Chemoresistance of Head and Neck Cancer Stem Cells

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a frequent and deadly malignancy. Despite significant advances in understanding the pathobiology of this disease, patient morbidity associated with invasive treatment modalities along with a high frequency of tumor recurrence and metastasis typically result in unacceptably low patient survival and poor quality of life. The cancer stem cell (CSC) hypothesis provides an explanation for the observed disease relapse following current treatment paradigms, with pivotal malignant features of a cancer cell deriving from a shift towards stem-like features. CSCs, identified as ALDHhighCD44high cells, constitute only around 5% of total tumor cells but function as drivers of tumor initiation, mediate therapeutic evasion, and promote recurrence and metastases of HNSCC. We previously demonstrated that interleukin-6 (IL-6) secreted by endothelial cells within the perivascular niche in the tumor microenvironment enhances CSC survival, self-renewal, and tumorigenic potential. Cisplatin, the most common chemotherapy for HNSCC, activates the IL-6 pathway, which potentiates Cisplatin-induction of the CSC fraction and Bmi-1 expression. Bmi-1 is a master regulator of stem cell self-renewal and highly expressed in CSCs. Notably, cytotoxic chemotherapy enhances Bmi-1 expression and increases the CSC fraction in HNSCC. By virtue of their high tumorigenic potential and resistance to a multitude of therapies, CSCs present an imperative target for developmental therapeutics in HNSCC. This project sought to understand signaling mechanisms underlying chemoresistance of CSCs. Our central hypothesis is that therapeutic blockade of the IL-6 and Bmi-1 signaling axis suppresses chemotherapy-induced CSC self-renewal and inhibits CSC resistance. In this work, we aimed to: 1) determine the effect of IL-6/STAT3 inhibition on Cisplatin-induced phenotypic changes in the cancer cell population and downstream signaling effectors, 2) determine the therapeutic effect of IL-6/STAT3 inhibition on resistance to conventional Cisplatin therapy in HNSCC, and 3) elucidate the role of Bmi-1 in increasing the fraction of CSCs in response to cytotoxic chemotherapy. We demonstrate that blockade of IL-6R via lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, sphere formation, and to decrease the CSC fraction even in a Cisplatin-resistant HNSCC model. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in the CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant cells. Our results from human tumor specimens of 216 HNSCC patients show that high Bmi-1 expression correlates with decreased recurrence-free survival time. Moreover, both genetic knockdown of Bmi-1 as well as pharmacologic inhibition using the small molecule inhibitor PTC596 abrogates the increase of CSC fraction, sphere formation, and DNA damage response by cytotoxic chemotherapy. Treatment with PTC596 also suppressed Cisplatin-mediated increase of the CSC fraction in a scaffold xenograft model in vivo. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function, thus inhibiting cancer stemness. We showed that Bmi-1 contributes to chemoresistance of CSCs, which may occur through induction of DNA damage repair mechanisms as Bmi-1 plays an important role in the DNA repair response. Further investigation into the role of Bmi-1 in DNA damage elicited by platinum-based chemotherapy is still necessary. This work unveils important molecular mechanisms underlying CSC maintenance and therapeutic resistance, which have the potential to inform novel mechanism-based therapies targeting CSCs to improve survival and quality of life of patients with head and neck cancer.