Proteomic Profiling of Iron-Treated Ovarian Cells Identifies AKT Activation that Modulates the CLEAR Network.

Although iron is essential for cell survival, dysregulated levels can contribute to cancer development or even cell death. The underlying mechanisms mediating these events remain unclear. Herein, proteomic alterations are assessed in iron-treated ovarian cell lines using reverse phase protein array (RPPA) technology and potential functional responses via ingenuity pathway analysis (IPA). Using these approaches, upregulation of pathways modulating organismal death with alterations in mTOR, MAPK, and AKT signaling in HEY ovarian cancer cells in contrast to T80 non-malignant ovarian cells is noted. Since modulation of cell death is mediated in part via microphthalmia-associated transcription factor (MiTF) family, which regulates lysosomal biogenesis and autophagosome formation by upregulating expression of coordinated lysosomal expression and regulation (CLEAR) network, expression changes in these factors in response to iron are investigated. Increased transcription factor EB (TFEB) in T80 (relative to HEY), accompanied by its nuclear translocation and increased CLEAR network gene expression with iron, is identified. Inhibition of AKT alters these responses in contrast to mTOR inhibition, which has little effect. Collectively, these findings support use of RPPA/IPA technology to predict functional responses to iron and further implicate AKT pathway and MiTF members in iron-induced cellular responses in ovarian cells.

Iron overload and altered iron metabolism in ovarian cancer.

Iron is an essential element required for many processes within the cell. Dysregulation in iron homeostasis due to iron overload is detrimental. This nutrient is postulated to contribute to the initiation of cancer; however, the mechanisms by which this occurs remain unclear. Defining how iron promotes the development of ovarian cancers from precursor lesions is essential for developing novel therapeutic strategies. In this review, we discuss (1) how iron overload conditions may initiate ovarian cancer development, (2) dysregulated iron metabolism in cancers, (3) the interplay between bacteria, iron, and cancer, and (4) chemotherapeutic strategies targeting iron metabolism in cancer patients.

Iron alters cell survival in a mitochondria-dependent pathway in ovarian cancer cells.

The role of iron in the development of cancer remains unclear. We previously reported that iron reduces cell survival in a Ras/mitogen-activated protein kinase (MAPK)-dependent manner in ovarian cells; however, the underlying downstream pathway leading to reduced survival was unclear. Although levels of intracellular iron, ferritin/CD71 protein and reactive oxygen species did not correlate with iron-induced cell survival changes, we identified mitochondrial damage (via TEM) and reduced expression of outer mitochondrial membrane proteins (translocase of outer membrane: TOM20 and TOM70) in cell lines sensitive to iron. Interestingly, Ru360 (an inhibitor of the mitochondrial calcium uniporter) reversed mitochondrial changes and restored cell survival in HEY ovarian carcinoma cells treated with iron. Further, cells treated with Ru360 and iron also had reduced autophagic punctae with increased lysosomal numbers, implying cross-talk between these compartments. Mitochondrial changes were dependent on activation of the Ras/MAPK pathway since treatment with a MAPK inhibitor restored expression of TOM20/TOM70 proteins. Although glutathione antioxidant levels were reduced in HEY treated with iron, extracellular glutamate levels were unaltered. Strikingly, oxalomalate (inhibitor of aconitase, involved in glutamate production) reversed iron-induced responses in a similar manner to Ru360. Collectively, our results implicate iron in modulating cell survival in a mitochondria-dependent manner in ovarian cancer cells.

Genetic ablation of Immt induces a lethal disruption of the MICOS complex.

The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.

Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells.

Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis.

Global miRNA/proteomic analyses identify miRNAs at 14q32 and 3p21, which contribute to features of chronic iron-exposed fallopian tube epithelial cells.

Malignant transformation of fallopian tube secretory epithelial cells (FTSECs) is a key contributing event to the development of high-grade serous ovarian carcinoma (HGSOC). Our recent findings implicate oncogenic transformative events in chronic iron-exposed FTSECs, including increased expression of oncogenic mediators, increased telomerase transcripts, and increased growth/migratory potential. Herein, we extend these studies by implementing an integrated transcriptomic and mass spectrometry-based proteomics approach to identify global miRNA and protein alterations, for which we also investigate a subset of these targets to iron-induced functional alterations. Proteomic analysis identified > 4500 proteins, of which 243 targets were differentially expressed. Sixty-five differentially expressed miRNAs were identified, of which 35 were associated with the "top" proteomic molecules (> fourfold change) identified by Ingenuity Pathway Analysis. Twenty of these 35 miRNAs are at the 14q32 locus (encoding a cluster of 54 miRNAs) with potential to be regulated by DNA methylation and histone deacetylation. At 14q32, miR-432-5p and miR-127-3p were ~ 100-fold downregulated whereas miR-138-5p was 16-fold downregulated at 3p21 in chronic iron-exposed FTSECs. Combinatorial treatment with methyltransferase and deacetylation inhibitors reversed expression of these miRNAs, suggesting chronic iron exposure alters miRNA expression via epigenetic alterations. In addition, PAX8, an important target in HGSOC and a potential miRNA target (from IPA) was epigenetically deregulated in iron-exposed FTSECs. However, both PAX8 and ALDH1A2 (another IPA-predicted target) were experimentally identified to be independently regulated by these miRNAs although TERT RNA was partially regulated by miR-138-5p. Interestingly, overexpression of miR-432-5p diminished cell numbers induced by long-term iron exposure in FTSECs. Collectively, our global profiling approaches uncovered patterns of miRNA and proteomic alterations that may be regulated by genome-wide epigenetic alterations and contribute to functional alterations induced by chronic iron exposure in FTSECs. This study may provide a platform to identify future biomarkers for early ovarian cancer detection and new targets for therapy.

Chronic iron exposure and c-Myc/H-ras-mediated transformation in fallopian tube cells alter the expression of EVI1, amplified at 3q26.2 in ovarian cancer.

Mechanisms underlying the pathogenesis of high-grade serous epithelial ovarian cancers (HGSOC) are not yet well defined although key precursor cells have been identified (including fimbriated fallopian tube epithelium, FTSECs). Since iron is elevated in endometriotic cysts and the pelvic cavity, it is suggested that this source of redox-active iron may contribute to ovarian cancer pathogenesis. Specifically, sources of nontransferrin-bound iron (NTBI) within the pelvic cavity could arise from ovulation, retrograde menstruation, follicular fluid, or iron overload conditions (i.e., hemochromatosis). Herein, we investigated the cellular response of p53-inactivated and telomerase-expressing (immortalized) FTSECs (Pax8+/FoxJ1-) to NTBI (presented as ferric ammonium citrate (FAC), supplemented in media for >2 months) in order to assess its ability to promote the transition to a tumor-like phenotype; this cellular response was compared with immortalized FTSECs transformed with H-RasV12A and c-MycT58A. Both approaches resulted in increased cell numbers and expression of the oncogenic transcriptional regulator, ecotropic virus integration site 1 (EVI1, a gene most frequently amplified at 3q26.2 in HGSOC, represented by multiple variants), along with other oncogenic gene products. In contrast to the transformed cells, FAC-exposed FTSECs elicited elevated migratory capacity (and epithelial-mesenchymal transition mRNA profile) along with increased expression of DNA damage response proteins (i.e., FANCD2) and hTERT mRNA relative to controls. Interestingly, in FAC-exposed FTSECs, EVI1 siRNA attenuated hTERT mRNA expression, whereas siRNAs targeting ß-catenin and BMI1 (both elevated with chronic iron exposure) reduced Myc and Cyclin D1 proteins. Collectively, our novel findings provide strong foundational evidence for potential iron-induced initiation events, including EVI1 alterations, in the pathogenesis of HGSOC, warranting further in depth investigations. Thus, these findings will substantially advance our understanding of the contribution of iron enriched within the pelvic cavity, which may identify patients at risk of developing this deadly disease.

Expression and function of nuclear receptor coactivator 4 isoforms in transformed endometriotic and malignant ovarian cells.

Iron is proposed to contribute to the transition from endometriosis to specific subtypes of ovarian cancers (OVCAs). Regulation of intracellular iron occurs via a ferritinophagic process involving NCOA4 (Nuclear Receptor Coactivator 4), represented by two major isoforms (NCOA4α and NCOA4ß), whose contribution to ovarian cancer biology remains uninvestigated. We thus generated transformed endometriotic cells (via HRASV12A, c-MYCT58A, and p53 inactivation) whose migratory potential was increased in response to conditioned media from senescent endometriotic cells. We identified elevated NCOA4 mRNA in transformed endometriotic cells (relative to non-transformed). Knockdown of NCOA4 increased ferritin heavy chain (FTH1) and p21 protein which was accompanied by reduced cell survival while NCOA4ß overexpression reduced colony formation. NCOA4α and NCOA4ß mRNA were elevated in malignant versus non-malignant gynecological cells; NCOA4α protein was increased in the assessed malignant cell lines as well as in a series of OVCA subtypes (relative to normal adjacent tissues). Further, NCOA4 protein expression was regulated in a proteasome- and autophagy-independent manner. Collectively, our results implicate NCOA4 in ovarian cancer biology in which it could be involved in the transition from precursors to OVCA.

Links Between Iron and Lipids: Implications in Some Major Human Diseases.

Maintenance of iron homeostasis is critical to cellular health as both its excess and insufficiency are detrimental. Likewise, lipids, which are essential components of cellular membranes and signaling mediators, must also be tightly regulated to hinder disease progression. Recent research, using a myriad of model organisms, as well as data from clinical studies, has revealed links between these two metabolic pathways, but the mechanisms behind these interactions and the role these have in the progression of human diseases remains unclear. In this review, we summarize literature describing cross-talk between iron and lipid pathways, including alterations in cholesterol, sphingolipid, and lipid droplet metabolism in response to changes in iron levels. We discuss human diseases correlating with both iron and lipid alterations, including neurodegenerative disorders, and the available evidence regarding the potential mechanisms underlying how iron may promote disease pathogenesis. Finally, we review research regarding iron reduction techniques and their therapeutic potential in treating patients with these debilitating conditions. We propose that iron-mediated alterations in lipid metabolic pathways are involved in the progression of these diseases, but further research is direly needed to elucidate the mechanisms involved.