Phenotypic rescue of a Drosophila model of mitochondrial ANT1 disease.

A point mutation in the Drosophila gene that codes for the major adult isoform of adenine nuclear translocase (ANT) represents a model for human diseases that are associated with ANT insufficiency [stress-sensitive B(1) (sesB(1))]. We characterized the organismal, bioenergetic and molecular phenotype of sesB(1) flies then tested strategies to compensate the mutant phenotype. In addition to developmental delay and mechanical-stress-induced seizures, sesB(1) flies have an impaired response to sound, defective male courtship, female sterility and curtailed lifespan. These phenotypes, excluding the latter two, are shared with the mitoribosomal protein S12 mutant, tko(25t). Mitochondria from sesB(1) adults showed a decreased respiratory control ratio and downregulation of cytochrome oxidase. sesB(1) adults exhibited ATP depletion, lactate accumulation and changes in gene expression that were consistent with a metabolic shift towards glycolysis, characterized by activation of lactate dehydrogenase and anaplerotic pathways. Females also showed downregulation of many genes that are required for oogenesis, and their eggs, although fertilized, failed to develop to the larval stages. The sesB(1) phenotypes of developmental delay and mechanical-stress-induced seizures were alleviated by an altered mitochondrial DNA background. Female sterility was substantially rescued by somatic expression of alternative oxidase (AOX) from the sea squirt Ciona intestinalis, whereas AOX did not alleviate developmental delay. Our findings illustrate the potential of different therapeutic strategies for ANT-linked diseases, based on alleviating metabolic stress.

Tumor hypoxia as a driving force in genetic instability.

Sub-regions of hypoxia exist within all tumors and the presence of intratumoral hypoxia has an adverse impact on patient prognosis. Tumor hypoxia can increase metastatic capacity and lead to resistance to chemotherapy and radiotherapy. Hypoxia also leads to altered transcription and translation of a number of DNA damage response and repair genes. This can lead to inhibition of recombination-mediated repair of DNA double-strand breaks. Hypoxia can also increase the rate of mutation. Therefore, tumor cell adaptation to the hypoxic microenvironment can drive genetic instability and malignant progression. In this review, we focus on hypoxia-mediated genetic instability in the context of aberrant DNA damage signaling and DNA repair. Additionally, we discuss potential therapeutic approaches to specifically target repair-deficient hypoxic tumor cells.

PTEN deletion in prostate cancer cells does not associate with loss of RAD51 function: implications for radiotherapy and chemotherapy.

PURPOSE: PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported. EXPERIMENTAL DESIGN: Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN(+/+), DU145-PTEN(+/-), PC3-PTEN(-/-)) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression. RESULTS: PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi among prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H(2)O(2), and methyl methanesulfonate but not to cisplatin, camptothecin, or paclitaxel. CONCLUSIONS: These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials.

The receptor tyrosine kinase inhibitor amuvatinib (MP470) sensitizes tumor cells to radio- and chemo-therapies in part by inhibiting homologous recombination.

BACKGROUND AND PURPOSE: RAD51 is a key protein involved in homologous recombination (HR) and a potential target for radiation- and chemotherapies. Amuvatinib (formerly known as MP470) is a novel receptor tyrosine kinase inhibitor that targets c-KIT and PDGFRα and can sensitize tumor cells to ionizing radiation (IR). Here, we studied amuvatinib mechanism on RAD51 and functional HR. MATERIALS AND METHODS: Protein and RNA analyses, direct repeat green fluorescent protein (DR-GFP) assay and polysomal fractioning were used to measure HR efficiency and global translation in amuvatinib-treated H1299 lung carcinoma cells. Synergy of amuvatinib with IR or mitomycin c (MMC) was assessed by clonogenic survival assay. RESULTS: Amuvaninib inhibited RAD51 protein expression and HR. This was associated with reduced ribosomal protein S6 phosphorylation and inhibition of global translation. Amuvatinib sensitized cells to IR and MMC, agents that are selectively toxic to HR-deficient cells. CONCLUSIONS: Amuvatinib is a promising agent that may be used to decrease tumor cell resistance. Our work suggests that this is associated with decreased RAD51 expression and function and supports the further study of amuvatinib in combination with chemotherapy and radiotherapy.

Tumor cell kill by c-MYC depletion: role of MYC-regulated genes that control DNA double-strand break repair.

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.

Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment.

Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.