Tin-Lap Lee

Nuclear Factor-κB is an Important Modulator of the Altered Gene Expression Profile and Malignant Phenotype in Squamous Cell Carcinoma

We reported previously that transcription factor nuclear factor (NF)-κB is constitutively activated in human and murine squamous cell carcinomas (SCCs). The role of NF-κB in the cumulative changes in gene expression with transformation and progression of the murine SCC Pam 212 and after switching off NF-κB by a dominant negative inhibitor κB mutant (IκBαM) was explored by profiling with a 15,000-element cDNA micoarrray. Remarkably, NF-κB modulated the expression of >60% of the 308 genes differentially expressed between normal keratinocytes and metastatic SCCs. NF-κB directly or indirectly modulated expression of programs of genes functionally linked to proliferation, apoptosis, adhesion, and angiogenesis. Among these, changes in expression of cyclin D1, inhibitor of apoptosis-1, mutant Trp53, and β-catenin detected with modulation of NF-κB by microarray were confirmed by Western and Northern blot. NF-κB DNA binding motifs were detected in the promoter of ∼63% of genes showing increased expression and 33% of the genes showing decreased expression. The ACTACAG motif implicated in the NF-κB-dependent down-regulation of mRNA expression of MyoD and Sox9 was detected in the coding portion of about 15% of genes showing increased or decreased expression. Inactivation of NF-κB inhibited malignant phenotypic features including proliferation, cell survival, migration, angiogenesis, and tumorigenesis. These results provide evidence that NF-κB is an important modulator of gene expression programs that contribute to the malignant phenotype of SCC.

Concurrent hypermethylation of multiple tumor-related genes in gastric carcinoma and adjacent normal tissues

Transcriptional silencing by CpG‐island hypermethylation now is believed to be an important mechanism of tumorigenesis. To date, studies on CpG‐island hypermethylation in gastric carcinoma and adjacent normal tissues are few.

Promoter hypermethylation of tumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer

Promoter hypermethylation is an alternative mechanism of gene silencing in human cancers including gastric cancer. While intestinal metaplasia (IM) is generally regarded as a precancerous lesion of the stomach, our study examines the presence of gene promoter hypermethylation in IM of patients with and without gastric cancer. We examined 31 samples of gastric cancer, 36 gastric IM (21 associated with gastric cancer and 15 from noncancer patients) and 10 normal gastric biopsies. Tissues containing foci of IM were carefully microdissected from paraffin‐embedded section. Bisulfite‐modifiedDNA was examined for gene promoter hypermethylation in DAP‐kinase, E‐cadherin, GSTP1, p14, p15, p16, RASSF1A and hMLH1 by methylation‐specific‐PCR. None of the control gastric tissues had hypermethylation detected, but gene promoter hypermethylation was frequently detected in gastric cancer and IM. The mean number of methylated genes in cancer and IM was 3.0 and 1.4, respectively (p < 0.0001). Methylation in IM from cancer patients was all associated with concurrent methylation in the corresponding tumor samples. The numbers of methylated genes were similar in IM obtained from cancer and noncancer patients. By examining the methylation patterns of these genes, 3 differential methylation patterns were recognized: hypermethylation was more frequent in cancer than in IM (DAP‐kinase, p14, p15 and p16); comparable frequencies of methylation in cancer and IM (E‐cadherin and hMLH1); and no methylation (GSTP1). Aberrant methylation in tumor‐related genes is frequently detected in gastric IM of both cancer and noncancer patients, suggesting their early involvement in the multistep progression of gastric carcinogenesis.

Epigenetic modification of SOCS-1 differentially regulates STAT3 activation in response to interleukin-6 receptor and epidermal growth factor receptor signaling through JAK and/or MEK in head and neck squamous cell carcinomas

Signal transducer and activator of transcription 3 (STAT3) has been reported to be activated by interleukin-6 receptor (IL-6R) or epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), which may have important implications for responsiveness to therapeutics targeted at EGFR, IL-6R, or intermediary kinases. Suppressor of cytokine signaling-1 (SOCS-1) has been implicated recently in the negative regulation of IL-6R/Janus-activated kinase (JAK)–mediated activation of STAT3, suggesting that SOCS-1 could affect alternative activation of STAT3 by EGFR, IL-6R, and associated kinases. We investigated whether epigenetic modification of SOCS-1 affects STAT3 activation in response to IL-6R-, EGFR-, JAK-, or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)–mediated signal activation. STAT3 was predominantly activated by IL-6R via Jak1/Jak2 in HNSCC lines UMSCC-9 and UMSCC-38 in association with transcriptional silencing of SOCS-1 by hypermethylation. In UMSCC-11A cells with unmethylated SOCS-1, STAT3 activation was regulated by both EGFR and IL-6R via a JAK-independent pathway involving MEK. Pharmacologic inhibitors of JAK and MEK and expression of SOCS-1 following demethylation or transient transfection inhibited STAT3 activation and cell proliferation and induced cell apoptosis in corresponding cell lines. Hypermethylation of SOCS-1 was found in about one-third of human HNSCC tissues, making it a potentially relevant marker for STAT-targeted therapy in HNSCC patients. We conclude that SOCS-1 methylation status can differentially affect STAT3 activation by IL-6R and EGFR through JAK or MEK in different HNSCC and response to pharmacologic antagonists. Identifying the potential factors and the regulatory pathways in STAT3 activation has important implications for the development and selection of molecularly targeted therapy in HNSCC. [Mol Cancer Ther 2006;5(1):8–19]

Methylation of an intronic region regulates miR-199a in testicular tumor malignancy.

In the testicular cancer cell line, NT2, we previously demonstrated that differentially methylated regions were located in introns or intergenic regions, and postulated these might regulate non-coding RNAs. Three microRNAs and three small nucleolar RNAs were differentially methylated; one, miR-199a, was associated with the progression and prognosis of gastric and ovarian cancers. In this report we document, by epigenomic profiling of testicular tissue, that miR-199a is transcribed as antisense of dynamin 3 (chromosome 1q24.3), and hypermethylation of this region is correlated with miR-199a-5p/3p repression and tumor malignancy. Re-expression of miR-199a in testicular cancer cells led to suppression of cell growth, cancer migration, invasion and metastasis. The miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with tumor malignancy. We further identified the embryonal carcinoma antigen podocalyxin-like protein 1 (PODXL), an anti-adhesive protein expressed in aggressive tumors, as a target of miR-199a-5p. We demonstrated PODXL is overexpressed in malignant testicular tumor, and cellular depletion of PODXL resulted in suppression of cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests PODXL is a downstream effector mediating the action of miR199a-5p. This report identifies DNA methylation, miR-199a dysregulation and PODXL as critical factors in tumor malignancy.

Yes-Associated Protein 1 Exhibits Oncogenic Property in Gastric Cancer and Its Nuclear Accumulation Associates with Poor Prognosis

Purpose: Yes-associated protein 1 (YAP1) is a multifunctional protein that can interact with different transcription factors to activate gene expression. The role of YAP1 in tumorigenesis is unclear. We aimed to investigate the functional role of YAP1 in tumorigenesis of gastric cancer. Experimental Design: YAP1 expresson in gastric adenocarcinoma was evaluated. The biological function was determined by proliferation assay, colony formation, cell invasion, and flow cytometric analysis through knocking down or ectopic expressing YAP1 in gastric cancer cell lines coupled with in vivo study. The possible downstream effectors of YAP1 were investigated by expression microarray. Results: YAP1 protein expression was upregulated in gastric cancer. Nuclear accumulation of YAP1 was associated with poor disease-specific survival (P = 0.021), especially in patients with early-stage diseases (P < 0.001). Knockdown YAP1 resulted in a significant reduction in proliferation, anchorage-dependent colony formation, cell invasion, and cell motility. Ectopic YAP1 expression promoted anchorage-independent colony formation, induced a more invasive phenotype, and accelerated cell growth both in vitro and in vivo. Microarray analysis highlighted the alteration of MAPK (mitogen-activated protein kinase) pathway by YAP1. We confirmed a constitutive activation of RAF/MEK/ERK (extracellular signal-regulated kinase) in YAP1-expressing MKN45 cells and further showed that YAP1 enhanced serum/epidermal growth factor–induced c-Fos expression in gastric cancer cells. Conclusions: Our findings supported that YAP1 exhibits oncogenic property in gastric cancer. We provided the first evidence that YAP1 exerted the oncogenic function by enhancing the capacity to activate the early-response gene pathway. YAP1 could be a prognostic biomarker and potential therapeutic target for gastric cancer. Clin Cancer Res; 17(8); 2130–9. ©2011 AACR.

Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer

Background:Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples.Methods:In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs).Results:We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system.Conclusion:This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.

DNA methylation of cancer genome

DNA methylation plays an important role in regulating normal development and carcinogenesis. Current understanding of the biological roles of DNA methylation is limited to its role in the regulation of gene transcription, genomic imprinting, genomic stability, and X chromosome inactivation. In the past 2 decades, a large number of changes have been identified in cancer epigenomes when compared with normals. These alterations fall into two main categories, namely, hypermethylation of tumor suppressor genes and hypomethylation of oncogenes or heterochromatin, respectively. Aberrant methylation of genes controlling the cell cycle, proliferation, apoptosis, metastasis, drug resistance, and intracellular signaling has been identified in multiple cancer types. Recent advancements in whole-genome analysis of methylome have yielded numerous differentially methylated regions, the functions of which are largely unknown. With the development of high resolution tiling microarrays and high throughput DNA sequencing, more cancer methylomes will be profiled, facilitating the identification of new candidate genes or ncRNAs that are related to oncogenesis, new prognostic markers, and the discovery of new target genes for cancer therapy.

A signal network involving coactivated NF-κB and STAT3 and altered p53 modulates BAX/BCL-XL expression and promotes cell survival of head and neck squamous cell carcinomas

Abrogation of apoptosis to sustain cell survival is an essential step in development of cancer. Aberrant activation of signal transcription factors NF‐κB or STAT3, alterations in p53 status, or BCL/BAX family expression have each been reported to affect cell survival in cancer, including head and neck squamous cell carcinomas (HNSCC). However, molecular targeting of these alterations individually has yielded disappointing results. In our study, we examined the hypothesis that alterations in a signal network involving NF‐κB, STAT3 and p53 modulates expression of proapoptotic BAX and antiapoptotic BCL‐XL proteins, and promotes cell survival of HNSCC. We found that NF‐κB and STAT3 are coactivated together, and with cytokine stimulation or siRNA knock‐down, both modulate BAX/BCL‐XL. Greater modulation among HNSCC lines expressing low wt p53 than those over‐expressing mt p53 protein suggested that decreased p53 expression might enhance activation of NF‐κB, STAT3 and BCL‐XL. Reexpression of wt p53 suppressed NF‐κB and STAT3 nuclear binding activity, and BCL‐XL expression, while inducing p21 and BAX. Over‐expression of p53 together with inhibition of NF‐κB or STAT3 induced greater increase in the BAX/BCL‐XL ratio and apoptosis than modulation of these transcription factors individually. Conversely, NF‐κB or STAT3 inducing cytokines decreased the BAX/BCL‐XL ratio. Thus, a network involving signal coactivation of NF‐κB and STAT3, differentially modified by p53 inactivation or mutation, promotes altered BAX/BCL‐XL expression and cell survival in HNSCC. Inhibition of signal activation of both NF‐κB and STAT3 together with reexpression of p53 could be the most effective strategy to restore BAX/BCL‐XL regulation and for cytotoxic therapy of HNSCC.

TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells

BackgroundTSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication.MethodsTo determine a possible cellular function for TSPY, we manipulated the TSPY expression in HeLa and NIH3T3 cells using the Tet-off system. Cell proliferation, colony formation assays and tumor growth in nude mice were utilized to determine the TSPY effects on cell growth and tumorigenesis. Cell cycle analysis and cell synchronization techniques were used to determine cell cycle profiles. Microarray and RT-PCR were used to investigate gene expression in TSPY expressing cells.ResultsOur findings suggest that TSPY expression increases cell proliferation in vitro and tumorigenesis in vivo. Ectopic expression of TSPY results in a smaller population of the host cells in the G2/M phase of the cell cycle. Using cell synchronization techniques, we show that TSPY is capable of mediating a rapid transition of the cells through the G2/M phase. Microarray analysis demonstrates that numerous genes involved in the cell cycle and apoptosis are affected by TSPY expression in the HeLa cells.ConclusionThese data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis.