Yen An Tang

The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients

DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in many human diseases, including cancer. The tobacco-specific carcinogen NNK also induces DNA methylation. However, the role of DNMT1-mediated methylation in tobacco carcinogenesis remains unclear. Here we used human and mouse lung cancer samples and cell lines to determine a mechanism whereby NNK induced DNMT1 expression and activity. We determined that in a human lung cell line, glycogen synthase kinase 3beta (GSK3beta) phosphorylated DNMT1 to recruit beta-transducin repeat-containing protein (betaTrCP), resulting in DNMT1 degradation, and that NNK activated AKT, inhibiting GSK3beta function and thereby attenuating DNMT1 degradation. NNK also induced betaTrCP translocation to the cytoplasm via the heterogeneous nuclear ribonucleoprotein U (hnRNP-U) shuttling protein, resulting in DNMT1 nuclear accumulation and hypermethylation of the promoters of tumor suppressor genes. Fluorescence immunohistochemistry (IHC) of lung adenomas from NNK-treated mice and tumors from lung cancer patients that were smokers were characterized by disruption of the DNMT1/betaTrCP interaction and DNMT1 nuclear accumulation. Importantly, DNMT1 overexpression in lung cancer patients who smoked continuously correlated with poor prognosis. We believe that the NNK-induced DNMT1 accumulation and subsequent hypermethylation of the promoter of tumor suppressor genes may lead to tumorigenesis and poor prognosis and provide an important link between tobacco smoking and lung cancer. Furthermore, this mechanism may also be involved in other smoking-related human diseases.

A Novel Sialyltransferase Inhibitor Suppresses FAK/Paxillin Signaling and Cancer Angiogenesis and Metastasis Pathways

Increased sialyltransferase (ST) activity promotes cancer cell metastasis, and overexpression of cell surface sialic acid correlates with poor prognosis in cancer patients. To seek therapies targeting metastasis for cancer treatment, we developed a novel ST inhibitor, Lith-O-Asp, and investigated its antimetastatic and antiangiogenic effects and mechanisms. We found that cells treated with Lith-O-Asp showed a reduction of activity on various ST enzymes by in vitro and cell-based activity analyses. Lith-O-Asp inhibited migration and invasion abilities in various cancer cell lines and showed inhibitory effect on the angiogenic activity of human umbilical vein endothelial cells. Indeed, Lith-O-Asp treatment consequently delayed cancer cell metastasis in experimental and spontaneous metastasis assays in animal models. Importantly, Lith-O-Asp decreased the sialic acid modification of integrin-β1 and inhibited the expression of phospho-FAK, phospho-paxillin, and the matrix metalloprotease (MMP) 2 and MMP9. Lith-O-Asp attenuated the Rho GTPase activity leading to actin dynamic impairment. In addition, 2DE-MS/MS and immunoblotting analyses showed that Lith-O-Asp altered the protein expression level and phosphorylation status of various proteins involved in crucial metastasis and angiogenesis pathways such as vimentin and ribonuclease/angiogenin inhibitor RNH1. Furthermore, Lith-O-Asp treatment significantly inhibited the invasive ability exerted by ectopic overexpression of various ST enzymes catalyzing α-2,6- or α-2,3-sialylation. Our results provide compelling evidence that the potential pan-ST inhibitor, Lith-O-Asp, suppressed cancer cell metastasis likely by inhibiting FAK/paxillin signaling and expressing antiangiogenesis factors. Lith-O-Asp is worthy for further testing as a novel antimetastasis drug for cancer treatment.

A Novel Histone Deacetylase Inhibitor Exhibits Antitumor Activity via Apoptosis Induction, F-Actin Disruption and Gene Acetylation in Lung Cancer

Background Lung cancer is the leading cause of cancer mortality worldwide, yet the therapeutic strategy for advanced non-small cell lung cancer (NSCLC) is limitedly effective. In addition, validated histone deacetylase (HDAC) inhibitors for the treatment of solid tumors remain to be developed. Here, we propose a novel HDAC inhibitor, OSU-HDAC-44, as a chemotherapeutic drug for NSCLC. Methodology/Principal Findings The cytotoxicity effect of OSU-HDAC-44 was examined in three human NSCLC cell lines including A549 (p53 wild-type), H1299 (p53 null), and CL1-1 (p53 mutant). The antiproliferatative mechanisms of OSU-HDAC-44 were investigated by flow cytometric cell cycle analysis, apoptosis assays and genome-wide chromatin-immunoprecipitation-on-chip (ChIP-on-chip) analysis. Mice with established A549 tumor xenograft were treated with OSU-HDAC-44 or vehicle control and were used to evaluate effects on tumor growth, cytokinesis inhibition and apoptosis. OSU-HDAC-44 was a pan-HDAC inhibitor and exhibits 3–4 times more effectiveness than suberoylanilide hydroxamic acid (SAHA) in suppressing cell viability in various NSCLC cell lines. Upon OSU-HDAC-44 treatment, cytokinesis was inhibited and subsequently led to mitochondria-mediated apoptosis. The cytokinesis inhibition resulted from OSU-HDAC-44-mediated degradation of mitosis and cytokinesis regulators Auroroa B and survivin. The deregulation of F-actin dynamics induced by OSU-HDAC-44 was associated with reduction in RhoA activity resulting from srGAP1 induction. ChIP-on-chip analysis revealed that OSU-HDAC-44 induced chromatin loosening and facilitated transcription of genes involved in crucial signaling pathways such as apoptosis, axon guidance and protein ubiquitination. Finally, OSU-HDAC-44 efficiently inhibited A549 xenograft tumor growth and induced acetylation of histone and non-histone proteins and apoptosis in vivo. Conclusions/Significance OSU-HDAC-44 significantly suppresses tumor growth via induction of cytokinesis defect and intrinsic apoptosis in preclinical models of NSCLC. Our data provide compelling evidence that OSU-HDAC-44 is a potent HDAC targeted inhibitor and can be tested for NSCLC chemotherapy.

MDM2 Overexpression Deregulates the Transcriptional Control of RB/E2F Leading to DNA Methyltransferase 3A Overexpression in Lung Cancer

Purpose: Overexpression of DNA 5′-cytosine-methyltransferase 3A (DNMT3A), which silences genes including tumor suppressor genes (TSG), is involved in many cancers. Therefore, we examined whether the transcriptional deregulation of RB/MDM2 pathway was responsible for DNMT3A overexpression and analyzed the therapeutic potential of MDM2 antagonist for reversing aberrant DNA methylation status in lung cancer. Experimental Design: The regulation of DNMT3A expression and TSG methylation status by RB/MDM2 was assessed in cancer cell lines and patients. The effects of Nutlin-3, an MDM2 antagonist, on tumor growth in relation to DNMT3A expression and TSG methylation status were examined by xenograft model. Results: We found that RB suppressed DNMT3A promoter activity and mRNA/protein expression through binding with E2F1 protein to the DNMT3A promoter, leading to the decrease of methylation level globally and TSG specifically. In addition, MDM2 dramatically induced DNMT3A expression by negative control over RB. In clinical study, MDM2 overexpression inversely correlated with RB expression, while positively associating with overexpression of DNMT3A in samples from patients with lung cancer. Patients with high MDM2 and low RB expression showed DNMT3A overexpression with promoter hypermethylation in TSGs. Treatment with Nutlin-3, an MDM2 antagonist, significantly suppressed tumor growth and reduced DNA methylation level of TSGs through downregulation of DNMT3A expression in xenograft studies. Conclusions: This study provides the first cell, animal, and clinical evidence that DNMT3A is transcriptionally repressed, in part, by RB/E2F pathway and that the repression could be attenuated by MDM2 overexpression. MDM2 is a potent target for anticancer therapy to reverse aberrant epigenetic status in cancers. Clin Cancer Res; 18(16); 4325–33. ©2012 AACR.

Oct4 transcriptionally regulates the expression of long non-coding RNAs NEAT1 and MALAT1 to promote lung cancer progression

BackgroundOct4, a key stemness transcription factor, is overexpressed in lung cancer. Here, we reveal a novel transcription regulation of long non-coding RNAs (lncRNAs) by Oct4. LncRNAs have emerged as important players in cancer progression.MethodsOct4 chromatin-immunoprecipitation (ChIP)-sequencing and several lncRNA databases with literature annotation were integrated to identify Oct4-regulated lncRNAs. Luciferase activity, qRT-PCR and ChIP-PCR assays were conducted to examine transcription regulation of lncRNAs by Oct4. Reconstitution experiments of Oct4 and downstream lncRNAs in cell proliferation, migration and invasion assays were performed to confirm the Oct4-lncRNAs signaling axes in promoting lung cancer cell growth and motility. The expression correlations between Oct4 and lncRNAs were investigated in 124 lung cancer patients using qRT-PCR analysis. The clinical significance of Oct4/lncRNAs signaling axes were further evaluated using multivariate Cox regression and Kaplan-Meier analyses.ResultsWe confirmed that seven lncRNAs were upregulated by direct binding of Oct4. Among them, nuclear paraspeckle assembly transcript 1 (NEAT1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and urothelial carcinoma-associated 1 (UCA1) were validated as Oct4 transcriptional targets through promoter or enhancer activation. We showed that lung cancer cells overexpressing NEAT1 or MALAT1 and the Oct4-silenced cells reconstituted with NEAT1 or MALAT1 promoted cell proliferation, migration and invasion. In addition, knockdown of NEAT1 or MALAT1 abolished Oct4-mediated lung cancer cell growth and motility. These cell-based results suggested that Oct4/NEAT1 or Oct4/MALAT1 axis promoted oncogenesis. Clinically, Oct4/NEAT1/MALAT1 co-overexpression was an independent factor for prediction of poor outcome in 124 lung cancer patients.ConclusionsOur study reveals a novel mechanism by which Oct4 transcriptionally activates NEAT1 via promoter and MALAT1 via enhancer binding to promote cell proliferation and motility, and led to lung tumorigenesis and poor prognosis.

Global Oct4 target gene analysis reveals novel downstream PTEN and TNC genes required for drug-resistance and metastasis in lung cancer

Overexpression of Oct4, a stemness gene encoding a transcription factor, has been reported in several cancers. However, the mechanism by which Oct4 directs transcriptional program that leads to somatic cancer progression remains unclear. In this study, we provide mechanistic insight into Oct4-driven transcriptional network promoting drug-resistance and metastasis in lung cancer cell, animal and clinical studies. Through an integrative approach combining our Oct4 chromatin-immunoprecipitation sequencing and ENCODE datasets, we identified the genome-wide binding regions of Oct4 in lung cancer at promoter and enhancer of numerous genes involved in critical pathways which promote tumorigenesis. Notably, PTEN and TNC were previously undefined targets of Oct4. In addition, novel Oct4-binding motifs were found to overlap with DNA elements for Sp1 transcription factor. We provided evidence that Oct4 suppressed PTEN in an Sp1-dependent manner by recruitment of HDAC1/2, leading to activation of AKT signaling and drug-resistance. In contrast, Oct4 transactivated TNC independent of Sp1 and resulted in cancer metastasis. Clinically, lung cancer patients with Oct4 high, PTEN low and TNC high expression profile significantly correlated with poor disease-free survival. Our study reveals a critical Oct4-driven transcriptional program that promotes lung cancer progression, illustrating the therapeutic potential of targeting Oc4 transcriptionally regulated genes.

Lack of association of C-Met-N375S sequence variant with lung cancer susceptibility and prognosis.

Background: Previously, we identified a sequence variant (N375S) of c-Met gene, however, its association with lung cancer risk and prognosis remain undefined. Patients and Methods: We investigated the genotype distribution of the c-Met-N375S sequence variant in 206 lung cancer patients and 207 non-cancer controls in the Taiwanese population by DNA sequencing. Results: Lung cancer patients with variant A/G and G/G genotypes showed 1.08-fold increased cancer risk when compared to patients with the wild-type A/A genotype (95% CI, 0.60-1.91). There were no significant differences in postoperative survival between c-Met-N375S and wild-type patients. In the cell model, the c-Met-N375S cells showed a decrease in cell death upon treatment with MET inhibitor SU11274 compared to wild-type cells. Conclusion: Our data suggest that the c-Met-N375S sequence variant may not play a significant role in cancer susceptibility and the prognosis of lung cancer patients. The correlation with chemoresponse of c-Met-N375S is worth further investigation in patients receiving MET therapy.

DNMT3B Overexpression by Deregulation of FOXO3a-Mediated Transcription Repression and MDM2 Overexpression in Lung Cancer

Introduction: DNA methyltransferase 3B (DNMT3B) contributes to de novo DNA methylation and its overexpression promotes tumorigenesis. However, whether DNMT3B is upregulated by transcriptional deregulation remains unclear. Methods: We studied the transcriptional repression of DNMT3B by forkhead O transcription factor 3a (FOXO3a) in lung cancer cell, animal, and clinical models. Results: The results of luciferase reporter assay showed that FOXO3a negatively regulated DNMT3B promoter activity by preferentially interacting with the binding element FOXO3a-E (+166 to +173) of DNMT3B promoter. Ectopically overexpressed FOXO3a or combined treatment with doxorubicin to induce FOXO3a nuclear accumulation further bound at the distal site, FOXO3a-P (−249 to −242) by chromatin-immunoprecipitation assay. Knockdown of FOXO3a resulted in an open chromatin structure and high DNMT3B mRNA and protein expression. Abundant FOXO3a repressed DNMT3B promoter by establishing a repressed chromatin structure. Note that FOXO3a is a degradation substrate of MDM2 E3-ligase. Cotreatment with doxorubicin and MDM2 inhibitor, Nutlin-3, further enforced abundant nuclear accumulation of FOXO3a resulting in decrease expression of DNMT3B leading to synergistic inhibition of tumor growth and decrease of methylation status on tumor suppressor genes in xenograft specimens. Clinically, lung cancer patients with DNMT3B high, FOXO3a low, and MDM2 high expression profile correlated with poor prognosis examined by immunohistochemistry and Kaplan-Meier survival analysis. Conclusions: We reveal a new mechanism that FOXO3a transcriptionally represses DNMT3B expression and this regulation can be attenuated by MDM2 overexpression in human lung cancer model. Cotreatment with doxorubicin and Nutlin-3 is a novel therapeutic strategy through epigenetic modulation.

Oncoprotein ZNF322A transcriptionally deregulates alpha-adducin, cyclin D1 and p53 to promote tumor growth and metastasis in lung cancer.

ZNF322A encoding a classical Cys2His2 zinc finger transcription factor was previously revealed as a potential oncogene in lung cancer patients. However, the oncogenic role of ZNF322A and its underlying mechanism in lung tumorigenesis remain elusive. Here we show ZNF322A protein overexpression in 123 Asian and 74 Caucasian lung cancer patients. Multivariate Cox regression analysis indicated that ZNF322A was an independent risk factor for a poor outcome in lung cancer, corroborating the Kaplan–Meier results that patients with ZNF322A protein overexpression had significantly poorer overall survival than other patients. Overexpression of ZNF322A promoted cell proliferation and soft agar growth by prolonging cell cycle in S phase in multiple lung cell lines, including the immortalized lung cell BEAS-2B. In addition, ZNF322A overexpression enhanced cell migration and invasion, whereas knockdown of ZNF322A reduced cell growth, invasion and metastasis abilities in vitro and in vivo. Quantitative proteomic analysis revealed potential ZNF322A-regulated downstream targets, including alpha-adducin (ADD1), cyclin D1 (CCND1), and p53. Using luciferase promoter activity assay combined with site-directed mutagenesis and sequential chromatin immunoprecipitation-PCR assay, we found that ZNF322A could form a complex with c-Jun and cooperatively activate ADD1 and CCND1 but repress p53 gene transcription by recruiting differential chromatin modifiers, such as histone deacetylase 3, in an AP-1 element dependent manner. Reconstitution experiments indicated that CCND1 and p53 were important to ZNF322A-mediated promotion of cell proliferation, whereas ADD1 was necessary for ZNF322A-mediated cell migration and invasion. Our results provide compelling evidence that ZNF322A overexpression transcriptionally dysregulates genes involved in cell growth and motility therefore contributes to lung tumorigenesis and poor prognosis.

A histone deacetylase inhibitor YCW1 with antitumor and antimetastasis properties enhances cisplatin activity against non-small cell lung cancer in preclinical studies

Many histone deacetylase (HDAC) inhibitors show limited therapeutic effects for solid tumors. Here, we develop a novel HDAC inhibitor YCW1 and verify the combination effect of YCW1 and cisplatin in lung cancer pre-clinical models. YCW1 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. YCW1 and cisplatin showed synergistic anti-tumor effects through impairment of DNA damage repair. YCW1 inhibited tumor growth in lung orthotopic and subcutaneously implanted xenograft models. YCW1 significantly suppressed lung metastases via inhibition of focal adhesion complex. Our findings suggested that YCW1 is a potential HDAC inhibitor for lung cancer treatment as single and in combination regimens with cisplatin.