Hypersensitive K303R oestrogen receptor-α variant not found in invasive carcinomas

INTRODUCTION: Genetic abnormalities or mutations in premalignant breast lesions may have a role in progression toward malignancy or influence the behaviour of subsequent disease. The A908G (Lys303→Arg) change in the gene encoding oestrogen receptor-α (ER-α) creates a hypersensitivity to oestradiol and would have significant consequences if present in breast carcinoma, especially those treated with endocrine therapy. We have therefore examined a panel of endocrine-treated invasive carcinomas for the presence of this mutation. METHODS: Sequencing of control DNA was shown to detect mutation present in as little as 15% of the starting material. Enrichment for the mutation by using MboII restriction digestion allowed the detection of mutant present at 1% or less. We applied these techniques to genomic DNA and cDNA from 136 invasive breast carcinomas. RESULTS: No evidence of the A908G mutation was found with either technique. The incidence of this mutation in our panel of tumours is therefore significantly less than previously reported. CONCLUSION: The fact that the mutation was not found leads us to believe that this mutation is absent from most cells in invasive carcinomas and furthermore that the major expression product of the ER-α gene in cancers does not contain the K303R mutation. It is therefore unlikely to influence the effectiveness of endocrine treatment

Association of oestrogen receptor beta 2 (ERβ2/ERβcx) with outcome of adjuvant endocrine treatment for primary breast cancer – a retrospective study

<p>Abstract</p> <p>Background</p> <p>Oestrogen receptor beta (ERβ) modulates ERα activity; wild type ERβ (ERβ1) and its splice variants may therefore impact on hormone responsiveness of breast cancer. ERβ2/ERβcx acts as a dominant negative inhibitor of ERα and expression of ERβ2 mRNA has been proposed as a candidate marker for outcome in primary breast cancer following adjuvant endocrine therapy. We therefore now assess ERβ2 protein by immunostaining and mRNA by quantitative RT-PCR in relation to treatment outcome.</p> <p>Methods</p> <p>ERβ2-specific immunostaining was quantified in 141 primary breast cancer cases receiving adjuvant endocrine therapy, but no neoadjuvant therapy or adjuvant chemotherapy. The expression of mRNA for ERβ2/ERβcx was measured in 100 cases by quantitative RT-PCR. Statistical analysis of breast cancer relapse and breast cancer survival was performed using Kaplan Meier log-rank tests and Cox's univariate and multivariate survival analysis.</p> <p>Results</p> <p>High ERβ2 immunostaining (Allred score >5) and high ERβ2 mRNA levels were independently associated with significantly better outcome across the whole cohort, including both ERα positive and negative cases (Log-Rank P < 0.05). However, only ERβ2 mRNA levels were significantly associated with better outcome in the ERα + subgroup (Log-Rank P = 0.01) and this was independent of grade, size, nodal status and progesterone receptor status (Cox hazard ratio 0.31 P = 0.02 for relapse; 0.17 P = 0.01 for survival). High ERβ2 mRNA was also associated with better outcome in node negative cases (Log Rank P < 0.001).</p> <p>ERβ2 protein levels were greater in ERα positive cases (T-test P = 0.00001), possibly explaining the association with better outcome. Levels of ERβ2 protein did not correlate ERβ2 mRNA levels, but 34% of cases had both high mRNA and protein and had a significantly better outcome (Log-Rank relapse P < 0.005).</p> <p>Conclusion</p> <p>High ERβ2 protein levels were associated with ERα expression. Although most cases with high ERβ2 mRNA had strong ERβ2 immunostaining, mRNA levels but not protein levels were independently predictive of outcome in tamoxifen-treated ERα + tumours. Post-transcriptional control needs to be considered when assessing the biological or clinical importance of ERβ proteins.</p

The estrogen receptor-α A908G (K303R) mutation occurs at a low frequency in invasive breast tumors: results from a population-based study

INTRODUCTION: Evidence suggests that alterations in estrogen signaling pathways, including estrogen receptor-α (ER-α), occur during breast cancer development. A point mutation in ER-α (nucleotide A908G), producing an amino acid change from lysine to arginine at codon 303 (K303R) results in receptor hypersensitivity to estrogen. This mutation was initially reported in one-third of hyperplastic benign breast lesions, although several recent studies failed to detect it in benign or malignant breast tissues. METHODS: We screened 653 microdissected, newly diagnosed invasive breast tumors from patients in the Carolina Breast Cancer Study, a population-based case-control study of breast cancer in African American and white women in North Carolina, for the presence of the ER-α A908G mutation by using single-strand conformational polymorphism (SSCP) analysis and (33)P-cycle sequencing. RESULTS: We detected the ER-α A908G mutation in 37 of 653 (5.7%) breast tumors. The absence of this mutation in germline DNA confirmed it to be somatic. Three tumors exhibited only the mutant G base at nucleotide 908 on sequencing, indicating that the wild-type ER-α allele had been lost. The ER-α A908G mutation was found more frequently in higher-grade breast tumors (odds ratio (OR) 2.83; 95% confidence interval (CI) 1.09 to 7.34, grade II compared with grade I), and in mixed lobular/ductal tumors (OR 2.10; 95% CI 0.86 to 5.12) compared with ductal carcinomas, although the latter finding was not statistically significant. CONCLUSION: This population-based study, the largest so far to screen for the ER-α A908G mutation in breast cancer, confirms the presence of the mutant in invasive breast tumors. The mutation was associated with higher tumor grade and mixed lobular/ductal breast tumor histology

Effects of two common polymorphisms in the 3' untranslated regions of estrogen receptor β on mRNA stability and translatability

Estrogen signaling is mediated by estrogen receptors (ERs), ERα and ERβ. Aberrant estrogen signaling is involved in breast cancer development. ERα is one of the key biomarkers for diagnosis and treatment of breast cancer. Unlike ERα, ERβ is still not introduced as a marker for diagnosis and established as a target of therapy. Numerous studies suggest antiproliferative effects of ERβ, however its role remains to be fully explored. Albeit important, ERα is not a perfect marker, and some aspects of ERα function are still unclear. This thesis aims to characterize distinct molecular facets of ER action relevant for breast cancer and provide valuable information for ER-based diagnosis and treatment design. In PAPER I, we analyzed the functionality of two common single nucleotide polymorphisms in the 3’ untranslated regions of ERβ, rs4986938 and rs928554, which have been extensively investigated for association with various diseases. A significant difference in allelic expression was observed for rs4986938 in breast tumor samples from heterozygous individuals. However, no difference in mRNA stability or translatability between the alleles was observed. In PAPER II, we provided a more comprehensive understanding of ERβ function independent of ERα. A global gene expression analysis in a HEK293/ERβ cell model identified a set of ERβ-regulated genes. Gene Ontology (GO) analysis showed that they are involved in cell-cell signaling, morphogenesis and cell proliferation. Moreover, ERβ expression resulted in a significant decrease in cell proliferation. In PAPER III, using the human breast cancer MCF-7/ERβ cell model, we demonstrated, for the first time, the binding of ERα/β heterodimers to various DNA-binding regions in intact chromatin. In PAPER IV, we investigated a potential cross-talk between estrogen signaling and DNA methylation by identifying their common target genes in MCF-7 cells. Gene expression profiling identified around 150 genes regulated by both 17β- estradiol (E2) and a hypomethylating agent 5-aza-2’-deoxycytidine. Based on GO analysis, CpG island prediction analysis and previously reported ER binding regions, we selected six genes for further analysis. We identified BTG3 and FHL2 as direct target genes of both pathways. However, our data did not support a direct molecular interplay of mediators of estrogen and epigenetic signaling at promoters of regulated genes. In PAPER V, we further explored the interactions between estrogen signaling and DNA methylation, with focus on DNA methyltransferases (DNMT1, DNMT3a and DNMT3b). E2, via ERα, up-regulated DNMT1 and down-regulated DNMT3a and DNMT3b mRNA expression. Furthermore, DNMT3b interacted with ERα. siRNA-mediated DNMT3b depletion increased the expression of two genes, CDKN1A and FHL2. We proposed that the molecular mechanism underlying regulation of FHL2 and CDKN1A gene expression involves interplay of DNMT3b and ERα. In conclusion, the studies presented in this thesis contribute to the knowledge of ERβ function, and give additional insight into the cross-talk mechanisms underlying ERα signaling with ERβ and with DNA methylation pathways

Genetic Susceptibility on CagA-Interacting Molecules and Gene-Environment Interaction with Phytoestrogens: A Putative Risk Factor for Gastric Cancer

OBJECTIVES: To evaluate whether genes that encode CagA-interacting molecules (SRC, PTPN11, CRK, CRKL, CSK, c-MET and GRB2) are associated with gastric cancer risk and whether an interaction between these genes and phytoestrogens modify gastric cancer risk. METHODS: In the discovery phase, 137 candidate SNPs in seven genes were analyzed in 76 incident gastric cancer cases and 322 matched controls from the Korean Multi-Center Cancer Cohort. Five significant SNPs in three genes (SRC, c-MET and CRK) were re-evaluated in 386 cases and 348 controls in the extension phase. Odds ratios (ORs) for gastric cancer risk were estimated adjusted for age, smoking, H. pylori seropositivity and CagA strain positivity. Summarized ORs in the total study population (462 cases and 670 controls) were presented using pooled- and meta-analysis. Plasma concentrations of phytoestrogens (genistein, daidzein, equol and enterolactone) were measured using the time-resolved fluoroimmunoassay. RESULTS: SRC rs6122566, rs6124914, c-MET rs41739, and CRK rs7208768 showed significant genetic effects for gastric cancer in both the pooled and meta-analysis without heterogeneity (pooled OR = 3.96 [95% CI 2.05-7.65], 1.24 [95% CI = 1.01-1.53], 1.19 [95% CI = 1.01-1.41], and 1.37 [95% CI = 1.15-1.62], respectively; meta OR = 4.59 [95% CI 2.74-7.70], 1.36 [95% CI = 1.09-1.70], 1.20 [95% CI = 1.00-1.44], and 1.32 [95% CI = 1.10-1.57], respectively). Risk allele of CRK rs7208768 had a significantly increased risk for gastric cancer at low phytoestrogen levels (p interaction<0.05). CONCLUSIONS: Our findings suggest that SRC, c-MET and CRK play a key role in gastric carcinogenesis by modulating CagA signal transductions and interaction between CRK gene and phytoestrogens modify gastric cancer risk

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations

A Systematic Screen for Micro-RNAs Regulating the Canonical Wnt Pathway

MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of â-catenin (β-cat), miR-1 and miR-613 act upstream of β-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive β-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer