Graduation Year

2021

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Meera Nanjundan, Ph.D.

Committee Member

Brant Burkhardt, Ph.D.

Committee Member

Sandy Westerheide, Ph.D.

Committee Member

Yu Chen, Ph.D.

Keywords

Fallopian tube secretory epithelial cells, Iron, Lysophosphatidic Acid, miRNA

Abstract

Elucidating molecular alterations underlying tumor development and chemoresistance are critical to expand our understanding of the disease pathophysiology. This dissertation is focused on analyzing the cellular and molecular alterations associated with LPA-induced chemoresistance in clear cell renal cell carcinoma (ccRCC) cells and chronic iron-induced deregulation of miRNA expression in fallopian tube secretory epithelial cells (FTSECs).

Kidney cancer is one of the ten most common cancers worldwide with <15% survival rate at advanced stage (American Cancer Society). ccRCC is the most common type of kidney cancer and is described as a metabolic disease characterized by deregulated lipid metabolism leading to increased intracellular lipid droplets [9, 10]. The current molecular-targeted treatment strategies involve VEGF/VEGFR and mTOR inhibition [9, 12]. However, there are limitations to these approaches leading to the reduced efficacy and/or increased resistance in ccRCC cells [13, 14]. Therefore, it is important to decipher the factors involved in compromising the chemosensitivity in these cells.

Lysophosphatidic acid (LPA), a bioactive phospholipid, was previously reported to increase resistance against Sunitinib (VEGFR/PDGFR inhibitor) in ccRCC cells and to increase migration and invasion in various tumors [15-17]. In Chapter 3 of this dissertation, we analyzed the role of LPA in mediating chemotherapeutic resistance in ccRCC cells by reversing the effects of an mTOR inhibitor Temsirolimus (TEMS). We further identified that LPA reverses the TEMS-induced changes in cellular viability, lipid droplets and mitochondrial networks, autophagy and PI3K/mTOR pathway markers in vitro [19]. We also observed that LPA increases lipid droplets partially in a MAPK-dependent manner [19]. Overall, these results indicate the role of LPA in inducing potential chemoresistance in ccRCC cells, which can be further explored for pre-clinical research applications in future.

Since epithelial ovarian cancer (EOC) is commonly diagnosed at advanced stage, it is imperative to investigate the molecular alterations associated with initiation of the disease [34-36]. HGSOC initiation from transformed fallopian tube stem cells was reported to be associated with iron addiction and deregulated iron homeostasis markers [40]. The contribution of chronic iron exposure in mediating oncogenic transformative events in FTSECs was previously studied in our lab [22]. We have extended these studies in Chapter 4 of this dissertation using a multi-omics approach to determine the global protein and miRNA alterations in chronic iron exposed FTSECs. Interestingly, ~57% of the altered miRNAs were located at chromosome 14q32 [43]. Chromosome 14q32 harbors a cluster of 54 miRNAs, which are deregulated in various tumor types, including ovarian cancer [25, 26, 422]. However, the role of iron-induced deregulation of 14q32 miRNAs in FTSECs had not been previously studied. We focused on the two most downregulated (~100-fold) 14q32 miRNAs – miR-432-5p and miR-127-3p and their common protein targets. We identified that overexpression of miR-432-5p reduced cell numbers induced by long-term iron exposure in FTSECs. We also identified that combinatorial treatment with methyltransferase and deacetylation inhibitors reversed the expression of miR-432-5p and miR-127-3p, suggesting that chronic iron treatment downregulates miRNA expression via epigenetic alterations.

Additionally, miR-138-5p was also downregulated in chronic iron exposed FTSECs. This miRNA is located at chromosome 3 (which also harbors EVI1, a common transcriptional activator in EOC). We identified a potential EVI1 binding site in miR-138 promoter region and therefore analyzed whether miR-138 transcription is regulated by EVI1. Although our studies indicated that miR-138-5p is regulated independently of EVI1, TERT RNA was found to be partially regulated by miR-138-5p [43]. Overall, this study reveals global molecular alterations induced by iron, potentially associated with transformation of FTSECs.

Collectively, these studies suggest the involvement of LPA in reversing ccRCC chemosensitivity and highlight the potential role of iron-induced 14q32 miRNA downregulation in FTSEC transformation. In future, a detailed study of the mechanism involved in LPA-mediated chemoresistance, and the role and regulation of miRNAs at 14q32 will expand our understanding of their functional consequences, which will also prove useful to design clinically improved therapeutic strategies.

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