Mucoepidermoid carcinoma (MEC) is the most common malignant salivary cancer. There is no effective therapy for advanced-stage MEC patients. Several studies show that cancer stem cells (CSC), a small sub-population of highly tumorigenic cells endowed with multipotency and self-renewal, play an important role in tumor progression, recurrence, and resistance to chemotherapy. Our group has unveiled the function of CSCs in MEC and that these cells present constitutive activation of the PI3K/AKT/mTOR pathway.
To investigate the effect of therapeutic inhibition of mTOR on the self-renewal of MEC CSCs and on the resistance of MEC CSCs to cisplatin.
Materials and Methods:
The effect of an mTOR inhibitor (temsirolimus) on resistance of MEC CSCs to cisplatin was evaluated in a panel of human MEC cell lines (UM-HMC-1, UM-HMC-3A, UM-HMC-3B), and using a patient-derived xenograft (PDX) model of MEC (UM-HMC-PDX-18). Western blots (WB) and immunohistochemical staining (IHC) were used to evaluate the impact of therapy on the activity of the PI3K/AKT/mTOR pathway and expression of p-S6K1 (downstream target of mTOR) and Bmi-1 (major regulator of stem cell self-renewal). The effect of therapy on self-renewal was evaluated with sphere assays in ultra-low attachment plates, and its effect on the fraction of CSCs by flow cytometry. The effect of therapy on CSCs in vivo was determined by treating mice bearing UM-HMC-PDX-18 tumors with temsirolimus and/or cisplatin.
Inhibition of mTOR signaling with temsirolimus decreased the expression of p-mTOR, p-S6K1, and Bmi-1, decreased the number of salispheres (P<0.05), and the fraction of CSCs (i.e. ALDHhighCD44high cells) (P<0.05). In contrast, cisplatin induced p-mTOR, p-S6K1, and Bmi-1 expression, increased the number and size of salispheres (P<0.05), and the fraction of CSCs in vitro (P<0.05). Combination treatment with temsirolimus prevented cisplatin-induced increase in the number of salispheres and in CSC fraction. We observed that single agent temsirolimus, and particularly combination of temsirolimus/cisplatin, suppressed MEC PDX tumor growth (P<0.05) and inhibited expression of p-S6K1 and Bmi-1 (P<0.05). Interestingly, we observed a strong correlation in number of pS6K1-positive and Bmi-1-positive cells in vivo, as measured by IHC (P<0.05). When the impact of treatment sequencing was evaluated, we observed that temsirolimus first followed by cisplatin was the most effective therapy in vivo(P<0.05).
Collectively, these results demonstrate that therapeutic inhibition of mTOR decreases CSC self-renewal, the fraction of CSCs, and tumor growth in preclinical models of MEC. These studies suggest that patients with salivary gland mucoepidermoid carcinoma might benefit from combination therapy involving temsirolimus and cisplatin.
This work was funded by grant from R01-DE021139 and R01-DE023220 from the NIH/NIDCR (J.E. Nör); the Uehara Memorial Foundation (T. Nakano); Soda Toyoji Memorial Foundation (T. Nakano).