Investigating the Role of Environmental Exposures and piRNA Expression in Breast Cancer Initiation and Progression

Abstract

Among women, breast cancer is the most prevalent form of cancer worldwide and has the second highest mortality rate of any cancer in the United States. Genetic predispositions are thought to account for 15-20% of all cases; therefore, 80 - 85% of cases occur in women with no family history of the disease. Consequently, the mechanisms of development of many breast cancers remain unknown. Breast tumors are heterogeneous, resulting from acquisition of morphological alterations and cancer hallmarks including stemness and cellular plasticity. Epigenetics is defined as mitotically heritable changes in gene function that do not alter the underlying DNA sequence, and epigenetic mechanisms regulate cellular plasticity and breast carcinogenesis. Epigenetic mechanisms such as DNA methylation and histone modifications have been extensively studied in cancer; however, small non-coding RNA and their role in cancer progression remain unclear. PIWI-interacting RNA (piRNA) are a class of small, non-coding RNAs which regulate transposons and repression of transposition. A growing number of studies show expression of PIWILs in breast tumors; however, the role of piRNAs in breast cancer development remains unclear. In the US, there are over 85,000 chemicals in use and only 3% of those are fully tested for human safety. These chemicals interact with our epigenome and may play an important role in carcinogenesis. Cadmium (Cd) is a naturally occurring heavy metal and a known lung carcinogen, however, its role in breast cancer remains controversial. In vitro studies show that breast cells exposed to Cd are malignantly transformed through estrogen receptor independent mechanisms. The overall goal of this dissertation is to examine the epigenomic, transcriptomic, and morphological changes linked to long term Cd exposure in breast cells and investigate how piRNAs are involved in breast carcinogenesis. In Aim 1, I define key players in piRNA biogenesis and machinery and use the Comparative Toxicogenomics Database (CTD) to assess what chemical exposures are associated with changes in piRNA-associated machinery expression as well as disease states linked to such changes in expression. My results indicate that aldehydes, metals, personal care products, pesticides, and polybrominated diphenyl ethers (PBDEs) impact expression of piRNA-associated genes. In Aim 2, I perform the first baseline characterization of the piRNA system in breast cells using two different cell lines (non-tumorigenic MCF10A and cancerous MCF7) in two culture conditions (2D-monolayer and 3D-mammospheres), with sodium periodate to identify piRNA transcripts. My results show distinct piRNA profiles in the two cell lines, as well as distinct piRNA expression patterns for 2D vs 3D culture conditions. In Aim 3, I investigate the role of long term (40-week), low dose cadmium chloride exposure (0.25uM and 2.5uM) on cancer-associated morphological alterations and cellular plasticity. My results show that the luminal marker, Keratin 8, decreases over time in both the control and treated groups, while the myoepithelial marker, Keratin 14, increases over time in the controls but decreases in the treated groups, with a divergence from the controls observed at week 30 and 40. RNA sequencing data indicated activation of the MYC oncogene, suggesting a potential shift in cellular behavior and increased proliferation associated with long-term, low dose cadmium exposure. Taken together, these assessments contribute to our understanding of the piRNA profile of breast cancer and highlight novel new mechanisms by which cadmium may promote breast cancer progression.