TY - JOUR T1 - Knockdown of cullin 4A inhibits growth and increases chemosensitivity in lung cancer cells. JF - J Cell Mol Med Y1 - 2016 A1 - Hung, Ming-Szu A1 - Chen, I-Chuan A1 - You, Liang A1 - Jablons, David M A1 - Li, Ya-Chin A1 - Jiang-Hua Mao A1 - Xu, Zhidong A1 - Lung, Jr-Hau A1 - Yang, Cheng-Ta A1 - Liu, Shih-Tung AB -

Cullin 4A (Cul4A) has been observed to be overexpressed in various cancers. In this study, the role of Cul4A in the growth and chemosensitivity in lung cancer cells were studied. We showed that Cul4A is overexpressed in lung cancer cells and tissues. Knockdown of the Cul4A expression by shRNA in lung cancer cells resulted in decreased cellular proliferation and growth in lung cancer cells. Increased sensitivity to gemcitabine, a chemotherapy drug, was also noted in those Cul4A knockdown lung cancer cells. Moreover, increased expression of p21, transforming growth factor (TGF)-β inducible early gene-1 (TIEG1) and TGF beta-induced (TGFBI) was observed in lung cancer cells after Cul4A knockdown, which may be partially related to increased chemosensitivity to gemcitabine. G0/G1 cell cycle arrest was also noted after Cul4A knockdown. Notably, decreased tumour growth and increased chemosensitivity to gemcitabine were also noted after Cul4A knockdown in lung cancer xenograft nude mice models. In summary, our study showed that targeting Cul4A with RNAi or other techniques may provide a possible insight to the development of lung cancer therapy in the future.

VL - 20 IS - 7 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26969027?dopt=Abstract ER - TY - JOUR T1 - Analysis of lung tumor initiation and progression in transgenic mice for Cre-inducible overexpression of Cul4A gene. JF - Thorac Cancer Y1 - 2015 A1 - Wang, Yang A1 - Xu, Zhidong A1 - Jiang-Hua Mao A1 - Hung, Ming-Szu A1 - Hsieh, David A1 - Au, Alfred A1 - Jablons, David M A1 - You, Liang AB -

BACKGROUND: Lung cancer is the leading cause of morbidity and death worldwide. Although the available lung cancer animal models have been informative and further propel our understanding of human lung cancer, they still do not fully recapitulate the complexities of human lung cancer. The pathogenesis of lung cancer remains highly elusive because of its aggressive biologic nature and considerable heterogeneity, compared to other cancers. The association of Cul4A amplification with aggressive tumor growth and poor prognosis has been suggested. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied.

METHODS: Viral delivery approaches have been used extensively to model cancer in mouse models. In our experiments, we used Cre-recombinase induced overexpression of the Cul4A gene in transgenic mice to study the role of Cul4A on lung tumor initiation and progression and have developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A.

RESULTS: Here we show that the use of a recombinant adenovirus expressing Cre-recombinase ("AdenoCre") to induce Cul4A overexpression in the lungs of mice allows controls of the timing and multiplicity of tumor initiation. Following our mouse models, we are able to study the potential role of Cul4A in the development and progression in pulmonary adenocarcinoma as well.

CONCLUSION: Our findings indicate that Cul4A is oncogenic in vivo, and this mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

VL - 6 IS - 4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26273405?dopt=Abstract ER - TY - JOUR T1 - Knockdown of Cul4A increases chemosensitivity to gemcitabine through upregulation of TGFBI in lung cancer cells. JF - Oncol Rep Y1 - 2015 A1 - Hung, Ming-Szu A1 - Chen, I-Chuan A1 - You, Liang A1 - Jablons, David M A1 - Li, Ya-Chin A1 - Jiang-Hua Mao A1 - Xu, Zhidong A1 - Hsieh, Meng-Jer A1 - Lin, Yu-Ching A1 - Yang, Cheng-Ta A1 - Liu, Shin-Tung A1 - Tsai, Ying-Huang KW - Animals KW - Cell Line, Tumor KW - Cell Transformation, Neoplastic KW - Cullin Proteins KW - Deoxycytidine KW - Drug Resistance, Neoplasm KW - Extracellular Matrix Proteins KW - Gene Expression Regulation, Neoplastic KW - Gene Knockdown Techniques KW - Humans KW - Lung Neoplasms KW - Mice KW - Transforming Growth Factor beta KW - Xenograft Model Antitumor Assays AB -

Cullin 4A (Cul4A) promotes oncogenesis through overexpression and then ubiquitination‑mediated proteolysis of tumor suppressors in various types of cancers. Transforming growth factor β‑induced (TGFBI) has been implicated as a tumor suppressor, which enhances gemcitabine chemosensitivity in lung cancer cells. The present study aimed to investigate the association of TGFBI and Cul4A and the mechanism by which Cul4A regulates TGFBI. In addition, we also evaluated the therapeutic value of Cul4A RNAi using adenoviral transfection of Cul4A RNAi in nude mouse xenograft models. We observed that knockdown of Cul4A was associated with increased sensitivity to gemcitabine through upregulation of TGFBI in lung cancer cells. Cul4A regulated TGFBI through direct interaction and then ubiquitin‑mediated protein degradation. In the nude mouse xenograft models, adenoviral transfection of Cul4A RNAi in combination with gemcitabine chemotherapy inhibited lung cancer tumor growth. As the result, combination of Cul4A RNAi with chemotherapy may provide a new approach to lung cancer treatment.

VL - 34 IS - 6 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26503734?dopt=Abstract ER - TY - JOUR T1 - Lung tumourigenesis in a conditional Cul4A transgenic mouse model. JF - J Pathol Y1 - 2014 A1 - Yang, Yi-Lin A1 - Hung, Ming-Szu A1 - Wang, Yang A1 - Ni, Jian A1 - Jiang-Hua Mao A1 - Hsieh, David A1 - Au, Alfred A1 - Kumar, Atul A1 - Quigley, David A1 - Fang, Li Tai A1 - Yeh, Che-Chung A1 - Xu, Zhidong A1 - Jablons, David M A1 - You, Liang KW - Adenocarcinoma KW - Animals KW - Antineoplastic Agents KW - Cell Cycle Proteins KW - Cell Line, Tumor KW - Cell Survival KW - Cell Transformation, Neoplastic KW - Cisplatin KW - Cullin Proteins KW - Cyclin-Dependent Kinase Inhibitor p16 KW - Cyclin-Dependent Kinase Inhibitor p19 KW - Cyclin-Dependent Kinase Inhibitor p21 KW - Cyclin-Dependent Kinase Inhibitor p27 KW - Disease Models, Animal KW - DNA-Binding Proteins KW - Dose-Response Relationship, Drug KW - Drug Resistance, Neoplasm KW - Gene Expression Regulation, Neoplastic KW - Genomic Instability KW - Humans KW - Lung KW - Lung Neoplasms KW - Mice KW - Mice, Transgenic KW - Neoplasm Grading KW - Proliferating Cell Nuclear Antigen KW - RNA Interference KW - Time Factors KW - Transfection KW - Up-Regulation AB -

Cullin4A (Cul4A) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes and regulates many cellular events, including cell survival, development, growth and cell cycle control. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Here, we used a Cul4A transgenic mouse model to study the potential oncogenic role of Cul4A in lung tumour development. After Cul4A over-expression was induced in the lungs for 32 weeks, atypical epithelial cells were observed. After 40 weeks, lung tumours were visible and were characterized as grade I or II adenocarcinomas. Immunohistochemistry (IHC) revealed decreased levels of Cul4A-associated proteins p21(CIP1) and tumour suppressor p19(ARF) in the lung tumours, suggesting that Cul4A regulated their expression in these tumours. Increased levels of p27(KIP1) and p16(INK4a) were also detected in these tumours. Moreover, the protein level of DNA replication licensing factor CDT1 was decreased. Genomic instability in the lung tumours was further analysed by the results from pericentrin protein expression and array comparative genomic hybridization analysis. Furthermore, knocking down Cul4A expression in lung cancer H2170 cells increased their sensitivity to the chemotherapy drug cisplatin in vitro, suggesting that Cul4A over-expression is associated with cisplatin resistance in the cancer cells. Our findings indicate that Cul4A is oncogenic in vivo, and this Cul4A mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

VL - 233 IS - 2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24648314?dopt=Abstract ER - TY - JOUR T1 - Hematein, a casein kinase II inhibitor, inhibits lung cancer tumor growth in a murine xenograft model. JF - Int J Oncol Y1 - 2013 A1 - Hung, Ming-Szu A1 - Xu, Zhidong A1 - Chen, Yu A1 - Smith, Emmanuel A1 - Jiang-Hua Mao A1 - Hsieh, David A1 - Lin, Yu-Ching A1 - Yang, Cheng-Ta A1 - Jablons, David M A1 - You, Liang KW - Animals KW - Apoptosis KW - Blotting, Western KW - Casein Kinase II KW - Enzyme Inhibitors KW - Female KW - Hematoxylin KW - Humans KW - Immunoenzyme Techniques KW - Lung Neoplasms KW - Mice KW - Mice, Inbred BALB C KW - Phosphorylation KW - Proto-Oncogene Proteins c-akt KW - T Cell Transcription Factor 1 KW - Tumor Cells, Cultured KW - Wnt Proteins KW - Xenograft Model Antitumor Assays AB -

Casein kinase II (CK2) inhibitors suppress cancer cell growth. In this study, we examined the inhibitory effects of a novel CK2 inhibitor, hematein, on tumor growth in a murine xenograft model. We found that in lung cancer cells, hematein inhibited cancer cell growth, Akt/PKB Ser129 phosphorylation, the Wnt/TCF pathway and increased apoptosis. In a murine xenograft model of lung cancer, hematein inhibited tumor growth without significant toxicity to the mice tested. Molecular docking showed that hematein binds to CK2α in durable binding sites. Collectively, our results suggest that hematein is an allosteric inhibitor of protein kinase CK2 and has antitumor activity to lung cancer.

VL - 43 IS - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24008396?dopt=Abstract ER - TY - JOUR T1 - Cul4A is an oncogene in malignant pleural mesothelioma. JF - J Cell Mol Med Y1 - 2011 A1 - Hung, Ming-Szu A1 - Jiang-Hua Mao A1 - Xu, Zhidong A1 - Yang, Cheng-Ta A1 - Yu, Jau-Song A1 - Harvard, Chansonette A1 - Lin, Yu-Ching A1 - Bravo, Dawn Therese A1 - Jablons, David M A1 - You, Liang KW - Cell Cycle KW - Cell Line, Tumor KW - Cullin Proteins KW - Humans KW - Mesothelioma KW - Oncogenes KW - Pleural Neoplasms KW - Proliferating Cell Nuclear Antigen KW - rho GTP-Binding Proteins KW - RNA Interference KW - RNA, Small Interfering KW - Signal Transduction AB -

Cullin 4A (Cul4A) is important in cell survival, development, growth and the cell cycle, but its role in mesothelioma has not been studied. For the first time, we identified amplification of the Cul4A gene in four of five mesothelioma cell lines. Consistent with increased Cul4A gene copy number, we found that Cul4A protein was overexpressed in mesothelioma cells as well. Cul4A protein was also overexpressed in 64% of primary malignant pleural mesothelioma (MPM) tumours. Furthermore, knockdown of Cul4A with shRNA in mesothelioma cells resulted in up-regulation of p21 and p27 tumour suppressor proteins in a p53-independent manner in H290, H28 and MS-1 mesothelioma cell lines. Knockdown of Cul4A also resulted in G0/G1 cell cycle arrest and decreased colony formation in H290, H28 and MS-1 mesothelioma cell lines. Moreover, G0/G1 cell cycle arrest was partially reversed by siRNA down-regulation of p21 and/or p27 in Cul4A knockdown H290 cell line. In the contrary, overexpression of Cul4A resulted in down-regulation of p21 and p27 proteins and increased colony formation in H28 mesothelioma cell line. Both p21 and p27 showed faster degradation rates in Cul4A overexpressed H28 cell line and slower degradation rates in Cul4A knockdown H28 cell line. Our study indicates that Cul4A amplification and overexpression play an oncogenic role in the pathogenesis of mesothelioma. Thus, Cul4A may be a potential therapeutic target for MPM.

VL - 15 IS - 2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19929949?dopt=Abstract ER - TY - JOUR T1 - Transgenic mice for cre-inducible overexpression of the Cul4A gene. JF - Genesis Y1 - 2011 A1 - Li, Tong A1 - Hung, Ming-Szu A1 - Wang, Yucheng A1 - Jiang-Hua Mao A1 - Tan, Jia-Li A1 - Jahan, Kenneth A1 - Roos, Hannah A1 - Xu, Zhidong A1 - Jablons, David M A1 - You, Liang KW - Adenoviridae KW - Animals KW - Cullin Proteins KW - Gene Expression Regulation KW - Genes, Reporter KW - Genetic Vectors KW - Integrases KW - Lac Operon KW - Mice KW - Mice, Knockout KW - Mice, Transgenic KW - Models, Animal KW - Promoter Regions, Genetic KW - Recombination, Genetic KW - Transgenes AB -

The Cullin 4A(Cul4A) gene is important in cell survival, development, growth, and cell cycle control and is amplified in breast and hepatocellular cancers. Recently, we reported that Cul4A plays an oncogenic role in the pathogenesis of mesothelioma. An important strategy for studying Cul4A in different tissues is targeted overexpression of this gene in vivo. Studies of Cul4A in mice have been restricted to the loss-of-function studies using Cul4A knockout mice; gain-of-function studies of Cul4A using transgenic mice have not been reported. We, therefore, generated a gain-of-function transgenic mouse model that overexpresses Cul4A in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development in most adult tissues. After Cre-mediated excision of the LacZ reporter, the transfected Cul4A gene is expressed along with a C-terminal Myc-tag in different tissues. In this study, Cre-excision was induced in mouse lungs by inhalation of an adenovirus vector encoding Cre recombinase. This mouse model provides a valuable resource for investigating the significance of Cul4A overexpression in various tissues.

VL - 49 IS - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/21381181?dopt=Abstract ER - TY - JOUR T1 - Functional polymorphism of the CK2alpha intronless gene plays oncogenic roles in lung cancer. JF - PLoS One Y1 - 2010 A1 - Hung, Ming-Szu A1 - Lin, Yu-Ching A1 - Jiang-Hua Mao A1 - Kim, Il-Jin A1 - Xu, Zhidong A1 - Yang, Cheng-Ta A1 - Jablons, David M A1 - You, Liang KW - Animals KW - Blotting, Western KW - Casein Kinase II KW - Cell Line KW - Cell Line, Tumor KW - Gene Expression Regulation, Neoplastic KW - Humans KW - Immunoprecipitation KW - In Situ Hybridization, Fluorescence KW - In Vitro Techniques KW - Lung Neoplasms KW - Mice KW - NIH 3T3 Cells KW - Nuclear Proteins KW - Polymorphism, Genetic KW - Protein Binding KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Small Interfering KW - Sequence Analysis, DNA KW - Transcription Factors KW - Tumor Suppressor Proteins AB -

Protein kinase CK2 is frequently up-regulated in human cancers, although the mechanism of CK2 activation in cancer remains unknown. In this study, we investigated the role of the CK2alpha intronless gene (CSNK2A1P, a presumed CK2alpha pseudogene) in the pathogenesis of human cancers. We found evidence of amplification and over-expression of the CSNK2A1P gene in non-small cell lung cancer and leukemia cell lines and 25% of the lung cancer tissues studied. The mRNA expression levels correlated with the copy numbers of the CSNK2A1P gene. We also identified a novel polymorphic variant (398T/C, I133T) of the CSNK2A1P gene and showed that the 398T allele is selectively amplified over the 398C allele in 101 non-small cell lung cancer tissue samples compared to those in 48 normal controls (p = 0.013<0.05). We show for the first time CSNK2A1P protein expression in transfected human embryonic kidney 293T and mouse embryonic fibroblast NIH-3T3 cell lines. Both alleles are transforming in these cell lines, and the 398T allele appears to be more transforming than the 398C allele. Moreover, the 398T allele degrades PML tumor suppressor protein more efficiently than the 398C allele and shows a relatively stronger binding to PML. Knockdown of the CSNK2A1P gene expression with specific siRNA increased the PML protein level in lung cancer cells. We report, for the first time, that the CSNK2A1P gene is a functional proto-oncogene in human cancers and its functional polymorphism appears to degrade PML differentially in cancer cells. These results are consistent with an important role for the 398T allele of the CSNK2A1P in human lung cancer susceptibility.

VL - 5 IS - 7 U1 - http://www.ncbi.nlm.nih.gov/pubmed/20625391?dopt=Abstract ER - TY - JOUR T1 - Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library. JF - BMC Cancer Y1 - 2009 A1 - Hung, Ming-Szu A1 - Xu, Zhidong A1 - Lin, Yu-Ching A1 - Jiang-Hua Mao A1 - Yang, Cheng-Ta A1 - Chang, Pey-Jium A1 - Jablons, David M A1 - You, Liang KW - Adenosine Triphosphate KW - Apoptosis KW - Casein Kinase II KW - Cell Growth Processes KW - Cell Line, Tumor KW - Dose-Response Relationship, Drug KW - HCT116 Cells KW - HeLa Cells KW - Hematoxylin KW - Humans KW - Lung Neoplasms KW - Neoplasms KW - Protein Kinase Inhibitors KW - Proto-Oncogene Proteins c-akt KW - Substrate Specificity AB -

BACKGROUND: Casein kinase 2 (CK2) is dysregulated in various human cancers and is a promising target for cancer therapy. To date, there is no small molecular CK2 inhibitor in clinical trial yet. With the aim to identify novel CK2 inhibitors, we screened a natural product library.

METHODS: We adopted cell-based proliferation and CK2 kinase assays to screen CK2 inhibitors from a natural compound library. Dose-dependent response of CK2 inhibitors in vitro was determined by a radioisotope kinase assay. Western blot analysis was used to evaluate down stream Akt phosphorylation and apoptosis. Apoptosis was also evaluated by annexin-V/propidium iodide (PI) labeling method using flow cytometry. Inhibition effects of CK2 inhibitors on the growth of cancer and normal cells were evaluated by cell proliferation and viability assays.

RESULTS: Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases. It appears to be an ATP non-competitive and partially reversible CK2 inhibitor with an IC50 value of 0.55 muM. In addition, hematein inhibited cancer cell growth partially through down-regulation of Akt phosphorylation and induced apoptosis in these cells. Furthermore, hematein exerted stronger inhibition effects on the growth of cancer cells than in normal cells.

CONCLUSION: In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor. Hematein showed stronger growth inhibition effects to cancer cells when compared to normal cells. This compound may represent a promising class of CK2 inhibitors.

VL - 9 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19419583?dopt=Abstract ER -