Josephine Mun Yee Ko

Identification of a tumor suppressive critical region mapping to 3p14.2 in esophageal squamous cell carcinoma and studies of a candidate tumor suppressor gene, ADAMTS9.

A gene critical to esophageal cancer has been identified. Functional studies using microcell-mediated chromosome transfer of intact and truncated donor chromosomes 3 into an esophageal cancer cell line and nude mouse tumorigenicity assays were used to identify a 1.61 Mb tumor suppressive critical region (CR) mapping to chromosome 3p14.2. This CR is bounded by D3S1600 and D3S1285 microsatellite markers. One candidate tumor suppressor gene, ADAMTS9, maps to this CR. Further studies showed normal expression levels of this gene in tumor-suppressed microcell hybrids, levels that were much higher than observed in the recipient cells. Complete loss or downregulation of ADAMTS9 gene expression was found in 15 out of 16 esophageal carcinoma cell lines. Promoter hypermethylation was detected in the cell lines that do not express this gene. Re-expression of ADAMTS9 was observed after demethylation drug treatment, confirming that hypermethylation is involved in gene downregulation. Downregulation of ADAMTS9 was also found in 43.5 and 47.6% of primary esophageal tumor tissues from Hong Kong and from the high-risk region of Henan, respectively. Thus, this study identifies and provides functional evidence for a CR associated with tumor suppression on 3p14.2 and provides the first evidence that ADAMTS9, mapping to this region, may contribute to esophageal cancer development.

Genomic instability and alterations in Apc, Mcc and Dcc in Hong Kong patients with colorectal carcinoma

Our aim was to reveal the significance of tumor‐suppressor genes and genomic instability in 99 Hong Kong Chinese colorectal carcinoma (CRC) patients by PCR‐LOH analysis and PCR‐PTT assay. The frequencies of allelic loss of Apc, Mcc and Dcc and of APC truncation were 31.3% (15/48), 11.6% (5/43), 44.4% (20/45) and 46/93 (49.5%), respectively. The frequency of Apc LOH was similar to, the Mcc LOH was lower than, and the Dcc LOH was higher than that reported for Caucasians and Japanese. In Hong Kong CRC patients, the replication error‐positive (RER+) phenotype occurred with a frequency of 10% (10/99), which was similar to other results using microsatellite markers where RER+ frequencies ranged from 11% to 28%. The rates of genetic alteration in RER+ tumors were lower in tumors harboring p53, Mcc and Dcc alterations; similar in Apc; and higher in Ki‐ras tumors compared with RER– tumors, though these differences did not achieve statistical significance. None of the biomarkers examined were predictive of survival independently, but strong trends confirming earlier observations of associations between RER+ phenotypes with proximal tumor location and poorly differentiated tumor status were noted. The RER+ phenotype was correlated significantly to the less aggressive Dukes stage B and improved prognosis. Additionally, tumors with RER+ phenotypes were positively correlated with young age and sex. Our results support the observation that a subset of younger male CRC patients in Hong Kong may develop CRC via the RER pathway and show differences in RER status and sex. A significantly higher percentage of older Hong Kong Chinese CRC patients had APC truncations. Int. J. Cancer (Pred. Oncol.) 84:404–409, 1999.

Cysteine-rich intestinal protein 2 (CRIP2) acts as a repressor of NF-κB–mediated proangiogenic cytokine transcription to suppress tumorigenesis and angiogenesis

Chromosome 14 was transferred into tumorigenic nasopharyngeal carcinoma and esophageal carcinoma cell lines by a microcell-mediated chromosome transfer approach. Functional complementation of defects present in the cancer cells suppressed tumor formation. A candidate tumor-suppressor gene, cysteine-rich intestinal protein 2 (CRIP2), located in the hot spot for chromosomal loss at 14q32.3, was identified as an important candidate gene capable of functionally suppressing tumor formation. Previous studies have shown that CRIP2 is associated with development. To date, no report has provided functional evidence supporting a role for CRIP2 in tumor development. The present study provides unequivocal evidence that CRIP2 can functionally suppress tumorigenesis. CRIP2 is significantly down-regulated in nasopharyngeal carcinoma cell lines and tumors. CRIP2 reexpression functionally suppresses in vivo tumorigenesis and angiogenesis; these effects are induced by its transcription-repressor capability. It interacts with the NF-κB/p65 to inhibit its DNA-binding ability to the promoter regions of the major proangiogenesis cytokines critical for tumor progression, including IL6, IL8, and VEGF. In conclusion, we provide compelling evidence that CRIP2 acts as a transcription repressor of the NF-κB–mediated proangiogenic cytokine expression and thus functionally inhibits tumor formation and angiogenesis.

Tumor suppressive role of a 2.4 Mb 9q33-q34 critical region and DEC1 in esophageal squamous cell carcinoma.

The key genes involved in the development of esophageal squamous cell carcinoma (ESCC) remain to be elucidated. Previous studies indicate extensive genomic alterations occur on chromosome 9 in ESCC. Using a monochromosome transfer approach, this study provides functional evidence and narrows down the critical region (CR) responsible for chromosome 9 tumor suppressing activity to a 2.4 Mb region mapping to 9q33–q34 between markers D9S1798 and D9S61. Interestingly, a high prevalence of allelic loss in this CR is also observed in primary ESCC tumors by microsatellite typing. Allelic loss is found in 30/34 (88%) tumors and the loss of heterozygosity (LOH) frequency ranges from 67 to 86%. Absent to low expression of a 9q32 candidate tumor suppressor gene (TSG), DEC1 (deleted in esophageal cancer 1), is detected in four Asian ESCC cell lines. Stably expressing DEC1 transfectants provide functional evidence for inhibition of tumor growth in nude mice and DEC1 expression is decreased in tumor segregants arising after long-term selection in vivo. There is 74% LOH in the DEC1 region of ESCC primary tumors. This study provides the first functional evidence for the presence of critical tumor suppressive regions on 9q33–q34. DEC1 is a candidate TSG that may be involved in ESCC development.

FINE MAPPING OF THE 11Q22-23 TUMOR SUPPRESSIVE REGION AND INVOLVEMENT OF TSLC1 IN NASOPHARYNGEAL CARCINOMA

Previous studies transferring an intact chromosome 11 into HONE1 cells demonstrated the functional significance of chromosome regions, 11q13 and 11q22–23, in nasopharyngeal carcinoma (NPC) development. In our study the 11q22–23 region was comprehensively re‐investigated by detailed microsatellite and single nucleotide polymorphism genotyping and by fluorescence in situ hybridization to map precisely the regions containing tumor suppressive activity. We observed 3 chromosomal intervals within 11q22–23 that were commonly lost in the tumor segregants derived from HONE1/chromosome 11 hybrids. One critical region of 0.36 Mb was mapped near the marker D11S2000 and a second 0.44 Mb region was located around the markers D11S1300 and D11S1391. In a third region high allelic loss was also observed at marker D11S4484, where a newly cloned tumor suppressor gene, TSLC1 (tumor suppressor in lung cancer 1), is located. The gene expression analysis showed absence or low expression levels of TSLC1 mRNA in 4 highly tumorigenic NPC cell lines. In addition, the methylation study results show that the TSLC1 promoter region was hypermethylated in all 4 NPC cell lines and re‐expression of the gene occurs in HONE1 cells after 5‐aza‐2′‐deoxycytidine treatment. Hence, the mode of silencing of this candidate TSG in NPC may be attributed to promoter hypermethylation. We have obtained functional evidence for multiple critical tumor suppressive regions in 11q22–23 by fine deletion mapping and for inactivation of TSLC1 being one of these candidate TSGs in NPC development.

Monochromosome Transfer and Microarray Analysis Identify a Critical Tumor-Suppressive Region Mapping to Chromosome 13q14 and THSD1 in Esophageal Carcinoma

Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG. (Mol Cancer Res 2008;6(4):592–603)

Frequent loss of heterozygosity on multiple chromosomes in Chinese esophageal squamous cell carcinomas

Analysis of the loss of heterozygosity (LOH) detected by polymerase chain reaction techniques using 18 polymorphic markers localized to chromosomes 3p, 5, 17, and 18q in 40 Hong Kong Chinese esophageal squamous cell carcinoma (ESC) patients showed that multiple alterations on several chromosomes are involved in ESC development. The LOH rates detected for markers on chromosome 3 ranged from 44.0 to 85.7%, for chromosome 5 from 40.9 to 61.9%, for chromosome 17 from 40.0 to 100%, and for chromosome 18 from 38.9 to 58.3%. No significant association was observed between LOH and the clinical and histopathological parameters.

Functional evidence of decreased tumorigenicity associated with monochromosome transfer of chromosome 14 in esophageal cancer and the mapping of tumor‐suppressive regions to 14q32

Despite the abundant evidence of high allelic loss of chromosome arm 14q in human cancers, tumor‐suppressor genes mapped to this chromosome have yet to be identified. To narrow the search for candidate genes, we performed monochromosome transfer of chromosome 14 into an esophageal carcinoma cell line, SLMT‐1 S1. Statistically significant suppression of the tumorigenic potential of microcell hybrids containing the transferred chromosome 14 provided functional evidence that tumor‐suppressive regions of chromosome 14 are essential for esophageal cancer. Tumor segregants emerging in nude mice during the tumorigenicity assay were analyzed by detailed PCR‐microsatellite typing to identify critical nonrandomly eliminated regions (CRs). A 680‐kb CR mapped to 14q32.13 and an ∼2.2‐Mb CR mapped to 14q32.33 were delineated. Dual‐color BAC FISH analysis of microcell hybrids and tumor segregants verified the selective loss of the 14q32.13 region. In contrast, similar transfers of an intact chromosome 11 into SLMT‐1 S1 did not significantly suppress tumor formation. These functional complementation studies showing the correlation of tumorigenic potential with critical regions of chromosome 14 validated the importance of the 14q32 region in tumor suppression in esophageal cancer. The present study also paved the path for further identification of novel tumor‐suppressor genes that are relevant to the molecular pathogenesis of esophageal cancer.

Monochromosome transfer provides functional evidence for growth-suppressive genes on chromosome 14 in nasopharyngeal carcinoma

In many cancers, including nasopharyngeal carcinoma (NPC), extensive and multiple regions of allelic loss occur on chromosome 14. However, to date no functionally conclusive tumor suppressor genes have yet been identified on this chromosome. Through use of the monochromosome transfer technique, this study provides functional evidence for the importance of two discrete regions of chromosome 14. A newly established A9 mouse donor cell line containing an intact copy of chromosome 14 was used for transfer of this intact chromosome into the NPC HONE1 cell line. Twelve independently established microcell hybrids demonstrated uniform loss of specific chromosome 14 loci from both endogenous and exogenous alleles. By microsatellite typing and fluorescence in situ hybridization with BAC probes, the two critical regions were localized to 14q11.2–13.1 and 14q32.1. Selective elimination of these regions during hybrid selection was strongly associated with both hybrid survival and tumor growth in vivo. This functional evidence now narrows down the candidate areas for further studies and suggests that at least two hitherto unidentified growth‐related genes localized on two critical regions of chromosome arm 14q play an important role in tumorigenesis.

The interplay of host genetic factors and Epstein-Barr virus in the development of nasopharyngeal carcinoma.

The interplay between host cell genetics and Epstein-Barr virus (EBV) infection contributes to the development of nasopharyngeal carcinoma (NPC). Understanding the host genetic and epigenetic alterations and the influence of EBV on cell signaling and host gene regulation will aid in understanding the molecular pathogenesis of NPC and provide useful biomarkers and targets for diagnosis and therapy. In this review, we provide an update of the oncogenes and tumor suppressor genes associated with NPC, as well as genes associated with NPC risk including those involved in carcinogen detoxification and DNA repair. We also describe the importance of host genetics that govern the human leukocyte antigen (HLA) complex and immune responses, and we describe the impact of EBV infection on host cell signaling changes and epigenetic regulation of gene expression. High-power genomic sequencing approaches are needed to elucidate the genetic basis for inherited susceptibility to NPC and to identify the genes and pathways driving its molecular pathogenesis.