Colorectal cancer (CRC) is one of the most common cancer and a leading cause of cancer related deaths both in men and in women worldwide including US. A positive family history is strongly correlated with an increased risk of CRC diagnosis during lifetime. However, CRC is an indolent disease in its early stages and usually becomes symptomatic when it progresses to more advanced stages. Conventional screening methods have several limitations and currently, there is no screening procedure available to detect CRC at a very early stage. Under normal physiological condition, the epithelial lining of the gut gets breached by various factors including food, that creates local wound. The surviving epithelial cells around the wound lose their differentiated structure and undergoes epithelial mesenchymal transition (EMT) and migrate and proliferate to cover the injured area. Once the wound is covered, the cells start differentiating back into their normal cell type and become a part of the epithelium. If the cells fail to stop proliferating following wound closure by overriding the switch to differentiating that can lead to colonic polyp, or tumor, and if not detected in a timely manner than, it leads ultimately to CRC development. Previously, we reported that β-catenin; an adherence junction protein gets phosphorylated by Janus kinase 3 (Jak3), a non-receptor tyrosine kinase, where Jak3 directly phosphorylates three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. Now, our data show that human biopsies from colonic polyps not only associate with EMT, but also with only those mutation in β-catenin where Jak3, fail to phosphorylate β-catenin thereby making the cells override the switch and undergo uncontrolled proliferation and polyp formation. Based on this mechanistic understanding, the present study demonstrates the development of probe that detects the transition of colonic cells from proliferative stage to a differentiated (non-proliferative) stage that can diagnose CRC at the pre-polyp stage. The probe can not only play an important role in the early detection and/or treatment of patients with colorectal cancer but also increase life expectancy post detection. For this study, we collected de-identified and discarded specimen from normal and/or polyps (n=40 cases; 20 normal and 20 polyps) through our collaboration with clinicians from local hospitals. Briefly, snap-frozen tissues were used to isolate DNA with the QIAamp DNA mini kit (Qiagen) as reported before by our group. All DNA samples were quantified using UV spectrophotometer and analyzed through agarose gel electrophoresis. The DNA sample was used as template in PCR reactions using the β-catenin-probes to amplify the regions encompassing Y30, Y64, and Y86 followed by sequencing of PCR samples. This was followed by histological analysis of colonic polyp sections from each patient stained with H&E and images were taken with phase contrast microscope. Our results show strongly correlation between mutations and histological analysis indicating the aforementioned detection of intactness of EMT switch as potential novel biomarker for the early detection of colorectal cancer.
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