Heparanase2 (Hpa2) Promotes a Higher Degree of Cell Differentiation by Inducing Sox2 Expression in Head and Neck Carcinoma

Introduction. Heparanase is an endoglycosidase that cleaves heparan sulfate (HS) side chains of proteoglycans, activity that is highly implicated in tumor metastasis and angiogenesis. Heparanase2 (Hpa2) is a close homolog of heparanase that lacks intrinsic HS-degrading activity but retains the capacity to bind HS with high affinity, possibly leading to inhibition of heparanase activity. In head and neck cancer patients, Hpa2 expression is markedly elevated, correlating with prolonged time to disease recurrence and inversely correlating with tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions as a tumor suppressor. The molecular mechanism associated with favorable prognosis following Hpa2 induction is unclear. We found that Hpa2 regulates selected genes that affect tumor vascularity, tumor fibrosis, ER-stress, and apoptosis, together resulting in tumor suppression. Furthermore, Hpa2 overexpression induces cellular differentiation thereby maintaining a more normal epithelial phenotype correlated with low tumor grade and better prognosis.

Materials and Methods. In order to better elucidate the mode of Hpa2 action as a tumor suppressor in head and neck cancer, we applied gene array, gene silencing, CRISPR/Cas9 and RNA seq methodologies.

Results and discussion. RNA was extracted from control (Vo) and Hpa2-overexpressing FaDu cells and gene array was utilized to asses differences in gene transcription. This system was preferred because tumor growth was markedly attenuated in FaDu cells overexpressing Hpa2 (Gross-Cohen et al, Cancer Res, 76: 2791-801, 2016). Among other genes, we found and validated that the expression of sex-determining region Y-box 2 (Sox2) was markedly increased in cells overexpressing Hpa2. Moreover, Hpa2 gene editing (CRISPR/Cas9) was associated with reduced Sox2 levels, further supporting the notion that Hpa2 regulates Sox2 expression.

To reveal the significance of Sox2 for Hpa2-mediated tumor attenuation, we applied si-, and shRNA to silence Sox2 expression. Interestingly, Sox2 gene silencing was associated with reduced levels of cytokeratins 13 and 15 shown to be induced in FaDu cells overexpressing Hpa2. This suggests that the pro-differentiation function of Hpa2 is mediated, at least in part, by Sox2. Furthermore, Sox2 overexpression decreased while Sox2 silencing increased xenograft growth by 2.

Although Sox2 may have proto-oncogenic roles in various malignancies, several studies indicate that Sox2 is down-regulated in some malignancies and is often associated with better prognosis of head and neck cancer patients.

Conclusions. Our results further elucidate the mechanism by which Hpa2 functions, apparently involving the regulation of selected genes that affect tumor vascularity, tumor fibrosis, and cell differentiation, in part via up-regulation of Sox2, thus expanding the repertoire of Hpa2 functions as a tumor suppressor.