Pooled Shrna Screening Using Mouse Xenografts Of Human Hepatocellular Carcinoma Cells Identifies Cdk5 As A Potential Mechanism Mediating Sorafenib Resistance

Abstract Background: Sorafenib is the only approved systemic agent for hepatocellular carcinoma (HCC). However, the efficacy of sorafenib is modest for HCC with low objective response rate and short time to tumor progression, indicating that the inherent and acquired resistance underlies the limited success of sorafenib in HCC. We conducted a pooled shRNA screening in mouse xenografts of human HCC cells treated with sorafenib, and intended to identify genes that are involved in the therapeutic efficacy of sorafenib. Materials and Methods: We introduced a pool of 3920 shRNAs targeting 1960 genes, including various kinases, transcriptional factors, phosphatase and cytokine, into Huh7 HCC cells by retrovirus infection. The Huh7 cells after infection with pooled shRNAs were implanted subcutaneously in immunocompromised mice, and were treated with vehicle or sorafenib at the dose of 30mg/kg/day for 4 weeks. Genomic DNAs were extracted from HCC xenografts, PCR-amplified of the shRNA regions, and deep-sequenced of the PCR products to quantify the abundance of each shRNA. Candidate genes were identified from those shRNAs with statistically different frequencies between vehicle-treated and sorafenib-treated xenografts, and were further validated by their impacts on sorafenib-sensitivity in HCC cells. Results: By calculating the abundance frequencies of each shRNA of sorafenib- treated xenografts versus that of vehicle-treated xenografts, we found that 3 candidate sorafenib- resistance genes because their shRNA abundance was significantly decreased in sorafenib-treated xenografts. Among them, only the cyclin dependent kinase 5 (CDK5) was further investigated because its expression was confirmed to be increased in sorafenib- treated xenografts. In PLC5 and a sorafenib-resistant subline of Huh7 cells (Huh7-SR), downregulation of CDK5 by RNA interference or roscovitine, a CDK5 inhibitor, enhanced the sensitivity to sorafenib- induced growth-suppression. On the other hand, overexpression of CDK5 increased the resistance to sorafenib in Huh7 cells. Mechanistically, sorafenib combined with shRNA-mediated CDK5 downregulation augmented the induction of apoptosis, and resulted in an increased downregulation of multiple anti-apoptosis molecules such as XIAP and survivin in PLC5 and Huh7-SR cells. In 8 paired human HCC tissues, we found 3 of them have unequivocally increased expression of CDK5 in HCC cells of post-sorafenib HCC tissues compared with those of pre-treatment tissues. Conclusions: Our results suggest that increased CDK5 expression may mediate resistance to sorafenib in HCC in vivo, and targeting CDK5 is warranted further studies in the treatment of HCC. (This study was supported by grants MOST 103-2314-B-002 -181 -MY2, MOST 105-2314-B-002-194-, MOST 103-2314-B-002 -090 - & MOST 104-2314-B-002 -073). Citation Format: Tzu-Hsuan Lin, Wen-Chi Feng, Yu-Yun Shao, Li-Chun Lu, Ya-Jhen Chen, Ann-Lii Cheng, Chih-Hung Hsu. Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance [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 80. doi:10.1158/1538-7445.AM2017-80