ECRG1, a novel candidate of tumor suppressor gene in the esophageal carcinoma, triggers a senescent program in NIH3T3 cells

Esophageal cancer-related gene 1 (ECRG1) is a novel tumor-suppressor gene candidate identified from the human esophagus. Previous studies showed that ECRG1 overexpression could inhibit cell growth and induce G1 cell cycle arrest and p15(INK4b) expression by interacting with Miz-1 (Myc-interacting zinc finger protein). Such evidence suggests the alterations in ECRG1 may play an important role in tumorigenesis. To further study the biological function of the ECRG1 gene, we transfected ECRG1 into NIH3T3 cells. Expression of ECRG1 in these cells caused senescence-like changes characterized in terms of altered cell morphology, cell cycle arrest at the G1/S phase, and significantly impaired cell proliferation (P < 0.01). Moreover, NIH3T3 cells transfected with ECRG1 stained positive for SA-beta-gal staining (pH 6.0), which is a specific marker of cellular senescence. We also studied changes in telomerase activity and the related senescence genes, such as p21 and p16. The results indicated that when ECRG1 induced a senescence-like state, telomerase activity was markedly decreased (P < 0.05), and expression of p21 was distinctly increased, whereas no changes were detected in p16 and telomerase-component RNA levels. These findings suggest that ECRG1 may be of importance in murine cell senescence, promoting senescence by regulating expression of p21.     

ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma,interacts directly with metallothionein 2A and links to apoptosis

Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus. To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library. Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins. The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy. Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells. In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups. When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced. When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared. The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis. ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis. The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.     

抑癌基因D L C-1 的研究进展

DLC-1（frequently deletedin liver cancer）基因是新发现的一个肿瘤抑制基因。它的失活有可能参与肿瘤的发生和发展。本文拟就DLC-1基因的结构功能及其在遗传和表遗传方面的失活机制作一综述。     

Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas

Frequent chromosome 3 losses have been described in several tumors types, which strongly suggest the presence of one or several tumor suppressor genes. Recently, a novel candidate tumor suppressor gene termed Ris-1 (for Ras-induced senescence 1) has been identified at chromosomal position 3p21.3. Ris-1 has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras. To analyze the role of Ris-1 as a putative tumor suppressor gene in human breast cancer, we have performed a real-time quantitative analysis of its mRNA expression in 60 patients. Moreover, we carried out a first approach to evaluate the most common inactivation mechanism that can affect expression levels of tumor suppressor genes (mutation, promoter hypermethylation and allelic losses). Furthermore, a correlation study between expression as well as inactivating mechanisms of Ris-1 and several clinico-pathological parameters of the tumors was designed, with the objective of appraising the prognostic value of Ris-1 status. Decreased expression of Ris-1 was observed in 23% of the cases and overexpressed Ris-1 was detected in 15% of the primary breast tumors. Our data showed high frequency of LOH (30%) at one of the markers used. Nevertheless, a polymorphism related with the expression levels was described. Statistically significant correlations were found between decreased Ris-1 expression and negative progesterone receptors, as well as between overexpressing Ris-1 tumors and high histological grade. Despite all these data, we conclude that the suggested role of Ris-1 as tumor suppressor gene is not evident, at least in breast cancer. Future and larger series studies in different tumor types are necessary to clarify Ris-1 function in human cancer.     

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.     

AZU-1: a candidate breast tumor suppressor and biomarker for tumor progression

To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT-3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up-regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis.     

DLC1在乳腺癌中的肿瘤抑制作用机理

DLC1是1998年在对原发性肝癌进行研究时首次分离和报道的,它不仅在肝癌中表达缺失,而且在人类多种恶性肿瘤中也表达低下或缺失,是近年来研究较热门的肿瘤抑制基因。乳腺癌是女性常见的恶性肿瘤,极大影响女性身心健康,在乳腺癌等恶性肿瘤中,DLC1具有抑制癌细胞增殖、迁移并诱导凋亡的作用。     

The human homolog of yeast SEP1 is a novel candidate tumor suppressor gene in osteogenic sarcoma

The hSEP1 gene is the human homolog of yeast SEP1. Yeast SEP1 is a multifunctional gene that regulates a variety of nuclear and cytoplasmic functions including homologous recombination, meiosis, telomere maintenance, RNA metabolism and microtubule assembly. The function of hSEP1 is not known. We show loss or reduced expression of hSEP1 messenger RNA (mRNA) in three of four primary osteogenic sarcoma (OGS)-derived cell lines and in eight of nine OGS biopsy specimen. In addition, we find a heterozygous missense mutation (Valine(1484)>Alanine) at a conserved amino acid in the primary OGS-derived cell line U2OS. Importantly, we identified a homozygous missense mutation involving a CG-dinucleotide leading to a change in a conserved amino acid, aspartic acid(1137) >asparagine, in the primary OGS-derived cell line, TE85. hSEP1 mRNA expression was nearly undetectable in TE85 and low in U2OS cell lines. None of these mutations were identified in 20 normal samples consisting of bone, cartilage and fibroblast. The hSEP1 gene is located in chromosome 3 at 3q25-26.1 between markers D3S1309 and D3S1569. An adjacent locus defined by the polymorphic markers D3S1212 and D3S1245 has previously been reported to undergo loss of heterozygosity (LOH) at a >70% frequency in OGS and claimed to harbor an important tumor suppressor gene in osteosarcoma. The homozygous mutation in the hSEP1 mRNA in TE85 cell line suggest that this gene itself is subject to LOH. Taken together, these results suggest that hSEP1 acts as a tumor suppressor gene in OGS.     

Cell-specific processing and release of the hormone-like precursor and candidate tumor suppressor gene product, Ecrg4

The human open reading frame C2orf40 encodes esophageal cancer-related gene-4 (Ecrg4), a newly recognized neuropeptide-like precursor protein whose gene expression by cells in vitro, over-expression in mice in vivo, and knock-down in zebrafish affects cell proliferation, migration and senescence, progenitor cell survival and differentiation, and inflammatory function. Unlike traditionally secreted neuropeptide precursors, however, we find that Ecrg4 localizes to the epithelial cell surface and remains tethered after secretion. Here, we used cell surface biotinylation to establish that 14-kDa Ecrg4 localizes to the cell surface of prostate (PC3) or kidney (HEK) epithelial cells after transfection. Accordingly, this Ecrg4 is resistant to washing cells with neutral, high salt (2 M NaCl), acidic (50 mM glycine, pH 2.8), or basic (100 mM Na(2)CO(3), pH 11) buffers. Mutagenesis of Ecrg4 established that cell tethering was mediated by an NH(2)-terminus hydrophobic leader sequence that enabled both trafficking to the surface and tethering. Immunoblotting analyses, however, showed that different cells process Ecrg4 differently. Whereas PC3 cells release cell surface Ecrg4 to generate soluble Ecrg4 peptides of 6-14 kDa, HEK cells do neither, and the 14-kDa precursor resembles a sentinel attached to the cell surface. Because a phorbol ester treatment of PC3 cells stimulated Ecrg4 release from, and processing at, the cell surface, these data are consistent with a multifunctional role for Ecrg4 that is dependent on its cell of origin and the molecular form produced.     

Deletions in chromosome 4 differentially associated with the development of cervical cancer: evidence of slit2 as a candidate tumor suppressor gene

The aim of this study was to locate the candidate tumor suppressor genes (TSGs) loci in the chromosomal 4p15-16, 4q22-23 and 4q34-35 regions associated with the development of uterine cervical carcinoma (CA-CX). Deletion mapping of the regions by microsatellite markers identified six discrete areas with high frequency of deletions, viz. 4p16.2 (D1: 40%), 4p15.31 (D2: 35–38%), 4p15.2 (D3: 37–40%), 4q22.2 (D4: 34%), 4q34.2-34.3 (D5: 37–59%) and 4q35.1 (D6: 40–50%). Significant correlation was noted among the deleted regions D1, D2 and D3. The deletions in D1, D2, D5 and D6 regions are suggested to be associated with the cervical intraepithelial neoplasia (CIN), and deletions in the D2, D3, D5 and D6 regions seems to be associated with progression of CA-CX. The deletions in the D2 and D6 regions showed significant prognostic implications (P = 0.001; 0.02). The expression of the candidate TSG SLIT2 mapped to D2 region gradually reduced from normal cervix uteri →CIN → CA-CX. SLIT2 promoter hypermethylation was seen in 28% CIN samples and significantly increased with tumor progression (P = 0.04). Significant correlation was seen between SLIT2 deletion and its promoter methylation (P = 0.001), indicating that both these phenomena could occur simultaneously to inactivate this gene. Immunohistochemical analysis showed reduced expression of SLIT2 in cervical lesions and CA-CX cell lines. Although no mutation was detected in the SLIT2 promoter region (−432 to + 55 bp), CC and AA haplotypes were seen in −227 and −195 positions, respectively. Thus, it indicates that inactivation of SLIT2-ROBO1 signaling pathway may have an important role in CA-CX development.     

WW domain-containing oxidoreductase: a candidate tumor suppressor

Common fragile site gene WWOX encodes a candidate tumor suppressor WW domain-containing oxidoreductase. Alteration of this gene, along with dramatic downregulation of WWOX protein, is shown in the majority of invasive cancer cells. Ectopic WWOX exhibits proapoptotic and tumor inhibitory functions in vitro and in vivo, probably interacting with growth regulatory proteins p53, p73 and others. Hyaluronidases regulate WWOX expression, increase cancer invasiveness and seem to be involved in the development of hormone-independent growth of invasive cancer cells. Estrogen and androgen stimulate phosphorylation and nuclear translocation of WWOX, although binding of WWOX to these sex hormones is unknown. We propose that suppression of WWOX expression by overexpressed hyaluronidases might contribute in part to the development of hormone independence in invasive cancer.     

Inactivated tumor suppressor Rb by nitric oxide promotes mitosis in human breast cancer cells

Inactivation of tumor suppressor protein retinoblastoma (Rb) is important mechanism for the G1/S transition during cell cycle progression. Human breast cancer cells T47D release great amount of nitric oxide (NO), but its relation to tumor suppressor Rb is unknown. In this study, it is shown that NO induces phosphorylation and inactivation of Rb tumor suppressor protein, increasing G2/M phase and cell proliferation of breast cancer cells T47D. NO did not induce changes in p53 ser-15 phosphorylation, the most phosphorylated site of p53 during its activation. These data indicate that NO induces cell proliferation through the Rb pathway. NO phosphorylates and inactivates tumor suppressor protein Rb inducing mitosis by the p53 independent pathway in breast cancer cell.