Abstract 1259: Identification of p97 as a prime target to inhibit cancer growth and stemness

Abstract

Abstract Purpose: The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is an adaptive mechanism for cancer cell survival manifested in many different tumors. In UPR, p97 is an AAA ATPase essential for the degradation of the misfolded proteins, a process also known as the ER-associated protein degradation (ERAD). Previous studies have shown that small molecule inhibitors targeting the key players of UPR and ERAD can inhibit tumor growth. However, the efficacy of these inhibitors has not been compared, and it is unknown whether they affect the population of cancer stem cells (CSC). The purpose of this study is to systematically study the effect of these inhibitors on the growth and differentiation of multiple tumors. Methods: Immunohistochemistry was performed to verify the upregulation of UPR and ERAD proteins in multiple cancers. Immunoblotting and reserve transcription PCR were used to examine the protein expression and the splicing of XBP-1. MTS, Annexin V-PI and Transwell assays were used to measure the proliferation, apoptosis and invasion of cancer cells in vitro. The sphere culture and flow cytometry were used to evaluate the percentage of mammary CSCs. Xenograft tumor model in nude mice was used to evaluate the growth and differentiation of breast cancer cells in vivo. Results: We found that p97, together with IRE1α, BiP/GRP78, ATF6α and ubiquitin, is up-regulated in multiple cancers when compared with the surronding non-cancerous tissues, and these proteins are expressed to different extent in eight solid and four blood tumor cell lines, which may reflect different degrees of UPR activation in these tumors. PERK and eIF2α are also phosphorylated in several tumor cells under normal culture condition, although no splicing of XBP-1 was detected. When cultured in low glucose condition, UPR activation is significantly increased. The inhibitors against IRE1α, PERK, ATF6α and p97 retard the proliferation of different tumor cells with different efficacy, while only p97 inhibitor induces significant apoptosis under normal culture condition. When combined with cisplatin or bortezomib, p97 inhibitor significantly increases cytotoxicity. Among all the inhibitors targeting UPR and ERAD, only p97 inhibitor significantly decreases the percentage of mammary CSC subpopulation of in vitro cultured MDA-MB-231 cells. P97 inhibitor also decreases tumor growth in vivo and reduces the CSC percentage in the tumor isolated from nude mice. Mechanistically, p97 inhibitor impairs the mammosphere forming and the cancer cell invasion through the inactivation of NF-κB signaling. Conclusion: Our comprehensive study demonstrates that the p97 inhibitor not only significantly reduces the growth of multiple tumors but also the CSC subpopulation, the major cause of tumor metastasis, recurrence and chemotherapy resistance, thus establishing p97 as a prime druggable target in the UPR pathway for interventional cancer therapy. Citation Format: Chuang Li, Meng Nie, Hongyang Quan, Qianqian Fan, Nan Zhang, Quancai Cui, Lin Wang. Identification of p97 as a prime target to inhibit cancer growth and stemness. [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 1259. doi:10.1158/1538-7445.AM2015-1259