Han Shui Hsu

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.

Epigenetic inactivation of the chromosomal stability control genes BRCA1, BRCA2, and XRCC5 in non-small cell lung cancer.

Purpose: Lung cancer cells frequently exhibit marked chromosome instability. We postulated that alterations of the double-strand break repair genes (BRCA1, BRCA2, and XRCC5) might be involved in lung cancer. Patients and Methods: We examined the loss of protein and mRNA expression and the 5′CpG hypermethylation and allelic imbalance of the BRCA1, BRCA2, and XRCC5 genes in 98 non–small cell lung cancer (NSCLC) samples. Anchorage-dependent growth after reexpression of these genes was examined in a lung cancer cell line that originally lacked BRCA1 and BRCA2 expression. Results: The data indicated that low protein expression of BRCA1 and BRCA2 was frequent in lung adenocarcinomas (42-44%), whereas low XRCC5 protein expression was more prevalent among squamous cell carcinoma (32%). In addition, low BRCA1 expression was significantly associated with low RB expression, especially in lung adenocarcinoma. Concurrent alterations in XRCC5 and p53 were the most frequent profiles in smoking patients. Importantly, low mRNA and protein expressions of BRCA1, BRCA2, and XRCC5 were significantly associated with their promoter hypermethylation. 5-Aza-2′-deoxycytidine treatment of NSCLC cells showed demethylation and reexpression of the BRCA1 and BRCA2 genes and reduced anchorage-independent growth. Conclusions: Our retrospective study provides compelling evidence that low mRNA and protein expression in the BRCA1/BRCA2 and XRCC5 genes occur in lung adenocarcinoma and squamous cell carcinoma, respectively, and that promoter hypermethylation is the predominant mechanism in deregulation of these genes. Alteration of the double-strand break repair pathway, perhaps by interacting with p53 and RB deregulation, is important in the pathogenesis of a subset of NSCLC.

Prognostic significance of global histone modifications in resected squamous cell carcinoma of the esophagus

Patterns of global histone modifications have been recently suggested as outcome predictors in cancer patients. To date, there has been no report on the prognostic significance of global histone modifications in esophageal squamous cell carcinoma. We investigated the role of global histone modification as outcome predictor in patients undergoing esophagectomy for esophageal squamous cell carcinoma. A retrospective clinicopathologic analysis was undertaken of 97 patients with esophageal squamous cell carcinoma who recovered from esophagectomy. Immunohistochemical expression of five histone modification markers, acetylated histone 3 lysine 18 (H3K18Ac), acetylated histone 4 lysine 12 (H4K12Ac), dimethylated histone 4 arginine 3 (H4R3diMe), dimethylated histone 3 lysine 4 (H3 K4diMe), and trimethylated histone 3 lysine 27 (H3K27triMe) was assessed in paraffin-embedded tumor samples. Results were analyzed in relation to patients’ clinicopathologic parameters. There was a positive relationship between tumor differentiation and H3K18Ac (P<0.001), H4R3diMe (P=0.003), and H3K27triMe (P<0.001). Expression of H3K27triMe correlated positively with nodal (N) status (P=0.012) and stage (P=0.025). Univariate analysis showed that better survival in patients with low expression of H3K18Ac (P=0.038) and H3K27triMe (P=0.003). Multivariate analysis showed that nodal status, metastasis status (M), and expression of H3K27triMe predicted survival independently (P<0.001, P=0.016, and 0.048, respectively). Low expression of H3K18Ac and H3K27triMe correlated with better prognosis of patients with esophageal squamous cell carcinoma, especially for those of early stages. We hypothesize that expression of H3K27triMe may be considered as a significant survival predictor for patients with esophageal squamous cell carcinoma.

hNaa10p contributes to tumorigenesis by facilitating DNMT1-mediated tumor suppressor gene silencing

Hypermethylation-mediated tumor suppressor gene silencing plays a crucial role in tumorigenesis. Understanding its underlying mechanism is essential for cancer treatment. Previous studies on human N-alpha-acetyltransferase 10, NatA catalytic subunit (hNaa10p; also known as human arrest-defective 1 [hARD1]), have generated conflicting results with regard to its role in tumorigenesis. Here we provide multiple lines of evidence indicating that it is oncogenic. We have shown that hNaa10p overexpression correlated with poor survival of human lung cancer patients. In vitro, enforced expression of hNaa10p was sufficient to cause cellular transformation, and siRNA-mediated depletion of hNaa10p impaired cancer cell proliferation in colony assays and xenograft studies. The oncogenic potential of hNaa10p depended on its interaction with DNA methyltransferase 1 (DNMT1). Mechanistically, hNaa10p positively regulated DNMT1 enzymatic activity by facilitating its binding to DNA in vitro and its recruitment to promoters of tumor suppressor genes, such as E-cadherin, in vivo. Consistent with this, interaction between hNaa10p and DNMT1 was required for E-cadherin silencing through promoter CpG methylation, and E-cadherin repression contributed to the oncogenic effects of hNaa10p. Together, our data not only establish hNaa10p as an oncoprotein, but also reveal that it contributes to oncogenesis through modulation of DNMT1 function.

Lung cancer susceptibility and prognosis associated with polymorphisms in the nonhomologous end-joining pathway genes: A multiple genotype-phenotype study

Nonsmall cell lung cancer (NSCLC) frequently exhibits genomic instability, such as high fractional allelic loss (FAL). Genomic instability may result from unrepaired or misrepaired double‐strand breaks (DSBs). The authors of this report postulated that polymorphisms in genes of the nonhomologous end‐joining (NHEJ) pathway, which is the major DSB repair pathway in mammalian cells, may modulate lung cancer susceptibility and prognosis.

Multiple genetic and epigenetic biomarkers for lung cancer detection in cytologically negative sputum and a nested case–control study for risk assessment†

The purpose of this study was to define a biomarker panel for detection of cancer cells in cytologically negative sputum and to evaluate the panel for assessment of lung cancer risk. We examined 19 genetic and epigenetic markers using a sensitive fluorescence‐based method in cytologically negative sputum and in lung tumour tissues from 82 lung cancer patients. We also used these markers to test the sputum of 37 cancer‐free individuals who were matched by age, sex, and smoking habit. Based on the concordance of biomarkers in lung tumours and corresponding sputum, and the low prevalence in cancer‐free individuals, we selected seven markers for a nested case–control study: microsatellite instability of D9S942; loss of heterozygosity of D9S286, D9S942, GATA49D12, and D13S170; and methylation of p16INK4a and RARβ. Based on the assumption that a lung cancer cell has alterations in two or more of the seven biomarkers, we compared the pattern of biomarker alteration in lung tumours and corresponding sputum. Our comparison yielded a sensitivity of 82%, specificity of 75%, and concordance of 79%. Three cancer‐free individuals were considered to have an elevated risk based on the criterion that their sputum showed alteration in two of the seven biomarkers. One individual was indeed diagnosed as having lung cancer 18 months after sputum collection. In the nested case–control study, six biomarkers showed significantly increased odds ratios ranging from 3.14 to 11.24. Our study defines a biomarker panel for detection of cancer cells in cytologically negative sputum and verifies its use for risk assessment of lung cancer. In combination with conventional diagnostic tools, this multiple genetic and epigenetic panel should improve the detection or risk assessment of lung cancer. Copyright

5'Cytosine-phospho-guanine island methylation is responsible for p14ARF inactivation and inversely correlates with p53 overexpression in resected non-small cell lung cancer

Purpose and Experimental Design: The molecular mechanisms by which the p14ARF gene is altered in non–small cell lung cancer (NSCLC) are complex and unclear. Using genetic and epigenetic analyses, we examined various molecular alterations including the loss of protein and mRNA expression, and 5′CpG hypermethylation, allelic imbalance, and mutation of the p14ARF gene in a series of 102 NSCLC samples, in parallel with clinicopathological and prognostic analyses. To clarify the biological significance of p14ARF alterations, its relationship with p16INK4a and p53 alterations was also examined. Results: We found that 34% of NSCLC patients had aberrant P14ARF protein expression, which was more frequent in adenocarcinomas (AD; 44%) than in squamous cell carcinomas (22%; P = 0.024). A high concordance was observed between alterations in protein and mRNA expression and 5′CpG hypermethylation (P ≤ 0.001). The p14ARF hypermethylation inversely correlated with P53 overexpression (P = 0.001). This mutually exclusive relationship for alteration between p14ARF and p53 was also supported by a worse prognosis of AD patients with positive P14ARF expression (P = 0.01) and of AD patients with P53 overexpression (P = 0.006). Our data also indicated that hemizygous/homozygous deletion and mutation in the p14ARF gene occurred at 26%, 9%, and 0%, respectively, of microdissected NSCLCs. Conclusions: Our data suggest that p14ARF 5′CpG hypermethylation is the predominant mechanism involved in the aberrant expression of the p14ARF gene. In addition, p14ARF 5′CpG hypermethylation occurs inversely to P53 overexpression.

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.

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.