A New Model for Raf Kinase Inhibitory Protein Induced Chemotherapeutic Resistance

Therapeutic resistance remains the most challenging aspect of treating cancer. Raf kinase inhibitory protein (RKIP) emerged as a molecule capable of sensitizing cancerous cells to radio- and chemotherapy. Moreover, this small evolutionary conserved molecule, endows significant resistance to cancer therapy when its expression is reduced or lost. RKIP has been shown to inhibit the Raf-MEK-ERK, NFκB, GRK and activate the GSK3β signaling pathways. Inhibition of Raf-MEK-ERK and NFκB remains the most prominent pathways implicated in the sensitization of cells to therapeutic drugs. Our purpose was to identify a possible link between RKIP-KEAP 1-NRF2 and drug resistance. To that end, RKIP-KEAP 1 association was tested in human colorectal cancer tissues using immunohistochemistry. RKIP miRNA silencing and its inducible overexpression were employed in HEK-293 immortalized cells, HT29 and HCT116 colon cancer cell lines to further investigate our aim. We show that RKIP enhanced Kelch-like ECH-associated protein1 (KEAP 1) stability in colorectal cancer tissues and HT29 CRC cell line. RKIP silencing in immortalized HEK-293 cells (termed HEK-499) correlated significantly with KEAP 1 protein degradation and subsequent NRF2 addiction in these cells. Moreover, RKIP depletion in HEK-499, compared to control cells, bestowed resistance to supra physiological levels of H2O2 and Cisplatin possibly by upregulating NF-E2-related nuclear factor 2 (NRF2) responsive genes. Similarly, we observed a direct correlation between the extent of apoptosis, after treatment with Adriamycin, and the expression levels of RKIP/KEAP 1 in HT29 but not in HCT116 CRC cells. Our data illuminate, for the first time, the NRF2-KEAP 1 pathway as a possible target for personalized therapeutic intervention in RKIP depleted cancers.治疗耐药性仍是癌症治疗中最具挑战性的难题。Raf激酶抑制蛋白（Raf kinase inhibitory protein，RKIP）作为一种可使癌细胞对放疗与化疗产生增敏效应的分子受到广泛关注。此外，这种进化保守的小分子在表达下调或缺失时，可显著增强癌细胞的治疗耐药性。已有研究证实，RKIP可抑制Raf-MEK-ERK、核因子κB（NFκB）以及G蛋白偶联受体激酶（GRK）介导的信号通路，并激活糖原合成激酶3β（GSK3β）信号通路。其中，抑制Raf-MEK-ERK与NFκB信号通路是介导癌细胞对治疗药物产生增敏效应的最核心途径。 本研究旨在探究RKIP-KEAP1-NRF2通路与化疗耐药之间的潜在关联。为此，我们采用免疫组化技术检测了人类结直肠癌组织中RKIP与KEAP1的相互结合情况。同时，在HEK-293永生化细胞、HT29与HCT116结肠癌细胞系中，分别通过miRNA沉默RKIP以及诱导型过表达RKIP的实验手段，对本研究的核心科学问题开展进一步验证。 研究结果表明，在结直肠癌组织与HT29结直肠癌细胞系中，RKIP可提升Kelch样ECH相关蛋白1（Kelch-like ECH-associated protein1，KEAP1）的稳定性。在命名为HEK-499的永生化HEK-293细胞中，RKIP沉默与KEAP1蛋白降解及后续出现的NRF2成瘾现象显著相关。此外，与对照组细胞相比，HEK-499细胞中RKIP的敲除可通过上调核因子红细胞2相关因子2（NF-E2-related nuclear factor 2，NRF2）的靶基因表达，使细胞对超生理水平的过氧化氢与顺铂产生耐药性。类似地，在阿霉素处理后，HT29细胞的凋亡程度与RKIP/KEAP1的表达水平呈直接正相关，但该关联未在HCT116结直肠癌细胞中观察到。 本研究首次揭示，NRF2-KEAP1通路可作为RKIP缺失型癌症实施个性化治疗干预的潜在靶点。