Joseph Kwong

Dysregulation of microRNA-204 mediates migration and invasion of endometrial cancer by regulating FOXC1

MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. Differential miRNA expression signatures have been documented in many human cancers but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. This study identifies significantly dysregulated miRNAs of EEC cells, and characterizes their impact on the malignant phenotype. We studied the expression of 365 human miRNAs using Taqman low density arrays in EECs and normal endometriums. Candidate differentially expressed miRNAs were validated by quantitative real‐time PCR. Expression of highly dysregulated miRNAs was examined in vitro through the effect of anti‐/pre‐miRNA transfection on the malignant phenotype. We identified 16 significantly dysregulated miRNAs in EEC and 7 of these are novel findings with respect to EEC. Antagonizing the function of miR‐7, miR‐194 and miR‐449b, or overexpressing miR‐204, repressed migration, invasion and extracellular matrix‐adhesion in HEC1A endometrial cancer cells. FOXC1 was determined as a target gene of miR‐204, and two binding sites in the 3′‐untranslated region were validated by dual luciferase reporter assay. FOXC1 expression was inversely related to miR‐204 expression in EEC. Functional analysis revealed the involvement of FOXC1 in migration and invasion of HEC1A cells. Our results present dysfunctional miRNAs in endometrial cancer and identify a crucial role for miR‐204‐FOXC1 interaction in endometrial cancer progression. This miRNA signature offers a potential biomarker for predicting EEC outcomes, and targeting of these cancer progression‐ and metastasis‐related miRNAs offers a novel potential therapeutic strategy for the disease.

RASSF1A is a target tumor suppressor from 3p21.3 in nasopharyngeal carcinoma

Deletion on the short arm of chromosome 3 is one of the most important genetic abnormalities in the tumorigenesis of nasopharyngeal carcinoma (NPC). Both physical mapping and functional studies have targeted an NPC‐related tumor suppressor gene(s) to chromosome 3p21.3. We have reported recently that RASSF1A gene, located on a 120‐kb minimal deletion region on 3p21.3, was frequently inactivated by promoter hypermethylation in NPC. We further confirmed that RASSF1A is the critical target tumor suppressor from 3p21.3, with the evidence that loss of expression and aberrant methylation of the other 8 candidate genes/transcripts (HYAL2, FUS1, RASSF1C, BLU, NPRL2, 101F6, PL6 and CACNA2D2) in this 120‐kb region were rare in NPC samples. The contribution of RASSF1A in NPC tumorigenesis was investigated by restoring its expression in a RASSF1A deficient cell line, C666‐1. Transient transfection of wild‐type RASSF1A resulted in marked growth inhibition in NPC cells. Isolated stable clones expressing wild‐type RASSF1A demonstrated retarded cell proliferation in vitro. Soft‐agar assay also showed decreased number and sizes of colony formed in these clones. In vivo nude mice assay demonstrated the dramatic reduction of tumorigenic potential in the RASSF1A‐transfected clones. Our results provide strong evidence to support RASSF1A as a target tumor suppressor gene on 3p21.3 in NPC.

Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma.

To identify the epigenetic changes in nasopharyngeal carcinoma (NPC), we performed methylation‐sensitive restriction fingerprinting (MSRF) analysis on NPC cell lines and xenografts. A 190 bp sequence methylated in NPC tumors was isolated and showed high homology to the 5′ CpG island of the endothelin receptor B (EDNRB) gene. Since the EDNRB gene is commonly inactivated in prostate and bladder cancers, it may be a candidate target gene involved in NPC tumorigenesis. By bisulfite sequencing, we have confirmed that hypermethylation of the 5′ CpG island of EDNRB occurred in both xenografts and all 4 cell lines but not in 2 normal nasopharyngeal outgrowths. RT‐PCR demonstrated that only original EDNRB transcripts, but not the splicing transcripts, were expressed in normal nasopharyngeal epithelial cells. Loss of the original EDNRB expression was consistently found in 2 xenografts and 3 cell lines with dense methylation patterns. Treatment of these 3 cell lines with 5′‐aza‐2′‐deoxycytidine led to re‐expression of the EDNRB transcript and demethylation of its promoter regions. Our results demonstrate that silencing of EDNRB gene expression in NPC is associated with promoter hypermethylation. Using methylation‐specific PCR, we also detected methylation of the 5′ CpG island of EDNRB in 19/21 (90.5%) primary tumors, while no methylation was found in all 6 normal nasopharyngeal epithelia. The high frequencies of promoter hypermethylation suggest that repression of the EDNRB gene may play a role in the development of NPC.

Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma

Tazarotene‐induced gene 1 (TIG1) and Tazarotene‐induced gene 3 (TIG3) are retinoid acid (RA) target genes as well as candidate tumor suppressor genes in human cancers. In our study, we have investigated the expression of TIG1 and TIG3 in nasopharyngeal carcinoma (NPC). Loss of TIG1 expression was found in 80% of NPC cell lines and 33% of xenografts, whereas TIG3 was expressed in all NPC samples and immortalized nasopharyngeal epithelial cells. In order to elucidate the epigenetic silencing of TIG1 in NPC, the methylation status of TIG1 promoter was examined by genomic bisulfite sequencing and methylation‐specific PCR (MSP). We have detected dense methylation of TIG1 5′CpG island in the 5 TIG1‐negative NPC cell lines and xenograft (C666‐1, CNE1, CNE2, HONE1 and X666). Partial methylation was observed in 1 NPC cell line HK1 showing dramatic decreased in TIG1 expression. Promoter methylation was absent in 2 TIG1‐expressed NPC xenografts and the normal epithelial cells. Restoration of TIG1 expression and unmethylated alleles were observed in NPC cell lines after 5‐aza‐2′‐deoxycytidine treatment. Moreover, the methylated TIG1 sequence was detected in 39 of 43 (90.7%) primary NPC tumors by MSP. In conclusion, our results showed that TIG1 expression is lost in the majority of NPC cell lines and xenografts, while promoter hypermethylation is the major mechanism for TIG1 silencing. Furthermore, the frequent epigenetic inactivation of TIG1 in primary NPC tumors implied that it may play an important role in NPC tumorigenesis.

An antagonist of the chemokine receptor CXCR4 induces mitotic catastrophe in ovarian cancer cells

The chemokine receptor CXCR4 is expressed by malignant cells in ovarian cancer and is implicated in their growth and spread. We report here a unique mechanism of action of a small peptide antagonist of CXCR4 on ovarian cancer cells: induction of cell death by mitotic catastrophe. CTCE-9908 inhibited ovarian cancer cell migration to CXCL12, but on longer incubation, caused cell death in CXCR4-positive cells. CTCE-9908 did not cause apoptosis or cellular senescence, but induced multinucleation, G2-M arrest, and abnormal mitosis in ovarian cancer cells. This suggests that cell death was caused by mitotic catastrophe. Using microarray and Western blot analysis, we showed that CTCE-9908 deregulated DNA damage checkpoint proteins and spindle assembly checkpoint proteins at G2-M phases of the cell cycle. Combination treatment of CTCE-9908 and the drug paclitaxel led to an additive cytotoxicity that also involved mitotic catastrophe. We conclude that CTCE-9908 has a unique mechanism of action in ovarian cancer cells that seems to be CXCR4 specific. [Mol Cancer Ther 2009;8(7):1893–905]

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non‐small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9‐bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK‐1 and CNE‐2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5‐aza‐2′‐deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.

Epigenetic inactivation of the deleted in lung and esophageal cancer 1 gene in nasopharyngeal carcinoma.

Deletion of the short arm of chromosome 3 is a common event in nasopharyngeal carcinoma (NPC), suggesting that one or more tumor suppressor genes at 3p are involved in this cancer. DLEC1, Deleted in Lung and Esophageal Cancer 1, located at 3p22.2, was recently identified as a candidate tumor suppressor gene in lung, esophageal, and renal cancers. In this study, we investigated the involvement of DLEC1 in the development of NPC. Down‐regulation of DLEC1 and promoter hypermethylation were observed in all NPC cell lines and xenografts but not in normal nasopharyngeal epithelial cells. Promoter hypermethylation of DLEC1 was also detected in 30 of 42 (71%) NPC primary tumors. Treatment of NPC cell lines with demethylating agent or histone deacetylase inhibitor resulted in restoration of DLEC1 expression. Overexpression of DLEC suppressed growth and reduced invasiveness of NPC cells. Furthermore, the tumorigenic potential of DLEC1 expressing NPC cells was highly reduced in nude mice. Taken together, our results strongly suggest that silencing of DLEC1 expression by promoter hypermethylation and histone deacetylation may be important in NPC tumorigenesis.

cDNA, genomic cloning, and gene expression analysis of mouse PSP94 (prostate secretory protein of 94 amino acids).

The potential use of prostate secretory protein of 94 amino acids (PSP94) as a diagnostic biomarker or a therapeutic agent for prostate cancer has been reported. In order to establish an animal model to further elucidate on its biological role, we cloned the mouse PSP94 cDNA (approximately 500 bp) by reverse transcriptase-polymerase chain reaction (RT-PCR) and disclosed its genomic structure. The whole mouse PSP94 gene (approximately 23 kb) was amplified by long and accurate-PCR and also cloned by screening of a mouse embryo stem-cell genomic library. Computational and statistical analyses have demonstrated several highly conserved characteristics of PSP94 among different species. Comparison of PSP94 from human, two primates, pig, and rodents revealed that the most significant feature is that PSP94 is rich in cysteines (10% of the total sequence) and their positions are highly conserved. The three intron-four exon structure of the human PSP94 gene and the consensus sequence (....GT-intron-AG...) for mRNA splicing are also strongly conserved. A high divergence in cDNA sequence in the protein-coding region and also in the genomic sequence of PSP94 was also observed among these species. Comparing with alpha-globin, a typical evolutionally conserved gene, with the PSP94 gene, the rate of nonsynonymous changes per site per year (kN) is 2 to 6 times higher, indicating that PSP94 gene has been under far fewer evolutionary constraints than other genes and has a potential role as a species barrier in reproductive biology. In order to test this hypothesis, we investigated the gene expression of PSP94 and its tissue distribution in various rodent tissues by RT-PCR and in situ hybridization (ISH). Gene expression was found only in the prostate, suggesting that PSP94 is probably more tissue specific in the prostate of rodents than in mammals. The ISH analysis also revealed a prostate lobe-specific expression of the PSP94 gene in both mice and rats. It was strongly expressed in the lateral prostate, but the findings were negative in the dorsal and ventral lobe. Therefore, it is hypothesized that one of the primary functions of rodent PSP94, as a major prostate secretory protein, is related to reproductive biology.

A novel miR-193a-5p-YY1-APC regulatory axis in human endometrioid endometrial adenocarcinoma

Aberrant expression and altered function of transcription factors (TFs) have vital roles in many aspects of tumor development and progression. In this study, we investigated the functional significance of a TF, Yin Yang1 (YY1) in tumorigenesis of endometrioid endometrial carcinoma (EEC). We demonstrated that YY1 is upregulated in EEC cell lines and primary tumors; and its expression is associated with tumor stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo, whereas overexpression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an oncogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis through inhibition of APC expression. Further mechanistic investigation uncovered a new epigenetic silencing mode of APC by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 on its promoter region. Moreover, YY1 overexpression was found to be a consequence of miR-193a-5p downregulation through direct miR-193a-5p-YY1 interplay. Our results therefore establish a novel miR-193a-5p-YY1-APC axis, which contributes to EEC development, and may serve as future intervention target.

RUNX3 protein is overexpressed in human epithelial ovarian cancer

OBJECTIVE RUNX family genes, including RUNX3, are developmental regulators that are important in human cancers. The purpose of this study was to evaluate expression and oncogenic potential of RUNX3 in ovarian carcinoma. METHODS Immunohistochemical staining was performed on 60 malignant, 14 borderline, and 5 normal ovarian specimens. Correlation between RUNX3 expression with tumor histology was performed. RUNX3 expression was evaluated by quantitative real-time polymerase chain reaction (QRT-PCR) in microdissected normal and malignant epithelial ovarian tissues. Cell proliferation and viability studies were performed on cells expressing RUNX3 by lentiviral infection and cells with silenced RUNX3 expression by siRNA. RESULTS RUNX3 expression by immunohistochemistry was higher in serous ovarian carcinomas versus normal ovarian epithelium (P<0.001). Immunofluorescent staining confirmed upregulation of cytoplasmic RUNX3 in ovarian cancer cell lines and tissues. QRT-PCR showed higher RUNX3 mRNA expression in microdissected borderline and malignant ovarian tumor tissues compared with the normal ovarian surface epithelial cells (HOSE) (P=0.006 and P=0.023). Forced RUNX3 expression by lentiviral gene delivery in ovarian cancer cells, SKOV3, that initially showed undetectable RUNX3 expression, resulted in increased cell viability (P=0.043). Silencing RUNX3 expression by siRNA transfection into ovarian cancer cells, OVCAR429, initially expressing high levels of endogenous RUNX3 resulted in a decrease in proliferation (P=0.021). CONCLUSION These results suggest that RUNX3 has a role in cell proliferation and viability in ovarian cancer.