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

Characterization of the Autophagic-Iron Axis in the Pathophysiology of Endometriosis and Epithelial Ovarian Cancers

For women in the United States, ovarian cancer (OVCA) represents the deadliest gynecological cancer with over 14,000 deaths predicted to occur in 2019 [13]. Although innovative treatment strategies have improved the 5-year outcome for these patients, the overall survival has not changed in over 40 years [14]. This is largely due to the challenges in detecting this heterogenous disease at an early stage, as there are multiple potential precursors that may give rise to different histological subtypes [14]. This dissertation is thus focused on investigating pro-survival pathways involved in the pathogenesis of endometriosis (a proposed precursor) and epithelial ovarian cancers. Endometriosis occurs when glands (comprised of both epithelial and stromal cells) from the endometrium are located in the pelvic cavity [15]. A cellular homeostasis process known as autophagy is characterized as being dysregulated in patients with endometriosis [16-20], but the contribution of this pathway to the formation of endometriotic lesions has been uncertain. Furthermore, studies have identified that patients diagnosed with endometriosis also have an increased risk for developing more rare epithelial OVCA (EOC), namely endometrioid and clear cell OVCAs [21, 22], which suggests that endometriosis is a likely precursor. In Chapter 2 of this dissertation, we characterized the mRNA and protein expression of autophagy markers in endometriosis-induced mice and from patient specimens. We further identified that inhibiting the autophagy pathway with an FDA-approved anti-malarial drug, hydroxychloroquine (HCQ), significantly reduced human endometriotic cell viability in vitro and reduced the number of endometriotic lesions that developed within an in vivo endometriosis model. These results suggest that autophagy may be an important contributor to the establishment of endometriosis. EOCs are characterized by genomic aberrations which can help in distinguishing the different histological subtypes [23-25]. Furthermore, recent studies suggest that iron dependency may be an important characteristic of OVCA [26], but further research into the contribution of iron dysregulation to ovarian cancer pathogenesis is needed. Thus, in Chapter 3, we generated transformed human endometriotic cell lines by inhibiting p53 (which is commonly inactivated via mutations in ~95% of high-grade serous OVCA (HGSOC) [25]) and overexpressing human telomerase reverse transcriptase (hTERT, which is commonly amplified in HGSOC [27]). These cells were also expressing oncogenic c-Myc (which is amplified at 8q24 in HGSOC [28]) as well as mutated (constitutively activated) H-Ras (which is commonly activated in OVCA [29]). We characterized increased colony forming ability and reduced senescence compared to non-transformed control cells, as expected, but also identified mRNA expression changes in key regulators of the iron pathway. One such regulator, NCOA4 (nuclear receptor coactivator 4), promotes the release of iron from the ferritin storage complex through a targeted autophagy process, ferritinophagy [3, 30]. Interestingly, NCOA4 mRNA and protein expression were elevated in transformed endometriotic cells relative to controls. We further identified increased NCOA4 mRNA and protein in malignant OVCA cell lines relative to non-malignant cells, as well as increased NCOA4 protein in human OVCA tumors relative to control adjacent tissues. These findings suggest a potential role for NCOA4 and iron dysregulation in OVCA pathogenesis. Iron is an essential metal required for many cellular processes [31-33], but it can also be detrimental through its ability to produce reactive oxygen species (ROS) [34, 35]. Excess iron has been implicated in the development of epithelial cancers, such as hepatocellular cancer [36]. Recent evidence also supports a role for iron in ovarian cancer pathogenesis [26], but the molecular events mediating this process have not been identified. We thus investigated the contribution of iron to the transition of precursors to OVCA in Chapter 4 by chronically exposing fallopian tube secretory epithelial cells (FTSECs, another proposed precursor to OVCA [14]) to a source of non-transferrin bound iron (NTBI). In addition, we generated transformed FTSECs and compared the functional and expressional changes between the two cell lines. We found that both chronic iron exposure and cellular transformation increased cell numbers and observed altered protein and mRNA expression of MECOM genes, which are commonly aberrant in OVCA [14]. Specifically, the expression of tumor suppressive MDS1/EVI1 was reduced while the expression of oncogenic EVI1 and EVI1Del190-515 was elevated in both conditions. Interestingly, we also observed differences between these cell lines, such as increased migratory capacity and elevated b-catenin protein expression with chronic iron treatment (but not with oncogenic transformation). Together, these findings demonstrate that iron may mediate the transition of fallopian tube precursors to a transformed-like state. Altogether, these findings have improved our knowledge of pathways that may be involved in the pathogenesis of endometriosis and epithelial ovarian cancers, namely the autophagy and iron regulatory pathways. Additional research is needed, however, in order to identify the respective underlying mechanisms. Such studies would also expand our understanding of the contribution of iron to the transition of endometriotic and fallopian tube precursor cells to OVCA. Understanding these mechanisms will promote innovative strategies to detect these diseases early and help improve current treatment strategies, thus improving on the clinical outcome for women diagnosed with ovarian cancer

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

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 NCOA4β), 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 NCOA4β overexpression reduced colony formation. NCOA4α and NCOA4β 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

Effect of Hydroxychloroquine and Characterization of Autophagy in a Mouse Model of Endometriosis

In endometriosis, the increased survival potential of shed endometrial cells (which normally undergo anoikis) is suggested to promote lesion development. One mechanism that may alter anoikis is autophagy. Using an autophagic flux inhibitor hydroxychloroquine (HCQ), we identified that it reduces the in vitro survival capacity of human endometriotic and endometrial T-HESC cells. We also identified that HCQ could decrease lesion numbers and disrupt lesion histopathology, as well as increase the levels of peritoneal macrophages and the IP-10 (10 kDa interferon-γ-induced protein) chemokine in a mouse model of endometriosis. We noted that RNA levels of a subset of autophagic markers were reduced in lesions relative to uterine horns from endometriosis-induced (untreated) mice. In addition, the RNA levels of autophagic markers were decreased in uterine horns of endometriosis-induced mice compared with those from controls. However, we noted that protein expression of LC3B (microtubule-associated protein 1 light-chain 3β; an autophagic marker) was increased in uterine horns of endometriosis-induced mice compared with uterine horns of controls. By immunohistochemical staining of a human endometriosis-focused tissue microarray, we observed LC3B expression predominantly in epithelial relative to stromal cells in both eutopic and ectopic endometria. Via transmission electron microscopy, cells from eutopic endometria of endometriosis-induced mice contained more lipid droplets (rather than autophagosomes) compared with uterine horns from controls. Collectively, our findings indicate that the autophagic pathway is dysregulated in both ectopic and eutopic endometrium in a murine model of endometriosis and that HCQ has potential as a therapeutic agent for women afflicted with endometriosis

Effect of Hydroxychloroquine and Characterization of Autophagy in a Mouse Model of Endometriosis

In endometriosis, the increased survival potential of shed endometrial cells (which normally undergo anoikis) is suggested to promote lesion development. One mechanism that may alter anoikis is autophagy. Using an autophagic flux inhibitor hydroxychloroquine (HCQ), we identified that it reduces the in vitro survival capacity of human endometriotic and endometrial T-HESC cells. We also identified that HCQ could decrease lesion numbers and disrupt lesion histopathology, as well as increase the levels of peritoneal macrophages and the IP-10 (10 kDa interferon-γ-induced protein) chemokine in a mouse model of endometriosis. We noted that RNA levels of a subset of autophagic markers were reduced in lesions relative to uterine horns from endometriosis-induced (untreated) mice. In addition, the RNA levels of autophagic markers were decreased in uterine horns of endometriosis-induced mice compared with those from controls. However, we noted that protein expression of LC3B (microtubule-associated protein 1 light-chain 3β; an autophagic marker) was increased in uterine horns of endometriosis-induced mice compared with uterine horns of controls. By immunohistochemical staining of a human endometriosis-focused tissue microarray, we observed LC3B expression predominantly in epithelial relative to stromal cells in both eutopic and ectopic endometria. Via transmission electron microscopy, cells from eutopic endometria of endometriosis-induced mice contained more lipid droplets (rather than autophagosomes) compared with uterine horns from controls. Collectively, our findings indicate that the autophagic pathway is dysregulated in both ectopic and eutopic endometrium in a murine model of endometriosis and that HCQ has potential as a therapeutic agent for women afflicted with endometriosis

Presentation_3_Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells.pptx

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.</p