Immanuel Lerner

Significance of Heparanase in Cancer and Inflammation

Heparan sulfate proteoglycans (HSPGs) are primary components at the interface between virtually every eukaryotic cell and its extracellular matrix. HSPGs not only provide a storage depot for heparin-binding molecules in the cell microenvironment, but also decisively regulate their accessibility, function and mode of action. As such, they are intimately involved in modulating cell invasion and signaling loops that are critical for tumor growth, inflammation and kidney function. In a series of studies performed since the cloning of the human heparanase gene, we and others have demonstrated that heparanase, the sole heparan sulfate degrading endoglycosidase, is causally involved in cancer progression, inflammation and diabetic nephropathy and hence is a valid target for drug development. Heparanase is causally involved in inflammation and accelerates colon tumorigenesis associated with inflammatory bowel disease. Notably, heparanase stimulates macrophage activation, while macrophages induce production and activation of latent heparanase contributed by the colon epithelium, together generating a vicious cycle that powers colitis and the associated tumorigenesis. Heparanase also plays a decisive role in the pathogenesis of diabetic nephropathy, degrading heparan sulfate in the glomerular basement membrane and ultimately leading to proteinuria and kidney dysfunction. Notably, clinically relevant doses of ionizing radiation (IR) upregulate heparanase expression and thereby augment the metastatic potential of pancreatic carcinoma. Thus, combining radiotherapy with heparanase inhibition is an effective strategy to prevent tumor resistance and dissemination in IR-treated pancreatic cancer patients. Also, accumulating evidence indicate that peptides derived from human heparanase elicit a potent anti-tumor immune response, suggesting that heparanase represents a promising target antigen for immunotherapeutic approaches against a broad variety of tumours. Oligosaccharide-based compounds that inhibit heparanase enzymatic activity were developed, aiming primarily at halting tumor growth, metastasis and angiogenesis. Some of these compounds are being evaluated in clinical trials, targeting both the tumor and tumor microenvironment.

Heparanase powers a chronic inflammatory circuit that promotes colitis-associated tumorigenesis in mice.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Expression of the enzyme heparanase is clearly linked to colon carcinoma progression, but its role in UC is unknown. Here we demonstrate for what we believe to be the first time the importance of heparanase in sustaining the immune-epithelial crosstalk underlying colitis-associated tumorigenesis. Using histological specimens from UC patients and a mouse model of dextran sodium sulfate-induced colitis, we found that heparanase was constantly overexpressed and activated throughout the disease. We demonstrate, using heparanase-overexpressing transgenic mice, that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors. We found that highly coordinated interactions between the epithelial compartment (contributing heparanase) and mucosal macrophages preserved chronic inflammatory conditions and created a tumor-promoting microenvironment characterized by enhanced NF-κB signaling and induction of STAT3. Our results indicate that heparanase generates a vicious cycle that powers colitis and the associated tumorigenesis: heparanase, acting synergistically with the intestinal flora, stimulates macrophage activation, while macrophages induce production (via TNF-α-dependent mechanisms) and activation (via secretion of cathepsin L) of heparanase contributed by the colon epithelium. Thus, disruption of the heparanase-driven chronic inflammatory circuit is highly relevant to the design of therapeutic interventions in colitis and the associated cancer.

Function of heparanase in prostate tumorigenesis: potential for therapy.

Purpose: Heparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. Whereas the role of heparanase in sustaining the pathology of human cancer is well documented, its association with prostate carcinoma remains uncertain. Our research was undertaken to elucidate the significance of heparanase in prostate tumorigenesis and bone metastasis. Experimental Design: We applied immunohistochemical analysis of tissue microarray, in vitro adhesion and invasion assays, as well as mouse models of intraosseous growth and spontaneous metastasis of prostate cancer, monitored by whole-body bioluminescent imaging. Electroporation-assisted administration of anti-heparanase small interfering RNA in vivo was applied as a therapeutic approach. Results: We report a highly statistically significant (P < 0.0001) prevalence of heparanase overexpression in prostate carcinomas versus noncancerous tissue, as well as strong correlation between tumor grade and the extent of heparanase expression. We observed >5-fold increase in the metastatic potential of PC-3 prostate carcinoma cells engineered to overexpress heparanase. Notably, overexpression of a secreted form of the enzyme also led to a dramatic increase in intraosseous prostate tumor growth. Local in vivo silencing of heparanase resulted in a 4-fold inhibition of prostate tumor growth, representing the first successful application of anticancer therapy based on heparanase small interfering RNA and validating the potential of heparanase as a target for prostate cancer treatment. Conclusions: Heparanase directly contributes to prostate tumor growth in bone and its ability to metastasize to distant organs. Thus, anti-heparanase strategy may become an important modality in the treatment of prostate cancer patients, particularly those with bone metastases.

Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis.

The coupling of voltage‐gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization‐evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion‐free solution. Cd2+, nifedipine, or verapamil inhibited depolarization‐evoked secretion in La3+, indicating specific binding of La3+ at the pore of L‐type VGCC, probably at the poly‐glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+ > Ce3+ > Pr3+, consistent with a size‐selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura‐2 imaging assay (∼1 pm) detected no La3+ entry, cytosolic La3+ build‐up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build‐up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build‐up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.

Role of Heparanase in Radiation-Enhanced Invasiveness of Pancreatic Carcinoma

Pancreatic cancer is characterized by very low survival rates because of high intrinsic resistance to conventional therapies. Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as one mechanism responsible for the limited benefit of radiotherapy in pancreatic cancer. In this study, we establish the role of heparanase-the only known mammalian endoglycosidase that cleaves heparan sulfate-in modulating the response of pancreatic cancer to radiotherapy. We found that clinically relevant doses of IR augment the invasive capability of pancreatic carcinoma cells in vitro and in vivo by upregulating heparanase. Changes in the levels of the transcription factor Egr-1 occurred in pancreatic cancer cells following radiation, underlying the stimulatory effect of IR on heparanase expression. Importantly, the specific heparanase inhibitor SST0001 abolished IR-enhanced invasiveness of pancreatic carcinoma cells in vitro, whereas combined treatment with SST0001 and IR, but not IR alone, attenuated the spread of orthotopic pancreatic tumors in vivo. Taken together, our results suggest that combining radiotherapy with heparanase inhibition is an effective strategy to prevent tumor resistance and dissemination, observed in many IR-treated pancreatic cancer patients. Further, the molecular mechanism underlying heparanase upregulation in pancreatic cancer that we identified in response to IR may help identify patients in which radiotherapeutic intervention may confer increased risk of metastatic spread, where antiheparanase therapy may be particularly beneficial.

Heparanase enzyme in chronic inflammatory bowel disease and colon cancer

Heparanase is the sole mammalian endoglycosidase that cleaves heparan sulfate, the key polysaccharide of the extracellular matrix and basement membranes. Enzymatic cleavage of heparan sulfate profoundly affects a variety of physiological and pathological processes, including morphogenesis, neovascularization, inflammation, and tumorigenesis. Critical involvement of heparanase in colorectal tumor progression and metastatic spread is widely documented; however, until recently a role for heparanase in the initiation of colon carcinoma remained underappreciated. Interestingly, the emerging data that link heparanase to chronic inflammatory bowel conditions, also suggest contribution of the enzyme to colonic tumor initiation, at least in the setting of colitis-associated cancer. Highly coordinated interplay between intestinal heparanase and immune cells (i.e., macrophages) preserves chronic inflammatory conditions and creates a tumor-promoting microenvironment. Here we review the action of heparanase in colon tumorigenesis and discuss recent findings, pointing to a role for heparanase in sustaining immune cell-epithelial crosstalk that underlies intestinal inflammation and the associated cancer.

Heparanase is preferentially expressed in human psoriatic lesions and induces development of psoriasiform skin inflammation in mice

Heparanase is the sole mammalian endoglycosidase that selectively degrades heparan sulfate, the key polysaccharide associated with the cell surface and extracellular matrix of a wide range of tissues. Extensively studied for its capacity to promote cancer progression, heparanase enzyme was recently implicated as an important determinant in several inflammatory disorders as well. Applying immunohistochemical staining, we detected preferential expression of heparanase by epidermal keratinocytes in human psoriatic lesions. To investigate the role of the enzyme in the pathogenesis of psoriasis, we utilized heparanase transgenic mice in a model of 12-O-tetradecanoyl phorbol 12-myristate 13-acetate-induced cutaneous inflammation. We report that over-expression of the enzyme promotes development of mouse skin lesions that strongly recapitulate the human disease in terms of histomorphological appearance and molecular/cellular characteristics. Importantly, heparanase of epidermal origin appears to facilitate abnormal activation of skin-infiltrating macrophages, thus generating psoriasis-like inflammation conditions, characterized by induction of STAT3, enhanced NF-κB signaling, elevated expression of TNF-α and increased vascularization. Taken together, our results reveal, for the first time, involvement of heparanase in the pathogenesis of psoriasis and highlight a role for the enzyme in facilitating abnormal interactions between immune and epithelial cell subsets of the affected skin. Heparanase inhibitors (currently under clinical testing in malignant diseases) could hence turn highly beneficial in psoriatic patients as well.

Abstract 2335: Role of heparanase in colitis associated cancer

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Here we report that heparanase enzyme acts as an important mediator of colitis-associated tumorigenesis. Heparanase is an only known mammalian enzyme that cleaves heparan sulfate, the major polysaccharide of the extracellular matrix, and plays multiple roles in inflammation and cancer progression. Applying histological specimens from UC patients and a mouse model of dextran sulfate sodium (DSS)-induced colitis, we found that heparanase is constantly overexpressed and activated during the course of the disease, both in the active and inactive phases of inflammation. Employing heparanase-overexpressing transgenic mice in the model of colitis-associated cancer, induced by carcinogen azoxymethane followed by repeated DSS administration, we demonstrated that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors, enabling faster tumor take, angiogenic switch and enhanced tumor progression. Notably, DSS-induced colitis alone (without azoxymethane pretreatment) lead to formation of colonic tumors in heparanase-transgenic, but not wild type mice, positioning heparanase as important physiological determinant in inflammation-driven colon carcinoma, replacing the need for carcinogen. Investigating molecular mechanisms underlying heparanase induction in colitis, we found that macrophage-derived cytokines (i.e., TNFalfa) are responsible for continuous overexpression of heparanase by chronically-inflamed colonic epithelium. Moreover, our results suggest the occurrence of heparanase-driven vicious cycle that power colitis and associated tumorigenesis: heparanase activity in inflamed colon, acting synergistically with the local cytokine milieu, stimulates macrophage activation, and the activated macrophages secrete TNFalfa which stimulate further production of heparanase by colonic epithelium. In addition, activated macrophages secrete cathepsin L - a cysteine protease responsible for proteolytic activation of latent heparanase enzyme. Altogether, our results identify heparanase as a key factor in pathogenesis of colitis-associated cancer and attest macrophages as both heparanase regulators and cellular target for heparanase action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2335.

Abstract C251: Heparanase mechanistically links chronic colitis and tumorigenesis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Here we report that heparanase enzyme acts as an important mediator of colitis‐associated tumorigenesis. Heparanase is an only known mammalian enzyme that cleaves heparan sulfate, the major polysaccharide of the extracellular matrix, and plays multiple roles in inflammation and cancer progression. Applying histological specimens from UC patients and a mouse model of dextran sulfate sodium (DSS)‐induced colitis, we found that heparanase is constantly overexpressed and activated during the course of the disease, both in the active and inactive phases of inflammation. Employing heparanase‐overexpressing transgenic mice in the model of colitis‐associated cancer, induced by carcinogen azoxymethane followed by repeated DSS administration, we demonstrated that heparanase overexpression markedly increased the incidence and severity of colitis‐associated colonic tumors, enabling faster tumor take, angiogenic switch and enhanced tumor progression. Notably, DSS‐induced colitis alone (without azoxymethane pretreatment) lead to formation of colonic tumors in heparanase‐transgenic, but not wild type mice, positioning heparanase as important physiological determinant in inflammation‐driven colon carcinoma, replacing the need for carcinogen. Investigating molecular mechanisms underlying heparanase induction in colitis, we found that TNF‐alpha is responsible for continuous overexpression of heparanase by chronically‐inflamed colonic epithelium. Moreover, our results suggest the occurrence of heparanase‐driven vicious cycle that powers colitis and associated tumorigenesis: heparanase activity in inflamed colon, acting synergistically with the local cytokine milieu, stimulates macrophage activation, and the activated macrophages secrete TNF‐alpha which stimulate further production of heparanase by colonic epithelium. In addition, activated macrophages secrete cathepsin L ‐ a cysteine protease responsible for proteolytic activation of latent heparanase enzyme. Altogether, our results identify heparanase as a key factor in pathogenesis of colitis‐associated cancer and attest the inhibition of heparanase as a promising mean to disrupt the vicious cycle that fuels chronic colitis and the associated tumorigenesis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C251.