparanase inhibitor Roneparstat diminished the uptake and the downstream effects of soluble heparanase. Together, these discoveries reveal a novel mechanism whereby chemotherapy upregulates heparanase, a known promoter of myeloma growth, and suggest that therapeutic targeting of heparanase during anti-cancer therapy may improve patient outcome. [ABSTRACT FROM AUTHOR]

Background Heparanase is an endo-β-D-glucuronidase that cleaves heparan sulfate chains of proteoglycans, resulting in the disassembly of the extracellular matrix. Heparanase has a central role in the development of various tumors, and its expression has been associated with increased tumor growth, angiogenesis and metastasis, but there is insufficient information about the function of heparanase in sarcomas. Study aims 1) To evaluate heparanase levels in adult soft tissue sarcomas (STS); 2) To examine the correlation between heparanase levels and pathological and clinical parameters and treatment outcome. Methods Pathological specimens of primary or metastatic STS were subjected to immunohistochemical analysis applying an anti-heparanase antibody. The clinical and the pathological data, together with the data of heparanase levels, were evaluated in a logistic regression model for tumor recurrence and survival. Results One hundred and one samples were examined, 55 from primary tumors and 46 from metastatic sites. A high expression of heparanase was observed in 29 (52.7%) and 22 specimens (47.8%), respectively. There was no statistically significant difference between heparanase expressions in the primary vs. metastatic sites of tumors. Moreover, no correlation was observed between heparanase staining and tumor aggressiveness, tumor recurrence or patient survival in various groups of patients. Conclusion Expression of heparanase was observed in 50% of the STS, in various histological subtypes. A larger study with homogenous groups of specific sub-types of STS or stages of disease is required to validate over-expression of heparanase as a marker of disease aggressiveness. [ABSTRACT FROM AUTHOR]

Heparanase is an endoglycosidase that degrades heparan sulfate (HS) at the cell surface and in the extracellular matrix. Heparanase is expressed mainly by cancer cells, and its expression is correlated with increased tumor aggressiveness, metastasis, and angiogenesis. Here, we report the cloning of a unique splice variant (splice 36) of heparanase from the subterranean blind mole rat (Spalax). This splice variant results from skipping part of exon 3, exons 4 and 5, and part of exon 6 and functions as a dominant negative to the wild-type enzyme. It inhibits HS degradation, suppresses glioma tumor growth, and decreases experimental B16-BL6 lung colonization in a mouse model. Intriguingly, Spalax splice variant 7 of heparanase (which results from skipping of exon 7) is devoid of enzymatic activity, but unlike splice 36 it enhances tumor growth. Our results demonstrate that alternative splicing of heparanase regulates its enzymatic activity and might adapt the heparanase function to the fluctuating normoxic-hypoxic subterranean environment that Spalax experiences. Development of anticancer drugs designed to suppress tumor growth, angiogenesis, and metastasis is a major challenge, of which heparanase inhibition is a promising approach. We anticipate that the heparanase splicing model, evolved during 40 million years of Spalacid adaptation to underground life, would pave the way for the development of heparanase-based therapeutic modalities directed against angiogenesis, tumor growth, and metastasis. [ABSTRACT FROM AUTHOR]

• Hpa2 attenuates sarcoma tumor growth. • Tumor growth inhibition by Hpa2 involves increased stress conditions (ER stress, hypoxia, JNK phosphorylation), induction of p63, and attenuation of stemness. • In leiomyosarcoma biopsies, nuclear localization of Hpa2 was associated with low stage tumors. This finding opens a new direction in Hpa2 research. The pro-tumorigenic properties of heparanase are well documented and established. In contrast, the role of heparanase 2 (Hpa2), a close homolog of heparanase, in cancer is not entirely clear. In carcinomas, Hpa2 is thought to attenuate tumor growth, possibly by inhibiting heparanase enzymatic activity. Here, we examine the role of Hpa2 in sarcoma, a group of rare tumors of mesenchymal origin, accounting for approximately 1% of all malignant tumors. Consistently, we found that overexpression of Hpa2 attenuates tumor growth while Hpa2 gene silencing results in bigger tumors. Mechanistically, attenuation of tumor growth by Hpa2 was associated with increased tumor stress conditions, involving ER stress, hypoxia, and JNK phosphorylation, leading to increased apoptotic cell death. In addition, overexpression of Hpa2 induces the expression of the p53 family member, p63 which, in sarcoma, functions to attenuate tumor growth. Moreover, we show that Hpa2 profoundly reduces stem cell characteristics of the sarcoma cells (stemness), most evident by failure of Hpa2 cells to grow as spheroids typical of cancer stem cells. Likewise, expression of CD44, a well-established cancer stem cell marker, was prominently decreased in Hpa2 cells. CD44 is also a cell surface receptor for hyaluronic acid (HA), a nonsulfated glycosaminoglycan that is enriched in connective tissues. Reduced expression of CD44 by Hpa2 may thus represent impaired cross-talk between Hpa2 and the extracellular matrix. Clinically, we found that Hpa2 is expressed by leiomyosarcoma tumor biopsies. Interestingly, nuclear localization of Hpa2 was associated with low-stage tumors. This finding opens a new direction in Hpa2 research. [ABSTRACT FROM AUTHOR]

• We report that Hpa2 attenuates head and neck tumor growth, associating with induction of Sox2 expression. • We provide evidence that silencing of Sox2 resulted in bigger tumors endowed with reduced cytokeratin levels, whereas smaller tumors were developed by cells overexpressing Sox2, suggesting that in head and neck carcinoma, Sox2 functions to inhibit tumor growth. • Hpa2-null cells engineered by Crispr/Cas 9, produced bigger tumors vs control cells, and rescue of Hpa2 attenuated tumor growth. • These results strongly imply that Hpa2 functions as a tumor suppressor in head and neck cancer, involving Sox2 upregulation mediated, in part, by the high-affinity interaction of Hpa2 with heparan sulfate. The pro-tumorigenic properties of heparanase are well documented, and heparanase inhibitors are being evaluated clinically as anti-cancer therapeutics. In contrast, the role of heparanase 2 (Hpa2), a close homolog of heparanase, in cancer is largely unknown. Previously, we have reported that in head and neck cancer, high levels of Hpa2 are associated with prolonged patient survival and decreased tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions to restrain tumorigenesis. Also, patients with high levels of Hpa2 were diagnosed as low grade and exhibited increased expression of cytokeratins, an indication that Hpa2 promotes or maintains epithelial cell differentiation and identity. To reveal the molecular mechanism underlying the tumor suppressor properties of Hpa2, and its ability to induce the expression of cytokeratin, we employed overexpression as well as gene editing (Crispr) approaches, combined with gene array and RNAseq methodologies. At the top of the list of many genes found to be affected by Hpa2 was Sox2. Here we provide evidence that silencing of Sox2 resulted in bigger tumors endowed with reduced cytokeratin levels, whereas smaller tumors were developed by cells overexpressing Sox2, suggesting that in head and neck carcinoma, Sox2 functions to inhibit tumor growth. Notably, Hpa2-null cells engineered by Crispr/Cas 9, produced bigger tumors vs control cells, and rescue of Hpa2 attenuated tumor growth. These results strongly imply that Hpa2 functions as a tumor suppressor in head and neck cancer, involving Sox2 upregulation mediated, in part, by the high-affinity interaction of Hpa2 with heparan sulfate. [ABSTRACT FROM AUTHOR]

• Our findings indicate that Hpa2 attenuates pancreatic tumor growth and highlight a feedback mechanism by which Hpa2 enhances ER stress which, in turn, induces Hpa2 expression. This results in consistent and severe ER stress, leading to increased apoptotic cell death and attenuated tumor growth. Thus, compounds designed to elicit ER stress may turn beneficial therapeutics in pancreatic cancer. • Hpa2 attenuates pancreatic tumor growth by exerting a feedback mechanism where Hpa2 enhances ER stress that, in turn, induces Hpa2 expression. • This results in consistent and severe ER stress, leading to increased apoptotic cell death and attenuated tumor growth. • Compounds designed to elicit ER stress may turn beneficial therapeutics in pancreatic cancer. While the pro-tumorigenic properties of the ECM-degrading heparanase enzyme are well documented, the role of its close homolog, heparanase 2 (Hpa2), in cancer is largely unknown. We examined the role of Hpa2 in pancreatic cancer, a malignancy characterized by a dense fibrotic ECM associated with poor response to treatment and bad prognosis. We show that pancreatic ductal adenocarcinoma (PDAC) patients that exhibit high levels of Hpa2 survive longer than patients with low levels of Hpa2. Strikingly, overexpression of Hpa2 in pancreatic carcinoma cells resulted in a most prominent decrease in the growth of tumors implanted orthotopically and intraperitoneally, whereas Hpa2 silencing resulted in bigger tumors. We further found that Hpa2 enhances endoplasmic reticulum (ER) stress response and renders cells more sensitive to external stress, associating with increased apoptosis. Interestingly, we observed that ER stress induces the expression of Hpa2, thus establishing a feedback loop by which Hpa2 enhances ER stress that, in turn, induces Hpa2 expression. This leads to increased apoptosis and attenuated tumor growth. Altogether, Hpa2 emerges as a powerful tumor suppressor in pancreatic cancer. [ABSTRACT FROM AUTHOR]

• Mice overexpressing heparanase (Hpa-tg) were applied to reveal the contribution of host heparanase to tumor growth and metastasis. • Xenograft tumors transplanted in Hpa-tg mice exhibited accelerated tumor progression and shorter survival time. • Accelerated tumor growth was attributed to increased levels of pro-tumorigenic factors in the plasma and bone marrow of the Hpa-tg mice. • Host heparanase impaired the overall anti-tumor activity of immune T-cell subpopulations. • Heparanase mediates the pro-tumorigenic crosstalk between the tumor, tumor microenvironment, immune cells and host factors. Heparanase, the sole heparan sulfate degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, angiogenesis and metastasis. Much of the impact of heparanase on tumor progression is related to its function in mediating tumor-host crosstalk, priming the tumor microenvironment to better support tumor growth and metastasis. We have utilized mice over-expressing (Hpa-tg) heparanase to reveal the role of host heparanase in tumor initiation, growth and metastasis. While in wild type mice tumor development in response to DMBA carcinogenesis was restricted to the mammary gland, Hpa-tg mice developed tumors also in their lungs and liver, associating with reduced survival of the tumor-bearing mice. Consistently, xenograft tumors (lymphoma, melanoma, lung carcinoma, pancreatic carcinoma) transplanted in Hpa-tg mice exhibited accelerated tumor growth and shorter survival of the tumor-bearing mice compared with wild type mice. Hpa-tg mice were also more prone to the development of metastases following intravenous or subcutaneous injection of tumor cells. In some models, the growth advantage was associated with infiltration of heparanase-high host cells into the tumors. However, in other models, heparanase-high host cells were not detected in the primary tumor, implying that the growth advantage in Hpa-tg mice is due to systemic factors. Indeed, we found that plasma from Hpa-tg mice enhanced tumor cell migration and invasion attributed to increased levels of pro-tumorigenic factors (i.e., RANKL, SPARC, MIP-2) in the plasma of Hpa-Tg vs. wild type mice. Furthermore, tumor aggressiveness and short survival time were demonstrated in wild type mice transplanted with bone marrow derived from Hpa-tg but not wild type mice. These results were attributed, among other factors, to upregulation of pro-tumorigenic (i.e., IL35+) and downregulation of anti-tumorigenic (i.e., IFN-γ+) T-cell subpopulations in the spleen, lymph nodes and blood of Hpa-tg vs. wild type mice and their increased infiltration into the primary tumor. Collectively, our results emphasize the significance of host heparanase in mediating the pro-tumorigenic and pro-metastatic interactions between the tumor cells and the host tumor microenvironment, immune cells and systemic factors. [ABSTRACT FROM AUTHOR]

The endoglycosidase heparanase specifically cleaves the heparan sulfate (HS) side chains on proteoglycans, an activity that has been implicated strongly in tumor metastasis and angiogenesis. Heparanase-2 (Hpa2) is a close homolog of heparanase that lacks intrinsic HS-degrading activity but retains the capacity to bind HS with high affinity. In head and neck cancer patients, Hpa2 expression was markedly elevated, correlating with prolonged time to disease recurrence and inversely correlating with tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions as a tumor suppressor. The molecular mechanism associated with favorable prognosis following Hpa2 induction is unclear. Here we provide evidence that Hpa2 overexpression in head and neck cancer cells markedly reduces tumor growth. Restrained tumor growth was associated with a prominent decrease in tumor vascularity (blood and lymph vessels), likely due to reduced Id1 expression, a transcription factor highly implicated in VEGF-A and VEGF-C gene regulation. We also noted that tumors produced by Hpa2- overexpressing cells are abundantly decorated with stromal cells and collagen deposition, correlating with a marked increase in lysyl oxidase expression. Notably, heparanase enzymatic activity was unimpaired in cells overexpressing Hpa2, suggesting that reduced tumor growth is not caused by heparanase regulation. Moreover, growth of tumor xenografts by Hpa2-overexpressing cells was unaffected by administration of a mAb that targets the heparin-binding domain of Hpa2, implying that Hpa2 function does not rely on heparanase or heparan sulfate. Cancer Res; 76(9); 2791-801. [ABSTRACT FROM AUTHOR]

Heparanase is the only enzyme inmammalscapable of cleaving heparan sulfate, an activity implicated in tumor inflammation, angiogenesis, and metastasis. Heparanase is secreted as a latent enzyme that is internalized and subjected to proteolytic processing and activation in lysosomes. Its role under normal conditions has yet to be understood. Here, we provide evidence that heparanase resides within autophagosomes, where studies in heparanase-deficient or transgenic mice established its contributions to autophagy. The protumorigenic properties of heparanase were found to be mediated, in part, by its proautophagic function, as demonstrated in tumor xenograft models of human cancer and through use of inhibitors of the lysosome (chloroquine) and heparanase (PG545), both alone and in combination. Notably, heparanase-overexpressing cells were more resistant to stress and chemotherapy in a manner associated with increased autophagy, effects that were reversed by chloroquine treatment. Collectively, our results establish a role for heparanase in modulating autophagy in normal and malignant cells, thereby conferring growth advantages under stress as well as resistance to chemotherapy. [ABSTRACT FROM AUTHOR]

Heparanase has been implicated in cancer but its contribution to the early stages of cancer development is uncertain. In this study, we utilized nontransformed human MCF10A mammary epithelial cells and two genetic mouse models [Hpa-transgenic (Hpa-Tg) and knockout mice] to explore heparanase function at early stages of tumor development. Heparanase overexpression resulted in significantly enlarged asymmetrical acinar structures, indicating increased cell proliferation and decreased organization. This phenotype was enhanced by coexpression of heparanase variants with a mutant H-Ras gene, which was sufficient to enable growth of invasive carcinoma in vivo. These observations were extended in vivo by comparing the response of Hpa-Tg mice to a classical two-stage 12-dimethylbenz(a)anthracene (DMBA)/12-o-tetradecanoylphorbol-13-acetate (TPA) protocol for skin carcinogenesis. Hpa-Tg mice overexpressing heparanase were far more sensitive than control mice to DMBA/TPA treatment, exhibiting a 10-fold increase in the number and size of tumor lesions. Conversely, DMBA/ TPA-induced tumor formation was greatly attenuated in Hpa-KO mice lacking heparanase, pointing to a critical role of heparanase in skin tumorigenesis. In support of these observations, the heparanase inhibitor PG545 potently suppressed tumor progression in this model system. Taken together, our findings establish that heparanase exerts protumorigenic properties at early stages of tumor initiation, cooperating with Ras to dramatically promote malignant development. [ABSTRACT FROM AUTHOR]

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth. [ABSTRACT FROM AUTHOR]