Abstract 2854: Adaptation of colorectal cancer cells to the brain microenvironment: the role of IRS2
Authors: Greenberg Inbal , Klein Anat , Grossman Rachel , Sokol Ethan , Yeini Eilam , Ofek Paula , Satchi-Fainaro Ronit , Liang Bertrand , Reuveni Hadas , Rubinek Tami , Wolf Ido 
Published: July 2021

Abstract
Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM), with rising incidence. Yet, molecular mechanisms supporting the formation of these lesions from CRC are unknown. We aimed to explore drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of insulin receptor substrate 2 (IRS2) gene amplification was noted in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. We subsequently constructed an in vitro system mimicking the brain microenvironment using cultured human astrocytes or their conditioned media (CM). Under these conditions using InSphero system, IRS2-overexpressed CRC cells survived better and formed larger 3D spheres. IRS2-silenced CRC cells showed a mirror image. Moreover, in an intracranial CRC surgical implantation BMs mouse model, IRS2-overexpressed CRC cells generated larger brain lesions, while silencing IRS2 in IRS2-amplified CRC cells dramatically decreased tumor outgrowth and survival. Interestingly, transcriptomic analysis revealed significant enrichment of the oxidative phosphorylation pathway by IRS2. Indeed, IRS2-expressing CRC cells showed increased mitochondrial activity using Seahorse extracellular flux analysis and glycolysis-independent viability. Furthermore, IRS2-expressing cells showed enhanced AKT phosphorylation and inhibition of PI3K or AKT using Alpelisib or iAKT, respectively, slightly decreased their proliferation in CM, suggesting that mechanisms additional to the AKT pathway may mediate IRS2 activity. The Wnt/β-catenin pathway was among the most significantly enriched pathways in the transcriptome. Indeed, IRS2-expressing cells had increased transcriptional activity of the β-catenin. In addition, iAKT or Alpelisib, and most significantly the IRS2 inhibitor (NT219), decreased the transcriptional activity of β-catenin in IRS2-expressing CRC cells, suggesting relevance of IRS2 in activating β-catenin. Moreover, NT219 demonstrated significant and dose-dependent inhibition of CRC cells viability. These data reveal, for the first time, the unique genomic profile of CRC BMs and imply the IRS2 role in promoting CRC BMs. These effects may be mediated, at least in part, by modulation of the AKT and β-catenin pathway. Given the molecular signature described, the approach to patients with BMs may be significantly impacted by agents such as NT219.
Citation Format: Inbal Greenberg, Anat Klein, Rachel Grossman, Ethan Sokol, Eilam Yeini, Paula Ofek, Ronit Satchi-Fainaro, Bertrand Liang, Hadas Reuveni, Tami Rubinek, Ido Wolf. Adaptation of colorectal cancer cells to the brain microenvironment: the role of IRS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2854.