Abstract 1644: Exploring the role of a <i>TERT</i> intronic tandem repeat in regulating cell proliferative responses to environmental factors and cancer risk
Authors: Michelle Ho, Oscar Florez-Vargas, Maxwell Hogshead, Brenen Papenberg, Chia Han Lee, Chi Zhang, Kaitlin Forsythe, Wusheng Yan, Raj Chari, Ludmila Prokunina-Olsson
Journal: Cancer Research (2024)

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.