ID proteins regulate IL-6 expression and cancer stem-like cell features following chemotherapy in ovarian cancer

Ovarian cancer (OC) remains the most lethal gynecological cancer. Discreet OC symptoms lead to late stage diagnosis, limiting treatment options. Most patients are initially responsive to chemotherapy, but over 80% of advanced cases relapse with treatment-resistant disease within 2 years. Cancer recurrence has been attributed to a small subpopulation of cancer stem-like cells (CSCs) that possess the ability to resist chemotherapy, self-renew, and asymmetrically divide. ID1-4 proteins, also known as inhibitors of differentiation, regulate cell fate and differentiation in normal stem and progenitor cells. In gliomas, ID1-4 proteins have been identified as master transcriptional regulators of CSC identity, signaling to downstream pathways important for self-renewal, tumor initiation, and survival; however, their role in OC CSCs and recurrence is unclear. Recently, we found that ID1-3 gene expression significantly increased following chemotherapy treatment in OC clinical samples and cell lines, and higher gene expression of ID1 and ID3 was associated with worse clinical outcomes. We hypothesize that ID proteins are required for OC CSC survival, self-renewal, and tumor recurrence following chemotherapy. In a subcutaneous xenograft mouse model, we found that cancer cell ID2 and ID4 protein expression significantly increased within 3 days following carboplatin treatment compared to vehicle groups, but this trend waned at later time points. Furthermore, we saw ID1-4 gene expression significantly increased stepwise following one and two cycles of chemotherapy in OVCAR8, OVCAR4, OVCAR5, and OV90 OC cell lines. Using a pan-ID inhibitor, AGX51, we have significantly knocked down ID expression in the four OC cell lines, and quantified CSC presence and function. Our data shows ID inhibition in OC cell lines reduces chemotherapy-dependent increases in CSC marker expression by flow cytometry (ALDH), spheroid formation, and gene expression of SOX2 and IL-6. Future studies will utilize siRNA silencing to elucidate the ID-dependent mechanisms of chemotherapy-induced SOX2 and IL-6 expression and how this contributes to OC survival. These studies highlight a novel target important in CSC maintenance that contributes to tumor progression and relapse. Understanding these mechanisms will enable development of therapies to overcome chemotherapy resistance and relapse, ultimately improving clinical outcomes.