Chandra M. Das

Valproic acid induces p21 and topoisomerase-II (α/β) expression and synergistically enhances etoposide cytotoxicity in human glioblastoma cell lines

ObjectEtoposide, a topoisomerase-II inhibitor promotes DNA damage and apoptosis of cancer cells. In this study, we have examined the ability of the histone deacetylase inhibitor, valproic acid (VPA) to modulate gene expression and sensitize glioblastoma cell lines to the cytotoxic effects of etoposide in vitro.MethodsThe effect of VPA and etoposide alone or a combination of the two drugs on the growth of three different glioblastoma cell lines (U87, LN18, and U251) were measured by MTT assays. Drug treated cells were analyzed for their cell cycle profile, gene expression, differentiation status, and induction of apoptosis by flow-cytometry, western blotting, immunofluorescence assays, and caspase activity measurements.ResultsWe observed that while VPA and etoposide independently inhibited the growth of U87, U251, and LN18 cells, exposure of tumor cells to both drugs significantly enhanced the cytotoxicity of etoposide in all cell lines. VPA promoted a G1 accumulation of U87, while an increase in the G2/M population of U251 and LN18 cells was observed upon exposure to the drug. Treatment with etoposide resulted in a G2/M arrest of U87, U251, and LN18 cells, whereas, exposure to both drugs increased the fraction of cells with a G2/M and sub-G1 DNA content. Further, VPA and not etoposide, promoted acetylation of histone H4 and induced the expression of the cyclin-dependent kinase inhibitor (CDKI), p21/WAF1. VPA also up-regulated the expression of the α and β isoforms of topoisomerase-II, as well as the glial differentiation marker, glial fibrillary acidic protein. Finally, a significant increase in caspase-3 activity and apoptosis was observed in the presence of both VPA and etoposide compared to either agent alone.ConclusionOur study demonstrates that VPA sensitizes U87, U251, and LN18 cells to the cytotoxic effects of etoposide in vitro by inducing differentiation and up-regulating the expression of p21/WAF1 and both isoforms of topoisomerase-II.

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL

Object Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. Methods Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. Results Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. Conclusion Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.

The deubiquitylase USP37 links REST to the control of p27 stability and cell proliferation

The RE1 silencing transcription factor (REST) is a repressor of neuronal differentiation and its elevated expression in neural cells blocks neuronal differentiation. In this study, we demonstrate a role for REST in the control of proliferation of medulloblastoma cells. REST expression decreased the levels of cyclin-dependent kinase (CDK)NIB/p27, a CDK inhibitor and a brake of cell proliferation in these cells. The reciprocal relationship between REST and p27 was validated in human tumor samples. REST knockdown in medulloblastoma cells derepessed a novel REST target gene encoding the deubiquitylase ubiquitin (Ub)-specific peptidase 37 (USP37). Ectopically expressed wild-type USP37 formed a complex with p27, promoted its deubiquitination and stabilization and blocked cell proliferation. Knockdown of REST and USP37 prevented p27 stabilization and blocked the diminution in proliferative potential that normally accompanied REST loss. Unexpectedly, wild-type USP37 expression also induced the expression of REST-target neuronal differentiation genes even though REST levels were unaffected. In contrast, a mutant of USP37 carrying a site-directed change in a conserved cysteine failed to rescue REST-mediated p27 destabilization, maintenance of cell proliferation and blockade to neuronal differentiation. Consistent with these findings, a significant correlation between USP37 and p27 was observed in patient tumors. Collectively, these findings provide a novel connection between REST and the proteasomal machinery in the control of p27 and cell proliferation in medulloblastoma cells.

Chromatin remodelling at the topoisomerase II-beta promoter is associated with enhanced sensitivity to etoposide in human neuroblastoma cell lines

Etoposide, an inhibitor of topoisomerase II, promotes DNA damage and apoptosis of cancer cells and is a component of standard therapy for neuroblastoma. Resistance to etoposide has been observed in neural tumour cells expressing lower levels of topoisomerase II. In the present study, we have examined the contribution of epigenetic modulation of gene expression in the potentiation of etoposide-mediated cytotoxicity in neuroblastoma cells. Specifically, we studied the effects of histone deacetylase inhibition with valproic acid on topoisomerase II gene expression and apoptosis in response to etoposide. Using human neuroblastoma cell lines SK-N-AS and SK-N-SH, we show that although the combination of valproic acid and etoposide promoted a reduction in growth compared to either drug alone in both cells, the effect was substantially enhanced in SK-N-AS compared to SK-N-SH cells. An increase in histone H3 acetylation and p21 expression was observed in both cell lines, however, upregulation of topoisomerase II-beta gene expression and an increase in PARP cleavage was observed in SK-N-AS cells only. Furthermore, chromatin immunoprecipitation assays revealed an increase in acetylation of histone H3 at the cognate topoisomerase II-beta gene after treatment with valproic acid in SK-N-AS cells. These results suggest a potential epigenetic mechanism of regulation of the topoisomerase II-beta gene and a possible role for its increased expression in the sensitivity of SK-N-AS neuroblastoma cells to etoposide.

Histone acetylation resulting in resistance to methotrexate in choroid plexus cells

Choroid plexus carcinomas are rare tumors that typically occur in young children. Prognosis is poor, and very little information is available to optimize treatment protocols. We used a cell culture model to evaluate whether combining chemotherapeutic agents such as methotrexate with histone deacetylase inhibitors (HDACI) such as valproic acid and MS-275 could improve efficacy. Valproic acid increased the cytotoxicity of radiation and of all the chemotherapeutic agents in Z310 and SV11 mouse choroid plexus cell lines, with the exception of methotrexate. Both HDACIs made choroid plexus cells resistant to this folate antagonist. Searching for a molecular explanation, we found that thymidylate synthase was up regulated when the cells were incubated with HDACI. We also confirmed this finding in human choroid plexus carcinoma cells. Methotrexate should not be combined with HDACI in the treatment of choroid plexus carcinoma.

Regulation of USP37 Expression by REST-Associated G9a-Dependent Histone Methylation

The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo. Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma. Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073–84. ©2017 AACR.

Abstract B2: Epigenetic deregulation of a novel deubiquitylase promotes medulloblastoma genesis

Medulloblastoma is a poorly differentiated and hyperproliferative malignant pediatric brain tumor. It frequently arises in the cerebellum. Current therapies while efficacious against primary tumors not only cause severe neurologic deficits in children with brain tumors, but are also ineffective against recurrences. The overall goal of our work is to gain a better understanding of tumor biology so that therapies that target tumor specific events can be developed. This approach would also spare the normal brain from therapy-related toxicity. The RE1 Silencing Transcription Factor (REST) is an epigenetic modulator of gene expression and a repressor of neuronal differentiation. While normal cerebellar neurons did not express REST, its levels were significantly elevated in human medulloblastoma samples, and was associated with poor prognosis for patients. To examine the feasibility of developing REST as a therapeutic target, we performed genetic and pharmacological experiments to explore its role in driving medulloblastoma formation. We observed that REST loss in human tumor cells abrogates their tumorigenic potential in mouse intracranial models. Conversely, REST elevation in neural progenitors promoted tumor formation in the context of Myc overexpression. REST-expressing tumor cells failed to undergo differentiation and had sustained proliferation. REST is a known repressor of genes required for terminal neurogenesis. Interestingly, we made a novel observation that REST also controlled tumor cell proliferation through direct transcriptional repression of a gene encoding a novel deubiquitylase called USP37. Using purified USP37 protein, we demonstrated that it deubiquitylated the cell cycle regulator p27, a critical mediator of normal cell cycle exit during neurogenesis. In patient samples, we noted a significant correlation between loss of USP37, decreased p27 protein levels and poor prognosis. Importantly, constitutive expression of wildtype USP37, but not mutant USP37, promoted a decrease in tumor cell proliferation in vitro and a loss of tumorigenic potential in mouse orthotopic models. Since REST-mediated transcriptional silencing is effected by its associated histone deacetylase (HDAC) and histone methyltransferase (HMT)-G9a chromatin remodeling complexes, we performed genetic and pharmacological assays to identify the specific epigenetic activity promoting USP37 down-regulation in medulloblastoma cells. These studies revealed that G9a loss or inhibition of its activity upregulated USP37 expression and blocked tumor cell proliferation in vitro and tumor growth in vivo. Together, these findings suggest that USP37 is a novel tumor suppressor gene in medulloblastoma and that its epigenetic modulation in REST-high tumors may have potential therapeutic application. Citation Format: Rashieda Jonine Hatcher, Vrushali Datar, Chandra M. Das, Jason Fangusaro, Stewart Goldman, Rishi Lulla, Veena Rajaram, Vidya Gopalakrishnan. Epigenetic deregulation of a novel deubiquitylase promotes medulloblastoma genesis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B2.

Abstract LB-320: Identification of USP37 as a novel tumor suppressive p27-specific deubiquitylase

Medulloblastoma is a malignant pediatric brain tumor. Current treatments based on clinical features-centered stratification into standard- and high-risk groups, has vastly improved patient survival. However, a subset of patients with standard-risk features exhibit recurrence and leptomeningeal spread. Unfortunately, the outcome for such patients is dismal. This highlights the need for new stratification approaches and therapeutic decisions based on a better understanding of tumor biology. Since medulloblastomas are poorly differentiated tumors, we asked if the RE1 silencing transcription factor (REST), a repressor of neuronal differentiation genes, played a role in tumor etiology. Relative to normal cerebella, we observed an increase in REST levels in islands of tumor cells in over 70% of samples studied. Interestingly, patients with very high REST expression in their tumors exhibited poor overall and event free survival, identifying REST as a poor prognostic factor. The requirement for REST in tumor maintenance and progression was then established using knockdown and overexpression studies in mouse orthotopic models. To dissect the role of REST in medulloblastoma genesis, we knocked down its expression in cell lines and neurosphere cultures established from patient tissue. To our surprise, we observed a decrease in tumor cell proliferation following REST loss that was associated with destabilization of the cyclin-dependent kinase inhibitor, p27. Previous studies have shown a contribution of p27 loss to medulloblastoma development, however the underlying mechanisms were unclear. To investigate if REST contributed to a decline in p27 levels, we studied the expression of a panel of genes (a) whose expression was upregulated upon REST loss (b) that had the potential to be a direct REST target gene based on bioinformatics and (c) could potentially regulate p27 degradation. A gene encoding a deubiquitylase USP37 was further investigated and validated as a novel REST target, and its role in blocking p27 proteasomal degradation established by genetic and biochemical assays. Importantly, constitutive expression of wild type USP37 in tumor cells with high REST expression promoted p27 accumulation and blocked in vitro cell proliferation and tumor growth in the mouse brain. In contrast, a catalytically inactive mutant of USP37 failed to promote p27 stabilization and inhibit tumor growth in vivo. A significant negative correlation between REST and p27 and a positive correlation between USP37 and p27 was also seen in human tumors. Thus, our studies have identified REST as a novel prognostic factor in medulloblastoma. We have also provided evidence that its elevated expression blocks neuronal differentiation and contributes to sustained tumor cell proliferation through repression of USP37 expression and destabilization of p27. Our findings suggest that USP37 has a tumor suppressive role in medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-320. doi:1538-7445.AM2012-LB-320