CD109 modulates tumorigenicity, cancer aggressiveness and PD-L1-mediated CD8+ T cell function via regulation of EGFR and STAT3 signaling in cervical squamous cell carcinoma

Abstract

Cervical cancer is a common genital tract cancer associated with cancer-related deaths among women worldwide. Depending on the carcinoma stage, treatment includes surgery, chemotherapy, radiotherapy and immunotherapy, or combinations thereof. The recent development of immune checkpoint inhibitors is a future direction in cancer immunotherapy, because blockade of immune checkpoints unleashes CD8+ T cell-dominant antitumor immunity. Although extensive screening and vaccination are beneficial in decreasing disease burden, the molecular mechanisms underlying carcinogenesis and aggressiveness are not fully understood. CD109 is a cell surface glycoprotein frequently detected in various squamous cell carcinoma (SCCs, included cervical cancer) although its pathogenic role in SCCs remains unclear. In a previous study, we found higher CD109 expression in spheroid cells than in their attached counterparts, both of which were derived from primary cervical tumor tissues. Therefore, elucidating the functional role of CD109 in the regulation of tumorigenic and aggressive properties and immunotherapy might provide a potential target for a novel treatment strategy. In this study, CD109 expression was examined by immunohistochemistry with cervical tissue microarrays, and CD109 upregulation was observed in the cell membranes of cervical SCCs. Accordingly, four cervical cancer cell lines C33A, C4-1, CaSki and SiHa were used to functionally characterize the role of CD109 in cervical cancer. First, CD109(-) and CD109(+) subpopulations were isolated by fluorescence-activated cell sorting from C4-1 and CaSki cells. Compared with the CD109(-) subpopulation, CD109(+) cells displayed enhanced migration, cell proliferation, sphere-forming and anchorage-independent cell growth ability. Cervical cancer cell lines with high CD109 expression were then chosen for functional studies with siRNA knockdown and CRISPR/Cas9 knockout. In contrast to CD109-expressing cells, cells with silenced CD109 expression reversed the in vitro and in vivo tumorigenic and aggressive properties in cervical SCCs. Furthermore, the expression of EGFR and STAT3 phosphorylation has been determined in CD109-expressing cells and CD109-silenced cells. The mechanism of this potential signal regulation has been examined in CD109-expressing cells through treatment with anti-EGFR antibody and knockdown of STAT3. CD109 induced EGFR-mediated STAT3 phosphorylation is believed to be responsible for cell migration, proliferation and the maintenance of a cancer stem-like cell (CSC) phenotype. Additionally, CD109 upregulated PD-L1 expression in cervical cancer. Using CD8+ T cell/cervical cancer cell co-culture system, the effects of CD109 on CD8+ T cell apoptosis and cytokine secretion were evaluated by flow cytometric analysis. Blockade of CD109 has been found to suppress the apoptotic rate in CD8+ T cells and to enhance IFN-γ secretion, whereas CD109-expressing cancer cells inhibited IFN-γ secretion from CD8+ T cell. Furthermore, CD109 is positively correlated with EGFR and PD-L1 expression in cervical SCCs. CD109 facilitated PD-L1 expression via modulation of EGFR expression. In summary, abundant CD109(+) populations in cervical cancer cells potentially contribute to carcinogenesis and aggressiveness, whereas silencing of CD109 expression reverses those properties. CD109 mediates cervical tumorigenicity and aggressiveness through CD109/EGFR/STAT3 signaling. Moreover, CD109 affects PD-L1-mediated CD8+ T cell function via regulation of EGFR in cervical SCCs. These findings may provide insight into potential molecular targets for a combined CSC-related and immunotherapeutic strategy against cervical SCCs.