Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers

Reported here is the application of three protein folding
stability
profiling techniques (including the stability of proteins from rates
of oxidation, thermal protein profiling, and limited proteolysis approaches)
to identify differentially stabilized proteins in six patient-derived
colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities
and eight CRC patient-derived xenografts (PDXs) derived from two of
the patient derived cell lines with different oxaliplatin sensitivities.
Compared to conventional protein expression level analyses, which
were also performed here, the stability profiling techniques identified
both unique and novel proteins and cellular components that differentiated
the sensitive and resistant samples including 36 proteins that were
differentially stabilized in at least two techniques in both the cell
line and PDX studies of oxaliplatin resistance. These 36 differentially
stabilized proteins included 10 proteins previously connected to cancer
chemoresistance. Two differentially stabilized proteins, fatty acid
synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with
biological functions that can be modulated to improve the efficacy
of CRC chemotherapy. These results add to our understanding of CRC
oxaliplatin resistance, suggest biomarker candidates for predicting
oxaliplatin sensitivity in CRC, and inform new strategies for overcoming
chemoresistance in CRC.

本研究报道了三种蛋白质折叠稳定性谱分析技术的应用：涵盖基于氧化速率的蛋白质稳定性分析、热蛋白质组分析（thermal protein profiling）与有限蛋白水解法（limited proteolysis），用于在6株具有不同奥沙利铂敏感性的患者来源结直肠癌（colorectal cancer, CRC）细胞系，以及源自其中2株细胞系的8株具有不同奥沙利铂敏感性的CRC患者来源异种移植模型（patient-derived xenografts, PDXs）中，鉴定差异稳定性蛋白质。相较于本研究同步开展的传统蛋白质表达水平分析，稳定性谱分析技术可鉴定出区分敏感与耐药样本的独有、新型蛋白质及细胞组分，其中包含36种在奥沙利铂耐药的细胞系与PDX模型研究中，至少通过2种技术检测到稳定性发生显著改变的蛋白质。上述36种差异稳定性蛋白质中，有10种此前已被证实与肿瘤化疗耐药相关。其中2种差异稳定性蛋白质——脂肪酸合酶（fatty acid synthase）与延伸因子2（elongation factor 2）——经体外功能验证，被证实为可药用蛋白质靶点，其生物学功能可被靶向调控以提升结直肠癌化疗疗效。本研究结果深化了学界对结直肠癌奥沙利铂耐药机制的认知，可为预测结直肠癌患者奥沙利铂敏感性提供候选生物标志物，并为破解结直肠癌化疗耐药困境提供全新干预策略。