Inactivation of VRK1 sensitizes ovarian cancer to PARP inhibition through regulating DNA-PK stability.

Ovarian cancer is the leading cause of gynecologic cancer death. Among the most innovative anti-cancer approaches, the genetic concept of synthetic lethality is that mutations in multiple genes work synergistically to effect cell death. Previous studies found that although vaccinia-related kinase-1 (VRK1) associates with DNA damage repair proteins, its underlying mechanisms remain unclear. Here, we found high VRK1 expression in ovarian tumors, and that VRK1 depletion can significantly promote apoptosis and cell cycle arrest. The effect of VRK1 knockdown on apoptosis was manifested by increased DNA damage, genomic instability, and apoptosis, and also blocked non-homologous end joining (NHEJ) by destabilizing DNA-PK. Further, we verified that VRK1 depletion enhanced sensitivity to a PARP inhibitor (PARPi), olaparib, promoting apoptosis through DNA damage, especially in ovarian cancer cell lines with high VRK1 expression. Proteins implicated in DNA damage responses are suitable targets for the development of new anti-cancer therapeutic strategies, and their combination could represent an alternative form of synthetic lethality. Therefore, normal protective DNA damage responses are impaired by combining olaparib with elimination of VRK1 and could be used to reduce drug dose and its associated toxicity. In summary, VRK1 represents both a potential biomarker for PARPi sensitivity, and a new DDR-associated therapeutic target, in ovarian cancer.

The Immune Suppressor IGSF1 as a Potential Target for Cancer Immunotherapy.

The development of first-generation immune-checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 ushered in a new era in anticancer therapy. Although immune-checkpoint blockade therapies have shown clinical success, a substantial number of patients yet fail to benefit. Many studies are under way to discover next-generation immunotherapeutic targets. Immunoglobulin superfamily member 1 (IGSF1) is a membrane glycoprotein proposed to regulate thyroid function. Despite containing 12 immunoglobin domains, a possible role for IGSF1, in immune response, remains unknown. Here, our studies revealed that IGSF1 is predominantly expressed in tumors but not normal tissues, and increased expression is observed in PD-L1low non-small cell lung cancer (NSCLC) cells as compared with PD-L1high cells. Subsequently, we developed and characterized an IGSF1-specific human monoclonal antibody, WM-A1, that effectively promoted antitumor immunity and overcame the limitations of first-generation immune-checkpoint inhibitors, likely via a distinct mechanism of action. We further demonstrated high WM-A1 efficacy in humanized peripheral blood mononuclear cells (PBMC), and syngeneic mouse models, finding additive efficacy in combination with an anti-PD-1 (a well-characterized checkpoint inhibitor). These findings support IGSF1 as an immune target that might complement existing cancer immunotherapeutics.

Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25.

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

TCOF1 promotes the colorectal cancer progression by stabilizing ß-catenin.

Colorectal cancer (CRC) is one of the highest mortality rates worldwide, and various studies reported to the occurrence of CRC. In particular, the Wnt/ß-catenin pathway is known to be a major factor in the progression of CRC and ß-catenin involved in the expression of its downstream target genes. We searched for TCOF1 through sliver staining to identify a new binding partner for ß-catenin and to investigate the role of the gene involved in CRC. Treacle Ribosome Biogenesis Factor 1 (TCOF1) is a nucleolar protein that regulates the transcription of ribosomal DNA (rDNA). There are many reports of genetic studies on TCOF1 mutations and defects, but its function in CRC remains unknown. We demonstrated that TCOF1 and ß-catenin expression in tissue microarray (TMA) containing 101 individual CRC and 17 adjacent normal samples. Additionally, the effects of TCOF1 knockdown or overexpression were examined proliferation, colony formation assay, western blot, and quantitative real-time PCR (qRT-PCR). TCOF1 knockdown or overexpression regulates cell proliferation about three-fold and the phosphorylation of ß-catenin, cyclin D1 expression levels. Besides, we discovered the mechanism through which TCOF1 regulates the stability of ß-catenin was involved in degradation through proteasome using ubiquitination assay. Finally, we confirmed the interaction of TCOF1 with the tankyrase inhibitor NVP-TNKS656, which destabilizes ß-catenin through in vitro and in vivo. Collectively, this study shows that significantly correlation was observed that TCOF1 and ß-catenin were risk factor for tumor progression. The stability of ß-catenin via regulating TCOF1 expression could be a potential strategy for therapeutic with CRC.

Targeting isoforms of RON kinase (MST1R) drives antitumor efficacy.

Recepteur d'origine nantais (RON, MST1R) is a single-span transmembrane receptor tyrosine kinase (RTK) aberrantly expressed in numerous cancers, including various solid tumors. How naturally occurring splicing isoforms of RON, especially those which are constitutively activated, affect tumorigenesis and therapeutic response, is largely unknown. Here, we identified that presence of activated RON could be a possible factor for the development of resistance against anti-EGFR (cetuximab) therapy in colorectal cancer patient tissues. Also, we elucidated the roles of three splicing variants of RON, RON Δ155, Δ160, and Δ165 as tumor drivers in cancer cell lines. Subsequently, we designed an inhibitor of RON, WM-S1-030, to suppress phosphorylation thereby inhibiting the activation of the three RON variants as well as the wild type. Specifically, WM-S1-030 treatment led to potent regression of tumor growth in solid tumors expressing the RON variants Δ155, Δ160, and Δ165. Two mechanisms for the RON oncogenic activity depending on KRAS genotype was evaluated in our study which include activation of EGFR and Src, in a trimeric complex, and stabilization of the beta-catenin. In terms of the immunotherapy, WM-S1-030 elicited notable antitumor immunity in anti-PD-1 resistant cell derived mouse model, likely via repression of M1/M2 polarization of macrophages. These findings suggest that WM-S1-030 could be developed as a new treatment option for cancer patients expressing these three RON variants.

Mutant PIK3CA as a negative predictive biomarker for treatment with a highly selective PIM1 inhibitor in human colon cancer.

Significant improvement in targeted therapy for colorectal cancer (CRC) has occurred over the past few decades since the approval of the EGFR inhibitor cetuximab. However, cetuximab is used only for patients possessing the wild-type oncogene KRAS, NRAS, and BRAF, and even most of these eventually acquire therapeutic resistance, via activation of parallel oncogenic pathways such as RAS-MAPK or PI3K/Akt/mTOR. The two aforementioned pathways also contribute to the development of therapeutic resistance in CRC patients, due to compensatory and feedback mechanisms. Therefore, combination drug therapies (versus monotherapy) targeting these multiple pathways may be necessary for further efficacy against CRC. In this study, we identified PIK3CA mutant (PIK3CA MT) as a determinant of resistance to SMI-4a, a highly selective PIM1 kinase inhibitor, in CRC cell lines. In CRC cell lines, SMI-4a showed its effect only in PIK3CA wild type (PIK3CA WT) cell lines, while PIK3CA MT cells did not respond to SMI-4a in cell death assays. In vivo xenograft and PDX experiments confirmed that PIK3CA MT is responsible for the resistance to SMI-4a. Inhibition of PIK3CA MT by PI3K inhibitors restored SMI-4a sensitivity in PIK3CA MT CRC cell lines. Taken together, these results demonstrate that sensitivity to SMI-4a is determined by the PIK3CA genotype and that co-targeting of PI3K and PIM1 in PIK3CA MT CRC patients could be a promising and novel therapeutic approach for refractory CRC patients.

DGG-300273, a novel WNT/ß-catenin inhibitor, induces apoptotic cell death by activating ROS-BIM signaling in a Wnt-dependent manner in colon cancer cells.

Dysregulated Wnt signaling is associated with malignant oncogenic transformation, especially in colon cancer. Recently, numerous drugs have been developed based on tumorigenesis biomarkers, thus having high potential as drug targets. Likewise, WNT/ß-catenin pathway members are attractive therapeutic targets for colon cancer and are currently in various stages of development. However, although inhibitors of proteins regulating the WNT/ß-catenin signaling pathway have been extensively studied, they have yet to be clinically approved, and the underlying molecular mechanism(s) of their anticancer effects remain poorly understood. Herein, we show that a novel WNT/ß-catenin inhibitor, DGG-300273, inhibits colon cancer cell growth in a Wnt-dependent manner due to upregulation of the BCL2-family protein Bim and caspase-dependent apoptotic cell death. Additionally, DGG-300273-mediated cell death occurs by increased reactive oxygen species (ROS), as shown by abrogation of apoptotic cell death and ROS production following pretreatment with the antioxidant N-acetylcysteine. These results suggest that DGG-300273 represents a promising investigational drug for the treatment of Wnt-associated cancer, thus warranting further characterization and study.

CJ14939, a Novel JAK Inhibitor, Increases Oxaliplatin-induced Cell Death Through JAK/STAT Pathway in Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer is reported to have the highest mortality rate among human malignancies. Although many research results for the treatment of colorectal cancer have been reported, there is no suitable treatment when resistance has developed. Therefore, it is necessary to develop new therapeutic agents. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling plays an essential role in cell differentiation, proliferation, and survival. Abnormal activation of the JAK/STAT signaling pathway, by gene mutation or amplification, may induce cancer development, and sustained JAK/STAT activation is involved in chemoresistance. While many therapeutic agents have been developed to treat colon cancer, there remains no drug to overcome resistance to chemotherapies. The purpose of this study was to determine the potential of CJ14939 as a novel JAK inhibitor for the treatment of colorectal cancer. MATERIALS AND METHODS: In this study, cell culture, cell death assay, 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, colony formation assay, immunoblot analysis and tumor xenograft were applied. RESULTS: CJ14939 induced cell death, and inhibited phosphorylation of JAK1 and STAT3 in colorectal cancer cells. Furthermore, CJ14939 also promoted oxaliplatin-induced cell death, up-regulated expression of cleaved caspase-3, and down-regulated expression of phospho-JAK1 and phospho-STAT3. In vivo, co-treatment with CJ14939 and oxaliplatin notably reduced tumor growth when compared with CJ14939 or oxaliplatin treatment alone. CONCLUSION: This study identifies the important potential of CJ14939 in colorectal cancer treatment and suggests that combining CJ14939 with oxaliplatin might be a novel therapeutic strategy for patients with colorectal cancer.

Mutation SVCT2 promotes cell proliferation, invasion and migration in colorectal cancer.

The sodium-dependent vitamin C transporter 2 (SVCT2) surface glycoprotein regulates ascorbate accumulation in the plasma, often resulting in the induction of cancer cell death. Therefore, high expression of this gene associates with increased overall survival in several cancers. However, in colorectal cancer (CRC), high (likely mutated) SVCT2 expression relates to poor overall survival, and its functional significance has not been studied. Thus, we hypothesize that mutant SVCT2 expression could affect CRC patient survival. According to biological databases, SVCT2 has been found to be mutated frequently, and SVCT2 E264K has a particularly high pathogenic score (0.98), compared to other SVCT2 mutant sites, in CRC patients. Interestingly, our results reveal expression of SVCT2 E264K in many CRC tissues and cells. Also, we found wild-type SVCT2 expression to be largely localized to the cytoplasm and membrane, while SVCT2 E264K was restricted to the cytoplasm. We further found that SVCT2 E264K overexpression increases cell growth. By contrast, SVCT2 E264K knockdown significantly reduced cell proliferation and promoted cell apoptosis, resulting in inhibition of cell invasion and migration. Taken together, SVCT2 E264K plays a critical role in proliferation in CRC. Our results suggest that SVCT2 E264K could be a promising novel therapeutic target in CRC.

Role of p53 in transcriptional repression of SVCT2.

SVCT2, Sodium-dependent Vitamin C Transporter 2, uniquely transports ascorbic acid (also known as vitamin C and ascorbate) into all types of cells. Vitamin C is an essential nutrient that must be obtained through the diet and plasma levels are tightly regulated by transporter activity. Vitamin C plays an important role in antioxidant defenses and is a cofactor for many enzymes that enable hormone synthesis, oxygen sensing, collagen synthesis and epigenetic pathways. Although SVCT2 has various functions, regulation of its expression/activity remains poorly understood. We found a p53-binding site, within the SVCT2 promoter, using a transcription factor binding-site prediction tool. In this study, we show that p53 can directly repress SVCT2 transcription by binding a proximal- (~-185 to -171 bp) and a distal- (~-1800 to -1787 bp) p53-responsive element (PRE), Chromatin immunoprecipitation assays showed that PRE-bound p53 interacts with the corepressor-histone deacetylase 3 (HDAC3), resulting in deacetylation of histones Ac-H4, at the proximal promoter, resulting in transcriptional silencing of SVCT2. Overall, our data suggests that p53 is a potent transcriptional repressor of SVCT2, a critical transporter of diet-derived ascorbic acid, across the plasma membranes of numerous essential tissue cell types.

Cell fate decisions by c-Myc depend on ZBTB5 and p53.

The oncoprotein, c-Myc, not only promotes cell proliferation, but can also induce or sensitize cells to apoptosis. However, how c-Myc decides cell fate and which c-Myc downstream target genes are involved remain unknown. Previously, we showed that ZBTB5 (zinc finger and BTB domain-containing 5) is a proto-oncogene that stimulates cell proliferation. ZBTB5 represses p21/CDKN1A by competing with p53 and recruiting corepressor histone deacetylase complexes. Herein, we found that c-Myc directly activates the transcription of ZBTB5. In the absence of p53, ZBTB5 is acetylated at K597 by interacting with p300, and activates transcription of NOXA, which induces apoptosis. In contrast, in the presence of p53, ZBTB5 interacts with p53 and acetylation at ZBTB5 K597 is blocked. ZBTB5 without K597 acetylation interacts with mSin3A/HDAC1 to repress p21/CDKN1A transcription and promote cell proliferation. Cell fate decisions by c-Myc depend on ZBTB5, p53 and p300, and acetylation of ZBTB5 K597.

Inhibition of JAK1/2 can overcome EGFR-TKI resistance in human NSCLC.

Non-small lung cancer (NSCLC) is the most common cancer in the world. The epidermal growth factor receptor (EGFR) gene is mutated in approximately 10% of lung cancer cases in the US and 50% of lung cancer in Asia. The representative target therapeutic agent, erlotinib (EGFR tyrosine kinase inhibitor; EGFR TKI), is effective in inactivating EGFR in lung cancer patients. However, approximately 50-60% of patients are resistant to EGFR TKI. These populations are associated with the EGFR mutation. To overcome resistance to EGFR TKI, we discovered a JAK1 inhibitor, CJ14939. We investigated the efficacy of CJ14939 in human NSCLC cell lines in vitro and in vivo. Our results showed that CJ14939 induced the inhibition of cell growth. Moreover, we demonstrated that combination treatment with erlotinib and CJ14939 induced cell death in vitro and inhibited tumor growth in vivo. In addition, we confirmed the suppression of phosphorylated EGFR, JAK1, and Stat3 expression in erlotinib and CJ14939-treated human NSCLC cell lines. Our results provide evidence that JAK inhibition overcomes resistance to EGFR TKI in human NSCLCs.

Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells.

Inhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.

Temporal and differential regulation of KAISO-controlled transcription by phosphorylated and acetylated p53 highlights a crucial regulatory role of apoptosis.

Transcriptional regulator KAISO plays a critical role in cell cycle arrest and apoptosis through modulation of p53 acetylation by histone acetyltransferase p300. KAISO potently stimulates apoptosis in cells expressing WT p53, but not in p53-mutant or p53-null cells. Here, we investigated how KAISO transcription is regulated by p53, finding four potential p53-binding sites (p53-responsive DNA elements; p53REs) located in a distal 5'-upstream regulatory element, intron 1, exon 2 coding sequence, and a 3'-UTR region. Transient transcription assays of pG5-p53RE-Luc constructs with various p53REs revealed that p53 activates KAISO (ZBTB33) transcription by acting on p53RE1 (-4326 to -4227) of the 5'-upstream region and on p53RE3 (+2929 to +2959) of the exon 2 coding region during early DNA damage responses (DDRs). ChIP and oligonucleotide pulldown assays further disclosed that p53 binds to the p53RE1 and p53RE3 sites. Moreover, ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) kinase-mediated p53 phosphorylation at Ser-15 or Ser-37 residues activated KAISO transcription by binding its p53RE1 or p53RE3 sites during early DDR. p53RE1 uniquely contained three p53-binding half-sites, a structural feature important for transcriptional activation by phosphorylated p53 Ser-15·Ser-37. During the later DDR phase, a KAISO-mediated acetylated p53 form (represented by a p53QRQ acetyl-mimic) robustly activated transcription by acting on p53RE1 in which this structural feature is not significant, but it provided sufficient KAISO levels to confer a p53 "apoptotic code." These results suggest that the critical apoptosis regulator KAISO is a p53 target gene that is differently regulated by phosphorylated p53 or acetylated p53, depending on DDR stage.

Hypoxia-induced RelA/p65 derepresses SLC16A3 (MCT4) by downregulating ZBTB7A.

Overexpressed Solute Carrier Family 16 Member 3 (SLC16A3, also called MCT4) plays a critical role in hypoxic cancer cell growth and proliferation, by expelling glycolysis-derived lactate across the plasma membrane. However, how SLC16A3 expression is regulated, under hypoxic conditions, is poorly understood. FBI-1, encoded by ZBTB7A, is a proto-oncoprotein. Interestingly, under hypoxic conditions, expression of SLC16A3, and hypoxia-inducible factor-1 (HIF-1), increased gradually, while FBI-1 expression decreased, suggesting a negative correlation between SLC16A3/HIF-1 and FBI-1 expression. Consequently, we hypothesized that FBI-1 might regulate SLC16A3 and/or HIF-1 expression. Transient transfection and transcription assays of SLC16A3 promoter reporter fusion constructs, oligonucleotide-pulldowns, and ChIP assays, showed that HIF-1α activates SLC16A3 by binding to a hypoxia-response element (HRE), while ectopic FBI-1 potently repressed SLC16A3, by binding to both FBI-1-response elements (FREs) and HREs, during hypoxia. Further evidence for this model was downregulation of ZBTB7A, correlated with SLC16A3 upregulation, in hypoxic colon cancer cells. We also investigated how FBI-1 expression is downregulated during hypoxia. The 5'-upstream regulatory region of ZBTB7A contains two NF-κB-binding sites and two HREs. Interestingly, hypoxia activated NF-κB (RelA/p65) and also increased its nuclear translocation. NF-κB repressed ZBTB7A by binding NF-κB-binding elements, and downregulated the repressor FBI-1, thereby increasing SLC16A3 transcription. While transcriptional repression of SLC16A3 by FBI-1 inhibited lactate efflux, repression of ZBTB7A and activation of lactate efflux by NF-κB, increased colon cancer cell growth and proliferation.

Zbtb7c is a critical gluconeogenic transcription factor that induces glucose-6-phosphatase and phosphoenylpyruvate carboxykinase 1 genes expression during mice fasting.

Gluconeogenesis is essential for blood glucose homeostasis during fasting and is regulated by various enzymes, which are encoded by gluconeogenic genes. Those genes are controlled by various transcription factors. Zinc finger and BTB domain-containing 7c (Zbtb7c, also called Kr-pok) is a BTB-POZ family transcription factor with proto-oncogenic activity. Previous findings have indicated that Zbtb7c is involved in the regulation of fatty acid biosynthesis, suggesting an involvement also in primary metabolism. We found here that fasting induced Zbtb7c expression in the mouse liver and in primary liver hepatocytes. We also observed that Zbtb7c-knockout mice have decreased blood glucose levels, so we investigated whether Zbtb7c plays a role in gluconeogenesis. Indeed, differential gene expression analysis of Zbtb7c-knockout versus wild type mouse livers showed downregulated transcription of gluconeogenic genes encoding the glucose 6-phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), while Zbtb7c expression upregulated these two genes, under fasting conditions. Mechanistically, we found that when complexed with histone deacetylase 3 (Hdac3), Zbtb7c binds insulin response elements (IREs) within the G6pc and Pck1 promoters. Moreover, complexed Zbtb7c deacetylated forkhead box O1 (Foxo1), thereby increasing Foxo1 binding to the G6pc and Pck1 IREs, resulting in their transcriptional activation. These results demonstrate Zbtb7c to be a crucial metabolic regulator of blood glucose homeostasis, during mammalian fasting.

Reciprocal negative regulation between the tumor suppressor protein p53 and B cell CLL/lymphoma 6 (BCL6) via control of caspase-1 expression.

Even in the face of physiological DNA damage or expression of the tumor suppressor protein p53, B cell CLL/lymphoma 6 (BCL6) increases proliferation and antagonizes apoptotic responses in B cells. BCL6 represses TP53 transcription and also appears to inactivate p53 at the protein level, and additional findings have suggested negative mutual regulation between BCL6 and p53. Here, using Bcl6-/- knockout mice, HEK293A and HCT116 p53-/- cells, and site-directed mutagenesis, we found that BCL6 interacts with p53 and thereby inhibits acetylation of Lys-132 in p53 by E1A-binding protein p300 (p300), a modification that normally occurs upon DNA damage-induced cellular stress and whose abrogation by BCL6 diminished transcriptional activation of p53 target genes, including that encoding caspase-1. Conversely, we also found that BCL6 protein is degraded via p53-induced, caspase-mediated proteolytic cleavage, and the formation of a BCL6-p53-caspase-1 complex. Our results suggest that p53 may block oncogenic transformation by decreasing BCL6 stability via caspase-1 up-regulation, whereas aberrant BCL6 expression inactivates transactivation of p53 target genes, either by inhibiting p53 acetylation by p300 or repressing TP53 gene transcription. These findings have implications for B cell development and lymphomagenesis.

Zbtb7c is a molecular 'off' and 'on' switch of Mmp gene transcription.

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that play roles in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. The expression of MMP gene is tightly regulated and shows cell- and tissue-specific expression patterns. Despite their differential expression, MMP genes have AP-1 (activator protein-1) binding elements within their promoters. Interestingly, c-JUN phosphorylation by cytokine signaling decreased its interaction with NCoR, but increased its interaction with p300, resulting in activation of MMP gene transcription. Here, we found that Zbtb7c (Kr-pok) is a critical component of a transcriptional repressor complex containing c-Jun and NCoR. c-Jun, bound at AP-1, interacts with Zbtb7c, which in turn recruits an NCoR/Hdac3 complex to repress several Mmp (-8, -10, -13, and -16) genes. The molecular interaction between c-Jun and Zbtb7c also prevents phosphorylation of c-Jun by p-Jnk, However, Zbtb7c phosphorylation by p-Jnk (induced by TNFα), and its (Zbtb7c) subsequent degradation by the ubiquitin-mediated proteasomal pathway, leads to c-Jun phosphorylation by p-Jnk. Promoter-bound p-c-Jun then recruits the coactivator p300 to upregulate Mmp gene. Overall, these findings show that Zbtb7c is a key molecule that recruits an NCoR/Hdac3 complex to inhibit phosphorylation of c-Jun, and thereby repress Mmp gene expression.

Role of MIZ-1 in AMELX gene expression.

Amelogenin (AMELX) is the main component of the developing tooth enamel matrix and is essential for enamel thickness and structure. However, little is known about its transcriptional regulation. Using gene expression analysis, we found that MIZ-1, a potent transcriptional activator of CDKN1A, is expressed during odontoblastic differentiation of hDPSCs (human dental pulp stem cells), and is essential for odontoblast differentiation and mineralization. We also investigated how MIZ-1 regulates gene expression of AMELX. Oligonucleotide-pull down assays showed that MIZ-1 binds to an MRE (MIZ-1 binding element) of the AMELX proximal promoter region (bp, -170 to -25). Combined, our ChIP, transient transcription assays, and promoter mutagenesis revealed that MIZ-1 directly binds to the MRE of the Amelx promoter recruits p300 and induces Amelx gene transcription. Finally, we show that the zinc finger protein MIZ-1 is an essential transcriptional activator of Amelx, which is critical in odontogenesis and matrix mineralization in the developing tooth.

Transcriptional activation of APAF1 by KAISO (ZBTB33) and p53 is attenuated by RelA/p65.

KAISO, a member of the POK protein family, is induced by DNA-damaging agents to enhance apoptosis in a p53-dependent manner. Previously, we found that p53 interacts with KAISO, and acetylation of p53 lysine residues by p300 is modulated by KAISO. APAF1, the core molecule of the apoptosome, is transcriptionally activated by KAISO only in cells expressing p53, which binds to APAF1 promoter p53-response elements (p53REs). APAF1 transcriptional upregulation is further enhanced by KAISO augmentation of p53 binding to the APAF1 promoter distal p53RE#1 (bp, -765 to -739). Interestingly, a NF-κB response element, located close to the p53RE#1, mediates APAF1 transcriptional repression by affecting interaction between KAISO and p53. Ectopic RelA/p65 expression led to depletion of nuclear KAISO, with KAISO being mainly detected in the cytoplasm. RelA/p65 cytoplasmic sequestration of KAISO prevents its nuclear interaction with p53, decreasing APAF1 transcriptional activation by a p53-KAISO-p300 complex in cells exposed to genotoxic stresses. While KAISO enhances p53-dependent apoptosis by increasing APAF1 gene expression, RelA/p65 decreases apoptosis by blocking interaction between KAISO and p53. These findings have relevance to the phenomenon of cancer cells' diminished apoptotic capacity and the onset of chemotherapy resistance.