Abstract 2276: Proteomic analysis of chemoresistance in colorectal cancer cells: potential paracrine mechanisms of resistance
Authors: Bose Debashish , Fan Fan , Tozzi Federico , Zimmerman Lisa , Herynk Matthew H. , Pierobon Mariaelena , Samuel Shaija , Slebos Robbert , Parikh Alexander , Petricoin Emmanuel , Liebler Daniel , Ellis Lee M. 
Published: February 2011

Background: Chemoresistance occurs in nearly all patients with metastatic colorectal cancer (CRC), and mechanisms to reverse chemoresistance remain elusive. We tested the hypothesis that CRC cells resistant to 5-fluorouracil (5FU-R) and oxaliplatin (Ox-R) exhibit proteomic profiles that may identify previously unrecognized mediators of resistance. In prior studies, we found that conditioned media from oxaliplatin-resistant HT29 cells (Ox-R) could mediate growth and chemoresistance in chemonaive parental HT29 cells in vitro. We sought to identify soluble factors in conditioned media that are potential mediators of the paracrine cell survival mechanisms.

Methods: Parental HT29 (Par) cells were grown in increasing concentrations of 5-FU and oxaliplatin to generate 5FU-R and Ox-R cells. Protein from cell lysates and conditioned media (CM) were analyzed by liquid chromatography-mass spectrometry (LC-MS), and spectral counts were compared. Antibody-conjugated bead technology and ELISAs were used to obtain cytokine profiles of CM. Reverse phase proteomic arrays (RPMA) were used to determine signal transduction pathways activated in cells treated with CM. Ox-R cells were injected into nude mice to determine the paracrine effect on Par cells growing on the opposite flank.

Results: Chemoresistant cells displayed significantly different proteomic profiles. In 5FU-R cells, pathways involving oxidative phosphorylation, inositol metabolism, actin cytoskeleton signaling, regulation of actin-based motility by Rho, and ATM signaling were significantly altered vs Par cells. In Ox-R cells, pathways mediating pyruvate metabolism, integrin signaling, caveolar-mediated endocytosis signaling, and mitochondrial dysfunction were among the most altered vs Par cells. Comparison of 5FU-R and Ox-R cells revealed differences in RNA post-transcriptional modification, ERK/MAPK, RAN, and chemokine signaling. Cytokine profiling demonstrated a significant increase in stem cell factor/c-Kit ligand (p=0.02) and a decrease in TRAIL (p=0.008) and IL-10 (p=0.04). RPPA analysis demonstrated early phosphorylation of EGFR and MEK1 followed by GSK, and mTOR activation. Ox-R tumors growing in vivo induced faster and larger tumor growth of contralateral Par tumors indicating a systemic effect. Conclusions: Chemoresistant CRC cells exhibit proteomes that reflect specific survival pathways, with many previously unrecognized potential mediators of resistance. Analysis of soluble factors from chemoresistant CRC cells demonstrates the presence of numerous potential mediators of cancer cell survival that may act, not only in an autocrine/paracrine manner, but also systemically.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2276.