Targeting of the PI3K/AKT/mTOR signaling pathway as an effective radiosensitizing strategy for the treatment of human oral squamous cell carcinoma in vitro and in vivo

• Main Authors: YU, CHIH-CHIA, 余芝嘉
• Other Authors: HUANG, HSIEN-BIN
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/62775039735888503311

博士 === 國立中正大學 === 分子生物研究所 === 104 === Background: Radiation therapy (RT) is the current standard adjuvant approach for oral squamous cell carcinoma (OSCC) patients. Activation of the PI3K/AKT/mTOR signaling pathway has been linked to the decreased radiation responsiveness in human oral cancer. Thus, it limits efficacy of radiotherapy. In addition, this pathway has been considered to be a major contributor in radiation resistance in various tumors. Although a number of studies suggest that blockage of this pathway can in vitro and in vivo enhance the radiation response, its role in radioresistance of OSCC is not fully explored and little is known about the radiosensitization capacity of patient-derived OSCCs. In this study, we used patient-derived OSCCs and a radiation-resistant cell line for two purposes: to in vitro and in vivo evaluate the anti-tumor effects and to understand the mechanisms for the targeting of PI3K/AKT/mTOR signaling pathway in regulation of radiosensitization. Methods: We have established primary cells isolated from patient-derived OSCC tumor tissue for further molecular analyses. In this study, we used patient-derived OSCCs, OSCC-derived cell lines, and a radiation-resistant cell line in vitro and in vivo to examine the radiosensitization effects of single or dual PI3K/AKT/mTOR inhibitors and to explore the possible mechanism for the PI3K/AKT/mTOR inhibitor to serve as a potential candidate drug for radiosensitization. We carried out clonogenic survival assays, cell cycle analysis, apoptotic and autophagic assays and Western blotting to evaluate the possible mechanism. Results: The effect of the allosteric mTORC1-specific inhibitor, RAD001, and the dual mTORC1/mTORC2 inhibitor, AZD2014, on the sensitivity of OSCC cells toward radiation was investigated. We found that the mTORC1/mTORC2 inhibitor combined with radiation resulted in a significant reduction in the colony formation of OSCC cell lines and patient-derived OSCCs. By contrast, treatment with RAD001 plus radiation only slightly enhanced radiosensitization in SCC4. However, treatment with RAD001 increased AKT phosphorylation, which may limit effectiveness of mTORC1-targeting therapies for suppressing tumor growth. Pre-treatment with AZD2014 in irradiated oral cancer cells induced cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing the levels of caspase-3 and LC3 in patient-derived OSCCs. Therefore, inhibition of mTORC1/mTORC2 can sensitize OSCC cells to radiation, leading to cell-growth inhibition. Although the dual mTORC1/ mTORC2 inhibitor significantly enhanced the radiosensitivity of OSCC cells, no additional effects of this inhibitor were observed in radiation-resistant OSCC cells. Next, we further examined the effect of PI3K and mTOR inhibition on the irradiated oral cancer cells. Compared to a combination of PI3K or mTOR inhibitors with radiation, a dual blockade of the PI3K and mTOR kinases significantly improved radiation efficacy in OSCC-derived cell lines, patient-derived OSCC cells and radiation-resistant OML1-R cells. The dual PI3K/mTOR inhibition enhanced the effect of radiation by inhibiting AKT/mTOR signaling pathways and caused cell cycle arrest at the G1 phase, which is associated with downregulation of cyclin D1/CDK4 activity, leading to growth inhibition. Nevertheless, inhibition of these signaling pathways slightly increased the autophagy modulator, LC3. In nude mice xenografted with radiation-resistant OML1-R cells, the combined treatment was more effective than RT alone in reducing tumor growth. Immunohistochemistry showed that the combination therapy can suppress the S6 kinase activity and down-regulate the expression of eIF4E. These findings indicate that activation of the PI3K/AKT/mTOR signaling pathway has a role in radioresistance of OSCC. Conclusion: We determined that either mTORC1/mTORC2 or PI3K/mTOR inhibitor in combination with radiation exhibits a synergistic inhibition of the AKT/mTOR axis and induces cell cycle arrest. This treatment also triggers autophagy, resulting in sensitization of OSCC cells to radiation and cell-growth inhibition. More importantly, BEZ235 is a strong radiosensitizer to overcome radioresistance in treatment of OSCC with RT. Our results show the therapeutic potential of drugs targeting the PI3K/AKT/mTOR signaling pathway, which should be new candidate drugs for radiosensitization in radiotherapy for OSCC patient.