Poor Outcome In Breast Cancer Research Articles

Hypoxia promotes stem cell phenotypes and poor prognosis through epigenetic regulation of DICER.

MicroRNAs are small regulatory RNAs that post-transcriptionally control gene expression. Reduced expression of DICER, the enzyme involved in microRNA processing, is frequently observed in cancer and is associated with poor clinical outcome in various malignancies. Yet the underlying mechanisms are not well understood. Here, we identify tumor hypoxia as a regulator of DICER expression in large cohorts of breast cancer patients. We show that DICER expression is suppressed by hypoxia through an epigenetic mechanism that involves inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B and results in silencing of the DICER promoter. Subsequently, reduced miRNA processing leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and ultimately results in the acquisition of stem cell phenotypes in human mammary epithelial cells. Our study uncovers a previously unknown relationship between oxygen-sensitive epigenetic regulators, miRNA biogenesis and tumor stem cell phenotypes that may underlie poor outcome in breast cancer.

Abstract 3466: Mediated by cancer cell and stroma cell TGF-β signaling, the periostin / β-Ig-H3 expression ratio is altered during breast cancer progression

Abstract Involved in the maintenance of normal breast tissue, the extracellular matrix (ECM) also supports the progression of several human cancers. In particular, both periostin and TGF-β-induced (β-Ig-H3) whose expression is regulated by transforming growth factor-β (TGF-β) signaling, are differentially expressed in many human cancers. While a link between increased periostin expression and poor breast cancer outcomes has been shown, our recent data suggest that overall periostin expression is variable and is poorly correlated with human breast cancer progression. Therefore, in this study we investigated the ratio between periostin and β-Ig-H3 expression and the relationship between TGF-β signaling and expression of these ECM proteins by cancer cells and stromal cells during human breast cancer progression. Expressions of periostin and β-Ig-H3 were determined in biopsy specimens collected from 61 breast cancer patients by immunohistochemistry and correlated with clinico-pathologic features. Additionally, periostin, β-Ig-H3, and TGF-β expression in multiple stroma and tumor cells were evaluated in vitro using western-blots and ELISAs. Increases in the periostin / ß-Ig-H3 ratio correlated with increased the AJCC stage and progesterone positive status of the breast cancer specimens tested (p<0.05). In vitro, murine L929 fibroblasts and 4T1 mammary cancer cells secreted higher periostin concentrations compared to J774 and RAW macrophages (p<0.05). Furthermore, the periostin / ß-Ig-H3 protein expression ratio of 4T1 cells was higher than those of J774 and RAW cells. Following treatment with TGF-ß that activated the SMAD pathway, L929 and 4T1 cells expressed higher periostin concentrations (p<0.05). In contrast, the high ß-Ig-H3 secretion by L929 fibroblasts and J774 macrophages remained unchanged following TGFß treatment (n.s.). These observations support a role for TGF-ß and periostin signaling loops between stroma and cancer cells in the microenvironment and may indicate a role of the periostin / ß-Ig-H3 ratio as a predictor of breast cancer aggressiveness. [This works was supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007)] Citation Format: Michelle M. Coleman, Anindita Das Burman, Amanda L. Lance, Didier Dreau. Mediated by cancer cell and stroma cell TGF-β signaling, the periostin / β-Ig-H3 expression ratio is altered during breast cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2014-3466

Abstract 4458: PDLIM2 is essential for feedback regulation of the B1 integrin-FAK-RhoA signalling pathway to maintain epithelial cell polarity and suppress transformation

Abstract PDLIM2 is a PDZ-LIM domain protein that regulates the stability of transcription factors (NFkB and STATs) in the nucleus. It is also associated with the cytoskeleton at cell adhesions and cell junctions and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT). Moreover, PDLIM2 expression is associated with poor outcome in basal breast cancers and its expression can be enhanced by vitamin D-induced differentiation of breast cancer cells. Since PDLIM2 expression repression may facilitate oncogenic transformation and its expression is modulated by the differentiation status of breast cancer cells we asked whether it is essential for maintaining a non-transformed epithelial phenotype. To address this we suppressed PDLIM2 in MCF10A cells and assessed the phenotype and mechanism in 3D Matrigel cultures. We found that PDLIM2 suppression is sufficient to disrupt cell polarization and acinar formation in Matrigel cultures. MCF10A cells with suppressed PDLIM2 formed spheres of proliferating cells that exhibited reduced apoptosis in the luminal space, compared to control acini with hollow or apoptotic lumina. Spheroids with suppressed PDLIM2 had increased expression of cell-cell adhesion proteins including E cadherin and β-catenin. Proteins associated with cell-matrix adhesion were also up-regulated, including activated beta 1 (β1) integrin, laminin, and fibronectin production. Interestingly, both the IGF-1 Receptor and RACK1, which scaffolds IGF-1R to β1 integrin, were increased, indicating a transformed phenotype. FAK and cofilin phosphorylation, and RhoA activity were also enhanced in spheroids with PDLIM2 suppressed compared to controls. Importantly, pharmacological inhibition of either FAK or Rho Kinase (ROCK) was sufficient to rescue the polarity defect and abrogate all the phenotypic changes in MCF10A cell spheroids caused by PDLIM2 suppression. We conclude that PDLIM2 mediates feedback regulation of the β1-integrin signalling pathway through FAK and RhoA that is essential for maintenance of polarized breast epithelial acinar structures, and for suppression of uncontrolled proliferation and cellular transformation. Citation Format: Ravi Kiran-Deevi, Orla T. Cox, Rosemary O'Connor. PDLIM2 is essential for feedback regulation of the B1 integrin-FAK-RhoA signalling pathway to maintain epithelial cell polarity and suppress transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4458. doi:10.1158/1538-7445.AM2014-4458

Abstract 2217: Genomic alteration of bromodomain protein ATAD2 in cancer

Abstract ATPase family, AAA domain containing 2 (ATAD2), is a member of the bromodomain-containing protein family. ATAD2 associates with co-factors such as hormone receptors, E2Fs and c-Myc to control a specific subset of genes. Overexpression and copy number alteration of ATAD2 are observed in many types of cancer, and these alterations are correlated with poor clinical outcome in breast, lung, and endometrial cancers. Here, we present an integrated genomic analysis of ATAD2 using the Oncomine™ database that shows cancer-associated features of ATAD2. In The Cancer Genome Atlas, amplifications of ATAD2 are observed in 63% of ovarian, 52% of breast, 39% of colorectal, and 18% of lung cancer patients. By contrast, no amplification of ATAD2 is observed in corresponding normal tissues. In most cases, amplifications are modest ranging up to a copy number of six. Moreover, a high correlation between ATAD2 mRNA expression levels and its copy number is observed in cultured cell lines. In order to investigate the role of the bromodomain in ATAD2, we conducted a cell-free physicochemical binding analysis and in vitro physiological studies using human colon cancer cells (HCT-116). To identify binding partners for the ATAD2 bromodomain, we constructed ATAD2 AlphaScreen systems and examined the binding activity of the ATAD2 bromodomain protein to various acetylated or non-acetylated histone peptides. The ATAD2 bromodomain interacted with histone H4 acetylated at lysine 5, 8 or 12, but not with other acetylated and non-acetylated histone peptides. Subcellular localization analysis by immunofluorescence, demonstrated that ATAD2 is mainly localizes to nucleus and that treatment with trichostatin A, an inhibitor of histone deacetylases increased nuclear ATAD2 protein levels, suggesting that the acetylation level of histone tails may affect ATAD2 expression or localization. In in silico expression analysis using the Cancer Cell Line Encyclopedia, showed most genes that correlated with ATAD2 expression have a role in proliferation or survival. To determine if the ATAD2 protein directly controls transcription of these genes, we performed ChIP-PCR assays and found that ATAD2 protein occupies the DNA promoter regions of downstream targets, eg. TOP2A, but does not occupy the promoters of non-target negative controls, such as β-actin. In conclusion, we show ATAD2 copy number alteration in a large number of cancer patients, suggesting that ATAD2 expression, or genomic alteration, may be useful prognostic markers of cancer. We also show that physicochemical binding of the ATAD2 bromodomain to specific acetylated histone peptides suggesting a physiological function of ATAD2 in nucleus as a transcriptional regulator of tumor-related genes. These results suggest that a blockade of ATAD2 bromodomain binding to specific acetylated histone tails may provide a novel approach to anti-tumor drug development. Citation Format: Tomoyo Takeda, Yuta Matsumura, Hirotoshi Nagasaki, Ken Eguchi, Miki Takatsuka, Yoichi Shinkai, Hiroyuki Nakagawa, Hitoshi Kawashima, Hiroyuki Ueno, Chiang J. Li. Genomic alteration of bromodomain protein ATAD2 in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2217. doi:10.1158/1538-7445.AM2014-2217

Abstract 3274: SNP-SNP interaction analyses of NQO1 and NF-κB signaling pathway genes on breast cancer survival and treatment outcome

Abstract To improve individualized therapy, new prognostic and predictive markers are needed to identify patients with expected poor breast cancer outcome and patients who profit most from a given treatment. NF-κB pathway plays a pervasive role in the pathogenesis of cancer through its implication in distinct hallmarks of tumorigenesis. Previous studies have indicated the role of NF-κB transcription factor in promoting cell survival and resistance to chemotherapy in breast carcinoma. We have previously found that NQO1-rs1800566 predicts poor survival among breast cancer patients, particularly after anthracycline-based adjuvant chemotherapy and in patients with p53-aberrant tumors. We have also reported the inverse relationship between NQO1 protein expression and NF-κB activation. We have here analyzed the interaction of SNPs residing within the NF-κB signaling pathway genes, and NQO1-rs6499255 (tagging NQO1-rs1800566 with r2> 0.90) on breast cancer patient survival and treatment outcome. The panel of markers included the NF-κB pathway SNPs available in a custom Illumina iSelect genotyping array designed for Collaborative Oncological Gene-Environment Study (iCOGS) as well as NQO1-rs6499255. For the SNP-SNP interaction analysis on patient survival we conducted a likelihood ratio test that compares Cox's regression model with and without an interaction term to evaluate whether the interaction model is a better fit for the prognostic data. The results obtained from this study will be reported. Identification of interactive SNPs/genes contribute to the elucidation of the mechanisms underlying complex diseases such as breast cancer. Citation Format: Maral Jamshidi, Rainer Fagerholm, Sofia Khan, Kristiina Aittomäki, Carl Blomqvist, Marjanka K. Schmidt, Heli Nevanlinna, BCAC: Breast Cancer Association Consortium. SNP-SNP interaction analyses of NQO1 and NF-κB signaling pathway genes on breast cancer survival and treatment outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3274. doi:10.1158/1538-7445.AM2014-3274

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Mesothelin expression is associated with poor outcomes in breast cancer.

Mesothelin is a potential therapeutic target and prognostic marker in breast cancer. However, results on its prognostic value in breast cancer have been equivocal and warranted further evaluation. We analyzed clinical data from two breast cancer patient cohorts comprising of 141 patients treated at our institution (discovery cohort) and 844 patients from The Cancer Genome Atlas (TCGA) (validation cohort). Mesothelin expression was quantified by immunohistochemistry or by RNA transcript levels as measured by whole-transcriptome sequencing in the discovery and validation cohorts respectively. Univariate analyses of data from the discovery cohort demonstrated that tumor size [hazard ratio (HR) = 1.30, 95 % confidence interval (CI) 1.11-1.51], positive (+) axillary lymph nodes (HR = 3.34; 95 % CI 1.51-7.39), and mesothelin expression (HR = 2.03; 95 % CI 1.10-3.74) were associated with disease-specific survival. Multivariate analyses demonstrated that mesothelin expression was significantly associated with worse survival (HR = 3.06, 95 % CI 1.40-6.68) after adjusting for (+) axillary lymph nodes and tumor size. Using TCGA cohort as validation dataset, mesothelin-expressing tumors were indeed significantly associated with worse overall survival with HR = 1.46; 95 % CI 1.05-2.03 and HR = 1.69; 95 % CI 1.17-2.42 in univariate and multivariate analyses respectively. Our results suggest that mesothelin is a prognostic breast tumor marker whose expression is highly enriched in triple negative breast cancer (TNBC) tumors. As there is no existing targeted therapy for TNBC, mesothelin may be a promising drug target for TNBC. Future work is needed to evaluate the efficacy of mesothelin directed targeted therapy in the treatment of breast cancer.

High EGFR gene copy number predicts poor outcome in triple-negative breast cancer

Epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer and is emerging as a therapeutic target. EGFR gene copy number alteration and mutation are highly variable and scientists have been challenged to define their prognostic significance in triple-negative breast cancer. We examined EGFR protein expression, EGFR gene copy number alteration and mutation of exon 18 to 21 in 151 cases of triple-negative breast cancer and correlated these findings with clinical outcomes. In addition, intratumoral agreement of EGFR protein overexpression and gene copy number alteration was evaluated. EGFR overexpression was found in 97 of 151 cases (64%) and high EGFR gene copy number was detected in 50 cases (33%), including 3 gene amplification (2%) and 47 high polysomy (31%). Five EGFR mutations were detected in 4 of 151 cases (3%) and included G719A in exon 18 (n=1), V786M in exon 20 (n=1), and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, correlated with EGFR protein overexpression. Intratumoral heterogeneity of EGFR protein overexpression and EGFR copy number alteration was not significant. In survival analyses, high EGFR copy number was found to be an independent prognostic factor for poor disease-free survival in patients with triple-negative breast cancer. Our findings showed that EGFR mutation was a rare event, but high EGFR copy number was relatively frequent and correlated with EGFR overexpression in triple-negative breast cancer. Moreover, high EGFR copy number was associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR copy number may be useful for predicting outcomes in patients with triple-negative breast cancer and for selecting patients for anti-EGFR-targeted therapy.

Circulating tumor cells in non-metastatic triple-negative breast cancer.

Circulating tumor cells (CTCs) can be identified in approximately 25 % of stage I-III breast cancer patients; CTCs presence is a predictor of poor outcome in metastatic breast cancer, but little is known regarding the prognostic significance of CTCs in non-metastatic triple-negative breast cancer (TNBC) patients. The aim of this study was to determine whether CTCs predict worse outcome in non-metastatic TNBC patients. We evaluated CTCs in 113 patients with stages I-III TNBC at the time of definitive surgery. CTCs were assessed using the CellSearch System®. Progression-free and overall survival were defined as time elapsed between date of diagnosis and either date of clinical disease progression, death, or last follow-up. Log-rank test and Cox regression analysis were used to determine associations of CTCs with progression-free and overall survival. The median follow-up was 40 months. CTCs were identified in 23/113 (20 %) of patients. No primary tumor characteristic or lymph node status predicted the presence of CTCs. The identification of ≥2 CTCs predicted shorter progression-free (log rank P ≤ 0.001; hazard ratio 8.30, 95 % CI 2.61–26.37) and overall survival (log rank P = 0.0004; hazard ratio 7.19, 95 % CI 1.98–26.06) versus survival for patients with <2 CTCs. Two or more CTCs predict shorter progression-free and overall survival in TNBC patients. Larger studies are needed to determine whether CTC assessment provides beneficial information that could be used in stratifying TNBC patients at increased risk for disease progression. Finally, CTCs characterization could facilitate the development of novel treatment approaches for TNBC.

The co-factor of LIM domains (CLIM/LDB/NLI) maintains basal mammary epithelial stem cells and promotes breast tumorigenesis.

Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans.

The Emerging Role of QSOX1 in Cancer

Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme that oxidizes thiols during protein folding, reducing molecular oxygen to hydrogen peroxide. Tumor cells may take advantage of oxidative environments at different stages of tumorigenesis, but QSOX1 may also serve additional functions in tumors. Several groups have reported the over-expression of QSOX1 in breast, pancreas, and prostate cancers. A consensus is building that QSOX1 over-expression is important during tumor cell invasion, facilitating tumor cell migration at the tumor-stroma interface. As such, QSOX1 may be considered a prognostic indicator of metastatic potential or even indicate that cancer is present in a host. However, some controversy exists between QSOX1 as a marker of poor or favorable outcome in breast cancer. More studies are required to reveal what advantage QSOX1 provides to breast and other types of cancer. More specifically, it is critical to learn which tumor types over-express QSOX1 and use its enzymatic activity to their advantage. As interest increases in understanding the mechanisms of tumorigenesis within the extracellular matrix and how tumor cells influence fibroblasts and other stromal cells, QSOX1 may be revealed as an important player in cancer detection and prognosis. Defining the mechanism(s) of QSOX1 activity in tumors and in in vivo models will provide important insights into how to target QSOX1 with anti-neoplastic agents.

Significant survival impact of MACC1 polymorphisms in HER2 positive breast cancer patients

BackgroundDeregulation of hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (MET) signalling has been associated with poor clinical outcome in breast cancer and other cancers. The recently discovered metastasis-associated in colon cancer-1 (MACC1) gene is a key regulator of the HGF/MET pathway. Potential links between genetic variants of the MACC1 gene and survival in breast cancer patients are unknown. In the present study, we therefore aimed to investigate the influence of MACC1 polymorphisms on event-free and overall survival in patients with human epidermal growth factor 2 (HER2)-positive breast cancer. MethodsThe present study included 164 consecutive white patients with HER2-positive breast cancer. Three MACC1 polymorphisms, rs1990172, rs975263 and rs3735615, already associated with cancer prognosis or with potential functional effects, were genotyped by the 5′ nuclease assay. ResultsMultivariate Cox regression analysis adjusted for age and tumour stage showed increased risk for progression or death for carriers of the rare allele (G-allele) of single nucleotide polymorphism (SNP) rs1990172 (hazard ratios (HR)=2.26; p=0.004 and HR=3.13; p=0.001 for event-free survival and overall survival, respectively). In addition, we were able to demonstrate an adverse effect on cancer prognosis for carriers of the rare allele (T-allele) of SNP rs975263 (HR=2.17; p=0.007 and HR=2.80; p=0.003 for event-free survival and overall survival, respectively). The rare allele (C-allele) of SNP rs3735615 showed a significant protective impact on event-free survival as well as overall survival (HR=0.25; p=0.001, and HR=0.16; p=0.001, respectively). ConclusionsThis study provides first evidence that MACC1 polymorphisms are associated with clinical outcome for HER2-positive breast cancer patients. Further studies are warranted to validate these findings.

Prognostic associations of 25OH vitamin D in NCIC CTG MA.21, a phase III adjuvant RCT of three chemotherapy regimens (EC/T, CEF, AC/T) in high-risk breast cancer (BC).

504 Background: Low vitamin D (VitD) has been associated with poor outcomes in BC. We examined in MA.21 the association of VitD blood levels with relapse-free-survival (RFS) in all patients, and in...

42P - Low Expression of FGD3, a Putative Guanine Nucleotide Exchange Factor for CDC42, is Prognostic of Poor Outcome in Breast Cancer

Identification of single gene biomarkers that are prognostic of outcome can gain insight into the molecular mechanisms that drive breast cancer. Exploratory analysis of single gene biomarkers in Metabric discovery and validation cohort indicates that low expression of FGD3 mRNA is prognostic of poor outcome. FGD3 has been shown to induce morphological changes in HeLa Tet-Off cells where high expression induces sheet-like protrusions and inhibits cell migration (Hayakawa et al. 2008). FGD3 is found to have high expression in immune response cells using curated data in 79 human and 61 mouse tissues from the GeneAtlas (Wu et al.

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer.

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.

Aberrant Expression of CXCR4 Significantly Contributes to Metastasis and Predicts Poor Clinical Outcome in Breast Cancer

Triple negative breast cancer is known for its visceral metastasis. We have found that CXCR4 is overexpressed in triple negative breast cancer and is associated with visceral metastasis. We further investigated whether CXCR4 is a prognostic factor affecting survival following visceral metastasis in breast cancer patients. Our results indicate that increased CXCR4 expression among breast cancer patients with visceral metastasis was positively correlated with poor overall survival (P<0.001). Silencing of CXCR4 was associated with a decrease in the tumorigenic properties of MDA-MB-231 breast cancer cells, caused reversion of EMT and suppression of MMP-9, increased apoptosis, and caused a reduced incidence of tumor lung metastasis in mice. These results are indicative of CXCR4 having a predictive role in patients with visceral metastasis and indicate that shRNA knock down of CXCR4 might be a novel therapeutic strategy to prevent breast cancer metastasis when CXCR4 is overexpressed.

Decreased mitochondrial SIRT3 expression is a potential molecular biomarker associated with poor outcome in breast cancer.

SIRT3 is a genomically expressed, mitochondrial localized tumor suppressor protein where it directs multiple metabolic processes by deacetylating downstream protein substrates. Genetic deletion of Sirt3 in mice leads to the spontaneous development of mammary tumors starting at 1 year, and decreased SIRT3 messenger RNA has been observed in several human tumors including breast malignancies. In this investigation, we assessed SIRT3 expression in human breast cancer tissue microarray and examined the relationship between SIRT3 expression and outcome in patients with breast cancer. SIRT3 protein expression is significantly lower in neoplastic compared with normal breast epithelium, including normal epithelium adjacent to tumors. Patients with breast cancer in the lowest quartile of SIRT3 expression had a significantly shorter locoregional relapse-free survival (hazard ratio, 0.53 [0.47-0.61]; log-rank P = 0). Notably, low SIRT3 expression was associated with reduced locoregional relapse-free survival in all breast cancer subtypes analyzed, including ER+, ER-, HER2+, and basal subtypes (hazard ratios, 0.44-0.65; log-rank P = 0-.0019). These results highlight the importance of the SIRT3 as a tumor suppressor protein in breast cancer and suggest that SIRT3 may be a potential molecular biomarker to identify high-risk patients across all molecular subtypes of breast cancer.

Abstract S6-05: High levels of APOBEC3B, a DNA deaminase and an enzymatic source of C-to-T transitions, are a validated marker of poor outcome in estrogen receptor-positive breast cancer

Abstract Two recent observations have connected the innate immune DNA cytosine deaminase APOBEC3B to the genetic evolution of breast cancer. First, APOBEC3B was shown to be up-regulated in the majority of breast cancers, and, in breast cancer cell lines, its activity was causally linked to a doubling of the number of C-to-T transitions over time and to a delay in cell cycle progression (1). Second, sequencing of the complete genome of 21 breast cancers independently suggested that APOBEC deaminase activity could be responsible for 2 of 5 mutational imprints identified, which involved clustered (also called kataegis) and dispersed C-to-T transition mutations in the context of 5’TC dinucleotide motifs (2). In the current study, we addressed a possible association of APOBEC3B expression with outcome in clinical breast cancer. For this we measured using real-time RT-PCR APOBEC3B mRNA levels in 1,491 primary invasive breast cancers and correlated these levels with disease-free survival (DFS), metastasis-free survival (MFS) and overall survival (OS) using univariate and multivariable Cox regression analysis. In addition, we independently validated our findings in available gene expression datasets with appropriate follow-up. In univariate analyses including all patients, increasing levels of APOBEC3B mRNA analyzed as a continuous variable were significantly associated with shorter DFS, MFS and OS (Hazard Ratio [HR] = 1.29, 1.31 and 1.36, respectively, all P&amp;lt;0.001). To determine the relation of APOBEC3B mRNA expression with the natural course of the disease without the potential confounding effects of systemic adjuvant therapy, we restricted our next analyses to MFS in 829 patients with lymph node-negative disease who had not received any (neo)adjuvant systemic therapy. This analysis showed that APOBEC3B mRNA expression was, in univariate, and in multivariable analysis, including the traditional prognostic factors (age, menopausal status, tumor size, grade and steroid hormone receptors), a marker of pure prognosis specifically in patients with estrogen receptor-positive (ER+) disease (univariate HR = 1.30; P = 0.003; multivariate HR = 1.22, P = 0.042). To substantiate and validate our findings, we analysed 4 independent available datasets containing in total 5,760 breast cancer cases in which APOBEC3B mRNA expression was measured by probes on microarrays and found that higher APOBEC3B mRNA expression (dichotomised by mean) was significantly associated with poor outcome in all 4 cohorts ([Metabric, 1,491 ER+ cases, HR = 1.82; P&amp;lt;0.001], [Affymetrix compiled dataset-1, 2,407 cases, HR = 2.22; P = 0.001], and [BIG 1-98; 1,207 cases, HR = 2.13; P&amp;lt;0.001 of late recurrence&amp;gt;5 years], and [Affymetrix dataset-2, 643 ER+ cases, HR = 2.04; P = 0.001]). Altogether, our analyses show that APOBEC3B mRNA - and as a result likely DNA deamination – is a validated predictor of poor outcome in breast cancer, supporting the notion that APOBEC3B is a potentially interesting clinical target for therapeutic intervention to prevent breast cancer progression and metastasis, particularly in ER+ disease. 1. Burns, M.B. et al. Nature 494, 366-70 (2013); 2. Nik-Zainal, S. et al. Cell 149, 979-93 (2012). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-05.

Abstract P6-06-47: The association between EGFR and cMET expression and phosphorylation and its prognostic implication in patients with breast cancer

Abstract Background: It has been shown that EGFR, cMET and their cross-talk play an important role in the progression of breast cancer and development of resistance to different targeted therapies. cMET expression and phosphorylation is linked with poor survival outcome in breast cancer. Recently, EGFR and cMET dual blockade has been proposed as an attractive targeted therapy for breast cancer. However, little is known about the co-expression patterns of EGFR and cMET in human breast cancer and its prognostic significance. We hypothesized that co-expression or co-activation of EGFR and cMET is linked with adverse survival outcome. Methods: We measured protein levels of EGFR, cMET and their phosphorylated proteins in 825 breast cancers using reverse phase protein array. Given unimodal distribution of proteins, median was selected as a cut-off after sensitivity analyses. Kaplan-Meier survival curves were used to estimate relapse-free (RFS) and overall survival (OS). Cox proportional hazards models were fit to determine associations of EGFR and cMET with outcomes after adjustment for other clinical characteristics such as age, tumor stage, nodal status, receptor subtype and histologic grade. Results: Mean age was 58 years. There were 457 (55%) hormone receptor (HR) positive, 211 (26%) triple-negative (TN) and 148 (18%) HER2 positive tumors. HER2 subgroup showed higher EGFR expression and phosphorylation compared to HR and TN subtypes (p&amp;lt;0.05). High EGFR expression group was associated with higher phosho-cMET (pcMET) but not cMET compared to low expression group (ANOVA pcMET p&amp;lt;0.001, MET p = 0.34). The same association was found with High phospho-EGFR (pEGFR) group at Try 992 and Tyr1068 (both p&amp;lt;0.001). In HR subtype, similar association was observed between EGFR/pEGFRs and pcMET (all p&amp;lt;0.01). High expression groups in either of two pEGFRs were linked with higher cMET as well (all p&amp;lt;0.001). In TN subtype, High expression group in EGFR and pEGFR at Tyr992 but not at Tyr1068 was associated with higher pcMET (p&amp;lt;0.00, p = 0.012, p = 0.4, respectively). Only high expression group in pEGFR at Tyr 992 was linked with higher expression of cMET (p = 0.02). In contrast, among HER2 subtype, high expression group in pEGFR at Tyr 1068 but not at Tyr992 was associated with higher cMET and pcMET (cMET p = 0.023, pcMET p&amp;lt;0.001). Four subgroups of patients defined by dichotomized EGFR (or pEGFR) and cMET (or pcMET) level demonstrated no significant differences in RFS and OS. In multivariate analyses, only cMET expression level was associated with adverse survival (RFS p = 0.06, OS p = 0.035) while levels of pcMET, EGFR, and pEGFRs did not show significant association with outcomes. Conclusion: We report that EGFR and cMET are either frequently co-expressed or co-activated in human breast cancer. HER2 subtype showed higher EGFR expression and activation compared to other subtypes. cMET protein expression level was found to be an independent prognostic factor irrespective of the EGFR expression or phosphorylation status. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-47.

The Association Between Adiposity and Breast Cancer Recurrence and Survival: A Review of the Recent Literature

Breast Cancer (BC) is the most common and second deadliest malignancy among American women. Many factors contribute to BC prognosis but a key modifiable lifestyle factor is body weight. In this review, we update the reader on the association between adiposity and poor BC outcomes. We summarize the findings from studies that show obesity to be a risk factor for BC recurrence and reduced survival, including research that shows that treatment with aromatase inhibitors in hormone-receptor positive BC survivors who are obese may not be as effective as in normal weight women. In addition, we summarize the findings from studies that show that obesity-induced changes in glucose metabolism, type-2 diabetes and metabolic syndrome contribute to negative outcomes in BC survivors. Given the evidence, there is a critical need to determine whether weight loss can improve outcomes in BC survivors.