Irit Cohen

Heparanase mediates cell adhesion independent of its enzymatic activity

Heparanase is an endo‐β‐D‐glucuronidase that cleaves heparan sulfate and is implicated in diverse physiological and pathological processes. In this study we report on a novel direct involvement of heparanase in cell adhesion. We demonstrate that expression of heparanase in nonadherent lymphoma cells induces early stages of cell adhesion, provided that the enzyme is expressed on the cell surface. Heparanasemediated cell adhesion to extracellular matrix (ECM) results in integrin‐dependent cell spreading, tyrosine phosphorylation of paxillin, and reorganization of the actin cytoskeleton. The surface‐bound enzyme also augments cell invasion through a reconstituted basement membrane. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating that heparanase functions as an adhesion molecule independent of its endoglycosidase activity. The combined feature of heparanase as an ECM‐degrading enzyme and a cell adhesion molecule emphasizes its significance in processes involving cell adhesion, migration, and invasion, including embryonic development, neovascularization, and cancer metastasis.— Goldshmidt, O., Zcharia, E., Cohen, M., Aingorn, H., Cohen, I., Nadav, L., Katz, B.‐Z., Geiger, B., Vlodavsky, I. Heparanase mediates cell adhesion independent of its enzymatic activity. FASEB J. 17, 1015–1025 (2003)

Heparanase promotes growth, angiogenesis and survival of primary breast tumors

Despite great strides toward diagnosis and therapy, breast cancer remains a most threatening disease in its incidence, morbidity and mortality; therefore, additional knowledge regarding the molecular mechanisms contributing to breast cancer progression, as well as new targets for drug discovery are highly needed. Heparanase is the predominant enzyme involved in cleavage of heparan sulfate, the main polysaccharide component of the extracellular matrix. Experimental and clinical data indicate that heparanase plays important roles in cancer metastasis and angiogenesis. In breast carcinoma patients, heparanase expression correlates with the metastatic potential of the tumor. The present study was undertaken to investigate the role of heparanase in local growth and angiogenesis of primary breast tumors. MCF‐7 breast carcinoma cells were stable transfected with the human heparanase (H‐hpa) cDNA, or empty vector (mock), and injected into the mammary pad of nude mice. MRI was applied to monitor progression of tumor growth and angiogenesis. We demonstrate that tumors produced by cells overexpressing heparanase grew faster and were 7‐fold larger than tumors produced by mock transfected cells. This enhanced growth was accompanied by increased tumor vascularization and a higher degree of vessel maturation. Histological examination ascribed the differences in tumor growth to heparanase‐stimulated cell proliferation and survival. In‐vitro experiments reinforced heparanase role as a survival factor under stress conditions. Moreover, H‐hpa tumor cells infiltrate into the adjacent stroma, promoting formation of highly vascularized fibrous bands. Our results emphasize the significance and clarify the involvement of heparanase in primary breast cancer progression by generating a supportive microenvironment that promotes tumor growth, angiogenesis and survival.

Human Protease-Activated Receptor 1 Expression in Malignant Epithelia: A Role in Invasiveness

While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.

Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and is considered to be the outcome of chronic liver inflammation. Currently, the main treatment for HCC is surgical resection. However, survival rates are suboptimal partially because of tumor recurrence in the remaining liver. Our aim was to understand the molecular mechanisms linking liver regeneration under chronic inflammation to hepatic tumorigenesis. Mdr2-KO mice, a model of inflammation-associated cancer, underwent partial hepatectomy (PHx), which led to enhanced hepatocarcinogenesis. Moreover, liver regeneration in these mice was severely attenuated. We demonstrate the activation of the DNA damage-response machinery and increased genomic instability during early liver inflammatory stages resulting in hepatocyte apoptosis, cell-cycle arrest, and senescence and suggest their involvement in tumor growth acceleration subsequent to PHx. We propose that under the regenerative proliferative stress induced by liver resection, the genomic unstable hepatocytes generated during chronic inflammation escape senescence and apoptosis and reenter the cell cycle, triggering the enhanced tumorigenesis. Thus, we clarify the immediate and long-term contributions of the DNA damage response to HCC development and recurrence.

Identification of a novel functional androgen response element within hPar1 promoter: implications to prostate cancer progression

Human protease‐activated receptor‐1 (hPar1) plays a role in malignant and physiological invasion processes. We have identified a functional androgen response element (ARE) located in the hPar1 promoter upstream of the transcription start site at ‐1791 to ‐1777. Dihydrotestosterone treatment of the prostate cancer cell line LNCaP increased endogenous hPar1 mRNA levels, consistent with the threefold increase in promoter activity of hPar1‐luciferase reporter con‐ struct. Specific binding of the hPar1‐derived ARE to LNCaP nuclear extracts was demonstrated by electro‐ phoretic mobility shift assay. This binding was abro‐ gated by antiandrogen receptor (anti‐AR) antibodies or excess cold oligonucleotide but not by a mutated oligonucleotide. Moreover, using chromatin immuno‐ precipitation assays, we confirm the in vivo interaction between the AR and ARE domain of the hPar1 pro‐ moter. In parallel, we show that hormone ablation therapy markedly reduces the otherwise high hPar1 expression levels in prostate cancer biopsy specimens. We suggest that the hPar1 gene is regulated transcrip‐ tionally by androgens, representing one of several target genes effectively reduced during hormone abla‐ tion therapy. A major limitation of hormonal depriva‐ tion is that it causes only a temporary remission, and the cancer eventually reappears in a more malignant, androgen‐independent form. hPar1 is also overex‐ pressed in CL1 cells, an aggressively metastasizing, hormone‐independent subclone of LNCaP, and in PC3 prostate adenocarcinoma lacking AR in a mechanism yet to be fully elucidated. These data may imply that hPar1 expression correlates with prostate cancer pro‐ gression in androgen‐dependent and ‐independent phases and therefore, provides an instrumental, thera‐ peutic target for treatment in prostate cancer. FASEBJ. 19, 62‐72 (2005)

PAR1 plays a role in epithelial malignancies: transcriptional regulation and novel signaling pathway.

Protease‐activated receptor1 (PAR1) is the first and prototype member of an established PAR family comprising four members. The role of PAR1 in tumor biology has been established, and is characterized by a consistent direct correlation between overexpression of its levels and epithelial tumor aggressiveness. We have found that high expression of the human Par1 (hPar1) gene in epithelial tumors is controlled largely at the transcriptional level. This led us to assign Egr‐1, a transcription activator, as an inducer of hPar1, and p53, a tumor suppressor gene, as an inhibitor, both acting to achieve fine tuning of hPar1 in prostate carcinoma. High PAR1 levels maintain prosurvival signals in tumor cells while silencing or ablation of the gene induce apoptosis. Studies of our hPar1 transgenic mice, which overexpress hPar1 in the mammary glands, revealed a novel PAR1‐induced β‐catenin stabilization function. The components connecting PAR1 to β‐catenin stabilization have been determined, assigning at first Gα13 as a selective immediate component. The PAR1‐Gα13 axis recruits disheveled (DVL), an upstream signaling partner of the canonical Wnt signaling pathway. Silencing of DVL by siRNA‐DVL potently abrogates PAR1‐induced β‐catenin stabilization, demonstrating its critical role in the process. We, thus, propose that transcriptional regulation of hPar1 gene over expression in epithelia malignancies initiates a novel signaling pathway, directly connecting to β‐catenin stabilization, a core event in both tumorigenesis and developmental processes.

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

Background While protease-activated-receptor 1 (PAR1) plays a central role in tumor progression, little is known about the cell signaling involved. Methodology/Principal Findings We show here the impact of PAR1 cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR1 activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR1 C-tail did not prevent Shc-PAR1 association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR1 C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR1-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR1 -C-tail, hPar1 oncogenic properties are abrogated. Conclusions/Significance This is the first demonstration that a cytoplasmic portion of the PAR1 C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR1 -associating region in the breast cancer signaling niche.

Tamoxifen Induces Heparanase Expression in Estrogen Receptor–Positive Breast Cancer

Purpose: Mammalian heparanase degrades heparan sulfate, the main polysaccharide of the basement membrane. Heparanase is an important determinant in cancer progression, acting via the breakdown of extracellular barriers for invasion, as well as release of heparan sulfate–bound angiogenic and growth-promoting factors. The present study was undertaken to elucidate molecular mechanisms responsible for heparanase overexpression in breast cancer. Experimental Design: To characterize heparanase regulation by estrogen and tamoxifen and its clinical relevance for breast tumorigenesis, we applied immunohistochemical analysis of tissue microarray combined with chromatin immunoprecipitation assay, reverse transcription-PCR, and Western blot analysis. Results: A highly significant correlation (P < 0.0001) between estrogen receptor (ER) positivity and heparanase overexpression was found in breast cancer. Binding of ER to heparanase promoter accompanied estrogen-induced increase in heparanase expression by breast carcinoma cells. Surprisingly, heparanase transcription was also stimulated by tamoxifen, conferring a proliferation advantage to breast carcinoma cells grown on a naturally produced extracellular matrix. Heparanase overexpression was invariably detected in ER-positive second primary breast tumors, developed in patients receiving tamoxifen for the initial breast carcinoma. The molecular mechanism of the estrogenlike effect of tamoxifen on heparanase expression involves recruitment of transcription coactivator AIB1 to the heparanase promoter. Conclusions: Heparanase induction by ligand-bound ER represents an important pathway in breast tumorigenesis and may be responsible, at least in part, for the failure of tamoxifen therapy in some patients. Our study provides new insights on breast cancer progression and endocrine therapy resistance, offering future strategies for delaying or reversing this process.

Regulation of human protease-activated receptor 1 (hPar1) gene expression in breast cancer by estrogen

A pivotal role is attributed to the estrogen‐receptor (ER) pathway in mediating the effect of estrogen in breast cancer progression. Yet the precise mechanisms of cancer development by estrogen remain poorly understood. Advancing tumor categorization a step forward, and identifying cellular gene fingerprints to accompany histopathological assessment may provide targets for therapy as well as vehicles for evaluating the response to treatment. We report here that in breast carcinoma, estrogen may induce tumor development by eliciting protease‐activated receptor‐1 (PAR1) gene expression. Induction of PAR1 was shown by electrophoretic mobility shift assay, luciferase reporter gene driven by the hPar1 promoter, and chromatin‐immunoprecipitation analyses. Functional estrogen regulation of hPar1 in breast cancer was demonstrated by an endothelial tube‐forming network. Notably, tissue‐microarray analyses from an established cohort of women diagnosed with invasive breast carcinoma exhibited a significantly shorter disease‐free (P= 0.006) and overall (P=0.02) survival of patients that were positive for ER and PAR1, compared to ER‐positive but PAR1‐negative patients. We propose that estrogen transcriptionally regulates hPar1, culminating in an aggressive gene imprint in breast cancer. While ER+ patients are traditionally treated with hormone therapy, the presence of PAR1 identifies a group of patients that requires additional treatment, such as anti‐PAR1 biological vehicles or chemotherapy.—Salah, Z., Uziely, B., Jaber, M., Maoz, M., Cohen, I., Hamburger, T., Maly, B., Peretz, T., B.‐S, R. Regulation of human protease‐activated receptor 1 (hPar1) gene expression in breast cancer by estrogen. FASEB J. 26, 2031‐2042 (2012). www.fasebj.org

PAR Genes: Molecular Probes to Pathological Assessment in Breast Cancer Progression

Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond differently to a given treatment. Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR1 and PAR2 play a central role in breast cancer. The release of N-terminal peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to serve as a “mirror-image” index reflecting the level of cell surface PAR1&2-in body fluids. It is possible to use the levels of PAR-released peptide in patients and accordingly determine the choice of treatment. We have both identified PAR1 C-tail as a scaffold site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR1 induced breast cancer. Altogether, both PAR1 and PAR2 may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.