Heparanase Augments Epidermal Growth Factor Receptor Phosphorylation: Correlation with Head and Neck Tumor Progression

Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size (∼4–7 kDa). Heparanase activity has long been correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage, and remodeling of the extracellular matrix (ECM) barrier (1, 2). More recently, heparanase up-regulation was documented in an increasing number of human carcinomas and hematologic malignancies (3, 4). In many cases, heparanase induction correlated with increased tumor metastasis, vascular density, and shorter postoperative survival of cancer patients, thus providing a strong clinical support for the prometastatic and proangiogenic functions of the enzyme and positioning heparanase as an attractive target for the development of anticancer drugs (5–7). Apart of the well-studied catalytic feature of the enzyme, heparanase was noted to exert biological functions apparently independent of its enzymatic activity. Nonenzymatic functions of heparanase include enhanced cell adhesion (8–11) and induction of p38 and Akt phosphorylation (8, 11–13). Moreover, enzymatically active and inactive heparanase were noted to induce VEGF expression in a Src-dependent manner (14), thus providing, among other mechanisms (15), a molecular basis for the potent proangiogenic capacity of heparanase. We hypothesized that Src stimulation by heparanase will facilitate the phosphorylation and activation of Src substrates such as epidermal growth factor receptor (EGFR). Here, we provide evidence that heparanase overexpression or exogenous addition enhances EGFR phosphorylation. Enhanced EGFR phosphorylation correlated with increased cell migration and proliferation, which was attenuated by Src inhibitors. Similarly, heparanase gene silencing was associated with reduced Src and EGFR phosphorylation levels and decreased cell proliferation. Moreover, heparanase expression correlated with increased phospho-EGFR levels and progression of head and neck carcinoma, thus providing a strong clinical support for EGFR modulation by heparanase.

Antibodies and reagents. The following antibodies were purchased from Santa Cruz Biotechnology: anti-Src (sc-18 and sc-19), anti-phosphotyrosine (PY; sc-7020), anti-Akt (sc-5298), anti-EGFR (sc-03), and anti-pEGFR (Tyr1173, sc-12351R). Polyclonal antibodies to phospho-Src (Tyr416), phospho-Akt (Ser473), and phospho-EGFR (Tyr845, Tyr1068, Tyr1148) were purchased from Cell Signaling. Anti-actin antibody was purchased from Sigma, and anti-p120^cat was purchased from Becton Dickinson. Bromodeoxyuridine (BrdUrd) was purchased from GE Healthcare, and anti-BrdUrd monoclonal antibody-horseradish peroxidase–conjugated was purchased from Roche. Anti-heparanase #1453 and #733 antibodies have previously been characterized (16). The selective p38 (SB 203580), PI 3-kinase (LY 294002), mitogen-activated protein kinase (MAPK; PD 98059), Src (PP1, PP2, Src inhibitor I), and EGFR (AG1478) inhibitors were purchased from Calbiochem and were dissolved in DMSO as stock solutions. DMSO was added to the cell culture as a control.

Cell culture and transfection. Human U87-MG glioma, Daoy meduloblastoma, LNCaP prostate carcinoma, MDA-MB-231 breast carcinoma, and A431 epidermoid carcinoma cells were purchased from the American Type Culture Collection. Cells were cultured in DMEM supplemented with glutamine, pyruvate, antibiotics, and 10% FCS in a humidified atmosphere containing 8% CO₂ at 37°C. For stable transfection, cells were transfected with heparanase gene constructs using the FuGene reagent according to the manufacturer's (Roche) instructions, selected with Zeocin (Invitrogen) for 2 wk, expanded, and pooled. Wild-type and double-mutated (DM; glutamic acid residues 225 and 343) recombinant heparanase proteins were purified from the conditioned medium of transfected HEK 293 cells, as described elsewhere (11).

Cell lysates, immunoprecipitation, and protein blotting. Preparation of cell lysates, immunoprecipitation (IP), and protein blotting was done essentially as described (8, 11, 16, 17) and detailed in Supplementary Materials and Methods section.

Cell proliferation. For growth curves, cells (5 × 10⁴) were seeded into 6-cm culture dishes in duplicates. Cells were dissociated with trypsin/EDTA and counted every other day using a Coulter Counter, and cell numbers were further confirmed by counting with a hemacytometer. Additionally, cell proliferation was analyzed by BrdUrd incorporation using cell proliferation labeling reagent (1:1,000; GE Healthcare), as described (11). At least 1,000 cells were counted for each cell type.

Colony formation in soft agar. Three milliliters of DMEM containing 0.5% Low Melt Agarose (Bio-Rad) and 10% FCS were poured into 60-mm Petri dish. The layer was covered with cell suspension (0.5 × 10⁴ cells) in 1.5 mL DMEM containing 0.3% Low Melt Agarose and 10% FCS, followed by addition of 2 mL DMEM containing 10% FCS. Medium was exchanged every 3 d. Colonies were visualized and counted under a microscope after 3 wk, as described (11).

Heparanase silencing and PCR analysis. Transfection and analysis of cells with anti-heparanase siRNA and control siRNA vectors were carried out essentially as described (12, 14, 18, 19). Anti-heparanase, anti-Src, anti-Yes, and control anti-green fluorescent protein (GFP) siRNA oligonucleotides (siGENOME ON-TARGET plus SMART pool duplex) were purchased from Dharmacon (Thermo Fisher Scientific, Inc.) and transfection was carried out with DharmaFECT reagent, according to the manufacturer's (Dharmacon) instruction. Total RNA was extracted with TRIzol (Life Technologies Bethesda Research Laboratories) and RNA (1 μg) was amplified using one step PCR amplification kit, according to the manufacturer's (ABgene) instructions. The PCR primer sets were as follows: Heparanase F-5′-AGGTCTGCATATGGAGGCGG-3′, Heparanase R-5′-TGAACTTCCTGGCCGGAGAG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) F-5′ CCAGCCGAGCCACATCGCTC-3′, and GAPDH R-5′ATGAGCCCCAGCCTTCTCCAT-3′.

Immunocytochemistry and cell migration. Immunofluorescent and cell migration assays were performed essentially as described (8, 11, 16) and detailed in Supplementary Materials and Methods section.

Immunostaining. Staining of formalin-fixed, paraffin-embedded 5-μm sections for phospho-EGFR (Tyr1173) was performed essentially as described (20, 21) and detailed in supplementary Materials and Methods section.

Tumorigenicity. Cells from exponential cultures of control (Vo) or heparanase-transfected U87 cells were dissociated with trypsin/EDTA, washed with PBS, and brought to a concentration of 5 × 10⁷cells/mL. Cell suspension (5 × 10⁶/0.1 mL) was inoculated s.c. at the right flank of 5-wk-old female Balb/C nude mice (n = 7). Xenograft size was determined twice a week by externally measuring tumors in two dimensions using a caliper. Tumor volume (V) was determined by the equation V = L × W² × 0.5, where L is the length and W the width of the xenograft. At the end of the experiment, mice were sacrificed and xenografts were resected, weighted, and fixed in formalin. Paraffin-embedded 5-μm sections were stained with anti–phospho-Src (Tyr416) and anti–phospho-EGFR (Tyr1173) antibodies, using the Envision kit, according to the manufacturer's instructions (Dako), as described (20, 21). All animal experiments were approved by the Animal Care Committee of the Technion, Haifa, Israel.

Heparanase activity assay. Preparation of ECM-coated 35-mm dishes and determination of heparanase activity were performed as described in detail elsewhere (22, 23) and in Supplementary Materials and Methods section.

Statistics. Univariate association between heparanase variables (intensity and extent of staining) and clinical and pathologic variables as well as patients' outcome, were analyzed using χ² tests (Pearson, Fisher exact test). Multivariable logistic regression was performed to detect independent variables that affect patients' status and to estimate relevant odds ratio with 95% confidence interval. A multivariable Cox's proportional hazard model was perform with stepwise selection, to identify independent predictors of survival (P value for enter and P value to stay were set as 0.1). The model included all variables with a P value of <0.2 by the univariate analysis.

All experiments were repeated at least thrice, with similar results. EGFR and Src phosphorylation is presented as mean + SE of at least five independent experiments quantified by densitometry analysis.

Heparanase facilitates EGFR phosphorylation. To explore the ability of heparanase to activate signaling cascades, heparanase was stably transfected into LNCaP prostate carcinoma cells and high levels of expression were confirmed by heparanase activity assay (Fig. 1A,, top) and immunoblotting (Fig. 1A,, second panel). Increased EGFR phosphorylation levels were observed in cells overexpressing heparanase by subjecting cell lysates to IP with an antibody directed against PY, followed by immunoblotting with anti-EGFR antibody (Fig. 1A,, third panel), or by using the reciprocal experiment (i.e., IP for EGFR, blot for PY; Fig. 1A,, fourth panel). Moreover, a marked elevation of EGFR phosphorylation was observed by using an antibody directed against the phosphorylated state of tyrosine 1173 localized at the protein COOH terminus (Fig. 1A,, bottom). We further applied this antibody and confirmed elevation of EGFR phosphorylation levels after heparanase overexpression in Daoy medulloblastoma (Fig. 1B), U87 glioma (Fig. 1C), and A431 epidermoid carcinoma (Fig. 1D) cells (third panels). A consistent, 3- to 5-fold increase in EGFR phosphorylation after heparanase overexpression was calculated by densitometry analysis of at least 5 independent experiments (Supplementary Fig. S1A; Fig. 1B,–D, bottom). Immunofluorescent staining of heparanase-transfected A431 (Fig. 2A,, top) and LNCaP cells (data not shown) further revealed elevation of EGFR phosphorylation levels and, moreover, localization of the phosphorylated receptors to the cell surface of cultured cells (Fig. 2A,, top), and tumor xenografts (Fig. 2A,, bottom). To further substantiate these findings, purified recombinant latent heparanase (wild-type) was exogenously added to U87 glioma cells and EGFR phosphorylation was examined by immunoblotting. Increased EGFR phosphorylation on Tyr1173 was noted already 15 minutes after heparanase addition, peaked at 30 minutes, and decreased thereafter (Supplementary Fig. S1B, left). Similar results were observed after addition of heparanase to A431 and Daoy cells (data not shown). Importantly, elevation of EGFR phosphorylation after heparanase addition correlated with a comparable ∼5-fold increase in Akt phosphorylation levels (Supplementary Fig. S1B, left; Fig. 2B,, third panel), a prominent signaling component downstream the EGFR, thus indicating EGFR activation (24–26). Although the latent 65 kDa heparanase protein was used in these experiments, contamination with active (50+8 kDa) enzyme, and the consequent release of HS-bound growth factors (i.e., HB-EGF) cannot be excluded. To verify this aspect, heparanase point mutated at amino acids critical for enzymatic catalysis (glutamic acids 225 and 343; DM; ref. 27) was similarly used. Addition of the DM heparanase facilitated EGFR phosphorylation 30 minutes after its addition to a magnitude comparable with the wild-type heparanase protein (Supplementary Fig. S1B, right; Fig. 2B,, DM, right), indicating that EGFR activation by heparanase does not involve enzymatic aspects but likely protein secretion (see below). To further substantiate the correlation between heparanase expression and EGFR phosphorylation levels noted in heparanase-transfected cells (Fig. 1A and B), we undertook the opposite approach and inhibited heparanase expression by means of anti-heparanase siRNA oligonucleotides. This approach was successfully applied to study the role of heparanase in tumor metastasis, angiogenesis, inflammation, and activation of signaling cascades (12, 14, 19, 28). As shown in Figure 2C, anti-heparanase siRNA effectively inhibited heparanase expression compared with control anti-GFP siRNA. Notably, heparanase down-regulation correlated with a marked, 5-fold decrease in EGFR phosphorylation (Fig. 2C , third and bottom panels), indicating that endogenous heparanase is intimately involved in EGFR regulation.

EGFR activation by heparanase involves Src. We have previously reported that heparanase induces the expression of VEGF, and that this effect is mediated by Src (14). We therefore suspected that EGFR activation by heparanase is similarly mediated by Src and examined this possibility by exposing heparanase-transfected U87 (Supplementary Fig. S2A, top two panels) and A431 (Supplementary Fig. S2A, bottom two panels) cells to specific inhibitors of key signaling molecules. No significant decrease in EGFR phosphorylation levels was observed after cell treatment with PI 3-kinase (LY), p38 (SB), or MAPK (PD) inhibitors (Supplementary Fig. S2A, top and third panels). In contrast, EGFR phosphorylation was markedly reduced in cells treated with the Src inhibitor PP2 (Supplementary Fig. S2A), and a similar decrease was noted in cells treated with two additional pharmacologic inhibitors of Src (PP1 and Src inhibitor I; data not shown). As little as 5 μmol/L PP2 resulted in a significant inhibition of EGFR phosphorylation on tyrosine 1173 (Supplementary Fig. S2B, top) and correlated with decreased Akt phosphorylation (Supplementary Fig. S2B, fourth panel). These findings suggest that heparanase enhances EGFR phosphorylation via activation of Src. This notion is supported by the following experiments. Enhanced phosphorylation of Src on tyrosine 416, thought to represent activated Src kinase, was observed after heparanase overexpression in U87 (Hepa; Fig. 3A,, third panel), A431, and LNCaP cells (Supplementary Fig. S2C, middle), and correlated with a comparable elevation of EGFR phosphorylation (Supplementary Fig. S2C, top; Fig. 3A,, top). Notably, EGFR and Src activation requires heparanase secretion. Thus, heparanase variant that fail to get secreted (Fig. 3A,, Δ15; ref. 18) did not induce EGFR and Src phosphorylation. In contrast, enzymatically inactive heparanase variant, which is secreted and accumulates extracellularly to high levels due to deletion of its heparin-binding domain (Fig. 3A,, Δ10; ref. 18), effectively stimulated EGFR and Src phosphorylation, clearly deciphering HS-independent heparanase function. Likewise, Src (Fig. 3B,, third panels) and EGFR (Fig. 3B,, top) phosphorylation were noted to be induced after heparanase overexpression (Fig. 2B,, left, 0), or exogenous addition of wild-type (Fig. 3B,, middle, WT, 0) or mutated, enzymatically inactive (DM; Fig. 3B,, right, 0) heparanase proteins, and this effect was markedly attenuated in cells treated with 5 μmol/L of the Src inhibitor, PP2 (Supplementary Fig. S3; Figs. 3B and 5). In agreement with this notion, we found reduced Src phosphorylation levels after heparanase gene silencing by means of anti-heparanase siRNA (Supplementary Fig. S2E; Fig. 3C,, left), further supporting a role of endogenous heparanase in Src and EGFR (Fig. 2C) regulation. Similarly, treatment of heparanase-transfected LNCaP cells with anti-Src siRNA oligonucleotides resulted in a marked decrease in Src (Fig. 3C,, middle and top) and EGFR (Fig. 3C,, middle and third panel) phosphorylation levels. Likewise, induction of Src and EGFR phosphorylation was evident after exogenous addition of heparanase to LNCaP cells transfected with control siRNA oligonucleotides (Fig. 3C,, right, si-GFP), and this induction was neglected in cells transfected with anti-Src si-RNA oligos (Fig. 3C , right, si-Src). In contrast, treatment with anti-Yes siRNA did not affect EGFR phosphorylation levels (data not shown). Moreover, heparanase overexpression (Supplementary Fig. S2F) or exogenous addition (data not shown) was noted to induce the phosphorylation of p120 catenin on tyrosine residues, a protein originally identified as a Src substrate (29), in agreement with our previous findings (14).

Ligands induce dimerization of EGFR, resulting in rapid autophosphorylation of tyrosine residues at positions 992, 1068, 1086, 1148, and 1173 (30). Src activation, however, can lead to phosphorylation of additional tyrosine residues of the EGFR (i.e., at positions 703, 845, 1101, 1148, and 1173; ref. 30). Among these, tyrosine 845 is considered most important to EGFR signaling (30–32). We, therefore, used antibodies directed against the phosphorylated state of tyrosine residue 845 and examined EGFR phosphorylation after heparanase addition or overexpression. We found that tyrosine 845 is phosphorylated upon heparanase addition, in magnitude and kinetics comparable with tyrosine 1173 (Supplementary Fig. S4A). Moreover, heparanase overexpression induced the phosphorylation of both tyrosine residues to a similar extent, and this effect was attenuated by the Src inhibitor PP2 (Supplementary Fig. S4B), or by Src gene silencing (Fig. 3C , fourth panels). In contrast, no significant elevation in the phosphorylation levels of tyrosine residues 1068 or 1148 could be detected after heparanase stimulation (data not shown). We concluded that heparanase stimulates EGFR on selected tyrosine residues such as 845 and 1173, likely involving Src activation.

Heparanase stimulates cell proliferation that is mediated by Src and EGFR. Next, we examined the cellular consequences of Src and EGFR activation by heparanase. Heparanase overexpression in LNCaP prostate carcinoma cells resulted in a nearly 2-fold increase in cell number (Fig. 4A,, Hepa), and treatment with EGFR (Fig. 4A,, Hepa+1478) or Src (Supplementary Fig. S5A, Hepa+PP2) inhibitors brought cell number to the level of control (Vo). Enhanced, 2.5-fold increase in cell proliferation was further confirmed by BrdUrd incorporation (Fig. 4B). Notably, heparanase-transfected LNCaP cells placed in soft agar produced significantly larger colonies compared with control (Vo) cells (Fig. 4C,, Hepa), whereas treatment with EGFR (Fig. 4C,, Hepa+1478) or Src (Fig. 4C,, Hepa+PP2) inhibitors brought colony size to the level of control. Exposing control (Vo) cells to PP2 or 1478 compounds did not affect colony size (Supplementary Fig. S5B). To further explore the involvement of heparanase in cell proliferation we examined BrdUrd incorporation after treatment with anti-heparanase siRNA oligonucleotides. We found a consistent, 2- to 3-fold decrease in BrdUrd incorporation after heparanase gene silencing in LNCaP (Fig. 4D,, left), MDA-MB-231 (Fig. 4D,, middle), and U87 (Fig. 4D , right) cells. These results suggest that endogenous heparanase plays a role in the regulation of cancer cell proliferation, likely by modulating the phosphorylation levels and activity of Src kinase and EGFR.

An in vivo support for this notion is shown in a tumor xenograft model. Heparanase overexpression in U87 glioma cells resulted in a 4- and 2.5-fold increase in tumor volume and weight, respectively (Fig. 5A), in agreement with previous findings (11). Notably, immunostaining of tumor xenograft sections revealed a marked enhancement of phospho Src (Tyr416) and phospho EGFR (Tyr1173) reactivity (Fig. 5B), further supporting a causal relationship between heparanase, the phosphorylation status of Src and EGFR, and tumor progression.

Heparanase expression correlates with the phosphorylation status of EGFR in squamous cell carcinoma of the head and neck. We have recently reported that heparanase expression by head and neck carcinomas correlates with tumor progression and inversely correlates with patients' status (21). To investigate the clinical significance of our findings, we subjected this cohort of tumor biopsies to immunostaining with phospho-specific EGFR antibodies (Tyr1173) and correlated the staining intensity and extent (i.e., percent of positively stained cells) with heparanase staining (21) and clinical variables. Among the 67 biopsies available for staining, 22 (33%) stained negative for phospho-EGFR (Tyr1173; Fig. 5C,, top) and 45 (67%) were positive. The phospho-EGFR–positive group was further categorized according to the intensity and extent of staining. Thus, weak staining (+1; Fig. 5C,, middle) was found in 64% (29 of 45) of positive specimens, whereas 36% (16 of 45) stained strongly (+2; Fig. 5C,, bottom) for phospho-EGFR. According to the extent criteria, 51% (23 of 45) of the specimens that positively stained for phospho-EGFR were scored as low extent (+1), and 49% (22 of 45) were scored as high extent (+2). Phospho-EGFR staining intensity correlated with tumor size (T; P = 0.006; Supplementary Table S1), and a similar correlation was found between phospho-EGFR staining extent and tumor size (T; P = 0.01; Supplementary Table S2), in agreement with the critical role of EGFR activation in head and neck tumor progression (33), and similar to the correlation found between heparanase expression and head and neck tumor progression (21). Notably, statistically significant correlation was found between heparanase expression and phospho-EGFR staining (P = 0.05). Even more significant was the correlation between the cellular localization of heparanase and phospho-EGFR staining intensity. Thus, although most cases (73%) exhibiting nuclear localization of heparanase were found negative for phospho-EGFR (Tyr1173), 69% of the cases with cytoplasmic heparanase stained strongly (+2) for phospho-EGFR (P = 0.03; Table 1). This finding is in agreement with a significant association between nuclear localization of heparanase and good prognosis of head and neck cancer patients (29). Logistic regression analysis revealed that both heparanase staining extent and EGFR phosphorylation (Tyr1173) are significant variables for tumor T stage (P = 0.04 and 0.01, respectively). We further used multivariate logistic regression analysis to examine the contribution of different variables to patients' status, including heparanase cellular localization and EGFR phosphorylation (Supplementary Table S3). The most significant variable that influenced the status of patients was heparanase localization (cytoplasmic versus nuclear, P = 0.003), whereas EGFR phosphorylation (Tyr1173) was not significant (P = 0.23). When tumor T stage (T0-2 versus T3-4) was included, heparanase localization continued to be the most significant variable for patients' status (P = 0.006), followed by the T stage (P = 0.03), whereas EGFR phosphorylation seemed insignificant (P = 0.69; Supplementary Table S4). Similar results were obtained applying Cox's Proportional Hazard Model (data not shown). These findings support our in vitro studies and suggest that heparanase facilitates the progression of head and neck carcinomas, and possibly other human malignancies, by modulating the phosphorylation levels and activity of selected signaling molecules such as Src and EGFR.

Heparanase activity has long been correlated with the metastatic potential of tumor-derived cells, a notion that is now well-supported experimentally and clinically, urging the development of heparanase inhibitors (3–7, 34). Heparanase up-regulation was found in most human carcinomas (3), yet its role in primary tumor progression is poorly understood. In some cases, heparanase up-regulation correlated with tumors larger in size (3, 21), a finding that was recapitulated in tumor xenograft models (11, 35, 36). Enhanced tumor progression is likely due, at least in part, to the induction of an angiogenic response. Elevation of microvessel density correlated with heparanase induction in solid (3) and hematologic (37) malignancies, and was also evident in tumor xenografts produced by cells overexpressing heparanase (11, 35, 36) and in heparanase-treated wounds (38, 39). The angiogenic capacity of heparanase has been traditionally attributed to its enzymatic activity, facilitating the sprouting of endothelial cells through the underlying basement membrane to form new capillaries, and releasing HS-bound angiogenic growth factors such as basic fibroblast growth factor and VEGF (15). More recently, heparanase was noted to induce the expression of angiogenic mediators such as tissue factor and VEGF in a manner that involves no enzymatic activity and is mediated by p38 and Src activation (13, 14), thus expanding the scope of heparanase function. Here, we report the ability of heparanase to activate Src in a variety of human tumor–derived cell lines. This was shown by overexpressing or after exogenous addition of heparanase and, moreover, by using heparanase gene silencing approach (Supplementary Figs. S2–4; Fig. 3), indicating that the endogenous levels of heparanase are intimately engaged in Src modulation. Activated Src, in turn, can induce the phosphorylation of EGFR on selected tyrosine residues (i.e., 845 and 1173), leading to enhanced cell proliferation (Fig. 4) and migration (Supplementary Fig. S5C and D). Significantly, heparanase gene silencing by means of siRNA oligonucleotides was associated with reduced BrdUrd incorporation in several cancer cell lines (Fig. 4), critically supporting a role of endogenous heparanase in tumor cell proliferation. This notion is best exemplified by the strong clinical correlation found between heparanase expression, EGFR phosphorylation, and tumor progression in head and neck cancer patients (Supplementary Tables S1–4; Table 1; ref. 21), depending on heparanase localization. Thus, although cytoplasmic heparanase correlated with intense staining of phospho-EGFR, most cases exhibiting nuclear localization of heparanase were scored negative for phospho-EGFR (73%; Table 1), in agreement with favorable outcome of these patients (21). These findings further support the good prognosis associated with nuclear heparanase in gastric and esophageal carcinomas (40, 41), due, possibly, to reduced EGFR activation. Overexpression of EGFR has been reported in ∼80% of head and neck tumors compared with low levels in normal mucosa, representing an early event in head and neck carcinogenesis (33, 42). However, although EGFR up-regulation is well-implicated in tumor aggressiveness, it may under signify the role of EGFR in primary tumor growth. Indeed, progress in the field has uncovered multiple mechanisms that lead to EGFR activation by various proteins and stimuli including, among others, cytokine receptors, integrins, cellular stress such as UV irradiation and oxidants, and G protein–coupled receptors (32, 43, 44). The latter was shown to be mediated by shedding of membrane-tethered HB-EGF by metalloproteases (44, 45). Notably, EGFR activation by heparanase does not involve enzymatic aspects because phosphorylation of EGFR to comparable levels was observed after the addition of enzymatically active or inactive heparanase proteins (Supplementary Fig. S1B; Figs. 2B and 3B). The ability of exogenously added heparanase to stimulate EGFR and Src phosphorylation suggests that heparanase secretion, rather than activity, is required. This is best exemplified by cells transfected with a gene construct lacking the heparin-binding domains of heparanase (Fig. 2A). Thus, deletion of the region Lys¹⁵⁸-Asn¹⁷¹, which results in a protein variant that is not secreted (18), failed to induce EGFR phosphorylation (Fig. 3A,, Δ15), whereas deletion of Gln²⁷⁰-Lys²⁸⁰, which results in accumulation of the protein to high levels in the culture medium (18), efficiently stimulated the phosphorylation of both Src and EGFR (Fig. 3A , Δ10). The ability of heparanase protein lacking the heparin binding domain (Δ10) to induce EGFR phosphorylation also excludes the release of HB-EGF, or other HS-bound growth factors, by nonenzymatic means (i.e., competition for HS binding sites).

In other cases, transactivation of EGFR was considered ligand-independent and was therefore attributed to intracellular mechanisms. c-Src has been shown to mediate ligand-independent EGFR phosphorylation on multiple sites, most notably tyrosine 845 (30, 32, 44, 46). Stimulation with heparanase induced EGFR phosphorylation on tyrosine 845 and 1173 to comparable levels (Supplementary Fig. S4; Fig. 3), which was suppressed by Src inhibitors (Supplementary Figs. S3 and 4; Fig. 3). This is in agreement with the role of Src in coupling various extracellular and intracellular signals to EGFR activation (47–49).

EGFR activation seems to be more prevalent in tumor cells that over express a variety of proteins, whereas increase in EGFR levels is not evident (50). Heparanase overexpression by human carcinomas emerges as such a protein that affects EGFR phosphorylation. Indeed, most of our studies were carried out with cells lines that express relatively low levels of EGFR (i.e., U87, LNCaP), with the exception of A431 cells. Thus, EGFR phosphorylation, rather than expression levels, evident by staining with phospho-specific antibodies such as those directed against Tyr845 or Tyr1173 (Fig. 5), may be used for tumor diagnosis and determination of patients prevalence for treatment with anti-EGFR small molecule inhibitors such as gefitinib or erlotinib.

Taken together, our results emphasize the notion that heparanase facilitates signaling cascades and modulates the activation state of Src, independent of its enzymatic activity. Activated Src, in turn, phosphorylates EGFR on selected tyrosine residues, leading to enhanced cell migration, cell proliferation, and tumor growth. Thus, apart of its enzymatic activity involved in cancer metastasis and angiogenesis (3–9), heparanase seems to modulate two critical systems responsible for tumor progression, namely VEGF (14) and EGFR. The ability of heparanase to function in an apparently enzymatic-independent manner and to activate signaling molecules such as Akt, p38, and Src (8–14) is intriguing and affects the way the protein is envisioned. Thus, whereas attention was mainly focused on compounds that inhibit heparanase enzymatic activity, no information is available on protein domains responsible for the nonenzymatic functions of heparanase. In this respect, identification of a putative heparanase receptor, possibly residing in lipid rafts (12) is a major future challenge. Neutralizing heparanase enzymatic and nonenzymatic functions is therefore expected to profoundly affect tumor progression and metastasis. Similarly, coadministration of EGFR and heparanase inhibitors may be more efficacious than each inhibitor alone. Studies examining this possibility are currently underway.

No potential conflicts of interest were disclosed.

Grant support: Israel Science Foundation (grant 549/06); National Cancer Institute, NIH (grant RO1-CA106456); the DKFZ-MOST cooperation program in cancer research; and the Rappaport Family Institute Fund. I. Vlodavsky is a Research Professor of the Israel Cancer Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.