Abstract 1970: Validation of candidate gastrointestinal cancer genes with ion channel functions, identified from Sleeping Beauty transposon-mediated mutagenesis screens in mice.
Authors: Than Bich L. N. , O'Sullivan M. Gerald , Starr Tim , Largaespada David , Cormier Robert T. , Scott Patricia M. 
Published: August 2014

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US. To identify the genetic alterations contributing to CRC, our labs have used Sleeping Beauty (SB)-transposon-mediated mutagenesis screens in Apc+/+ and ApcMin mice. These screens have identified a set of common insertion site (CIS) associated candidate cancer genes, that when dysregulated, may contribute to CRC development. Depletion of several CIS genes identified from an SB screen in ApcMin mice resulted in a significant decrease in human CRC cell viability. The SB screen in Apc+/+ mice has generated a list of CIS genes including two ion-channel encoding genes: Kcnq1 (potassium voltage-gated channel, KQT-like subfamily, member 1) and Cftr (cystic fibrosis transmembrane conductance regulator, ATP-binding cassette sub-family C, member 7) which act together to promote chloride ion secretion in the normal colon. We hypothesize that these candidate genes, when dysregulated, contribute to the development of CRC. The function of the candidate genes is being tested in a mouse model of Kcnq1 haploinsufficiency that was introgressed into the ApcMin model of GI cancer and in human CRC cell line DLD-1. In the mouse model, Kcnq1 expression is abrogated by targeted germline mutagenesis, resulting in a null allele. In cell culture, expression of each candidate gene is depleted by siRNA knockdown, confirmed by quantitative real time PCR and followed by measurements of cancer-related phenotypes which include cell viability measured by the MTT assay. We found that haploinsufficiency for Kcnq1 significantly enhances tumorigenesis in ApcMin mice. In support of this result, a 60% knockdown of KCNQ1 in DLD-1 cells resulted in ∼1.4x increase in cell viability, compared with a control siRNA treatment at day seven after transfection. The normal physiological functions of KCNQ1 indicate it may work with CFTR to prevent inflammation in the GI tract, thus suggesting that loss of KCNQ1 or CFTR may be oncogenic via an inflammatory mechanism. To begin testing this idea, colon tissues of Kcnq1+/+ and Kcnq1−/− mice were compared for expression of the inflammatory mediator Nfkb. Nfkb mRNA level was increased by ∼1.6x in Kcnq1−/− mice. To begin testing the potential connections between KCNQ1 and CFTR, CFTR was successfully depleted by 40% in DLD-1 cells and the effect on cell viability and inflammatory mediators’ expression is being determined. In summary, our results of both in vivo and in vitro studies confirm a tumor suppressor role for Kcnq1 in the GI tract. Current work is focused on investigating the model that genetic alterations of KCNQ1 and CFTR promote oncogenesis by a common pathway, possibly by an inflammatory mechanism.

Citation Format: Bich L. N. Than, M. Gerald O'Sullivan, Tim Starr, David Largaespada, Robert T. Cormier, Patricia M. Scott. Validation of candidate gastrointestinal cancer genes with ion channel functions, identified from Sleeping Beauty transposon-mediated mutagenesis screens in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1970. doi:10.1158/1538-7445.AM2013-1970