Characterisation of the BRCT domains of the breast cancer susceptibility gene product BRCA1

The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.     

乳腺癌易感基因研究进展

Ｂｒｃａ１和Ｂｒｃａ２是乳腺癌易感基因,在家族性乳腺癌患者中的突变是可以遗传的,在散发性乳腺癌患者中有杂合性丢失（ＬＯＨ）,而且表达水平下降。体外实验证明,Ｂｒｃａ１能抑制乳腺癌和卵巢癌细胞的增殖。Ｂｒｃａ１和Ｂｒｃａ２基因分别定位于１７ｑ１２－２１和１３ｑ１２－１３,编码序列分别为５７１１ｂｐ和１０９８７ｂｐ,其表达有一定的组织特异性。ＢＲＣＡ１和ＢＲＣＡ２蛋白分别由１８６３个氨基酸和３４１８个氨基酸组成,这两个蛋白都具有Ｇｒａｎｉｎ蛋白的某些特征。它们的功能目前还不是很清楚,但有证据表明这两个基因为生长发育所必须,并参与细胞增殖分化和ＤＮＡ损伤修复等生命活动。     

Identifying cancer genes from cancer mutation profiles by cancer functions

It is of great importance to identify new cancer genes from the data of large scale genome screenings of gene mutations in cancers. Considering the alternations of some essential functions are indispensable for oncogenesis, we define them as cancer functions and select, as their approximations, a group of detailed functions in GO (Gene Ontology) highly enriched with known cancer genes. To evaluate the efficiency of using cancer functions as features to identify cancer genes, we define, in the screened genes, the known protein kinase cancer genes as gold standard positives and the other kinase genes as gold standard negatives. The results show that cancer associated functions are more efficient in identifying cancer genes than the selection pressure feature. Furthermore, combining cancer functions with the number of non-silent mutations can generate more reliable positive predictions. Finally, with precision 0.42, we suggest a list of 46 kinase genes as candidate cancer genes which are annotated to cancer functions and carry at least 3 non-silent mutations.     

Urinary concentration of endocrine-disrupting phthalates and breast cancer risk in Indian women: A case-control study with a focus on mutations in phthalate-responsive genes

BackgroundPhthalates are known endocrine-disrupting chemicals used indiscriminately as constituents in consumer products including food processing, and packaging, cosmetics, personal care and household items. Although, few studies have assessed the risk of breast cancer on exposure to phthalates, their association with breast cancer risk in Indian women have not yet been evaluated.MethodsWe conducted a case-control study involving 171 participants. Urinary concentrations of six phthalate dieters; DMP (Dimethyl phthalate), DEP (Diethyl phthalate), DBP (Dibutyl phthalate), BBP (benzyl butyl phthalate), DEHP (Di-2-ethyl-hexyl phthalate), DINOP (Di-n-octyl phthalate) were estimated by GC-MS and geometric means were calculated. Univariate and multivariable logistic regression was performed to assess breast cancer risk on exposure to phthalates. Genes responsive to phthalates were identified through literature search and matched with NGS data, and gene-enrichment analysis was performed.ResultsSignificant associations were observed between urinary phthalate concentrations and increased risk of breast cancer for di-butyl phthalate (OR=1.5, 95% CI; 1.06, 2.11, p = 0.002) and di-2-ethyl-hexyl phthalate (>median vs ≤ median; OR=2.97, 95% CI; 1.18, 7.47, p = 0.005) in multivariable analyses. We also found several phthalate-responsive gene mutations in paired breast tumor tissues, which include PTPRD (76.19%), AR (42.86%), CYP1A1 (42.86%), CYP19A1 (23.81%), AHRR (19.05%), PIK3CA (19.05%), CYP1B1 (9.52%), RB1 (9.52%) and MMP9 (9.52%). Gene-enrichment analysis revealed that these genes form a major part of ER/PR, PPAR and HIF-1α-TGF-β signaling cascades involved in breast cancerConclusionAlthough the sample size is small, in this first case-control study from India, DBP and DEHP were found to be associated with increased risk of invasive breast cancer and tumor tissues revealed mutations in several phthalate-responsive genes. It is, therefore suggested that human biomonitoring in India and larger studies evaluating the early life genetic and epigenetic alterations on phthalates exposure are required to establish their role in breast carcinogenesis.     

MCPH1 protein expression and polymorphisms are associated with risk of breast cancer

Microcephalin 1 (MCPH1) has a crucial role in the DNA damage response by promoting the expression of checkpoint kinase 1 (CHK1) and breast cancer susceptibility gene 1 (BRCA1). MCPH1 containing BRCT domain has been suggested as a tumor suppressor in breast and ovarian cancers. We analyzed the effect of both protein expression and MCPH1 polymorphisms in breast cancer patients. Low nuclear expression of microcephalin was present in 52.4% of breast cancers and was associated with allele T in rs2912010 (p = 0.046). However, cytoplasmic microcephalin expression increased with increasing grade (p = 0.010). An association between low nucleus microcephalin expression and allele T was identified in rs2912010 (p = 0.046). After data analysis, allele distribution of the MCPH1 polymorphisms was not different between breast cancer patients and healthy controls. But the polymorphism was associated with negative status for ER (rs2912010/C2302T; p = 0.032, rs1057090/C2358T; p = 0.027, rs2912016/C2494A; p = 0.024), and allele T in both rs2912010 and rs1057090 was associated with increasing tumor grade (rs2912010; p = 0.040, rs1057090; p = 0.043) in breast cancer. We are first to report that association of MCPH1 protein expression and its polymorphisms in breast cancer. The MCPH1 polymorphisms and protein expression were associated with tumorigenesis in breast cancer and may be a useful biomarker for identification of the aggressive types of breast cancer.     

Polymorphisms in the promoter region of ESR2 gene and breast cancer susceptibility

Genetic variations like single nucleotide polymorphisms (SNPs) in genes involved in estrogen biosynthesis, metabolism and signal transduction have been suggested to affect breast cancer susceptibility. In this study we tested the hypothesis that polymorphisms in the promoter of ESR2 gene may be associated with increased risk for breast cancer. We analyzed three SNPs in the promoter region of human ESR2 gene by means of allele-specific tetra-primer PCR. A total of 318 sporadic breast cancer cases and 318 age-matched controls were included in the study. With regard to homozygous genotypes, women with sporadic breast cancer more frequently carried the CC genotype of ESR2 promoter SNP rs2987983 (OR 1.99, p = 0.005). Calculation of allele positivity demonstrated that presence of T allele of this SNP was more frequent in healthy women. Our data suggest that a SNP in the promoter region of ESR2 gene might be able to affect breast cancer risk. These results further support the emerging hypothesis that ERβ is an important factor in breast cancer development.     

Fibroblast growth factor receptor 2 gene (FGFR2) rs2981582T/C polymorphism and susceptibility to breast cancer in Saudi women

Fibroblast growth factor receptor 2 is a protein encoded by FGFR2 gene and plays an important role in cellular growth. This study was conducted to investigate a potential association of FGFR2 rs2981582 with breast cancer. DNA was obtained from 137 Formalin-fixed, paraffin-embedded tumors and 98 normal breast tissue samples. Genotypes were carried out with PCR-RFLP. The odds ratio and 95% confidence interval (CI) were used to evaluate the power of the associations. A significant association between FGFR2 rs2981582 C allele and susceptibility to breast cancer was found (p-value < 0.0001, Odds Ratio = 2.3, %95 CI (1.5–3.0). No significant differences in FGFR2 rs2981582 genotypes and alleles distribution among breast patients with different hormonal receptor status (p > 0.05) were detected. However, a significant difference was found in genotypes and alleles distribution in ER+, PR- and HER2 between breast cancer cases and controls. This study showed an association of FGFR2 rs2981582T/C with breast cancer in Saudi women, further large study is required to validate the results.     

Purification and characterisation of a soluble N-terminal fragment of the breast cancer susceptibility protein BRCA1

The BRCA1 gene encodes a large multidomain protein of 1863 residues, mutations in which lead to breast cancer. Studies to elucidate the mechanisms by which BRCA1 prevents tumour formation have been restricted by the size of the protein. Unable to purify large amounts of the full-length protein, we have identified a fragment of BRCA1, amino acid residues 230-534, that when cloned into the expression vector pET 22b and expressed in Escherichia coli is found predominantly in the soluble portion of the cell lysate. The resulting protein was purified to homogeneity and studies reveal that BRCA1 230-534 binds specifically to four-way junction DNA when compared to duplex and single-stranded DNA.     

Reproductive factors and breast cancer risk among BRCA1 or BRCA2 mutation carriers: Results from ten studies

Although reproductive factors are among the most well-established risk factors for breast cancer in the general population, it is still a matter for debate whether these factors act as risk modifiers among BRCA1 or BRCA2 mutation carriers. This meta-analysis is the first to be performed to determine the relationship between reproductive factors and breast cancer risk among BRCA1 and BRCA2 mutation carriers. We searched the PubMed database up to February 2013. A total of ten studies met the inclusion criteria. The results showed that the reproductive factors may be associated with breast cancer risk only among BRCA1 mutation carriers. No association was found between parity and breast cancer risk. Compared with women at the youngest age in the first-birth category, women in the oldest age category were at a 38% lower risk of breast cancer (RR = 0.62, 95%CI = 0.45–0.85). Breastfeeding for at least 1 or 2 years was associated with a 37% reduction in breast cancer risk (RR = 0.63, 95%CI = 0.46–0.86). Women at the oldest age in the menarche category were at a 34% lower risk of breast cancer (RR = 0.66, 95%CI = 0.53–0.81) than women in the youngest age category. However, none of the reproductive factors were associated with breast cancer risk among BRCA2 mutation carriers. In conclusion, late age at first birth, breastfeeding, and late age at menarche protect against breast cancer in BRCA1 mutation carriers only. Further studies are needed to explore the mechanisms.     

Breast cancer susceptibility loci in association with age at menarche,age at natural menopause and the reproductive lifespan

Background: Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with breast cancer risk. Some of these loci have unknown functional significance and may mediate the effects of hormonal exposures on breast cancer risk. We examined relationships between breast cancer susceptibility variants and menstrual/reproductive factors using data from two population-based studies. Methods: The first analysis was based on a sample of 1328 women age 20–74 who participated as controls in a case–control study of breast cancer conducted in three U.S. states. We evaluated the associations between age at menarche, age at natural menopause and the reproductive lifespan with 13 previously identified breast cancer variants. Associations were also examined with a genetic score created as the sum of at-risk alleles across the 13 variants. For validation, significant results were evaluated in a second dataset comprised 1353 women age 43–86 recruited as part of a cohort study in Wisconsin. Results: Neither the genetic score nor any of the 13 variants considered individually were associated with age at menarche or reproductive lifespan. Two SNPs were associated with age at natural menopause; every increase in the minor allele (A) of rs17468277 (CASP8) was associated with a 1.12 year decrease in menopause age (p = 0.02). The minor allele (G) of rs10941679 (5p12) was associated with a 1.01 year increase in age at natural menopause (p = 0.01). The results were not replicated in the validation cohort (B = −0.61, p = 0.14 and B = −0.01, p = .0.98, respectively). Conclusions: The evaluated variants and reproductive experiences may work through separate pathways to influence breast cancer risk.     

A fine balance between CCNL1 and TIMP1 contributes to the development of breast cancer cells

Cyclin L1 (CCNL1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) are candidate genes involved in several types of cancer. However, the expression of CCNL1 and the relationship between CCNL1 and TIMP1 in breast cancer cells is unknown. Using patients’ breast cancer tissues, the expression of CCNL1 and TIMP1 was measured by cDNA microarray and further confirmed by real-time RT-PCR and western blotting. Overexpression or repression of CCNL1 and TIMP1, individually or together, was performed in breast cancer MDA-MB-231 cells by transient transformation methods to investigate their role in breast cancer cell growth. Simultaneously, mRNA and protein expression levels of CCNL1 and TIMP1 were also measured. CCNL1 and TIMP1 expression was significantly elevated in breast cancer tissues compared with that in peri-breast cancer tissues of patients by cDNA microarray and these results were further confirmed by real-time RT-PCR and western blotting. Interestingly, in vitro experiments showed a stimulatory effect of TIMP1 and an inhibitory effect of CCNL1 on growth of MDA-MB-231 cells. Co-expression or co-repression of these two genes did not affect cell growth. Overexpression of CCNL1 and TIMP1 individually induced overexpression of each other. These data demonstrate that there is a fine balance between CCNL1 and TIMP1, which may contribute to breast cancer development.     

Identification of eight genes that are potentially involved in tamoxifen sensitivity in breast cancer cells

Although the antiestrogen agent tamoxifen has long been used to treat women with hormone receptor positive invasive breast carcinoma, the mechanisms of its action and acquired resistance to tamoxifen during treatment are largely unknown. A number of studies have revealed that over-activation of some signaling pathways can cause tamoxifen resistance; however, very little information is available regarding the genes whose loss-of-function alternation contribute to tamoxifen resistance. Here we used a forward genetic approach in vitro to generate tamoxifen resistant cells from the tamoxifen sensitive breast cancer cell line ZR-75-1, and further identified the disrupted gene in different tamoxifen resistant clones. Retinol binding protein 7, DNA polymerase-transactivated protein 3, γ-glutamyltransferase-like activity 1,slit-robo RhoGTPase-activating protein, tetraspan NET-4, HSPC194, amiloride-sensitive epithelial sodium channel gene,and Notch2, were the eight mutated genes identified in different tamoxifen resistant clones, suggesting their requirement for tamoxifen sensitivity in ZR-75-1 cells. Since the functions of these genes are not related to each other, it suggests that multiple pathways can influence tamoxifen sensitivity in breast cancer ceils.