Abstract IA01: Recent advances in studying EGFR and its ligands in colorectal cancer

Abstract

Abstract At Vanderbilt, we have fashioned a three-pronged approach to study colorectal cancer (CRC): from in vitro studies to those both in mouse and humans. In vitro, we have analyzed trafficking and cell-surface delivery of EGFR and its ligands in a battery of CRC cell lines that retain the ability to form uniform polarizing monolayers on Transwell filters. In the mouse, we have discovered that the pan-ERBB inhibitor, LRIG1, marks a largely quiescent population of colonic stem cells distinct from LGR5 and that administration of tamoxifen to Lrig1-CreET2;Apcfl/+ mice eliminates one allele of Apc and results in highly penetrant, multiple, histologically advanced adenomas in the distal colon 50-100 days later; these adenomas show the expected loss of the 2nd Apc allele. Later this month, we will submit the 4th renewal of our GI Specialized Programs of Research Excellence (SPORE) that focuses on CRC. An advance in each of these areas will be discussed. Based on our trafficking studies, we have discovered that EGFR ligands can be released in endosome-derived small vesicles called exosomes. The ligands are signaling competent and bind to EGFR on recipient cells. We have termed this new mode of signaling, Exosomal Targeted Receptor Activation (ExTRAcrine). We have recently advanced this work to studying EGFR in exosomes isolated from mouse and human plasma using a method we have developed, fluorescence-based vesicle sorting (FAVS). Second, a limitation to studying EGFR in the mouse is the lack of robust reagents to monitor EGFR by immunohistochemistry. To circumvent this limitation, we have used CRISPR gene editing to target the endogenous mouse locus to create a chimeric EGFR-Emerald (green) reporter mouse. We will present data showing the value of this mouse in monitoring EGFR in the normal colon and in colonic tumors generated in our Lrig1-CreERT2;Apcfl/+ mouse model. Finally, we have developed a new 3D system to study CRC. Using this system, we have discovered a novel mode of cetuximab resistance and will show its relevance to human CRC. 1. Singh B, Coffey RJ. Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells. Annu Rev Physiol 76: 275-300, 2014. PMC4180094. 2. Higginbotham JN, Demory Beckler M, Gephart JD, Franklin JL, Bogatcheva G, Kremers GJ, Piston DW, Ayers GD, McConnell RE, Tyska MJ, Coffey RJ. Amphiregulin exosomes increase cancer cell invasion. Curr Biol 21: 779-86, 2011. PMC3417320. 3. Higginbotham JN, Zhang Q, Jeppesen DK, Scott AM, Manning HC, Ochieng J, Franklin JL, Coffey RJ. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J Extracell Vesicles 5: 29254, 2016. PMC4921784. 4. Powell AE, Wang Y, Li Y, Poulin EJ, Means AL, Washington MK, Higginbotham JN, Juchheim A, Prasad N, Levy SE, Guo Y, Shyr Y, Aronow BJ, Haigis KM, Franklin JL, Coffey RJ. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 149: 146-58, 2012. PMCPMC3563328. 5. Powell AE, Vlacich G, Zhao ZY, McKinley ET, Washington MK, Manning HC, Coffey RJ. Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis. Am J Physiol Gastrointest Liver Physiol 307: G16-23, 2014. PMC4080164. 6. Wang Y, Shi C, Lu Y, Poulin EJ, Franklin JL, Coffey RJ. Loss of Lrig1 leads to expansion of Brunner glands followed by duodenal adenomas with gastric metaplasia. Am J Pathol 185: 1123-34, 2015. PMC4380843. 7. Poulin EJ, Powell AE, Wang Y, Li Y, Franklin JL, Coffey RJ. Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine. Stem Cell Res 13: 422-430, 2014. PMC638408. 8. Kondo J, Powell AE, Wang Y, Musser MA, Southard-Smith EM, Franklin JL, Coffey RJ. LRIG1 Regulates Ontogeny of Smooth Muscle-Derived Subsets of Interstitial Cells of Cajal in Mice. Gastroenterology 149: 407-19 e8, 2015. PMC4527342. Citation Format: Robert J. Coffey. Recent advances in studying EGFR and its ligands in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr IA01.