Impaired OXPHOS Complex III in Breast Cancer

We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis

Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin

The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho0) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho0 derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho0 breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2

Mitochondrial DNA Polymerase POLG1 Disease Mutations and Germline Variants Promote Tumorigenic Properties.

Germline mutations in mitochondrial DNA polymerase gamma (POLG1) induce mitochondrial DNA (mtDNA) mutations, depletion, and decrease oxidative phosphorylation. Earlier, we identified somatic mutations in POLG1 and the contribution of these mutations in human cancer. However, a role for germline variations in POLG1 in human cancers is unknown. In this study, we examined a role for disease associated germline variants of POLG1, POLG1 gene expression, copy number variation and regulation in human cancers. We analyzed the mutations, expression and copy number variation in POLG1 in several cancer databases and validated the analyses in primary breast tumors and breast cancer cell lines. We discovered 5-aza-2'-deoxycytidine led epigenetic regulation of POLG1, mtDNA-encoded genes and increased mitochondrial respiration. We conducted comprehensive race based bioinformatics analyses of POLG1 gene in more than 33,000 European-Americans and 5,000 African-Americans. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 protein at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans (allele frequency 0.03777) when compared to African-American (allele frequency 0.00151) population. We identified T251I and P587L missense variations in exonuclease and linker region of POLG1 also to be more prevalent in European-Americans. Expression of these variants increased glucose consumption, decreased ATP production and increased matrigel invasion. Interestingly, conditional expression of these variants revealed that matrigel invasion properties conferred by these germline variants were reversible suggesting a role of epigenetic regulators. Indeed, we identified a set of miRNA whose expression was reversible after variant expression was turned off. Together, our studies demonstrate altered genetic and epigenetic regulation of POLG1 in human cancers and suggest a role for POLG1 germline variants in promoting tumorigenic properties

Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome

<div><p>To date, mitochondrial DNA polymerase γ (POLG) is the only polymerase known to be present in mammalian mitochondria. A dogma in the mitochondria field is that there is no other polymerase present in the mitochondria of mammalian cells. Here we demonstrate localization of REV3 DNA polymerase in the mammalian mitochondria. We demonstrate localization of REV3 in the mitochondria of mammalian tissue as well as cell lines. REV3 associates with POLG and mitochondrial DNA and protects the mitochondrial genome from DNA damage. Inactivation of <i>Rev3</i> leads to reduced mitochondrial membrane potential, reduced OXPHOS activity, and increased glucose consumption. Conversely, inhibition of the OXPHOS increases expression of <i>Rev3</i>. <i>Rev3</i> expression is increased in human primary breast tumors and breast cancer cell lines. Inactivation of <i>Rev3</i> decreases cell migration and invasion, and localization of <i>Rev3</i> in mitochondria increases survival and the invasive potential of cancer cells. Taken together, we demonstrate that REV3 functions in mammalian mitochondria and that mitochondrial REV3 is associated with the tumorigenic potential of cells.</p></div

Lack of UQCRFS1/RISP expression in ρ⁰ cells.

<p><b>A.</b> Western blot analysis of RISP expression in 143B and 143B ρ⁰ osteosarcoma cells. Tubulin served as a loading control. <b>B.</b> RISP knock-down in 143B cells transduced with either pSiren (control) or shRNA directed against RISP (clone #2). <b>C.</b> Resistance to apoptosis was measured by analyzing SubG1 fraction by DNA staining with propidium iodide. <b>D</b>. <i>In vitro</i> invasion was measured by a Matrigel invasion assay in RISP knockdown cells. ** <i>p</i><0.005 *** <i>p</i><0.0005.</p

UQCRFS1/RISP expression in primary breast tumors.

<p><b>A.</b> Representative images of IHC performed on a breast tissue array (I. Benign breast tissue incubated with secondary antibody used as a negative control; II. Benign breast tissue with no carcinoma; III. Ductal carcinoma in situ (DCIS); IV. Breast invasive ductal carcinoma (IDC); V. Invasive lobular carcinoma (ILC); VI. Metastatic breast adenocarcinoma in lymph nodes (LNM)). IHC was done with an anti-RISP antibody and visualized using DAB with hematoxylin counterstain. <b>B.</b> Graph representing semi-quantitative scoring of immunoreactivity for RISP expression in benign breast, ductal carcinoma in situ (DCIS), invasive carcinomas and metastatic breast carcinoma in lymph nodes (LN metastasis). IHC analysis was done on tissue array containing 54 breast tissue cores with anti-RISP antibody. Note high expression (score +++) of RISP protein in DCIS, invasive primary tumor, and metastatic breast carcinoma compared to benign breast tissue. RISP protein was visualized using DAB with hematoxylin counterstain.</p

Primers for PCR of Complex III subunits.

<p>Primers for PCR of Complex III subunits.</p

UQCRFS1/RISP knock-down decreases expression of NADPH oxidase (NOX) genes.

<p>RT-PCR analyses of NOX genes in RISP knockdown cells. Actin served as a loading control.</p

Altered Complex III gene expression in matched normal and tumor human breast tumors.

<p><b>A. </b><i>UQCRFS1</i>-Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1; <i>UQCRH</i>-Ubiquinol-cytochrome c reductase hinge protein; <i>CYC1</i>-Cytochrome c-1; <i>UCRC</i>-Ubiquinol-cytochrome c reductase subunit X.; <i>UQCRQ</i>-Ubiquinol-cytochrome c reductase subunit VII; <i>UQCR</i>-Ubiquinol-cytochrome c reductase subunit XI; <i>CYTB</i>-Cytochrome b; <i>UQCRB</i>-Ubiquinol-cytochrome c reductase binding protein; <i>UQCRC1</i>-Ubiquinol-cytochrome c reductase core protein I; <i>UQCRC2</i>-Ubiquinol-cytochrome c reductase core protein II. Actin serves as a loading control. Subunits labeled in green are located in the intermembrane space, subunits labeled in red are transmemebrane, and subunits labeled in blue are located in the matrix. <b>B.</b> Coregulation of <i>UQCRFS1</i> and <i>UQCRH</i> expression in matched normal and tumor breast tissue. <b>C.</b> Quantitative RT-PCR analyses of UQCRFS1 expression in matched normal and tumor breast tissue.</p