Bmi-1 Inhibition Overcomes Cancer Stem Cell Resistance to Cytotoxic Therapy

Objectives: Conventional chemotherapy agents induce self-renewal and increase the fraction of cancer stem cells (CSC) in head and neck squamous cell carcinoma (HNSCC), priming the tumor for a more aggressive phenotype and evasive resistance. Cisplatin preferentially increases IL-6/STAT3 signaling and downstream expression of Bmi-1 in CSC, which drive therapeutic resistance and recurrence. Our hypothesis is that Bmi-1 inhibition will suppress the Cisplatin-induced CSC fraction and self-renewal.
Methods: Human HNSCC cells were treated with Cisplatin and/or Bmi-1 inhibitor (PTC-596) in vitro. shRNA-mediated Bmi-1 knockdown cells were generated and treated with Cisplatin or Carboplatin. The resulting CSC fraction was analyzed by flow cytometry (ALDH, CD44, Annexin V, DAPI), whereas expression of key signaling effectors was analyzed by western blot. Orospheres were generated using an automated liquid handler and subjected to stemness analyses. Immunocytochemistry (Bmi-1, pH2A, DAPI) was performed in chamber slides and analyzed via Leica THUNDER imaging system.
Results: Inhibition of Bmi-1 with PTC-596 and shRNA-Bmi-1 cells suppressed the conventional chemotherapy-induced increase in CSC fraction, and decreased IL-6/STAT3 signaling in both orospheres as well as cells grown in regular attachment conditions. Conventional chemotherapy induced dose-dependent Bmi-1 expression and DNA damage (pH2A). Bmi-1 inhibition induced apoptosis in HNSCC cells (Annexin V, cleaved-Caspase3). Immunofluorescence demonstrated co-staining of Bmi-1 and pH2A. Both pharmacologic inhibition and lentiviral knockdown prevented Cisplatin-induced orosphere formation and decreased orosphere size.
Conclusions: Collectively, these results demonstrate that Bmi-1 signaling plays an important role in HNSCC CSC chemoresistance and elucidate the mechanistic relationship between Cisplatin and the IL-6/STAT3 pathway. The cytotoxic effect of Cisplatin is elicited through DNA damage and Bmi-1 is a key mediator in the DNA damage response, suggesting a potential mechanism for the Cisplatin-mediated induction of Bmi-1 and increase in CSC fraction. Our results suggest a novel treatment paradigm for HNSCC patients based on combination of cytotoxic therapy and a Bmi-1 inhibitor.