Down-regulation of ECRG4, a candidate tumor suppressor gene, in human breast cancer

Introduction

ECRG4/C2ORF40 is a potential tumor suppressor gene (TSG) recently identified in esophageal carcinoma. Its expression, gene copy number and prognostic value have never been explored in breast cancer.

Methods

Using DNA microarray and array-based comparative genomic hybridization (aCGH), we examined ECRG4 mRNA expression and copy number alterations in 353 invasive breast cancer samples and normal breast (NB) samples. A meta-analysis was done on a large public retrospective gene expression dataset (n = 1,387) in search of correlations between ECRG4 expression and histo-clinical features including survival.

Results

ECRG4 was underexpressed in 94.3% of cancers when compared to NB. aCGH data revealed ECRG4 loss in 18% of tumors, suggesting that DNA loss is not the main mechanism of underexpression. Meta-analysis showed that ECRG4 expression was significantly higher in tumors displaying earlier stage, smaller size, negative axillary lymph node status, lower grade, and normal-like subtype. Higher expression was also associated with disease-free survival (DFS; HR = 0.84 [0.76–0.92], p = 0.0002) and overall survival (OS; HR = 0.72 [0.63–0.83], p = 5.0E-06). In multivariate analysis including the other histo-clinical prognostic features, ECRG4 expression remained the only prognostic factor for DFS and OS.

Conclusions

Our data suggest that ECRG4 is a candidate TSG in breast cancer, the expression of which may help improve the prognostication. If functional analyses confirm this TSG role, restoring ECRG4 expression in the tumor may represent a promising therapeutic approach.     

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。     

Cell-specific modulation of papovavirus replication by tumor suppressor protein p53

Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.     

The protein product of the tumor suppressor gene,melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24

The melanoma differentiation-associated gene 7 (mda-7) has been studied primarily in the context of its tumor suppressor activity. Although mda-7 has been designated as IL-24 based on its gene location in the IL-10 locus and its mRNA expression in leukocytes, no functional evidence supporting this cytokine designation exists. To further characterize MDA-7/IL-24 expression patterns in the human immune system, MDA-7/IL-24 protein levels were examined in human PBMC. MDA-7/IL-24 was detected in PHA- and LPS-stimulated whole PBMC lysate by Western blot and in PHA-activated CD56 and CD19 subsets by immunohistochemistry. The biological function of MDA-7/IL-24, secreted from Ad-MDA7-transfected HEK 293 cells, was assessed by examining the effect of MDA-7/IL-24 on the cytokine secretion profile of PBMC. Within 48 h MDA-7/IL-24 induced secretion of high levels of IL-6, TNF-alpha, and IFN-gamma and low levels of IL-1beta, IL-12, and GM-CSF from human PBMC as measured by ELISA. The MDA-7/IL-24-mediated induction of these Th1-type cytokines was inhibited by the addition of IL-10 to the PBMC cultures, suggesting that these two related protein family members may provide antagonistic functions. Therefore, because human blood leukocytes can be stimulated to produce MDA-7/IL-24, as well as respond to MDA-7/IL-24 by expressing secondary cytokines, MDA-7/IL-24 has the expression profile and major functional attributes that justify its designation as an IL.     

Methylation of the promoter region of the RASSF1A gene, a candidate tumor suppressor, in primary epithelial tumors

Methylation of the promoter CpG-islands of the candidate tumor suppressor gene RASSF1A (3p21.31) was studied in primary tumors of kidney, breast and ovary (172 cases). Methylation-specific PCR (MSP) and methyl-sensitive restriction endonuclease digestion followed by PCR (MSRA) were applied. Statistically significant correlation (P < 10(-6)) was shown for the results of the MSP and MSRA, and the data of bisulfite sequencing reported earlier. The frequency of RASSF1A methylation according to MSP and MSRA was 86% (25/29) and 94% (50/53) in renal cell carcinoma (RCC) and 64% (18/28) and 78% (32/41)--in breast carcinoma (BC) samples, and 59% (17/29) and 73% (33/45) in ovarian epithelial tumors (OET), respectively. The use of several methyl-sensitive restriction enzymes (HpaII, HhaI, Bsh12361, AciI) enhanced the sensitivity of MSRA and allowed to analyze methylation status of 18 CpG-pairs in the RASSF1A CpG-island. Density of methylation of the RASSF1A CpG-island was 72% (644/900) in RCC, 63% (361/576) in BC, and 58% (346/594) in OET samples (18 CpG-pairs multiplied to the number of samples shown methylation were assumed as 100%). The RASSF1A gene methylation was also observed in samples of morphologically normal tissues adjacent to corresponding tumors (11-35%), but it was not detected in blood DNAs of healthy donors (0/15). The RASSF1A methylation frequency did not show significant correlation to tumor stage, grade and metastases (P = 0.3-1.0). The RASSF1A gene methylation was observed more frequently than other investigated aberrations--hemi- and homozygous deletions inside or around this gene. These observations are consistent with the hypothesis that the RASSF1A gene methylation is an early event in the carcinogenesis and one of the dominant ways of its inactivation.     

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.     

WWOX, a new potential tumor suppressor gene

BACKGROUND: WWOX (WW domain-containing oxidoreductase) gene, located on chromosome 16q 23.3-24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene involved in various cancers: breast, ovarian, prostate, esophageal, lung, pancreatic, gastric and hepatic. The aim of this study was to describe (i) putative protein interactions of WWOX (ii) the molecular mechanisms of tumor suppressor activity (iii) present an overview of WWOX in relation to nervous system and breast, prostate and ovarian cancers. METHODS AND RESULTS: WWOX expression is up-regulated in endocrine organs indicating its importance in these tissues. In many cancers WWOX expression is down-regulated and low WWOX expression is related to poor prognosis. CONCLUSION: All the evidence suggest that WWOX can be considered as a new tumor suppressor gene and target for gene therapy due to the association of high WWOX expression with improved disease free survival.     

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.     

Cloning of a novel gene (ING1L) homologous to ING1, a candidate tumor suppressor

The ING1 gene encodes p33(ING1), a putative tumor suppressor for neuroblastomas and breast cancers, which has been shown to cooperate with p53 in controlling cell proliferation. We have isolated a novel human gene, ING1L, that potentially encodes a PHD-type zinc-finger protein highly homologous to p33(ING1). Fluorescence in situ hybridization and radiation-hybrid analyses assigned ING1L to human chromosome 4. Both ING1 and ING1L are expressed in a variety of human tissues, but we found ING1L expression to be significantly more pronounced in tumors from several colon-cancer patients than in normal colon tissues excised at the same surgical sites. Although the significance of this observation with respect to carcinogenesis remains to be established, the data suggest that ING1L might be involved in colon cancers through interference with signal(s) transmitted through p53 and p33(ING1).     

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.