Abstract 2056: MYC and HIF-2alpha mediates resistance to Epidermal Growth Factor Receptor (EGFR) antagonism in oral squamous carcinoma cells
Authors: Sukanya Gayan, Reza Bayat-Mokhtari, Bidisha Pal, Anupam Sarma, Joyeeta Talkudar, Sorra Sandhya, Rashmi Bhuyan, Seema Bhuyan, Jaishree Garhyan, Debabrat Baishya, Amal Kataki, Rakesh Bhatnagar, Herman Yeger, Bikul Das
Journal: Cancer Research (2017)

Abstract Background: Oral squamous cell carcinoma (OSCC) is a devastating disease. Recent findings suggest that the EGFR antagonist including erlotinib may induce tumor regression alone or in combination with chemo/radiation. Our preliminary clinical study conducted at KaviKrishna laboratory indicates that many poor patients are taking the highly expensive drug Erlotinib, and or equivalent EGFR antagonists. However, oral cancer lesions are often hypoxic, and the hypoxia-induced cellular mechanisms might contribute to drug resistance. Here, we investigated, whether resistance to EGFR antagonists may involve MYC and HIF-2alpha, two transcription factors upregulated during hypoxia by using established oral cancer cell lines. We have also obtained primary oral cancer cells from patients living in the Kamrup district of Assam, where KaviKrishna laboratory is located. We intend to evaluate the drug sensitivity of these oral cancer cells towards EGFR antagonists. Method: We used SSC-25 and SCC-9 cell lines, as well patient derived primary oral cancer cells (n=5) for the study. Immunomagnetic sorting was performed to obtain ABCG2+ population. The self-renewal was studied using in vitro clonogenic and in vivo serial transplantation assay in NOD/SCID mice. Results: First, we identified a rare ABCG2+ expressing, highly tumorigenic cell population in SSC-25, and SCC-9 having cancer stem cell (CSC) like characteristics. Second, we found that the ABCG2+ cells exhibited sensitivity to PD158780 (10 uM; 62% inhibition within 48 hours), and AG1478 (10 uM; 56% inhibition within 48 hours), two small molecular inhibitors of EGFR tyrosine kinase. These small molecular inhibitors significantly inhibited the clonogenic capacity of the ABCG2+ cells. Next, we found that ABCG2+ cells also showed sensitivity to Erlotinib in the in vitro clonogenic assay. Third, the ABCG2+ cells, when exposed to hypoxia (&amp;lt;0.1% O2, 24 hours), exhibited enhanced expression and transcriptional activity of MYC, and HIF-2alpha. The post-hypoxia ABCG2+ cells exhibited complete resistance to PD158780, AG1478 and Erlotinib treatment, which could be reversed by siRNA silencing of MYC and or HIF-2alpha. ChIP assay revealed that HIF-2alpha directly binds to MYC in ABCG2+ cells. We found similar results in ABCG2+ cells obtained from primary oral cancer samples (n=4). Importantly, ABCG2+ cells directly isolated from patients exhibited hypoxic phenotype, including the high expression of HIF-1alpha, and HIF-2alpha, as well as resistance to erlotinib. Furthermore, erlotinib enhanced the stemness of ABCG2+ cells by activation of the MYC/HIF-2alpha self-renewal pathway (1). Conclusion: These data indicate that MYC and HIF-2alpha co-operate to mediate intrinsic resistance of oral squamous cancer cells to EGFR antagonist in the hypoxia microenvironment. (1). Bhuyan et al. Cancer Research, volume 76 (14), abstract 935; 2016 Note: This abstract was not presented at the meeting. Citation Format: Sukanya Gayan, Reza Bayat-Mokhtari, Bidisha Pal, Anupam Sarma, Joyeeta Talkudar, Sorra Sandhya, Rashmi Bhuyan, Seema Bhuyan, Jaishree Garhyan, Debabrat Baishya, Amal Kataki, Rakesh Bhatnagar, Herman Yeger, Bikul Das. MYC and HIF-2alpha mediates resistance to Epidermal Growth Factor Receptor (EGFR) antagonism in oral squamous carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2056. doi:10.1158/1538-7445.AM2017-2056