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...

Disruption of the BRCA1 tumor suppressor can be caused not only by inherited mutations in familial cancers but also by BRCA1 gene silencing in sporadic cancers. Hypoxia, a key feature of the tumor microenvironment, has been shown to downregulate BRCA1 at the transcriptional level via repressive E2F4/p130 complexes. Here we showed that hypoxia also drives epigenetic modification of the BRCA1 promoter, with decreased H3K4 methylation as a key repressive modification produced by the lysine-specific histone demethylase LSD1. We also observed increased H3K9 methylation coupled with decreased H3K9 acetylation. Similar modifications were seen in the RAD51 promoter, which is also downregulated by hypoxia, whereas exactly opposite changes were seen in the promoter of the hypoxia-inducible gene VEGF. In cells containing the BRCA1 promoter driving a selectable HPRT gene, long-term silencing of the promoter was observed following exposure to hypoxic stress. Clones with silenced BRCA1 promoters were detected at frequencies of 2% or more following hypoxia, but at less than 6 ؋ 10 ؊5 without hypoxia. The silenced clones showed decreased H3K4 methylation and decreased H3K9 acetylation in the BRCA1 promoters, consistent with the acute effects of hypoxic stress. Hypoxia-induced BRCA1 promoter silencing persisted in subsequent normoxic conditions but could be reversed by treatment with a histone deacetylase (HDAC) inhibitor but not with a DNA methylation inhibitor. Interestingly, treatment of cells with inhibitors of poly(ADP-ribose) polymerase (PARP) can cause short-term repression of BRCA1 expression, but such treatment does not produce H3K4 or H3K9 histone modification or BRCA1 promoter silencing. These results suggest that hypoxia is a driving force for long-term silencing of BRCA1, thereby promoting genome instability and tumor progression.Solid tumors constitute a unique tissue type, characterized by hypoxia, low pH, and nutrient deprivation. Previous work has shown that hypoxic stress is a source of genetic instability in tumors (3,5,7,40,58), causing increased point mutations (40), gene amplification (11, 58), and fragile-site induction (12). Our previous work revealed that BRCA1 and RAD51, key genes in the homology-dependent repair (HDR) pathway, and MLH1, a key DNA mismatch (MMR) repair gene, are downregulated at the mRNA and protein levels in response to hypoxia via specific pathways of transcriptional regulation (3-5, 7). Moreover, BRCA1 and MLH1 have been found to be silenced in many sporadic cancers of multiple sites (8,14,16). The silencing of BRCA1 and MLH1 has been attributed primarily to promoter DNA hypermethylation at CpG sites (14). However, recent studies suggest that silenced promoters in cancer cells are also marked by characteristic histone modifications (9, 33, 48), and evidence is emerging that histone methylation may be a mediator of silencing that is independent of DNA methylation (26,29,30).Posttranslational modification of histones is widely recognized as an important epigenetic mechanism in the orga...

SUMMARY Silencing of the MLH1 gene is frequently seen in sporadic cancers. We report that hypoxia causes decreased H3K4 methylation at the MLH1 promoter via the H3K4 demethylases, LSD1 and PLU-1, and promotes long-term silencing of the promoter in a pathway that requires LSD1. Knockdown of LSD1 or its co-repressor, CoREST, also prevents the re-silencing (and cytosine DNA methylation) of the endogenous MLH1 promoter in RKO colon cancer cells following transient reactivation by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC). The results demonstrate that hypoxia is a critical driving force for silencing of MLH1 through chromatin modification and indicate that the LSD1/CoREST complex is essential for MLH1 silencing.

The development of small-molecule tyrosine kinase inhibitors (TKIs) specific for epidermal growth factor receptors (EGFRs) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis due, in part, to acquired resistance to conventional therapy. This study, documents that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line, HCC827, which harbors an activating EGFR mutation. Following hypoxic growth conditions, HCC827 cells treated with gefitinib upregulated N- cadherin, Fibronectin and Vimentin expression and downregulated E-cadherin, characteristic of an epithelial-mesenchymal transition (EMT) which prior studies have linked to EGFR TKI resistance. Mechanistically, knockdown of the histone demethylases, LSD1 and PLU-1, prevented and reversed hypoxia-induced gefitinib resistance, with inhibition of the associated EMT, suggesting that LSD1 and PLU-1 play key roles in hypoxia-induced gefitinib resistance and EMT. Moreover, hypoxia-treated HCC827 cells demonstrated more aggressive tumor growth in vivo compared to cells grown in normoxia, but inhibition of LSD1 function by shRNA- mediated knockdown or by the small-molecular inhibitor, SP2509, suppressed tumor growth and enhanced gefitinib response in vivo. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through epigenetic change and coordination of EMT in NSCLC. This study suggests that combination of therapy with EGFR TKIs and LSD1 inhibitors may offer an attractive therapeutic strategy for NSCLCs.

◥Development of resistance remains the key obstacle to the clinical efficacy of EGFR tyrosine kinase inhibitors (TKI). Hypoxia is a key microenvironmental stress in solid tumors associated with acquired resistance to conventional therapy. Consistent with our previous studies, we show here that long-term, moderate hypoxia promotes resistance to the EGFR TKI osimertinib (AZD9291) in the nonsmall cell lung cancer (NSCLC) cell line H1975, which harbors two EGFR mutations including T790M. Hypoxia-induced resistance was associated with development of epithelial-mesenchymal transition (EMT) coordinated by increased expression of ZEB-1, an EMT activator. Hypoxia induced increased fibroblast growth factor receptor 1 (FGFR1) expression in NSCLC cell lines H1975, HCC827, and YLR086, and knockdown of FGFR1 attenuated hypoxia-induced EGFR TKI resistance in each line. Upregulated expression of FGFR1 by hypoxia was mediated through the MAPK pathway and attenuated induction of the proapoptotic factor BIM. Consistent with this, inhibition of FGFR1 function by the selective small-molecule inhibitor BGJ398 enhanced EGFR TKI sensitivity and promoted upregulation of BIM levels. Furthermore, inhibition of MEK activity by trametinib showed similar effects. In tumor xenografts in mice, treatment with either BGJ398 or trametinib enhanced response to AZD9291 and improved survival. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased expression of FGFR1. The combination of EGFR TKI and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLC.