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Table S1. Cell line authentication. Cell line DNA profiling was carried out using ATCC MCF7 DNA profile as reference. Table S2: List of ChIP and mRNA primers used for DNA and mRNA quantification on the Lightcycler (Roche). Table S3: Patient information for endocrine treated patients. Table S4. Over represented TF binding motifs in 666 ER ChIPseq peaks from LetR cells (using the Universe as background, q-value

Purpose: Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumor cells to evade therapy and colonize distant organs remain unclear. We sought to characterize global expression changes occurring with metastatic disease progression in the endocrine-resistant setting.Experimental Design: Here, for the first time, RNAsequencing has been performed on matched primary, nodal, and liver metastatic tumors from tamoxifen-treated patients following disease progression. Expression of genes commonly elevated in the metastases of sequenced patients was subsequently examined in an extended matched patient cohort with metastatic disease from multiple sites. The impact of tamoxifen treatment on endocrine-resistant tumors in vivo was investigated in a xenograft model.Results: The extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumors were common to all patients. Larger divergence was observed between primary and liver tumors than between primary and nodal tumors, reflecting both the latency to disease progression and the genetic impact of intervening therapy. Furthermore, an endocrine-resistant in vivo mouse model demonstrated that tamoxifen treatment has the potential to drive disease progression and establish distant metastatic disease. Common functional pathways altered during metastatic, endocrine-resistant progression included extracellular matrix receptor interactions and focal adhesions.Conclusions: This novel global analysis highlights the influence of primary tumor biology in determining the transcriptomic profile of metastatic tumors, as well as the need for adaptations in cell–cell communications to facilitate successful tumor cell colonization of distant host organs. Clin Cancer Res; 21(23); 5371–9. ©2015 AACR.

Purpose: Acquired resistance to aromatase inhibitor (AI) therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumor cells develop this resistance remain unclear. Here, the adapted function of estrogen receptor (ER) to an estrogen-depleted environment following AI treatment is reported.Experimental Design: Global ER chromatin immuno-precipitation (ChIP)-seq analysis of AI-resistant cells identified steroid-independent ER target genes. Matched patient tumor samples, collected before and after AI treatment, were used to assess ER activity.Results: Maintained ER activity was observed in patient tumors following neoadjuvant AI therapy. Genome-wide ER–DNA-binding analysis in AI-resistant cell lines identified a subset of classic ligand-dependent ER target genes that develop steroid independence. The Kaplan–Meier analysis revealed a significant association between tumors, which fail to decrease this steroid-independent ER target gene set in response to neoadjuvant AI therapy, and poor disease-free survival and overall survival (n = 72 matched patient tumor samples, P = 0.00339 and 0.00155, respectively). The adaptive ER response to AI treatment was highlighted by the ER/AIB1 target gene, early growth response 3 (EGR3). Elevated levels of EGR3 were detected in endocrine-resistant local disease recurrent patient tumors in comparison with matched primary tissue. However, evidence from distant metastatic tumors demonstrates that the ER signaling network may undergo further adaptations with disease progression as estrogen-independent ER target gene expression is routinely lost in established metastatic tumors.Conclusions: Overall, these data provide evidence of a dynamic ER response to endocrine treatment that may provide vital clues for overcoming the clinical issue of therapy resistance. Clin Cancer Res; 22(11); 2765–77. ©2016 AACR.

PDF file, 14KB, Supplementary Table 1: Demographics of patients according to AIB1 status.

PDF file, 320KB, Supplementary Figures 1-9 Supplementary Figure 1: Expression levels of aromatase in our cell lines and aromatase activity assay Supplementary Figure 2: Proliferation of Aro and LetR in response to treatment Supplementary Figure 3: Kaplan Meier estimates of disease free survival in the entire ER positive population Supplementary Figure 4:Interactions between ERalpha and AIB1 in letrozole sensitive Aro cells Supplementary Figure 5: Analysis of protein levels in Anastrozole resistant cell line (ANAR) Supplementary Figure 6: ChiP showing recruitment of ERalpha and AIB1 to distal region promoters Supplementary Figure 7: Basal protein levels of Myc and cyclinD1 protein and basal mRNA levels of pS2 Supplementary Figure 8:Effect of Lapatinib treatment on pS2 and Myc mRNA levels Supplementary Figure 9: Pathway analysis of ERalpha-AIB1 target genes.

Supplementary Table S4: Patient information for patients A-L

Purpose: The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy.Experimental Design: Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole.Results: Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH2-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter.Conclusion: This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype. Clin Cancer Res; 18(12); 3305–15. ©2012 AACR.

Supplementary Figures S1-7. Supplementary Figure S1: Gene expression profiles of classic biomarkers in matched tumour samples from 3 patients. Supplementary Figure S2: Network map showing the enriched KEGG pathways from upregulated differentially expressed genes (DEGs). Supplementary Figure S3: Network map showing the enriched KEGG pathways from downregulated differentially expressed genes (DEGs). Supplementary Figure S4: Protein expression in matched primary and metastatic tissue from endocrine treated breast cancer patients. Supplementary Figure S5: Phosphorylated mTOR signaling in endocrine resistant breast cancer tumours. Supplementary Figure S6: Interaction plot showing functional roles for 21 genes commonly upregulated in metastatic liver tumours of all three patients. Supplementary Figure S7: Genes upregulated in liver metastases are also strongly expressed in breast metastases to the brain.

Figure S1. Ligand independent ER drives growth of AI resistant cells. Figure S2. Confirmation of transient knockdown using siRNA. Figure S3: pSer118 ER expression significantly increases during neoadjuvant AI therapy. Figure S4: Androgen Receptor (AR) expression in patient tumours before and after endocrine therapy. Figure S5. Reduced ligand dependency is a feature of multiple endocrine resistant cells compared to endocrine sensitive MCF7 cells. Figure S6. FoxA1 expression and interactions with ER are comparable between MCF7 and LetR cells treated with either estrogen (E2) or androstenedione (A). Figure S7. Steroid-sensitive recruitment of ER to a selection of target genes in MCF7-Aro cells. q-PCR of ER ChIP in MCF7-Aro cells treated with vehicle (V) or steroid (S) for 45 minutes. IgG was used as an internal control. Bar charts show relative recruitment to each target gene with n=3. Heat map shows relative recruitment of ER to each target gene as a fold increase above IgG control. Figure S8. ER ChIPseq binding peak profiles in 7 genes of the ER target gene signature. ER binding activity is observed at the same location in both LY2 and LetR cells. Images were captured from UCSC Genome Browser. V, vehicle; E, estrogen; A, androstenedione. Figure S9. ER binding does not affect expression of PEBP4. Figure S10. A, EGR3 knockdown decreases cell growth in LetR cells. Figure S11: ER target gene signature is more accurate than ESR1 alone for predicting outcome on AI therapy.

PDF file, 55KB, Supplementary table 2: Rate of incidence of disease recurrence according to AIB1, HER2, tumour grade, tumour size and nodal status in breast cancer patients treated with first-line AI.

PDF file, 213KB, Supplementary Table 3b ER/AIB1 overlap genelist - partial ERE.

Supplementary Materials and Methods

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To prevent the development of endocrine-resistant breast cancer, additional targeted therapies are increasingly being trialled in combination with endocrine therapy. The molecular mechanisms facilitating cancer cell survival during endocrine treatment remain unknown but could help direct selection of additional targeted therapies. We present a novel proteomic timecourse dataset, profiling potential drug targets in a population of MCF7 cells during 1 year of tamoxifen treatment. Reverse phase protein arrays profiled >70 proteins across 30 timepoints. A biphasic response to tamoxifen was evident, which coincided with changes in growth rate. Tamoxifen strongly impeded cell growth for the first 160 days, followed by gradual growth recovery and eventual resistance development. The growth-impeded phase was distinguished by the phosphorylation of Stat3 (y705) and Src (y527). Tumour tissue from patients treated with neo-adjuvant endocrine therapy (

Women consistently show lower incidence and mortality rates for colorectal cancer (CRC) compared to men. Epidemiological evidence supports a pivotal role for estrogen in protecting women against CRC. Estrogen protective effects in CRC have been mainly attributed to the estrogen receptor beta (ERβ) however its expression is lost during CRC progression. The role of the G-protein coupled membrane estrogen receptor (GPER/GPER1/GPR30), which remains expressed after ERβ loss in CRC, is currently under debate. We hypothesise that estrogen can protect against CRC progression via GPER by modulating the Wnt/β-catenin proliferative pathway which is commonly hyperactivated in CRC. We sought evidence of sexual dimorphism within the Wnt/β-catenin pathway by conducting Kaplan–Meier analyses based on gene expression of the Wnt receptor FZD1 (Frizzled 1) in multiple public domain CRC patient data sets. High expression of FZD1 was associated with poor relapse-free survival rates in the male but not the female population. In female-derived HT29 CRC cell lines, we show that β-catenin nuclear translocation was not affected by treatment with the GPER agonist G1. However, G1 prevented the Wnt pathway-induced upregulation of the JUN oncogene. These novel findings indicate a mechanistic role for GPER in protecting against CRC progression by selectively reducing the tumorigenic effects of hyperactive Wnt/β-catenin signalling pathways in CRC.

// Viviana Bustos 1 , Aine M. Nolan 1 , Anke Nijhuis 2 , Harry Harvey 1 , Alexandra Parker 2 , Richard Poulsom 2 , Jean McBryan 1 , Warren Thomas 1 , Andrew Silver 2 and Brian J. Harvey 1 1 Department of Molecular Medicine, Education and Research Centre, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin, Ireland 2 Centre for Digestive Diseases, National Centre for Bowel Research and Surgical Innovation, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Correspondence to: Brian J. Harvey, email: brianharvey@rcsi.ie Keywords: estrogen, GPER, VEGF, hypoxia, colorectal cancer Received: March 29, 2017 Accepted: July 26, 2017 Published: September 06, 2017 ABSTRACT The estrogen receptor ERβ is the predominant ER subtype expressed in normal well-differentiated colonic epithelium. However, ERβ expression is lost under the hypoxic microenvironment as colorectal cancer (CRC) malignancy progresses. This raises questions about the role of signalling through other estrogen receptors such as ERα or G-protein coupled estrogen receptor (GPER, GPR30) by the estrogen 17β-estradiol (E2) under hypoxic conditions after ERβ is lost in CRC progression. We tested the hypothesis that E2 or hypoxia can act via GPER to contribute to the altered phenotype of CRC cells. GPER expression was found to be up-regulated by hypoxia and E2 in a panel of CRC cell lines. The E2-modulated gene, Ataxia telangiectasia mutated ( ATM ), was repressed in hypoxia via GPER signalling. E2 treatment enhanced hypoxia-induced expression of HIF1-α and VEGFA, but repressed HIF1-α and VEGFA expression under normoxic conditions. The expression and repression of VEGFA by E2 were mediated by a GPER-dependent mechanism. E2 treatment potentiated hypoxia-induced CRC cell migration and proliferation, whereas in normoxia, cell migration and proliferation were suppressed by E2 treatment. The effects of E2 on these cellular responses in normoxia and hypoxia were mediated by GPER. In a cohort of 566 CRC patient tumor samples, GPER expression significantly associated with poor survival in CRC Stages 3-4 females but not in the stage-matched male population. Our findings support a potentially pro-tumorigenic role for E2 in ERβ-negative CRC under hypoxic conditions transduced via GPER and suggest a novel route of therapeutic intervention through GPER antagonism.

Eosinophils account for 1–3% of peripheral blood leukocytes and accumulate at sites of allergic inflammation, where they play a pathogenic role. Studies have shown that treatment with mepolizumab (an anti-IL-5 monoclonal antibody) is beneficial to patients with severe eosinophilic asthma, however, the mechanism of precisely how eosinophils mediate these pathogenic effects is uncertain. Eosinophils contain several cationic granule proteins, including Eosinophil Peroxidase (EPO). The main significance of this work is the discovery of EPO as a novel ligand for the HER2 receptor. Following HER2 activation, EPO induces activation of FAK and subsequent activation of β1-integrin, via inside-out signaling. This complex results in downstream activation of ERK1/2 and a sustained up regulation of both MUC4 and the HER2 receptor. These data identify a receptor for one of the eosinophil granule proteins and demonstrate a potential explanation of the proliferative effects of eosinophils., Highlights • Eosinophil peroxidase (EPO) is confirmed as a ligand for the HER2 receptor. • EPO activation of HER2 leads to activation of FAK, ERK and β1 integrin. • EPO induces a sustained upregulation of MUC4 and HER2. • Possible mechanism for the proliferative effects of eosinophils is uncovered.

Purpose: Acquired resistance to aromatase inhibitor (AI) therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumor cells develop this resistance remain unclear. Here, the adapted function of estrogen receptor (ER) to an estrogen-depleted environment following AI treatment is reported. Experimental Design: Global ER chromatin immuno-precipitation (ChIP)-seq analysis of AI-resistant cells identified steroid-independent ER target genes. Matched patient tumor samples, collected before and after AI treatment, were used to assess ER activity. Results: Maintained ER activity was observed in patient tumors following neoadjuvant AI therapy. Genome-wide ER–DNA-binding analysis in AI-resistant cell lines identified a subset of classic ligand-dependent ER target genes that develop steroid independence. The Kaplan–Meier analysis revealed a significant association between tumors, which fail to decrease this steroid-independent ER target gene set in response to neoadjuvant AI therapy, and poor disease-free survival and overall survival (n = 72 matched patient tumor samples, P = 0.00339 and 0.00155, respectively). The adaptive ER response to AI treatment was highlighted by the ER/AIB1 target gene, early growth response 3 (EGR3). Elevated levels of EGR3 were detected in endocrine-resistant local disease recurrent patient tumors in comparison with matched primary tissue. However, evidence from distant metastatic tumors demonstrates that the ER signaling network may undergo further adaptations with disease progression as estrogen-independent ER target gene expression is routinely lost in established metastatic tumors. Conclusions: Overall, these data provide evidence of a dynamic ER response to endocrine treatment that may provide vital clues for overcoming the clinical issue of therapy resistance. Clin Cancer Res; 22(11); 2765–77. ©2016 AACR.

Purpose: Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumor cells to evade therapy and colonize distant organs remain unclear. We sought to characterize global expression changes occurring with metastatic disease progression in the endocrine-resistant setting. Experimental Design: Here, for the first time, RNAsequencing has been performed on matched primary, nodal, and liver metastatic tumors from tamoxifen-treated patients following disease progression. Expression of genes commonly elevated in the metastases of sequenced patients was subsequently examined in an extended matched patient cohort with metastatic disease from multiple sites. The impact of tamoxifen treatment on endocrine-resistant tumors in vivo was investigated in a xenograft model. Results: The extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumors were common to all patients. Larger divergence was observed between primary and liver tumors than between primary and nodal tumors, reflecting both the latency to disease progression and the genetic impact of intervening therapy. Furthermore, an endocrine-resistant in vivo mouse model demonstrated that tamoxifen treatment has the potential to drive disease progression and establish distant metastatic disease. Common functional pathways altered during metastatic, endocrine-resistant progression included extracellular matrix receptor interactions and focal adhesions. Conclusions: This novel global analysis highlights the influence of primary tumor biology in determining the transcriptomic profile of metastatic tumors, as well as the need for adaptations in cell–cell communications to facilitate successful tumor cell colonization of distant host organs. Clin Cancer Res; 21(23); 5371–9. ©2015 AACR.

Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumour cells to evade therapy and colonise distant organs remain unclear. Here, for the first time, RNAsequencing has been performed on matched primary, nodal and liver metastatic tumours from three tamoxifen-treated patients following metastatic disease progression. Despite all primary tumours being of a luminal subtype and all cancers metastasising to the liver, the extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumours were common to all patients. Larger divergence was observed between primary and liver tumours than between primary and nodal tumours, reflecting both the latency time to disease progression and the genetic impact of endocrine therapy. Furthermore, a xenograft model demonstrated the ability of tamoxifen to drive disease progression and establish distant metastatic disease in the endocrine resistant setting. Common functional pathways altered during metastatic, endocrine-resistant progression included ECM receptor interactions and focal adhesions. This novel global analysis highlights the influence of primary tumour biology in determining the transcriptomic profile of metastatic tumours, as well as the need for adaptations in cell-cell communications in order for tumour cells to successfully colonise distant host organs. Citation Format: Jean McBryan, Ailis Fagan, Damian McCartan, Jarlath C Bolger, Fiona T Bane, Christopher Byrne, Marie McIlroy, Arnold D Hill, Leonie S Young. Transcriptomic profiling of patient sequential tumours provides cutting edge view of global metastatic expression changes following endocrine therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-13.

The incidence of brain metastasis (BM) among women with metastatic breast cancer (MBC) is currently on the rise, and it is estimated that between 10-30% of patients diagnosed with breast cancer may also develop BM (Weil 2005). BM is considered a late complication in the clinical setting and is an infrequent occurrence even in mice studies that readily metastasise to other organs (Valiente 2014). While pre-menopausal status and negative estrogen receptor (ER) status are seen as main risk factors (Gil-Gil 2013), it is now recognised that BM is emerging as an increasing clinical problem across the various MBC subtypes. BM patients have very poor overall survival and as a result it is crucial to identify the factors that promote the survival and adaptation of breast cancer cells to allow them to colonise distant sites such as the brain. The ability to predict the BM potential could be of great clinical importance and in this study we decided to investigate the mechanisms underlying BM across the various MBC subtypes. To our surprise, in a cohort of breast cancer patients we found a comparable number of ER positive and ER negative BM. We established that ER positive patients (n=10) had a substantially longer disease latency period (median disease free survival (DFS) 36 months, range 8-85 months) in comparison to ER negative patients (n=8, HER2 positive, n=2, triple negative, n=6), (median DFS 24 months, range 9-60 months). To understand and elucidate the gene alterations required for successful colonisation of the brain we undertook RNAseq of sequential primary and brain metastatic tumours from ER positive (n=4) and ER negative (n=3) breast cancer patients. Significant elevations in gene expression were observed in the brain metastatic tumours in comparison to the matched primary. Though a large degree of patient heterogeneity was observed, common ER-specific metastatic pathways were identified. This study highlights the requirement of unique gene sets for the colonisation to the brain and that functional characterisation of the differentially expressed genes will enable the identification of novel molecular targets for prevention and treatment of breast cancer BM. Citation Format: Damir Vareslija, Ailis Fagan, Jean McBryan, Patrick Buckley, Michael Farrell, Arnold DK Hill, Leonie Young. Global analysis of the transcriptome in matched primary and metastatic tumours defines ER specific gene alterations [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-26.

The K+ channel KCNQ1 has been proposed as a tumor suppressor in colorectal cancer (CRC). We investigated the molecular mechanisms regulating KCNQ1:β-catenin bidirectional interactions and their effects on CRC differentiation, proliferation, and invasion. Molecular and pharmacologic approaches were used to determine the influence of KCNQ1 expression on the Wnt/β-catenin signaling and epithelial-to-mesenchymal transition (EMT) in human CRC cell lines of varying stages of differentiation. The expression of KCNQ1 was lost with increasing mesenchymal phenotype in poorly differentiated CRC cell lines as a consequence of repression of the KCNQ1 promoter by β-catenin:T-cell factor (TCF)-4. In well-differentiated epithelial CRC cell lines, KCNQ1 was localized to the plasma membrane in a complex with β-catenin and E-cadherin. The colocalization of KCNQ1 with adherens junction proteins was lost with increasing EMT phenotype. ShRNA knock-down of KCNQ1 caused a relocalization of β-catenin from the plasma membrane and a loss of epithelial phenotype in CRC spheroids. Overexpression of KCNQ1 trapped β-catenin at the plasma membrane, induced a patent lumen in CRC spheroids, and slowed CRC cell invasion. The KCNQ1 ion channel inhibitor chromanol 293B caused membrane depolarization, redistribution of β-catenin into the cytosol, and a reduced transepithelial electrical resistance, and stimulated CRC cell proliferation. Analysis of human primary CRC tumor patient databases showed a positive correlation between KCNQ1:KCNE3 channel complex expression and disease-free survival. We conclude that the KCNQ1 ion channel is a target gene and regulator of the Wnt/β-catenin pathway, and its repression leads to CRC cell proliferation, EMT, and tumorigenesis.

Acquired resistance to aromatase inhibitor therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumour cells develop this resistance remain unclear. Here, discovery of pro-survival adaptations to the estrogen receptor (ER) signalling pathway in response to AI treatment is reported. Global ChIPseq analysis shows that the ER retains binding activity in AI resistant cells under steroid-depleted conditions. In AI treated patients, evidence of steroid independent adaptive ER signalling was demonstrated by the ER target gene Early Growth Response 3 (EGR3). Expression of EGR3 decreased initially upon AI treatment but subsequently recovered following as little as 12 weeks of AI treatment. In vitro data indicates that this increased expression of EGR3 may enhance cell growth and motility of tumour cells. Finally, evidence from established metastatic tumours suggests that the ER signalling network may undergo further adaptations with disease progression as EGR3 expression is routinely lost in the established metastatic tumour. Overall, these data provide evidence of a dynamic ER response to AI treatment which may provide vital clues for overcoming the clinical issue of AI resistance. Citation Format: Damir Vareslija, Jean McBryan, Ailis Fagan, Aisling Redmond, Yuan Hao, Arnold DK Hill, Leonie S Young. Adaptation to AI therapy in breast cancer can induce dynamic alterations in ER activity resulting in estrogen independent metastatic tumours [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-24.

Treatment with tyrosine kinase inhibitors (TKIs) including trastuzumab has revolutionized the management of HER2-positive breast cancer. Recent evaluation of clinical trial data suggests that a subset of HER2/ER double-positive cancers may not receive significant benefit from the TKI therapy. Here we investigate the cross talk between HER2 and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc. In HER2-positive breast cancer patients treated with neoadjuvant trastuzumab, steroid receptor-negative status (ER and PR negative) of pre-treatment biopsies predicted pathological complete response (pCR) (n=31 patients, P=0.0486), whereas elevated Myc protein inversely associated with pCR (P=0.0446). Liquid chromatography mass spectrometry identified the corepressor SMRT as a novel Myc-interacting protein. Trastuzumab treatment enhanced Myc-SMRT interactions in HER2-overexpressing breast cancer cells (LCC1) and inhibited expression of the Myc target gene survivin. In HER2-low, ER-positive steroid-dominant cells (MCF7), trastuzumab therapy repressed Myc-SMRT interactions and upregulated survivin expression. Trastuzumab treatment induced ER-CBP interactions, enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2. The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients (n=25, P=0.0089) and pS2 expression associated with residual cancer burden (P=0.0196). Furthermore, metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive. In HER2-overexpressing cells, trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable. However, with co-expression of the steroid pathway, this inhibition is lost and response to treatment is often poor.

During the past 25 years, the combination of increasingly sophisticated gene targeting technology with transplantation techniques has allowed researchers to address a wide array of questions about postnatal mammary gland development. These in turn have significantly contributed to our knowledge of other branched epithelial structures. This review chapter highlights a selection of the mouse models exhibiting a pubertal mammary gland phenotype with a focus on how they have contributed to our overall understanding of in vivo mammary morphogenesis. We discuss mouse models that have enabled us to assign functions to particular genes and proteins and, more importantly, have determined when and where these factors are required for completion of ductal outgrowth and branch patterning. The reason for the success of the mouse mammary gland model is undoubtedly the suitability of the postnatal mammary gland to experimental manipulation. The gland itself is very amenable to investigation and the combination of genetic modification with accessibility to the tissue has allowed an impressive number of studies to inform biology. Excision of the rudimentary epithelial structure postnatally allows genetically modified tissue to be readily transplanted into wild type stroma or vice versa, and has thus defined the contribution of each compartment to particular phenotypes. Similarly, whole gland transplantation has been used to definitively discern local effects from indirect systemic effects of various growth factors and hormones. While appreciative of the power of these tools and techniques, we are also cognizant of some of their limitations, and we discuss some shortcomings and future strategies that can overcome them.

During the past 25 years, the combination of increasingly sophisticated gene targeting technology with transplantation techniques has allowed researchers to address a wide array of questions about postnatal mammary gland development. These in turn have significantly contributed to our knowledge of other branched epithelial structures. This review chapter highlights a selection of the mouse models exhibiting a pubertal mammary gland phenotype with a focus on how they have contributed to our overall understanding of in vivo mammary morphogenesis. We discuss mouse models that have enabled us to assign functions to particular genes and proteins and, more importantly, have determined when and where these factors are required for completion of ductal outgrowth and branch patterning. The reason for the success of the mouse mammary gland model is undoubtedly the suitability of the postnatal mammary gland to experimental manipulation. The gland itself is very amenable to investigation and the combination of genetic modification with accessibility to the tissue has allowed an impressive number of studies to inform biology. Excision of the rudimentary epithelial structure postnatally allows genetically modified tissue to be readily transplanted into wild type stroma or vice versa, and has thus defined the contribution of each compartment to particular phenotypes. Similarly, whole gland transplantation has been used to definitively discern local effects from indirect systemic effects of various growth factors and hormones. While appreciative of the power of these tools and techniques, we are also cognizant of some of their limitations, and we discuss some shortcomings and future strategies that can overcome them.

Warner-Bratzler shear force (WBSF) and % intramuscular fat content (IMF) are objective meat quality measurements that are significantly correlated with aspects of palatability such as tenderness, flavour and juiciness. Using cDNA microarrays, Musculus longissimus transcriptomic profiles at slaughter were compared in samples displaying lower or higher IMF (n=8) and WBSF values on day 1 post mortem (n=8). 101 identified genes were differentially expressed in relation to WBSF, while 160 genes were associated with differences in IMF. Reduced expression of protein synthesis genes and enhanced expression of genes involved in protein degradation were associated with lower WBSF values on day 1. Pathways including oxidative phosphorylation and the citrate cycle were significantly associated with higher IMF. Many lipid oxidation and fatty acid metabolism pathway genes were down-regulated in high IMF tissue, suggesting a suppression of fatty acid turnover in muscle with higher fat content. Identified genes provide targets for the discovery of novel genetic variation influential on pork palatability.

The role of intercellular tight junctions in breast epithelial cells is traditionally thought to be in maintaining polarity and barrier function. However, claudin-4, a tight junction protein, is overexpressed in breast tumour cells compared to normal epithelial cells, which generally corresponds to a loss in polarity. The aim of this study was to investigate the distribution and potential clinical value of claudin-4 in breast cancer, and to evaluate its usefulness as a prognostic and predictive biomarker. Expression of claudin-4 was initially examined by Western blot analysis in a cohort of 88 breast tumours, and was found to correlate positively with tumour grade and negatively with ER. Claudin-4 expression was then evaluated by immunohistochemistry in a larger cohort of 299 tumours represented on a tissue microarray. Claudin-4 expression correlated positively with tumour grade and Her2, and negatively with ER. High claudin-4 expression was also associated with worse breast cancer-specific survival (p = 0.003), recurrence-free survival (p = 0.025) and overall survival (p = 0.034). Multivariate analysis revealed that claudin-4 independently predicted survival in the entire cohort (HR 1.95; 95%CI 1.01-3.79; p = 0.047) and in the ER positive subgroup treated with adjuvant tamoxifen (HR 4.34; 95%CI 1.14-16.53; p = 0.032). This relationship between increased claudin-4 expression and adverse outcome was validated at the mRNA level in a DNA microarray dataset of 295 breast tumours. We conclude that high levels of claudin-4 protein are associated with adverse outcome in breast cancer patients, including the subgroup of patients treated with adjuvant tamoxifen.

During puberty the mammary gland develops from a rudimentary tree to a branched epithelial network of ducts which can support alveolar development and subsequent milk production during pregnancy and lactation. This process involves growth, proliferation, migration, branching, invasion, apoptosis and above all, tight regulation which allows these processes to take place simultaneously during the course of just a few weeks to create an adult gland. The process is under hormonal control and is thus coordinated with reproductive development. Mouse models, with overexpressed or knocked-out genes, have highlighted a number of pubertal mammary gland phenotypes and given significant insight into the regulatory mechanisms controlling this period of development. Here we review the published findings of the wide range of gene-manipulated mammary mouse models, documenting the common pubertal mammary gland phenotypes observed, and summarizing their contribution to our current understanding of how pubertal mammary gland development occurs.

Expression microarray analysis identified CITED1 among a group of genes specifically upregulated in the pubertal mouse mammary gland. At puberty, CITED1 localizes to the luminal epithelial cell population of the mammary ducts and the body cells of the terminal end buds. Generation of CITED1 gene knockout mice showed that homozygous null mutants exhibit retarded mammary ductal growth at puberty and, in addition, dilated ductal structures with a lack of spatial restriction of the subtending branches. Analysis of CITED1 homozygous null and heterozygous null mammary gland gene expression using microarrays suggested that the mammary-specific phenotype seen in the homozygous null females is due to a disturbance in the transcription of a number of key mediators of pubertal ductal morphogenesis. These include estrogen and TGFbeta responsive genes, such as the EGFR/ErbB2 ligand, amphiregulin, whose transcription we suggest is directly or indirectly regulated by CITED1.

Aromatase inhibitors (AIs) target the production of estrogens with the intention of reducing estrogen receptor signalling in breast cancer. One mechanism by which tumour cells can evade AI therapy is to find alternative ways to activate the estrogen receptor in the absence of ligand. Here we discuss mechanisms of ligand-independent receptor activation including growth factor cross talk, kinase induced phosphorylation, the involvement of co-factors as well as hypersensitivity of the estrogen receptor. Understanding how cell signalling pathways regulate estrogen receptor activity is helping to identify biomarkers of AI resistance. The research discussed here has also led to the development of a number of new treatment strategies to help combat AI resistant disease.

peer-reviewed Background: Intramuscular fat (IMF) content is positively correlated with aspects of pork palatability, including flavour, juiciness and overall acceptability. The ratio of energy to protein in the finishing diet of growing pigs can impact on IMF content with consequences for pork quality. The objective of this study was to compare gene expression profiles of Musculus semimembranosus (SM) of animals divergent for IMF as a consequence of protein dietary restriction in an isocaloric diet. The animal model was derived through the imposition of low or high protein diets during the finisher stage in Duroc gilts. RNA was extracted from post mortem SM tissue, processed and hybridised to Affymetrix porcine GeneChip® arrays. Results: IMF content of SM muscle was increased on the low protein diet (3.60 ± 0.38% versus 1.92 ± 0.35%). Backfat depth was also greater in animals on the low protein diet, and average daily gain and feed conversion ratio were lower, but muscle depth, protein content and moisture content were not affected. A total of 542 annotated genes were differentially expressed (DE) between animals on low and high protein diets, with 351 down-regulated and 191 up-regulated on the low protein diet. Transcript differences were validated for a subset of DE genes by qPCR. Alterations in functions related to cell cycle, muscle growth, extracellular matrix organisation, collagen development, lipogenesis and lipolysis, were observed. Expression of adipokines including LEP, TNFα and HIF1α were increased and the hypoxic stress response was induced. Many of the identified transcriptomic responses have also been observed in genetic and fetal programming models of differential IMF accumulation, indicating they may be robust biological indicators of IMF content. Conclusion: An extensive perturbation of overall energy metabolism in muscle occurs in response to protein restriction. A low protein diet can modulate IMF content of the SM by altering gene pathways involved in lipid biosynthesis and degradation; however this nutritional challenge negatively impacts protein synthesis pathways, with potential consequences for growth. Department of Agriculture, Food and the Marine, Ireland - Food Institutional Research Measure

Purpose: The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy. Experimental Design: Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole. Results: Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH2-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter. Conclusion: This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype. Clin Cancer Res; 18(12); 3305–15. ©2012 AACR.

Introduction: The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUPTFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer. Results: FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARb2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERa, SRC1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade. Conclusions: Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.

Aromatase inhibitors (AI) are a standard-of-care treatment for postmenopausal, estrogen receptor–positive breast cancers. Although tumor recurrence on AI therapy occurs, the mechanisms underlying acquired resistance to AIs remain unknown. In this study, we examined a cohort of endocrine-treated breast cancer patients and used a cell line model of resistance to the AI letrozole. In patients treated with a first-line AI, hormone receptor switching between primary and resistant tumors was a common feature of disease recurrence. Resistant cells exhibited a switch from steroid-responsive growth to growth factor–responsive and endocrine-independent growth, which was accompanied by the development of a more migratory and disorganized phenotype. Both the resistant cells and tumors from AI-resistant patients showed high expression of the steroid receptor coactivator SRC-1. Direct interactions between SRC-1 and the transcription factor Ets2 regulated Myc and MMP9. SRC-1 was required for the aggressive and motile phenotype of AI-resistant cells. Interestingly, SRC-1 expression in primary and/or recurrent tumors was associated with a reduction in disease-free survival in treated patients. Moreover, there was a significant association between SRC-1 and Ets2 in the recurrent tissue compared with the matched primary tumor. Together, our findings elucidate a mechanism of AI-specific metastatic progression in which interactions between SRC-1 and Ets2 promote dedifferentiation and migration in hormone-dependent breast cancer. Cancer Res; 72(2); 548–59. ©2011 AACR.

The aim of this study was to identify a set of stably expressed endogenous control genes for quantitative PCR analysis of mRNA expression in the porcine LTL muscle and to subsequently perform expression analysis of potential candidate genes associated with drip loss. Expression stability of seven commonly used reference genes was examined in n = 60 pigs from three independent populations of different genetic backgrounds. The genes examined were: ACTB, ATP5G1, B2M, GPX1, RPL4, TBP and YWHAZ. GeNorm analysis of expression stability identified B2M, RPL4 and TBP as consistently stable in each breed examined. Analysis of meat samples divergent for water holding capacity identified positive and negative associations between drip loss and gene expression using B2M, RPL4 and TBP as endogenous controls. Specifically, expression of COL1A1 increased significantly with increasing drip loss while expression of CAST decreased significantly with increasing drip loss. This study therefore indicates the use of B2M, RPL4 and TBP as suitable endogenous controls for gene expression analysis of the porcine LTL muscle. Further study is recommended to identify the detailed roles of COL1A1 and CAST with respect to the development of drip loss.

Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.

Extensive epithelial cell proliferation underlies the ductal morphogenesis of puberty that generates the mammary tree that will eventually fill the fat pad. This estrogen-dependent process is believed to be essentially dependent on locally produced growth factors that act in a paracrine fashion. EGF-like growth factor ligands, acting through EGF receptors are some of the principal promoters of pubertal ductal morphogenesis. Amphiregulin is the most abundant EGF-like growth factor in the pubertal mammary gland. Its gene is transcriptionally regulated by ERalpha, and recent evidence identifies it as a key mediator of the estrogen-driven epithelial cell proliferation of puberty: The pubertal deficiency in mammary gland ductal morphogenesis in ERalpha, amphiregulin, and EGFR knockout mice phenocopy each other. As a prognostic indicator in human breast cancer, amphiregulin indicates an outcome identical to that predicted by ERalpha presence. Despite this, a range of studies both on preneoplastic human breast tissue and on cell culture based models of breast cancer, suggest a possibly significant role for amphiregulin in driving human breast cancer progression. Here we summarise our current understanding of amphiregulin's contribution to mammary gland development and breast cancer progression.

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.

Aromatase inhibitors (AI) have evolved over the last decade into an effective therapeutic regime for postmenopausal women with primary or advanced breast cancer. Despite their remarkable success in the clinic, intrinsic resistance to therapy occurs in a proportion of patients, while other patients who respond initially to treatment will relapse with recurrent disease. Previous studies suggest that this may be due, at least in part to estrogen receptor (ER) hypersensitivity. We undertook ER ChIPseq analysis on AI resistant cell model and data for this suggests that ER transcriptional regulation alone may not be responsible for the development of the resistant phenotype. We examined the role of the established ER coactivator protein AIB1. AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy. In tamoxifen treated patients, expression of AIB1 in conjunction with an activated HER2 cascade has been associated with treatment resistance and early disease recurrence. By contrast, we have observed that AIB1 alone can predict response to AIs. In our TMA the expression of AIB1 associated with disease recurrence (p = 0.025) and reduced disease free survival time (p = 0.0471) in patients treated with AIs as first-line therapy. Reflecting increased growth factor activity reported in AI resistance, AIB1 expression associated with the growth factor second messenger signaling proteins, p-Src and pERK1/2, but not the receptor HER2. These results suggest that AIB1 may utilize additional transcription factors other than ER to drive endocrine resistance. Additionally, we show that AIB1 is highly expressed in AI resistant metastases; therefore, monitoring AIB1 expression may be useful to screen for disease progression and detect disease advancement before metastases appear. Our studies of cell line models of AI resistance suggest that AIB1 may play a functional role in aggressive, migratory, phenotype of AI resistance. We have generated cell line models of resistance to letrozole (LetR) and anastrozole (AnaR). Our resistance models have higher levels of AIB1 and have increased migratory capacity. Interestingly, knockdown of AIB1 reduces the migratory capacity of the resistant cells. Furthermore, we have observed that AIB1 regulation of ER target genes is selectively enhanced in AI resistant cells in a promoter specific context. AIB1 recruitment to ER target genes such as pS2 and Myc becomes insensitive to letrozole. By contrast, AIB1 recruitment to cyclinD1 retained letrozole sensitivity. Our evidence suggests that steroidal regulation of transcription factors such as Jun and Fos may contribute to this promoter-specific regulation of ER target genes. We establish a role for AIB1 in AI-resistant breast cancer and describe a new mechanism of ERalpha/AIB1 gene regulation which could contribute to the development of an aggressive tumour phenotype. We provide evidence of a central role for AIB1 in regulating selective ER transcriptional activity and driving tumour recurrence in AI treated patients. Tackling the emerging problem of AI resistance in a timely fashion will enable us to tailor existing therapies and improve outcome in specific patient groups before disease recurrence becomes a clinical issue. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-21.

Human cancers utilise telomerase to maintain telomeres and prohibit cell senescence. Human telomerase reverse transcriptase (hTERT), an essential component of this complex, is regulated at the level of gene transcription. Using SILAC-proteomic analysis and molecular studies, we identified the AAA+ ATPase, RuvBl2 as a transcriptional regulator of hTERT and established that this regulation is through cooperation with Ets-2. In colon cancer patients, nuclear expression of RuvBl2 associated with nuclear expression of hTERT, pEts2 and advanced nodal disease (P < 0.01, P = 0.05 and P = 0.03 respectively, n = 170). These data firmly implicate RuvBl2 in Ets2 mediated regulation of hTERT in colon cancer which has functional and clinical consequences.

Background The PRKAG3 gene encodes the γ3 subunit of adenosine monophosphate activated protein kinase (AMPK), a protein that plays a key role in energy metabolism in skeletal muscle. Non-synonymous single nucleotide polymorphisms (SNPs) in this gene such as I199V are associated with important pork quality traits. The objective of this study was to investigate the relationship between gene expression of the PRKAG3 gene, SNP variation in the PRKAG3 promoter and meat quality phenotypes in pork. Results PRKAG3 gene expression was found to correlate with a number of traits relating to glycolytic potential (GP) and intramuscular fat (IMF) in three phenotypically diverse F1 crosses comprising of 31 Large White, 23 Duroc and 32 Pietrain sire breeds. The majority of associations were observed in the Large White cross. There was a significant association between genotype at the g.-311A>G locus and PRKAG3 gene expression in the Large White cross. In the same population, ten novel SNPs were identified within a 1.3 kb region spanning the promoter and from this three major haplotypes were inferred. Two tagging SNPs (g.-995A>G and g.-311A>G) characterised the haplotypes within the promoter region being studied. These two SNPs were subsequently genotyped in larger populations consisting of Large White (n = 98), Duroc (n = 99) and Pietrain (n = 98) purebreds. Four major haplotypes including promoter SNP’s g.-995A>G and g.-311A>G and I199V were inferred. In the Large White breed, HAP1 was associated with IMF% in the M. longissmus thoracis et lumborum (LTL) and driploss%. HAP2 was associated with IMFL% GP-influenced traits pH at 24 hr in LTL (pHULT), pH at 45 min in LTL (pH45LT) and pH at 45 min in the M. semimembranosus muscle (pH45SM). HAP3 was associated with driploss%, pHULT pH45LT and b* Minolta. In the Duroc breed, associations were observed between HAP1 and driploss% and pHUSM. No associations were observed with the remaining haplotypes (HAP2, HAP3 and HAP4) in the Duroc breed. The Pietrain breed was monomorphic in the promoter region. The I199V locus was associated with several GP-influenced traits across all three breeds and IMF% in the Large White and Pietrain breed. No significant difference in promoter function was observed for the three main promoter haplotypes when tested in vitro. Conclusion Gene expression levels of the porcine PRKAG3 are associated with meat quality phenotypes relating to glycolytic potential and IMF% in the Large White breed, while SNP variation in the promoter region of the gene is associated with PRKAG3 gene expression and meat quality phenotypes.