KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs.

Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML. SIGNIFICANCE: AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a "writer-reader" epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML. This article is highlighted in the In This Issue feature, p. 587.

The ion channel TRPM7 regulates zinc-depletion-induced MDMX degradation.

Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn2+ concentration to promote tumorigenesis.

AKT3 Is a Novel Regulator of Cancer-Associated Fibroblasts in Head and Neck Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAFs) play vital roles in tumor progression by promoting epithelial-to-mesenchymal transition, angiogenesis, and immunosuppression. In the present study, we sought to identify the key regulators of the pro-tumoral functions of CAFs in head and neck squamous cell carcinoma (HNSCC). mRNA expression data obtained from The Cancer Genome Atlas revealed that CAF-specific mRNA expression correlated with genes that relate to an immunosuppressive microenvironment in a HNSCC cohort. RNA sequencing of CAFs and normal fibroblasts isolated from HNSCC specimens identified 1127 differentially expressed genes (DEGs) and several upregulated pathways in CAFs. Among the 1127 DEGs, we identified 13 immune function-related genes and focused on AKT3 as a potential regulator of CAFs. The targeted depletion of AKT3 in CAFs revealed that AKT3 promotes their myofibroblastic phenotype. AKT3-transduced CAFs exhibited downregulated the expression of immunosuppressive cytokine genes, impairing T-cell suppression and pro-tumoral macrophage induction. The immunohistochemistry of 72 HNSCC patients showed that AKT3 expression in CAFs positively correlated with tumor infiltration by CAFs, tumor-associated macrophages, dendritic cells, and T cells. Moreover, AKT3 expression in CAFs was an independent prognostic factor for overall survival. In conclusion, AKT3 is a potential target for cancer therapy that inhibits the pro-tumoral function of CAFs and reverses CAF-mediated immunosuppression.

AKT3 is a key regulator of head and neck squamous cell carcinoma.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway plays a vital role in cell proliferation, apoptosis, metabolism, and angiogenesis in various human cancers, including head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to clarify the role of AKT, which is a major downstream effector of the PI3K-AKT-mTOR pathway, in HNSCC. We first investigated the mRNA expression of AKT isoforms using RNA-sequencing data from The Cancer Genome Atlas database. We observed a specific elevation of AKT3 expression in HNSCC tissues when compared with that in normal tissues. Furthermore, AKT3 expression correlated with genes related to the immunosuppressive microenvironment more than the other AKT isoforms and PIK3CA. Accordingly, we focused on AKT3 and performed a knockdown approach using an HNSCC cell line. AKT3 knockdown cells exhibited impaired proliferation, a shift in the cell cycle from G2/M to G1/G0 phase, an increase in apoptotic cells, and downregulation of gene expression related to immunosuppression, as well as the knockdown of its upstream regulator PIK3CA. We also performed immunohistochemistry for both AKT3 and PIK3CA using surgical specimens from 72 patients with HNSCC. AKT3 expression in tumor cells correlated with immune cell infiltration and unfavorable prognosis when compared with PIK3CA. These findings suggested that AKT3 expression is a potential biomarker for predicting the immunoreactivity and prognosis of HNSCC. Furthermore, the isoform-specific inhibition of AKT3 could be developed as a novel cancer therapy that efficiently suppresses the PI3K-AKT-mTOR pathway.

Distinctive roles of syntaxin binding protein 4 and its action target, TP63, in lung squamous cell carcinoma: a theranostic study for the precision medicine.

BACKGROUND: Lung squamous cell carcinoma (LSCC) remains a challenging disease to treat, and further improvements in prognosis are dependent upon the identification of LSCC-specific therapeutic biomarkers and/or targets. We previously found that Syntaxin Binding Protein 4 (STXBP4) plays a crucial role in lesion growth and, therefore, clinical outcomes in LSCC patients through regulation of tumor protein p63 (TP63) ubiquitination. METHODS: To clarify the impact of STXBP4 and TP63 for LSCC therapeutics, we assessed relevance of these proteins to outcome of 144 LSCC patients and examined whether its action pathway is distinct from those of currently used drugs in in vitro experiments including RNA-seq analysis through comparison with the other putative exploratory targets and/or markers. RESULTS: Kaplan-Meier analysis revealed that, along with vascular endothelial growth factor receptor 2 (VEGFR2), STXBP4 expression signified a worse prognosis in LSCC patients, both in terms of overall survival (OS, p = 0.002) and disease-free survival (DFS, p = 0.041). These prognostic impacts of STXBP4 were confirmed in univariate Cox regression analysis, but not in the multivariate analysis. Whereas, TP63 (ΔNp63) closely related to OS (p = 0.013), and shown to be an independent prognostic factor for poor OS in the multivariate analysis (p = 0.0324). The action pathway of STXBP4 on suppression of TP63 (ΔNp63) was unique: Ingenuity pathway analysis using the knowledge database and our RNA-seq analysis in human LSCC cell lines indicated that 35 pathways were activated or inactivated in association with STXBP4, but the action pathway of STXBP4 was distinct from those of other current drug targets: STXBP4, TP63 and KDR (VEGFR2 gene) formed a cluster independent from other target genes of tumor protein p53 (TP53), tubulin beta 3 (TUBB3), stathmin 1 (STMN1) and cluster of differentiation 274 (CD274: programmed cell death 1 ligand 1, PD-L1). STXBP4 itself appeared not to be a potent predictive marker of individual drug response, but we found that TP63, main action target of STXBP4, might be involved in drug resistance mechanisms of LSCC. CONCLUSION: STXBP4 and the action target, TP63, could afford a key to the development of precision medicine for LSCC patients.

High-Throughput RNA Interference Screen Targeting Synthetic-Lethal Gain-of-Function of Oncogenic Mutant TP53 in Triple-Negative Breast Cancer.

TNBC is an aggressive and metastatic subtype of breast cancer in which TP53 mutation occurs frequently and is associated with particularly poor outcome. Mutations in TP53 can disrupt the intrinsic function of the tumor suppressor as well as acquire oncogenic gain-of-function (GOF) activities. However, little is known about its oncogenic GOF mediators and functions. Targeted therapy for TNBC patients is thus one of the most urgent needs in breast cancer therapeutics, and identifying genes that have synthetic lethal interactions with mutant TP53 may be a promising approach. In this chapter, we present procedures on sequential analysis of RNA-seq followed by high-throughput RNA interference screening (HTS-RNAi screening). This approach has been utilized to identify genes with synthetic lethality of mutant TP53, providing a promising strategy for the treatment of mutant TP53 in TNBC and determining its impact on tumorigenesis.

Carbonic anhydrase 9 expression is associated with poor prognosis, tumor proliferation, and radiosensitivity of thymic carcinomas.

INTRODUCTION: Thymic epithelial tumors (TETs) comprise several histologies of thymoma and thymic carcinomas (TCs), and TC frequently metastasizes and causes death. We therefore aimed here to identify key molecules closely related to prognosis and their biological roles in high-risk TETs, particularly TCs. RESULTS: RNA sequence analysis demonstrated that hypoxia-related genes were highly expressed in TETs. The expression of the hypoxia-related gene CA9 was noteworthy, particularly in TCs. Immunohistochemical analysis revealed that CA9 was expressed in 81.0% of TCs and 20.7% of all TET samples. CA9 expression was significantly associated with Masaoka stage, WHO classification, and recurrence-free survival after tumor resection (P = 0.005). The down-regulation of CA9 transcription in TC cell lines by small interfering RNAs significantly inhibited CA9 expression, which inhibited proliferation and increased sensitivity to irradiation. CONCLUSIONS: CA9 expression may serve as a significant prognostic marker of TETs and therefore represents a potential target for the development of novel drugs and radiation-sensitizing therapy designed to improve the outcomes of patients with TCs. MATERIALS AND METHODS: We performed comprehensive transcriptome sequencing of 23 TETs and physiologic thymic specimens to identify genes highly and specifically expressed in high-risk TETs, particulary TCs. We performed immunohistochemical analysis of 179 consecutive surgically resected TETs to evaluate the significance of the association of protein expression with clinicopathological features and prognosis. The biological significance of the most promising prognostic marker was further studied using the TC cell lines, Ty-82 and MP57.

Mutant TP53 modulates metastasis of triple negative breast cancer through adenosine A2b receptor signaling.

PURPOSE: The identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis. EXPERIMENTAL DESIGN: TNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53. A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression. Functional assays were performed in vitro and in vivo to assess the role of ADORA2B in TNBC. RESULTS: Transcriptome profiling identified ADORA2B as up-regulated in basal-like TNBC cell lines with R248Q-mutated TP53, with shRNA-screening suggesting the potential for a synthetic-lethal interaction between these genes. In clinical samples, ADORA2B was highly expressed in 39.4% (26/66) of TNBC patients. ADORA2B-expression was significantly correlated with ER (P < 0.01), PgR (P = 0.027), EGFR (P < 0.01), and tumor size (P = 0.037), and was an independent prognostic factor for outcome (P = 0.036). In line with this, ADORA2B-transduced TNBC cells showed increased tumorigenesis, and ADORA2B knockdown, along with mutant p53 knockdown, decreased metastasis both in vitro and in vivo. Notably, the cytotoxic cyclic peptide SA-I suppressed ADORA2B expression and tumorigenesis in TNBC cell lines. CONCLUSIONS: ADORA2B expression increases the oncogenic potential of basal-like TNBC and is an independent factor for poor outcome. These data suggest that ADORA2B could serve as a prognostic biomarker and a potential therapeutic target for basal-like TNBC.

STXBP4 regulates APC/C-mediated p63 turnover and drives squamous cell carcinogenesis.

Levels of the N-terminally truncated isoform of p63 (ΔN p63), well documented to play a pivotal role in basal epidermal gene expression and epithelial maintenance, need to be strictly regulated. We demonstrate here that the anaphase-promoting complex/cyclosome (APC/C) complex plays an essential role in the ubiquitin-mediated turnover of ΔNp63α through the M-G1 phase. In addition, syntaxin-binding protein 4 (Stxbp4), which we previously discovered to bind to ΔNp63, can suppress the APC/C-mediated proteolysis of ΔNp63. Supporting the physiological relevance, of these interactions, both Stxbp4 and an APC/C-resistant version of ΔNp63α (RL7-ΔNp63α) inhibit the terminal differentiation process in 3D organotypic cultures. In line with this, both the stable RL7-ΔNp63α variant and Stxbp4 have oncogenic activity in soft agar and xenograft tumor assays. Notably as well, higher levels of Stxbp4 expression are correlated with the accumulation of ΔNp63 in human squamous cell carcinoma (SCC). Our study reveals that Stxbp4 drives the oncogenic potential of ΔNp63α and may provide a relevant therapeutic target for SCC.

Elevated expression of ΔNp63 in advanced esophageal squamous cell carcinoma.

This study aims to explore the expression level of ΔNp63 in esophageal squamous cell carcinoma (ESCC). To investigate the association between ΔNp63 (p40) expression and ESCC biology, we compared the levels of ΔNp63 expression in normal and tumor tissues, with a specific focus on the diagnostic value of ΔNp63 in ESCC. We analyzed 160 consecutive patients with ESCC who underwent surgical resection without neoadjuvant chemotherapy at Gunma University Hospital (Maebashi, Japan) between September 2000 and January 2010. The clinicopathological characteristics and survival of patients were subclassified based on the expression of ΔNp63 as determined by immunohistochemistry, indicating that ΔNp63 was highly expressed in 75.6% (121/160) of ESCC patients. Clinicopathological analysis of ΔNp63 expression showed that ΔNp63-positive tumors significantly correlated with two important clinical parameters: T factor (P = 0.0316) and venous invasion (P = 0.0195). The 5-year overall survival rates of advanced ESCC patients with positive and negative expression of ΔNp63 were 35.6% and 71.7%, respectively. Multivariate analysis revealed that the expression of ΔNp63 was identified as an independent prognostic factor (P = 0.0049) in advanced ESCC. In line with this, ΔNp63α-transduced ESCC cell lines increased tumor growth in a soft agar colony formation assay. We report here for the first time that ΔNp63 expression increases the oncogenic potential of ESCC and is an independent marker for predicting poor outcome in advanced ESCC. Our findings suggest that ΔNp63 could serve as a new diagnostic marker for ESCC and might be a relevant therapeutic target for the treatment of patients with this disease.

Caspase14 expression is associated with triple negative phenotypes and cancer stem cell marker expression in breast cancer patients.

BACKGROUND AND OBJECTIVES: The Caspase14 (CASP14) was reported that the low expression of CASP14 in ovarian cancer and colon cancer was associated with cancer progression, on the other hand, that the CASP14 expression in breast cancer was higher than that of non-cancerous tissues. The purpose of this study is to determine the clinical significance of CASP14 in breast cancer. METHODS: We performed immunohistochemistry for CASP14, ER, PgR, HER2, Ki67, EGFR, CK5/6, CD44, CD24, ALDH1, claudins, and androgen receptor in 222 breast cancer patients including 55 TNBC cases, and evaluated the relationship of CASP14, above-mentioned markers, and prognosis. Using public microarray database of breast cancer, the prognostic value of CASP14 was calculated. RESULTS: High CASP14 expression was significantly associated with TNBC subtype (P = 0.015), nuclear grade (P = 0.006), Ki67, EGFR (P < 0.001, P = 0.016), ALDH1, CD44 and CD24 (P < 0.001, P < 0.001, P = 0.001) in 222 breast cancer cases, and the high expression of claudin1 (P = 0.017), and androgen receptor (P = 0.002) in TNBC cases was related to the high CASP14. According to the public database, survival in the high CASP14 breast cancer patients was shorter than low CASP14 patients. CONCLUSIONS: High CASP14 expression is a marker of breast cancer aggressiveness in association with proliferation, TNBC phenotype, and cancer stemness.

Inhibition of Ubiquitin-conjugating Enzyme E2 May Activate the Degradation of Hypoxia-inducible Factors and, thus, Overcome Cellular Resistance to Radiation in Colorectal Cancer.

BACKGROUND: NSC697923, a ubiquitin-conjugating enzyme E2 (UBE2) inhibitor, was suggested as an agent to degrade hypoxia-inducible factor 1 alpha subunit (HIF1α), a key factor in radiation resistance. We attempted to clarify whether NSC697923 could overcome radiation resistance. MATERIALS AND METHODS: Radiation resistance and expression of HIFs were evaluated in radiation-sensitive HCT116 and -resistant SW480 cells treated with or without NSC697923 and radiation under normoxia and hypoxia in vitro and in vivo. We examined NSC697923-regulated genes using RNA sequencing. RESULTS: HIF expression significantly increased under hypoxia with an increase of cellular radiation resistance in vitro and in vivo. The therapeutic activity of NSC697923 was higher in radiation-resistant SW480 than radiation-sensitive HCT116 in vivo. Next-generation RNA sequencing revealed that NSC697923 regulated the expression of cell migration-inducing protein, hyaluronan binding (CEMIP) and apelin (APLN) genes, that are related to HIF pathways. CONCLUSION: NSC697923 might effectively regulate HIF families, and be a promising partner with radiation to overcome resistance.

STXBP4 Drives Tumor Growth and Is Associated with Poor Prognosis through PDGF Receptor Signaling in Lung Squamous Cell Carcinoma.

Purpose: Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be an SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis.Experimental Design: We carried out a clinicopathologic analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft-agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells.Results: Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman rank correlation ρ = 0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P < 0.001), disease stage (P = 0.030), and pleural involvement (P = 0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death, and survival in cancer. Platelet-derived growth factor receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft-agar and xenograft assays.Conclusions: STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC. Clin Cancer Res; 23(13); 3442-52. ©2017 AACR.

APOBEC3B high expression status is associated with aggressive phenotype in Japanese breast cancers.

BACKGROUND: The members of AID/APOBEC protein family possess cytidine deaminase activity that converts cytidine residue to uridine on DNA and RNA. Recent studies have shown the possible influence of APOBEC3B (A3B) as DNA mutators of breast cancer genome. However, the clinical significance of A3B expression in Japanese breast cancer has not been studied in detail. METHODS: Ninety-three primary breast cancer tissues (74 estrogen-receptor (ER) positive, 3 ER and HER2 positive, 6 HER2 positive, and 10 triple negative) including 37 tumor-normal pairs were assessed for A3B mRNA expression using quantitative real-time RT-PCR. We analyzed the relation between A3B expression, mutation analysis of TP53 and PIK3CA by direct sequencing, polymorphic A3B deletion allele and human papillomavirus (HPV) infection in tumors. RESULTS: A3B mRNA was overexpressed in tumors compared with normal tissue. Patients with high A3B expression were associated with subtype and progression of lymph node metastasis and pathological nuclear grade. However, the expression was not related to any other clinicopathological factors, including mutation of TP53 and PIK3CA, polymorphic A3B deletion allele, HPV infection and survival time. CONCLUSION: The expression of A3B in breast cancer was higher than in non-cancerous tissues and was related to the lymph node metastasis and nuclear grade, which are reliable aggressive phenotype markers in breast cancer. Evaluation of A3B expression in tumor may be a marker for breast cancer with malignant potential.

Nuclear PROX1 is Associated with Hypoxia-Inducible Factor 1α Expression and Cancer Progression in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Transcription factor prospero homeobox 1 (PROX1) has been identified as a master regulator of lymphangiogenesis associated with metastasis. Although PROX1 expression has been investigated in several cancers, its clinical significance remains controversial and needs further validation. In this study, we investigated the clinical and functional significance of PROX1 and PROX1 regulator hypoxia-inducible factor 1α (HIF1α) in esophageal squamous cell carcinoma (ESCC). METHODS: A total of 117 samples from ESCC patients were analyzed for PROX1, HIF1α, and E-cadherin expression by immunohistochemistry; correlation with clinicopathological characteristics was determined. PROX1 function was evaluated in PROX1 small interfering RNA (siRNA)-transfected human ESCC cells in vitro by assessing cell proliferation and migration. RESULTS: PROX1 expression was higher in ESCC than in normal tissues. Patients with higher PROX1 expression (n = 26) had increased nuclear accumulation of HIF1α (p = 0.004) and more advanced metastasis, both lymph node (N factor; p = 0.09) and hematogenous (M factor; p = 0.04), than those with lower PROX1 expression (n = 91). In addition, high PROX1 and HIF1α expression correlated with low levels of E-cadherin, an epithelial cell marker. Analysis of overall and cancer-specific survival indicated that elevated PROX1 expression was significantly correlated with poor prognosis (p = 0.0064). PROX1 downregulation in ESCC cells inhibited cellular proliferation and migration (p < 0.05). Hypoxia restored PROX1 levels that were reduced by PROX1-specific siRNA. CONCLUSION: Our data suggest that high expression of PROX1 in ESCC could be used as an indicator of poor prognosis, and that PROX1 is a promising candidate molecular target for ESCC treatment.

Immunosuppressive activity of cancer-associated fibroblasts in head and neck squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) have been shown to play an important role in angiogenesis, invasion, and metastasis. In the present study, we determined whether CAFs within the tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) contributed to promoting immunosuppression and evasion from immune surveillance. Six pairs of CAFs and normal fibroblasts (NFs) were established from the resected tumor tissues of patients with HNSCC. The effects of CAFs and NFs on the functions of T cells were comparatively analyzed. CAFs expressed the co-regulatory molecules, B7H1 and B7DC, whereas NFs did not. The expression levels of cytokine genes, including those for IL6, CXCL8, TNF, TGFB1, and VEGFA, were higher in CAFs. T cell proliferation was suppressed more by CAFs or their supernatants than by NFs. Moreover, PBMCs co-cultured with the supernatants of CAFs preferentially induced T cell apoptosis and regulatory T cells over those co-cultured with the supernatants of NFs. A microarray analysis revealed that the level of genes related to the leukocyte extravasation and paxillin signaling pathways was higher in CAFs than in NFs. These results demonstrated that CAFs collaborated with tumor cells in the TME to establish an immunosuppressive network that facilitated tumor evasion from immunological destruction.

MOZ increases p53 acetylation and premature senescence through its complex formation with PML.

Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)- and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML.

Monocytic leukemia zinc finger (MOZ) interacts with p53 to induce p21 expression and cell-cycle arrest.

Upon DNA damage, p53 can induce either cell-cycle arrest or apoptosis. Here we show that monocytic leukemia zinc finger (MOZ) forms a complex with p53 to induce p21 expression and cell-cycle arrest. The levels of the p53-MOZ complex increased in response to DNA damage to levels that induce cell-cycle arrest. MOZ(-/-) mouse embryonic fibroblasts failed to arrest in G1 in response to DNA damage, and DNA damage-induced expression of p21 was impaired in MOZ(-/-) cells. These results suggest that MOZ is involved in regulating cell-cycle arrest in the G1 phase. Screening of tumor-associated p53 mutants demonstrated that the G279E mutation in p53 disrupts interactions between p53 and MOZ, but does not affect the DNA binding activity of p53. The leukemia-associated MOZ-CBP fusion protein inhibits p53-mediated transcription. These results suggest that inhibition of p53/MOZ-mediated transcription is involved in tumor pathogenesis and leukemogenesis.

Involvement of FKHR-dependent TRADD expression in chemotherapeutic drug-induced apoptosis.

Chemotherapeutic drugs exhibit their cytotoxic effect by inducing apoptosis in tumor cells. Because the serine/threonine kinase Akt is involved in apoptosis suppression, we investigated the relationship between Akt activity and drug sensitivity. We discovered that certain chemotherapeutic drugs induced apoptosis with caspase activation only when Akt was inactivated after drug treatment, while inactivation of Akt was not observed when tumor cells showed resistance to the drug-induced caspase activation. So, turn-off of the Akt-mediated survival signal is correlated with the sensitivity of the cells to chemotherapy. With a cDNA microarray, we revealed that tumor necrosis factor receptor-associated death domain (tradd) gene expression was elevated in response to Akt inactivation. Reportedly, Forkhead family transcription factors are phosphorylated by Akt, which results in their nuclear exit and inactivation. Analysis of the tradd promoter revealed that it contains at least one potential Forkhead family transcription factor-responsive element, and we confirmed that this element was involved in chemotherapeutic drug-induced TRADD expression. Overexpression of mutant TRADD proteins to block its apoptosis-inducing capability attenuated chemotherapeutic drug-induced apoptosis. Thus, chemotherapeutic drugs exhibited their cytotoxic effects in part by down-regulating Akt signaling following TRADD expression. These results indicate that Akt kinase activity after drug treatment is a hallmark of sensitivity of the cells to chemotherapeutic drugs.