CHD5 is a tumor suppressor at human 1p36

Cancer gene discovery has relied extensively on analyzing tumors for gains and losses to reveal the location of oncogenes and tumor suppressor genes, respectively. Deletions of 1p36 are extremely common genetic lesions in human cancer, occurring in malignancies of epithelial, neural, and hematopoietic origin. Although this suggests that 1p36 harbors a gene that drives tumorigenesis when inactivated, the identity of this tumor suppressor has remained elusive. Here we use chromosome engineering to generate mouse models with gain and loss of a region corresponding to human 1p36. This approach functionally identifies chromodomain helicase DNA binding domain 5 (Chd5) as a tumor suppressor that controls proliferation, apoptosis, and senescence via the p19(Arf)/p53 pathway. We demonstrate that Chd5 functions as a tumor suppressor in vivo and implicate deletion of CHD5 in human cancer. Identification of this tumor suppressor provides new avenues for exploring innovative clinical interventions for cancer.     

Data on the Recurrence of Breast Tumors Fit a Model in which Dormant Cells are Subject to Slow Attrition but Can Randomly Awaken to Become Malignant

We successfully modeled the recurrence of tumors in breast cancer patients, assuming that:(i) A breast cancer patient is likely to have some circulating metastatic cells, even after initial surgery. (ii) These metastatic cells are dormant. (iii) The dormant cells are subject to attrition by the body’s immune system, or by random apoptosis or senescence.(iv) Recurrence suppressor mechanisms exist. (v) When such genes are disabled by random mutations, the dormant metastatic cell is activated, and will develop to a cancer recurrence. The model was also fitted to data on the survival of pancreatic cancer patients. The time course of cancer recurrence in a group of poor prognosis breast cancer patients could not be linked to the over- (or under-) expression of any gene in the primary tumors from which the recurrent tumors derived. Thus, the recurrence of the tumor in breast cancer patients appears to be a random event. Inasmuch as the kinetics of cancer recurrence in published data sets closely follows the model found for the appearance of sporadic retinoblastoma, tumor recurrence could be triggered by mutations in awakening-suppressor mechanisms. The retinoblastoma tumor suppressor gene was identified by tracing its occurrence in familial retinoblastoma pedigrees. Will it be possible to track the postulated cancer recurrence, awakening suppressor gene(s) in early recurrence breast cancer patients?     

miR-126 调控前列腺癌机制研究

miR-126通过靶向作用于表皮生长因子域7(EGFL7)、同源框A9(HOXA9)、胰岛素受体底物-1(IlLS-1)、p85-B基因等,在转录后水平调控靶基因表达,在肿瘤形成中起重要作用。前列腺癌细胞中高表达miR-126,能明显下调VEGF-A、EGLF7、HOXA9、VCAM-l等与肿瘤生长、转移密切相关的蛋白分子。miR-126作为抑癌因子,在多种肿瘤中均下调。其抑癌作用及机制在肺癌、白血病、乳腺癌、宫颈癌等中均已得到证实。本课题拟对miR-126调控前列腺癌机制做一综述。     

miR-126调控前列腺癌机制研究

miR-126通过靶向作用于表皮生长因子域7（EGFL7）、同源框A9（HOXA9）、胰岛素受体底物-1（11LS-1）、p85-B基因等,在转录后水平调控靶基因表达,在肿瘤形成中起重要作用。前列腺癌细胞中高表达miR,126,能明显下调VEGF—A、EGLF7、HOXA9、VCAM—1等与肿瘤生长、转移密切相关的蛋白分子。miR-126作为抑癌因子,在多种肿瘤中均下调。其抑癌作用及机制在肺癌、白血病、乳腺癌、宫颈癌等中均已得到证实。本课题拟对miR-126调控前列腺癌机制做一综述。     

miR-7 and miR-218 epigenetically control tumor suppressor genes RASSF1A and Claudin-6 by targeting HoxB3 in breast cancer

Many microRNAs have been implicated as key regulators of cellular growth and differentiation and have been found to dysregulate proliferation in human tumors, including breast cancer. Cancer-linked microRNAs also alter the epigenetic landscape by way of DNA methylation and post-translational modifications of histones. Aberrations in Hox gene expression are important for oncogene or tumor suppressor during abnormal development and malignancy. Although recent studies suggest that HoxB3 is critical in breast cancer, the putative role(s) of microRNAs impinging on HoxB3 is not yet fully understood. In this study, we found that the expression levels of miR-7 and miR-218 were strongly and reversely associated with HoxB3 expression. Stable overexpression of miR-7 and miR-218 was accompanied by reactivation of tumor suppressor genes including RASSF1A and Claudin-6 by means of epigenetic switches in DNA methylation and histone modification, giving rise to inhibition of the cell cycle and clone formation of breast cancer cells. The current study provides a novel link between overexpression of collinear Hox genes and multiple microRNAs in human breast malignancy.     

Real-time PCR quantification of full-length and exon 11 spliced BRCA1 transcripts in human breast cancer cell lines

Germline alterations of the BRCA1 tumor suppressor gene have been implicated at least in half of familial breast cancers. Nevertheless, in sporadic breast cancer no mutation of this gene has been characterized to date. In sporadic breast tumors, other BRCA1 gene loss of function mechanisms, such as down-regulation of gene expression, have been suggested. In an effort to better understand the relationship between BRCA1 expression and malignant transformation, we have adapted the new real-time quantitative PCR method based on a 5' nuclease assay and the use of doubly labeled fluorescent TaqMan probes to quantify BRCA1 mRNA. We have compared expression of BRCA1 mRNA with or without exon 11 in the normal breast epithelial cell line MCF10a and in three cancer cell lines (MCF-7, MDA-MB231 and HBL100) by comparing two methods of quantification: the comparative C(T) and the standard curve. We found that the full length BRCA1 mRNA, which encodes the functional nuclear protein, was down-regulated in tumor cells when compared with MCF10a cells.     

MicroRNA-224 targets RKIP to control cell invasion and expression of metastasis genes in human breast cancer cells

The Raf kinase inhibitor protein (RKIP) is a tumor suppressor that protects against metastasis and genomic instability. RKIP is downregulated in many types of tumors, although the mechanism for this remains unknown. MicroRNAs silence target genes via translational inhibition or target mRNA degradation, and are thus important regulators of gene expression. In the current study, we found that miR-224 expression is significantly upregulated in breast cancer cell lines, and especially in highly invasive MDA-MB-231 cells, compared to human normal breast epithelial cells. In addition, miR-224 inhibits RKIP gene expression by directly targeting its 3'-untranslated region (3'-UTR). Moreover, metastasis, as assayed by Transwell migration, 3D growth in Matrigel, and wound healing, was enhanced by ectopic expression of miR-224 and inhibited by miR-224 downregulation. Promotion of metastasis in response to miR-224 downregulation was associated with derepression of the stroma-associated RKIP target genes, CXCR4, MMP1, and OPN, which are involved in breast tumor metastasis to the bone. Taken together, our data indicate that miR-224 play an important role in metastasis of human breast cancer cells to the bone by directly suppressing the RKIP tumor suppressor.     

Yes-associated protein (YAP) functions as a tumor suppressor in breast

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.     

DNA methylation changes in murine breast adenocarcinomas allow the identification of candidate genes for human breast carcinogenesis

Epigenetic inactivation due to aberrant promoter methylation is a key process in breast tumorigenesis. Murine models for human breast cancer have been established for nearly every important human oncogene or tumor suppressor gene. Mouse-to-human comparative gene expression and cytogenetic profiling have been widely investigated for these models; however, little is known about the conservation of epigenetic alterations during tumorigenesis. To determine if this key process in human breast tumorigenesis is also mirrored in a murine breast cancer model, we mapped cytosine methylation changes in primary adenocarcinomas and paired lung metastases derived from the polyomavirus middle T antigen mouse model. Global changes in methylcytosine levels were observed in all tumors when compared to the normal mammary gland. Aberrant methylation and associated gene silencing was observed for Hoxa7, a gene that is differentially methylated in human breast tumors, and Gata2, a novel candidate gene. Analysis of HOXA7 and GATA2 expression in a bank of human primary tumors confirms that the expression of these genes is also reduced in human breast cancer. In addition, HOXA7 hypermethylation is observed in breast cancer tissues when compared to adjacent tumor-free tissue. Based on these studies, we present a model in which comparative epigenetic techniques can be used to identify novel candidate genes important for human breast tumorigenesis, in both primary and metastatic tumors.     

Loss of heterozygosity studies in tumors from families with breast-ovarian cancer syndrome

A gene (BRCA1) predisposing for familial breast and ovarian cancer has been mapped to chromosome band 17q12-21. Based on the observation that ovarian tumors from families with breast and ovarian cancer lose the wild-type allele in the region for the BRCA1 locus, it has been suggested that the gene functions as a tumor suppressor gene. We have studied chromosomal deletions in the BRCA1 region in seven breast tumors, three ovarian tumors, one bladder cancer, and one colon cancer from patients in six families with breast-ovarian cancer, in order to test the hypothesis of the tumor suppressor mechanism at this locus. We have found a low frequency of loss of heterozygosity at this region, and our results do not support the idea that BRCA1 is a tumor suppressor gene. Alternatively, the disease segregating in these families is linked to one or more different loci.     

Negative Regulation of NF-κB by the ING4 Tumor Suppressor in Breast Cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.     

Characterization of MTAP Gene Expression in Breast Cancer Patients and Cell Lines

MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors´ MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNBC.