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

As soybean trypsin inhibitor type I (SBTI; Sigma-Aldrich catalog no. T6522) (29), the urinary trypsin inhibitor ulinastatin (30) , and SPINK1, which is also known as pancreatic secretory trypsin inhibitor or tumorassociated trypsin inhibitor (TATI) (31, 32) , suppress trypsin activity, we considered whether these agents could inhibit the PRSS1-mediated proteolytic cleavage of mAbs. SBTI, ulinastatin, and recombinant human SPINK1 suppressed the proteolytic cleavage of bevacizumab by recombinant PRSS1 in phosphate-buffered saline (PBS); SPINK1 was the strongest inhibitor, while ulinastatin was the weakest (Fig. 4, A and B) . We also tested the inhibitory effect of SBTI on the proteolytic cleavage of cetuximab by a high concentration of PRSS1 in the HT-29 cell culture supernatant. Immunoblotting showed that SBTI barely suppressed the PRSS1-mediated cleavage of cetuximab in the cell culture supernatant (fig. S5, A and B) . As SPINK1 is overexpressed in multiple human cancers and increased serum SPINK1 levels have been correlated with aggressive disease, we assessed SPINK1 expression in colon cancer cell lines. SPINK1 expression was highest in SW480 cells (Fig. 4, C and D, and fig. S5C ), which may be why cetuximab cleavage in the SW480 cell culture supernatant was very weak although PRSS1 expression was highest in the SW480 cell line among the colon cancer cell lines (Fig. 1, C and D, and fig. S5 , D and E).