Exploring tsRNA Function in Aggressive Breast Cancer Cell Models

Abstract Breast cancer is highly prevalent in the United States with an estimated 260,000 women diagnosed with invasive breast cancer in 2018 alone. There is a growing need to identify the molecular drivers of metastatic breast cancer as the molecular mechanisms responsible for the transition from normal mammary epithelial cells to aggressive cancer cells remain poorly understood. Understanding this transition may reveal a therapeutic target for aggressive breast cancer. Small, noncoding RNAs (ncRNA), such as microRNAs (miRNAs), have recently been discovered to promote initiation, progression, and metastasis of breast cancer. Similar in size to miRNAs, tRNA-derived small RNAs (tsRNAs) are a novel class of small ncRNA whose expression may differentiate between cancer types and cancer cell lines. TsRNAs are created during the maturation process of primary tRNA transcripts, where the 3-prime end of the tRNA is cleaved by RNaseZ, resulting in a 16-48 nucleotide long strand of RNA. Although similar in size to miRNA, the functions of tsRNA are largely unknown. Previously identified two tsRNA, ts-2 and ts-112, that are expressed at 10-fold higher levels in the MCF10CA1a aggressive breast cancer cell line than the normal-like MCF10A mammary epithelial cell line. Further, ts-2 and ts-112 are detected at similarly high levels in female human embryonic cells, displaying oncofetal expression. For these reasons we hypothesize that ts-2 and ts-112 promote breast cancer characteristics. Custom inhibitors of ts-2 and ts-112 were transfected into the aggressive breast cancer cell line MCF10CA1a in vitro. The following phenotypic assays were conducted to determine the function of ts-2 and ts-112 in the MCF10CA1a cell line: proliferation, cell cycle, and wound healing. Following transfection of ts-2 inhibitors, in the proliferation assay showed a 15-20% reduction in growth of aggressive cancer cells. Ts-2 inhibition also saw an increase in population doubling time of 7% from 12 to 14 hours. These results suggest that ts-2 may play a role in cell cycle progression. Following ts-112 inhibition in the aggressive MCF10CA1a breast cancer cell line, cell cycle analysis revealed a statistically significant decrease in the number of cells in G1 phase and an increase in S phase. Using a candidate approach we analyzed the effect of ts-2 and ts-112 inhibition on G1/S phase and S/G2 phase checkpoint markers by qPCR. The inhibition of these tsRNA showed no effect on the chosen genes. Our data are in support for ts-2 and ts-112 having a role in aggressive breast cancer and may be tumor promoting In on-going studies, the capacity of tsRNAs to act as predictive biomarkers of long-term breast cancer risk is being evaluated in serum collected from women at elevated risk. Analyzing ts-2 and ts-112 biological consequences following inhibition furthers our understanding of the molecular mechanisms that are responsible for aggressive breast cancer. Continued study of tsRNA function could produce a model of their role in aggressive breast cancer and thus metastasis. Understanding tsRNA function in metastatic breast cancer in turn could lead to their use as a possible biomarker or therapeutic target