JmjC‐domain containing histone demethylase 1B‐mediated p15Ink4b suppression promotes the proliferation of leukemic progenitor cells through modulation of cell cycle progression in acute myeloid leukemia

The histone demethylase JHDM1B has been implicated in cell cycle regulation and tumorigenesis. In addition, it has been reported that JHDM1B is highly expressed in various human tumors, including leukemias. However, it is not clearly understood how JHDM1B contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of JHDM1B in AML cells. In AML cell lines and AML‐derived ALDH^hi (high aldehyde dehydrogenase activity)/CD34⁺ cells, the levels of JHDM1B mRNA were significantly higher than in normal ALDH^hi/CD34⁺ cells. Reduction of JHDM1B expression in AML cells inhibited cell proliferation compared to control cells, through induction of G1 cell cycle arrest, an increase in the p15^Ink4b mRNA and protein expression. JHDM1B mRNA was overexpressed in all 133 AML clinical specimens tested (n = 22, 57, 34, and 20 for M1, 2, 4, and 5 subtypes respectively). Compared to normal ALDH^hi/CD34⁺ cells, JHDM1B gene expression was 1.57‐ to 1.87‐fold higher in AML‐derived ALDH^hi/CD34⁺cells. Moreover, the JHDM1B protein was more strongly expressed in AML‐derived ALDH^hi/CD34⁺ cells from compared to normal ALDH^hi/CD34⁺ cells. In addition, depletion of JHDM1B reduced colony formation of AML‐derived ALDH^hi/CD34⁺ cells due to induction of p15^Ink4b expression through direct binding to p15^Ink4b promoter and loss of demethylation of H3K36me2. In summary, we found that JHDM1B mRNA is predominantly expressed in AML‐derived ALDH^hi/CD34⁺ cells, and that aberrant expression of JHDM1B induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of JHDM1B expression represents an attractive target for AML therapy. © 2011 Wiley Periodicals, Inc.     

KDM1 is a novel therapeutic target for the treatment of gliomas

Glioma development is a multistep process, involving alterations in genetic and epigenetic mechanisms. Understanding the mechanisms and enzymes that promote epigenetic changes in gliomas are urgently needed to identify novel therapeutic targets. We examined the role of histone demethylase KDM1 in glioma progression. KDM1 was overexpressed in gliomas and its expression positively correlated with histological malignancy. Knockdown of KDM1 expression or its pharmacological inhibition using pargyline or NCL-1 significantly reduced the proliferation of glioma cells. Inhibition of KDM1 promoted up regulation of the p53 target genes p21 and PUMA. Patient-derived primary GBM cells expressed high levels of KDM1 and pharmacological inhibition of KDM1 decreased their proliferation. Further, KDM1 inhibition reduced the expression of stemness markers CD133 and nestin in GBM cells. Mouse xenograft assays revealed that inhibition of KDM1 significantly reduced glioma xenograft tumor growth. Inhibition of KDM1 increased levels of H3K4-me2 and H3K9-Ac histone modifications, reduced H3K9-me2 modification and promoted expression of p53 target genes (p21 and PUMA), leading to apoptosis of glioma xenograft tumors. Our results suggest that KDM1 is overexpressed in gliomas and could be a potential therapeutic target for the treatment of gliomas.     

Bisindole-PBD regulates breast cancer cell proliferation via SIRT-p53 axis

In a previous study we reported the role of potent bisindole-PBD conjugate as an inclusion in the arsenal of breast cancer therapeutics. In breast cancer cell proliferation, PI3K/AKT/mTOR pathway plays a crucial role by prosurvival mechanism that inhibits programmed cell death. Here, 2 breast cancer cells lines, MCF-7 and MDA-MB-231 were treated with Vorinostat (suberoylanilide hydroxamic acid / SAHA) and bisindole-PBD (5b). We have investigated the effect on PI3K/AKT/mTOR pathway and SIRT expression including epigenetic regulation. There was consistent decrease in the level of PI3K, AKT, mTOR proteins upon treatment of 5b in both MCF-7 and MDA-MB-231 cell lines compared to untreated controls. Treatment with caspase inhibitor (Q-VD-OPH) confirmed that the effect of 5b on PI3K signaling was ahead of apoptosis. Real time PCR and western blot analysis showed profound reduction in the mRNA and protein levels of SIRT1 and SIRT2. Molecular docking studies also supported the interaction of 5b with various amino acids of SIRT2 proteins. Treatment with 5b caused epigenetic changes that include increase of acetylated forms of p53, increase of histone acetylation at p21 promoter as well as decrease in methylation state of p21 gene. Compound 5b thus acts as SIRT inhibitor and cause p53 activation via inhibition of growth factor signaling and activation of p53 dependent apoptotic signaling. This present study focuses bisindole-PBD on epigenetic alteration putting 5b as a promising therapeutic tool in the realm of breast cancer research.     

Inhibition of histone demethylase,LSD2 (KDM1B), attenuates DNA methylation and increases sensitivity to DNMT inhibitor-induced apoptosis in breast cancer cells

Increasing evidence suggests that dysfunction of histone lysine demethylase is associated with abnormal chromatin remodeling and gene silencing, contributing to breast tumorigenesis. In silico analysis shows that the newly identified histone demethylase lysine-specific demethylase 2 is highly expressed in breast cancer, especially in invasive tumors. However, it is currently unknown how LSD2 regulates chromatin remodeling and gene expression regulation in breast cancer. Using short hairpin RNA, we stably knocked down LSD2 (LSD2-KD) in MDA-MB-231 breast cancer cells. LSD2-KD led to accumulation of H3K4me1/2 without changing methylation levels of other key histone lysine residues, suggesting that LSD2 acts as a bona fide H3K4 demethylase in breast cancer cells. LSD2-KD resulted in decreased colony formation and attenuated global DNA methylation in MDA-MB-231 cells. Additionally, treatment with the DNMT inhibitor, 5-aza-deoxycytidine (DAC), synergistically increased mRNA expression of aberrantly silenced genes important in breast cancer development, including PR, RARβ, ERα, SFRP1, SFRP2, and E-cadherin in LSD2-KD cells. Furthermore, LSD2-KD cells are more susceptible to cell death than scramble controls, and combined treatment with tranylcypromine, an LSD2 inhibitor, and DAC resulted in synergistic growth inhibition of breast cancer cells. DNMT inhibition by DAC in LSD2-KD cells led to internucleosomal DNA fragmentation, enhanced PARP cleavage and increased sub-G1 apoptotic cell population. These results demonstrate an important role for LSD2 in regulation of DNA methylation and gene silencing in breast cancer, and suggest that inhibition of LSD2 in combination with DNA methyltransferase inhibition represents a novel approach for epigenetic therapy of breast cancer.     

Identification and functional analysis of 9p24 amplified genes in human breast cancer

Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2?Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16(INK4a), p21(Waf1/Cip1) and p27(Kip1). Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis.     

Targeted cancer therapy with ribosome biogenesis inhibitors: a real possibility?

The effects of many chemotherapeutic drugs on ribosome biogenesis have been underestimated for a long time. Indeed, many drugs currently used for cancer treatment – and which are known to either damage DNA or hinder DNA synthesis – have been shown to exert their toxic action mainly by inhibiting rRNA synthesis or maturation. Moreover, there are new drugs that have been proposed recently for cancer chemotherapy, which only hinder ribosome biogenesis without any genotoxic activity. Even though ribosome biogenesis occurs in both normal and cancer cells, whether resting or proliferating, there is evidence that the selective inhibition of ribosome biogenesis may, in some instances, result in a selective damage to neoplastic cells. The higher sensitivity of cancer cells to inhibitors of rRNA synthesis appears to be the consequence of either the loss of the mechanisms controlling the cell cycle progression or the acquisition of activating oncogene and inactivating tumor suppressor gene mutations that up-regulate the ribosome biogenesis rate. This article reviews those cancer cell characteristics on which the selective cancer cell cytotoxicity induced by the inhibitors of ribosome biogenesis is based.     

Mutant p53: a novel target for the treatment of patients with triple‐negative breast cancer?

The identification and validation of a targeted therapy for patients with triple‐negative breast cancer (TNBC) is currently one of the most urgent needs in breast cancer therapeutics. One of the key reasons for the failure to develop a new therapy for this subgroup of breast cancer patients has been the difficulty in identifying a highly prevalent, targetable molecular alteration in these tumors. Recently however, the p53 gene was found to be mutated in approximately 80% of basal/TNBC, raising the possibility that targeting the mutant p53 protein product might be a new approach for the treatment of this form of breast cancer. In this study, we investigated the anti‐cancer activity of PRIMA‐1 and PRIMA‐1^MET (APR‐246), two compounds which were previously reported to reactivate mutant p53 and convert it to a form with wild‐type (WT) properties. Using a panel of 18 breast cancer cell lines and 2 immortalized breast cell lines, inhibition of proliferation by PRIMA‐1 and PRIMA‐1^MET was found to be cell‐line dependent, but independent of cell line molecular subtype. Although response was independent of molecular subtype, p53 mutated cell lines were significantly more sensitive to PRIMA‐1^MET than p53 WT cells (p = 0.029). Furthermore, response (measured as IC₅₀ value) correlated significantly with p53 protein level as measured by ELISA (p = 0.0089, r= ?0.57, n = 19). In addition to inhibiting cell proliferation, PRIMA‐1^MET induced apoptosis and inhibited migration in a p53 mutant‐dependent manner. Based on our data, we conclude that targeting mutant p53 with PRIMA‐1^MET is a potential new approach for treating p53‐mutated breast cancer, including the subgroup with triple‐negative (TN) disease.     

Acetylation of the Cell-Fate Factor Dachshund Determines p53 Binding and Signaling Modules in Breast Cancer

Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21^CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.     

Epigenetic regulation of the cell type-specific gene 14-3-3sigma

Epigenetic control participates in processes crucial in mammalian development, such as X-chromosome inactivation, gene imprinting, and cell type-specific gene expression. We provide evidence that the p53-inducible gene 14-3-3sigma is a new example of a gene important to human cancer, where epigenetic mechanisms participate in the control of normal cell type-specific expression, as well as aberrant gene silencing in cancer cells. Like a previously identified cell type-specific gene maspin, 14-3-3sigma is a p53-inducible gene; however, it participates in G2/M arrest in response to DNA-damaging agents. 14-3-3Sigma expression is restricted to certain epithelial cell types, including breast and prostate, whereas expression is absent in nonepithelial tissues such as fibroblasts and lymphocytes. In this report, we show that in normal cells expressing 14-3-3sigma, the 14-3-3sigma CpG island is unmethylated; associated with acetylated histones, unmethylated histone H3 lysine 9; and an accessible chromatin structure. By contrast, normal cells that do not express 14-3-3sigma have a methylated 14-3-3sigma CpG island with hypoacetylated histones, methylated histone H3 lysine 9, and an inaccessible chromatin structure. These findings extend the spectrum of cell type-specific genes controlled, partly, by normal epigenetic mechanisms, and suggest that this subset of genes may represent important targets of epigenetic dysregulation in human cancer.     

Id-1 regulates Bcl-2 and Bax expression through p53 and NF-κB in MCF-7 breast cancer cells

Although increasing evidence supports the protective role of inhibitor of differentiation and DNA binding-1 (Id-1) against anticancer drug-induced apoptosis, the underlying molecular mechanisms seem to vary depending on the tumor system. Here, we examined the direct role of Id-1 in MCF-7 breast cancer cells by ectopically overexpressing Id-1 under serum-free condition, where the endogenous Id-1 expression was suppressed. Id-1 expression resulted in increased number of viable cells, reduced Bax expression, enhanced Bcl-2 expression, but no change in Bcl-xL expression. The expression of nuclear factor-κB (NF-κB) was augmented, while those of p53 and IκB were reduced. Such changes in p53 and NF-κB pathways were also functional, as assessed by real-time polymerase chain reactions and reporter assays of their known downstream targets, p21 and Il-6, as well as Bax and Bcl-2 genes. Finally, Id-1 played a protective role against taxol-induced apoptosis in breast cancer cells as assessed by MTT assay and apoptotic cell count upon taxol treatment (0–200 nM). Reduced Bax expression and enhanced Bcl-2 expression by Id-1 were also noted in the presence of taxol. Taken together, we present a molecular mechanism where Id-1 regulates p53 and NF-κB pathways, which in turn regulates Bax and Bcl-2 genes, thus providing a survival advantage under exogenous stress such as serum-free or taxol treatment in MCF-7 breast cancer cells. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer progression and anti-cancer drug resistance. Hwan Kim and Heekyoung Chung equally contributed to this work.     

Ribosome biogenesis and control of cell proliferation: p53 is not alone

Cell growth is a prerequisite for cell proliferation, and ribosome biogenesis is a limiting factor for cell growth. In mammalian cells, the tumor suppressor p53 has been shown to induce cell-cycle arrest in response to impaired ribosome biogenesis. Recently, p53-independent mechanisms of cell-cycle arrest in response to alterations of ribosome biogenesis have been described. These findings provide a rational basis for the use of drugs that specifically impact ribosome biogenesis for the treatment of cancers lacking active p53 and extend the scenario of mechanisms involved in the relationship between cell growth and cell proliferation.     

p33^ING1和p53基因蛋白在乳腺癌中的表达及其意义

目的 探讨p23ING1、p53基因蛋白在乳腺增生及乳腺癌中表达的意义及相互关系. 方法 应用Envision免疫组化法对正常乳腺组织、单纯增生、非典型增生、乳腺癌各50例标本进行p33ING1、p53基因蛋白表达检测. 结果 p33ING1蛋白在乳腺正常组织、单纯增生、非典型增生、乳腺癌中阳性表达率分别为100.0%(50/50)、100.0%(50/50)、96.0%(48/50)和62.0%(31/50),阳性表达逐渐下降;p53蛋白阳性表达率分别为0(0/50)、0(0/50)、24.0%(12/50)和54.0%(27/50),阳性表达率逐渐上升.非典型增生与正常组织相比,差异有显著性(P＜0.05);乳腺癌与正常组织相比,差异有非常显著性(P＜0.01). 结论 p33ING1阳性者p53阳性表达率远低于p33ING1阴性者,p33ING1与p53呈负相关.p33ING1和野生型p53基因都是抑癌基因,p33ING1是p53的分子伴侣.