Regulation of KCNQ1 and CFTR in Colorectal Cancer

Abstract

Colorectal cancer is the 2nd deadliest cancer in the US with more than 50,000 deaths in 2017. Our group has identified KCNQ1 and CFTR as colorectal cancer tumor suppressors. KCNQ1-low as well as CFTR-low expressing tumors have significantly poorer prognosis than high expressers: KCNQ1-low in stages II, III and IV colorectal cancer; CFTR-low in stage II. Thus, it is important to understand how KCNQ1 and CFTR are downregulated in these poor prognosis cancers. KCNQ1 and CFTR are expressed at the base of the intestinal crypt, the site of the stem cell compartment and origin of colorectal cancer. The Wnt/ β-catenin signaling pathway is important in determining stemness and is dysregulated in >85% of human colon cancers. Therefore, I am testing two hypotheses involving Wnt/ β-catenin and these tumors suppressors: 1) KCNQ1 is regulated through a putative enhancer region containing a β-catenin binding site and 2) KCNQ1 and CFTR regulate β-catenin activity. Our putative enhancer region was identified through a survey of the KCNQ1 genomic sequence for eQTLs (SNPs associated with altered expression of KCNQ1) which identified SNP rs2283155 in KCNQ1 intron 2. SNPs are markers for potential polymorphic variants associated with altered gene expression. The enhancer region includes the SNP rs2283155, a TCF7L2 binding site (the binding partner of transcriptionally active β-catenin) and a larger DNase I hypersensitivity enhancer region. Luciferase reporter assays showed regulation by our enhancer region of a reporter gene. Follow-up data has suggested that KCNQ1 is the gene being regulated. Second, to investigate the effect of Kcnq1 and Cftr on β-catenin signaling, I am examining β-catenin in organoids, a well-established model of the stem cell compartment. Activation of β-catenin is characterized by its movement from the cytoplasm or membrane to the nucleus. I developed an immunofluorescence assay to quantitatively measure nuclear β-catenin localization in Kcnq1 deficient/expressing and Cftr deficient/expressing organoids. These organoids had increased β-catenin activity in Kcnq1-deficient and Cftr-deficient organoids, suggesting a mechanism for tumor suppressor activity of Kcnq1 and Cftr. This work may provide insight into the role of Kcnq1 and Cftr within the cellular environment and potential ways to manipulate their expression to improve survival.