PRSS contributes to cetuximab resistance in colorectal cancer

Tan, Zhaoli; Gao, Lihua; Wang, Yan; Yin, Huihui; Xi, Yongyi; Wu, Xiaojie; Shao, Yong; Qiu, Weiyi; Du, Peng; Shen, Wenlong; Fu, Ling; Jia, Ru; Zhao, Chuanhua; Zhang, Yun; Zhao, Zhihu; Sun, Zhiwei; Chen, Hongxing; Hu, Xianwen; Xu, Jianming; Wang, Youliang

To confirm the role of PRSS1 in cetuximab resistance, stable PRSS1silenced HT-29 cells and LoVo cells were generated using a lentivirusbased PRSS1 short hairpin RNA (shRNA) approach. The knockdown efficiency of the PRSS1-specific shRNA in five batches of pooled shPRSS1 cells (shPRSS1-a, shPRSS1-b, shPRSS1-c, shPRSS1-d, and shPRSS1-e) was compared with that in nontargeted control cells. Pooled shPRSS1-a and shPRSS1-c HT-29 cells demonstrated >90% knockdown of PRSS1 at the mRNA level as shown by qPCR ( fig. S1C ) and RT-PCR (fig. S1D) and at the secreted protein level as shown by ELISA using an anti-PRSS1 antibody (Fig. 1F) . The shPRSS1-a and shPRSS1-c cells were also termed shPRSS1-1 and shPRSS1-2 cells, respectively. All pooled shPRSS1-1 and shPRSS1-2 LoVo cells demonstrated >85% knockdown of PRSS1 mRNA expression, as shown by qPCR ( fig. S1E ), and >80% knockdown of the secreted protein level (Fig. 1G ). Next, to confirm the response of PRSS1-silenced HT-29 and LoVo cells to cetuximab, we performed an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay using pooled shPRSS1-1, shPRSS1-2 (showing maximum PRSS1 expression knockdown), and control cells. Under cetuximab treatment, compared with control cells, both PRSS1 expression knockdown cell lines exhibited significantly decreased proliferation (Fig. 1, H