To address Focus Areas advocated by the Fiscal Year 2021 Rare Cancers Research Program, this study will investigate biology, etiology, and therapy of cholangiocarcinoma (CCA). CCA is an aggressive type of bile duct cancer characterized by a high mortality rate due to difficulty in early diagnosis and ineffective treatment. The pathogenesis and progression of CCA is not fully understood yet. Many researchers suggest that overexpression of Notch genes and their response elements (NREs) promote development and progression of CCA. Thus investigation of Notch signaling pathway could be a novel approach in revealing mechanism, diagnosis, and treatment of CAA. It is known that there are RNA molecules named non-coding RNAs (ncRNAs) that do not encode proteins. However, emerging evidence suggests that a number of ncRNAs can indeed encode peptides that play roles in normal cell functions or human diseases, including cancers.
Whether the peptides can be derived from these ncRNAs from CCA cells and regulate the expression of Notch genes and their related elements in association of occurrence, progression, and clinical outcomes of patients with CCA remains unclear.
It is speculated that micropeptides derived from ncRNAs can regulate notch signaling pathways in association with pathogenesis and progression of CCA. Many advanced biological molecular technologies will be used in this study to demonstrate the speculation with following approaches, briefly, to generate a CRISPR single guide RNA (sgRNA) library against small open reading frames of selected lncRNA; to generate NRE reporter cell line; to perform library screening through infection of SNU-1079 (one of CCA cell lines) carrying the reporter with this CRISPR sgRNA library; to recover guide RNA (gRNA) sequences through PCR amplification, deep sequencing for PCR products, and identify enriched gRNA sequences by bioinformatics analysis; and to functionally characterize genes corresponding to the gRNA clones by manipulating their expression through KO/rescue or ectopic expression; and determining whether the peptides in SNU-1079 cells play roles in regulating expression of Notch genes, genes mediated by Notch, and products from these genes by RTPCR, western blot, and immunoassays.
At this preliminary point, this study will not have any risk to patients because the study will be conducted in cell lines in vitro. Once this preliminary study succeeds, further studies will be quickly conducted with animal model, human samples, and clinical trial to confirm finding in the preliminary studies. Success in this study will benefit patients with CCA and people with hepatobiliary disorders in terms of early diagnosis/differential diagnosis of CCA, revealing mechanisms in occurrence and metastasis, new therapeutic intervention to CCA patients, and assurance to people without CCA.
Completion of this preliminary study will take 1-2 years, and it might take another 3-5 years to be clinically used in diagnosis and treatment of patients. Success in this study will also shed light on other rare cancers, as well as common cancers, in understanding mechanisms, revealing new diagnostic/prognostic biomarkers, and discovering novel therapeutic targets in Notch signaling pathways. |
