Event In Breast Tumorigenesis Research Articles

Acetylation of Steroidogenic Acute Regulatory Protein Sensitizes 17β-Estradiol Regulation in Hormone-Sensitive Breast Cancer Cells.

An imbalance in estrogen signaling is a critical event in breast tumorigenesis. The majority of breast cancers (BCs) are hormone-sensitive; they majorly express the estrogen receptor (ER+) and are activated by 17β-estradiol (E2). The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in steroid biosynthesis. The dysregulation of the epigenetic machinery, modulating E2 levels, is a primary occurrence for promoting breast tumorigenesis. StAR expression, concomitant with E2 synthesis, was reported to be aberrantly high in human and mouse hormone-dependent BC cells compared with their non-cancerous counterparts. However, the mechanism of action of StAR remains poorly understood. We discovered StAR as an acetylated protein and have identified a number of lysine (K) residues that are putatively acetylated in malignant and non-malignant breast cells, using LC-MS/MS (liquid chromatography-tandem mass spectrometry), suggesting they differently influence E2 synthesis in mammary tissue. The treatment of hormone-sensitive MCF7 cells with a variety of histone deacetylase inhibitors (HDACIs), at therapeutically and clinically relevant doses, identified a few additional StAR acetylated lysine residues. Among a total of fourteen StAR acetylomes undergoing acetylation and deacetylation, K111 and K253 were frequently recognized either endogenously or in response to HDACIs. Site-directed mutagenesis studies of these two StAR acetylomes, pertaining to K111Q and K253Q acetylation mimetic states, resulted in increases in E2 levels in ER+ MCF7 and triple negative MB-231 BC cells, compared with their values seen with human StAR. Conversely, these cells carrying K111R and K253R deacetylation mutants diminished E2 biosynthesis. These findings provide novel and mechanistic insights into intra-tumoral E2 regulation by elucidating the functional importance of this uncovered StAR post-translational modification (PTM), involving acetylation and deacetylation events, underscoring the potential of StAR as a therapeutic target for hormone-sensitive BC.

Inhibition of HAS2 and hyaluronic acid production by 1,25-Dihydroxyvitamin D3 in breast cancer.

1,25-Dihydroxyvitamin D3 (1,25D3) induces growth arrest and apoptosis in breast cancer cells in vivo and in vitro, however the exact mechanisms are unclear. Although the vitamin D receptor (VDR), a ligand dependent transcription factor, is required for growth regulation by vitamin D, the specific target genes that trigger these effects are unknown. Genomic profiling of murine mammary tumor cells with differential VDR expression identified 35 transcripts that were altered by the 1,25D3-VDR complex including Hyaluronan Synthase-2 (Has2). Here we confirmed that 1,25D3 reduces both HAS2 gene expression and hyaluronic acid (HA) synthesis in multiple models of breast cancer. Furthermore, we show that the growth inhibitory effects of 1,25D3 are partially reversed in the presence of high molecular weight HA. HAS2 expression and HA production are elevated in immortalized human mammary epithelial cells induced to undergo epithelial-mesenchymal transition (EMT) through stable expression of TGFβ, SNAIL or TWIST and in those expressing oncogenic H-RASV12, indicating that deregulation of HA production may be an early and frequent event in breast tumorigenesis. 1,25D3 also reduces HA secretion and acts additively with an HA synthesis inhibitor to slow growth of cells expressing TGFβ, SNAIL and TWIST. Analysis of mammary gland and tumors from Vdr knockout mice suggest that loss of VDR is associated with enhanced HAS2 expression and HA production in vivo. These data define a novel role for 1,25D3 and the VDR in control of HA synthesis in epithelial tissues that likely contributes to its anti-cancer actions.

The novel TP53 3'-end methylation pattern associated with its expression would be a potential biomarker for breast cancer detection.

Deficiency or silence of TP53 is an early event in breast tumorigenesis. Aberrant methylation and mutation in regulatory regions were considered as crucial regulators of gene expression. Using multiplex-PCR and next-generation sequencing technology, we analyzed TP53 mutation spectrum in its promoter region. Using PCR target sequence enrichment and next-generation bisulfite sequencing technology, we analyzed the methylation profile of the promoter and 3'-end regions of TP53 gene in paired breast tumor and normal tissues from 120 breast cancer patients. The expression of TP53 and the flanking gene ATP1B2 was explored with qPCR method in the same cohort. No promoter mutation of TP53 gene was found in the cohort of the 120 breast cancer patients. The 3'-end of TP53 gene was hyper-methylated (average 78.71%) compared with the promoter region (average less than 1%) in breast tumor tissues. TP53 was significantly lower expressed (P = 1.68E-15) and hyper-methylated in 3'-end (P = 1.82E-18) in tumor. Negative cis correlation was found between the TP53 expression and its 3'-end methylation (P = 9.02E-8, R = 0.337). TP53 expression was significantly associated with PR status (P = 0.0128), Ki67 level (P = 0.0091), and breast cancer subtypes (P = 0.0109). TP53 3'-end methylation and expression showed a good performance in discriminating breast cancer and normal tissues with an AUC of 0.930. The 3'-end methylation of TP53 might be a crucial regulator for its expression in breast cancer, suggesting that TP53 3'-end hyper-methylation associated with its lower expression could be a potential biomarker for breast cancer diagnosis.

Promoter methylation of BRCA1 in the prognosis of breast cancer: a meta-analysis

The inactivation of BRCA1 by epigenetic alterations is a critical event in breast tumorigenesis, which may potentially be used as a prognostic marker for patients with breast cancer. The present study systematically reviewed the promoter methylation of BRCA1 and its relationship to the clinical outcomes of breast cancer patients. We performed a meta-analysis following the PRISMA guideline. Relevant articles were identified by searching PubMed, Web of Science and Embase database until August 2013. The pooled hazard ratio (HR) and 95 % confidence interval (CI) were applied to estimate the effect of BRCA1 methylation. Random or fixed effect model was chosen based on the heterogeneity analysis. A total of 3,205 patients from nine eligible studies were included in the meta-analysis. BRCA1 methylation was found to be significantly correlated with a poor overall survival of breast cancer, with the combined HR (95 % CI) of 2.02 (1.35-3.03). After adjusting for potential confounders using the Cox regression model, the pooled HR (95 % CI) of BRCA1 methylation on patients' overall survival was 1.38 (1.04-1.84). If we used the disease-free survival as the outcome, the combined HR (95 % CI) was 2.89 (1.73-4.83) for univariate analysis and 3.92 (95 % CI 1.49-10.32) for multivariate analysis, respectively. Subgroup analysis of specimen types revealed that the pooled HR (95 % CI) for overall survival was 1.48 (1.22-1.81) when using formalin-fixed paraffin-embedded (FFPE) specimen and 1.38 (0.16-11.84) when using fresh frozen tissues. As for the disease-free survival, the pooled HR (95 % CI) was 2.47 (1.33-4.58) when using FFPE specimen and 2.78 (1.47-5.28) when using fresh frozen tissues. As a conclusion, the present meta-analysis provides evidence that BRCA1 methylation is associated with a poor survival of breast cancer patients. Our findings underscore the clinical relevance of aberrant epigenetic alteration as a promising biomarker for the prognosis of human cancers.

The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor GeneBRCA1through GABP Beta

Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the β-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPβ interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk.

Down-regulation of the Tumor Suppressor Protein 14-3-3σ Is a Sporadic Event in Cancer of the Breast

14-3-3 proteins comprise a family of highly conserved and broadly expressed multifunctional regulatory proteins that are involved in various cellular processes such as cell cycle progression, cell growth, differentiation, and apoptosis. Transcriptional expression of the sigma isoform of 14-3-3 is frequently impaired in human cancers, including carcinomas of the breast, which has led to the suggestion that this protein might be involved in the neoplastic transformation of breast epithelial cells. Here we report on the analysis of 14-3-3sigma expression in primary breast tumors using a proteomic approach complemented by immunohistochemical analysis by means of specific antibodies against this isoform. We show that the levels of expression of 14-3-3sigma were similar in non-malignant breast epithelial tissue and matched malignant tissue with only sporadic loss of expression observed in 3 of the 68 tumors examined. Moreover we show that 14-3-3sigma immunoreactivity was restricted to epithelial cells and significantly stronger in the myoepithelial cells that line the mammary ducts and lobules. The lack of expression of 14-3-3sigma in the three breast carcinomas was not associated with high levels of expression of the dominant-negative transcriptional regulator DeltaNp63 or with increased expression of estrogen-responsive finger protein, a ubiquitin-protein ligase (E3) that targets 14-3-3sigma for proteolysis. Validation of the results was performed retrospectively on an independent clinical tumor sample set using a tissue microarray containing 65 primary tumors. Our data suggest that, contrary to what was previously thought, loss of expression of 14-3-3sigma protein is not a frequent event in breast tumorigenesis.

Upregulation of focal adhesion kinase (FAK) expression in ductal carcinoma in situ (DCIS) is an early event in breast tumorigenesis

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in a subset of invasive breast cancers. FAK transmits signals that mediate several functions including tumor cell proliferation, migration, adhesion and survival. We used immunohistochemical techniques to assess FAK expression in patients with fibrocystic disease (FCD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC). Formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained from 119 patients (12 FCD, 38 ADH, 51 DCIS and 18 IDC). The anti-FAK 4.47 monoclonal antibody was used to detect FAK expression. FAK expression was scored as high (3 or 4 intensity and > or =90% positive cells) or low. The DCIS tissue sections demonstrated high FAK expression in 34/51 (66%) of the sections. High FAK expression was demonstrated in 6/18 (33%) of the IDC tissue sections and 8/38 (21%) of the ADH tissue sections. None (0/12) of the FCD tissues sections stained high for FAK. The pattern of FAK expression in DCIS was significantly higher than ADH (p < 0.0001) and IDC (p = 0.02). We conclude that FAK overexpression in preinvasive, DCIS tumors precedes tumor cell invasion or metastasis, suggesting that FAK may function as a survival signal and be an early event in breast tumorigenesis.

Loss of SFRP1 is associated with breast cancer progression and poor prognosis in early stage tumors

Aberrant activation of the Wnt signaling pathway plays an important role in the development of solid tumors such as breast and colon cancer. Secreted Frizzled-related protein 1 (SFRP1) is a negative regulator of the Wnt pathway. It has been described that SFRP1 mRNA is strongly down-regulated in breast cancer and a putative tumor suppressor function has been postulated. We have generated and characterized an SFRP1 specific antibody to analyze its expression on protein level and to investigate the association of SFRP1 expression with clinicopathological parameters and patient survival. Analysis of >2000 invasive breast tumors and 56 carcinoma in situ revealed similar frequencies of SFRP1 loss in these tumors (46% and 43% respectively). Therefore, we propose that loss of SFRP1 expression is an early event in breast tumorigenesis. SFRP1 expression was inversely correlated with tumor stage (p<0.001) but not with tumor grade (p=0.14) or lymph node status (p=0.84). Performing a multivariate analysis we could confirm the association between tumor stage and SFRP1 expression (p=0.029). In particular, loss of SFRP1 expression in early stage breast tumors (pT1) was associated with poor prognosis (p=0.04). In conclusion, expression of SFRP1 is commonly lost in breast cancer. SFRP1 expression might be useful as a novel prognostic marker in early stage breast cancer.

TP53 and breast cancer.

The TP53 gene (p53) is found altered in breast carcinomas in approximately 20-40% of all cases depending on tumor size and stage of the disease. It seems to be an early event in breast tumorigenesis. Several polymorphisms in the TP53 gene have been detected and their possible roles in breast cancer risk and association to type of cancer developed are discussed. The different mutation spectra seen in geographical and ethnic populations may be used to identify environmental exposure contributing to breast cancer development. The role of TP53 mutation as a prognostic marker is reviewed as well as its role as a predictor for therapy response. All data available on TP53 mutation analyses of human breast carcinomas, as well data from transgenic animal studies and experimental cell studies, support an important role for TP53 in mammary carcinogenesis.

Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients.

The newly identified 3p21.3 tumour suppressor gene RASSF1A is methylated in the majority of primary lung tumours, lung tumour cell lines and in a variable percentage of breast tumours. To determine the extent of RASSF1A promoter hypermethylation in early lung tumorigenesis, we analysed sputum samples from lung cancer patients and from current and former smokers using a sensitive methylation-specific PCR (MSP) technique. We also analysed RASSF1A promoter region hypermethylation in trios of normal breast/invasive ductal breast carcinoma/ductal carcinoma in situ (DCIS) from breast cancer patients and DCIS without invasive cancer. We found that 50% of small cell lung cancer (SCLC) and 21% of non-small cell lung cancer (NSCLC) patients had RASSF1A methylation, while one of two former smokers and four of 13 current smokers demonstrated RASSF1A methylation in sputum. Furthermore, two of the four current smokers and one former smoker showing RASSF1A methylation in their sputum developed cancer within 12-14 months of bronchoscopy. In our breast cancer trios, RASSF1A promoter hypermethylation was detected in 65% of invasive cancers, in 42% of corresponding DCIS but in none of the normal breast samples. In addition, we found that three out of 10 DCIS without invasive breast cancer also underwent RASSF1A promoter hypermethylation. Our findings suggest that RASSF1A promoter region hypermethylation may be a useful molecular marker for early detection of lung cancer. Furthermore, since RASSF1A promoter hypermethylation was detected in ductal carcinoma in situ, inactivation of RASSF1A may be an early event in breast tumorigenesis.

Abnormal Expression of Cell Cycle Regulatory Proteins in Ductal and Lobular Carcinomas of the Breast

In a previous study, we demonstrated that the G1 cell cycle checkpoint in carcinomas of the breast is frequently abrogated by loss of p16, the product of the CDKN2/INK4A gene, and, to a lesser extent, by loss of pRB, the product of the retinoblastoma gene. The purpose of the present study was to determine whether other mechanisms of cell cycle deregulation exist in breast cancers which have retained RB and p16 function. Paraffin sections of 81 invasive breast carcinomas (49 ductal, 26 lobular, 6 mixed) were reacted with monoclonal antibodies against cyclin D1 and p53, using optimized immunohistochemical staining protocols. The staining results were correlated with the expression of p16 and pRB, and with a variety of pathological parameters and DNA ploidy. Twenty-five tumors (31%) accumulated (presumably mutant) p53 and 28 (35%) overexpressed cyclin D1; 7 carcinomas (not including any pure lobular cancers) abnormally expressed both proteins. p53 accumulation correlated with nuclear, mitotic, and overall grade, but not with tumor size, lymph node involvement, or DNA ploidy. Overexpression of cyclin D1 was not associated with any of the patho-biological variables. There was an inverse correlation between loss of p16 and high levels of p53, but not cyclin D1. The G1 cell cycle checkpoint, which is controlled by RB, cyclin D1, and p16, was abrogated in 65% of carcinomas, and only p53 was abnormal in an additional 17%. The number of abnormally expressed genes correlated with mitotic activity and overall tumor grade, but not with tumor histology, size, or nodal status, suggesting that cell cycle deregulation is an early event in breast tumorigenesis. Only 18% of the carcinomas showed a normal level of expression of the four genes tested, and p16 appeared to be the most common target of cell cycle deregulation. These data point to the importance of cell cycle regulatory protein abnormalities in human breast cancer.

Complex role of tumor cell transforming growth factor (TGF)-beta s on breast carcinoma progression.

Growth inhibition by the TGF-beta s has been extensively studied in both normal and transformed mammary epithelial cells. It has been proposed that loss of autocrine TGF-beta mediated growth regulation is a critical event in breast tumorigenesis and several lines of in vitro and in vivo data support this hypothesis. However, a positive association between the expression of TGF-beta s by tumor cells and the progression or maintenance of breast cancinoma cells has been observed in many studies in in vivo tumor models. Possible mechanisms for these growth enhancing effects of TGF-beta include immunosuppression mediated by tumor TGF-beta s, enhanced angiogenesis, increased peritumoral stroma formation, and cell adhesion. The net effect of tumor cell TGF-beta on the biology of breast carcinogenesis would depend on the balance between autocrine growth inhibition of mammary epithelial cells and these growth enhancing effects.

452 Loss of heterozygosity on 7Q31 occurs early during breast tumorigenesis

Although human breast tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumor suppressor genes, the identity of the genetic alterations that are critical in the early stages of the breast tumorigenic process remains obscure. A high frequency (27–41%) of LOH occurrence has been shown at the <i>MET</i> locus on chromosome band 7q31 and this specific alteration is associated with poorer survival. Here, we report that RFLP analysis on 221 informative (heterozygous) primary breast tumors and 57 informative relapses (13 local recurrences and 44 distant metastases) revealed a similar frequency of 7q31 LOH as tumors progress from primary cancer to relapse, in marked contrast to other changes such as 11p15.5 LOH. This finding suggests that inactivation of a putative tumor suppressor gene located in 7q31 is a very early event in breast tumorigenesis. Our results also show that metastatic potential is an induced phenomenon that occurs at a relatively early stage, rather than a marker of tumor progression.

Loss of heterozygosity on 7q31 occurs early during breast tumorigenesis

Although human breast tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumor suppressor genes, the identity of the genetic alterations that are critical in the early stages of the breast tumorigenic process remains obscure. A high frequency (27-41%) of loss of heterozygosity (LOH) occurrence has been shown at the MET locus on chromosome band 7q31 and this specific alteration is associated with poorer survival. Here, we report that restriction fragment length polymorphism (RFLP) analysis on 221 informative (heterozygous) primary breast tumors and 57 informative relapses (13 local recurrences and 44 distant metastases) revealed a similar frequency of 7q31 LOH as tumors progress from primary cancer to relapse, in marked contrast to other changes such as 11p15.5 LOH. This finding suggests that inactivation of a putative tumor suppressor gene located in 7q31 is a very early event in breast tumorigenesis. Our results also show that metastatic potential is an induced phenomenon that occurs at a relatively early stage, rather than a marker of tumor progression.