BACKGROUND Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas. METHODS We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi. RESULTS We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway. CONCLUSIONS Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.)

Genetic instability is a hallmark of cancer; the hypoxic tumor microenvironment has been implicated as a cause of this phenomenon. MicroRNAs (miR) are small nonprotein coding RNAs that can regulate various cellular pathways. We report here that two miRs, miR-210 and miR-373, are up-regulated in a hypoxia-inducible factor-1A-dependent manner in hypoxic cells. Bioinformatics analyses suggested that these miRs could regulate factors implicated in DNA repair pathways. Forced expression of miR-210 was found to suppress the levels of RAD52, which is a key factor in homology-dependent repair (HDR); the forced expression of miR-373 led to a reduction in the nucleotide excision repair (NER) protein, RAD23B, as well as in RAD52. Consistent with these results, both RAD52 and RAD23B were found to be down-regulated in hypoxia, but in both cases, the hypoxia-induced down-regulation could be partially reversed by antisense inhibition of miR-210 and miR-373. Importantly, luciferase reporter assays indicated that miR-210 is capable of interacting with the 3 ¶ untranslated region (UTR) of RAD52 and that miR-373 can act on the 3 ¶ UTR of RAD23B. These results indicate that hypoxia-inducible miR-210 and miR-373 play roles in modulating the expression levels of key proteins involved in the HDR and NER pathways, providing new mechanistic insight into the effect of hypoxia on DNA repair and genetic instability in cancer. [Cancer Res 2009;69(3):1221-9]

BackgroundHypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis.Methods and FindingsIn human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.ConclusionsInduction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

Emerging evidence indicates that the tumor microenvironmental stress of hypoxia can induce genetic instability in cancer cells. We and others have found that the expression levels of key genes within the DNA mismatch repair (MMR) and homologous recombination (HR) pathways are coordinately repressed by hypoxia. These decreases are associated with functional impairments in both MMR and HR repair under hypoxic conditions, and thus they represent a possible mechanistic explanation for the observed phenomenon of hypoxia-induced genetic instability. In parallel, studies also indicate that several DNA damage response factors are activated in response to hypoxia and subsequent reoxygenation, including ATM/ATR, Chkl/Chk2 and BRCA1. Taken together, these findings reveal that hypoxia induces a unique cellular stress response involving an initial, acute DNA damage response to hypoxia and reoxygenation, followed by a chronic response to prolonged hypoxia in which selected DNA repair pathways are coordinately suppressed. In this review, we discuss these pathways and the possible mechanisms involved, as well as the consequences for genetic instability and tumor progression within the tumor microenvironment.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy, on the basis of the role of PARP in recruitment of base excision repair (BER) factors to sites of DNA damage. Here we show that PARP inhibition to block BER is toxic to hypoxic cancer cells, in which homology-dependent repair (HDR) is known to be down-regulated. However, we also report the unexpected finding that disruption of PARP, itself, either via chemical PARP inhibitors or siRNAs targeted to PARP-1, can inhibit HDR by suppressing expression of BRCA1 and RAD51, key factors in HDR of DNA breaks. Mechanistically, PARP inhibition was found to cause increased occupancy of the BRCA1 and RAD51 promoters by repressive E2F4/p130 complexes, a pathway prevented by expression of HPV E7, which disrupts p130 activity, or by siRNAs to knock down p130 expression. Functionally, disruption of p130 by E7 expression or by siRNA knockdown also reverses the cytotoxicity and radiosensitivity associated with PARP inhibition, suggesting that the down-regulation of BRCA1 and RAD51 is central to these effects. Direct measurement of HDR using a GFP-based assay demonstrates reduced HDR in cells treated with PARP inhibitors. This work identifies a mechanism by which PARP regulates DNA repair and suggests new strategies for combination cancer therapies.DNA repair | hypoxia P oly(ADP-ribose) polymerases (PARPs) comprise a family of enzymes that catalyze ADP ribosylation of a variety of cellular factors (1-4). PARP-1 is thought to play a key role in DNA repair, primarily by modifying chromatin factors at sites of DNA damage and thereby recruiting repair factors. Inhibitors of PARP have attracted interest for cancer therapy because cancer cells deficient in BRCA1 or BRCA2 due to inactivating mutations are sensitive to PARP inhibition (5-8). This has been attributed to the role of PARP in recruiting base excision repair (BER) factors that remove damaged bases and fix single-strand breaks (SSBs) (1). SSBs persisting into S-phase produce replication fork collapse, requiring BRCA1-and BRCA2-mediated homology-dependent repair (HDR) for resolution (5, 9, 10).In prior work, we found that hypoxia suppresses HDR in human cells via transcriptional down-regulation of BRCA1 and RAD51 (11-15). Hence, we hypothesized that cancer cells in hypoxia, with acquired deficiency in HDR, might have increased sensitivity to PARP inhibition. Work presented here confirms this hypothesis, showing that PARP inhibitors are more cytotoxic to hypoxic than to normoxic cells. Because hypoxia causes BRCA1 and RAD51 down-regulation by stimulating E2F4/p130 occupancy of the BRCA1 and RAD51 promoters, we asked whether disruption of p130 function via expression of human papillomavirus (HPV) E7 would reverse the sensitivity of hypoxic cells to PARP inhibition. We found that E7 expression, as predicted, does confer resistance to PARP inhibitors on hypoxic cells, but surprisingly, it also blocks the toxicity of PARP inhibition in normoxic cells.As a basis for this effect, we present eviden...