Quantitative Immunohistochemical Analysis Reveals Association between Sodium Iodide Symporter and Estrogen Receptor Expression in Breast Cancer

Background

Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images.

Methods

hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images.

Results

Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2–ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method.

Conclusion

The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted ¹³¹I radio-ablative therapy is aimed.     

Assessing SNP-SNP Interactions among DNA Repair,Modification and Metabolism Related Pathway Genes in Breast Cancer Susceptibility

Genome-wide association studies (GWASs) have identified low-penetrance common variants (i.e., single nucleotide polymorphisms, SNPs) associated with breast cancer susceptibility. Although GWASs are primarily focused on single-locus effects, gene-gene interactions (i.e., epistasis) are also assumed to contribute to the genetic risks for complex diseases including breast cancer. While it has been hypothesized that moderately ranked (P value based) weak single-locus effects in GWASs could potentially harbor valuable information for evaluating epistasis, we lack systematic efforts to investigate SNPs showing consistent associations with weak statistical significance across independent discovery and replication stages. The objectives of this study were i) to select SNPs showing single-locus effects with weak statistical significance for breast cancer in a GWAS and/or candidate-gene studies; ii) to replicate these SNPs in an independent set of breast cancer cases and controls; and iii) to explore their potential SNP-SNP interactions contributing to breast cancer susceptibility. A total of 17 SNPs related to DNA repair, modification and metabolism pathway genes were selected since these pathways offer a priori knowledge for potential epistatic interactions and an overall role in breast carcinogenesis. The study design included predominantly Caucasian women (2,795 cases and 4,505 controls) from Alberta, Canada. We observed two two-way SNP-SNP interactions (APEX1-rs1130409 and RPAP1-rs2297381; MLH1-rs1799977 and MDM2-rs769412) in logistic regression that conferred elevated risks for breast cancer (P _interaction<7.3×10⁻³). Logic regression identified an interaction involving four SNPs (MBD2-rs4041245, MLH1-rs1799977, MDM2-rs769412, BRCA2-rs1799943) (P _permutation = 2.4×10⁻³). SNPs involved in SNP-SNP interactions also showed single-locus effects with weak statistical significance, while BRCA2-rs1799943 showed stronger statistical significance (P _{correlation/trend} = 3.2×10⁻⁴) than the others. These single-locus effects were independent of body mass index. Our results provide a framework for evaluating SNPs showing statistically weak but reproducible single-locus effects for epistatic effects contributing to disease susceptibility.     

Ethnic Features of Genetic Susceptibility to Breast Cancer

The results of screening for BRCA1, BRCA2, ATM, NBN, CHEK2, PALB2, BLM gene mutations in 1000 breast cancer (BC) patients from the Republic of Bashkortostan (RB) are presented. Germline mutations in these genes accounted for 7.5% of breast cancer patients. The wide spectrum of mutations was found in women of Slavic origin, including: c.5266dupC, c.181T>G, and c.4034delA in BRCA1; c.5932G>T in ATM; c.657_661del5 in NBN; c.444+1G>A, c.1100delC, and dele9,10(5kb) in CHEK2; c.509_510delGA and c.172_175delTTGT in PALB2; and c.1642C>T in BLM gene.     

mRNA Profiling Reveals Determinants of Trastuzumab Efficiency in HER2-Positive Breast Cancer

Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs) affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we called and analyzed SNPs. These results contribute to a better understanding of trastuzumab action and resistance mechanisms.     

过表达ER恢复雌激素对子宫内膜癌KLE细胞中Notch信号通路的调控

目的：通过观察雌激素对子宫内膜癌KLE细胞中Notch信号通路的影响,探讨过表达雌激素核受体（estrogenreceptor,ER）是否可以恢复雌激素对Notch信号通路的调控作用,继而调节细胞增殖活性。方法：MTT检测雌激素及Notch信号通路对细胞增殖活性的影响;RT．PCR及Westem．blotting检测雌激素及Notch通路抑制剂DAPT对Notch表达的影响;质粒的抽提及转染使KLE细胞中的雌激素核受体ER过表达。结果：雌激素呈剂量依赖效应促进KLE细胞的增殖活性,其中以雌激素浓度为1．0×10-9M时最明显（相对于对照组为1．25±0．026,P〈0．05）;抑制Notch信号通路的表达可以明显下调KLE细胞的增殖活性（0．76±0．02,P〈0．05）;在KLE细胞中,雌激素对Notch的表达没有明显的调控作用,但是将其雌激素核受体过表达后,雌激素可明显上调Notch的表达,并显著促进细胞的增殖活性（1．24±0．02,P〈0．05）。结论：在ER阴性的子宫内膜癌细胞中过表达ER,可以恢复雌激素对Notch信号通路的调控,从而进一步的调控细胞增殖活性。     

过表达ER恢复雌激素对子宫内膜癌KLE细胞中Notch信号通路的调控

目的:通过观察雌激素对子宫内膜癌KLE细胞中Notch信号通路的影响,探讨过表达雌激素核受体（estrogen receptor,ER）是否可以恢复雌激素对Notch信号通路的调控作用,继而调节细胞增殖活性。方法:MTT检测雌激素及Notch信号通路对细胞增殖活性的影响;RT-PCR及Western-blotting检测雌激素及Notch通路抑制剂DAPT对Notch表达的影响;质粒的抽提及转染使KLE细胞中的雌激素核受体ER过表达。结果:雌激素呈剂量依赖效应促进KLE细胞的增殖活性,其中以雌激素浓度为1.0×10^-9M时最明显（相对于对照组为1.25±0.026,P<0.05）;抑制Notch信号通路的表达可以明显下调KLE细胞的增殖活性（0.76±0.02,P<0.05）;在KLE细胞中,雌激素对Notch的表达没有明显的调控作用,但是将其雌激素核受体过表达后,雌激素可明显上调Notch的表达,并显著促进细胞的增殖活性（1.24±0.02,P<0.05）。结论:在ER阴性的子宫内膜癌细胞中过表达ER,可以恢复雌激素对Notch信号通路的调控,从而进一步的调控细胞增殖活性。     

Genome-Wide Association Study to Identify the Genetic Determinants of Otitis Media Susceptibility in Childhood

Background

Otitis media (OM) is a common childhood disease characterised by middle ear inflammation and effusion. Susceptibility to recurrent acute OM (rAOM; ≥3 episodes of AOM in 6 months) and chronic OM with effusion (COME; MEE ≥3 months) is 40–70% heritable. Few underlying genes have been identified to date, and no genome-wide association study (GWAS) of OM has been reported.

Methods and Findings

Data for 2,524,817 single nucleotide polymorphisms (SNPs; 535,544 quality-controlled SNPs genotyped by Illumina 660W-Quad; 1,989,273 by imputation) were analysed for association with OM in 416 cases and 1,075 controls from the Western Australian Pregnancy Cohort (Raine) Study. Logistic regression analyses under an additive model undertaken in GenABEL/ProbABEL adjusting for population substructure using principal components identified SNPs at CAPN14 (rs6755194: OR = 1.90; 95%CI 1.47–2.45; P_adj-PCA = 8.3×10⁻⁷) on chromosome 2p23.1 as the top hit, with independent effects (rs1862981: OR = 1.60; 95%CI 1.29–1.99; P_adj-PCA = 2.2×10⁻⁵) observed at the adjacent GALNT14 gene. In a gene-based analysis in VEGAS, BPIFA3 (P_Gene = 2×10⁻⁵) and BPIFA1 (P_Gene = 1.07×10⁻⁴) in the BPIFA gene cluster on chromosome 20q11.21 were the top hits. In all, 32 genomic regions show evidence of association (P_adj-PCA<10⁻⁵) in this GWAS, with pathway analysis showing a connection between top candidates and the TGFβ pathway. However, top and tag-SNP analysis for seven selected candidate genes in this pathway did not replicate in 645 families (793 affected individuals) from the Western Australian Family Study of Otitis Media (WAFSOM). Lack of replication may be explained by sample size, difference in OM disease severity between primary and replication cohorts or due to type I error in the primary GWAS.

Conclusions

This first discovery GWAS for an OM phenotype has identified CAPN14 and GALNT14 on chromosome 2p23.1 and the BPIFA gene cluster on chromosome 20q11.21 as novel candidate genes which warrant further analysis in cohorts matched more precisely for clinical phenotypes.     

Analysis of Interleukin-8 Gene Variants Reveals Their Relative Importance as Genetic Susceptibility Factors for Chronic Periodontitis in the Han Population

Interleukin (IL)-8, an important chemokine that regulates the inflammatory response, plays an important role in periodontitis. Previous studies indicate that certain IL-8 gene polymorphisms are associated with periodontitis susceptibility in some populations. However, the literature is somewhat contradictory, and not all IL-8 polymorphisms have been examined, particularly in Han Chinese individuals. The aim of this study was to investigate the association of every IL-8 SNP with chronic periodontitis in Han Chinese individuals. We analyzed 23 SNPs with minor allele frequency (MAF)≥0.01, which were selected from 219 SNPs in the NCBI dbSNP and preliminary HapMap data analyses from a cohort of 400 cases and 750 controls from genetically independent Han Chinese individuals. Single SNP, haplotype and gender-specific associations were performed. We found that rs4073 and rs2227307 were significantly associated with chronic periodontitis. Further haplotype analysis indicated that a haplotype block (rs4073-rs2227307-rs2227306) that spans the promoter and exon1 of IL-8 was highly associated with chronic periodontitis. Additionally, the ATC haplotype in this block was increased 1.5-fold in these cases. However, when analyzing the samples by gender, no significant gender-specific associations in IL-8 were observed, similar to the results of haplotype association analyses in female and male subgroups. Our results provide further evidence that IL-8 is associated with chronic periodontitis in Han Chinese individuals. Furthermore, our results confirm previous reports suggesting the intriguing possibilities that IL-8 plays a role in the pathogenesis of chronic periodontitis and that this gene may be involved in the etiology of this condition.     

Association of Genetic Polymorphisms in CDH1 and CTNNB1 with Breast Cancer Susceptibility and Patients' Prognosis among Chinese Han Women

This study aims to investigate whether the germline variants in CDH1 and CTNNB1 would affect breast cancer susceptibility and patients’ prognosis among Chinese Han women using a haplotype-based association analysis. We genotyped 12 haplotype-tagging single nucleotide polymorphisms (htSNPs) in CDH1 and CTNNB1 among 1,160 BC cases and 1,336 age-matched cancer-free controls using the TaqMan^® Genotyping Assay. For association analyses of germline variants with breast cancer susceptibility, the results showed that rs7200690, rs7198799, rs17715799, rs13689 and diplotype CGC/TGC (rs7200690 + rs12185157 + rs7198799) in CDH1 as well as rs2293303 in CTNNB1 were associated with increased breast cancer risk. In addition, the Generalized Multifactor Dimensionality Reduction (GMDR) and logistic regression analysis predicted an interaction on breast cancer risk between rs17715799 and rs13689 as well as rs13689 and menarche-FFTP (First Full-Term Pregnancy) interval. For survival analyses, the results demonstrated that the minor allele homozygotes of rs13689 and haplotype TGC in CDH1 were linked with unfavorable event-free survival of breast cancer, whereas, rs4783689 of CDH1 showed the opposite effect under dominant model. Notably, the stratified analysis revealed that rs7186053 was associated with favorable event-free survival among patients with estrogen receptor (ER)-positive, progesterone receptor (PR)-positive or lymph node metastasis negative patients. Moreover, rs7200690 and rs7198799 in CDH1 as well as rs4533622 in CTNNB1 were associated with worse event-free survival among patients with clinical stage 0-I tumors. This study indicated that the genetic polymorphisms of CDH1 and CTNNB1 were associated with breast cancer susceptibility and patients’ prognosis.     

Hedgehog信号通路与乳腺癌

近年来的研究表明,Hedgehog信号通路在肿瘤的发生发展中具有重要的作用,该通路基因突变或异常表达将导致多种器官肿瘤的发生,并与Wnt、MAPK等信号通路相互作用,共同调节肿瘤的发生发展。我们简要综述了Hedgehog信号通路在乳腺癌发生发展中的重要作用,旨在了解乳腺癌发生、发展的分子机制.     

Simultaneous Inhibition of Estrogen Receptor and the HER2 Pathway in Breast Cancer: Effects of HER2 Abundance

The estrogen receptor (ER) pathway and the epidermal growth factor receptor (EGFR) pathway play pivotal roles in breast cancer progression. Targeted therapies able to intercept ER or signaling downstream to EGFR and its kin, HER2, are routinely used to treat distinct groups of breast cancer patients. However, patient responses are limited by resistance to endocrine therapy, which may be due to compensatory HER2/EGFR signaling. This raises the possibility that simultaneous interception of HER2 and ER may enhance therapeutic efficacy. To address the question, we treated breast cancer cells with both fulvestrant (ICI 182780), an ER antagonist with no agonist effects, and lapatinib, an orally available tyrosine kinase inhibitor specific to EGFR and HER2. Our results indicate that the combination of drugs is especially effective when applied to HER2-overexpressing, ER-positive cancer cells. Interestingly, fulvestrant activated the mitogen-activated protein kinase (MAPK) pathway of these cells, but complete inhibition of MAPK signaling was observed on cotreatment with lapatinib. Taken together, our observations reinforce the possibility that the effectiveness of combining anti-ER and anti-HER2/EGFR drugs may be especially effective on a relatively small subtype of HER2-overexpressing, ER-positive tumors of the breast.     

RAD51 and Breast Cancer Susceptibility: No Evidence for Rare Variant Association in the Breast Cancer Family Registry Study

Background

Although inherited breast cancer has been associated with germline mutations in genes that are functionally involved in the DNA homologous recombination repair (HRR) pathway, including BRCA1, BRCA2, TP53, ATM, BRIP1, CHEK2 and PALB2, about 70% of breast cancer heritability remains unexplained. Because of their critical functions in maintaining genome integrity and already well-established associations with breast cancer susceptibility, it is likely that additional genes involved in the HRR pathway harbor sequence variants associated with increased risk of breast cancer. RAD51 plays a central biological function in DNA repair and despite the fact that rare, likely dysfunctional variants in three of its five paralogs, RAD51C, RAD51D, and XRCC2, have been associated with breast and/or ovarian cancer risk, no population-based case-control mutation screening data are available for the RAD51 gene. We thus postulated that RAD51 could harbor rare germline mutations that confer increased risk of breast cancer.

Methodology/Principal Findings

We screened the coding exons and proximal splice junction regions of the gene for germline sequence variation in 1,330 early-onset breast cancer cases and 1,123 controls from the Breast Cancer Family Registry, using the same population-based sampling and analytical strategy that we developed for assessment of rare sequence variants in ATM and CHEK2. In total, 12 distinct very rare or private variants were characterized in RAD51, with 10 cases (0.75%) and 9 controls (0.80%) carrying such a variant. Variants were either likely neutral missense substitutions (3), silent substitutions (4) or non-coding substitutions (5) that were predicted to have little effect on efficiency of the splicing machinery.

Conclusion

Altogether, our data suggest that RAD51 tolerates so little dysfunctional sequence variation that rare variants in the gene contribute little, if anything, to breast cancer susceptibility.     

Obesity Suppresses Estrogen Receptor Beta Expression in Breast Cancer Cells via a HER2-Mediated Pathway

Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ) expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB): ≥30 kg/m²; normal weight (N): 18.5–24.9 kg/m²). Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231) and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu) following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s) mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease.     

Association between PARP-1 V762A Polymorphism and Breast Cancer Susceptibility in Saudi Population

Genetic aberrations of DNA repair enzymes are known to be common events and to be associated with different cancer entities. Aim of the following study was to analyze the genetic association of rs1136410 (Val762Ala) in PARP1 gene with the risk of breast cancer using genotypic assays and insilico structural predictions. Genotypic analysis of individual locus showed statistically significant association of Val762Ala with increased susceptibility to breast cancer. Protein structural analysis was performed with Val762Ala variant allele and compared with the predicted native protein structure. Protein prediction analysis showed that this nsSNP may cause changes in the protein structure and it is associated with the disease. In addition to the native and mutant 3D structures of PARP1 were also analyzed using solvent accessibility models for further protein stability confirmation. Taken together, this the first study that confirmed Val762Ala variant has functional effect and structural impact on the PARP1 and may play an important role in breast cancer progression in Saudi population.     

APOBEC3基因拷贝数变异与乳腺癌易感性的关联

为了探讨APOBEC3基因缺失拷贝数变异的多态性与汉族女性乳腺癌易感性的关联性,本研究应用聚合酶链式反应-限制性多态性内切酶技术检测281例乳腺癌和292例正常对照组。我们发现APOBEC3基因拷贝数变异位点的等位基因、基因型频率分布在乳腺癌和对照组之间比较有显著性差异(p<0.05),校正混杂因素后基因型Del/Del,Del/Ins,显性模型和加性模型下对乳腺癌的风险预测值分别为2.753 (95%Cl:1.363～3.560; p=0.005)、1.462(95%Cl:1.021～2.094; p=0.038)、2.282(95%Cl:1.155～3.508; p=0.018)和1.596(95%Cl:1.129～2.256; p=0.008)。研究表明APOBEC3基因的缺失变异位点多态性与乳腺癌的发病风险存在关联,等位基因del是乳腺癌发病的危险因素。本研究的结果可以为本地区群体的乳腺癌易感基因的筛选以及评估易感基因对患病的风险程度提供参考数据,为乳腺癌的防治、诊断和个体化治疗研究提供了帮助。