E2F Regulates Bmi-1 Expression in Salivary Gland Mucoepidermoid Carcinoma

Objectives: The CRTC1/MAML2 gene fusion is a major driver of mucoepidermoid carcinoma (MEC) tumorigenesis. Cancer stem cells (CSC) promote MEC tumorigenesis and might be involved in therapeutic resistance. We have shown that Bmi-1, a master regulator of self-renewal, is highly expressed in MEC CSCs. Here, we hypothesized that E2F regulates Bmi-1 expression and MEC stemness.
Methods: To examine a potential link between CRTC1/MAML2 and MEC stemness, we silenced the fusion-positive MEC cells (UM-HMC-1,-3A,-3B) and evaluated impacts on Bmi-1 and CSC fraction. Additionally, we used an inhibitor of E2F and evaluated impacts on Bmi-1 and MEC stemness. shRNA-mediated CRTC1/MAML2-silenced MEC cells were generated, characterized, and seeded in biodegradable scaffolds to generate xenograft tumors in SCID mice. The silencing of CRTC1/MAML2 was confirmed in vitro and in vivo via western blot. Salispheres were also generated from control and CRTC1/MAML2-silenced cells in ultra-low attachment conditions. Finally, MEC cells were exposed to 0-80 µmol/L HLM006474 (small molecule inhibitor of E2F) for 24-72 hours, and the effect on Bmi-1 expression was evaluated via western blot.
Results: CRTC1/MAML2-silenced MEC cells showed enhanced Bmi-1 expression in vitro and in vivo. Interestingly, CRTC1/MAML2 silencing did not affect Bmi-1 expression in salispheres. Tumors generated from CRTC1/MAML2-silenced MEC cells exhibited enhanced tumor growth rate. Time course and dose dependence assays using HLM006474 showed inhibition of Bmi-1 expression in all MEC cell lines.
Conclusions: Collectively, these results demonstrate that the CRTC1/MAML2 fusion and the transcriptional factor E2F regulate Bmi-1 expression and tumor growth in models of mucoepidermoid carcinoma. These data suggest that a E2F inhibitor might be beneficial in the treatment of patients with salivary gland mucoepidermoid carcinoma.