Oestrogen deprivation induces chemokine production and immune cell recruitment in in vitro and in vivo models of oestrogen receptor-positive breast cancer.

BACKGROUND: Oestrogen receptor-positive (ER+) breast cancer is commonly treated using endocrine therapies such as aromatase inhibitors which block synthesis of oestradiol, but the influence of this therapy on the immune composition of breast tumours has not been fully explored. Previous findings suggest that tumour infiltrating lymphocytes and immune-related gene expression may be altered by treatment with aromatase inhibitors. However, whether these changes are a direct result of impacts on the host immune system or mediated through tumour cells is not known. We aimed to investigate the effect of oestrogen deprivation on the expression of chemokines and immune infiltration in vitro and in an ER+ immunocompetent mouse model. METHODS: RT-qPCR and a bead-based Bioplex system were used to investigate the expression of chemokines in MCF-7 breast cancer cells deprived of oestrogen. A migration assay and flow cytometry were used to measure the migration of human peripheral blood mononuclear cells (PBMCs) to MCF-7 cells grown without the main biologically active oestrogen, oestradiol. Using flow cytometry and immunohistochemistry, we examined the immune cell infiltrate into tumours created by injecting SSM3 ER+ breast cancer cells into wild-type, immunocompetent 129/SvEv mice. RESULTS: This study demonstrates that oestrogen deprivation increases breast cancer secretion of TNF, CCL5, IL-6, IL-8, and CCL22 and alters total human peripheral blood mononuclear cell migration in an in vitro assay. Oestrogen deprivation of breast cancer cells increases migration of CD4+ T cells and decreases migration of CD11c+ and CD14+ PBMC towards cancer cells. PBMC migration towards breast cancer cells can be reduced by treatment with the non-steroidal anti-inflammatory drugs, aspirin and celecoxib. Treatment with endocrine therapy using the aromatase inhibitor letrozole increases CD4+ T cell infiltration into ER+ breast cancer tumours in immune competent mice. CONCLUSIONS: These results suggest that anti-oestrogen treatment of ER+ breast cancer cells can alter cytokine production and immune cells in the area surrounding the cancer cells. These findings may have implications for the combination and timing of anti-oestrogen therapies with other therapies.

Increased gene expression variability in BRCA1-associated and basal-like breast tumours.

PURPOSE: Inherited variants in the cancer susceptibility genes, BRCA1 and BRCA2 account for up to 5% of breast cancers. Multiple gene expression studies have analysed gene expression patterns that maybe associated with BRCA12 pathogenic variant status; however, results from these studies lack consensus. These studies have focused on the differences in population means to identified genes associated with BRCA1/2-carriers with little consideration for gene expression variability, which is also under genetic control and is a feature of cellular function. METHODS: We measured differential gene expression variability in three of the largest familial breast cancer datasets and a 2116 breast cancer meta-cohort. Additionally, we used RNA in situ hybridisation to confirm expression variability of EN1 in an independent cohort of more than 500 breast tumours. RESULTS: BRCA1-associated breast tumours exhibited a 22.8% (95% CI 22.3-23.2) increase in transcriptome-wide gene expression variability compared to BRCAx tumours. Additionally, 40 genes were associated with BRCA1-related breast cancers that had ChIP-seq data suggestive of enriched EZH2 binding. Of these, two genes (EN1 and IGF2BP3) were significantly variable in both BRCA1-associated and basal-like breast tumours. RNA in situ analysis of EN1 supported a significant (p = 6.3 × 10-04) increase in expression variability in BRCA1-associated breast tumours. CONCLUSION: Our novel results describe a state of increased gene expression variability in BRCA1-related and basal-like breast tumours. Furthermore, genes with increased variability may be driven by changes in DNA occupancy of epigenetic effectors. The variation in gene expression is replicable and led to the identification of novel associations between genes and disease phenotypes.

Fatty acid oxidation is associated with proliferation and prognosis in breast and other cancers.

BACKGROUND: Altered cellular metabolism is a hallmark of cancer but the association between utilisation of particular metabolic pathways in tumours and patient outcome is poorly understood. We sought to investigate the association between fatty acid metabolism and outcome in breast and other cancers. METHODS: Cox regression analysis and Gene Set Enrichment Analysis (GSEA) of a gene expression dataset from primary breast tumours with well annotated clinical and survival information was used to identify genesets associated with outcome. A geneset representing fatty acid oxidation (FAO) was then examined in other datasets. A doxycycline-inducible breast cancer cell line model overexpressing the rate-limiting enzyme in FAO, carnitine palmitoyl transferase 1A (CPT1A) was generated and analysed to confirm the association between FAO and cancer-associated characteristics in vitro. RESULTS: We identified a gene expression signature composed of 19 genes associated with fatty acid oxidation (FAO) that was significantly associated with patient outcome. We validated this observation in eight independent breast cancer datasets, and also observed the FAO signature to be prognostic in other cancer types. Furthermore, the FAO signature expression was significantly downregulated in tumours, compared to normal tissues from a variety of anatomic origins. In breast cancer, the expression of CPT1A was higher in oestrogen receptor (ER)-positive, compared to ER-negative tumours and cell lines. Importantly, overexpression of CPT1A significantly decreased the proliferation and wound healing migration rates of MDA-MB231 breast cancer cells, compared to basal expression control. CONCLUSIONS: Our findings suggest that FAO is downregulated in multiple tumour types, and activation of this pathway may lower cancer cell proliferation, and is associated with improved outcomes in some cancers.

Integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer.

INTRODUCTION: Aromatase inhibitors (AIs) are a vital component of estrogen receptor positive (ER+) breast cancer treatment. De novo and acquired resistance, however, is common. The aims of this study were to relate patterns of copy number aberrations to molecular and proliferative response to AIs, to study differences in the patterns of copy number aberrations between breast cancer samples pre- and post-AI neoadjuvant therapy, and to identify putative biomarkers for resistance to neoadjuvant AI therapy using an integrative analysis approach. METHODS: Samples from 84 patients derived from two neoadjuvant AI therapy trials were subjected to copy number profiling by microarray-based comparative genomic hybridisation (aCGH, n=84), gene expression profiling (n=47), matched pre- and post-AI aCGH (n=19 pairs) and Ki67-based AI-response analysis (n=39). RESULTS: Integrative analysis of these datasets identified a set of nine genes that, when amplified, were associated with a poor response to AIs, and were significantly overexpressed when amplified, including CHKA, LRP5 and SAPS3. Functional validation in vitro, using cell lines with and without amplification of these genes (SUM44, MDA-MB134-VI, T47D and MCF7) and a model of acquired AI-resistance (MCF7-LTED) identified CHKA as a gene that when amplified modulates estrogen receptor (ER)-driven proliferation, ER/estrogen response element (ERE) transactivation, expression of ER-regulated genes and phosphorylation of V-AKT murine thymoma viral oncogene homolog 1 (AKT1). CONCLUSIONS: These data provide a rationale for investigation of the role of CHKA in further models of de novo and acquired resistance to AIs, and provide proof of concept that integrative genomic analyses can identify biologically relevant modulators of AI response.

Identification of chemokine receptors as potential modulators of endocrine resistance in oestrogen receptor-positive breast cancers.

INTRODUCTION: Endocrine therapies target oestrogenic stimulation of breast cancer (BC) growth, but resistance remains problematic. Our aims in this study were (1) to identify genes most strongly associated with resistance to endocrine therapy by intersecting global gene transcription data from patients treated presurgically with the aromatase inhibitor anastrazole with those from MCF7 cells adapted to long-term oestrogen deprivation (LTED) (2) to assess the clinical value of selected genes in public clinical data sets and (3) to determine the impact of targeting these genes with novel agents. METHODS: Gene expression and Ki67 data were available from 69 postmenopausal women with oestrogen receptor-positive (ER+) early BC, at baseline and 2 weeks after anastrazole treatment, and from cell lines adapted to LTED. The functional consequences of target genes on proliferation, ER-mediated transcription and downstream cell signalling were assessed. RESULTS: By intersecting genes predictive of a poor change in Ki67 with those upregulated in LTED cells, we identified 32 genes strongly correlated with poor antiproliferative response that were associated with inflammation and/or immunity. In a panel of LTED cell lines, C-X-C chemokine receptor type 7 (CXCR7) and CXCR4 were upregulated compared to their wild types (wt), and CXCR7, but not CXCR4, was associated with reduced relapse-free survival in patients with ER+ BC. The CXCR4 small interfering RNA variant (siCXCR4) had no specific effect on the proliferation of wt-SUM44, wt-MCF7 and their LTED derivatives. In contrast, siCXCR7, as well as CCX733, a CXCR7 antagonist, specifically suppressed the proliferation of MCF7-LTED cells. siCXCR7 suppressed proteins associated with G1/S transition and inhibited ER transactivation in MCF7-LTED, but not wt-MCF7, by impeding association between ER and proline-, glutamic acid- and leucine-rich protein 1, an ER coactivator. CONCLUSIONS: These data highlight CXCR7 as a potential therapeutic target warranting clinical investigation in endocrine-resistant BC.

ESR1 is co-expressed with closely adjacent uncharacterised genes spanning a breast cancer susceptibility locus at 6q25.1.

Approximately 80% of human breast carcinomas present as oestrogen receptor α-positive (ER+ve) disease, and ER status is a critical factor in treatment decision-making. Recently, single nucleotide polymorphisms (SNPs) in the region immediately upstream of the ER gene (ESR1) on 6q25.1 have been associated with breast cancer risk. Our investigation of factors associated with the level of expression of ESR1 in ER+ve tumours has revealed unexpected associations between genes in this region and ESR1 expression that are important to consider in studies of the genetic causes of breast cancer risk. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with an aromatase (oestrogen synthase) inhibitor was analyzed on Illumina 48K microarrays. Multiple-testing corrected Spearman correlation revealed that three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1, C6ORF96, C6ORF97, and C6ORF211 were highly correlated with ESR1 (Rs =  0.67, 0.64, and 0.55 respectively, FDR<1 × 10(-7)). Publicly available datasets confirmed this relationship in other groups of ER+ve tumours. DNA copy number changes did not account for the correlations. The correlations were maintained in cultured cells. An ERα antagonist did not affect the ORFs' expression or their correlation with ESR1, suggesting their transcriptional co-activation is not directly mediated by ERα. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells, and C6ORF211 positively correlated with a proliferation metagene in tumours. In contrast, C6ORF97 expression correlated negatively with the metagene and predicted for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1. Our observations suggest that some of the biological effects previously attributed to ER could be mediated and/or modified by these co-expressed genes. The co-expression and function of these genes may be important influences on the recently identified relationship between SNPs in this region and breast cancer risk.

Prediction of late distant recurrence in patients with oestrogen-receptor-positive breast cancer: a prospective comparison of the breast-cancer index (BCI) assay, 21-gene recurrence score, and IHC4 in the TransATAC study population.

BACKGROUND: Biomarkers to improve the risk-benefit of extended adjuvant endocrine therapy for late recurrence in patients with oestrogen-receptor-positive breast cancer would be clinically valuable. We compared the prognostic ability of the breast-cancer index (BCI) assay, 21-gene recurrence score (Oncotype DX), and an immunohistochemical prognostic model (IHC4) for both early and late recurrence in patients with oestrogen-receptor-positive, node-negative (N0) disease who took part in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) clinical trial. METHODS: In this prospective comparison study, we obtained archival tumour blocks from the TransATAC tissue bank from all postmenopausal patients with oestrogen-receptor-positive breast cancer from whom the 21-gene recurrence score and IHC4 values had already been derived. We did BCI analysis in matched samples with sufficient residual RNA using two BCI models-cubic (BCI-C) and linear (BCI-L)-using previously validated cutoffs. We assessed prognostic ability of BCI for distant recurrence over 10 years (the primary endpoint) and compared it with that of the 21-gene recurrence score and IHC4. We also tested the ability of the assays to predict early (0-5 years) and late (5-10 years) distant recurrence. To assess the ability of the biomarkers to predict recurrence beyond standard clinicopathological variables, we calculated the change in the likelihood-ratio χ(2) (LR-Δχ(2)) from Cox proportional hazards models. FINDINGS: Suitable tissue was available from 665 patients with oestrogen-receptor-positive, N0 breast cancer for BCI analysis. The primary analysis showed significant differences in risk of distant recurrence over 10 years in the categorical BCI-C risk groups (p<0·0001) with 6·8% (95% CI 4·4-10·0) of patients in the low-risk group, 17·3% (12·0-24·7) in the intermediate group, and 22·2% (15·3-31·5) in the high-risk group having distant recurrence. The secondary analysis showed that BCI-L was a much stronger predictor for overall (0-10 year) distant recurrence compared with BCI-C (interquartile HR 2·30 [95% CI 1·62-3·27]; LR-Δχ(2)=22·69; p<0·0001). When compared with BCI-L, the 21-gene recurrence score was less predictive (HR 1·48 [95% CI 1·22-1·78]; LR-Δχ(2)=13·68; p=0·0002) and IHC4 was similar (HR 1·69 [95% CI 1·51-2·56]; LR-Δχ(2)=22·83; p<0·0001). All further analyses were done with the BCI-L model. In a multivariable analysis, all assays had significant prognostic ability for early distant recurrence (BCI-L HR 2·77 [95% CI 1·63-4·70], LR-Δχ(2)=15·42, p<0·0001; 21-gene recurrence score HR 1·80 [1·42-2·29], LR-Δχ(2)=18·48, p<0·0001; IHC4 HR 2·90 [2·01-4·18], LR-Δχ(2)=29·14, p<0·0001); however, only BCI-L was significant for late distant recurrence (BCI-L HR 1·95 [95% CI 1·22-3·14], LR-Δχ(2)=7·97, p=0·0048; 21-gene recurrence score HR 1·13 [0·82-1·56], LR-Δχ(2)=0·48, p=0·47; IHC4 HR 1·30 [0·88-1·94], LR-Δχ(2)=1·59, p=0·20). INTERPRETATION: BCI-L was the only significant prognostic test for risk of both early and late distant recurrence and identified two risk populations for each timeframe. It could help to identify patients at high risk for late distant recurrence who might benefit from extended endocrine or other therapy. FUNDING: Avon Foundation, National Institutes of Health, Breast Cancer Foundation, US Department of Defense Breast Cancer Research Program, Susan G Komen for the Cure, Breakthrough Breast Cancer through the Mary-Jean Mitchell Green Foundation, AstraZeneca, Cancer Research UK, and the National Institute for Health Research Biomedical Research Centre at the Royal Marsden (London, UK).

Restriction site associated DNA sequencing for tumour mutation burden estimation and mutation signature analysis.

BACKGROUND: Genome-wide measures of genetic disruption such as tumour mutation burden (TMB) and mutation signatures are emerging as useful biomarkers to stratify patients for treatment. Clinicians commonly use cancer gene panels for tumour mutation burden estimation, and whole genome sequencing is the gold standard for mutation signature analysis. However, the accuracy and cost associated with these assays limits their utility at scale. METHODS: WGS data from 560 breast cancer patients was used for in silico library simulations to evaluate the accuracy of an FDA approved cancer gene panel as well as restriction enzyme associated DNA sequencing (RADseq) libraries for TMB estimation and mutation signature analysis. We also transfected a mouse mammary cell line with APOBEC enzymes and sequenced resulting clones to evaluate the efficacy of RADseq in an experimental setting. RESULTS: RADseq had improved accuracy of TMB estimation and derivation of mutation profiles when compared to the FDA approved cancer panel. Using simulated immune checkpoint blockade (ICB) trials, we show that inaccurate TMB estimation leads to a reduction in power for deriving an optimal TMB cutoff to stratify patients for immune checkpoint blockade treatment. Additionally, prioritisation of APOBEC hypermutated tumours in these trials optimises TMB cutoff determination for breast cancer. The utility of RADseq in an experimental setting was also demonstrated, based on characterisation of an APOBEC mutation signature in an APOBEC3A transfected mouse cell line. CONCLUSION: In conclusion, our work demonstrates that RADseq has the potential to be used as a cost-effective, accurate solution for TMB estimation and mutation signature analysis by both clinicians and basic researchers.

Preclinical and clinical studies of estrogen deprivation support the PDGF/Abl pathway as a novel therapeutic target for overcoming endocrine resistance in breast cancer.

INTRODUCTION: The majority of breast tumors at primary diagnosis are estrogen receptor positive (ER+). Estrogen (E) mediates its effects by binding to the ER. Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ breast cancer. However, resistance remains a major clinical problem. METHODS: To identify molecular mechanisms associated with resistance to E-deprivation, we assessed the temporal changes in global gene expression during adaptation to long-term culture of MCF7 human breast cancer cells in the absence of estradiol (E2), long term estrogen deprived (LTED), that leads to recovery of proliferative status and models resistance to an aromatase inhibitor (AI). The expression levels of proteins were determined by western blotting. Proliferation assays were carried out using the dual platelet derived growth factor receptor (PDGFR)/Abelson tyrosine kinase (Abl) inhibitor nilotinib. Luciferase reporter assays were used to determine effects on ER-mediated transactivation. Changes in recruitment of cofactors to the gene regulated by estrogen in breast cancer 1 (GREB1) promoter were determined by chromatin immunoprecipitation (ChIP). Gene expression data were derived from 81 postmenopausal women with ER+ BC pre-treatment and at two-weeks post-treatment with single agent anastrozole in a neoadjuvant trial. RESULTS: The PDGF/Abl canonical pathway was significantly elevated as early as one week post E-deprivation (P = 1.94 E-04) and this became the top adaptive pathway at the point of proliferative recovery (P = 1.15 E-07). Both PDGFRß and Abl protein levels were elevated in the LTED cells compared to wild type (wt)-MCF7 cells. The PDGF/Abl tyrosine kinase inhibitor nilotinib, suppressed proliferation in LTED cells in the presence or absence of E. Nilotinib also suppressed ER-mediated transcription by destabilizing the ER and reducing recruitment of amplified in breast cancer-1 (AIB1) and the CREB binding protein (CBP) to the promoter of the E-responsive gene GREB1. High PDGFRß in primary ER+ breast cancer of 81 patients prior to neoadjuvant treatment with an AI was associated with poorer antiproliferative response. Additionally PDGFRß expression increased after two weeks of AI therapy (1.25 fold, P = 0.003). CONCLUSIONS: These preclinical and clinical data indicate that the PDGF/Abl signaling pathway merits clinical evaluation as a therapeutic target with endocrine therapy in ER+ breast cancer.

Structure vs. Function of TRIB1-Myeloid Neoplasms and Beyond.

The Tribbles family of proteins-comprising TRIB1, TRIB2, TRIB3 and more distantly related STK40-play important, but distinct, roles in differentiation, development and oncogenesis. Of the four Tribbles proteins, TRIB1 has been most well characterised structurally and plays roles in diverse cancer types. The most well-understood role of TRIB1 is in acute myeloid leukaemia, where it can regulate C/EBP transcription factors and kinase pathways. Structure-function studies have uncovered conformational switching of TRIB1 from an inactive to an active state when it binds to C/EBPα. This conformational switching is centred on the active site of TRIB1, which appears to be accessible to small-molecule inhibitors in spite of its inability to bind ATP. Beyond myeloid neoplasms, TRIB1 plays diverse roles in signalling pathways with well-established roles in tumour progression. Thus, TRIB1 can affect both development and chemoresistance in leukaemia; glioma; and breast, lung and prostate cancers. The pervasive roles of TRIB1 and other Tribbles proteins across breast, prostate, lung and other cancer types, combined with small-molecule susceptibility shown by mechanistic studies, suggests an exciting potential for Tribbles as direct targets of small molecules or biomarkers to predict treatment response.

Variable expression quantitative trait loci analysis of breast cancer risk variants.

Genome wide association studies (GWAS) have identified more than 180 variants associated with breast cancer risk, however the underlying functional mechanisms and biological pathways which confer disease susceptibility remain largely unknown. As gene expression traits are under genetic regulation we hypothesise that differences in gene expression variability may identify causal breast cancer susceptibility genes. We performed variable expression quantitative trait loci (veQTL) analysis using tissue-specific expression data from the Genotype-Tissue Expression (GTEx) Common Fund Project. veQTL analysis identified 70 associations (p < 5 × 10-8) consisting of 60 genes and 27 breast cancer risk variants, including 55 veQTL that were observed in breast tissue only. Pathway analysis of genes associated with breast-specific veQTL revealed an enrichment of four genes (CYP11B1, CYP17A1 HSD3B2 and STAR) involved in the C21-steroidal biosynthesis pathway that converts cholesterol to breast-related hormones (e.g. oestrogen). Each of these four genes were significantly more variable in individuals homozygous for rs11075995 (A/A) breast cancer risk allele located in the FTO gene, which encodes an RNA demethylase. The A/A allele was also found associated with reduced expression of FTO, suggesting an epi-transcriptomic mechanism may underlie the dysregulation of genes involved in hormonal biosynthesis leading to an increased risk of breast cancer. These findings provide evidence that genetic variants govern high levels of expression variance in breast tissue, thus building a more comprehensive insight into the underlying biology of breast cancer risk loci.

Emerging biomarkers and new understanding of traditional markers in personalized therapy for breast cancer.

The era of personalized medicine is likely to see an escalation in the use of biomarkers to ensure breast cancer patients receive optimal treatment. A combination of prognostic and predictive biomarkers should enable better quantification of the residual risk faced by patients and indicate the potential value of additional treatment. Established biomarkers such as estrogen receptor and progesterone receptor already play a significant role in the selection of patients for endocrine therapy. Human epidermal growth factor receptor 2 (HER2) is recognized as a strong predictor of response to trastuzumab whereas, more recently, the role of estrogen receptor and HER2 as negative and positive indicators for chemotherapy has also been explored. Ki67 has traditionally been recognized as a modest prognostic factor, but recent neoadjuvant studies suggest that on-treatment measurement may be a more effective predictor of treatment efficacy for both endocrine treatment and chemotherapy. The last decade has seen the emergence of numerous multigene expression profiles that aim to outdo traditional predictive and prognostic factors. The Oncotype DX assay and the MammaPrint profile are currently undergoing prospective clinical trials to clearly define their role. Other gene expression-based assays also show potential but are yet to be tested clinically. Rigorous comparison of these emerging markers with current treatment selection criteria will be required to determine whether they offer significant benefit to justify their use.

Destabilized adhesion in the gastric proliferative zone and c-Src kinase activation mark the development of early diffuse gastric cancer.

The initial development of diffuse gastric cancer (DGC) is poorly understood. The study of E-cadherin (CDH1) germ line mutation carriers predisposed to DGC provides a rare opportunity to elucidate the genetic and biological events surrounding disease initiation. Samples from various stages of hereditary and sporadic DGC were investigated to determine general mechanisms underlying early DGC development. Paraffin-embedded tissues from 13 CDH1 mutation carriers and from 10 sporadic early DGC cases were analyzed. Immunofluorescence and immunohistochemistry using differentiation, proliferation, and adhesion markers showed that DGC initiation seems to occur at the proliferative zone (the upper neck) of the gastric epithelium and correlates with absent or reduced expression of junctional proteins (beta-actin, p120, Lin-7). Slow proliferation of neoplastic cells at the upper gastric neck leads to the formation of intramucosal signet-ring cell carcinoma (SRCC) displaying differentiated features. As shown by immunolabeling, invasion from SRCC lesions beyond the gastric mucosa is associated with poor differentiation, increased proliferation, activation of the c-Src system, and an epithelial-mesenchymal transition. Our results provide a molecular description of the early development of DGC and explain the relationship between the two main DGC types, poorly differentiated carcinoma and SRCC: both share their origin, but SRCC develops following cancer cell differentiation and seems relatively indolent in its intramucosal stage.

Impact of estrogen deprivation on gene expression profiles of normal postmenopausal breast tissue in vivo.

Aromatase inhibitors play a key role in the clinical management of hormone receptor-positive breast cancer and have potential utility as chemopreventive agents. Further understanding of the molecular effects of estrogen and its deprivation in normal breast tissue may allow the development of biomarkers of risk of breast cancer and help to predict the value of chemoprevention with aromatase inhibitors. Core biopsies of normal breast tissue were taken before and after letrozole treatment from postmenopausal women in the LITMaS pilot prevention study. RNA was extracted from these samples and used for cDNA microarray analysis. Gene expression changes induced by letrozole treatment were much less extensive than observed in estrogen receptor-positive malignant tissue; however, overall, they correlated to a highly significant degree (rho = 0.511; P < 10(-20)). As well as some classically estrogen-associated genes, many genes associated with extracellular matrix remodeling were affected by estrogen deprivation in the normal breast in vivo. These data indicate for the first time that gene expression of normal breast tissue remains dependent on endogenous estrogens after the menopause. The modest degree of gene change suggests that intermediate markers of chemoprevention may be difficult to identify.

A novel diffuse gastric cancer susceptibility variant in E-cadherin (CDH1) intron 2: a case control study in an Italian population.

BACKGROUND: Inherited genetic factors such as E-cadherin (CDH1) promoter variants are believed to influence the risk towards sporadic diffuse gastric cancer (DGC). Recently, a new regulatory region essential for CDH1 transcription has been identified in CDH1 intron 2. METHODS: We genotyped all known polymorphisms located within conserved sequences of CDH1 intron 2 (rs10673765, rs9932686, rs1125557, rs9282650, rs9931853) in an Italian population consisting of 134 DGC cases and 100 healthy controls (55 patient relatives and 45 unrelated, matched individuals). The influence of individual variants on DGC risk was assessed using chi2-tests and logistic regression. The relative contribution of alleles was estimated by haplotype analysis. RESULTS: We observed a significant (p < 0.0004) association of the CDH1 163+37235G>A variant (rs1125557) with DGC risk. Odds ratios were 4.55 (95%CI = 2.09-9.93) and 1.38 (95%CI = 0.75-2.55) for AA and GA carriers, respectively. When adjusted for age, sex, smoking status, alcohol intake and H. pylori infection, the risk estimates remained largely significant for AA carriers. Haplotype analysis suggested the 163+37235A-allele contributes to disease risk independently of the other variants studied. CONCLUSION: The CDH1 163+37235G>A polymorphism may represent a novel susceptibility variant for sporadic DGC if confirmed in other populations. Considering the broad expression of E-cadherin in epithelia, this exploratory study encourages further evaluation of the 163+37235A-allele as a susceptibility variant in other carcinomas.

Substrate binding allosterically relieves autoinhibition of the pseudokinase TRIB1.

The Tribbles family of pseudokinases recruits substrates to the ubiquitin ligase COP1 to facilitate ubiquitylation. CCAAT/enhancer-binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid cell development. We found that the TRIB1 pseudokinase was able to recruit various C/EBP family members and that the binding of C/EBPß was attenuated by phosphorylation. To explain the mechanism of C/EBP recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα, which revealed that TRIB1 underwent a substantial conformational change relative to its substrate-free structure and bound C/EBPα in a pseudosubstrate-like manner. Crystallographic analysis and molecular dynamics and subsequent biochemical assays showed that C/EBP binding triggered allosteric changes that link substrate recruitment to COP1 binding. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation-by means of the activation loop and αC helix-and raise the possibility of small molecules targeting either the activation "loop-in" or "loop-out" conformations of Tribbles pseudokinases.

A PAM50-Based Chemoendocrine Score for Hormone Receptor-Positive Breast Cancer with an Intermediate Risk of Relapse.

Purpose: Hormone receptor-positive (HR+) breast cancer is clinically and biologically heterogeneous, and subgroups with different prognostic and treatment sensitivities need to be identified.Experimental Design: Research-based PAM50 subtyping and expression of additional genes was performed on 63 patients with HR+/HER2- disease randomly assigned to neoadjuvant multiagent chemotherapy versus endocrine therapy in a phase II trial. The biology associated with treatment response was used to derive a PAM50-based chemoendocrine score (CES). CES's predictive ability was evaluated in 4 independent neoadjuvant data sets (n = 675) and 4 adjuvant data sets (n = 1,505). The association of CES, intrinsic biology, and PAM50 risk of relapse (ROR) was explored across 6,007 tumors.Results: Most genes associated with endocrine sensitivity were also found associated with chemotherapy resistance. In the chemotherapy test/validation data sets, CES was independently associated with pathologic complete response (pCR), even after adjusting for intrinsic subtype. pCR rates of the CES endocrine-sensitive (CES-E), uncertain (CES-U), and chemotherapy-sensitive (CES-C) groups in both data sets combined were 25%, 11%, and 2%, respectively. In the endocrine test/validation data sets, CES was independently associated with response. Compared with ROR, >90% of ROR-low and ROR-high tumors were identified as CES-E and CES-C, respectively; however, each CES group represented >25% of ROR-intermediate disease. In terms of survival outcome, CES-C was associated with poor relapse-free survival in patients with ROR-intermediate disease treated with either adjuvant endocrine therapy only or no adjuvant systemic therapy, but not in patients treated with (neo)adjuvant chemotherapy.Conclusions: CES is a genomic signature capable of estimating chemoendocrine sensitivity in HR+ breast cancer beyond intrinsic subtype and risk of relapse. Clin Cancer Res; 23(12); 3035-44. ©2016 AACR.

Accurate Prediction and Validation of Response to Endocrine Therapy in Breast Cancer.

PURPOSE: Aromatase inhibitors (AIs) have an established role in the treatment of breast cancer. Response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Accurate biomarkers are urgently needed to predict response in these settings and to determine which individuals will benefit from adjuvant AI therapy. PATIENTS AND METHODS: Pretreatment and on-treatment (after 2 weeks and 3 months) biopsies were obtained from 89 postmenopausal women who had estrogen receptor-alpha positive breast cancer and were receiving neoadjuvant letrozole for transcript profiling. Dynamic clinical response was assessed with use of three-dimensional ultrasound measurements. RESULTS: The molecular response to letrozole was characterized and a four-gene classifier of clinical response was established (accuracy of 96%) on the basis of the level of two genes before treatment (one gene [IL6ST] was associated with immune signaling, and the other [NGFRAP1] was associated with apoptosis) and the level of two proliferation genes (ASPM, MCM4) after 2 weeks of therapy. The four-gene signature was found to be 91% accurate in a blinded, completely independent validation data set of patients treated with anastrozole. Matched 2-week on-treatment biopsies were associated with improved predictive power as compared with pretreatment biopsies alone. This signature also significantly predicted recurrence-free survival (P = .029) and breast cancer -specific survival (P = .009). We demonstrate that the test can also be performed with use of quantitative polymerase chain reaction or immunohistochemistry. CONCLUSION: A four-gene predictive model of clinical response to AIs by 2 weeks has been generated and validated. Deregulated immune and apoptotic responses before treatment and cell proliferation that is not reduced 2 weeks after initiation of treatment are functional characteristics of breast tumors that do not respond to AIs.

Effect of aromatase inhibition on functional gene modules in estrogen receptor-positive breast cancer and their relationship with antiproliferative response.

PURPOSE: To investigate potential associations between gene modules representing key biologic processes and response to aromatase inhibitors (AI) in estrogen receptor-positive (ER(+)) breast cancer. PATIENTS AND METHODS: Paired gene expression and Ki67 protein expression were available from 69 postmenopausal women with ER(+) early breast cancer, at baseline and 2 weeks post-anastrozole treatment, in the presurgical setting. Functional gene modules (n = 26) were retrieved from published studies and their module scores were computed before and after elimination of proliferation-associated genes (PAG). Ki67 and module scores were assessed at baseline and 2 weeks post-anastrozole. Unsupervised clustering was used to assess associations between modules and Ki67. RESULTS: Proliferation-based modules were highly correlated with Ki67 expression both pretreatment and on-treatment. At baseline with and without PAGs, Ki67 expression was significantly inversely correlated with ERG, ESR1.2, SET, and PIK3CA modules. Modules measuring estrogen signaling strongly predicted antiproliferative response to therapy with and without PAGs. Baseline expression of insulin-like growth factor-1 (IGF-I) module predicted a poor change in Ki67-implicating genes within the module as involved in de novo resistance to AIs. High expression of Immune.2.STAT1 module pretreatment predicted poor antiproliferative response to therapy. A significant association between estrogen-regulated genes modules (ESR1, ESR1-2, SET, and ERG) was evident post AI. CONCLUSIONS: Multiple processes and pathways are affected by AI treatment in ER(+) breast cancer. Modules closely associated with ESR1 expression were predictive of good antiproliferative response to AIs, but modules representing immune activity and IGF-I/MAPK were predictive of poor Ki67 response, supporting their therapeutic targeting in combination with AIs.

Differences in the transcriptional response to fulvestrant and estrogen deprivation in ER-positive breast cancer.

PURPOSE: Endocrine therapies include aromatase inhibitors and the selective estrogen receptor (ER) downregulator fulvestrant. This study aimed to determine whether the reported efficacy of fulvestrant over anastrozole, and high- over low-dose fulvestrant, reflect distinct transcriptional responses. EXPERIMENTAL DESIGN: Global gene expression profiles from ERα-positive breast carcinomas before and during presurgical treatment with fulvestrant (n = 22) or anastrozole (n = 81), and corresponding in vitro models, were compared. Transcripts responding differently to fulvestrant and estrogen deprivation were identified and integrated using Gene Ontology, pathway and network analyses to evaluate their potential significance. RESULTS: The overall transcriptional response to fulvestrant and estrogen deprivation was correlated (r = 0.61 in presurgical studies, r = 0.87 in vitro), involving downregulation of estrogen-regulated and proliferation-associated genes. The transcriptional response to fulvestrant was of greater magnitude than estrogen deprivation (slope = 0.62 in presurgical studies, slope = 0.63 in vitro). Comparative analyses identified 28 genes and 40 Gene Ontology categories affected differentially by fulvestrant. Seventeen fulvestrant-specific genes, including CAV1/2, SNAI2, and NRP1, associated with ERα, androgen receptor (AR), and TP53, in a network regulating cell cycle, death, survival, and tumor morphology. Eighteen genes responding differently to fulvestrant specifically predicted antiproliferative response to fulvestrant, but not anastrozole. Transcriptional effects of low-dose fulvestrant correlated with high-dose treatment, but were of lower magnitude (ratio = 0.29). CONCLUSIONS: The transcriptional response to fulvestrant has much in common with estrogen deprivation, but is stronger with distinctions potentially attributable to arrest of estrogen-independent ERα activity and involvement of AR signaling. Genes responding differently to fulvestrant may have predictive utility. These data are consistent with the clinical efficacy of fulvestrant versus anastrozole and higher dosing regimens.