Impact of heparanase-2 (Hpa2) on cancer and inflammation: Advances and paradigms.

HPSE2, the gene-encoding heparanase 2 (Hpa2), is mutated in urofacial syndrome (UFS), a rare autosomal recessive congenital disease attributed to peripheral neuropathy. Hpa2 lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase (Hpa1), yet it exhibits a high affinity toward HS, thereby inhibiting Hpa1 enzymatic activity. Hpa2 regulates selected genes that promote normal differentiation, tissue homeostasis, and endoplasmic reticulum (ER) stress, resulting in antitumor, antiangiogenic, and anti-inflammatory effects. Importantly, stress conditions induce the expression of Hpa2, thus establishing a feedback loop, where Hpa2 enhances ER stress which, in turn, induces Hpa2 expression. In most cases, cancer patients who retain high levels of Hpa2 survive longer than patients bearing Hpa2-low tumors. Experimentally, overexpression of Hpa2 attenuates the growth of tumor xenografts, whereas Hpa2 gene silencing results in aggressive tumors. Studies applying conditional Hpa2 knockout (cHpa2-KO) mice revealed an essential involvement of Hpa2 contributed by the host in protecting against cancer and inflammation. This was best reflected by the distorted morphology of the Hpa2-null pancreas, including massive infiltration of immune cells, acinar to adipocyte trans-differentiation, and acinar to ductal metaplasia. Moreover, orthotopic inoculation of pancreatic ductal adenocarcinoma (PDAC) cells into the pancreas of Hpa2-null vs. wild-type mice yielded tumors that were by far more aggressive. Likewise, intravenous inoculation of cancer cells into cHpa2-KO mice resulted in a dramatically increased lung colonization reflecting the involvement of Hpa2 in restricting the formation of a premetastatic niche. Elucidating Hpa2 structure-activity-relationships is expected to support the development of Hpa2-based therapies against cancer and inflammation.

Dual T cell receptor/chimeric antigen receptor engineered NK-92 cells targeting the HPV16 E6 oncoprotein and the tumor-associated antigen L1CAM exhibit enhanced cytotoxicity and specificity against tumor cells.

The human papillomavirus type 16 (HPV16) causes a large fraction of genital and oropharyngeal carcinomas. To maintain the transformed state, the tumor cells must continuously synthesize the E6 and E7 viral oncoproteins, which makes them tumor-specific antigens. Indeed, specific T cell responses against them have been well documented and CD8+ T cells engineered to express T cell receptors (TCRs) that recognize epitopes of E6 or E7 have been tested in clinical studies with promising results, yet with limited clinical success. Using CD8+ T cells from peripheral blood of healthy donors, we have identified two novel TCRs reactive to an unexplored E618-26 epitope. These TCRs showed limited standalone cytotoxicity against E618-26-HLA-A*02:01-presenting tumor cells. However, a single-signaling domain chimeric antigen receptor (ssdCAR) targeting L1CAM, a cell adhesion protein frequently overexpressed in HPV16-induced cancer, prompted a synergistic effect that significantly enhanced the cytotoxic capacity of NK-92/CD3/CD8 cells armored with both TCR and ssdCAR when both receptors simultaneously engaged their respective targets, as shown by live microscopy of 2-D and 3-D co-cultures. Thus, virus-specific TCRs from the CD8+ T cell repertoire of healthy donors can be combined with a suitable ssdCAR to enhance the cytotoxic capacity of the effector cells and, indirectly, their specificity.

Nuclear localization of heparanase 2 (Hpa2) attenuates breast carcinoma growth and metastasis.

Unlike the intense research effort devoted to exploring the significance of heparanase in cancer, very little attention was given to Hpa2, a close homolog of heparanase. Here, we explored the role of Hpa2 in breast cancer. Unexpectedly, we found that patients endowed with high levels of Hpa2 exhibited a higher incidence of tumor metastasis and survived less than patients with low levels of Hpa2. Immunohistochemical examination revealed that in normal breast tissue, Hpa2 localizes primarily in the cell nucleus. In striking contrast, in breast carcinoma, Hpa2 expression is not only decreased but also loses its nuclear localization and appears diffuse in the cell cytoplasm. Importantly, breast cancer patients in which nuclear localization of Hpa2 is retained exhibited reduced lymph-node metastasis, suggesting that nuclear localization of Hpa2 plays a protective role in breast cancer progression. To examine this possibility, we engineered a gene construct that directs Hpa2 to the cell nucleus (Hpa2-Nuc). Notably, overexpression of Hpa2 in breast carcinoma cells resulted in bigger tumors, whereas targeting Hpa2 to the cell nucleus attenuated tumor growth and tumor metastasis. RNAseq analysis was performed to reveal differentially expressed genes (DEG) in Hpa2-Nuc tumors vs. control. The analysis revealed, among others, decreased expression of genes associated with the hallmark of Kras, beta-catenin, and TNF-alpha (via NFkB) signaling. Our results imply that nuclear localization of Hpa2 prominently regulates gene transcription, resulting in attenuation of breast tumorigenesis. Thus, nuclear Hpa2 may be used as a predictive parameter in personalized medicine for breast cancer patients.

miR-184 represses ß-catenin and behaves as a skin tumor suppressor.

miR-184-knockout mice display perturbed epidermal stem cell differentiation. However, the potential role of miR-184 in skin pathology is unclear. Here, we report that miR-184 controls epidermal stem cell dynamics and that miR-184 ablation enhances skin carcinogenesis in mice. In agreement, repression of miR-184 in human squamous cell carcinoma (SCC) enhances neoplastic hallmarks of human SCC cells in vitro and tumor development in vivo. Characterization of miR-184-regulatory network, suggests that miR-184 inhibits pro-oncogenic pathways, cell proliferation, and epithelial to mesenchymal transformation. Of note, depletion of miR-184 enhances the levels of ß-catenin under homeostasis and following experimental skin carcinogenesis. Finally, the repression of ß-catenin by miR-184, inhibits the neoplastic phenotype of SCC cells. Taken together, miR-184 behaves as an epidermal tumor suppressor, and may provide a potentially useful target for skin SCC therapy.

A New Synthesized Dicarboxylated Oxy-Heparin Efficiently Attenuates Tumor Growth and Metastasis.

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.

Heparanase-A single protein with multiple enzymatic and nonenzymatic functions.

Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions extracellularly to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors, augmenting, among other effects, gene transcription, autophagy, exosome formation, and heparan sulfate (HS) turnover. 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, metastasis, and chemoresistance. The enzyme appears to fulfill some normal functions associated, for example, with vesicular traffic, lysosomal-based secretion, autophagy, HS turnover, and gene transcription. It activates cells of the innate immune system, promotes the formation of exosomes and autophagosomes, and stimulates signal transduction pathways via enzymatic and nonenzymatic activities. These effects dynamically impact multiple regulatory pathways that together drive tumor growth, dissemination, and drug resistance as well as inflammatory responses. The emerging premise is that heparanase expressed by tumor cells, immune cells, endothelial cells, and other cells of the tumor microenvironment is a key regulator of the aggressive phenotype of cancer, an important contributor to the poor outcome of cancer patients and a valid target for therapy. So far, however, antiheparanase-based therapy has not been implemented in the clinic. Unlike heparanase, heparanase-2 (Hpa2), a close homolog of heparanase (Hpa1), does not undergo proteolytic processing and hence lacks intrinsic HS-degrading activity, the hallmark of heparanase. Hpa2 retains the capacity to bind heparin/HS and exhibits an even higher affinity towards HS than heparanase, thus competing for HS binding and inhibiting heparanase enzymatic activity. It appears that Hpa2 functions as a natural inhibitor of Hpa1 regulates the expression of selected genes that maintain tissue hemostasis and normal function, and plays a protective role against cancer and inflammation, together emphasizing the significance of maintaining a proper balance between Hpa1 and Hpa2.

Heparanase 2 (Hpa2)- a new player essential for pancreatic acinar cell differentiation.

Heparanase 2 (Hpa2, HPSE2) is a close homolog of heparanase. Hpa2, however, lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase enzymatic activity. Mutations of HPSE2 were identified in patients diagnosed with urofacial syndrome (UFS), a rare genetic disorder that exhibits abnormal facial expression and bladder voiding dysfunction, leading to renal damage and eventually renal failure. In order to reveal the role of HPSE2 in tissue homeostasis, we established a conditional Hpa2-KO mouse. Interestingly, the lack of Hpa2 was associated with a marked decrease in the expression of key pancreatic transcription factors such as PTF1, GATA6, and Mist1. This was associated with a two-fold decrease in pancreas weight, increased pancreatic inflammation, and profound morphological alterations of the pancreas. These include massive accumulation of fat cells, possibly a result of acinar-to-adipocyte transdifferentiation (AAT), as well as acinar-to-ductal metaplasia (ADM), both considered to be pro-tumorigenic. Furthermore, exposing Hpa2-KO but not wild-type mice to a carcinogen (AOM) and pancreatic inflammation (cerulein) resulted in the formation of pancreatic intraepithelial neoplasia (PanIN), lesions that are considered to be precursors of invasive ductal adenocarcinoma of the pancreas (PDAC). These results strongly support the notion that Hpa2 functions as a tumor suppressor. Moreover, Hpa2 is shown here for the first time to play a critical role in the exocrine aspect of the pancreas.

Heparanase Modulates Chromatin Accessibility.

Heparanase is the sole endoglucuronidase that degrades heparan sulfate in the cell surface and extracellular matrix (ECM). Several studies have reported the localization of heparanase in the cell nucleus, but the functional role of the nuclear enzyme is still obscure. Subjecting mouse embryonic fibroblasts (MEFs) derived from heparanase knockout (Hpse-KO) mice and applying transposase-accessible chromatin with sequencing (ATAC-seq), we revealed that heparanase is involved in the regulation of chromatin accessibility. Integrating with genome-wide analysis of chromatin states revealed an overall low activity in the enhancer and promoter regions of Hpse-KO MEFs compared with wild-type (WT) MEFs. Western blot analysis of MEFs and tissues derived from Hpse-KO vs. WT mice confirmed reduced expression of H3K27ac (acetylated lysine at N-terminal position 27 of the histone H3 protein). Our results offer a mechanistic explanation for the well-documented attenuation of inflammatory responses and tumor growth in Hpse-KO mice.

Effect of HPSE and HPSE2 SNPs on the Risk of Developing Primary Paraskeletal Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell malignancy that is accompanied by hypercalcemia, renal failure, anemia, and lytic bone lesions. Heparanase (HPSE) plays an important role in supporting and promoting myeloma progression, maintenance of plasma cell stemness, and resistance to therapy. Previous studies identified functional single nucleotide polymorphisms (SNPs) located in the HPSE gene. In the present study, 5 functional HPSE SNPs and 11 novel HPSE2 SNPs were examined. A very significant association between two enhancer (rs4693608 and rs4693084), and two insulator (rs4364254 and rs4426765) HPSE SNPs and primary paraskeletal disease (PS) was observed. SNP rs657442, located in intron 9 of the HPSE2 gene, revealed a significant protective association with primary paraskeletal disease and lytic bone lesions. The present study demonstrates a promoting (HPSE gene) and protective (HPSE2 gene) role of gene regulatory elements in the development of paraskeletal disease and bone morbidity. The effect of signal discrepancy between myeloma cells and normal cells of the tumor microenvironment is proposed as a mechanism for the involvement of heparanase in primary PS. We suggest that an increase in heparanase-2 expression can lead to effective suppression of heparanase activity in multiple myeloma accompanied by extramedullary and osteolytic bone disease.

Heparin-Superparamagnetic Iron Oxide Nanoparticles for Theranostic Applications.

In this study, superparamagnetic iron oxide nanoparticles (SPIONs) were engineered with an organic coating composed of low molecular weight heparin (LMWH) and bovine serum albumin (BSA), providing heparin-based nanoparticle systems (LMWH@SPIONs). The purpose was to merge the properties of the heparin skeleton and an inorganic core to build up a targeted theranostic nanosystem, which was eventually enhanced by loading a chemotherapeutic agent. Iron oxide cores were prepared via the co-precipitation of iron salts in an alkaline environment and oleic acid (OA) capping. Dopamine (DA) was covalently linked to BSA and LMWH by amide linkages via carbodiimide coupling. The following ligand exchange reaction between the DA-BSA/DA-LMWH and OA was conducted in a biphasic system composed of water and hexane, affording LMWH@SPIONs stabilized in water by polystyrene sulfonate (PSS). Their size and morphology were investigated via dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The LMWH@SPIONs' cytotoxicity was tested, showing marginal or no toxicity for samples prepared with PSS at concentrations of 50 µg/mL. Their inhibitory activity on the heparanase enzyme was measured, showing an effective inhibition at concentrations comparable to G4000 (N-desulfo-N-acetyl heparin, a non-anticoagulant and antiheparanase heparin derivative; Roneparstat). The LMWH@SPION encapsulation of paclitaxel (PTX) enhanced the antitumor effect of this chemotherapeutic on breast cancer cells, likely due to an improved internalization of the nanoformulated drug with respect to the free molecule. Lastly, time-domain NMR (TD-NMR) experiments were conducted on LMWH@SPIONs obtaining relaxivity values within the same order of magnitude as currently used commercial contrast agents.

Macrophages Upregulate Estrogen Receptor Expression in the Model of Obesity-Associated Breast Carcinoma.

Breast cancer (BC) and obesity are two heterogeneous conditions with a tremendous impact on health. BC is the most commonly diagnosed neoplasm and the leading cause of cancer-related mortality among women, and the prevalence of obesity in women worldwide reaches pandemic proportions. Obesity is a significant risk factor for both incidence and worse prognosis in estrogen receptor positive (ER+) BC. Yet, the mechanisms underlying the association between excess adiposity and increased risk/therapy resistance/poorer outcome of ER+, but not ER-negative (ER-), BC are not fully understood. Tumor-promoting action of obesity, predominantly in ER + BC patients, is often attributed to the augmented production of estrogen in 'obese' adipose tissue. However, in addition to the estrogen production, expression levels of ER represent a key determinant in hormone-driven breast tumorigenesis and therapy response. Here, utilizing in vitro and in vivo models of BC, we show that macrophages, whose adverse activation by obesogenic substances is fueled by heparanase (extracellular matrix-degrading enzyme), are capable of upregulating ER expression in tumor cells, in the setting of obesity-associated BC. These findings underscore a previously unknown mechanism through which interplay between cellular/extracellular elements of obesity-associated BC microenvironment influences estrogen sensitivity-a critical component in hormone-related cancer progression and resistance to therapy.

Heparanase 2 (Hpa2) attenuates the growth of human sarcoma.

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.

Non-enzymatic heparanase enhances gastric tumor proliferation via TFEB-dependent autophagy.

Heparanase (HPA) is the predominant enzyme that cleaves heparan sulfate and plays a critical role in a variety of pathophysiological processes. HPA activity has been traditionally correlated with tumor metastasis due to participation in the cleavage and remodeling of the extracellular matrix (ECM). Apart from its well-characterized catalytic properties, HPA was noticed to exert biological functions not rely on its enzymatic activity. This feature is supported by studies showing induction of signaling events, such as Src and AKT, by nonenzymatic HPA mutant. We provide evidence here that active HPA and inactive HPA mutant proteins enhance gastric cancer cell growth, possibly attributed to TFEB-mediated autophagy. Similarly, HPA gene silencing resulted in decreased gastric cancer cell proliferation and autophagy. Besides, TFEB inhibition reduced cell growth and autophagy induced by nonenzymatic HPA. Notably, HPA and TFEB were significantly elevated in gastric carcinomas compared with the adjacent gastric tissue. Moreover, the elevation of HPA gene expression and upregulation of TFEB levels have been associated with advanced clinical stage and poor prognosis of gastric cancer, providing strong clinical support for a connection between TFEB and HPA. Thus, neutralizing the nonenzymatic function of HPA and the related TFEB-driven autophagy may profoundly impact gastric cancer progression.

Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Heparanase Expression Propagates Liver Damage in CCL4-Induced Mouse Model.

Heparanase is elevated in various pathological conditions, primarily cancer and inflammation. To investigate the significance and involvement of heparanase in liver fibrosis, we compared the susceptibility of wild-type (WT) and heparanase-overexpressing transgenic (Hpa-tg) mice to carbon tetrachloride (CCL4)-induced fibrosis. In comparison with WT mice, Hpa-tg mice displayed a severe degree of tissue damage and fibrosis, including higher necrotic tendency and intensified expression of smooth muscle actin. While damage to the WT liver started to recover after the acute phase, damage to the Hpa-tg liver was persistent. Recovery was attributed, in part, to heparanase-stimulated autophagic activity in response to CCL4, leading to increased apoptosis and necrosis. The total number of stellate cells was significantly higher in the Hpa-tg than the WT liver, likely contributing to the increased amounts of lipid droplets and smooth muscle actin. Our results support the notion that heparanase enhances inflammatory responses, and hence may serve as a target for the treatment of liver damage and fibrosis.

Induction of heparanase 2 (Hpa2) expression by stress is mediated by ATF3.

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.

CMV Seropositive Status Increases Heparanase SNPs Regulatory Activity, Risk of Acute GVHD and Yield of CD34+ Cell Mobilization.

Heparanase is an endo-ß-glucuronidase that is best known for its pro-cancerous effects but is also implicated in the pathogenesis of various viruses. Activation of heparanase is a common strategy to increase viral spread and trigger the subsequent inflammatory cascade. Using a Single Nucleotide Polymorphisms (SNP)-associated approach we identified enhancer and insulator regions that regulate HPSE expression. Although a role for heparanase in viral infection has been noticed, the impact of HPSE functional SNPs has not been determined. We investigated the effect of cytomegalovirus (CMV) serostatus on the involvement of HPSE enhancer and insulator functional SNPs in the risk of acute graft versus host disease (GVHD) and granulocyte-colony stimulating factor related CD34+ mobilization. A significant correlation between the C alleles of insulator rs4364254 and rs4426765 and CMV seropositivity was found in healthy donors and patients with hematological malignancies. The risk of developing acute GVHD after hematopoietic stem cell transplantation was identified only in CMV-seropositive patients. A significant correlation between the enhancer rs4693608 and insulator rs28649799 and CD34+ cell mobilization was demonstrated in the CMV-seropositive donors. It is thus conceivable that latent CMV infection modulates heparanase regulatory regions and enhances the effect of functional SNPs on heparanase function in normal and pathological processes.

The HPSE Gene Insulator-A Novel Regulatory Element That Affects Heparanase Expression, Stem Cell Mobilization, and the Risk of Acute Graft versus Host Disease.

The HPSE gene encodes heparanase (HPSE), a key player in cancer, inflammation, and autoimmunity. We have previously identified a strong HPSE gene enhancer involved in self-regulation of heparanase by negative feedback exerted in a functional rs4693608 single-nucleotide polymorphism (SNP) dependent manner. In the present study, we analyzed the HPSE gene insulator region, located in intron 9 and containing rs4426765, rs28649799, and rs4364254 SNPs. Our results indicate that this region exhibits HPSE regulatory activity. SNP substitutions lead to modulation of a unique DNA-protein complex that affects insulator activity. Analysis of interactions between enhancer and insulator SNPs revealed that rs4693608 has a major effect on HPSE expression and the risk of post-transplantation acute graft versus host disease (GVHD). The C alleles of insulator SNPs rs4364254 and rs4426765 modify the activity of the HPSE enhancer, resulting in altered HPSE expression and increased risk of acute GVHD. Moreover, rs4426765 correlated with HPSE expression in activated mononuclear cells, as well as with CD3 levels and lymphocyte counts following G-CSF mobilization. rs4363084 and rs28649799 were found to be associated with CD34+ levels. Our study provides new insight into the mechanism of HPSE gene regulation and its impact on normal and pathological processes in the hematopoietic system.

Role of heparanase 2 (Hpa2) in gastric cancer.

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.