Abstract 4238: Lung cancer stem cell targets sensitizing tumor cells to chemo therapy and radiation therapy by exploiting synthetic lethal relationships in an RNAi high-throughput screen
Authors: Chaudhary Kunal R. , Cheng Haiying , Halmos Balaz , Silva Jose M. , Chao K.S. Clifford , Hei Tom K. , Cheng Simon 
Published: August 2015

Platinum-based concurrent chemoradiation therapy is the mainstay of treatment for locally advanced lung cancer. However patient outcomes remain dismal and overall survival at 5 years is only 15%. The resistance mechanisms for concurrent chemoradiation therapy are poorly studied. Cancer stem cells have been proposed to be the driver for many cancers including lung cancer and may be also responsible for therapy resistance. We sought therefore to identify therapy resistance pathways in lung cancer by using small interfering RNAs (siRNAs) to increase the sensitivity of tumor cells to therapeutic challenge.

To identify novel genes that modulate the response to platinum chemotherapy and radiation therapy we have performed a genome-wide RNAi high-throughput screen via a shRNA viral library containing approx. 60,000 individual shRNAs on a human lung adenocarcinoma cell line (PC9). After two weeks of cisplatin and/or radiation treatment of PC9 cells with shRNA and controls, depleted versus enriched shRNAs were identified through library sequencing. We hypothesized that depleted shRNAs in the treated versus control cells might represent genes whose function is crucial for resistance to therapy-induced cell death.

From the cisplatin and radiation screen, analysis of out of top 100 potential hits (depleted shRNAs) interestingly showed several cancer stem cells markers including Sox, Lrg6, and members of the Hedgehog signaling pathway Patched and Bmi1. We further determined the effects of Bmi1 on therapy resistance with survival assays by treating PC9 cells with Bmi1 inhibitor PTC-09. MTT cell survival and colonogenic assays was performed by treating PC9 cells with PTC-09 in triplicate and then treated with increasing dosage of cisplatin (0.1, 1 and 10 μM) or X-ray radiation (2, 4 and 6 Gy). Significantly decreased cell survival was observed in PTC-09 treated PC9 cells treated with cisplatin or radiation compared to control and cisplatin or radiation alone treated cells. Further colonogenic assay of PC9 cells treated with 2Gy+1 um cisplatin and increasing dosage of PTC-09 showed significant decrease in the ability of cells to form colonies compared to control.

By performing an unbiased genome wide for therapeutic resistance, we have successfully identified and validated a target in the cancer stem cell pathways. We are further evaluating effect of Bmi1 using CRISPR-Cas9 knock out model.

Citation Format: Kunal R. Chaudhary, Haiying Cheng, Balaz Halmos, Jose M. Silva, K.S. Clifford Chao, Tom K. Hei, Simon Cheng. Lung cancer stem cell targets sensitizing tumor cells to chemo therapy and radiation therapy by exploiting synthetic lethal relationships in an RNAi high-throughput screen. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4238. doi:10.1158/1538-7445.AM2015-4238