Prostate Tumor Suppressor Research Articles

Thymoquinone Potentially Modulates the Expression of Key Onco- and Tumor Suppressor miRNAs in Prostate and Colon Cancer Cell Lines: Insights from PC3 and HCT-15 Cells.

Prostate cancer (PC) and colon cancer significantly contribute to global cancer-related morbidity and mortality. Thymoquinone (TQ), a naturally occurring phytochemical found in black cumin, has shown potential as an anticancer compound. This study aimed to investigate the effects of TQ on the expression profile of key tumor suppressor and onco-suppressor miRNAs in PC3 prostate cancer cells and HCT-15 colon cancer cells. Cell viability assays revealed that TQ inhibited the growth of both cell lines in a dose-dependent manner, with IC50 values of approximately 82.59 μM for HCT-15 and 55.83 μM for PC3 cells. Following TQ treatment at the IC50 concentrations, miRNA expression analysis demonstrated that TQ significantly downregulated miR-21-5p expression in HCT-15 cells and upregulated miR-34a-5p, miR-221-5p, miR-17-5p, and miR-21-5p expression in PC3 cells. However, no significant changes were observed in the expression levels of miR-34a-5p and miR-200a-5p in HCT-15 cells. The current findings suggest that TQ might exert its antiproliferative effects by modulating specific tumor suppressor and onco-suppressor miRNAs in prostate and colon cancer cells. Further investigations are warranted to elucidate the precise underlying mechanisms and to explore the therapeutic potential of TQ in cancer treatment. To the best of our knowledge, this is the first report regarding the effect of TQ on the miRNA expression profile in colon and prostate cancer cell lines.

Deregulation of SPOP in Cancer.

Speckle-type POZ protein (SPOP) is a substrate-binding adaptor of the CULLIN3/RING-box1 E3 ubiquitin ligase complex. SPOP is frequently mutated in prostate and endometrial cancers, whereas it is overexpressed in renal cell carcinoma (RCC). SPOP can mediate both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions such as androgen receptor (AR), SRC-3, TRIM24, BRD4, PD-L1, 53BP1, GLP/G9a, c-Myc, SENP7, among others. Cancer-associated SPOP mutants often impair SPOP binding and polyubiquitination of its substrates to influence various cancer-relevant pathways, which include androgen/AR signaling, DNA repair and methylation, cellular stress surveillance, cancer metabolism, and immunity. Although SPOP is recognized as a tumor suppressor in prostate and endometrial cancers, it acts like an oncoprotein in RCC. This review provides an overview of the recent progress in understanding of the upstream regulators of SPOP and its downstream targets, highlights the significant impact of SPOP mutations and overexpression on cancer pathogenesis, and discusses the potential of targeting SPOP for cancer treatment.

Forkhead Box Protein P3 (FOXP3) Represses ATF3 Transcriptional Activity.

Activating transcription factor 3 (ATF3), a transcription factor and acute stress sensor, is rapidly induced by a variety of pathophysiological signals and is essential in the complex processes in cellular stress response. FOXP3, a well-known breast and prostate tumor suppressor from the X chromosome, is a novel transcriptional repressor for several oncogenes. However, it remains unknown whether ATF3 is the target protein of FOXP3. Herein, we demonstrate that ATF3 expression is regulated by FOXP3. Firstly, we observed that overexpression of FOXP3 reduced ATF3 protein level. Moreover, knockdown FOXP3 by siRNA increased ATF3 expression. Secondly, FOXP3 dose-dependently reduced ATF3 promoter activity in the luciferase reporter assay. Since FOXP3 is regulated by post-translational modifications (PTMs), we next investigated whether PTMs affect FOXP3-mediated ATF3 expression. Interestingly, we observed that phosphorylation mutation on FOXP3 (Y342F) significantly abolished FOXP3-mediated ATF3 expression. However, other PTM mutations on FOXP3, including S418 phosphorylation, K263 acetylation and ubiquitination, and K268 acetylation and ubiquitination, did not alter FOXP3-mediated ATF3 expression. Finally, the FOXP3 binding site was found on ATF3 promoter region by deletion and mutagenesis analysis. Taken together, our results suggest that FOXP3 functions as a novel regulator of ATF3 and that this novel event may be involved in tumor development and progression.

Abstract 310A: Transcription factors FOXP3 regulate ATF3 expression

Abstract Activating transcription factor 3 (ATF3), a stress sensor, is rapidly induced by a variety of physiological and pathological signals, including those from cancer cells, and is essential in the complex processes of stress response. Several lines of evidence have demonstrated that ATF3 is involved in host defense and cancer development. Several studies also provide the relationship between ATF3 and p53 indicating that ATF3 binds to and stabilizes p53 while ATF3 is a negative regulator of TP53 gene expression. FOXP3, a well-known breast and prostate tumor suppressor, is a novel transcriptional repressor for several oncogenes including SKP2. However, whether ATF3 is the target protein of FOXP3 remains unknown. Herein, we demonstrate that ATF3 expression is regulated by FOXP3. First, we observed that overexpression of FOXP3 reduced ATF3 protein level in several cancer cell lines (H1299, HepG2, and Ishikawa cells). Secondly, FOXP3 dose-dependently reduced ATF3 promoter activity in gene reporter assay in several cell lines (MCF7, H1299, MDAMB231, and Saos2 cells). Since FOXP3 is regulated by post-translational modifications (PTMs), we next investigated whether PTMs affect FOXP3-mediated ATF3 expression. Interestingly, we observed that phosphorylation mutation on FOXP3 (Y342F) abolished FOXP3-mediated ATF3 expression. However, other PTM mutations on FOXP3, including S418 phosphorylation, K263 acetylation and ubiquitination, and K268 acetylation and ubiquitination, did not alter FOXP3-mediated ATF3 expression. Finally, FOXP3 binding site was found on ATF3 promoter region, around -870 bp region. Taken together, our results suggest that FOXP3 functions as a novel regulator of ATF3 and this novel event may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Chiung-Min Wang, William Yang. Transcription factors FOXP3 regulate ATF3 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 310A.

Loss of Ceacam1 promotes prostate cancer progression in Pten haploinsufficient male mice

ObjectivePTEN haploinsufficiency plays an important role in prostate cancer development in men. However, monoallelic deletion of Pten gene failed to induce high prostate intraepithelial neoplasia (PIN) until Pten+/− mice aged or fed a high-calorie diet. Because CEACAM1, a cell adhesion molecule with a potential tumor suppression activity, is induced in Pten+/− prostates, the study aimed at examining whether the rise of CEACAM1 limited neoplastic progression in Pten+/− prostates. MethodsPten+/− were crossbred with Cc1−/− mice harboring a null deletion of Ceacam1 gene to produce Pten+/−/Cc1−/− double mutants. Prostates from 7-month old male mice were analyzed histologically and biochemically for PIN progression. ResultsDeleting Ceacam1 in Pten+/− mice caused an early development of high-grade PIN in parallel to hyperactivation of PI3 kinase/Akt and Ras/MAP kinase pathways, with an increase in cell proliferation, epithelial-to-mesenchymal transition, angiogenesis and inflammation relative to Pten+/− and Cc1−/− individual mutants. It also caused a remarkable increase in lipogenesis in prostate despite maintaining insulin sensitivity. Concomitant Ceacam1 deletion with Pten+/− activated the IL-6/STAT3 signaling pathways to suppress Irf-8 transcription that in turn, led to a decrease in the expression level of promyelocytic leukemia gene, a well characterized tumor suppressor in prostate. ConclusionsCeacam1 deletion accelerated high-grade prostate intraepithelial neoplasia in Pten haploinsufficient mice while preserving insulin sensitivity. This demonstrated that the combined loss of Ceacam1 and Pten advanced prostate cancer by increasing lipogenesis and modifying the STAT3-dependent inflammatory microenvironment of prostate.

PGC1α Suppresses Prostate Cancer Cell Invasion through ERRα Transcriptional Control.

The PPARγ coactivator 1 alpha (PGC1α) is a prostate tumor suppressor that controls the balance between anabolism and catabolism. PGC1A downregulation in prostate cancer is causally associated with the development of metastasis. Here we show that the transcriptional complex formed by PGC1α and estrogen-related receptor 1 alpha (ERRα) controls the aggressive properties of prostate cancer cells. PGC1α expression significantly decreased migration and invasion of various prostate cancer cell lines. This phenotype was consistent with remarkable cytoskeletal remodeling and inhibition of integrin alpha 1 and beta 4 expression, both in vitro and in vivo. CRISPR/Cas9-based deletion of ERRα suppressed PGC1α regulation of cytoskeletal organization and invasiveness. Mechanistically, PGC1α expression decreased MYC levels and activity prior to inhibition of invasiveness. In addition, PGC1α and ERRα associated at the MYC promoter, supporting the inhibitory activity PGC1α. The inverse correlation between PGC1α-ERRα activity and MYC levels was corroborated in multiple prostate cancer datasets. Altogether, these results support that PGC1α-ERRα functions as a tumor-suppressive transcriptional complex through the regulation of metabolic and signaling events. SIGNIFICANCE: These findings describe how downregulation of the prostate tumor suppressor PGC1 drives invasiveness and migration of prostate cancer cells.

Conditional Deletion of Eaf1 Induces Murine Prostatic Intraepithelial Neoplasia in Mice

ELL-associated factor 1 is a transcription elongation factor that shares significant homology and functional similarity to the androgen-responsive prostate tumor suppressor ELL-associated factor 2. EAF2 is frequently down-regulated in advanced prostate cancer and Eaf2 deletion in the mouse induced the development of murine prostatic intraepithelial neoplasia. Here we show that similar to EAF2, EAF1 is frequently down-regulated in advanced prostate cancer. Co-downregulation of EAF1 and EAF2 occurred in 40% of clinical specimens with Gleason score >7. We developed and characterized a murine model of prostate-epithelial specific deletion of Eaf1 in the prostate and crossed it with our previously generated mouse with conventional deletion of Eaf2. The prostates of Eaf1 deletion mice displayed murine prostatic intraepithelial neoplasia lesions with increased proliferation and inflammation. Combined deletion of Eaf1 and Eaf2 in the murine model induced an increased incidence in mPIN lesions characterized by increased proliferation and CD3+ T cells and CD19+ B cells infiltration compared to individual deletion of either Eaf1 or Eaf2 in the murine prostate. These results suggest that EAF1 may play a tumor suppressive role in the prostate. Cooperation between EAF1 and EAF2 may be important for prostate maintaining prostate epithelial homeostasis, and concurrent loss of these two tumor suppressors may promote prostate tumorigenesis and progression.

IGFBP7 As a Potential Therapeutic Target for AML

Insulin-like growth factor binding proteins (IGFBPs) are secretory factors that play essential roles in regulation of insulin-like growth factors (IGFs) in tissue as well as in modulating IGF binding to its receptors. IGFBP7, known as IGFBP-related protein 1 (IGFBP-rP1), mac25/angiomodulin, function as a potential tumor suppressor in various human solid cancers, including breast, prostate, gastric and liver cancer. We have reported the overexpression of IGFBP7 in the context of acute myeloid leukemia (AML), showing that IGFBP7 expression level in AML patients is significantly increased compared with controls (P<0.001). IGFBP7 expression was obviously decreased in AML patients achieving complete remission (P<0.01), and was significantly increased in relapsed AML patients (P<0.01). In addition, AML patients with high expression of IGFBP7 had shorter overall survival. Here, we investigate the role and mechanism of IGFBP7 in the development and progression of AML.In order to study the role of IGFBP7 in AML, stable cell lines expressing IGFBP7 and control in AML cells were constructed using lentiviral packaging system. Expression microarray assay was carried out to analyze the global gene level changes driven by IGFBP7. MTT and transwell assays were performed to evaluate the effect of IGFBP7 on cell proliferation and migration. Bioinformatics results found that IGFBP7 appeared to utilize multiple cellular processes for its oncogenic roles, including adhesion, migration, and proliferation. Experimental data showed overexpression of IGFBP7 in K562 cells resulted in a 2-3 fold increase in migration in contrast to control cells. Moreover, enforced expression of IGFBP7 also led to phosphorylation of Akt and Erk, whose activities inactivation by pharmacologically inhibitors resulted in the loss of ability to migrating. Finally, knockdown of IGFBP7 in cells with high IGFBP7 level, their migration abilities were significantly decreased.To assess the role of IGFBP7 in leukemogenesis in vivo, the same numbers of K562/IGFBP7 and K562-Vector cells, U937-shIGFBP7 and U937-shNEG cells were injected into NOD-SCID mice by tail vein injection, respectively. About two weeks later, it was showed that mice of K562/IGFBP7 and U937 groups displayed higher white blood cell counts compared with mice of K562-Vector and U937-shIGFBP7 groups, respectively. Mice of K562/IGFBP7 and U937 groups had more severe splenomegaly and hepatomagaly compared with its corresponding control groups.We further characterized the molecular mechanism underlying leukemogenesis driven by IGFBP7 in AML cell lines. The global expression profiling and molecular biological experiments showed PI3K/AKT signaling was activated by overexpression of IGFBP7, and knockdown of IGFBP7 in AML cells led to a decrease of PI3K/AKT activity in PTEN-dependent manner. IGFBP7 promotes proliferation and migration of AML cells, the promotion could be suppressed by both RNA interference and pharmacological inhibition of PI3K/AKT pathway. Immuno-precipitation assay showed that IGFBP7 associated with AXAN2 and induced PTEN degradation. The expression of ANXA2 was significantly positive correlated with the expression ANXA2 in AML patients.The expression of IGFBP7 in AML, overexpression as well as knockdown of IGFBP7 in leukemia cells and in mice model, all suggest that IGFBP7 is a potential proto-oncogene. Collectively this work suggests that targeting IGFBP7 activity may be an effective therapeutic strategy for AML. DisclosuresNo relevant conflicts of interest to declare.

Integrative analysis of transcriptomics and clinical data uncovers the tumor-suppressive activity of MITF in prostate cancer

The dysregulation of gene expression is an enabling hallmark of cancer. Computational analysis of transcriptomics data from human cancer specimens, complemented with exhaustive clinical annotation, provides an opportunity to identify core regulators of the tumorigenic process. Here we exploit well-annotated clinical datasets of prostate cancer for the discovery of transcriptional regulators relevant to prostate cancer. Following this rationale, we identify Microphthalmia-associated transcription factor (MITF) as a prostate tumor suppressor among a subset of transcription factors. Importantly, we further interrogate transcriptomics and clinical data to refine MITF perturbation-based empirical assays and unveil Crystallin Alpha B (CRYAB) as an unprecedented direct target of the transcription factor that is, at least in part, responsible for its tumor-suppressive activity in prostate cancer. This evidence was supported by the enhanced prognostic potential of a signature based on the concomitant alteration of MITF and CRYAB in prostate cancer patients. In sum, our study provides proof-of-concept evidence of the potential of the bioinformatics screen of publicly available cancer patient databases as discovery platforms, and demonstrates that the MITF-CRYAB axis controls prostate cancer biology.

Abstract 1403: Modeling of Abi1 loss using spheroid cultures to investigate mechanisms of prostate tumorigenesis

Abstract American Cancer Society predicts over 160,000 new prostate cancer cases in 2017, which amounts to about 20 percent of all cancer diagnoses in men. Despite several treatment options being available, poor prognosis of high-risk and metastasis remains a major concern for prostate cancer (PCa) patients. Therefore, it is important to understand molecular mechanisms of PCa progression to aggressive disease. Our previous research identified Abelson interactor 1 (Abi1), a member of WAVE complex, as a tumor suppressor in human prostate. Not only did we find mutations and deletions in the gene in prostate tumor patient samples, but WAVE complex levels were also found to be inversely correlated to disease recurrence and castration resistance. Moreover, Abi1 is downregulated in majority of prostate cancer cell lines, and patient organoid cultures derived from metastatic bone and lymph node biopsies. These data point to the clinical significance of defining Abi1-dependent tumor suppression mechanisms. To understand these mechanisms we generated CRISPR-mediated Abi1 KO in the RWPE-1 non-tumor human prostate cell line. When plated in 3D cultures in matrigel, we observed striking phenotypic differences in the appearance and behavior of the Abi1 KO spheroids compared to the parental cells. While the parental RWPE-1 grew into spherical organoids with tight cell-cell boundaries, the Abi1 KO cells grew as loose irregular-shaped spheres, with many cells migrating out of the organoids. Hence, the loss of Abi1 in these non-tumor cells lead to gain of an invasive phenotype. Western Blots of Abi1-KO cells demonstrated concurrent disruption of WAVE complex and deregulation of WAVE complex protein levels. Moreover, cell-cell adhesion proteins such as E-cadherin and β-catenin showed modest decrease and/or a substantial loss of membrane localization. Rescue experiments of Abi1 re-expression in the KO cells reversed the phenotype indicating that the observed phenotype is Abi1-dependent. Analyses of RNA sequencing of cells obtained from 3D cultures demonstrated differences in expression signatures characteristic of altered cell-cell and cell-matrix adhesion pathways and indicated upregulation of pathways associated with the invasive phenotype of cancer cells. We propose that Abi1 loss and WAVE complex deregulation represents key mediators of invasive phenotype in PCa. [Supported by NCI R01 CA161018 and NYS Department of Health Prostate Cancer Hypothesis Development RFA #1410200115] Citation Format: Disharee Das, Ming Chen, Claudia Mondragon, Dawn Post, Alexander Nappi, Heidi Hehnly, Anita Hryniewicz-Jankowska, Pier Paolo Pandolfi, Gennady Bratslavsky, Leszek Kotula. Modeling of Abi1 loss using spheroid cultures to investigate mechanisms of prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1403.

Protocols for Studies on TMPRSS2/ERG in Prostate Cancer.

TMPRSS2/ERG is the most common type of gene fusions found in human prostate cancer. There are two important features of TMPRSS2/ERG fusions. One is that these gene fusions lead to ectopic expression of ERG, an ETS family transcription factor, in prostate epithelial cells from the 5' control region of an androgen/estrogen dual-responsive gene, TMPRSS2; the other is that ~60% of these fusions are generated via intrachromosomal deletion of the interstitial region between TMPRSS2 and ERG. To recapitulate these important aspects of TMPRSS2/ERG fusions, we generated several TMPRSS2/ERG knockin mouse models based on the endogenous Tmprss2 locus. We found that TMPRSS2/ERG represents an early event in prostate tumorigenesis, by sensitizing prostate cells for cooperation with other oncogenic events, such as PTEN-deficiency. We also found that the interstitial region between TMPRSS2 and ERG harbors at least one prostate tumor suppressor, ETS2, whose loss contributes to prostate cancer progression. In this protocol, we describe how these knockin mouse models can be utilized to study roles of TMPRSS2/ERG fusions in prostate cancer development both in vivo and in vitro.

Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia.

Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivoEll2-knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2-knockout mice. Microarray analysis of prostates from Ell2-knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer.

1665P - Dual targeting of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) by miR-4317 displays a synergistic efficacy in repressing breast cancer progression

Background: Recently, hydrogen sulphide (H2S) and its synthesizing enzymes, CBS and CSE, have been casted as pleiotropic regulators in the malignant transformation process. H2S paradoxically acts as oncogenic mediator in ovarian and liver cancers, and as tumor suppressor in prostate and gastric carcinomas. However, the link between H2S and Breast cancer (BC) remains unclear. Thus we aimed at unraveling the association between H2S and its synthesizing enzymes in BC progression. Furthermore, it was essential to evaluate their possible adoption as therapeutic targets in BC through their dual targeting by short non-coding RNAs. Methods: Breast tissues were collected from 30 BC patients. Ki67 levels were quantified using immunohistochemistry. MDA-MB-231 and MCF7 cells were cultured and transfected with different oligonucleotides and/or treated with NaHS, an exogenous source of H2S. Total RNA was extracted and quantified by qRT-PCR. Cellular viability, proliferation, and migation were measured using MTT, BrdU and scratch assays respectively. Bioinformatic analysis was performed to predict novel miRNAs that could target both CBS and CSE. Results: CBS and CSE were significantly upregulated in BC tissues. Patients with high Ki67 scores showed the highest expression levels of CBS and CSE. Knocking down of CBS and CSE using siRNAs resulted in a significant attenuation of different hallmarks of BC. On the other hand, NaHS resulted in an increase in BC progression. miR-4317 was found to putatively target both CBS and CSE oncogenes with high binding scores. Ectopic expression of miR-4317 in BC cell lines resulted in a simultaneous reduction of CBS and CSE transcripts which was associated with a concomitant reduction in cellular viability, proliferation and migration. Finally, co-treatment of miR-4317 and NaHS resulted in abrogation of miR-4317 tumor suppressor activity. Conclusions: This study showed a marked upregulation of CBS and CSE in BC tissues and characterized them as aggressive oncogenic drivers in BC. Moreover, miR-4317, a novel tumor suppressor in BC, displayed a synergistic effect in halting BC progression via twin-targeting CBS and CSE and diminishing H2S levels in BC cell lines. Legal entity responsible for the study: German University in Cairo Funding: None Disclosure: All authors have declared no conflicts of interest.

Convergent Effects of Resveratrol and PYK2 on Prostate Cells

Resveratrol, a dietary polyphenol, is under consideration as chemopreventive and chemotherapeutic agent for several diseases, including cancer. However, its mechanisms of action and its effects on non-tumor cells, fundamental to understand its real efficacy as chemopreventive agent, remain largely unknown. Proline-rich tyrosine kinase 2 (PYK2), a non-receptor tyrosine kinase acting as signaling mediator of different stimuli, behaves as tumor-suppressor in prostate. Since, PYK2 and RSV share several fields of interaction, including oxidative stress, we have investigated their functional relationship in human non-transformed prostate EPN cells and in their tumor-prone counterpart EPN-PKM, expressing a PYK2 dead-kinase mutant. We show that RSV has a strong biological activity in both cell lines, decreasing ROS production, inducing morphological changes and reversible growth arrest, and activating autophagy but not apoptosis. Interestingly, the PYK2 mutant increases basal ROS and autophagy levels, and modulates the intensity of RSV effects. In particular, the anti-oxidant effect of RSV is more potent in EPN than in EPN-PKM, whereas its anti-proliferative and pro-autophagic effects are more significant in EPN-PKM. Consistently, PYK2 depletion by RNAi replicates the effects of the PKM mutant. Taken together, our results reveal that PYK2 and RSV act on common cellular pathways and suggest that RSV effects on prostate cells may depend on mutational-state or expression levels of PYK2 that emerges as a possible mediator of RSV mechanisms of action. Moreover, the observation that resveratrol effects are reversible and not associated to apoptosis in tumor-prone EPN-PKM cells suggests caution for its use in humans.

The chromatin regulator chd8 is a context-dependent mediator of cell survival in murine hematopoietic malignancies

Aberrant chromatin regulation is a frequent driver of leukemogenesis. Mutations in chromatin regulators often result in more stem-like cells that seed a bulk leukemic population. Inhibitors targeting these proteins represent an emerging class of therapeutics, and identifying further chromatin regulators that promote disease progression may result in additional drug targets. We identified the chromatin-modifying protein CHD8 as necessary for cell survival in a mouse model of BCR-Abl+ B-cell acute lymphoblastic leukemia. This disease has a poor prognosis despite treatment with kinase inhibitors targeting BCR-Abl. Although implicated as a risk factor in autism spectrum disorder and a tumor suppressor in prostate and lung cancer, the mechanism of CHD8’s activity is still unclear and has never been studied in the context of hematopoietic malignancies. Here we demonstrate that depletion of CHD8 in B-ALL cells leads to cell death. While multiple B cell malignancies were dependent on CHD8 expression for survival, T cell malignancies displayed milder phenotypes upon CHD8 knockdown. In addition, ectopic expression of the Notch1 intracellular domain in a T cell malignancy partially alleviated the detrimental effect of CHD8 depletion. Our results demonstrate that CHD8 has a context-dependent role in cell survival, and its inhibition may be an effective treatment for B lymphoid malignancies.

Nkx3.1 controls the DNA repair response in the mouse prostate.

BACKGROUNDThe human prostate tumor suppressor NKX3.1 mediates the DNA repair response and interacts with the androgen receptor to assure faithful completion of transcription thereby protecting against TMPRSS2‐ERG gene fusion. To determine directly the effect of Nkx3.1 in vivo we studied the DNA repair response in prostates of mice with targeted deletion of Nkx3.1.METHODSUsing both drug‐induced DNA damage and γ‐irradiation, we assayed expression of γ‐histone 2AX at time points up to 24 hr after induction of DNA damage.RESULTSWe demonstrated that expression of Nkx3.1 influenced both the timing and magnitude of the DNA damage response in the prostate.CONCLUSIONSNkx3.1 affects the DNA damage response in the murine prostate and is haploinsufficient for this phenotype. Prostate 76:402–408, 2016. © 2015 The Authors. The Prostate published by Wiley Periodicals, Inc.

The Chromatin Regulator CHD8 Is a Context-Dependent Mediator of Cell Survival in Murine Hematopoietic Malignancies.

Aberrant chromatin regulation is a frequent driver of leukemogenesis. Mutations in chromatin regulators often result in more stem-like cells that seed a bulk leukemic population. Inhibitors targeting these proteins represent an emerging class of therapeutics, and identifying further chromatin regulators that promote disease progression may result in additional drug targets. We identified the chromatin-modifying protein CHD8 as necessary for cell survival in a mouse model of BCR-Abl+ B-cell acute lymphoblastic leukemia. This disease has a poor prognosis despite treatment with kinase inhibitors targeting BCR-Abl. Although implicated as a risk factor in autism spectrum disorder and a tumor suppressor in prostate and lung cancer, the mechanism of CHD8’s activity is still unclear and has never been studied in the context of hematopoietic malignancies. Here we demonstrate that depletion of CHD8 in B-ALL cells leads to cell death. While multiple B cell malignancies were dependent on CHD8 expression for survival, T cell malignancies displayed milder phenotypes upon CHD8 knockdown. In addition, ectopic expression of the Notch1 intracellular domain in a T cell malignancy partially alleviated the detrimental effect of CHD8 depletion. Our results demonstrate that CHD8 has a context-dependent role in cell survival, and its inhibition may be an effective treatment for B lymphoid malignancies.

Abstract 1951: Identification of novel molecular targets for treatment of castration-resistant prostate cancer

Abstract One of the major limitations of current therapies for treatment of prostate cancer is the acquisition of castration-resistance by tumor cells. Castration-resistant prostate cancer (CRPC) proceeds through multiple signaling pathways that utilize the PI3K/AKT axis to direct anti-apoptotic and pro-survival responses in CRPC cells. We performed in-depth genomic and phenotypic analysis which revealed that two transcription factors, Cyclic AMP Response Element Binding protein (CREB) and Forkhead Box O3 (FOXO3a), were inversely expressed in CRPC cells. Specifically, an upregulation of CREB in stage-specific manner in CRPC tumor cells was suggestive of its proliferative role in tumor growth. Conversely, downregulation of FOXO3a expression suggested inhibition of this transcription factor's pro-apoptotic function, which may also facilitate tumor growth. In pre-clinical models of CRPC, both CREB and FOXO3a transcriptionally regulated the function of prostate tumor suppressor, Par-4 (prostate apoptotic response gene-4). Promoter bashing studies identified consensus regulatory target binding sites of CREB and FOXO3a on Par-4 promoter. The sequential deletion of three CREB binding sites in the Par-4 promoter relieved CREB-mediated suppression of Par-4, whereas sequential deletion of FOXO3a binding sites in Par-4 promoter abrogated Par-4 promoter activity. We further dissected upstream events of CREB and FOXO3a, which are regulated by protein kinase-B AKT using a small-molecule AKT inhibitor (SMAI) drug. Overexpression of AKT in CRPC (DU-145/AKT, C4-2B/AKT and PC-3/AKT) cells induced pro-survival machinery by activating CREB and inhibiting pro-apoptotic function of FOXO3a and resulting in resistance of cells to drug. The in vivo efficacy of our SMAI drug was evaluated in DU-145/AKT and C4-2B/AKT tumor xenografts in nude mouse model. AKT overexpressing tumors exhibited aggressive growth rates and exhibited high vascularisation and angiogenesis as compared to vector-transfected tumors. Daily oral administration of SMAI drug significantly inhibited tumor growth in both DU-145 and C4-2B AKT-overexpressing tumors. IHC analysis of these tumor tissues revealed reduced FOXO3a expression and increased pCREB expression following treatment with SMAI drug in AKT versus control tumors. The cooperative effects of CREB and FOXO3a in AKT-mediated signaling suggests that the development of small molecule inhibitors targeting AKT/CREB function while simultaneously inducing FOXO3a/Par-4 activation in CRPC tumors, represents an attractive target for drug development. Citation Format: Jim Moselhy, Ram V. Roy, Suman Suman, Trinath P. Das, Murali Ankem, Chendil Damodaran. Identification of novel molecular targets for treatment of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1951. doi:10.1158/1538-7445.AM2015-1951

NKX3.1 Suppresses TMPRSS2-ERG Gene Rearrangement and Mediates Repair of Androgen Receptor-Induced DNA Damage.

TMPRSS2 gene rearrangements occur at DNA breaks formed during androgen receptor-mediated transcription and activate expression of ETS transcription factors at the early stages of more than half of prostate cancers. NKX3.1, a prostate tumor suppressor that accelerates the DNA repair response, binds to androgen receptor at the ERG gene breakpoint and inhibits both the juxtaposition of the TMPRSS2 and ERG gene loci and also their recombination. NKX3.1 acts by accelerating DNA repair after androgen-induced transcriptional activation. NKX3.1 influences the recruitment of proteins that promote homology-directed DNA repair. Loss of NKX3.1 favors recruitment to the ERG gene breakpoint of proteins that promote error-prone nonhomologous end-joining. Analysis of prostate cancer tissues showed that the presence of a TMPRSS2-ERG rearrangement was highly correlated with lower levels of NKX3.1 expression consistent with the role of NKX3.1 as a suppressor of the pathogenic gene rearrangement.

Genetic interaction between Tmprss2-ERG gene fusion and Nkx3.1-loss does not enhance prostate tumorigenesis in mouse models.

Gene fusions involving ETS family transcription factors (mainly TMPRSS2-ERG and TMPRSS2-ETV1 fusions) have been found in ~50% of human prostate cancer cases. Although expression of TMPRSS2-ERG or TMPRSS2-ETV1 fusion alone is insufficient to initiate prostate tumorigenesis, they appear to sensitize prostate epithelial cells for cooperation with additional oncogenic mutations to drive frank prostate adenocarcinoma. To search for such ETS-cooperating oncogenic events, we focused on a well-studied prostate tumor suppressor NKX3.1, as loss of NKX3.1 is another common genetic alteration in human prostate cancer. Previous studies have shown that deletions at 8p21 (harboring NKX3.1) and 21q22 (resulting in TMPRSS2-ERG fusion) were both present in a subtype of prostate cancer cases, and that ERG can lead to epigenetic silencing of NKX3.1 in prostate cancer cells, whereas NKX3.1 can in turn negatively regulate TMPRSS2-ERG fusion expression via suppression of the TMPRSS2 promoter activity. We recently generated knockin mouse models for TMPRSS2-ERG and TMPRSS2-ETV1 fusions, utilizing the endogenous Tmprss2 promoter. We crossed these knockin models to an Nkx3.1 knockout mouse model. In Tmprss2-ERG;Nkx3.1+/- (or -/-) male mice, although we observed a slight but significant upregulation of Tmprss2-ERG fusion expression upon Nkx3.1 loss, we did not detect any significant cooperation between these two genetic events to enhance prostate tumorigenesis in vivo. Furthermore, retrospective analysis of a previously published human prostate cancer dataset revealed that within ERG-overexpressing prostate cancer cases, NKX3.1 loss or deletion did not predict biochemical relapse after radical prostatectomy. Collectively, these data suggest that although TMPRSS2-ERG fusion and loss of NKX3.1 are among the most common mutational events found in prostate cancer, and although each of them can sensitize prostate epithelial cells for cooperating with other oncogenic events, these two events themselves do not appear to cooperate at a significant level in vivo to enhance prostate tumorigenesis.