A different methylation profile of circadian genes promoter in breast cancer patients according to clinicopathological features

ABSTRACT

One of the supposed mechanisms that may lead to breast cancer (BC) is an alteration of circadian gene expression and DNA methylation. We undertook an integrated approach to identify methylation pattern of core circadian promoter regions in BC patients with regard to clinical features. We performed a quantitative methylation-specific real-time PCR analysis of a promoter methylation profile in 107 breast tumor and matched non-tumor tissues. A panel of circadian genes CLOCK, BMAL1, PERIOD (PER1, 2, 3), CRYPTOCHROME (CRY1, 2) and TIMELESS as well as their association with clinicopathological characteristics were included in the analysis. Three out of the eight analyzed genes exhibited marked hypermethylation (PER1, 2, 3), whereas CLOCK, BMAL1, CRY2 showed significantly lower promoter CpG methylation in the BC tissues when compared to the non-tumor tissues. Among variously methylated genes we found an association between the elevated methylation level of PERs promoter region and molecular subtypes, histological subtypes and tumor grading of BC. Methylation status may be associated with a gene expression level of circadian genes in BC patients. An aberrant methylation pattern in circadian genes in BC may provide information that could be used as novel biomarkers in clinics and molecular epidemiology as well as play an important role in BC etiology.     

DNA methylation profiling to assess pathogenicity of BRCA1 unclassified variants in breast cancer

Germline pathogenic mutations in BRCA1 increase risk of developing breast cancer. Screening for mutations in BRCA1 frequently identifies sequence variants of unknown pathogenicity and recent work has aimed to develop methods for determining pathogenicity. We previously observed that tumor DNA methylation can differentiate BRCA1-mutated from BRCA1-wild type tumors. We hypothesized that we could predict pathogenicity of variants based on DNA methylation profiles of tumors that had arisen in carriers of unclassified variants. We selected 150 FFPE breast tumor DNA samples [47 BRCA1 pathogenic mutation carriers, 65 BRCAx (BRCA1-wild type), 38 BRCA1 test variants] and analyzed a subset (n=54) using the Illumina 450K methylation platform, using the remaining samples for bisulphite pyrosequencing validation. Three validated markers (BACH2, C8orf31, and LOC654342) were combined with sequence bioinformatics in a model to predict pathogenicity of 27 variants (independent test set).  Predictions were compared with standard multifactorial likelihood analysis. Prediction was consistent for c.5194-12G>A (IVS 19-12 G>A) (P>0.99); 13 variants were considered not pathogenic or likely not pathogenic using both approaches. We conclude that tumor DNA methylation data alone has potential to be used in prediction of BRCA1 variant pathogenicity but is not independent of estrogen receptor status and grade, which are used in current multifactorial models to predict pathogenicity.     

Aberrant expression of Arpin in human breast cancer and its clinical significance

Arpin (Arp2/3 complex inhibitor), a novel protein found in 2013, plays a pivotal role in cell motility and migration. However, the precise role of Arpin in cancer is unclear. This study investigated the expression of Arpin in breast cancer and evaluated its correlation with the characteristics of clinical pathology and prognosis of breast cancer patients. Immunohistochemistry (IHC) for Arpin protein was performed on formalin‐fixed, paraffin‐embedded 176 breast cancer tissues and 43 normal breast tissues while qRT‐PCR for Arpin mRNA with 104 paired tumour and paratumoural tissues from breast cancer patients respectively. The association of Arpin expression with clinical pathological features and survival was assessed in a retrospective cohort analysis of patients. The results showed that the expression of Arpin protein in cancer tissues was lower compared to that in normal breast and the expression of Arpin mRNA was also lower in cancer tissues than that in the matched paratumoural tissues. Among the 176 breast cancer patients, the lower expression of Arpin was significantly associated with advanced tumour, nodes and metastasis system stage, and the reduced Arpin expression was strongly associated with axillary lymph node metastasis using univariate and multivariate logistic regression analysis [odds ratio: 3.242; 95% confidence interval (CI): 1.526, 6.888; P < 0.05]. Furthermore, Arpin expression was an independent risk factor for recurrence‐free survival (HR: 0.373; 95% CI: 0.171, 0.813; P < 0.05). As Arpin expression was first examined in human breast cancer tissues with qRT‐PCR and IHC, our results suggest that Arpin downregulation may contribute to the initiation and development of breast cancer metastasis. Therefore, as a potential predictive marker, Arpin deserves future studies.     

Expression and clinicopathological significance of lipin-1 in human breast cancer and its association with p53 tumor suppressor gene

Breast cancer (BC) is an important cause of female cancer-related death. It has recently been demonstrated that metabolic disorders including lipid metabolism are a hallmark of cancer cells. Lipin-1 is an enzyme that displays phosphatidate phosphatase activity and regulates the rate-limiting step in the pathway of triglycerides and phospholipids synthesis. The objective of this study was to evaluate lipin-1 expression, its prognostic significance, and its correlation with p53 tumor suppressor in patients with BC. In this study, 55 pairs of fresh samples of BC and adjacent noncancerous tissue were used to analyze lipin-1, using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other clinicopathological variables and p53 was also examined using IHC technique. The cell migration was studied in MCF-7 and MDA-MB231 cells following the inhibition of lipin-1 by propranolol. Our results show that the relative expression of lipin-1 messenger RNA was significantly higher in BC tissues compared with the adjacent normal tissue and its inhibition reduced cell migration in cancer cells. This upregulation was negatively correlated with histological grade of tumor and p53 status (p = .001 and p = .034) respectively and positively correlated with the tumor size (p = .006). Our results also seem to indicate that the high lipin-1 expression is related to a good prognosis in patients with BC. The expression of lipin-1 may be considered as a novel independent prognostic factor. The inhibition of lipin-1 may also have therapeutic significance for patients with BC. The correlation between lipin-1 and p53 confirms the role of p53 in the regulation of lipid metabolism in cancer cells.     

乳腺癌相关基因的甲基化

肿瘤组织中常伴随基因组整体甲基化水平降低和(或)某些基因CpG岛甲基化水平异常升高,这两种变化在肿瘤发生和发展中都扮演着重要的角色。近年来诸多研究报道了CpG岛高甲基化可导致乳腺癌相关的一系列关键基因的表达缺失。由于表观遗传变化存在潜在可逆性,因此,通过检测患者特定基因甲基化状态早期诊断乳腺癌,以及运用甲基化抑制剂来治疗乳腺癌,已成为国内外研究的热点和新思路。     

DNA methylation and breast tumor clinicopathological features: The Western New York Exposures and Breast Cancer (WEB) study

We evaluated the association between methylation of 9 genes, SCGB3A1, GSTP1, RARB, SYK, FHIT, CDKN2A, CCND2, BRCA1, and SFN in tumor samples from 720 breast cancer cases with clinicopathological features of the tumors and survival. Logistic regression was used to estimate odds ratios (OR) of methylation and Cox proportional hazards models to estimate hazard ratios (HR) between methylation and breast cancer related mortality. Estrogen receptor (ER) and progesterone receptor (PR) positivity were associated with increased SCGB3A1 methylation among pre- and post-menopausal cases. Among premenopausal women, compared with Stage 0 cases, cases of invasive cancer were more likely to have increased methylation of RARB (Stage I OR = 4.7, 95% CI: 1.1–19.0; Stage IIA/IIB OR = 9.7, 95% CI: 2.4–39.9; Stage III/IV OR = 5.6, 95% CI: 1.1–29.4) and lower methylation of FHIT (Stage I OR = 0.2, 95% CI: 0.1–0.9; Stage IIA/IIB OR = 0.2, 95% CI: 0.1–0.8; Stage III/IV OR = 0.6, 95% CI: 0.1–3.4). Among postmenopausal women, methylation of SYK was associated with increased tumor size (OR = 1.7, 95% CI: 1.0–2.7) and higher nuclear grade (OR = 2.0, 95% CI 1.2–3.6). Associations between methylation and breast cancer related mortality were observed among pre- but not post-menopausal women. Methylation of SCGB3A1 was associated with reduced risk of death from breast cancer (HR = 0.41, 95% CI: 0.17–0.99) as was BRCA1 (HR = 0.41, 95% CI: 0.16–0.97). CCND2 methylation was associated with increased risk of breast cancer mortality (HR = 3.4, 95% CI: 1.1–10.5). We observed differences in methylation associated with tumor characteristics; methylation of these genes was also associated with breast cancer survival among premenopausal cases. Understanding of the associations of DNA methylation with other clinicopathological features may have implications for prevention and treatment.     

DNA methylation in breast cancers: Differences based on estrogen receptor status and recurrence

DNA methylation plays a role in the etiology of primary breast cancers. We analyzed paired primary and second breast tumors to elucidate the role of methylation in recurrence. Methylation profiles from paired primary and second breast tumors of 23 women were assessed using the HumanMethylation450 BeadChip. Twelve women had estrogen receptor positive (ERpos) primary and second tumors, five had estrogen receptor negative (ERneg) primary and second tumors, and six had an ERpos primary tumor but an ERneg second tumor. Stratifying tumors by occurrence revealed that the greater methylation previously associated with ERpos tumors, is more pronounced in primary tumors than in second tumors. Further, ERneg second tumors are more methylated than ERpos second tumors among women who had ERpos primary tumors. Pathway analyses using gene lists generated from comparisons of methylation in ERpos primary tumors from the paired sets with ERpos tumors from six women without recurrences, identified differences between groups based on the ER status of the second tumor. Hypermethylated genes of significantly enriched pathways were differentially associated with survival. DNA methylation profiles of ERpos primary breast tumors support the development and use of tumor methylation profiles for stratifying women with breast cancer both for prognosis and therapy.     

Epigenetic regulation of estrogen signaling in breast cancer

Estrogen signaling is mediated by ERα and ERβ in hormone dependent, breast cancer (BC). Over the last decade the implication of epigenetic pathways in BC tumorigenesis has emerged: cancer-related epigenetic modifications are implicated in both gene expression regulation, and chromosomal instability. In this review, the epigenetic-mediated estrogen signaling, controlling both ER level and ER-targeted gene expression in BC, are discussed: (1) ER silencing is frequently observed in BC and is often associated with epigenetic regulations while chemical epigenetic modulators restore ER expression and increase response to treatment;(2) ER-targeted gene expression is tightly regulated by co-recruitment of ER and both coactivators/corepressors including HATs, HDACs, HMTs, Dnmts and Polycomb proteins.     

Retrospective assessment of cyclin-dependent kinase 5 mRNA and protein expression and its association with patient survival in breast cancer

Cyclin-dependent kinase 5 (Cdk5) is an atypical member of the cyclin-dependent kinase family and functions as a serine/threonine kinase that can be activated by non-cyclin binding activators p35 or p39. Cdk5 expression and activity has been linked with the development and progression of cancer; however, its expression in breast cancer has not been fully described. Protein expression of Cdk5 was determined in a large cohort of early-stage invasive breast cancer tumours (n = 1110) with long-term follow-up data using immunohistochemistry. Expression of CDK5 mRNA was assessed in the METABRIC cohort (n = 1980). Low nuclear and cytoplasmic expression of Cdk5 expression was significantly associated with shorter breast cancer-specific survival (P = .004 and P = .001, respectively). Importantly, low nuclear and cytoplasmic expression of Cdk5 remained associated with survival in multivariate analysis, including potentially confounding factors (hazard ratio (HR) = 0.612, 95% confidence interval (CI) = 0.418-0.896, P = .011 and HR = 0.507, 95% CI = 0.318-0.809, P = .004, respectively). In addition, low nuclear and cytoplasmic expression of Cdk5 was significantly associated with clinicopathological criteria associated with adverse patient prognosis. Low CDK5 mRNA expression was associated with shorter patient survival (P = .005) in the METABRIC cohort; no associations between copy gain or loss and survival were observed. These data suggest that low Cdk5 expression is associated with poor clinical outcome of breast cancer patients and may be of clinical relevance.     

Plasma protein carbonyl levels and breast cancer risk

To study the role of oxidative stress in breast cancer risk, we analysed plasma levels of protein carbonyls in 1050 cases and 1107 controls. We found a statistically significant trend in breast cancer risk in relation to increasing quartiles of plasma protein carbonyl levels (OR = 1.2, 95% CI = 0.9-1.5; OR = 1.5, 95% CI = 1.2-2.0; OR = 1.6, 95% CI = 1.2-2.1, for the 2(nd), 3(rd) and 4(th) quartile relative to the lowest quartile, respectively, P for trend = 0.0001). The increase in risk was similar for younger (<50 years) and older women, more pronounced among women with higher physical activity levels (0.7 hrs/week for 4(th) quartile versus lowest quartile OR = 2.0, 95% CI = 1.4-3.0), higher alcohol consumption (> or = 15 grams/day for 4(th) quartile versus lowest quartile OR = 2.3, 95% CI = 1.1-4.7), and hormone replacement therapy use (HRT, OR = 2.6, 95% CI = 1.6-4.4 for 4(th) quartile versus lowest quartile). The multiplicative interaction terms were statistically significant only for physical activity and HRT. The positive association between plasma protein carbonyl levels and breast cancer risk was also observed when the analysis was restricted to women who had not received chemotherapy or radiation therapy prior to blood collection. Among controls, oxidized protein levels significantly increased with cigarette smoking and higher fruit and vegetable consumption, and decreased with alcohol consumption >30 grams per day. Women with higher levels of plasma protein carbonyl and urinary 15F(2t)-isoprostane had an 80% increase in breast cancer risk (OR = 1.8, 95% CI = 1.2-2.6) compared to women with levels below the median for both markers of oxidative stress. In summary, our results suggest that increased plasma protein carbonyl levels may be associated with breast cancer risk.     

LINE-1 methylation status in prostate cancer and non-neoplastic tissue adjacent to tumor in association with mortality

Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LINE-1 methylation in prostate cancer and non-neoplastic tissue adjacent to tumor (NTAT) in association with mortality from prostate cancer. We selected 157 prostate cancer patients with available NTAT from 2 cohorts of patients diagnosed between 1982–1988 and 1993–1996, followed up until 2010. An association between LINE-1 hypomethylation and prostate cancer mortality in tumor was suggested [hazard ratio per 5% decrease in LINE-1 methylation levels: 1.40, 95% confidence interval (CI): 0.95–2.01]. After stratification of the patients for Gleason score, the association was present only for those with a Gleason score of at least 8. Among these, low (<75%) vs. high (>80%) LINE-1 methylation was associated with a hazard ratio of 4.68 (95% CI: 1.03–21.34). LINE-1 methylation in the NTAT was not associated with prostate cancer mortality. Our results are consistent with the hypothesis that tumor tissue global hypomethylation may be a late event in prostate cancerogenesis and is associated with tumor progression.     

PTPH1 immunohistochemical expression and promoter methylation in breast cancer patients from India: A retrospective study

Protein Tyrosine Phosphatase H1/Protein Tyrosine Phosphatase Non receptor Type 3 (PTPH1/PTPN3) is upregulated and/or mutated in glioma, ovarian, gastric, and colorectal cancers. Previous studies have documented that PTPH1-associated breast cancers exhibit enhanced sensitivity to tamoxifen and tyrosine kinase inhibitors through dephosphorylation of ER and epidermal growth factor receptor, respectively. Owing to the key role that PTPH1 plays as a biomarker in predicting the response of chemotherapeutic drugs and lack of studies on Indian breast cancer patients, the present study investigated PTPH1 protein expression and its relationship to clinical features, ER/PR/HER2/neu statuses, and methylation of promoter in breast cancer tissues (n = 67) among Indian population by immunohistochemistry and methylation specific polymerase chain reaction. PTPH1 expression was upregulated in 58.21% (39/67) and downregulated in the rest of tumor specimens, and it correlated with ER, PR, and HER2/neu statuses with p values of <0.0001, 0.0113, and 0.0448, respectively. Additionally, we found that the 2 kb region upstream of PTPH1 gene harbored CpG sites within, and was ubiquitously methylated in breast cancer (n = 13), colon cancer tissue (n = 1), uterine cancer tissue (n = 1), normal breast tissue (n = 1) in addition to Hela and MCF7 cell lines. In conclusion, our data showed a strong correlation of the PTPH1 status with the ER and ubiquitous nature of PTPH1 promoter methylation at specific CpG sites irrespective of cancer types and protein expression. Our findings underscore the clinical relevance of PTPH1 expression in Indian patients and warrant additional studies to explore the importance of ubiquitously methylated promoter at specific CpG sites in upstream of the PTPH1 gene.     

Estrogen activates raf-1 kinase and induces expression of Egr-1 in MCF-7 breast cancer cells

We have demonstrated that in normal and b/b rat red blood cells (RBCs) hsp70-like protein (heat shock protein 70-like) is localized in the cytosol and it is exported via exosomes during in vivo reticulocytes maturation. As we have presumed, in the mutant (b/b) rat, hsp70-like protein transfers from cytosol to the RBC membrane. In the normal rat RBCs this happens when those cells are submitted to heat stress conditions. Our study indicates that the presence of hsp70-like protein in the b/b rat RBC plasma membrane is consistent with a primary defect and is not a consequence of life long stress, i.e. hypoxia.     

Nuclear NHERF1 expression as a prognostic marker in breast cancer

Our purpose was to investigate whether Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression could be linked to prognosis in invasive breast carcinomas. NHERF1, an ezrin-radixin-moesin (ERM) binding phosphoprotein 50, is involved in the linkage of integral membrane proteins to the cytoskeleton. It is therefore believed to have an important role in cell signaling associated with changes in cell cytoarchitecture. NHERF1 expression is observed in various types of cancer and is related to tumor aggressiveness. To date the most extensive analyses of the influence of NHERF1 in cancer development have been performed on breast cancer. However, the underlying mechanism and its prognostic significance are still undefined. NHERF1 expression was studied by immunohistochemistry (IHC) in a cohort of 222 breast carcinoma patients. Association of cytoplasmic and nuclear NHERF1 expression with survival was analyzed. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan–Meier method. Cytoplasmic NHERF1 expression was associated with negative progesterone receptor (PgR) (P=0.017) and positive HER2 expression (P=0.023). NHERF1 also showed a nuclear localization and this correlated with small tumor size (P=0.026) and positive estrogen receptor (ER) expression (P=0.010). Multivariate analysis identified large tumor size (P=0.011) and nuclear NHERF1 expression (P=0.049) to be independent prognostic variables for DFS. Moreover, the nuclear NHERF1(−)/ER(−) immunophenotype (27%) was statistically associated with large tumor size (P=0.0276), high histological grade (P=0.0411), PgR-negative tumors (P<0.0001) and high proliferative activity (P=0.0027). These patients had worse DFS compared with patients with nuclear NHERF1(+)/ER(+) tumors (75.4% versus 92.6% P=0.010). These results show that the loss of nuclear NHERF1 expression is associated with reduced survival, and the link between nuclear NHERF1 and ER expression may serve as a prognostic marker for the routine clinical management of breast cancer patients.     

E-cadherin deregulation in breast cancer

E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy.     

Differences in DNA methylation by extent of breast cancer family history in unaffected women

Breast cancer clusters within families but genetic factors identified to date explain only a portion of this clustering. Lower global DNA methylation in white blood cells (WBC) has been associated with increased breast cancer risk. We examined whether WBC DNA methylation varies by extent of breast cancer family history in unaffected women from high-risk breast cancer families. We evaluated DNA methylation levels in LINE-1, Alu and Sat2 in 333 cancer-free female family members of the New York site of the Breast Cancer Family Registry, the minority of which were known BRCA1 or BRCA2 mutation carriers. We used generalized estimated equation models to test for differences in DNA methylation levels by extent of their breast cancer family history after adjusting for age. All unaffected women had at least one sister affected with breast cancer. LINE-1 and Sat2 DNA methylation levels were lower in individuals with 3 or more (3+) first-degree relatives with breast cancer relative to women with only one first-degree relative. For LINE-1, Alu, and Sat2, having 3+ affected first-degree relatives was associated with a decrease of 23.4% (95%CI = ?46.8%, 0.1%), 17.9% (95%CI = ?39.5%, 3.7%) and 11.4% (95% CI = ?20.3%, ?2.5%), respectively, relative to individuals with only one affected first-degree relative, but the results were only statistically significant for Sat2. Individuals having an affected mother had 17.9% lower LINE-1 DNA methylation levels (95% CI = ?28.8%, ?7.1%) when compared with those not having an affected mother. No associations were observed for Alu or Sat2 by maternal breast cancer status. If replicated, these results indicate that lower global WBC DNA methylation levels in families with extensive cancer histories may be one explanation for the clustering of cancers in these families. Family clustering of disease may reflect epigenetic as well as genetic and shared environmental factors.     

Differences in DNA methylation by extent of breast cancer family history in unaffected women

Breast cancer clusters within families but genetic factors identified to date explain only a portion of this clustering. Lower global DNA methylation in white blood cells (WBC) has been associated with increased breast cancer risk. We examined whether WBC DNA methylation varies by extent of breast cancer family history in unaffected women from high-risk breast cancer families. We evaluated DNA methylation levels in LINE-1, Alu and Sat2 in 333 cancer-free female family members of the New York site of the Breast Cancer Family Registry, the minority of which were known BRCA1 or BRCA2 mutation carriers. We used generalized estimated equation models to test for differences in DNA methylation levels by extent of their breast cancer family history after adjusting for age. All unaffected women had at least one sister affected with breast cancer. LINE-1 and Sat2 DNA methylation levels were lower in individuals with 3 or more (3+) first-degree relatives with breast cancer relative to women with only one first-degree relative. For LINE-1, Alu, and Sat2, having 3+ affected first-degree relatives was associated with a decrease of 23.4% (95%CI = −46.8%, 0.1%), 17.9% (95%CI = −39.5%, 3.7%) and 11.4% (95% CI = −20.3%, −2.5%), respectively, relative to individuals with only one affected first-degree relative, but the results were only statistically significant for Sat2. Individuals having an affected mother had 17.9% lower LINE-1 DNA methylation levels (95% CI = −28.8%, −7.1%) when compared with those not having an affected mother. No associations were observed for Alu or Sat2 by maternal breast cancer status. If replicated, these results indicate that lower global WBC DNA methylation levels in families with extensive cancer histories may be one explanation for the clustering of cancers in these families. Family clustering of disease may reflect epigenetic as well as genetic and shared environmental factors.