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• 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]

Activity of the endo-beta-glucuronidase heparanase, capable of cleaving heparan sulfate (HS), is most often elevated in many types of tumors, associating with increased tumor metastasis and decreased patients' survival. Heparanase is therefore considered to be a valid drug target, and heparanase inhibitors are being evaluated clinically in cancer patients. Heparanase 2 (Hpa2) is a close homolog of heparanase that gained very little attention, likely because it lacks HS-degrading activity typical of heparanase. The role of Hpa2 in cancer was not examined in detail. In head and neck cancer, high levels of Hpa2 are associated with decreased tumor cell dissemination to regional lymph nodes and prolonged patients' survival, suggesting that Hpa2 functions to attenuate tumor growth. Here, we examined the role of Hpa2 in normal thyroid tissue and in benign thyroid tumor, non-metastatic, and metastatic papillary thyroid carcinoma (PTC) utilizing immunostaining in correlation with clinicopathological parameters. Interestingly, we found that Hpa2 staining intensity does not significantly change in the transition from normal thyroid gland to benign, non-metastatic, or metastatic thyroid carcinoma. Remarkably, we observed that in some biopsies, Hpa2 is accumulating on the membrane (envelop) of the nucleus and termed this cellular localization NM (nuclear membrane). Notably, NM localization of Hpa2 occurred primarily in metastatic PTC and was associated with an increased number of positive (metastatic) lymph nodes collected at surgery. These results describe for the first time unrecognized localization of Hpa2 to the nuclear membrane, implying that in PTC, Hpa2 functions to promote tumor metastasis. [ABSTRACT FROM AUTHOR]

Unlike the intense research effort devoted to exploring the significance of heparanase in human diseases, very little attention was given to its close homolog, heparanase 2 (Hpa2). The emerging role of Hpa2 in a rare autosomal recessive congenital disease called urofacial syndrome (UFS), clearly indicates that Hpa2 is not a pseudogene but rather a gene coding for an important protein. Hpa2 lacks the heparan sulfate (HS)-degrading activity typical of heparanase, yet exhibits high affinity to HS, affinity that is 10-fold higher than that of heparanase. The consequences of this high-affinity interaction of Hpa2 with plasma membrane HSPG has not been explored yet. Here, we used highly purified Hpa2 protein to examine this aspect. We provide evidence that cells adhere to and spread on dishes coated with Hpa2. We also show that cell migration is attenuated markedly by exogenous addition of Hpa2 to primary and transformed cells, a function that agrees with the anti-cancer properties of Hpa2. Interestingly, we found that exogenous addition of Hpa2 also disrupts the morphology of cell colonies, resulting in cell scattering. This implies that under certain conditions and experimental settings, Hpa2 may exhibit pro-tumorigenic properties. We further developed a panel of anti-Hpa2 monoclonal antibodies (mAb) and show that these properties of Hpa2 are prevented by some of the newly-developed mAb, thus providing new molecular tools to better appreciate the significance of Hpa2 in health and disease. [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]

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]

• 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]

• Expression of Hpa2 is markedly induced by stress. • Hpa2 induction by stress is mediated by ATF3. • High levels of Hpa2 and ATF3 are associated with longer survival of cancer patients. Activity of heparanase, endoglycosidase that cleaves heparan sulfate side chains in heparan sulfate proteoglycans, is highly implicated in tumor progression and metastasis. Heparanase inhibitors are therefore being evaluated clinically as anti-cancer therapeutics. Heparanase 2 (Hpa2) is a close homolog of heparanase that lacks HS-degrading activity and functions as an endogenous inhibitor of heparanase. As a result, Hpa2 appears to attenuate tumor growth but mechanisms that regulate Hpa2 expression and determine the ratio between heparanase and Hpa2 are largely unknown. We have recently reported that the expression of Hpa2 is induced by endoplasmic reticulum (ER) and proteotoxic stresses, but the mechanism(s) underlying Hpa2 gene regulation was obscure. Here we expand the notion that Hpa2 is regulated by conditions of stress. We report that while ER and hypoxia, each alone, resulted in a 3-7 fold increase in Hpa2 expression, combining ER stress and hypoxia resulted in a noticeable, over 40-fold increase in Hpa2 expression. A prominent induction of Hpa2 expression was also quantified in cells exposed to heat shock, proteotoxic stress, lysosomal stress, and chemotherapy (cisplatin), strongly implying that Hpa2 is regulated by conditions of stress. Furthermore, analyses of the Hpa2 gene promoter led to the identification of activating-transcription-factor 3 (ATF3) as a transcription factor that mediates Hpa2 induction by stress, thus revealing, for the first time, a molecular mechanism that underlies Hpa2 gene regulation. Induction of Hpa2 and ATF3 by conditions of stress that often accompany the rapid expansion of tumors is likely translated to improved survival of cancer patients. [ABSTRACT FROM AUTHOR]

The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized, encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here, we focused on the cross-talk between macrophages, chemotherapy, and heparanase and the combined effect on tumor progression. Macrophages were markedly activated by chemotherapeutics paclitaxel and cisplatin, evidenced by increased expression of proinflammatory cytokines, supporting recent studies indicating that chemotherapy may promote rather than suppress tumor regrowth and spread. Strikingly, cytokine induction by chemotherapy was not observed in macrophages isolated from heparanase-knockout mice, suggesting macrophage activation by chemotherapy is heparanase dependent. paclitaxel-treated macrophages enhanced the growth of Lewis lung carcinoma tumors that was attenuated by a CXCR2 inhibitor. Mechanistically, paclitaxel and cisplatin activated methylation of histone H3 on lysine 4 (H3K4) in wild-type but not in heparanase-knockout macrophages. Furthermore, the H3K4 presenter WDR5 functioned as a molecular determinant that mediated cytokine induction by paclitaxel. This epigenetic, heparanase-dependent host-response mechanism adds a new perspective to the tumor-promoting functions of chemotherapy, and offers new treatment modalities to optimize chemotherapeutics. SIGNIFICANCE: Chemotherapy-treated macrophages are activated to produce proinflammatory cytokines, which are blunted in the absence of heparanase., (©2019 American Association for Cancer Research.)

While the pro-tumorigenic function of heparanase is well taken, the role of its close homolog, heparanase 2 (Hpa2) in cancer is by far less investigated. Utilizing immunohistochemical analysis we found that Hpa2 is expressed by normal bladder transitional epithelium and its levels are decreased substantially in bladder cancer. Notably, tumors that retain high levels of Hpa2 were diagnosed as low grade (p=0.001) and low stage (p=0.002), suggesting that Hpa2 is required to preserve cell differentiation and halt cell motility. Indeed, migration of 5637 bladder carcinoma cells was attenuated significantly by exogenous addition of purified Hpa2, and over expression of Hpa2 in 5637 cells resulted in smaller tumors that were diagnosed as low grade. We also noted that tumors produced by Hpa2 over expressing cells are abundantly decorated with stromal cells and collagen deposition evident by Masson's/Trichrome staining, correlating with a marked increase in lysyl oxidase (LOX) staining. The association between Hpa2 and LOX was further confirmed clinically, because of the 16 cases that exhibited strong staining of Hpa2, 14 (87.5%) were also stained strongly for LOX (p=0.05). Collectively, our results suggest that Hpa2 functions as a tumor suppressor in bladder cancer, maintaining cellular differentiation and decreasing cell motility in a manner that appears to be independent of regulating heparanase activity., Competing Interests: The authors have no potential conflict of interest to declare.