Keshav K. Singh - US grants

Human mitochondrial DNA (mitDNA) is extremely vulnerable to attack by environmental carcinogens because it contains no introns, has no protective histones and is continuously exposed to reactive free radicals produced within the mitochondria. In the past decade it has been clearly recognized that mutations in mitDNA lead to pathogenesis of a variety of mitochondrial diseases. It is estimated that of the 4 million children born each year in the United States, up to 4,000 develop mitochondrial disease. However, there is very little information available on the effects of environmental carcinogens, and the molecular mechanisms of mitDNA mutagenesis important in the pathology of mitochondrial diseases. Many environmental carcinogens deaminate bases in DNA. Of these bases, deamination of cytosine to uracil is of particular importance because human mitDNA is rich in cytosine and uracil mispairs with guanine in DNA, thus causing cytosine to thymine mutations during replication. Deamination, therefore, can be an important mechanism for generating mutations in mitDNA. In fact, cytosine to thymine mutations in certain mitochondrial genes cause mitochondrial myopathy, fatal infantile cardiomyopathy minus, progressive external ophthalmoplegia and other multisystem disorders. A key enzyme of the base excision repair pathway, uracil-DNA glycosylase (UDG) removes uracil from nuclear DNA and protects cells from cytosine to thymine mutations in the nucleus. Interestingly, UDG is present in mitochondria as well. However, UDG's exact biological function in mitochondria is unknown. Furthermore, nothing is known about the impact of deamination on mitDNA and on the induction of mitochondrial disease. Our previous work has established first, that human UDG physically associates, both in vivo and in vitro, with a multifunctional nuclear protein, replication protein A (RPA), and second, the aminoterminal sequence that targets UDG to mitochondria is required for its interaction with RPA. These data suggest that UDG's interaction with RPA may be important for repairing damaged mitDNA. Based upon our results, we hypothesize that mitDNA of human cells are susceptible to mutations by deaminating carcinogens, and that UDG protects mitDNA from mutation induced by these carcinogens. To test this hypothesis, we will: 1. Determine whether deaminating carcinogens cause mutations in mitDNA and subsequently lead to mitochondrial dysfunction in human cells. 2. Determine the ability of a major DNA repair protein, UDG, to protect human mitDNA from deamination-induced mutations. 3. Characterize the nature of UDG-RPA interaction in repairing damaged human mitDNA. The proposed study will fill a major gap in our understanding of the mechanisms of mitDNA mutagenesis induced by environmental carcinogens which has direct relevance in on the induction of mitochondrial diseases.

[unreadable] DESCRIPTION (provided by applicant): A century of scientific research has established that mitochondria dysfunction is one of the most prevalent and profound phenotypes of human cancer cells. At the genetic level, the differences between normal and tumor cells include both the depletion of mitochondrial DNA (mtDNA) and somatic mtDNA mutations in all human cancers examined to date. The rate of mutation in mtDNA appears to be 20 times higher than that in nuclear DNA. This high rate of mutation is due to the high concentration of reactive oxygen species (ROS) produced by the mitochondria, limited DNA repair and lack of DNA protective histones in the mitochondria. Until now, studies have focused on the identification and characterization of mutations in mtDNA, with little insight into the contribution of these mutations to the etiology of cancer. To understand the contribution of mtDNA in etiology of cancer, we have generated the complete mtDNA- knockout ( rho(o)) in breast epithelial cells. We demonstrate that mtDNA knockout in epithelial cells leads to neoplastic transformation that shows increased invasiveness and anchorage-independent growth. Interestingly, these properties of (rho(o) epithelial cells are restored by reintroduction of mtDNA (transmitochondrial cybrid) suggesting a role for mtDNA in the induction of the cancer phenotype. We also demonstrate that mitochondrial gene knockout leads to i) gross chromosomal rearrangements (GCR) 2) resistance to apoptosis 3) altered expression of ROS generating NADPH oxidase (Nox1) involved in tumorigenesis. Based on these observations we hypothesize that mutant mtDNA contributes to the etiology of cancer. MtDNA knockout cells established in our laboratory will serve as a novel tool for transfer of mutant mtDNA in a uniform genetic background. The mtDNA mutations could contribute to neoplastic transformation of epithelial cells by increasing oxidative stress, modulating cell proliferation, modulating apoptosis, affecting the expression of ROS producing Noxi protein and increasing nuclear genome instability. To test this hypothesis we plan to: i) Construct a mtDNA mutator system and characterize those mutants that are found in primary breast tumors. 2) Generate isogenic mutant mtDNA cybrids, and determine the consequences of mutations on mitochondrial function. 3) Using isogenic transmitochondrial cybrids determine the consequences of mtDNA mutations on cell proliferation, apoptosis and nuclear genome instability. 4) Using isogenic transmitochondrial cybrids determine the importance of superoxide generating Nox1 in tumorigenesis. 5) Using isogenic mutant mtDNA cybrids assess the significance of mtDNA mutations on in vitro and in vivo tumorigenesis and metastasis in severe combined immunodeficient (SCID) mice. The proposed study will help determine the contribution of mutant mtDNA to the development of human cancer. [unreadable] [unreadable] [unreadable] [unreadable]

Mitochondrial dysfunction is one of the hallmarks of cancer. However, very little is known about how mitochondrial dysfunction leads to carcinogenesis. Our studies described in this proposal suggest a novel role for mitochondria in protecting cells from nuclear genome mutagenesis, an important event in human carcinogenesis. Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine resistant colonies as an indicator of nuclear genome mutagenesis. Our data demonstrate that mitochondrial dysfunction leads to mutation in the nuclear genome (i) in mutant strains lacking the entire mitochondrial genome (rho[unreadable] or p[unreadable]) or those with deleted mitochondrial DNA (rho" or p") (mitochondrial genetic dysfunction, MGD) and (ii) when oxidative phosphorylation is blocked in wild type yeast by antimycin A (mitochondrial metabolic dysfunction, MMD). The nuclear mutation frequencies in both MMD and MGD cells were higher compared to untreated control and wild type cells respectively. MGD led to decreased intracellular levels of ROS. In contrast MMD led to increased intracellular levels of reactive oxygen species (ROS). We demonstrate that nuclear genome mutagenesis due to MGD is dependent on REV1, REVS or REV7 gene products, all implicated in post-replication repair (PRR). However, nuclear genome mutagenesis due to MMD does not involve REV1, REVS or REV7 genes. Furthermore, we provide evidence that Rtg2 protein (retrograde 2) involved in mitochondria-to-nucleus retrograde response pathway protect cells from nuclear genome mutagenesis. Based on these observations we hypothesize that mitochondria protect and employ multiple pathways to guard the nuclear genome against mutagenesis. We propose 4 specific aims to test the proposed hypothesis. In all cases we have preliminary data that provide the basis for the proposed specific aims. These specific aims are:1) Establish the role of mitochondria-to-nucleus retrograde response pathway in nuclear genome mutagenesis due to mitochondrial dysfunction 2) Determine whether compromised oxidative stress response leads to nuclear genome mutagenesis due to mitochondrial dysfunction 3) Determine the role of post-replication repair pathway in nuclear genome mutagenesis due to mitochondrial dysfunction 4) Determine the nature of nuclear genome mutagenesis due to mitochondrial dysfunction Our proposed study should provide insight into the molecular genetic mechanisms of nuclear genome mutagenesis due to mitochondrial dysfunction that is of fundamental significance to human cancer and other diseases.

DESCRIPTION (provided by applicant): Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. We showed the presence of myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia (Genes Chromosome Cancer 25:222-229, 1999). We established a new cell line from one of the lymphoid cases, MUTZ5, that carries a single t(12;13) translocation (Leukemia 15:1471-1474, 2001). The molecular characterization of this translocation allowed us to identify a new gene called FLJ13639 that is disrupted and lost in the MUTZ5 cell line. This gene shares homologies with the large family of short-chain dehydrogenase reductase (SDR). This new protein localizes in the mitochondria. One of the consequences of the loss of FLJ13639 is the over-expression of CD24. CD24 over-expression has been linked with hypoxic conditions and poor prognostic in acute leukemia as well as chemoresistance. We therefore hypothesize that loss of the FLJ13639 expression leads to altered mitochondrial function as well as increase in CD24 expression that provides leukemia cells with a proliferation and invasiveness advantage as well as a certain degree of resistance to chemotherapy. The addition of exogenous FLJ13639 recombinant protein would lead to an increase in chemosensitivity and reduce chemoprotection provided to leukemia cells by altered mitochondrial function and over-expression of CD24, among other markers. Our three hypothesis-driven Specific aims will be as follows: Specific Aim 1: Hypothesis: The FLJ13639 gene is down regulated in acute leukemia by both genetic and epigenetic events;Specific Aim 2: Hypothesis: The FLJ13639-P1 protein has a dehydrogenase activity and is involved in electron transport in mitochondria and Specific Aim 3: Hypothesis: Delivery of FLJ13639 protein induces apoptosis in acute leukemia cells. Confirmation of the role of the loss of FLJ13639 in leukemia prognosis, increase of chemo resistance should lead us and others, to develop new prognostic tools as well as novel, innovative therapeutic strategies.

DESCRIPTION (provided by applicant): Mitochondria perform multiple cellular functions. These functions include ATP production via oxidative phosphorylation (OXPHOS), control of cell growth, cell death, development and various metabolic pathways. OXPHOS is coupled through a series of oxido-reduction reactions catalyzed by five mitochondrial OXPHOS complexes (Complex I, II, III, IV and V). Of the 86 subunits that make up the mitochondrial OXPHOS system, mitochondrial DNA (mtDNA) encodes 13 subunits. The rest are encoded by nuclear DNA (nDNA). Interestingly, studies in the past few years have described the risk of various cancers associated with polymorphism in mitochondrial OXPHOS genes. Studies show the 10398 G to A (G10398A) base substitution in the mtDNA-encoded ND3 gene (NADH dehydrogenase 3) subunit of complex I increases the risk of invasive breast cancer in African-American women (Canter et al 2005).The G to A substitution results in an amino acid change from alanine (encoded by the G allele) to threonine (encoded by the A allele). This G10398A substitution is found in 5% of the African-American population (Canter et al 2005). To understand the contribution of ND3 G10398A gene substitution associated with invasive breast cancer in African-American women, we generated a cell line completely devoid of mtDNA (mtDNA- knockout or rhoo or ?0). We used this rhoo cell line to develop a cybrid cell culture model for introducing the G10398A mtDNA variant into the isogenic nuclear background (trans-mitochondrial cybrid). We demonstrate that the G10398A variant confers increased complex I activity, resistance against cell death, and increased tumorigenicity. Based on these observations, we hypothesize that ND3 G10398A affects expression of nDNA encoded subunits of complex I, which contributes to increased complex I activity, resistance against cell death and tumorigenicity. To address the above hypothesis, we will: Aim 1: Determine G10398A-induced changes in subunit gene expression and composition of complex I. Mitochondrial complex I represents the largest and the least understood multimeric OXPHOS complex. It is composed of 45 subunits. Of these, 38 subunits are encoded by nDNA and 7 (including ND3) are encoded by mtDNA. To investigate whether increased complex I activity associated with G10398A (Kulawiec et al. 2009A) is due to increased expression of subunits, we will use a focused OXPHOS array developed in our laboratory to measure changes in the gene expression of all 45 subunits. To investigate whether alterations in complex I gene expression affect complex I composition, we will use blue native PAGE, which allows for measurement of the amounts of the respiratory complexes in their native state. Aim 2: Determine a role for NDUFA13/GRIM19 (an essential complex I regulatory subunit) in resistance to cell death and tumorigenesis. Of the 45 subunits that make up complex I, NDUFA13/GRIM19 (the gene associated with retinoid interferon induced mortality) acts as a death regulator protein (Zhang et al 2003, Lufei et al 2003) and is involved in tumorigenesis (Maximo et al 2008, Nallar et al 2008). NDUFA13/GRIM19 is also an essential component of complex I. It plays an indispensable role in the assembly and enzymatic activity of complex I (Huang et al 2004). Our study suggests that G10398A confers resistance against cell death and induces tumorigenesis (Kulawiec et al 2009). We will therefore determine whether the G10398A-associated resistance against cell death and tumorigenesis is due to changes in gene expression and/or posttranscriptional modification of GRIM19/NDUFA13. Our grant proposal addresses the two key objectives of the program announcement PAR-09-160 requesting exploratory/development grants program in basic cancer research in cancer health disparities (R21). These objectives include: 1) the development of "new cell culture models/systems designed to investigate cancer disparities" and 2) explores the susceptibility to breast cancer in African-American women due to "genetic differences" in mtDNA. The G10398A transmitochondrial cybrid system developed in our laboratory has the potential to enhance breast cancer research in the African-American population. PUBLIC HEALTH RELEVANCE: In the last few years, several epidemiological studies have reported the increased risk of breast (and other) cancer associated with mtDNA polymorphisms in the African-American population. However, a mechanistic understanding of how certain mtDNA polymorphisms induce cancer is lacking. The proposed study will help determine the mechanism underlying G10398A induced tumorigenicity in African-American women. Innovative aspects of this project include the development of a G10398A transmitochondrial cybrid cell line. This cell line will serve as an important tool for the proposed studies. It will permit the mechanistic analysis of African-American polymorphic mtDNA in an isogenic nuclear background. The proposed cybrid model will pave the way to analyze the relevance of other mtDNA polymorphisms and epistatic interaction (i.e. synergy) between individual polymorphic loci within the mitochondrial genome in the African-American population. .

DESCRIPTION (provided by applicant): Estrogen receptor-1 (ER) and tumor suppressor p53 play important, but opposite, roles in the onset and progression of breast cancer. Compared to other cancers, overall frequency of p53 mutation in breast cancer is about 20%; however, wild type p53 is functionally debilitated. Both ER and p53 are localized in the nuclei as well as mitochondria and are functionally important in both the compartments. ER has been reported to bind and inhibit wild type p53 function in the nucleus. There is a fundamental gap in understanding how mitochondrial p53 is antagonized by ER. This gap represents an important problem because deciphering the role of ER in suppressing mitochondrial p53 is essential in understanding the mechanisms by which wild type p53 is inactivated in breast cancer. The long-term goal is to understand the mechanisms by which nuclear and mitochondrial functions of p53 are compromised in breast cancer. The objective in this particular application is to analyze mitochondrial ER-p53 interaction and its functional consequences. The central hypothesis is that ER and p53 interact within mitochondria leading to important functional consequences. The rationale that underlies the proposed research is that given the importance of mitochondrial p53 in responding to oncogenic signaling and regulation of cellular metabolism, understanding the interaction between mitochondrial ER and p53 and its functional consequences would provide new therapeutic targets in addition to mechanistic insights that could be exploited for better intervention strategies. This hypothesis wil be tested by pursuing three specific aims: 1) Analyze ER-p53 interaction in mitochondria in breast cancer cells; 2) Investigate effect of ER- p53 interaction on oxidative phosphorylation (OXPHOS) in mitochondria; and 3) Determine the effect of ER- p53 interaction on mitochondrial gene transcription and apoptosis. Under the first aim, already proven immunoprecipitation (IP) assay, RNA interference (RNAi) approach, and gene transfection approaches, which have been established in the applicants' laboratories, will be used to characterize ER-p53 interaction within mitochondria. Under the second specific aim, effect of ER-p53 interaction on OXPHOS will be analyzed using approaches such as respiratory complex assays and quantitative real-time PCR (qPCR) assay that are established in the applicants' laboratories along with a OXPHOS-Chip expression array already developed and validated by the applicants. The third aim will address, using multiple technical approaches already optimized by the applicants, how ER-p53 interaction affect mitochondrial gene transcription and apoptosis. The research is innovative because it represents a new and substantive departure from the status quo, namely analyzing ER and p53 function in breast cancer cell mitochondria in an integrated manner instead of pursuing them as independent regulators of separate pathways. The proposal is significant because it is the first step in a continuum of research that is expected to lead to a better understanding of the roles of mitochondrial ER and p53 in breast cancer that could be exploited for developing new therapeutic strategies.

DESCRIPTION (provided by applicant): Arsenic is a well-known human carcinogen. Previous studies including human population studies provide an extensive and important link between the arsenic exposure and development of breast cancer. These studies suggest that arsenic accumulates in breast tissues and acts as an endocrine disruptor to promote development of breast cancer. Arsenic is one of the few human carcinogens that do not induce tumors in laboratory animals. Therefore, development of models for arsenic-induced breast cancer is critical for understanding the mechanism(s) underlying the tumorigenic process. We have developed a mammary epithelial cell model for arsenic-induced cancer. To replicate normal field exposure conditions, we exposed mammary epithelial cells to a low dose of arsenic for several months. We discovered that a five month continuous exposure of mammary epithelial cells results in increased cell proliferation, increased wound healing, increased anchorage independent growth, as well as increased matrigel invasion. These studies suggest a tumorigenic transformation of mammary epithelial cells by exposure to arsenic. Mitochondria control cell growth and cell death. Mitochondria also perform other cellular functions including ATP production via mitochondrial oxidative phosphorylation (mtOXPHOS). Consistent with this finding arsenic-transformed cells show 1) altered mtOXPHOS Complex I and IV activities; 2) an altered expression of subunit NDUFB8 comprising mtOXPHOS Complex I; and 3) altered expression of COXII subunit comprising mtOXPHOS complex IV. Interestingly, our study suggests that arsenic-treatment did not induce changes in mtOXPHOS Complex II and III activities. These preliminary studies revealed that arsenic targets mitochondria and induces mitochondrial stress. Recent studies suggest that human cells contain mitochondria specific stress response pathway in which transcription factor GADD153 (also known as CHOP or DDIT3) plays a key role. We measured the expression of GADD153 and found that arsenic represses GADD153 expression. GADD153 is described to play a critical role in cell death and suppression of GADD153 expression is known to protect cells from cell death. However, GADD153's role in arsenic induced carcinogenesis is unknown. We hypothesize that arsenic represses expression of GADD153/CHOP/DDIT3 to protect cells from arsenic induced cell death which contributes to tumorigenic transformation of mammary epithelial cells induced by arsenic. To address this hypothesis we will: Aim 1: Determine a role for GADD153 in protection against cell death and mitochondrial stress induced by arsenic. Aim 2: Determine whether arsenic repression of GADD153 expression contributes to tumorigenic transformation of breast epithelial cells in vitro and in vivo in mouse xenograft model. The proposed studies should provide insight in to the mechanism involved in arsenic induced breast tumorigenesis.

DESCRIPTION (provided by applicant): Mitochondria play a central role in cell proliferation, cell signaling and apoptosis. Mitochondria contain their own DNA (mtDNA). mtDNA serves as a determinant of human origin and ethnicity. Human mtDNA is a very small (16,569 bp) genome. The entire protein coding capacity of the mitochondrial genome is dedicated to the production of 13 protein subunits. These 13 proteins constitute various subunits that make up four oxidative phosphorylation (OXPHOS) complexes essential for energy (ATP) production and other mitochondrial function. African-American men (AAM) have the highest rate of prostate cancer, develop prostate cancer at an early age, present with a higher tumor grade at time of diagnosis, and have a higher rate of metastasis and mortality than Caucasian American men (CAM). The genetic mechanism(s) underlying this racial diversity in prostate cancer is not well understood. Our studies demonstrate that in AAM reduced mtDNA content plays an important role in prostate carcinogenesis. We measured the mtDNA copy number in white blood cells (WBC) and prostate tumor obtained from AAM and CAM. Our preliminary studies suggest that WBC of both AAM and CAM contained similar amount of mtDNA (copy number). However, when adjusted for age, Gleason grade and PSA (prostate specific antigen) the prostate tumors of AAM contained >6 times less mtDNA than CAM tumors. Our preliminary data suggest that mutations in nuclear gene encoding mitochondrial DNA polymerase gamma (POLG1) induce depletion of mtDNA and increase tumorigenic potential of cancer cells in vitro. These studies suggest that mtDNA homeostasis must play an important role in carcinogenesis. Based on these observations we hypothesize that reduced mtDNA content plays a critical role in prostate tumorigenesis in AAM by significantly altering the mitochondrial OXPHOS. AIM 1: Determine whether mutational spectrum of POLG1 in AAM prostate tumors differs from POLG1 mutational spectrum of CAM. Identify distinctive mutations in POLG1 gene in primary prostate tumors of AAM and determine its functional significance on mtDNA depletion, OXPHOS activity and apoptosis. AIM 2: Determine the consequences of mutant POLG1 induced depletion of mtDNA on prostate tumorigenesis in vitro and in mouse xenograft model. The proposed development of mtDNA depletion in prostate cells has the significant potential to enhance our understanding of prostate tumorigenesis in the African American population. This proposal addresses the two key objectives of the PAR-09-160 requesting exploratory grants in basic cancer research in cancer health disparities (R21). These objectives include: 1) the development of new cell culture models/systems designed to investigate cancer disparities and 2) explores the susceptibility to prostate cancer in African-American men due to genetic differences in mtDNA content.

? DESCRIPTION (provided by applicant): The androgen receptor (AR) plays an important role in normal development of the prostate gland, in prostate carcinogenesis, and in the progression of prostate cancer to advanced metastatic disease. Traditional thinking is that AR localizes exclusively to the nucleus and that nuclear AR regulates genes that are essential to prostate cancer development. This is true. However, we demonstrate a previously unrecognized function of AR in mitochondria. We have discovered that AR 1) directly localizes into the mitochondria and 2) indirectly transcriptionally regulates nuclear genes whose products localize into mitochondria and perform mitochondrial functions. Our studies reveal that i) AR localizes into mitochondria in primary prostate tissues and cell lines, ii) AR is imported into isolated mitochondria, and iii) AR contains a mitochondrial localization signal (MLS) capable of targeting foreign proteins, such as green fluorescent protein, into mitochondria. Indirectly, AR controls expression of a variety of nuclear DNA (nDNA)-encoded mitochondrial oxidative phosphorylation (mtOXPHOS) subunits, including NDUFB8 (Complex I), SDHB (Complex II), UQCRC2 (Complex III), COXII subunit (Complex IV), and ATP5A (Complex V). AR also down-regulates the TFAM, GFM1, and GFM2 genes, which control mitochondrial DNA (mtDNA) content. Consistent with this, the mtDNA content and the expression of mtDNA-encoded COX II protein is significantly reduced in PC3-AR prostate cells expressing AR. Notably, we demonstrate that the mtDNA content in prostate tumors of African-Americans (AA) is >6 times less than in tumors of Caucasian-Americans (CA). mtDNA content was also lower in normal prostates of AA than CA. To identify the underlying mitochondrial basis of prostate cancer diversity, we conducted comprehensive, race-based bioinformatics analyses of variants in more than 6000 AA and 33,000 CA and discovered, in the AR gene of AA, missense variants located in two domains: the N-terminal domain containing the MLS and the DNA-binding domain. Of note, missense mutations in CA were found only in the hinge domain containing the nuclear localization signal (NLS) of AR. Expression of AR variant S598G in PC3 cells reduced more than the wild type the expression of TFAM, which controls mtDNA content. We hypothesize that AR missense variants/mutants present, solely in AA, contribute to the gain or loss of mitochondrial function and thereby to prostate cancer diversity in AA. AIM 1: Determine the prognostic significance of AR missense variants/mutants and mtDNA content on prostate cancer metastasis and reoccurrence in AA and CA. AIM 2: Evaluate the significance of mitochondrial AR missense variants/mutants as direct regulators of mitochondrial functions that affect composition, organization, stability, and activity of mtOXPHOS super-complexes and apoptosis. AIM 3: Evaluate the significance of nuclear AR missense variants/mutants as indirect regulators of mitochondrial function. AIM 4: Use mouse xenograft model to establish the significance of mitochondrial and nuclear AA- and CA-specific AR missense variants/mutants on prostate tumorigenesis and metastasis.