Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

MutT homolog 1 counteracts the effect of anti-neoplastic treatments in adult and pediatric glioblastoma cells.

Glioblastoma, a fatal disease in both adult and pediatric patients, currently has limited treatment options that offer no more than temporary relief. Our experiments with adult and pediatric glioblastoma cell lines showed that radiation induces a dose-dependent increase in the level of MutT homolog 1 (MTH1) - an enzyme that hydrolyzes oxidized purine nucleoside triphosphates. Similarly, the combination of vorinostat, which is a histone deacetylase inhibitor, and ABT-888, which is a PARP-1 inhibitor, enhanced clonogenic death and increased the MTH1 level, relative to each treatment alone. This result suggests that the MTH1 level is directly related to the damage that is inflicted upon the cells, and its activity protects them against anti-neoplastic therapy. Indeed, the MTH1 inhibitor TH588 and MTH1 siRNA increased glioblastoma's response to both radiation and the combination of vorinostat and ABT-888. TH588 also inhibited glioblastoma's capacity for migration and invasion. In normal fibroblasts, low radiation doses and the combination of vorinostat and ABT-888 decreased the level of the enzyme. TH588 did not alter the fibroblasts' response to radiation and only mildly affected their response to the combination of vorinostat and ABT-888. In summary, the inhibition of MTH1 is required to better realize the therapeutic potential of anti-neoplastic treatments in glioblastoma.

Correction: Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

[This corrects the article DOI: 10.1371/journal.pone.0155711.].

Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

Inhibitors of poly[ADP-ribose] polymerase 1 (PARPis) show promise for treatment of cancers which lack capacity for homologous recombination repair (HRR). However, new therapeutic strategies are required in order to overcome innate and acquired resistance to these drugs and thus expand the array of cancers that could benefit from them. We show that human cancer cell lines which respond poorly to ABT-888 (a PARPi), become sensitive to it when co-treated with vorinostat (a histone deacetylase inhibitor (HDACi)). Vorinostat also sensitized PARPis insensitive cancer cell lines to 6-thioguanine (6-TG)-a drug that targets PARPis sensitive cells. The sensitizing effect of vorinostat was associated with increased phosphorylation of eukaryotic initiation factor (eIF) 2α which in and of itself increases the sensitivity of cancer cells to ABT-888. Importantly, these drug combinations did not affect survival of normal fibroblasts and breast cells, and significantly increased the inhibition of xenograft tumor growth relative to each drug alone, without affecting the mice weight or their liver and kidney function. Our results show that combination of vorinostat and ABT-888 could potentially prove useful for treatment of cancer with innate resistance to PARPis due to active HRR machinery, while the combination of vorinostat and 6-TG could potentially overcome innate or acquired resistance to PARPis due to secondary or reversal BRCA mutations, to decreased PARP-1 level or to increased expression of multiple drug resistant proteins. Importantly, drugs which increase phosphorylation of eIF2α may mimic the sensitizing effect of vorinostat on cellular response to PARPis or to 6-TG, without activating all of its downstream effectors.

Eukaryotic initiation factor 2α--a downstream effector of mammalian target of rapamycin--modulates DNA repair and cancer response to treatment.

In an effort to circumvent resistance to rapamycin--an mTOR inhibitor--we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal--an inhibitor of eIF2α dephosphorylation--decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal--the phosphomimetic eIF2α variant--S51D--increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor--vorinostat. Finally, the catalytic competitive inhibitor of mTOR--Ku-0063794--increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway.

Cyclophosphamide triggers follicle activation and "burnout"; AS101 prevents follicle loss and preserves fertility.

Premature ovarian failure and infertility are major side effects of chemotherapy treatments in young cancer patients. A more thorough understanding of the mechanism behind chemotherapy-induced follicle loss is necessary to develop new methods to preserve fertility in these patients. We show that the alkylating agent cyclophosphamide (Cy) activates the growth of the quiescent primordial follicle population in mice, resulting in loss of ovarian reserve. Despite the initial massive apoptosis observed in growing, though not in resting, follicles of Cy-treated mice, differential follicle counts demonstrated both a decrease in primordial follicles and an increase in early growing follicles. Immunohistochemistry showed that granulosa cells were undergoing proliferation. Analysis of the phosphatidylinositol 3-kinase signaling pathway demonstrated that Cy increased phosphorylation of proteins that stimulate follicle activation in the oocytes and granulosa cells. Coadministration of an immunomodulator, AS101, reduced follicle activation, thereby increasing follicle reserve and rescuing fertility after Cy, and also increased the efficacy of Cy against breast cancer cell lines. These findings suggest that the mechanism in Cy-induced loss of ovarian reserve is accelerated primordial follicle activation, which results in a "burnout" effect and follicle depletion. By preventing this activation, AS101 shows potential as an ovarian-protective agent, which may be able to preserve fertility in female cancer patients.

Pathways that regulate autophagy and their role in mediating tumor response to treatment.

In addition to their role in cellular homeostasis, pathways that regulate autophagy affect both tumorigenesis and tumor response to treatment. Therefore, understanding the regulation of autophagy in treated cancer cells is relevant to the discovery of molecular targets for the development of anti-cancer drugs. Our recent report points to radiation-induced inactivation of the mTOR pathway as an underlying mechanism of radiation-induced autophagy in the human breast cancer cell line MCF-7. Most importantly, radiation-induced inactivation of this pathway was detrimental to cell survival and was associated with reversal of mitochondrial ATPase activity and mitochondrial hyperpolarization, decreased level of eukaryotic initiation factor 4G (eIF4G) and increased phosphorylation of p53. Future analysis of the interrelationship among these events and the role each of them plays in cell survival following radiation will increase our ability to employ the mTOR pathway in anti-cancer therapy.

Rapamycin-sensitive pathway regulates mitochondrial membrane potential, autophagy, and survival in irradiated MCF-7 cells.

Radiation-induced inhibition of rapamycin-sensitive pathway and its effect on the cellular response to radiation were studied in the human breast cancer cell line MCF-7. Both radiation and rapamycin shared molecular targets and induced similar physiologic responses. Each of these treatments increased immunostaining of mammalian target of rapamycin (mTOR) in the nucleus, and radiation led to decreased phosphorylation of its autophosphorylation site Ser2481. In addition to dephosphorylation of established mTOR downstream effectors 4E-binding protein 1 and p70 ribosomal S6 kinase, both treatments decreased the level of eukaryotic initiation factor 4G. Experiments with the potentiometric dye, JC-1, revealed an oligomycin-dependent increase in mitochondrial membrane potential following radiation or rapamycin treatment, suggesting that both lead to reversal of F0F1ATPase activity. Both radiation and rapamycin induced sequestration of cytoplasmic material in autophagic vacuoles. In both cases, appearance of autophagic vacuoles involved the participation of microtubule-associated protein 1 light chain 3 (LC3). Transient cotransfection of green fluorescent protein-LC3 with either wild-type or dominant-negative mTOR further showed that inactivation of mTOR pathway is sufficient to induce autophagy in these cells. Finally, administration of rapamycin in combination with radiation led to enhanced mitochondria hyperpolarization, p53 phosphorylation, and increased cell death. Taken together, these experiments show that radiation-induced inhibition of rapamycin-sensitive pathway in MCF-7 cells causes changes in mitochondria metabolism, development of autophagy, and an overall decrease in cell survival.

The relationship between twenty missense ATM variants and breast cancer risk: the Multiethnic Cohort.

Deficiencies in tasks of detecting and repairing DNA damage lead to mutations and chromosomal abnormalities, a hallmark of cancer. The gene mutated in ataxia-telangiectasia (A-T), ATM, is a proximal component in performing such tasks. Studies of A-T families have suggested an increased risk of breast cancer among obligate female heterozygous carriers of ATM mutations. Paradoxically, studies of sporadic and familial breast cancer have failed to demonstrate an elevated prevalence of mutations among breast cancer cases. We characterized the prevalence and distribution of 20 ATM missense mutations/polymorphisms in a population-based case-control study of 854 African-American, Latina, Japanese, and Caucasian women aged >/==" BORDER="0">45 years participating in the Multiethnic Cohort Study. The study population included 428 incident breast cancer cases and 426 controls. The prevalence of variants ranged from 0% to 13.6% among controls and varied by ethnicity (0-32.5%). Overall, these data provide little support for an association of ATM missense mutations with breast cancer among older women. We observed only one sequence variation (L546V), common among African-American women, to be overrepresented among all high-stage breast cancer cases (odds ratio, 3.35; 95% confidence interval, 1.27-8.84). After correction for multiple comparisons, this observed risk modification did not attain statistical significance. The distribution of ATM missense mutations and polymorphisms varied widely across the four ethnic groups studied. Although a single missense variant (L546V) appeared to act as a modest predictor of risk, the remaining variants were no more common in breast cancer cases as compared with controls.

Rare variants of ATM and risk for Hodgkin's disease and radiation-associated breast cancers.

PURPOSE: In this study, we first sought to evaluate whether individuals heterozygous for ATM mutations may have an increased susceptibility to radiation-induced breast cancer (BC) after treatment for Hodgkin's disease (HD). We next sought to determine the frequency of ATM variants in patients with Hodgkin's lymphoma, regardless of coexisting BC, compared with healthy volunteers. EXPERIMENTAL DESIGN: Full sequence analysis of ATM was performed on cDNA from peripheral blood lymphocytes from 37 cases of BC after therapeutic radiation therapy for HD and 27 comparison cases with HD and no BC treated during the same time period. The frequency of ATM variants was analyzed in the total group of 64 cases of HD and compared to allele frequencies in 128 ethnically matched controls from the same geographical region. RESULTS: No protein-truncating ATM mutations were observed in cases with HD with or without BC. Missense mutations were more frequent in the cohort with HD compared with patients with BC following HD (P = 0.02). The median time from HD to the development of BC was 18 years in patients with ATM variants compared with 16 years in those with no ATM variants (P = 0.04). Multiple ATM variants, including one homozygous mutation, were observed in 9 HD cases. CONCLUSIONS: Heterozygous protein-truncating or missense mutations of ATM were not associated with increased radiation-associated risk of BC after HD. The observation of multiple germ-line mutations and a homozygote suggests that rare ATM variants may constitute cancer-susceptibility alleles in a subset of cases.