Molecular profiling of clinical drug resistance

One of the greatest challenges in oncology is drug resistance. 5-Fluorouracil (5- FU) is the third most commonly used anti-neoplastic, so lack of initial or continued response to 5-FU represents a big clinical problem. Despite its prominence in cancer treatment, the mechanisms for its resistance remain largely undefined. The third leading cause of cancer-related deaths is colorectal cancer for which 5-FU is an essential part its therapeutic backbone. Resistance to 5-FU is a primary cause of treatment failure. Developing models explaining 5-FU resistance is imperative to advancing care. Quantitative proteomics is a rapidly emerging tool that when combined with functional studies can be valuable for mechanistic elucidation. Our combined modality approach utilizes colorectal tumors that are well-phenotyped with respect to 5-FU exposure (clinical resistance), demographics, and baseline disease characteristics. Expression of critical 5-FU pathway proteins is quantified within both tumors 5-FU exposed and unexposed, expression is then compared, and proteins with differential expression associated with 5-FU resistance are carried forward for functional validation. Then, augmentation of 5-FU sensitivity (IC50) after knockdown of the genes (DUT, UCK2, and DPYD) encoding the differentially expressed proteins was evaluated in colorectal cancer cell lines. DUT and UCK2 knockdown decreased IC50 by >2-fold in two or more cell lines while DPD knockdown yielded decreased IC50 by >2-fold in one cell line and nearly 2-fold in the others. This mechanistic validation supports overexpression of these targets as a mechanism for 5-FU resistance. Additionally, copy number gains in TYMS occurred 5 times more frequently in exposed compared to unexposed patients, suggesting that TYMS gains is also a mechanism for 5-FU resistance. This work could have a significant impact on defining mechanisms of drug resistance and designing rationale therapies for resistant patients. This model provides a strategy for not only screening multiple candidates potentially causing resistance but also a method for stratifying samples in a manner that enriches for variations associated with resistance and a means of credentialing these candidates for their putative mechanism