Usawadee Dier

Mitochondrial Superoxide Dismutase Has a Protumorigenic Role in Ovarian Clear Cell Carcinoma.

Epithelial ovarian cancer (EOC) is the fourth leading cause of death due to cancer in women and comprises distinct histologic subtypes, which vary widely in their genetic profiles and tissues of origin. It is therefore imperative to understand the etiology of these distinct diseases. Ovarian clear cell carcinoma (OCCC), a very aggressive subtype, comprises >10% of EOCs. In the present study, we show that mitochondrial superoxide dismutase (Sod2) is highly expressed in OCCC compared with other EOC subtypes. Sod2 is an antioxidant enzyme that converts highly reactive superoxide (O2 (•-)) to hydrogen peroxide (H2O2) and oxygen (O2), and our data demonstrate that Sod2 is protumorigenic and prometastatic in OCCC. Inhibiting Sod2 expression reduces OCCC ES-2 cell tumor growth and metastasis in a chorioallantoic membrane (CAM) model. Similarly, cell proliferation, migration, spheroid attachment and outgrowth on collagen, and Akt phosphorylation are significantly decreased with reduced expression of Sod2. Mechanistically, we show that Sod2 has a dual function in supporting OCCC tumorigenicity and metastatic spread. First, Sod2 maintains highly functional mitochondria, by scavenging O2 (•-), to support the high metabolic activity of OCCC. Second, Sod2 alters the steady-state ROS balance to drive H2O2-mediated migration. While this higher steady-state H2O2 drives prometastatic behavior, it also presents a doubled-edged sword for OCCC, as it pushed the intracellular H2O2 threshold to enable more rapid killing by exogenous sources of H2O2. Understanding the complex interaction of antioxidants and ROS may provide novel therapeutic strategies to pursue for the treatment of this histologic EOC subtype.

Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis

Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC.

Regulation of MMP-1 expression in response to hypoxia is dependent on the intracellular redox status of metastatic bladder cancer cells.

High steady-state reactive oxygen species (ROS) production has been implicated with metastatic disease progression. We provide new evidence that this increased intracellular ROS milieu uniquely predisposes metastatic tumor cells to hypoxia-mediated regulation of the matrix metalloproteinase MMP-1. Using a cell culture metastatic progression model we previously reported that steady-state intracellular H2O2 levels are elevated in highly metastatic 253J-BV bladder cancer cells compared to their non-metastatic 253J parental cells. 253J-BV cells display higher basal MMP-1 expression, which is further enhanced under hypoxic conditions (1% O2). This hypoxia-mediated MMP-1 increase was not observed in the non-metastatic 253J cells. Hypoxia-induced MMP-1 increases are accompanied by the stabilization of hypoxia-inducible transcription factors (HIFs)-1α and HIF-2α, and a rise in intracellular ROS in metastatic 253J-BV cells. RNA interference studies show that hypoxia-mediated MMP-1 expression is primarily dependent on the presence of HIF-2α. Further, hypoxia promotes migration and spheroid outgrowth of only the metastatic 253J-BV cells and not the parental 253J cells. The observed HIF stabilization, MMP-1 expression and migration under hypoxia are dependent on increases in intracellular ROS, as these effects are attenuated by treatment with the antioxidant N-acetyl-L-cysteine. These data show that ROS play an important role in hypoxia-mediated MMP-1 expression and that an elevated intracellular redox environment, as observed in metastasis, predisposes tumor cells to an enhanced hypoxic response. It further supports the notion that metastatic tumor cells are uniquely able to utilize intracellular increases in ROS to drive pro-metastatic signaling events and highlights the important interplay between ROS and hypoxia in malignancy.

Abstract MIP-059: MITOCHONDRIAL DYNAMICS AND DYSFUNCTION IN OVARIAN CANCER

Altered mitochondrial function remains a key feature of many tumor cells and drives pathways such gene expression, metabolic and stress responses, cell cycle progression and resistance to apoptosis. Many chemotherapeutics activate programmed cell death and it is thought that mitochondrial dysfunction may be one mechanism by which cancer cells evade killing by these compounds. Screening the metabolic profiles of ovarian cancer cell lines and patient ascites-derived tumor cells reveals that ovarian cancers fall into unique bioenergetic subgroups. For example, Ovarian Clear Cell Carcinomas (OCCC) display high oxygen consumption rate and glycolytic flux compared to the more common high grade serous adenocarcinoma (HGSA) subtype. In addition, we show that a portion of HGSAs have severe mitochondrial dysfunction, that is marked by a decrease in mitochondrial respiration, a lack of response to the uncoupler FCCP and a concomitant reliance on alternate metabolic pathways. Moreover, this is accompanied by enhanced chemoresistance to Cisplatin and Taxol. The cause of mitochondrial dysfunction has been attributed to a number of factors, including deregulation in mitochondrial fission/fusion dynamics. Moreover, fission is an integral component of apoptotic and authophagy pathways. Interestingly, the observed HGSA mitochondrial dysfunction correlates with aberrant fusion/fission dynamics and expression of a low molecular weight variant of the mitochondrial fission protein Drp1. The potential significance of Drp1 in ovarian cancer etiology is highlighted by TCGA data, where more than 15% of HGSA samples display significant increases in Drp1 mRNA levels, and associated amplification of the Drp1 gene DNM1L. Whether this represents the shorter, potentially dominant-negative Drp1 variant identified in our work is currently under active investigation. Our data suggest that compromised mitochondrial function and fission/fusion dynamics may be a hallmark of a previously unidentified subgroup of highly chemoresistant EOCs and that this is associated with aberrant expression of the fission protein Drp1. Studies are underway to identify the molecular identity and regulation of short Drp1, and the mechanistic links to alterations in fission, metabolic switching and chemoresistance. Citation Format: Dong-Hui Shin, Usawadee Dier, Patrick F. Timmins, Joshua Kesterson, Rebecca Phaeton and Nadine Hempel. MITOCHONDRIAL DYNAMICS AND DYSFUNCTION IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-059.

Abstract A12: Regulation of the mitochondrial stress response and metabolism by the intraperitoneal tumor environment during ovarian cancer transcoelomic metastasis

Transcoelomic metastasis represents one of the routes by which ovarian cancer cells colonize the omental tissues of the intraperitoneal (IP) cavity. Large quantities of viable tumor cells and spheroid aggregates can be isolated from ascites of advanced stage ovarian cancer patients, suggesting that cancer cells spend some time in anchorage independence under the physical and chemical influence of the IP tumor environment. Using ovarian cancer cell lines and patient ascites-derived tumor cells we demonstrate that levels of the mitochondrial stress response proteins, superoxide dismutase 2 (Sod2) and the deacetylase sirtuin 3 (SIRT3), are significantly increased when cultured in anchorage independent conditions, where cells rapidly form spheroid aggregates. Mimicking the ascites fluid current we show that expression of these proteins is further enhanced when spheroids are exposed to sheer forces, suggesting that the physical environment influences cellular stress adaptation. Presently, the combination of these physical cues and chemical stimuli emanating from ascites fluid are being interrogated in the co-regulation of the above mitochondrial proteins, focusing on the role of redox modulators and metabolite composition of ascites. It is our hypothesis that this stress response adaptation not only aids survival of cells in anchorage independence, but that it also drives a metabolic regulatory switch. Anchorage independent spheroids display a slowing in glycolysis and an increase in mitochondrial respiratory reserve capacity, as assessed by extracellular flux analysis. Sod2 expression is required for maintaining this mitochondrial respiratory function by scavenging damaging superoxide anion. Further, we observe decreases in global and mitochondrial acetylation, suggesting that deacetylase activity may be increased in spheroids. SIRT3 is a direct regulator of Sod2 activity, and is an important sensor of nutrient stress and modulator of metabolism, including the stimulation of fatty acid oxidation by deacetylation and activation of long-chain-specific acyl-CoA dehydrogenase. It has previously been demonstrated that metastatic ovarian cancer cells rely on fatty acid oxidation given their propensity to colonize the lipid-rich environments of the omentum. Studies are under way to test the hypothesis that SIRT3 expression stimulated by the IP tumor environment during anchorage independence primes ovarian cancer cells for fatty acid oxidation and is a necessary step before the colonization of the omentum. Citation Format: Dong-Hui Shin, Victoria Jones, Sara Shimko, Usawadee Dier, Trevor Shepherd, Timmins Patrick, Nadine Hempel. Regulation of the mitochondrial stress response and metabolism by the intraperitoneal tumor environment during ovarian cancer transcoelomic metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A12.

Abstract 1440: Mitochondrial superoxide dismutase (Sod2) modulates ovarian clear cell carcinoma transcoelomic metastatic pathway

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian clear cell carcinoma (OCCC) is a highly aggressive histological subtype that represents approximately 10% of all ovarian cancer cases and displays high expression of mitochondrial superoxide dismutase (Sod2), a major mitochondrial antioxidant enzyme. In the female body ovarian cancer cells metastasize via the transcoelomic route, where cells detach from the primary tumor into the intraperitoneal (IP) cavity and survive as unattached single cells or spheroid aggregates until they get carried via ascites fluid to the omental wall and surrounding organs for metastatic colonization. Ovarian cancer spheroids evade anoikis and display strong resistant to most anti-proliferative cancer drugs. This may contribute to the high recurrence rate and chemoresistance of ovarian cancer. The present study suggests that Sod2 is critically important for the key events of this transcoelomic metastatic route of OCCC. Levels of Sod2 are further increased in OCCC cell lines when these are cultured as anchorage-independent spheroid aggregates, suggesting that Sod2 may play a major role in spheroid stability and survival. When Sod2 levels are reduced using RNA interference, spheroid morphology of ES-2 OCCC cell line is compromised, suggesting a potential correlation between anoikis resistant and Sod2 levels in OCCC. Sod2 knockdown compromises mitochondrial metabolism, as a consequence of reduced superoxide scavenging, and enhances spheroid sensitivity to cisplatin. In addition, Sod2 is necessary for spheroid attachment and outgrowth on extracellular matrix components, representing the invasion of spheroids into the omental wall. Further, tumor cell metastasis is attenuated with reduced Sod2 levels when ES-2 spheroids are placed on the vasculature of the Chorioallantoic membrane (CAM). Scavenging H2O2 by exogenous addition of catalase inhibits the migration of OCCC and our data suggest that high levels of Sod2 in OCCC shift the intracellular redox balance to drive H2O2-dependent pro-metastatic signaling. These results indicate that Sod2 plays a major role in OCCC tumor metastasis, by regulating survival and chemoresistance of tumor spheroids in the IP cavity and by supporting attachment and migration at secondary metastatic sites. Inhibition of Sod2 can be a potential therapeutic target against OCCC transcoelomic metastasis. Citation Format: L.P.Madhubhani P. Hemachandra, Usawadee Dier, Nadine Hempel. Mitochondrial superoxide dismutase (Sod2) modulates ovarian clear cell carcinoma transcoelomic metastatic pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1440. doi:10.1158/1538-7445.AM2015-1440

Abstract B65: The mitochondrial antioxidant Superoxide Dismutase 2 is necessary for ovarian cancer spheroid formation

The formation of cellular spheroid aggregates in the Intraperitoneal Cavity (IP) is considered to be an important step in the metastatic progression of ovarian cancer. Spheroids may represent a unique morphological adaptation that enhances long-term survival of tumor cells in the IP cavity, reminiscent of dormant cell populations. Like other dormant tumor cells, in vivo-isolated and in vitro-cultured spheroids exhibit enhanced chemoresistance and display markers of quiescence. We show that expression of mitochondrial superoxide dismutase (Sod2), which regulates mitochondrial redox balance, is significantly increased during formation of dense spheroids from a number of ovarian cancer cell lines. This is accompanied by increases in the quiescence marker p27(kip1). Importantly, siRNA and shRNA knock-down of Sod2 compromise spheroid formation and integrity, and abrogate increases in p27(kip1) expression in anchorage-independent 3D cultures. Mechanistically, it is likely that increases in Sod2 expression protect cells from build-up of mitochondrial and IP cavity-derived ROS, enhancing their survival as dormant spheroid populations. In addition, bioenergetic data suggest that Sod2 is directly involved in enhancing the mitochondrial integrity of tumor cells within spheroid aggregates. This leads to a metabolic profile that is associated with a slowing in ATP demand and an enhanced respiratory reserve capacity, with Sod2 being intricately involved in manipulating these changes. Enhanced Sod2 expression, a concomitant decrease in mitochondrial ROS and changes in mitochondrial function are likely indicators of a quiescence adaptation of tumor spheroids, necessary for survival in the IP cavity. Manipulating mitochondrial function and redox balance by effectively targeting mitochondrial antioxidant enzymes may provide novel therapeutic strategies to combat recurrence and metastatic spread of ovarian cancer. Citation Format: LP Madhubhani Hemachandra, Usawadee Dier, Larissa M. Uusitalo, Nadine Hempel. The mitochondrial antioxidant Superoxide Dismutase 2 is necessary for ovarian cancer spheroid formation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B65.

Abstract B40: Mitochondrial superoxide dismutase (Sod2) confers a survival advantage to ovarian cancer cells under stress conditions by enhancing mitochondrial respiratory reserve capacity

The formation of cellular spheroids is considered to be an important step in the metastatic progression of ovarian cancer. These clusters of tumor cells are characterized by an enhanced survival within the peritoneal cavity, the ability to attach at secondary metastatic sites within the cavity and associated with increased chemoresistance. High expression of mitochondrial superoxide dismutase (Sod2), which regulates mitochondrial redox balance, has been observed in metastatic ovarian cancer, specifically ovarian clear cell carcinomas (CCC), and in vitro, following spheroid formation. To assess the role of Sod2 in spheroid formation and ovarian cancer metastasis we utilized siRNA knock-down to decrease Sod2 expression in Ovcar3 and the CCC cell line ES-2. Surprisingly, under normal culture conditions, decreasing Sod2 levels had little effect on proliferation and spheroid formation. To elucidate the role of Sod2 on ovarian cancer mitochondrial function an extracellular flux metabolic analyzer (Seahorse Bioscience) was utilized to measure rates of cellular respiration. While the basal oxygen consumption rate remained unchanged, a decrease in Sod2 expression resulted in a concomitant decrease in respiratory reserve capacity. This reserve capacity is an important determinant of a cell9s ability to increase metabolic rate under stress conditions. Given that ovarian cancer spheroids are exposed to the hypoxic environment of the peritoneal cavity, the role of Sod2 on ovarian cancer spheroid formation was assessed under low oxygen (1%) conditions. Hypoxia increased the clonogenicity and spheroid size of control cells, which was significantly abrogated following Sod2 knock-down. These data suggest that Sod2 confers a survival advantage to ovarian cancer spheroids under stress conditions by maintaining an adequate mitochondrial respiratory reserve. Manipulating mitochondrial function and redox balance by effectively targeting mitochondrial antioxidant enzymes may enhance chemosensitivity and allow for novel therapeutic strategies against ovarian cancer. Citation Format: Usawadee Dier, Larissa Uusitalo, Corinne Giroux, Nadine Hempel. Mitochondrial superoxide dismutase (Sod2) confers a survival advantage to ovarian cancer cells under stress conditions by enhancing mitochondrial respiratory reserve capacity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B40.