Abstract 1644: Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk

Abstract

Abstract The chr 5p15.33 genomic locus encoding telomerase reverse transcriptase (TERT) is structurally complex, with several highly polymorphic intronic variable number tandem repeats (VNTRs). A germline VNTR located in TERT intron 6 (VNTR6-1, 38 bp repeat unit) has previously been associated with TERT alternative splicing but its potential role in regulating cellular functions and disease risk is unclear. To functionally characterize VNTR6-1, we deleted this region (1-2 kb) using CRISPR/Cas9 in UMUC3 (bladder cancer cell line). Cell count growth tracking of parental and VNTR6-1 knockout (VNTR6-1 KO) cells via automated live-cell monitoring revealed different relative growth rates under serum starvation vs. normal conditions, suggesting that VNTR6-1 might be sensitive to growth factors present in culture media. Evaluation of growth in media with full vs. charcoal-stripped (CS) serum, showed significant differences, with higher cell counts in parental vs. VNTR6-1 KO in CS-serum (P=4.1e-3), parental vs. parental in full (P=1.57e-4) or CS-serum (P=4.76e-2), but not in parental vs. VNTR6-1 KO in full serum (P=0.378). After 5-6 days of growth, cells in CS-serum also rapidly began outgrowing cells in full serum. We observed that VNTR6-1 KO dampened the outgrowth response in cells lacking growth factors in CS-serum. Proliferation-specific growth assays tracking the dilution of an incorporated dye in cells by flow cytometry further showed that VNTR6-1 KO leads to more pronounced proliferation differences due to the presence or absence of serum-supplied factors. Overall, cell growth patterns appear to be sensitive to serum in interaction with VNTR6-1, suggesting a potential VNTR6-1-dependent response to extracellular signaling. In human populations, we have identified two VNTR6-1 groups: Short (24-27 copies) and Long (40.5-66.5 copies). In the PLCO dataset (n=100,445 cancer cases and controls), logistic regression showed that VNTR6-1-Long group was associated with a decreased risk in male-prevalent cancers such as bladder and prostate, but an increased risk in female-prevalent cancers such as breast, endometrial and ovarian. These results support a link between the TERT region, and potentially VNTR6-1, with hormone signaling and cancer risk. Motif analysis within VNTR6-1 predicted binding sites for several transcription factors, including those associated with development and cell growth. Together, these results suggest that VNTR6-1 may be involved in regulating cell growth, potentially by facilitating responses to extracellular signaling pathways. Additionally, given the location of VNTR6-1 within a multi-cancer GWAS region, our findings provide functional context for the role of this polymorphic repeat in differential risk and outcomes in certain cancers. Citation Format: Michelle Ho, Oscar Florez-Vargas, Maxwell Hogshead, Brenen Papenberg, Chia Han Lee, Chi Zhang, Kaitlin Forsythe, Wusheng Yan, Raj Chari, Ludmila Prokunina-Olsson. Exploring the role of a TERT intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1644.