Peter C. Hart

MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer

Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signaling. Here, we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppress the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical in support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumor cell metabolism.

Selected mitochondrial DNA landscapes activate the SIRT3 axis of the UPRmt to promote metastasis

By causing mitochondrial DNA (mtDNA) mutations and oxidation of mitochondrial proteins, reactive oxygen species (ROS) leads to perturbations in mitochondrial proteostasis. Several studies have linked mtDNA mutations to metastasis of cancer cells but the nature of the mtDNA species involved remains unclear. Our data suggests that no common mtDNA mutation identifies metastatic cells; rather the metastatic potential of several ROS-generating mutations is largely determined by their mtDNA genomic landscapes, which can act either as an enhancer or repressor of metastasis. However, mtDNA landscapes of all metastatic cells are characterized by activation of the SIRT/FOXO/SOD2 axis of the mitochondrial unfolded protein response (UPRmt). The UPRmt promotes a complex transcription program ultimately increasing mitochondrial integrity and fitness in response to oxidative proteotoxic stress. Using SOD2 as a surrogate marker of the UPRmt, we found that in primary breast cancers, SOD2 is significantly increased in metastatic lesions. We propose that the ability of selected mtDNA species to activate the UPRmt is a process that is exploited by cancer cells to maintain mitochondrial fitness and facilitate metastasis.

Caveolin-1 regulates cancer cell metabolism via scavenging Nrf2 and suppressing MnSOD-driven glycolysis

Aerobic glycolysis is an indispensable component of aggressive cancer cell metabolism. It also distinguishes cancer cells from most healthy cell types in the body. Particularly for this reason, targeting the metabolism to improve treatment outcomes has long been perceived as a potentially valuable strategy. In practice, however, our limited knowledge of why and how metabolic reprogramming occurs has prevented progress towards therapeutic interventions that exploit the metabolic peculiarities of tumors. We recently described that in breast cancer, MnSOD upregulation is both necessary and sufficient to activate glycolysis. Here, we focused on determining the molecular mechanisms of MnSOD upregulation. We found that Caveolin-1 (Cav-1) is a central component of this mechanism due to its suppressive effects of NF-E2-related factor 2 (Nrf2), a transcription factor upstream of MnSOD. In transformed MCF10A(Er/Src) cells, Cav-1 loss preceded the activation of Nrf2 and its induction of MnSOD expression. Consistently, with previous observations, MnSOD expression secondary to Nrf2 activation led to an increase in the glycolytic rate dependent on mtH2O2 production and the activation of AMPK. Moreover, rescue of Cav-1 expression in a breast cancer cell line (MCF7) suppressed Nrf2 and reduced MnSOD expression. Experimental data were reinforced by epidemiologic nested case-control studies showing that Cav-1 and MnSOD are inversely expressed in cases of invasive ductal carcinoma, with low Cav-1 and high MnSOD expression being associated with lower 5-year survival rates and molecular subtypes with poorest prognosis.

SOD2 and the Mitochondrial UPR: Partners Regulating Cellular Phenotypic Transitions

ATP and reactive oxygen species (ROS) are signaling molecules that control cellular function and phenotype. Mitochondria produce both ATP and ROS. Since the electrons needed to generate either ATP or ROS originate from NADH/FADH2, the mechanism through which electrons flow towards oxygen determines yields and whether ATP or ROS prevails. Alterations in the electron flow impact cells dramatically, such as by supporting specialization (which requires high ATP) or imposing dedifferentiation. High ROS, facilitated by enzymes such as superoxide dismutase 2 (SOD2) that enhance mitochondrial hydrogen peroxide (mtH2O2), are normally linked to dedifferentiation of somatic cells. Here we propose that combined high mtH2O2 and mitochondrial unfolded protein response (UPR(mt)) activation are essential for somatic dedifferentiation programs and the acquisition of stem-like properties in reparative processes and disease.

Allele-specific interaction between glutathione peroxidase 1 and manganese superoxide dismutase affects the levels of Bcl-2, Sirt3 and E-cadherin

Abstract Manganese superoxide dismutase (MnSOD) is a mitochondrial-resident enzyme that reduces superoxide to hydrogen peroxide (H2O2), which can be further reduced to water by glutathione peroxidase (GPX1). Data from human studies have indicated that common polymorphisms in both of these proteins are associated with the risk of several cancers, including breast cancer. Moreover, polymorphisms in MnSOD and GPX1 were shown to interact to increase the risk of breast cancer. To gain an understanding of the molecular mechanisms behind these observations, we engineered human MCF-7 breast cancer cells to exclusively express GPX1 and/or MnSOD alleles and investigated the consequences on the expression of several proteins associated with cancer aetiology. Little or no effect was observed on the ectopic expression of these genes on the phosphorylation of Akt, although allele-specific effects and interactions were observed for the impact on the levels of Bcl-2, E-cadherin and Sirt3. The patterns observed were not consistent with the steady-state levels of H2O2 determined in the transfected cells. These results indicate plausible contributing factors to the effects of allelic variations on cancer risk observed in human epidemiological studies.

Abstract 3062: Metformin inhibits TGFβ-induced stromal ECM remodeling to impede invasion in ovarian cancer

Recently, our group has elucidated that metformin, the commonly used medication for type 2 diabetes, has anti-ovarian cancer (OvCa) effects in preclinical models and use of the drug is associated with improved OvCa survival in retrospective epidemiological studies. While the cytotoxic effects of metformin in ovarian cancer cell lines is well demonstrated, the impact of metformin on tumor-stromal interactions in the tumor microenvironment is not well defined. Currently, our work has focused on the impact of metformin on the tumor microenvironment and cancer cell behavior in physiologic models of tumor metastasis. Using a 3D organotypic model, we demonstrate that OvCa cell invasion is inhibited by metformin. In evaluating the molecular mechanism mediating this effect we show that OvCa cells co-cultured with stromal cells or stimulated with TGFβ1 upregulate the production of the extracellular matrix (ECM) proteins fibronectin and collagen 1 and that this effect is attenuated by metformin. Interestingly, metformin’s inhibition of ECM production was not dependent on AMP-activated kinase (AMPK), a common metformin target. To expand on this finding we performed label-free shotgun proteomics and identified collagens, fibronectin and TGFBR2 to be among the proteins most highly repressed in response to metformin exposure in OvCa cells. Finally, we identified that this repression of ECM remodeling by metformin was associated with an overall reduction in TGFBR2 expression and consequent SMAD/STAT3 activity. Taken together, our data indicate that metformin may have novel targets in the tumor microenvironment where it targets pro-tumorigenic effects of the stromal compartment by repressing TGFβ-dependent stromal cell ECM secretion, thereby inhibiting ovarian cancer invasion and metastasis. Citation Format: Peter C. Hart, Shermeen Sheikh, Ernst Lengyel, Iris Romero. Metformin inhibits TGFβ-induced stromal ECM remodeling to impede invasion in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3062. doi:10.1158/1538-7445.AM2017-3062

Type 2 Diabetes Mellitus as a Risk Factor for Alzheimer's Disease

Abstract Type 2 diabetes mellitus (T2DM) is an acknowledged risk factor for the development of Alzheimer’s disease (AD). However, molecular mechanisms linking these disorders remain poorly defined. This chapter will review recent advancements in the understanding of the etiologic causes of AD occurring in a subgroup of diabetic patients based on studies using a variety of animal models of insulin resistance, and T2DM. We will also focus on vascular deterioration and defective insulin signaling in the brain as primary causes of molecular, vascular, and neuronal changes that translate into cognitive decline, memory loss, and other features characteristic of AD.

Abstract 225: Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins

MnSOD detoxifies superoxide and impacts tumor biology by generating H 2 O 2 which can diffuse through the mitochondrial membrane and affect metabolism and apoptosis. The selenium-dependent enzyme GPx-1 localizes to both the cytoplasm and the mitochondria and reduces H 2 O 2 to water and may therefore modulate the impact of MnSOD on carcinogenesis. A val to ala polymorphism in codon 16 of the MnSOD gene has been shown to be associated with increased prostate cancer risk in men with the lowest level of dietary antioxidant intake and individuals who consumed less dietary antioxidants, including selenium, had the greatest risk of prostate cancer (Li et al, 2005). A polymorphism in the GPx-1 gene resulting in a leucine (L) instead of a proline (P) at position 198 has frequently been reported to be associated with elevated cancer risk. To examine the molecular mechanism behind the epidemiological observation that genotypes in GPx-1 gene modify elevated risk of cancer associated with MnSOD genotypes, MCF-7 human breast cancer cells null for GPx-1 and having negligible levels of endogenous MnSOD were transfected with GPx-1 and MnSOD allele-specific expression constructs to determine outcomes related to cellular signaling and metabolism. MnSOD and GPx-1 alleles differentially interacted to modulate the expression of the anti-oxidant stress response regulator Nrf2, the cell adhesion protein E-Cadherin, the cell signaling protein pAkt, the anti-apoptotic protein Bcl-2 and the mitochondria located deacetylase Sirt3. Also, co-expression of the GPx-1 A7L and either MnSOD val or MnSOD ala increased oxidative phosphorylation as measured by O 2 uptake using the Seahorse XF24 XF analyzer, which simultaneously determines relative mitochondrial respiration and glycolysis. In order to assess whether GPx-1 and MnSOD allelic variants modulate mitochondrial membrane potential, CMX-ROS fluorescence was measured. Independent and irrespective of which allele was evaluated, MnSOD and GPx-1 individually decreased mitochondrial potential as compared to that seen using MCF-7 control cells. In addition, co-expression of GPx-1 A7L with either MnSOD val or MnSOD ala further decreased membrane potential above that observed for either MnSOD and GPx-1 alone. In order to determine if MnSOD and GPx-1 levels are associated with a higher risk for biochemical recurrence of prostate cancer after radical prostatectomy, immunohistochemistry of human prostate tissue cores will be performed. Preliminary results indicate that a high MnSOD/ GPx-1 ratio was observed in prostate cancer tissue compared to adjacent normal tissue. Citation Format: Dede N. Ekoue, Soumen Bera, Emmanuel Ansong, Peter Hart, Virgilia Macias, Andre Kajdacsy-Balla, Marcelo Bonini, Alan M. Diamond. Allelic variations in MnSOD and GPx-1 affect metabolism, mitochondrial membrane potential and expression of signaling proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 225.