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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]

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]

We report that gastric cancer patients exhibiting high levels of heparanase 2 (Hpa2) survive longer. Similarly, mice administrated with gastric carcinoma cells engineered to overexpress Hpa2 produced smaller tumors and survived longer than mice administrated with control cells. These beneficial effects were found to associate with increased phosphorylation of AMP-activated protein kinase (AMPK) that play an instrumental role in cell metabolism and is situated at the center of a tumor suppressor network. We also found that MG132, an inhibitor of the proteasome that results in proteotoxic stress, prominently enhances Hpa2 expression. Notably, Hpa2 induction by MG132 appeared to be mediated by AMPK, thus establishing a loop that feeds itself where Hpa2 enhances AMPK phosphorylation that, in turn, induces Hpa2 expression, possibly leading to attenuation of gastric tumorigenesis. Heparanase is highly implicated in tumor metastasis due to its capacity to cleave heparan sulfate and, consequently, remodel the extracellular matrix underlying epithelial and endothelial cells. In striking contrast, only little attention was given to its close homolog, heparanase 2 (Hpa2), possibly because it lacks heparan sulfate-degrading activity typical of heparanase. We subjected sections of gastric carcinoma to immunostaining and correlated Hpa2 immunoreactivity with clinical records, including tumor grade, stage and patients' status. We over-expressed Hpa2 in gastric carcinoma cell lines and examined their tumorigenic properties in vitro and in vivo. We also evaluated the expression of Hpa2 by gastric carcinoma cells following inhibition of the proteasome, leading to proteotoxic stress, and the resulting signaling responsible for Hpa2 gene regulation. Here, we report that gastric cancer patients exhibiting high levels of Hpa2 survive longer. Similarly, mice administrated with gastric carcinoma cells engineered to over-express Hpa2 produced smaller tumors and survived longer than mice administrated with control cells. This was associated with increased phosphorylation of AMP-activated protein kinase (AMPK), a kinase that is situated at the center of a tumor suppressor network. We also found that MG132, an inhibitor of the proteasome that results in proteotoxic stress, prominently enhances Hpa2 expression. Notably, Hpa2 induction by MG132 appeared to be mediated by AMPK, and AMPK was found to induce the expression of Hpa2, thus establishing a loop that feeds itself where Hpa2 enhances AMPK phosphorylation that, in turn, induces Hpa2 expression, leading to attenuation of gastric tumorigenesis. These results indicate that high levels of Hpa2 in some tumors are due to stress conditions that tumors often experience due to their high rates of cell proliferation and high metabolic demands. This increase in Hpa2 levels by the stressed tumors appears critically important for patient outcomes. [ABSTRACT FROM AUTHOR]