Expression Of ZBTB7A Research Articles

ZBTB7A governs estrogen receptor alpha expression in breast cancer.

ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer.

Stable knockdown of ZBTB7A promotes cell proliferation and progression in nasopharyngeal carcinoma

Although high expression of ZBTB7A is positively relative to metastasis in nasopharyngeal carcinoma (NPC) patients, the association between its low expression and metastasis of NPC remains unclear. The present study aimed to definitely identify the association. The level of ZBTB7A was effectively knocked down by stable transfection of short hair RNA plasmid in NPC cell lines CNE2 and 5-8F (shRNA-CNE2 and shRNA-5-8F), compared with the cells that stably transfected empty plasmid (NC-CNE2 and NC-5-8F). The levels of ZBTB7A were assessed by real-time polymerase chain reaction and Western blot in the cell lines. MTT assay, colorimetric focus-formation assay, flow cytometry, wound healing assay, transwell assays, and xenograft model were performed to analyze cell vitality, proliferation, cell cycle, migration, invasion, and tumorigenicity. The levels of ZBTB7A were effectively reduced in shRNA-CNE2 and shRNA-5-8F. Their carcinogenicity was stronger separately than the abilities of NC-CNE2 and NC-5-8F. NC-CNE2 and shRNA-CNE2 were selected to establish the xenograft model because of their stronger tumorigenicity than NC-6-10B and shRNA-5-8F. The assay showed that shRNA-CNE2 had stronger tumorigenicity than NC-CNE2. The results demonstrated the reverse association between the expression of ZBTB7A and the tumorigenicity of NPC. We postulate that some oncogenic pathways, which are suppressed by ZBTB7A, will vicariously promote the proliferation and progression of NPC when ZBTB7A is decreased.

Differences in Zbtb7a expression cause heterogeneous changes in human nasopharyngeal carcinoma CNE3 sublines

The present study aimed to determine the association between changes in Zbtb7a expression levels and heterogeneity of nasopharyngeal carcinoma (NPC) CNE3 sublines. CNE3 sublines were established by screening of serial dilution and continuous passage. Proliferative ability and tumorigenicity of the sublines were analyzed separately by soft-agar colony formation and mouse studies. The NPC tissues from mice were analyzed by histological evaluation and immunohistochemistry. The expression levels of Zbtb7a mRNA and protein were analyzed separately by quantitative reverse transcription polymerase chain reaction and western blotting. According to findings from the soft-agar colony formation and mouse studies, two sublines with increased tumorigenicity compared with other sublines were transfected transiently with Zbtb7a short hairpin RNA (shRNA) recombinant plasmid. The changes in viability, migration and invasion abilities were evaluated separately by MTT, colorimetric focus-formation, Transwell migration and invasion assays. The sublines CNE3-GX6 and CNE3-GX11 were selected for subsequent study due to increased tumorigenicity and increased Zbtb7a expression levels compared with the other sublines. High metastatic potency was not observed in all of the sublines. Zbtb7a expression levels were positively associated with tumorigenic degree of the sublines. The growth, migration and invasion abilities of the sublines transfected with Zbtb7a shRNA plasmid were decreased compared with the cells transfected with empty vector in the negative control group. The findings suggest Zbtb7a expression levels may be associated with heterogeneity of CNE3 sublines. Therefore, Zbtb7a may have an important role in the regulatory mechanism of NPC heterogeneity.

KLF1 directly activates expression of the novel fetal globin repressor ZBTB7A/LRF in erythroid cells.

Genes encoding the human β-like hemoglobin proteins undergo a developmental switch from fetal γ-globin to adult β-globin expression around the time of birth. β-hemoglobinopathies, such as sickle-cell disease and β-thalassemia, result from mutations affecting the adult β-globin gene. The only treatment options currently available carry significant adverse effects. Analyses of heritable variations in fetal hemoglobin (HbF) levels have provided evidence that reactivation of the silenced fetal γ-globin genes in adult erythroid cells is a promising therapy. The γ-globin repressor BCL11A has become the major focus, with several studies investigating its regulation and function as a first step to inhibiting its expression or activity. However, a second repression mechanism was recently shown to be mediated by the transcription factor ZBTB7A/LRF, suggesting that understanding the regulation of ZBTB7A may also be useful. Here we show that Krüppel-like factor 1 (KLF1) directly drives expression of ZBTB7A in erythroid cells by binding to its proximal promoter. We have also uncovered an erythroid-specific regulation mechanism, leading to the upregulation of a novel ZBTB7A transcript in the erythroid compartment. The demonstration that ZBTB7A, like BCL11A, is a KLF1 target gene also fits with the observation that reduced KLF1 expression or activity is associated with HbF derepression.

High Expression of ZBTB7A at Diagnosis Associated with Inferior Outcome in Acute Myeloid Leukemia Patients Receiving Hematopoietic Stem Cell Transplantation

Introduction: The transcription factor ZBTB7A regulates early differentiation of hematopoietic progenitors & has been associated with oncogenic as well as oncosuppressive functions. While it was shown that ectopic overexpression of Zbtb7a in immature lymphocytes leads to the development of an aggressive T-cell lymphoblastic leukemia, high ZBTB7A expression in cytogenetically normal acute myeloid leukemia (CN-AML) is associated with improved outcomes. Furthermore, recently a leukemogenic cooperation between RUNX1/RUNX1T1 &ZBTB7A mutations in t(8;21)-associated AML was suggested.Here, we further evaluated the complex role of ZBTB7A expression in hematopoietic malignancies by assessing its potential prognostic impact in AML pts undergoing hematopoietic stem cell transplantation (HSCT) after non-myeloablative conditioning (NMA).Methods: We analyzed bone marrow (BM) at diagnosis of 140 pts (median age 63 years [y], range 37-75y) treated at our institution between 2000 & 2015. All pts received NMA conditioning (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation) followed by HSCT in complete remission with (CR; n=111; 79.3%) or without peripheral hematological recovery (CRi; n=29; 20.7%). Median follow-up for pts alive was 3.5y.Our cohort included pts with CN-AML (n=62, 44.3%), complex karyotype (n=17; P=12.1%) & other cytogenetic abnormalities (n=61; 43.6%). At diagnosis mutations in the genes CEBPA, DNMT3A, IDH1, IDH2, NPM1 & the presence of FLT3-ITD were determined. In diagnostic BM cytogenetics were analyzed using standard techniques for banding & fluorescence in-situ hybridization & the expression of common surface markers was analyzed using flow cytometry. The expression of ZBTB7A was assessed using quantitative RT-PCR & normalized to ABL1 as internal control. As a cut-off the third quartile of normalized gene expression was identified to group high & low ZBTB7A expressers.Results: At diagnosis pts with a high ZBTB7A expression more often had a complex karyotype (P=.02) & were less likely to have core-binding factor AML by trend (P=.18). Additionally, high ZBTB7A levels associated with significantly fewer blasts in peripheral blood (P=.008) & BM (P=.02). The BM mononuclear cells in high ZBTB7A expressers were to a smaller extent positive for myeloid markers (CD38 P=.03; CD33 P=.11; CD13 P=.13) & exhibited a higher percentage of erythroid (Glycophorin A P=.03) as well as monocytic (CD11b P=.04; CD14 P=.01) surface markers. We did not find any statistical associations between ZBTB7A levels & the mutation status of NPM1, CEBPA, IDH1, DNMT3A or the presence of FLT3-ITD. Yet, there was a trend for more IDH2 mutations in the group of high ZBTB7A expressers (P=.18).At diagnosis a high expression of ZBTB7A associated with a significantly higher cumulative incidence of relapse (CIR; P=.002, Figure 1A). This finding also translated into a significantly shorter overall survival (OS; P=.01; Figure 1B) for AML pts with high ZBTB7A levels at diagnosis. When we restricted our analyses to CN-AML, high ZBTB7A expression remained a negative prognostic factor by trend (CIR P=.16; OS P=.11).Conclusion: Expression of ZBTB7A associated with distinct biological features & surface marker pattern in AML. This underlines the results of recent studies which identified ZBTB7A as a novel player in leukemogenesis. However, our findings are in contrast with the previously shown favorable prognostic impact of high ZBTB7A levels in a CN-AML cohort mainly treated with chemotherapy. In contract all pts included in our studies were consolidated with NMA-HSCT. Since this treatment regimen is mainly based on the graft versus leukemia effect a high ZBTB7A expression could potentially interfere with the immunological recognition of the AML blasts resulting in a reduced response to NMA-HSCT.Consequently, future functional studies & clinical trials should aim at further characterize the complex role of ZBTB7A in AML. [Display omitted] DisclosuresPoenisch:Mundipharma: Research Funding.

Abstract 3657: ZBTB7A is a tumor suppressor and novel therapeutic target in triple-negative breast cancer

Abstract Due to lack of targeted therapies triple-negative breast cancers (TNBC) are the deadliest form of breast cancer, raising a pressing need to develop new therapeutic strategies. Recent advances in tumor metabolism have created exciting therapeutic opportunities by unveiling many previously unrecognized therapeutic targets. Like other tumor types, TNBCs also feature elevated glucose and glutamine metabolism (Shen, L., et al. 2015; McCleland, M., et al. 2012). A better understanding of the underlying mechanisms may uncover novel therapeutic targets for TNBC. Our lab has recently discovered that ZBTB7A suppresses glycolysis by transcriptionally repressing key genes within the glycolytic pathway in various cancer types (Liu, X.S., et al. 2014). Currently, ZBTB7A is proposed as an oncogene in breast cancer (Zu, X., et al. 2011), however our data suggest that ZBTB7A acts as a tumor suppressor specifically in TNBC. The purpose of this study was to determine the potential therapeutic value of targeting ZBTB7A in TNBC. Using publicly available databases, we find that low ZBTB7A expression is associated with poor prognosis in patients with hormone receptor-negative breast cancers. Our studies corroborate with patient derived data by demonstrating that reduced expression of ZBTB7A in TNBC cell lines leads to increased proliferation and resistance to radiation and chemotherapy. Differential gene expression analysis across human breast cancer samples in TCGA database revealed an inverse correlation between ZBTB7A and glycolytic genes. Overexpression of ZBTB7A in TNBC cells leads to decreased MCT1, LDHB and GLUT3 expression at the transcript level, consistent with our published data. Modulation of ZBTB7A expression also leads to changes in lactate production and glucose consumption in TNBC cells. Results from this study support ZBTB7A as a novel tumor suppressor and perspective therapeutic target in TNBC. Citation Format: Mary Ellen Molloy, Monika Lewinska, Xue-Song Liu, Angela A. Wang, Zhi-Min Yuan. ZBTB7A is a tumor suppressor and novel therapeutic target in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3657.

A Zbtb7a proto-oncogene as a novel target for miR-125a.

In our previous study, we showed that miR-125a directly targeted a WT1 oncogene, which was overexpressed in leukemia and various kinds of solid tumors including lung, breast, gastric, and colon cancers, and brain tumors and was deeply involved in leukemogenesis and tumorigenesis and that miR-125a knockout mice overexpressed WT1 and developed myeloproliferative disease. It had been also reported that miR-125a is downregulated in leukemia and various types of solid tumors such as lung cancers, suggesting its tumor suppressor function. Therefore, it is important to elucidate what is target(s) of miR-125a for understandings of such functions although few target genes for it are known. In the present study, Zbtb7a oncogene was identified as a potential target for miR-125a by gene expression profiling in miR-125a knockout mice combined with bioinformatics target prediction. EGFP-3'UTR reporter assay showed that miR-125a suppressed Zbtb7a expression through its direct binding to the Zbtb7a-3'UTR. Zbtb7a knockdown by siRNA suppressed cell proliferation and induced G1 cell cycle arrest and apoptosis in lung cancer cells. Furthermore, miR-125a expression showed a negative correlation with Zbtb7a expression in non-small cell lung cancer tissues. The present study showed for the first time that Zbtb7a was a direct target for miR-125a and was involved in cell cycle progression and apoptosis of lung cancer cells. These results also demonstrated that deregulation of miR-125a-Zbtb7a signaling was associated with the development and progression of lung cancer. © 2015 Wiley Periodicals, Inc.

Somatic human ZBTB7A zinc finger mutations promote cancer progression.

We recently reported that ZBTB7A is a bona fide transcription repressor of key glycolytic genes and its downregulation in human cancer contributes to tumor metabolism. As reduced expression of ZBTB7A is found only in a subset of human cancers, we explored alternative mechanisms of its inactivation by mining human cancer genome databases. We discovered recurrent somatic mutations of ZBTB7A in multiple types of human cancers with a marked enrichment of mutations within the zinc finger domain. Functional characterization of the mutants demonstrated that mutations within the zinc finger region of ZBTB7A invariably resulted in loss of function. As a consequence, the glycolytic genes were markedly upregulated in cancer cells harboring ZBTB7A zinc finger mutation, leading to increased glycolysis and proliferation. Our study uncovers the loss-of-function mutation in ZBTB7A as a novel mechanism causing elevated glycolysis in human cancer, which carries important therapeutic implication.

C/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer

MicroRNAs have been reported to play key roles in various human cancers, including gastric cancer. However, understanding of the expression of miR-100 and its regulatory mechanisms in human gastric cancer remains elusive. In this study, we reveal that miR-100 is downregulated in gastric cancer samples and gastric cancer cell lines. Furthermore, lower miR-100 expression was found in primary gastric cancer samples with lymphatic metastasis compared to those without lymphatic metastasis. Overexpression of miR-100 suppressed tumor growth in vivo and inhibited gastric cancer invasion and metastasis in vitro and in vivo. Furthermore, we demonstrated that miR-100 reduced gastric cancer aggressiveness by directly targeting ZBTB7A. Knockdown of ZBTB7A by siRNA disrupted gastric cancer progression by impairing tumor invasion and metastasis. High expression of ZBTB7A was significantly correlated with poorer prognosis in gastric cancer patients. Our results also showed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) could induce the expression of miR-100 by binding to the putative promoter region of miR-100. This study demonstrated that miR-100 could be induced by C/EBPα and may act as a tumor suppressor gene by inhibiting ZBTB7A.

De novo development of dsa and autoantibodies to lung associated self-antigens is associated with upregulation of transcription factor Zbtb7a: Role in chronic rejection following human lung transplantation

Aim Administration of anti-MHC class I (H-2Kb) leads to induction of transcription factor Zinc finger and BTB domain containing 7A (Zbtb7a) leading to inflammatory cascade and chronic rejection in a murine obliterative airway disease model. Development of DSA and Abs to lung-associated self-antigens (K-alpla-1 Tubulin (K α 1T) and Collagen V (Col-V) are significant risk factors in development of bronchiolitis obliterans syndrome (BOS) following human lung transplantation (LTx). The current study focused on the role of Zbtb7a in development of BOS following human LTx. Methods We analyzed 25 LTx patients that developed BOS years after LTx as well as 10 stable LTx performed at Barnes-Jewish Washington University. Kinetics of Zbtb7a expression was studied in bronchoalveolar lavage (BAL) cells. Quantitative PCR analysis was performed to evaluate mRNA levels and data was compared with the DSA, autoantibody and immunosuppression. Results Increases in the Zbtb7a expression was observed in BAL cells collected at visits immediately prior to clinical BOS diagnosis compared with earlier visits. Zbtb7a expression was significantly (p 0.01) increased (150%) in pre-BOS BAL cells. On the contrary, stable patients did not demonstrate any change in Zbtb7a levels. All of the BOS+ patients had developed DSA and Abs to K α 1T and Col-V. Zbtb7a expression in 13 patients who received Ab directed therapy following DSA demonstrated significant reduction along with decrease in DSA and Abs to lung self-antigens. Therefore, Zbtb7a activation is an important inducer of the DSA and autoantibody. Conclusions Up regulation of Zbtb7a was positively associated with de novo DSA and Abs to lung-associated self-antigens. Ab directed therapy significantly reduced Zbtb7a expression. We propose that Zbtb7a may be used as a prognostic marker in evaluating Ab-induced inflammation in the lung as well as efficacy of Ab directed therapy following development of DSA.

OR7 Antibodies to MHC class I induce transcription factor Zbtb7a and regulate development of autoimmunity leading to chronic rejection

Aim Chronic rejection, bronchiolitis obliterans syndrome (BOS), is a major hurdle following human lung transplantation. Antibodies (Ab) to HLA (DSA) and lung-associated antigens (Collagen V (Col-V) and K-alpha-1 Tubulin (K α 1T) have been associated with development of BOS. Our goal was to demonstrate early events (genes and their role in inflammation) associated with administration of anti-MHC class I (H-2 Kb) into murine (C57BL/6) lungs that precede cellular and humoral autoimmunity and chronic rejection. Methods We analyzed molecular signatures arising from anti-MHC administration by genechip microarray. Zbtb7a was selected for functional analysis. siRNA-Lentivirus was used to knockdown Zbtb7a in lungs to study its role in Ab induced chronic rejection. Kinetics of Ab development and T cell responses was tested by ELISA and ELISPOT. Changes in leukocyte profile were analyzed by flow cytometry and gene expression levels by real-time PCR. Results In genechip assay, 12 genes including Zbtb7a were significantly ( p 1.5-fold) at 4 h post Ab administration. Use of siRNA-Lentivirus knocked down Zbtb7a expression. Following anti-MHC I, Zbtb7a knockdown demonstrated significant ( p α 1T and anti-Col-V, and remained free from inflammation and fibrosis. Further, anti-MHC elicited lower K α 1T and Col-V specific Th17 and Th1 cells. Moreover, less infiltration of neutrophil and B cell were seen in lungs and decreased levels of B cell (CXCL13) and neutrophil (CXCL15) chemoattractants were observed. Conclusions We demonstrate that DSA activates unique molecular signature involved in lung autoimmunity. Zbtb7a, as a transcription factor induced by DSA, is a “master regulator” of B and T cell development and has novel inflammatory functions leading to chronic rejection. By targeted knockdown, we established that Zbtb7a has an obligatory role in the amplification of inflammatory circuits where its loss rendered protection from Ab induced obliterative airway disease.

ZBTB7A Suppresses Melanoma Metastasis by Transcriptionally Repressing MCAM.

The excessive metastatic propensity of melanoma makes it the most deadly form of skin cancer, yet the underlying mechanism of metastasis remains elusive. Here, mining of cancer genome datasets discovered a frequent loss of chromosome 19p13.3 and associated downregulation of the zinc finger transcription factor ZBTB7A in metastatic melanoma. Functional assessment of ZBTB7A-regulated genes identified MCAM, which encodes an adhesion protein key to melanoma metastasis. Using an integrated approach, it is demonstrated that ZBTB7A directly binds to the promoter and transcriptionally represses the expression of MCAM, establishing ZBTB7A as a bona fide transcriptional repressor of MCAM. Consistently, downregulation of ZBTB7A results in marked upregulation of MCAM and enhanced melanoma cell invasion and metastasis. An inverse correlation of ZBTB7A and MCAM expression in association with melanoma metastasis is further validated with data from analysis of human melanoma specimens. Together, these results uncover a previously unrecognized role of ZBTB7A in negative regulation of melanoma metastasis and have important clinical implications.

Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis.

ABSTRACTThe deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. From our previous studies in the vinegar fly, Drosophila melanogaster, we have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib) and overexpression of the BTB-ZF protein Abrupt (Ab). Herein, we show that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, we show that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis has revealed that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems.

ZBTB7A acts as a tumor suppressor through the transcriptional repression of glycolysis.

Elevated glycolysis is a common metabolic trait of cancer, but what drives such metabolic reprogramming remains incompletely clear. We report here a novel transcriptional repressor-mediated negative regulation of glycolysis. ZBTB7A, a member of the POK (POZ/BTB and Krüppel) transcription repressor family, directly binds to the promoter and represses the transcription of critical glycolytic genes, including GLUT3, PFKP, and PKM. Analysis of The Cancer Genome Atlas (TCGA) data sets reveals that the ZBTB7A locus is frequently deleted in many human tumors. Significantly, reduced ZBTB7A expression correlates with up-regulation of the glycolytic genes and poor survival in colon cancer patients. Remarkably, while ZBTB7A-deficient tumors progress exceedingly fast, they exhibit an unusually heightened sensitivity to glycolysis inhibition. Our study uncovers a novel tumor suppressor role of ZBTB7A in directly suppressing glycolysis.

Insulin upregulates the expression of zinc finger and BTB domain-containing 7A in HepG2 cells

Zinc finger and BTB domain-containing 7A (Zbtb7A) is a proto-oncogene overexpressed in numerous cancers. In this study, we explored the mechanism of insulin-induced Zbtb7A expression. Real-time PCR and western blotting were used to detect Zbtb7A expression. Zbtb7A promoter activity was monitored by Luciferase reporter assay. It was shown that insulin elevates the mRNA and protein levels of the Zbtb7A gene in HepG2 cells. Using chemical inhibitors of insulin downstream pathways, we demonstrated that the insulin-induced Zbtb7A gene expression was completely blocked by LY294002, a PI3K/AKT inhibitor, and partially attenuated by the MAPK inhibitor PD98059. Transfection of HepG2 cells with a 1 kb Zbtb7A promoter-luciferase reporter construct revealed a dose-dependent activation of the Zbtb7A promoter by insulin, while mutation of the Sp1 binding site within the Zbtb7A promoter resulted in the failure of insulin-induced promoter activation, suggesting that insulin increases Zbtb7A expression through transcriptional regulation mediated by Sp1 in HepG2 cells.

P.2.a.028 Gene × environment interaction in depressive disorders: which environment is of risk?

ZBTB7A is a member of the POK family of transcription factors that possesses a POZ-domain at the N-terminus and Krüppel-like zinc-finger at the c-terminus. ZBTB7A was initially isolated as a protein that binds to the inducer of the short transcript of HIV-1 virus TAT gene promoter. The protein forms a homodimer through protein-protein interaction via the N-terminus POZ-domains. ZBTB7A typically binds to the DNA elements through its zinc-finger domains and represses transcription both by modification of the chromatin organization and through the direct recruitment of transcription factors to gene regulatory regions. ZBTB7A is involved in several fundamental biological processes including cell proliferation, differentiation, and development. It also participates in hematopoiesis, adipogenesis, chondrogenesis, cellular metabolism and alternative splicing of BCLXL, DNA repair, development of oligodendrocytes, osteoclast and unfolded protein response. Aberrant ZBTB7A expression promotes oncogenic transformation and tumor progression, but also maintains a tumor suppressive role depending on the type and genetic context of cancer. In this comprehensive review we provide information about the structure, function, targets, and regulators of ZBTB7A and its role as an oncogenic driver and transcriptional repressor in various human diseases.

Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis

Human Zbtb7A was proved to be an important molecular switch in oncogenesis. However, it is difficult to obtain its protein expression in prokaryotic system, due to high G + C content and rare codons in zbtb7a gene. Therefore, to further research the function and application of this protein, we optimized its coding sequence according to the codon bias of Pichia pastoris, synthesized the sequence with two-step PCR and confirmed the accuracy by DNA sequencing. The assembled fragment was introduced into P. pastoris expression vector pPIC9K and the resultant plasmid pPIC9K –zbtb7a–his 6 was transformed into the P. pastoris strain GS115 by electroporation. The products of the transformants induced by methanol were analyzed by 10% SDS–PAGE and identified by Western Blot assay. The expression conditions of the selected transformant were optimized. Additionally, a two-step purification protocol was applied to purify the recombinant protein. The results showed that the synthetic coding sequence of human Zbtb7A was successfully obtained and inserted into pPIC9K vector. Human Zbtb7A protein was expressed in P. pastoris and identified by western blot. The optimal conditions for its expression in P. pastoris were under a final concentration of 1% methanol and a time-course of 4d. Through the two-step purification, Zbtb7A protein was purified in high purity and its production reached up to as high as 18 mg/L. These results indicated that an effective procedure for expressing and purifying human Zbtb7A in P. pastoris was established.

Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non‐small cell lung cancer

ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14(ARF) inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non-small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two- to five-fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non-amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post-translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild-type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14(ARF)-MDM2-p53 axis. The concomitant expression of ZBTB7A with p14(ARF) (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the pathogenesis of lung cancer.