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The emerging role of heparanase in tumor initiation, growth, metastasis, and chemoresistance is well recognized and is encouraging the development of heparanase inhibitors as anticancer drugs. Unlike the function of heparanase in cancer cells, very little attention has been given to heparanase contributed by cells composing the tumor microenvironment. Here we used a genetic approach and examined the behavior and function of macrophages isolated from wild-type (WT) and heparanase-knockout (Hpa-KO) mice. Hpa-KO macrophages express lower levels of cytokines (e.g., TNFα, IL1-β) and exhibit lower motility and phagocytic capacities. Intriguingly, inoculation of control monocytes together with Lewis lung carcinoma (LLC) cells into Hpa-KO mice resulted in nearly complete inhibition of tumor growth. In striking contrast, inoculating LLC cells together with monocytes isolated from Hpa-KO mice did not affect tumor growth, indicating that heparanase is critically required for activation and function of macrophages. Mechanistically, we describe a linear cascade by which heparanase activates Erk, p38, and JNK signaling in macrophages, leading to increased c-Fos levels and induction of cytokine expression in a manner that apparently does not require heparanase enzymatic activity. These results identify heparanase as a key mediator of macrophage activation and function in tumorigenesis and cross-talk with the tumor microenvironment.

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 is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of the extracellular matrix (ECM). Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. The enzyme also releases angiogenic factors from the ECM and thereby induces an angiogenic response in vivo. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer patients. Heparanase is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. Heparanase exhibits also non-enzymatic activities, independent of its involvement in ECM degradation. Among these, are the enhancement of Akt signaling, stimulation of PI3K- and p38-dependent endothelial cell migration, and up regulation of VEGF, all contributing to its potent pro-angiogenic activity. Studies on relationships between structure and heparanase inhibition activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of N-acetylation, and glycol-splitting of both pre-existing nonsulfated uronic acid residues (prevalently D-glucuronic) and/or those (Liduronic acid/L-galacturonic acid) generated by graded 2-O-desulfation, have permitted to select effective inhibitors of the enzymatic activity of heparanase. N-acetylated, glycol-split heparins emerged as especially strong inhibitors of heparanase, exerting little or no release of growth factors from ECM. N-acetylated glycol-split species of heparin, as well as heparanase gene silencing inhibit tumor metastasis, angiogenesis and inflammation in experimental animal models. These observations and the unexpected identification of a single functional heparanase, suggest that the enzyme is a promising target for anti-cancer and anti-inflammatory drug development.

Heparanase is an endoglycosidase that cleaves heparan sulfate side chains of proteoglycans, resulting in disassembly of the extracellular matrix underlying endothelial and epithelial cells and associating with enhanced cell invasion and metastasis. Heparanase expression is induced in carcinomas and sarcomas, often associating with enhanced tumor metastasis and poor prognosis. In contrast, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. Here, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkin’s B-lymphomas, and that heparanase inhibitors restrain the growth of tumor xenografts produced by lymphoma cell lines. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies that inhibit cell invasion and tumor metastasis, the hallmark of heparanase activity. Using luciferase-labeled Raji lymphoma cells, we show that the heparanase-neutralizing monoclonal antibodies profoundly inhibit tumor load in the mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that Raji cells lack intrinsic heparanase activity, but tumor xenografts produced by this cell line exhibit typical heparanase activity, likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing monoclonal antibodies attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans. Utilizing an ELISA method capable of detection and quantification of heparanase, we examined heparanase levels in the plasma and urine of a cohort of 29 patients diagnosed with type 2 diabetes mellitus (T2DM), 14 T2DM patients who underwent kidney transplantation, and 47 healthy volunteers. We provide evidence that heparanase levels in the urine of T2DM patients are markedly elevated compared to healthy controls (1162 ± 181 vs. 156 ± 29.6 pg/ml for T2DM and healthy controls, respectively), increase that is statistically highly significant (P<0.0001). Notably, heparanase levels were appreciably decreased in the urine of T2DM patients who underwent kidney transplantation, albeit remained still higher than healthy individuals (P<0.0001). Increased heparanase levels were also found in the plasma of T2DM patients. Importantly, urine heparanase was associated with elevated blood glucose levels, implying that glucose mediates heparanase upregulation and secretion into the urine and blood. Utilizing an in vitro system, we show that insulin stimulates heparanase secretion by kidney 293 cells, and even higher secretion is observed when insulin is added to cells maintained under high glucose conditions. These results provide evidence for a significant involvement of heparanase in diabetic complications.

The semaphorin guidance factors receptor plexin-A2 transduces sema6A and sema6B signals and may mediate, along with plexin-A4, the anti-angiogenic effects of sema6A. When associated with neuropilins plexin-A2 also transduces the anti-angiogenic signals of sema3B. Here we show that inhibition of plexin-A2 expression in glioblastoma derived cells that express wild type p53 such as U87MG and A172 cells, or in primary human endothelial cells, strongly inhibits cell proliferation. Inhibition of plexin-A2 expression in U87MG cells also results in strong inhibition of their tumor forming ability. Knock-out of the plexin-A2 gene in U87MG cells using CRISPR/Cas9 inhibits cell proliferation which is rescued following plexin-A2 re-expression, or expression of a truncated plexin-A2 lacking its extracellular domain. Inhibition of plexin-A2 expression results in cell cycle arrest at the G2/M stage, and is accompanied by changes in cytoskeletal organization, cell flattening, and enhanced expression of senescence associated β-galactosidase. It is also associated with reduced AKT phosphorylation and enhanced phosphorylation of p38MAPK. We find that the pro-proliferative effects of plexin-A2 are mediated by FARP2 and FYN and by the GTPase activating (GAP) domain located in the intracellular domain of plexin-A2. Point mutations in these locations inhibit the rescue of cell proliferation upon re-expression of the mutated intracellular domain in the knock-out cells. In contrast re-expression of a plexin-A2 cDNA containing a point mutation in the semaphorin binding domain failed to inhibit the rescue. Our results suggest that plexin-A2 may represent a novel target for the development of anti-tumorigenic therapeutics.

Tumor metastasis, the leading cause of cancer patients’ death, is still insufficiently understood. While concepts and mechanisms of tumor metastasis are evolving, it is widely accepted that cancer metastasis is accompanied by orchestrated proteolytic activity executed by array of proteases. While matrix metalloproteinases (MMPs) attracted much attention, other proteases constitute the tumor milieu, of which a large family consists of cysteine proteases named cathepsins. Like MMPs, some cathepsins are often upregulated in cancer and, once secreted or localized to the cell surface, can degrade components of the extracellular matrix. In addition, cathepsin L is held responsible for processing and activation of heparanase, an endo-β-glucuronidase capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans, activity that is strongly implicated in cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. In this review, we discuss recent progress in heparanase research focusing on heparanase-related molecules namely, cathepsin L and heparanase 2 (Hpa2), a heparanase homolog.

Little attention was given to heparanase 2 (Hpa2) over the last two decades, possibly because it lacks a heparan sulfate (HS)-degrading activity typical of heparanase. Emerging results suggest, nonetheless, that Hpa2 plays a role in human pathologies, including cancer progression where it functions as a tumor suppressor. Here, we examined the role of Hpa2 in cervical carcinoma. We report that high levels of Hpa2 correlate with prolonged survival of cervical carcinoma patients. Strong staining intensity of Hpa2 also correlates with low tumor grade. Overexpression of Hpa2 in SiHa cervical carcinoma cells resulted in tumor xenografts that were two-fold smaller than control tumors. Interestingly, even smaller tumor xenografts were developed by SiHa cells overexpressing the Pro140Arg and Asn543Ile Hpa2 missense mutations that were identified in patients diagnosed with urofacial syndrome (UFS). Utilizing the Ras recruitment system, we identified bromodomain-containing protein 7 (BRD7) to interact with Hpa2 and found that both BRD7 and the Hpa2 mutants are translocated to the cell nucleus in tumors developed by the Pro140Arg and Asn543Ile Hpa2 mutants. Utilizing our newly developed conditional Hpa2-KO mice, we further show that Hpa2 plays a critical role in macrophage polarization; in the absence of Hpa2, macrophages are shifted towards pro-tumorigenic, M2 phenotype. Notably, implanting SiHa cervical carcinoma cells together with Hpa2-KO macrophages promoted tumor growth. These results support, and further expand, the notion that Hpa2 functions as a tumor suppressor, co-operating with another tumor suppressor, BRD7.

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 β-catenin under homeostasis and following experimental skin carcinogenesis. Finally, the repression of β-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.