Genes In Breast Cancer Patients Research Articles

Examination of methylation changes of VIM, CXCR4, DOK7, and SPDEF genes in peripheral blood DNA in breast cancer patients.

Studying whole blood DNA methylation as a risk marker has valuable applications in either diagnosis or staging of breast cancer. We investigated whole blood DNA methylation status of VIM, CXCR4, DOK7, and SPDEF genes in breast cancer patients in comparison to healthy control subjects. 60 patients with breast cancer and 40 healthy controls were examined. Genomic DNA isolated from peripheral blood and restriction enzyme polymerase chain reaction (REP) method was applied for analysis. Real-time PCR was used to confirm methylation status of the aforementioned genes and therefore to find out the methylation differences between normal and breast cancer subjects. Level of DOK7 promoter hypomethylation in normal and breast cancer samples was significant (P-value = 0.001). The study, also, showed that hypomethylation of VIM and CXCR4 genes are significant in patients compared with normal cases (P-value < 0.05). Furthermore, SPDEF promoter hypomethylation was not significantly differed between normal and breast cancer samples (P-value = 0.2). Hypermethylation of DOK7 gene in DNA from patients affected with breast cancer offers a biomarker for diagnosis of the breast cancer. This study indicates that methylation status of VIM and CXCR4 genes changes in breast cancer; so, they can be used as molecular biomarkers in breast cancer prognosis.

Method Development for Detection of E545A Mutation PIK3CA Gene in Breast Cancer Patients Using Tm Shift SYBR Green I qPCR

E545A is one of the point mutations, and its frequency is high in PIK3CA gene (3.8%), particularly breast cancer patients in Singapore (13.8%) and Mexico (11.5%). In addition to induce breast cancer, the mutation also caused resistance of anti-HER2 in HER2 cancer subtype. The tremendous effect of this mutation was not supported by affordable detection method. This study aimed to develop a feasible and sensitive method of E545A detection. The developing method used Tm and Ct to identify samples. Based on optimization, the best condition was obtained at optimization 2 at annealing temperature of 65°C. At this condition, Tm and Ct of each sample were (a) exon 9 (78.4°C and 13.005±0.007) and (b) E5454A (80.4°C and 10.07±0.1). This method also demonstrated good precision as observed in variance coefficient of intra and inter assay (0). Thus, method for E5454A detection mutation was successfully developed.

Abstract 2391: Integrated analysis of methylation and expression profile of telomere-related genes in breast cancer patients

Abstract Telomeres at the ends of chromosomes are critical in maintaining the integrity and stability of the genome. Aberrant telomere or telomerase dysfunction participates in tumorgenesis. A majority of human cancers exhibit critically aberrant telomere length, suggesting that tumors can arise from genetically instable cells with dysfunctional telomeres. Many genes are involved in the complex regulatory mechanisms of telomere length and telomerase activity. In addition, the methylation and expression pattern for most of telomere related genes in breast cancer are still unknown. Using microfluidic-PCR based target enrichment and next-generation bisulfite sequencing technology, we explored the promoter methylation profile of 29 telomere-related genes in 184 breast cancer patients with paired tumor and matched normal tissues. The average methylation level was significantly higher in tumor (8.13%) than that in matched normal tissues (7.08%) (P = 4.30E-21). Four genes showed significant hyper-methylation in the breast tumor tissues. All of these 4 genes are annotated with potential TFBSs in the promoter regions. In subtype analysis, RAD51D showed significant methylation difference among four subtypes. In analysis of the association between methylation and clinicopathologic characteristics, TERC showed significant difference between ER+/ER- tumors. The expression profile of the 4 significant hyper-methylated genes was explored in the same cohort using qPCR method. All of them showed significantly lower expression in breast tumor tissues compared with the matched normal tissues. Two genes showed significant and negative cis correlation between methylation and gene expression. These results were also validated in the TCGA database. The 4 genes panel showed a good performance in predicting breast cancer with ROC analysis. In summary, our results revealed the methylation pattern of telomere related genes in breast cancer and illustrated the epigenetic regulatory mechanism on expression of aberrant methylated genes. Our study provides a novel panel of telomere related genes which may be a valuable diagnostic biomarker for breast cancer prediction. Note: This abstract was not presented at the meeting. Citation Format: Jianfu Heng, Xinwu Guo, Lili Tang, Fan Zhang, Limin Peng, Ming Chen, Xipeng Luo, Xunxun Xu, Shouman Wang, Jun Wang. Integrated analysis of methylation and expression profile of telomere-related genes in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2391. doi:10.1158/1538-7445.AM2017-2391

Polymorphisms and Mutations in GSTP1, RAD51, XRCC1 and XRCC3 Genes in Breast Cancer Patients

Genotoxic factors, including ionizing radiation and oxidative stress, are associated with genomic instability and development of breast cancer (BC). The homologous recombination DNA repair (HRR) pathway, base excision repair (BER) mechanism, and antioxidative enzymes are required as defense mechanisms against these DNA damaging agents. GSTP1, XRCC1, XRCC3 and RAD51 proteins are essential components of antioxidation, BER and HRR of DNA, respectively. Deficiencies in BER, HRR and antioxidation pathways are involved in the progression of cancer. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and blood samples of BC patients of an Italian population. Genomic DNA was also extracted from blood specimens of a control group. DNA sequencing was performed for six single-nucleotide polymorphisms (SNPs) in the GSTP1, RAD51, XRCC1 and XRCC3 genes in BC patients and the control group. Two variants in the 5'-UTR of the XRCC3 (rs1799794 A/G) and RAD51 (rs1801321) genes showed a significant association with susceptibility to BC (OR = 4.125; 95% CI 1.057-16.102; p = 0.03 and OR = 2.04; 95% CI 0.4925-8.449; p = 0.007, respectively). Additionally, we reported 2 mutations in intron 7 of the XRCC3 gene, CTdel (rs543072564) and A/G (rs369703243). Our results underscored the existence of an association between XRCC3-5'-UTR-A/G (rs1799794) and RAD51-5'-UTR G172T (rs1801321) genotypes and BC risk in an Italian population. The presence of mutations in the intronic region of the XRCC3 gene highlights the importance of more sequence screening of DNA repair genes for possible genetic penetrance in BC.

P034 - The 3020insC allele of the NOD2 gene in breast cancer patients – a clinicopathological analysis

P034 - The 3020insC allele of the NOD2 gene in breast cancer patients – a clinicopathological analysis

P038 - Promoter region methylation of RASSF1A and PITX2 genes in breast cancer patients

P038 - Promoter region methylation of RASSF1A and PITX2 genes in breast cancer patients

Abstract P1-04-03: Cross-talk between RNA methyltransferase and demethylase regulates breast cancer growth and progression

Abstract The importance of RNA methylation in biological processes has just begun to emerge. However, our understanding of the role and mechanism by which RNA methylation may play a role in tumorigenesis is unclear. Here, we report that RNA N6-adenosine methyltransferase protein METTL14 supports breast cancer growth and progression. We show that METTL14 mediates its pro-tumorigenic function by activating the expression of RNA binding protein HuR and consequently inducing the transcriptional stability of key cell cycle-associated genes, TGFβ and its signaling partners as well as HMGA2, VEGF and PDGF that are known to play critical roles in cancer growth and metastasis. Importantly, m6 A RNA methylation levels of METTL14 target genes were significantly lower in breast cancer patients compared to normal matched controls. The decreased RNA methylation level resulting in increased activity of these target genes in breast cancer patients is regulated by a feedback loop between RNA demethylase ALKBH5 and METTL14. Our results reveal that ALKBH5 regulates the expression of METTL14 and HuR, which in turn transcriptionally stabilizes ALKBH5. Furthermore, we demonstrate that like METTL14, ALKBH5 also supports breast cancer growth and progression. Taken together, these findings suggest that a collaboration between RNA methylation and demethylation machinery may set up a threshold of RNA methylation that ensures the stability of genes that play a vital role in normal cell proliferation; and any perturbation of that threshold may lead to uncontrolled expression/activity of those genes resulting in tumor growth and progression. Citation Format: Subbarayalu P, Eedunuri VK, Timilsina S, Rajamanickam S, Viswanadhapalli S, Abdelfattah N, Onyeagucha BC, Cui X, Mohammad TA, Huang TH-M, Huang Y, Chen Y, Rao MK. Cross-talk between RNA methyltransferase and demethylase regulates breast cancer growth and progression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-03.

Study of the regulatory promoter polymorphism (-938C>A) of B-cell lymphoma 2 gene in breast cancer patients of Mazandaran province in Northern Iran.

Background:The incidence rate of breast cancer has been dramatically increasing since the last decade in Iran, and it is now one of the most common female malignant tumors. B-cell lymphoma 2 (BCL2) family is the most important regulator of apoptosis, and −938C>A single nucleotide polymorphism (SNP) of BCL2 gene promoter has been demonstrated to influence breast cancer susceptibility. In this research, we study the effect of −938C>A allelic variants on breast cancer risk in Mazandaran province at the North of Iran.Materials and Methods:This analysis performed on 120 breast cancer patients who underwent surgery in some referenced hospitals at Mazandaran province along with 130 healthy individuals as a control. DNA extracted from peripheral blood samples was applied in polymerase chain reaction-single-strand conformation polymorphism analysis to determine −938C>A genotype. The association of the −938C>A genotype and breast cancer risk as well as clinicopathological characters were analyzed by logistic regression method.Results:Results showed that genotype frequency of AA, AC, and CC genotypes was 10%, 62%, and 28% for case and 28%, 50%, and 22% in control group, respectively. In the logistic regression model, BCL2 − 938C/A variant genotype AA was associated with a decreased risk of breast cancer (P = 0.041) by 0.31-fold (odds ratio = 0.31, confidence interval = 0.091–0.909) compared to CC genotype. However, no significant association found between −938C>A genotype and clinicopathological characters.Conclusion:The study showed that AA genotype of BCL2 gene (−938C>A) is associated with decreased susceptibility to breast cancer. Hence, investigating the −938C>A SNP of BCL2 gene promoter could be an appropriate molecular marker to determine individual sensitivity to breast cancer.

Promoter Methylation and Loss of Expression of PTEN Gene in Breast Cancer Patients from Saudi Population

Objective: PTEN, a tumor-suppressor gene, located on chromosome 10q23.3, is implicated in various types of cancer including breast cancer. The aim of this study is to investigate the promoter methylation, loss of expression and significance of PTEN gene in breast cancer and to determine the correlation between promoter methylation and gene expression. Methods: Promoter methylation and loss of expression of PTEN gene were analyzed using methylation-specific PCR and immunohistochemical methods respectively. The chi square test is used to correlate the promoter methylation and gene expression with their clincopathologic parameters. Results: We examined 53 breast cancer specimens and 10 normal tissues adjacent to tumor. The results showed a 58.5% promoter methylation in PTEN gene and none in normal tissue. PTEN methylation was observed in advanced stages III-IV (81.8%, 18 of 22, P=0.015) and higher grades G2-G3 (71.4%, 20 of 28, P=0.043) of disease. The correlation of PTEN methylation with clinical stage and tumor grade was found to be statistically significant. Nuclear PTEN expression was detected in 73.6% (39 of 53) cases of breast cancer and in the remaining 26.4% (14 of 53) cases expressional loss was observed. The loss of PTEN expression was observed in all normal tissues (10 of 10). The loss of PTEN expression was significantly correlated with patient’s age (P=0.028) and clinical stage (P = 0.029). The expressional loss was observed in 12 (38.7%) cases among 31 methylation positive cases, whereas among 22 methylation- negative cases, only 2 (9.1%) cases were seen as immunostaining negative with the statistically significant value (P=0.016). Conclusion: Promoter methylation and loss of expression of PTEN gene occur frequently in breast cancer. Our results suggest that PTEN plays an important role in breast carcinogenesis.

Reserch of the gene polymorphism TOX3 / LOC643714 and the risk of breast cancer development in persons exposed to ionizing radiation after Chornobyl disaster

The objective of this work was to identify and compare the polymorphism of the rs3803662 polymorphism of the TOX3/LOC643714 gene in breast cancer patients who have undergone ionizing radiation due to the Chornobyl accident and in patients without ionizing radiation (IR) in the history. The determination of the rs3803662 polymorphism of the TOX3/LOC643714 gene was per formed by polymerase chain reaction (PCR) in 83 patients with breast cancer: 42 subjects who were exposed to ion izing radiation due to the Chornobyl accident, 41 people without ionizing radiation in history and 17 controls in Ukraine without cancer pathology. In order to compare the obtained data on spontaneous and radiation associated breast cancer and to calculate the differences in the frequencies of alleles and the risk of oncopathology, data from literature on control groups of the populations of the Russian Federation, Sweden, and the United Kingdom were used. Comparing with the literature data and the group of exposed subjects, the homozygous carriers of the minor alleles of the TOX3/LOC643714 ТТ gene revealed an increased risk of developing breast cancer: OR = 2.89, p = 0.02 (CI 95% 1.17 7,16). In subjects without the influence of IR in history, the carrier of homozygous minor axis of the gene TOX3/LOC643714 ТТ is also associated with the risk of breast cancer: OR = 3.83, p = 0.0002 (CI 95% 0.82-14.14). In the homozygous carriers of the minor alleles of the TOX3 / LOC643714 gene exposed to IR, there was no increase in the risk of developing breast cancer (OR = 0.65, p = 0.46, CI 95% 0.21-2.04) compared with the con trol group of Ukrainian population. The carrier of homozygous minor alleles of the TOX3/LOC643714 gene is not a risk factor for the devel opment of breast cancer under conditions of exposure to ionizing radiation in the study group of the Ukrainian population.

&amp;lt;i&amp;gt;BRCA1&amp;lt;/i&amp;gt; Promoter Hypermethylationas an Early Diagnostic Tool for Breast Cancer

<i>Background</i>: Breast cancer is the second most common cancer among women after cervical cancer. As Cancer development and progression is dictated by chain of alterations in genes. Over the past few years, the Kashmir valley has witnessed a tremendous increase in the incidence of breast cancer in its unexplored ethnic population. The aim of present study was to find out the role of Promoter Hypermethylation of BRCA1 gene in Breast cancer patients. <i>Material Methods</i>: The DNA was extracted from all the samples and was modified using bisulphite modification kit. Methylation-specific polymerase chain reaction was used for the analysis of the promoter hypermethylation status of BRCA1 gene. <i>Results</i>: The epigenetic analysis revealed that unlike other high risk regions, Kashmiri population has a different promoterhypermethylation profile of BRCA1 gene as 68% of the cases showed BRCA1 promoter hypermethylation in comparison to 20% of the normal cases, the association of promoter hypermethylation with breast cancer and normal cases was found to be significant (P=0.0006). The frequency of BRCA1 promoter hypermethylation was found to be certainly higher in Stage III/IV (75%) compared to Stage I/ II (62%) but the difference was not statistically significant(P =0.0674). The frequency of promoter methylation was foundhigher (77.1%) in age group above 60- years) than ages below 60 years. <i>Conclusion</i>: These results suggest that BRCA1 aberrant promoter hypermethylation in Kashmiri population contributes to the process of carcinogenesis in breast cancer and is reportedly one of the commonest epigenetic changes in the development of breast cancer.

Integrated analysis of gene expression and methylation profiles of 48 candidate genes in breast cancer patients.

Gene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management. Using high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters. Breast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient. Our results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

Abstract 4482: Expression profiling of SETD family genes in breast cancer patients

Abstract Epigenetic alterations have recently been recognized to play an important role in the progression of many tumor types. Protein methyltrasferases are among the most relevant epigenetic effectors. Dysregulation in the expression or activity of these enzymes is among the major epigenetic alterations involved in carcinogenesis. Methyltransferases of the SETD family share a SET domain which is involved in the methylation of lysine residues on histones and non-histone proteins. So far, little is known about the relation of SETD family genes with mammary carcinogenesis. In the present study we evaluated the relative expression of 10 SETD family genes in 18 breast tumor samples and non-tumor breast tissues from patients with breast cancer by real time qPCR. The comparative expression profile of each gene was correlated with the most relevant clinicopathologic prognostic information available from patients. We found a significant increase in the expression of SETMAR in tumor samples compared with the normal breast tissues. In addition, triple negative tumor subtype (n = 7), when compared with a pool of normal tissue samples (n = 13) presented an increased relative expression of SETMAR. Interestingly, SETD8 was 10 times overexpressed when the angiolymphatic invasion (n = 8) was present compared to the absence of this feature (n = 10). Patients with strong axillary tumor burden defined as the presence of at least 10 axillary lymph nodes with cancer (n = 6), showed an increased relative expression of SETD5 and SETD8 genes when compared to the absence of this feature (n = 12). Evaluating the expression on paired samples (normal tissue and tumor tissue from the same patient (n = 7), we observed an increased relative expression of SETD1B. Also in the paired samples, we found a strong correlation among the expression of the genes SETD1B, SETD5, SETD8 and SETMAR in tumor samples with no correlation in normal tissues. Furthermore, immunohistochemical analysis revealed a stronger expression of SETD5 and SETMAR in carcinomas in situ when compared to normal tissues and invasive tumors. Taken together, these data points to a likely role of SETMAR and SETD1B genes in mammary carcinogenesis, with relevant clinical implications and with the possible involvement of SETD5 and SETD8 genes in more advanced stages of the disease. The correlation on the expression of these genes with relevant clinical prognostic factors in breast cancer opens the way to exploit them not only as therapeutic targets, but also as new prognostic markers. Keywords: SETD gene family, expression profile, breast cancer Citation Format: Joao N. Matos Neto, Doralina do Amaral Rabello, Fabio Pittella-Silva. Expression profiling of SETD family genes in breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4482.

Abstract 1912: Combinatorial targeting of PI3K and MAPK signaling pathways using microRNAs to inhibit tumor growth and metastasis in breast cancer

Abstract PI3K/Akt and MAPK signaling pathways, regulating cancer cell proliferation, apoptosis and metastasis, are among the top most deregulated pathways in cancer. Current kinase inhibitors used in clinics have limited success since one of these pathways gets activated when the other is suppressed. Alternatively, simultaneous silencing of both pathways results in high toxicity. Here, we aimed to establish a microRNA-based, non-toxic approach to inhibit these pathways simultaneously in breast cancer. By analyzing our previously published Reverse Phase Protein Array (RPPA) data showing differential expression of 26 proteins upon overexpression of 733 different miRNAs, we selected top miRNAs significantly deregulating PI3K and MAPK pathways as well as cell cycle. We narrowed down the list by selecting miRNAs whose predicted targets were among the PI3K and MAPK pathway elements. We came up with a tumor suppressor miRNA affecting both in vitro viability and migration/invasion of breast cancer cells. We identified the set of differentially expressed genes in breast cancer patients under low and high expression of the miRNA. A significant association was observed between the expression of the miRNA or its identified gene signature and clinico-pathological properties of breast cancer patients. Combinatorial knockdown of targets of the miRNA that are in PI3K and MAPK pathways as a cocktail phenocopied the tumor suppressive effects of the miRNA in breast cancer cell lines. Notably, low expression of the target cocktail predicts a better outcome in breast cancer patients. Overall, we identified a tumor suppressor miRNA to be used an alternative to current kinase inhibitor-based therapies for major subtypes of breast cancer by suppressing both PI3K and MAPK pathways and as a result inhibiting proliferation and migration/invasion in breast cancer. Citation Format: Merve Mutlu, Ozge Saatci, Erol Eyupoglu, Umar Raza, Ozgur Sahin. Combinatorial targeting of PI3K and MAPK signaling pathways using microRNAs to inhibit tumor growth and metastasis in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1912.

Abstract 3474: Prevalence of BRCA1 and BRCA2 small mutation and large genomic rearrangements in breast cancer patients visiting a genetic counseling clinic

Abstract BACKGROUND: This study was conducted to investigate mutation prevalence of the BRCA1 and BRCA2 genes in breast cancer patients, including both small mutations and large genomic rearrangements (LGRs) following the patients in the process of genetic counseling. METHODS: A total of 358 breast cancer patients who visited genetic counseling clinic and screened for BRCA1/2 mutation at the National Cancer Center were included. Among the non-carriers by direct sequencing, a subset of patients who were agreed on participation of the research were screened for the presence of LGRs using a multiple ligation-dependent probe amplification (MLPA) assay. Clinico-pathologic characteristics of cancer were evaluated thorough medical record review and characteristics for those who found BRCA mutation through MLPA were described. RESULTS: 53 (14.8%) mutation carriers were identified. The frequency of BRCA small mutations in specific subgroups was as follows: 18.0% of patients with a family history, 12.8% of early-onset breast cancer, and 20.6% of bilateral breast cancer patients. MLPA identified BRCA1 LGRs in 3 (1.5%) out of 204 patients with BRCA1 and BRCA2 small mutation-negative results. Those with LGRs showed higher probabilities of BRCA mutation carriers estimated by risk assessment models and receptor negatives of tumor immunohistochemistry. CONCLUSIONS: The prevalence of BRCA1/2 mutation prevalence in breast cancer patients with family history or personal risk factors were comparable to previous Korean studies. MLPA to screen for mutations in the BRCA1 gene could be recommended for breast cancer patients negative for small mutations. To specify proper targets of MLPA, probabilities of carrying BRCA1/2 mutation and tumor receptor status could be considered. Citation Format: Ji Yeon Sohn, Boyoung Park, Kyong-Ah Yoon, Soo Jin Park, Moo Hyun Lee, Eun Hae Cho, Keun Seok Lee, Myong Cheol Lim, Sun-Young Kong, Eun Sook Lee. Prevalence of BRCA1 and BRCA2 small mutation and large genomic rearrangements in breast cancer patients visiting a genetic counseling clinic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3474.

Evaluation of Mutations in Exons 7 and 8 of TP53 Gene in Breast Cancer Patients from Azarbaijan

BACKGROUND AND OBJECTIVE: Breast cancer is the most common type of malignancy in women, and TP53 tumor-suppressor gene is one of the most commonly transformed genes in human cancers. Accurate assessment of TP53 gene mutations in cancer patients can play an important role in diagnosis, prognosis, or treatment. This study aimed to identify mutations of this gene in breast cancer patients. METHODS: In this descriptive study, 102 tumor samples were obtained from female breast cancer patients from Azarbaijan, Iran. All the participants were referred to hospitals of Tabriz during 2007-2009, and their DNA was extracted by Proteinase K. TP53 gene mutations in exons 7 and 8 and intron 7 were investigated using polymerase chain reaction technique and direct sequencing. FINDINGS: Seven (6.86%) cases of mutation and 14 (13.72%) cases of polymorphisms were identified. Mutations (CGG → CAG) at codon 248 (in two cases) and (CTG → CCG) at codon 257 in exon 7 and G>T mutation in the first nucleotide of intron 7 were observed. In exon 8, GTG>ATG mutation at codon 272, CCT>TCT mutation at codon 278, and three nucleotide deletion at codon 262 were identified. CONCLUSION: The results of this study showed a different pattern of TP53 gene mutation in female breast cancer patients. Further studies could specify the role of TP53 mutations in the progression of breast cancer.

Abstract 4085: Depletion of Runx1 promotes epithelial to mesenchymal transition in breast cancer

Abstract Runx1 is an essential transcription factor for definitive hematopoiesis and Runx1 translocations are well established as the causative factor of various leukemias. Recent studies have identified Runx1 as one of the most frequently mutated genes in breast cancer patients, and also that Runx1 expression levels are decreased in high-grade primary breast tumors compared to low/mid-grade tumors. The recognition of Runx1 expression in epithelial lining cells of glands suggests that Runx1 functions in maintaining the integrity of that cell layer. Therefore we examined the role of Runx1 during cancer progression. We found that Runx1 is highly expressed in immortalized human mammary epithelial cells (MCF10A) compared with breast cancer epithelial cells (MCF7) and invasive breast cancer cells (MDA-MB-231). To test the hypothesis that Runx1 contributes to the epithelial to mesenchymal transition (EMT) in breast cancer, the EMT was induced in MCF10A cells which resulted in a 50% decrease of Runx1 mRNA, but complete loss of Runx1 protein. This finding suggests a role for Runx1 as an EMT suppressor. To further address the relationship between Runx1 and EMT, Runx1 depleted stable cell lines were generated in both MCF10A and MCF7 cells. We observed that Runx1 knockdown (90%) in MCF10A cells leads to a dramatic change in their morphology towards a mesenchymal state, with a concomitant decrease in expression of the epithelial marker E-cadherin. In addition Runx1 depletion in MCF7 cells directly induced EMT state by repression of E-cadherin, as well as increased expression of mesenchymal genes, including vimentin, N-cadherin and fibronectin. Our results highlight a crucial role for Runx1 in preventing epithelial to mesenchymal transition and tumor progression in breast cancer. This Runx1 mediated mechanism points to novel intervention strategies for early stage breast cancer. Citation Format: Deli Hong, Jane Lian, Janet Stein, Gary Stein. Depletion of Runx1 promotes epithelial to mesenchymal transition in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4085. doi:10.1158/1538-7445.AM2015-4085

Genotype and Haplotype Analyses of TP53 Gene in Breast Cancer Patients: Association with Risk and Clinical Outcomes.

Variations in the TP53 gene have been suggested to play a role in many cancers, including breast. We previously observed an association between TP53 haplotypes based on four polymorphisms (rs17878362, rs1042522, rs12947788, and rs17884306) and the risk of colorectal and pancreatic cancer. Based on these results, in the present study, we have investigated the same polymorphisms and their haplotypes in 705 breast cancer cases and 611 healthy controls in relation to the disease risk, histopathological features of the tumor and clinical outcomes. In comparison to the most common haplotype A1-G-C-G, all the other identified haplotypes were globally associated with a significantly decreased breast cancer risk (P = 0.006). In particular, the A2-G-C-G haplotype was associated with a marked decreased risk of breast cancer when compared with the common haplotype (P = 0.0001). Moreover, rs1042522 in patients carrying the GC genotype and receiving only the anthracycline-based chemotherapy was associated with both overall and disease-free survival (recessive model for overall survival HR = 0.30 95% CI 0.11–0.80, P = 0.02 and for disease-free survival HR = 0.42 95% CI 0.21–0.84, P = 0.01). Present results suggest common genetic features in the susceptibility to breast and gastrointestinal cancers in respect to TP53 variations. In fact, similar haplotype distributions were observed for breast, colorectal, and pancreatic patients in associations with cancer risk. Rs1042522 polymorphism (even after applying the Dunn-Bonferroni correction for multiple testing) appears to be an independent prognostic marker in breast cancer patients.

Association of folate and other one-carbon related nutrients with hypermethylation status and expression of RARB, BRCA1, and RASSF1A genes in breast cancer patients.

Dietary methyl group donors could influence the hypermethylation status of certain putative genes. The present study explored the possible associations of dietary intake of one-carbon metabolism-related nutrients with promoter hypermethylation status and expression of retinoic acid receptor-beta (RARB), breast cancer-1 (BRCA1), and Ras association domain family-1, isoform A (RASSF1A) genes in Iranian women with breast cancer (BC). The hypermethylation status was investigated in 146 dissected BC tissue samples using methylation-specific PCR. The expression level was evaluated by real-time RT-PCR. Dietary nutrients were estimated using a validated 136-item food frequency questionnaire. Expression levels of the genes were associated with the unmethylated status of related promoters (p < 0.05). The crude dietary folate and adjusted cobalamin intakes were inversely associated with methylated RARB and BRCA1. Low intake of residual folate and cobalamin was correlated with the methylated status of RARB for subjects at <48 years of age, and folate alone was linked to BRCA1 at >48 years of age. High dietary intake of riboflavin and pyridoxine was the only determinant of the methylated promoter of RARB at odds ratios (ORs) of 4.15 (95 % confidence interval (CI) 1.28-13.50) and 2.53 (95 % CI 1.14-3.83) in multivariate models, respectively. One-carbon nutrients most often correlated inversely with the methylation-influenced expression of RARB. Although high folate intake increased the chance of unmethylation-dependent overexpression of BRCA1 3-fold, cobalamin and methionine were inversely linked to methylation-mediated expression. Nutritional epigenomics less actively influenced RASSF1A. These findings provide new insights into and a basic understanding of the selective contributions of individual B vitamins on hypermethylation and methylation-related expression of RARB and BRCA1 in BC. Hypermethylation at promoters of RARB, BRCA1, and RASSF1A is associated with reduced transcript levels of the respective gene in primary breast cancer tissue samples. Dietary folate and cobalamin intake is inversely associated with methylated RARB and BRCA1. High dietary intake of riboflavin and pyridoxine is associated with increased methylation in the RARB promoter. There is evidence for the age-dependent effects of nutrient intake on promoter methylation status. Bioavailability to the pool of nutrients might determine selectivity.

The role of the UTS2 gene polymorphisms and plasma Urotensin-II levels in breast cancer.

Breast cancer is the most common malignancy predominantly affecting women. To date, numerous numbers of studies were reported novel genetic contributors with diagnostic, prognostic, and therapeutic potential for the breast carcinogenesis. However, the role of urotensin-II in breast carcinogenesis has not been elucidated yet. Urotensin-II is a somatostatin-like cyclic tiny peptide identified by its potent vasoconstrictor activity. Soon after its discovery, its involvement in many disease states as well as its expression in various tissues including the tumors have been demonstrated. Moreover, there is strong evidence that suggest urotensin-II as the significant contributor of angiogenesis as well as cell proliferation and tumor biology. In this study, enzyme-linked immunosorbent assay (ELISA) and restriction fragment length polymorphism analysis were used to evaluate plasma levels of urotensin-II and Thr21Met and Ser89Asn polymorphisms of UTS2 gene in breast cancer patients. In the present case-control study, we noticed a significant decrease in the levels of urotensin-II protein in the plasma of the breast cancer patients (p < 0.05). Also, Thr21Met polymorphism in the UTS2 gene was associated with the risk of developing breast cancer (p < 0.0001), whereas the genotype frequency of Ser89Asn was found to be similar in patients and controls (p > 0.05). In addition, we demonstrated the gradual decreasing of urotensin-II protein levels from TT and TM to MM genotypes. In conclusion, these results strongly suggest that urotensin-II could contribute to breast carcinogenesis and Thr21Met polymorphism can be an important risk factor in developing breast tumors.