Abstract 1806: Endothelial cells activate the cancer stem cell-associated Nanogp8 pathway in colorectal cancer cells in a paracrine fashion
Authors: Wang Rui , Bhattacharya Rajat , Ye Xiangcang , Boulbes Delphine R. , Fan Fan , Ellis Lee M. 
Published: July 2017

INTRODUCTION: Median survival of patients with metastatic colorectal cancer (mCRC) is ~2.5 years and patients often become resistance to systemic therapy within 1 year of the diagnosis of metastasis. There is accumulating evidence for the existence of cancer stem cells (CSCs) in CRC, which are now believed to mediate chemoresistance. The molecular mechanisms that regulate the CSC phenotype in CRC have not been fully elucidated. We have previously described that endothelial cells (ECs) from liver, the most common site of metastases of CRC, secreted soluble Jagged-1 peptides to activate Notch signaling and increase the CSC phenotype of CRC cells in a paracrine fashion. In the current project, we sought to further elucidate the paracrine roles of liver ECs and ECs from other organs in mediating specific CSC-associated pathways in CRC cells.

METHODS: Several EC lines from liver, lung, colon mucosa and kidney were established and used to determine if conditioned medium (CM) of ECs from distinct organs promotes the CSC phenotype and chemoresistance in CRC cells. CRC cells were incubated with either their own CM (control) or CM from ECs. The CSC phenotype was determined by sphere formation assays. Chemoresistance was determined by Western blotting for apoptotic markers and fluorescence-activated cell sorting (FACS)-based Annexin V-PI double staining assay for apoptosis after treating CRC cells with 5-fluorouracil (5-FU) either in control CM or CM of ECs. The effects of EC CM on CSC-associated genes were determined by unbiased qPCR array and validated by Western blotting.

RESULTS: CM from ECs from liver and other organs all significantly increased sphere formation in CRC cells. qPCR arrays and Western blotting showed that the CSC-associated Nanog and Oct4 genes were increased by EC CM. Moreover, CRC cells treated by liver EC CM became more resistant to 5-FU induced apoptosis. Luciferase reporter assays suggested that the Nanog proteins in CRC cells were encoded by the retrogene NANOGP8, and EC CM specifically induced the expression of NANOGP8 but not NANOG. The specific induction of NANOGP8 in CRC cells were confirmed by AlwNI digestion, an established technique to distinguish NANOG and NANOGP8 based on a single nucleotide polymorphism (SNP) between two genes. In addition, Akt was shown to mediate the induction of NANOGP8 in CRC cell, as the PI3K inhibitor wortmannin blocked liver EC CM-induced NanogP8 expression and blocked sphere formation in CRC cells.

CONCLUSION: Our studies demonstrated a paracrine role of ECs from several organs, including liver, in regulating the CSC phenotype in CRC cells, via increasing NanogP8 expression. This study elucidated the importance of ECs in the microenvironment in mediating the survival of metastatic CRC cells in the liver, and potentially other organs.

Citation Format: Rui Wang, Rajat Bhattacharya, Xiangcang Ye, Delphine R. Boulbes, Fan Fan, Lee M. Ellis. Endothelial cells activate the cancer stem cell-associated Nanogp8 pathway in colorectal cancer cells in a paracrine fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2017-1806