Graduation Year

2014

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Meera Nanjundan

Keywords

autophagy, endometriosis, lysosomes, mitochondria, ovarian cancer, Ras/MAPK

Abstract

Ovarian carcinoma afflicts over 22,000 women each year with a 5 year survival rate of only 18% for stage IV patients [23]. Current treatment options are limited due to high rates of drug resistance and recurrence. Further, the identity of "precursor lesions" which give rise to various subclasses of epithelial ovarian cancer has been evasive. This is due to discovery of the cancer at already an advanced stage. Interestingly, endometriosis a benign but invasive gynecological disease has been described as a "precursor lesion" in the development of specific subtypes of ovarian cancer. Endometriotic cyst development involves the accumulation of "old blood" components including iron-rich heme. Published evidence implicates excess iron that is involved in the transformation of normal surface epithelial cells inducing morphological characteristics of clear cell ovarian cancer cells [13, 34]. Due to excess iron in endometriotic cysts, this essential element may play a transformative role in the development of clear cell ovarian cancer and possibly other subtypes [13, 35-38]. Further, studies show increased risk of developing ovarian cancer, particularly clear cell and endometrioid ovarian subtypes, in patients diagnosed with endometriosis [36, 37, 39, 40] .

This thesis aims to initiate an investigation regarding the contribution of iron and endometriotic lesions in the development and progression of specific subtypes of epithelial ovarian cancers. Since there is a lack of well-validated and characterized endometriotic cell lines that could be used for endometriosis studies, we sought to develop an immortalized cell line for future endometriotic in vitro and in vivo studies. Thus, in Chapter 3 we present our efforts in developing a novel life-span extended epithelial endometriotic cell line. The cells were derived from the endometriotic tissue of a patient with endometriosis. We describe our attempts at immortalization and the characterization of this endometriotic cell line in relation to previously reported/available endometrial/endometriotic cell lines.

In Chapter 4 we investigated the role of iron in modulating functional aspects of various gynecological cell lines. Although our expectation was that iron could transform normal ovarian surface epithelial cells (OSE) to a carcinoma-like phenotype, we instead discovered that ovarian cell lines containing Ras mutations (or with H-Ras overexpression) responded to iron (presented as ferric ammonium citrate (FAC)) with a reduced growth response. Further treatment with iron induced an apoptotic/necrotic death response in the Ras mutated HEY ovarian carcinoma cell line. Interestingly, we identified that iron induced autophagic activation in all ovarian cell lines investigate, although autophagy contributed only modestly to the cell death event. Furthermore, we noted that iron activated the MAPK pathway and its inhibition (via U0126, a MAPK inhibitor) allowed survival of cells.

In Chapter 5, we briefly explore the role of iron in ovarian cell types growing under anchorage-independent conditions. We found that the cell lines displayed increased cleaved PARP and apoptosis when placed under these conditions. Treatment with iron led to a reduction in cleaved PARP suggesting that iron promotes cell survival in anchorage-independent conditions. Further, inhibition of autophagy via chloroquine led to increased cleaved PARP suggesting that autophagy may mediate a protective role against anchorage-independent apoptotic response

In Chapter 6, we attempted to elucidate the downstream mechanism following Ras/MAPK activation in response to iron. This study identified several signaling pathways including that involved in translational control, iron metabolism, as well as mitochondrial function. The inhibition of the iron regulatory and translation control pathway did not significantly lead to rescue of iron-induced cell death of Ras mutated/overexpressed cells. However, we noted mitochondrial stress and damage including altered expression of mitochondrial markers (TOM20/TOM70, outer membrane transporters) which occurred concurrently with iron-induced cell death. The inhibition of iron import into mitochondria using a calcium uniporter channel inhibitor (Ru360) led to a marked reversal of the cell death response. Collectively, these studies suggest that increased mitochondrial permeabilization may be responsible for the observed iron-induced cell death response.

Overall, the studies presented in this thesis have revealed novel responses to iron in the gynecological cell types investigated. We initially sought to understand the role of iron in precursor lesions which included the development of a novel life-span extended epithelial endometriotic cell type. Remarkably, our findings revealed a Ras driven sensitivity to excess iron. Treatment with iron caused decreased cell growth and increased cell death in cell types containing Ras mutation/overexpression. Further, we found that the mechanism leading to the iron-induced cell death events was mediated via the MAPK pathway. We then determined that the cell death response was associated with mitochondrial permeabilization. Loss of mitochondrial integrity occurred in Ras sensitive cell lines and inhibition of iron import into the mitochondria (via the calcium uniporter channel inhibitor, Ru360) led to reversal of this response. We show herein the cellular response of excess iron and its potential implication in ovarian cancer research.

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