Ming-Szu Hung

Cul4A is an oncogene in malignant pleural mesothelioma.

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.

Pulmonary cryptococcosis: Clinical, radiographical and serological markers of dissemination

Background and objective:  This study aimed to identify markers of disseminated infection in patients presenting with pulmonary cryptococcosis.

WIF-1 promoter region hypermethylation as an adjuvant diagnostic marker for non-small cell lung cancer-related malignant pleural effusions

PurposeMalignant pleural effusion is an important staging criterion in non-small cell lung cancer (NSCLC). Although cytologic examination remains the major diagnostic tool for NSCLC-related malignant pleural effusion, sometimes other invasive methods maybe required. Aberrant activation of Wnt signaling pathway due to Wnt inhibitory factor-1 (WIF-1) promoter region hypermethylation is common in NSCLC, and can be specifically detected by methylation-specific polymerase chain reaction (MSP). We hypothesized that WIF-1 promoter region MSP can be used to improve the diagnostic yield of NSCLC-related malignant pleural effusion.MethodsWe performed WIF-1 promoter region MSP in 36 definite malignant pleural effusions from consecutive NSCLC patients and 35 pleural effusion specimens of benign origin. Pleural effusion cells were collected for DNA extraction. After bisulfite treatment, DNA was amplified by methylation-specific and unmethylation-specific primers, respectively, to identify the methylation status of WIF-1 promoter region.ResultsThe results of WIF-1 promoter region MSP were positive in 25 (69.4%) of 36 NSCLC patients with malignant pleural effusion. In addition, the results of WIF-1 promoter region MSP were negative in all 35 patients with pleural effusion of benign origin. The age, gender, and smoking status of patients were not correlated with the methylation status of WIF-1 promoter region in NSCLC-related malignant pleural effusion.ConclusionsWIF-1 promoter region MSP might be used as an adjuvant tool to complement cytologic examination for the diagnosis of NSCLC-related malignant pleural effusion.

Frizzled-8 receptor is activated by the Wnt-2 ligand in non-small cell lung cancer

BackgroundWnt-2 plays an oncogenic role in cancer, but which Frizzled receptor(s) mediates the Wnt-2 signaling pathway in lung cancer remains unclear. We sought to (1) identify and evaluate the activation of Wnt-2 signaling through Frizzled-8 in non-small cell lung cancer, and (2) test whether a novel expression construct dominant negative Wnt-2 (dnhWnt-2) reduces tumor growth in a colony formation assay and in a xenograft mouse model.MethodsSemi-quantitative RT-PCR was used to identify the expression of Wnt-2 and Frizzled-8 in 50 lung cancer tissues from patients. The TCF reporter assay (TOP/FOP) was used to detect the activation of the Wnt canonical pathway in vitro. A novel dnhWnt-2 construct was designed and used to inhibit activation of Wnt-2 signaling through Frizzled-8 in 293T, 293, A549 and A427 cells and in a xenograft mouse model. Statistical comparisons were made using Student’s t-test.ResultsAmong the 50 lung cancer samples, we identified a 91% correlation between the transcriptional increase of Wnt-2 and Frizzled-8 (p<0.05). The Wnt canonical pathway was activated when both Wnt-2 and Frizzled-8 were co-expressed in 293T, 293, A549 and A427 cells. The dnhWnt-2 construct we used inhibited the activation of Wnt-2 signaling in 293T, 293, A549 and A427 cells, and reduced the colony formation of NSCLC cells when β-catenin was present (p<0.05). Inhibition of Wnt-2 activation by the dnhWnt-2 construct further reduced the size and mass of tumors in the xenograft mouse model (p<0.05). The inhibition also decreased the expression of target genes of Wnt signaling in these tumors.ConclusionsWe demonstrated an activation of Wnt-2 signaling via the Frizzled-8 receptor in NSCLC cells. A novel dnhWnt-2 construct significantly inhibits Wnt-2 signaling, reduces colony formation of NSCLC cells in vitro and tumor growth in a xenograft mouse model. The dnhWnt-2 construct may provide a new therapeutic avenue for targeting the Wnt pathway in lung cancer.

Functional Polymorphism of the CK2α Intronless Gene Plays Oncogenic Roles in Lung Cancer

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 CK2α intronless gene (CSNK2A1P, a presumed CK2α 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.

Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library

BackgroundCasein 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.MethodsWe 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.ResultsHematein 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 μM. 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.ConclusionIn 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.

Transgenic mice for cre-inducible overexpression of the Cul4A gene

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. genesis 49:134–141, 2011.

Lung tumourigenesis in a conditional Cul4A transgenic mouse model

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 p21CIP1 and tumour suppressor p19ARF in the lung tumours, suggesting that Cul4A regulated their expression in these tumours. Increased levels of p27KIP1 and p16INK4a 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. Published by John Wiley & Sons, Ltd

Statin improves survival in patients with EGFR-TKI lung cancer: A nationwide population-based study

Long-term use of statins has been reported to reduce the risk of death in patients with lung cancer. This study investigated the effect of statin use among patients with lung cancer receiving epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKIs) therapy. A nationwide, population-based case-control study was conducted using the Taiwan National Health Insurance Research Database. From January 1, 1997 to December 31, 2012, a total of 1,707 statin and 6,828 non-statin matched lung cancer cohorts with EGFR-TKIs treatment were studied. Statin use was associated with a reduced risk of death (HR: 0.58, 95% CI: 0.54–0.62, p < 0.001). In addition, statin use was associated with a significantly longer median progression-free survival (8.3 months, 95% CI: 7.6–8.9 vs. 6.1 months, 95% CI: 6.0–6.4, p < 0.001) and median overall survival (35.5 months, 95% CI: 33.8–38.1 vs. 23.9 months, 95% CI: 23.4–24.7, p < 0.001). In conclusion, statins might potentially enhance the therapeutic effect and increase survival in patients with lung cancer receiving EGFR-TKI therapy.

Hematein, a casein kinase II inhibitor, inhibits lung cancer tumor growth in a murine xenograft model

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.