Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination

<p>Abstract</p> <p>Background</p> <p>Activating Transcription Factor (ATF) 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC) inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor.</p> <p>Results</p> <p>The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) as well as chromatin immunoprecipitation (ChIP) assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells.</p> <p>Conclusion</p> <p>This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.</p

Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

AbstractThe mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3) as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK) pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38) resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs) were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects

The effect of the histone deacetylase inhibitor M344 on BRCA1 expression in breast and ovarian cancer cells

<p>Abstract</p> <p>Background</p> <p>The inhibition of Breast Cancer 1 (BRCA1) expression sensitizes breast and ovarian cancer cells to platinum chemotherapy. However, therapeutically relevant agents that target BRCA1 expression have not been identified. Our recent report suggested the potential of the histone deacetylase (HDAC) inhibitor, M344, to inhibit BRCA1 expression. In this study, we further evaluated the effect of M344 on BRCA1 mRNA and protein expression, as well as its effect on cisplatin-induced cytotoxicity in various breast (MCF7, T-47D and HCC1937) and ovarian (A2780s, A2780cp and OVCAR-4) cancer cell lines.</p> <p>Results</p> <p>With the addition of M344, the platinum-sensitive breast and ovarian cancer cell lines that displayed relatively high BRCA1 protein levels demonstrated significant potentiation of cisplatin cytotoxicity in association with a reduction of BRCA1 protein. The cisplatin-resistant cell lines, T-47D and A2780s, elicited increased cytotoxicity of cisplatin with M344 and down regulation of BRCA1 protein levels. A2780s cells subjected to combination platinum and M344 treatment, demonstrated increased DNA damage as assessed by the presence of phosphorylated H2A.X foci in comparison to either treatment alone. Using Chromatin Immunoprecipitation, A2780s and MCF7 cells exposed to M344 alone and in combination with cisplatin, did not demonstrate enhanced acetylated Histone 4 at the <it>BRCA1 </it>promoter, suggesting an indirect effect on this promoter.</p> <p>Conclusions</p> <p>The enhanced sensitivity of HDAC inhibition to platinum may be mediated through a BRCA1-dependent mechanism in breast and ovarian cancer cells. The findings of this study may be important in the future design of clinical trials involving HDAC inhibitors using BRCA1 as a tumour biomarker.</p

Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

BACKGROUND: In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics. METHODOLOGY/PRINCIPAL FINDINGS: The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. CONCLUSIONS/SIGNIFICANCE: These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors

An analysis of growth and differentiation of human neuroblastoma, identification of HMG-CoA reductase and a novel zinc finger gene as potential mediators

grantor: University of TorontoSeveral biological and clinical features of neuroblastoma suggest that this neural crest derived tumor retains the potential for further differentiation. Delineating these events may provide insights for eventual clinical management of this chemorefractory disease. The NUB-7 human neuroblastoma derived cell line, established in our laboratory, uniquely displayed features of both primitive neuronal and Schwannian cells and was further inducible along both cell lineages with dibutyryl cyclic AMP and retinoic acid respectively. This differentiation potential mimics the in vivo differentiation patterns of neuroblastoma. Using the novel molecular technique of differential display, we identified 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and a novel zinc finger gene as potential differentiation responsive genes in NUB-7. HMG-CoA reductase functions as the rate limiting enzyme of the mevalonate pathway involved in de novo cholesterol synthesis and cell cycle progression. Its expression and modulation during neuroblastoma induced differentiation implicated HMG-CoA reductase as a key regulator of neuroblastoma growth and a mediator of the biological effects of retinoic acid. Lovastatin, a non-reversible competitive inhibitor of HMG-CoA reductase induced marked cytotoxicity in neuroblastoma cell lines expressing P-glycoprotein, a well recognized drug efflux pump involved in chemoresistance. Moreover, this cytotoxicity was enhanced by the addition of dibutyryl cyclic AMP but not retinoic acid. These observations support the possible clinical utility of HMG-CoA reductase inhibitors in combination with differentiating agents in the treatment of neuroblastoma. A second novel clone, designated zf5-3, was localized to chromosome 19 and possessed 4 zinc finger DNA binding motifs characteristic of a wide variety of transcriptional regulators. The expression of zf5-3 was restricted to fetal neuronal, hepatic and renal tissues and their tumor derived cell lines. The expression of zf5-3 was downregulated during nervous tissue maturation as well as induced neuronal maturation of various neuroblastoma cell lines. These observations implicate zf5-3 as a potential mediator of differentiation in fetal neuronal tissues and their derived tumors including neuroblastoma through the maintenance of the undifferentiated state.Ph.D

miR-105 inhibits prostate tumour growth by suppressing CDK6 levels.

A significant role for micro (mi)RNA in the regulation of gene expression in tumours has been recently established. In order to further understand how miRNA expression may contribute to prostate tumour growth and progression, we evaluated expression of miRNA in two invasive prostate tumour lines, PC3 and DU145, and compared it to that in normal prostate epithelial cells. Although a number of miRNAs were differentially expressed, we focused our analysis on miR-105, a novel miRNA not previously linked to prostate cancer. miR-105 levels were significantly decreased in both tumour cell lines in comparison to normal prostate epithelial cells. To determine its potential role in prostate cancer pathogenesis, we overexpressed miR-105 in both PC3 and DU145 cells and determined its effect on various tumourigenic properties. miR-105 overexpression inhibited tumour cell proliferation, tumour growth in anchorage-independent three-dimensional conditions and tumour invasion in vitro, properties of highly aggressive tumour cells. Of potential clinical significance, miR-105 overexpression inhibited tumour growth in vivo in xenograft models using these cell lines. We further identified CDK6 as a putative target of miR-105 which is likely a main contributor to the inhibition of tumour cell growth observed in our assays. Our results suggest that miR-105 inhibits tumour cell proliferation and hence may represent a novel therapeutically relevant cellular target to inhibit tumour growth or a marker of aggressive tumours in prostate cancer patients

Does metformin usage improve survival in head and neck squamous cell carcinoma? A population-based study

Abstract Background We sought to expand upon preliminary data suggesting that metformin confers a survival benefit to patients with head and neck squamous cell carcinoma (HNSCC). Methods A large-scale retrospective cohort study of all patients in Ontario diagnosed with squamous cancer of the larynx, hypopharynx, and nasopharynx between Dec 1st 2007 to Dec 1st 2012 was undertaken. The Institute for Clinical and Evaluative Sciences was accessed to obtain patient demographic, treatment and outcome information. We included patients on metformin at the time of diagnosis. Kaplan Meier methods and Cox Regression models were used. Results Patients taking metformin at the time of diagnosis had a higher comorbid status but were otherwise similar to patients without metformin usage. Using multivariate analysis, neither overall survival nor disease specific survival was improved in patients on metformin (OS: HR 1.123, p = .338; DSS: HR 1.048, p = .792). Conclusions No survival advantage was observed in patients with HNSCC taking metformin at the time of diagnosis

Lovastatin in combination with VEGFR-2 TKIs inhibits ligand induced activation of AKT, S6K1 and 4EBP1.

<p>Control cells were serum starved for 24 hr followed by 2 hr treatments with either 10 µM KRN633, 10 nM rapamycin, 5 µM lovastatin or their combinations. All cells were then lysed after stimulation with 50 ng/ml VEGF₁₆₅ for 30 min. Results demonstrated that lovastatin in combination with KRN633 induced the most significant decrease in phosphorylation status of all three proteins in H28 cells. Expression levels of total AKT, S6K1 and 4EBP1 were assayed as loading controls.</p