Role of lncRNA UCA1 in anoikis resistantce and colorectal cancer metastasis

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Research Summary: Colorectal cancer (CRC) is the second deadliest cancer, and patients’ survival rate drops from 90% to 14% when cancer metastasizes to distant organs. Herein, we proposed investigating the role of a long noncoding RNA (LncRNA) in anoikis resistance and CRC metastasis. Metastasis is a multistep process, and one of the key steps is to acquire anoikis resistance to survive after detachment from the primary sites. Thus, understanding the molecular players involved in the anoikis process and metastasis could be vital for improving the survival of CRC patients. The aberrant expression of a long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been identified in CRC; however, its roles in metastasis processes are not yet well defined. Our preliminary results in the anchorage-independent growth (anoikis model) demonstrate increased lncRNA UCA1 and Glucose Transporter1 (GluT1) protein expression. Moreover, the overexpression of lncRNA UCA1 led to high Glut1 expression and higher glucose uptake in CRC cells, which indicates a potential mechanistic role of lncRNA UCA1 in anoikis resistance. In this study, we propose to elucidate the role(s) of UCA1 and its associated signaling pathways during anoikis resistance. We hypothesize that the overexpression of lncRNA UCA1 enhances CRC metastasis through anoikis resistance-associated signaling pathways. We will utilize Isogenic CRC cell lines SW480 (oncogenic) and SW620 (metastatic) to understand the mechanistic regulation of anoikis resistance. AIM 1 is proposed to elucidate lncRNA UCA1 associated molecular mechanisms involved in anoikis resistance and CRC metastasis. We propose to study the role of lncRNA UCA1 in anoikis resistance using Lentiviral transduced stable overexpression (SW480+UCA1//GFP) and knockdown (SW620+CRISPRgUCA1) cell lines using cell cycle, pro-survival, anti-apoptotic, stemness, glucose metabolism, kinase phosphorylation immunoprecipitation (IP), co-IP and Proximity Ligation Assay (PLA) assays. In AIM 2, we proposed investigating UCA1 linked proteins, RNAs, and pathways associated with anoikis resistance. We will use stable isogenic lncRNA UCA1 (OE and KD) cell line models and an unbiased approach to identify novel UCA1 associated/linked proteins and RNAs using Biotin-ReCLP (Reversible Cross-Linked Precipitation), in vivo RNA Antisense Proteomics (iRAP), and RNAseq analyses and validate them by RT-PCR, ddPCR, IP, co-IP and PLA studies. While AIM 3 is proposed to evaluate the functional impact of lncRNA UCA1 expression using a metastatic mouse model. In this aim, SCID adult male/female mice will be injected with Luciferase expressing validated stable isogenic human CRC cell lines (UCA1 OE and KD as in aim1) through the portal vein, and their metastatic potential will be evaluated by bioluminescence imaging. This study will provide new insights into CRC metastasis and will help in developing innovative therapeutics to improve patients’ survival, generate data for future NIH proposals, and training of Hispanic underserved minority students at the University of Texas Rio Grande Valley (UTRGV), which is a prominent Hispanic serving institution in South Texas Rio Grande Valley region.