Human Breast Cancer Subtypes Research Articles

Novel syngeneic mouse mammary carcinoma cell lines from aggressive ErbB2/Neu-overexpressing/PTEN-deficient tumors.

Breast cancer cell lines and mouse models are valuable tools for investigating the biology of and developing potential therapeutics for human breast carcinoma. The PTEN-/-/NIC mouse is a genetically engineered mouse model for ErbB2/Neu-overexpressing/‑PTEN deficient breast carcinoma with histopathological and molecular features relevant to the luminal subtype of primary human breast cancer. However, the PTEN-/-/NIC model develops multifocal and aggressive mammary tumors with a short life-span, which greatly impedes its preclinical usage. To complement the genetic engineering approach and to facilitate the future application of this model, in the present study, two newly established cell lines, NICP20 and NICP21, from PTEN-/-/NIC mammary tumors are described. These NICP20 and NICP21 cells retained the crucial molecular phenotype similar to the origin, as confirmed by genotyping and western blot analysis. These cells induced tumors in immunocompetent syngeneic mice by mammary fat pad injection and produced lung metastasis when injected intravenously. Tumors induced by these cells displayed luminal‑like histologic morphology and hyperactivation of Akt which are similar to PTEN-/-/NIC tumors. Immunohistochemical staining also revealed that tumors induced by the NICP20 and NICP21 cells showed a high proliferative level, comparable angiogenesis and T-cell infiltration properties similar to PTEN-/-/NIC tumors. Therefore, these NICP20 and NICP21 cells represent an alternative and useful model system to enhance our understanding of the nature of ErbB2-positive breast cancers, particularly accompanying PTEN loss and to facilitate further experimental therapeutic studies.

Abstract 1352: Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells

Abstract Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 was recently shown to be specifically expressed in basal-like and HER-2 positive but not in luminal subtypes of human breast cancer. Overexpression of galectin-7 in breast cancer cells also increases lung and bone metastases. However, the molecular mechanisms used by galectin-7 to increase the dissemination of breast cancer cells remain largely unknown. Although galectins are best known for their ability to bind cell surface receptors, there is increasing evidence that their intracellular forms have important functions in cancer cells. This is particularly true in the control of apoptosis. Up to now, regulation of apoptosis by intracellular galectins has been largely attributed to their ability to translocate to mitochondria, possibly following their interaction with bcl-2. Here, we report that a mutant of galectin-7 that is unable to translocate to mitochondria and nucleus induces resistance of human breast cancer cells to apoptosis induced by etoposide or by hypoxia-mimicking conditions. Surprisingly, this mutant and the wild-type form of galectin-7 bind equally well to bcl-2 in vitro and in vivo. Interestingly, both forms decreases the translocation of p53 to the nucleus and reduce the expression of p21 following treatment with doxorubicin. These results challenges the current paradigm that mitochondrial galectins are important for resistance to apoptosis and call for a greater focus on the role of cytosolic galectins in cancer cells. Our data also indicate that targeting cytosolic galectin-7 could improve the efficacy of anti-cancer drugs for the treatment of aggressive forms of breast cancer. Citation Format: Andrée-Anne Grosset, Donald Gagné, Marilyne Labrie, Maria Vladoiu, Louis Gaboury, Nicolas Doucet, Yves St-Pierre. Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells. [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 1352. doi:10.1158/1538-7445.AM2014-1352

Abstract SY20-02: Insights into mechanisms of breast cancer metastasis

Abstract SY20-02: Insights into mechanisms of breast cancer metastasis

Abstract 4333: Metabolite profiling reveals druggable metabolic distinctions between basal-like and non-basal-like triple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that represents about 15-20% of all breast cancers. Because TNBC tumors do not express the estrogen or progesterone receptor and lack HER2 amplification, the disease is not responsive to current targeted therapies. The development of therapeutic approaches specific for TNBC is hindered by genetic heterogeneity, and significant efforts are being made to subtype the disease. To this end, we performed metabolite profiling (metabolomics) to characterize metabolic fingerprints within TNBC in order to define metabolic subtypes, and identify molecular drivers for the development of targeted therapies. We profiled twelve well-characterized TNBC-derived cell lines as well as a non-transformed, immortalized breast cell line and two primary human mammary epithelial cell lines. Those cancer cell lines recapitulate all 7 genetic subtypes of TNBC which were proposed recently based on mRNA gene expression profiles (1). Our approaches used and data generated have implications for drug target discovery. Hierarchical clustering based on high quality intracellular metabolites clearly and reproducibly segregated the TNBC cell lines from the non-transformed lines. Alterations in energy utilization, lipid metabolism, and other pathways of importance to highly proliferative cells differed significantly from the control cell line MCF-10A. In addition, TNBC cell lines segregated into two discrete groups, suggesting the existence of two major metabolic subtypes of TNBC, which correlated with basal-like vs. non-basal-like gene expression. Metabolites like glutamate and glutamine, serine, glycine, trans-4-hydroxyproline, 5-oxoproline, several complex lipids (phosphatidylcholines and sphingomyelins), myo-inositol, polyamines spermidine and putrescine represented metabolites differing significantly between TNBC metabolic subtypes. Ongoing studies are evaluating whether these differences represent dependencies with therapeutic relevance. Metabolite profiling was also used to identify potential metabolic liabilities generated by treatment with clinical kinase inhibitors. Response of the metabolome to treatment with rapamycin, sorafenib, imatinib, and lapatinib in four genetically diverse TNBC cell lines and MCF-10A control cell lines revealed specific drug-induced metabolic alterations. Co-targeting kinases and metabolic targets may offer an approach to synthetic lethality with a reduced likelihood for the development of drug resistance. (1) (Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121) Citation Format: Alexander Beatty, Lauren Fink, Ulrike Rennefahrt, Alexander Strigun, Erik Peter, Hajo Schiewe, Regina Reszka, Jeffrey R. Peterson. Metabolite profiling reveals druggable metabolic distinctions between basal-like and non-basal-like triple-negative breast cancers. [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 4333. doi:10.1158/1538-7445.AM2014-4333

Subtype-specific overexpression of the Rac-GEF P-REX1 in breast cancer is associated with promoter hypomethylation.

IntroductionThe Rac-GEF P-REX1 is a key mediator of ErbB signaling in breast cancer recently implicated in mammary tumorigenesis and metastatic dissemination. Although P-REX1 is essentially undetectable in normal human mammary epithelial tissue, this Rac-GEF is markedly upregulated in human breast carcinomas, particularly of the luminal subtype. The mechanisms underlying P-REX1 upregulation in breast cancer are unknown. Toward the goal of dissecting the mechanistic basis of P-REX1 overexpression in breast cancer, in this study we focused on the analysis of methylation of the PREX1 gene promoter.MethodsTo determine the methylation status of the PREX1 promoter region, we used bisulfite genomic sequencing and pyrosequencing approaches. Re-expression studies in cell lines were carried out by treatment of breast cancer cells with the demethylating agent 5-aza-2′-deoxycitidine. PREX1 gene methylation in different human breast cancer subtypes was analyzed from the TCGA database.ResultsWe found that the human PREX1 gene promoter has a CpG island located between -1.2 kb and +1.4 kb, and that DNA methylation in this region inversely correlates with P-REX1 expression in human breast cancer cell lines. A comprehensive analysis of human breast cancer cell lines and tumors revealed significant hypomethylation of the PREX1 promoter in ER-positive, luminal subtype, whereas hypermethylation occurs in basal-like breast cancer. Treatment of normal MCF-10A or basal-like cancer cells, MDA-MB-231 with the demethylating agent 5-aza-2′-deoxycitidine in combination with the histone deacetylase inhibitor trichostatin A restores P-REX1 levels to those observed in luminal breast cancer cell lines, suggesting that aberrant expression of P-REX1 in luminal breast cancer is a consequence of PREX1 promoter demethylation. Unlike PREX1, the pro-metastatic Rho/Rac-GEF, VAV3, is not regulated by methylation. Notably, PREX1 gene promoter hypomethylation is a prognostic marker of poor patient survival.ConclusionsOur study identified for the first time gene promoter hypomethylation as a distinctive subtype-specific mechanism for controlling the expression of a key regulator of Rac-mediated motility and metastasis in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0441-7) contains supplementary material, which is available to authorized users.

SHON, a novel secreted protein, regulates epithelial-mesenchymal transition through transforming growth factor-β signaling in human breast cancer cells.

The epithelial-mesenchymal transition (EMT) is one of the main mechanisms contributing to the onset of cancer metastasis, and has proven to be associated with breast cancer progression. SHON is a novel secreted hominoid-specific protein we have previously identified; it is specifically expressed in all human cancer cell lines tested and is oncogenic for human mammary carcinoma cells. Here, we show that ectopic overexpression of SHON in immortalized human mammary epithelial cells is sufficient for cells to acquire the mesenchymal traits, as well as the enhanced cell migration and invasion, along with the epithelial stem cell properties characterized by increased CD44(high) /CD24(low) subpopulation and mammosphere-forming ability. Moreover, we demonstrate that SHON positively activates the autocrine transforming growth factor-β (TGF-β) pathway to contribute to EMT, while SHON itself is induced by TGF-β in mammary epithelial cells. These data are in favor of a SHON-TGFβ-SHON-positive feedback loop that regulates EMT program in breast cancer progression. Finally, examination of the human clinic breast cancer specimens reveals that tumor cells may extracellularly release SHON protein to promote the cancerization of surrounding cells. Together, our findings define an important function of SHON in regulation of EMT via TGF-β signaling, which is closely associated with the invasive subtypes of human breast cancer.

Expression of RUNX1 correlates with poor patient prognosis in triple negative breast cancer.

The RUNX1 transcription factor is widely recognised for its tumour suppressor effects in leukaemia. Recently a putative link to breast cancer has started to emerge, however the function of RUNX1 in breast cancer is still unknown. To investigate if RUNX1 expression was important to clinical outcome in primary breast tumours a tissue microarray (TMA) containing biopsies from 483 patients with primary operable invasive ductal breast cancer was stained by immunohistochemistry. RUNX1 was associated with progesterone receptor (PR)-positive tumours (P<0.05), more tumour CD4+(P<0.05) and CD8+(P<0.01) T-lymphocytic infiltrate, increased tumour CD138+plasma cell (P<0.01) and more CD68+macrophage infiltrate (P<0.001). RUNX1 expression did not influence outcome of oestrogen receptor (ER)-positive or HER2-positive disease, however on univariate analysis a high RUNX1 protein was significantly associated with poorer cancer-specific survival in patients with ER-negative (P<0.05) and with triple negative (TN) invasive breast cancer (P<0.05). Furthermore, multivariate Cox regression analysis of cancer-specific survival showed a trend towards significance in ER-negative patients (P<0.1) and was significant in triple negative patients (P<0.05). Of relevance, triple negative breast cancer currently lacks good biomarkers and patients with this subtype do not benefit from the option of targeted therapy unlike patients with ER-positive or HER2-positive disease. Using multivariate analysis RUNX1 was identified as an independent prognostic marker in the triple negative subgroup. Overall, our study identifies RUNX1 as a new prognostic indicator correlating with poor prognosis specifically in the triple negative subtype of human breast cancer.

Triple negative tumors accumulate significantly less methylglyoxal specific adducts than other human breast cancer subtypes.

Metabolic syndrome and type 2 diabetes are associated with increased risk of breast cancer development and progression. Methylglyoxal (MG), a glycolysis by-product, is generated through a non-enzymatic reaction from triose-phosphate intermediates. This dicarbonyl compound is highly reactive and contributes to the accumulation of advanced glycation end products. In this study, we analyzed the accumulation of Arg-pyrimidine, a MG-arginine adduct, in human breast adenocarcinoma and we observed a consistent increase of Arg-pyrimidine in cancer cells when compared with the non-tumoral counterpart. Further immunohistochemical comparative analysis of breast cancer subtypes revealed that triple negative lesions exhibited low accumulation of Arg-pyrimidine compared with other subtypes. Interestingly, the activity of glyoxalase 1 (Glo-1), an enzyme that detoxifies MG, was significantly higher in triple negative than in other subtype lesions, suggesting that these aggressive tumors are able to develop an efficient response against dicarbonyl stress. Using breast cancer cell lines, we substantiated these clinical observations by showing that, in contrast to triple positive, triple negative cells induced Glo-1 expression and activity in response to MG treatment. This is the first report that Arg-pyrimidine adduct accumulation is a consistent event in human breast cancer with a differential detection between triple negative and other breast cancer subtypes.

Abcc10 status affects mammary tumour growth, metastasis, and docetaxel treatment response.

Background:Resistance to chemotherapeutic agents is a major obstacle to cancer treatment. A group of ABC efflux pumps, the Multidrug Resistance Proteins, is a source of resistance. Herein, we investigated the role of ABCC10 in mammary tumours, given the important role we have defined for ABCC10 in transporting taxanes, and the recognition that some ABCC proteins have roles in tumour growth.Methods:ABCC10 expression was correlated to human breast cancer subtype using breast tissue microarrays. Real-time quantitative PCR and western blot analysis were used to examine ABCC10 expression in human breast cancer lines. Abcc10−/− mice were crossed to MMTV-PyVmT mice to produce Abcc10−/− vs Abcc10+/+ mammary tumours and derivative cell lines. We used allograft and cellular assays to perform baseline and drug sensitization analysis of tumours and cell lines.Results:Clinical sample analyses indicated that ABCC10 was more highly expressed in Her2+ and ER+ than in Her2−, ER−, and triple-negative breast cancer. Unexpectedly, PyVmT; Abcc10−/− tumours grew more rapidly than PyVmT; Abcc10+/+ tumours and were associated with significantly reduced apoptosis and metastasis. PyVmT; Abcc10−/− lines were less migratory than PyVmT; Abcc10+/+ lines. Finally, we showed increased survival of docetaxel-treated MMTV-PyVmT; Abcc10−/− mice compared with wild-type mice.Conclusions:These data identify roles for Abcc10 in breast cancer pathogenesis and in vivo docetaxel resistance.

Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models

The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER)-negative cells. Basal cell-origin tumours displayed similar histological phenotypes, regardless of the depleted gene. In contrast, luminal ER-negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER-negative and, strikingly, ER-positive invasive ductal carcinomas. Molecular profiling demonstrated that luminal ER-negative cell-origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal-like, luminal B and 'normal-like'. Furthermore, a subset of these tumours resembled the 'claudin-low' tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell of origin and driver genetic aberrations, but also multiple mammary tumour subtypes, including both ER-positive and -negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour aetiology.

XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization1. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE12 and its substrate XBP13. Previous studies report UPR activation in various human tumors4-6, but XBP1's role in cancer progression in mammary epithelial cells is largely unknown. Triple negative breast cancer (TNBC), a form of breast cancer in which tumor cells do not express the genes for estrogen receptor, progesterone receptor, and Her2/neu, is a highly aggressive malignancy with limited treatment options7, 8. Here, we report that XBP1 is activated in TNBC and plays a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumor growth and tumor relapse and reduced the CD44high/CD24low population. Hypoxia-inducing factor (HIF)1α is known to be hyperactivated in TNBCs 9, 10. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and imply that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

Radiation-Induced Gene Signature Predicts Pathologic Complete Response to Neoadjuvant Chemotherapy in Breast Cancer Patients

The identification of biomarkers predictive of neoadjuvant chemotherapy response in breast cancer patients would be an important advancement in personalized cancer therapy. In this study, we hypothesized that due to similarities between radiation- and chemotherapy-induced cellular response mechanisms, radiation-responsive genes may be useful in predicting response to neoadjuvant chemotherapy. Murine p53 null breast cancer cell lines representative of the luminal, basal-like and claudin-low human breast cancer subtypes were irradiated to identify radiation-responsive genes across subtypes. These murine tumor radiation-induced genes were then converted to their human orthologs, and subsequently tested as a predictor of pathologic complete response (pCR), which was validated on two independent published neoadjuvant chemotherapy datasets of genomic data with chemotherapy response. A radiation-induced gene signature consisting of 30 genes was identified on a training set of 337 human primary breast cancer tumor samples that was prognostic for survival. Mean expression of this signature was calculated for individual samples on two independent published datasets and was found to be significantly predictive of pCR. Multivariate logistic regression analysis in both independent datasets showed that this 30 gene signature added significant predictive information independent of that provided by standard clinical predictors and other gene expression-based predictors of pCR. This study provides new information for radiation-induced biology, as well as information regarding response to neoadjuvant chemotherapy and a possible means of improving the prediction of pCR.

Reverse phase protein array based tumor profiling identifies a biomarker signature for risk classification of hormone receptor-positive breast cancer

A robust subclassification of luminal breast cancer, the most common molecular subtype of human breast cancer, is crucial for therapy decisions. While a part of patients is at higher risk of recurrence and requires chemo-endocrine treatment, the other part is at lower risk and also poorly responds to chemotherapeutic regimens. To approximate the risk of cancer recurrence, clinical guidelines recommend determining histologic grading and abundance of a cell proliferation marker in tumor specimens. However, this approach assigns an intermediate risk to a substantial number of patients and in addition suffers from a high interobserver variability. Therefore, the aim of our study was to identify a quantitative protein biomarker signature to facilitate risk classification. Reverse phase protein arrays (RPPA) were used to obtain quantitative expression data for 128 breast cancer relevant proteins in a set of hormone receptor-positive tumors (n = 109). Proteomic data for the subset of histologic G1 (n = 14) and G3 (n = 22) samples were used for biomarker discovery serving as surrogates of low and high recurrence risk, respectively. A novel biomarker selection workflow based on combining three different classification methods identified caveolin-1, NDKA, RPS6, and Ki-67 as top candidates. NDKA, RPS6, and Ki-67 were expressed at elevated levels in high risk tumors whereas caveolin-1 was observed as downregulated. The identified biomarker signature was subsequently analyzed using an independent test set (AUC = 0.78). Further evaluation of the identified biomarker panel by Western blot and mRNA profiling confirmed the proteomic signature obtained by RPPA. In conclusion, the biomarker signature introduced supports RPPA as a tool for cancer biomarker discovery.

Abstract P6-06-05: RadiotypeDx: Identification and validation of a radiation sensitivity signature in human breast cancer

Abstract Purpose: An unmet clinical need in breast cancer (BC) management is the identification of which patients will respond to radiation therapy (RT). We hypothesized that the integration of post-RT clonogenic survival data with gene expression data across a large spectrum of BC cell lines would generate a BC-specific RT sensitivity signature predictive for RT response in BC patients and allow identification of patients with tumors refractive to conventional therapy. Methods: Using clonogenic survival assays, we identified the range of surviving fraction (SF) after 2 Gy of RT across 21 BC cell lines. Using SF as a continuous variable, the RT sensitivity score (RSS) was correlated to gene expression using a Spearman correlation method on an individual gene basis. Genes were selected for the signature based on positive or negative correlation with a p-value &amp;lt;0.05 and FDR of &amp;lt;0.01. Unsupervised hierarchical clustering identified differences in gene expression across resistant and sensitive cell lines to generate a radiation sensitivity (RS) signature. This signature was trained and validated in a separate human breast tumor dataset (185 pts) containing early stage, node-negative patients treated with surgery and RT alone without adjuvant chemotherapy to assess the predictive effect of RS signature on recurrence risk after RT. Gene function and potentially actionable targets from the signature were validated using clongenic survival and DNA damage assays. Results: Clonogenic survival identifies a range of radiation sensitivity in human BCC lines (SF 77%-17%) with no significant correlation (r value &amp;lt;0.3) to the intrinsic BC subtype. Using Spearmans correlation method, a total of 126 genes were identified as being associated with radiation sensitivity (72 positively correlated, 54 negatively correlated). Unsupervised hierarchical expression discriminates gene expression patterns in the RT resistant and RT sensitive cell lines and is enriched for genes involved in cell cycle arrest and DNA damage response (enrichment p-value 5.0 E-22). Knockdown of genes associated with the radioresistance signature identifies previously unreported radiation resistance genes, including TACC1 and RND3 with enhancement ratios of 1.25 and 1.37 in BCC lines. Application of this RS signature to an independent breast cancer dataset with clinical outcomes validates the signature and accurately identifies patients with decreased rates of recurrence compared to patients with high expression of the radioresistant signature (p-value &amp;lt;0.0001, misclassification error rate .31, 12/13 patients with locoregional recurrence accurately identified). Conclusion: In this study, we derive a human BC-specific RT sensitivity signature (RadiotypeDx) with biologic relevance from preclinical studies and validate this signature for prediction of recurrence in an independent clinical dataset. The signature is not correlated to the intrinsic subtypes of human breast cancer and thus provides useful information beyond traditional breast cancer subtyping. By identifying patients with tumors refractory to standard RT, this signature has the potential to allow for personalization of radiotherapy, particularly in patients for whom treatment intensification is needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-05.

Abstract P6-06-23: Glycogen-rich, clear cell breast cancer - An underestimated histopathological risk factor?

Abstract Introduction: Glycogen-rich clear cell carcinoma (GRCCC) is a rare tumour and there is only few data on the prognosis of this subtype, the largest cases series ever published in literature comprising of 21 cases only. (1) Patients and methods: In a case-control study of 56 GRCCC early breast cancer cases with matched pairs we analyzed the features of this subtype and survival data regarding DFS and OS. 56 patients with GRCCC have been identified from the period 1992 until 2010 in a multicenter retrospective case cohort trial. To determine the incidence of glycogen-rich clear cell carcinoma in various age groups were stratified in 3 age groups: &amp;lt; 45 years, &amp;lt; 65 years and &amp;gt; 65 years. This was compared to incidence of all breast cancer subtypes due to epidemiological data of Robert-Koch-Institute of the German Ministry of Health. Results: Patients were in a range of 33-87 years, median 59,5 years, distributed to the following age groups: 14% (n = 8) of patients were &amp;lt; 45 years, 71,4% (n = 40) &amp;lt; 65 years and 28,6% (n = 16) &amp;gt; 65 years. In the epidemiological data set 10% were &amp;lt; 45 years, 50% &amp;lt; 65 years and 50% &amp;gt; 65 years. Thus patients with glycogen-rich clear cell carcinoma were younger compared to the epidemiological data of all breast cancers registered nationally (&amp;lt; 65 years: 71,4% vs. 50%; p &amp;lt; 0,002). Stage distribution was as follows: Stage T1 a: no cases, T1b: 2, T1c: 19, T2: 20, T3: 4, T4: 4, unknown T-stage in 7 cases. Nodal status was in 22 nodal-positive, 23 nodal-negative and 11 with unknown nodal status. The Grading was in G1 none, G2 24 and G3 29 and unknown in 3 cases. Approximated intrinsic tumour subtype was: Luminal A: 10, Luminal B 3, Luminal Her2 positive 16, non-luminal Her2 negative: 14, Triple-Negative Breast Cancer: 7; not further specified: 6. Chemotherapy was applied in 35 out of 56 patients (62,5%) in our study cohort. 5-years-(OS) was 84% and 5-years (DFS) was 80%. Recurrence rates at a mean time of follow-up of 8,5 years were significantly higher (11 recurrences out of 56 cases) in this specific subtype than in the matched control group (p&amp;lt;0,005). OS was significantly lower in the group with a local recurrence, dropping down from a 5-years OS-rate of 92% in the recurrence-free group to 48% in the group with a loco-regional recurrence. Conclusion: GRCCC of the breast is associated with a highly proliferative and aggressive receptor profile and unfavourable course of disease with significantly higher risk of recurrence (p&amp;lt; 0,005). This is - to our best knowledge - the largest cohort published in literature on this rare histological subtype of human early breast cancer. Evidence derived from this analysis suggests effective chemotherapy treatment to reduce the risk of recurrence and mortality from this aggressive tumour subtype. The poor prognosis of 48% in 5-years-overall-survival in case of a loco-regional recurrences of glycogen-rich clear cell carcinom also endorses a role of chemo-therapy in case of recurrence, with regards to the fact that survival rates in this tumor subtype of our cohort were even lower than in the cohort of the CALOR trial.(2) Literature: 1) Hayes MMM, Am J Surg Pathol. 1995;19:904-911. 2)Aebi S, S3-2. San Antonio Breast Cancer Symposium 2012. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-23.

Collective Invasion in Breast Cancer Requires a Conserved Basal Epithelial Program

Carcinomas typically invade as a cohesive multicellular unit, a process termed collective invasion. It remains unclear how different subpopulations of cancer cells contribute to this process. We developed three-dimensional (3D) organoid assays to identify the most invasive cancer cells in primary breast tumors. Collective invasion was led by specialized cancer cells that were defined by their expression of basal epithelial genes, such as cytokeratin-14 (K14) and p63. Furthermore, K14+ cells led collective invasion in the major human breast cancer subtypes. Importantly, luminal cancer cells were observed to convert phenotypically to invasive leaders following induction of basal epithelial genes. Although only a minority of cells within luminal tumors expressed basal epithelial genes, knockdown of either K14 or p63 was sufficient to block collective invasion. Our data reveal that heterotypic interactions between epithelial subpopulations are critical to collective invasion. We suggest that targeting the basal invasive program could limit metastatic progression.

Cyclin D1 induction of Dicer governs microRNA processing and expression in breast cancer

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates the pRB protein and promotes G1/S cell cycle progression and oncogenesis. Dicer is a central regulator of miRNA maturation, encoding an enzyme that cleaves double strand RNA or stem-loop-stem RNA into 20–25 nucleotide long small RNA, governing sequence specific gene silencing and heterochromatin methylation. The mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Here we show that cyclin D1−/− cells are defective in pre-miRNA processing which is restored by cyclin D1a rescue. Cyclin D1 induces Dicer expression in vitro and in vivo. Dicer is transcriptionally targeted by cyclin D1, via a cdk-independent mechanism. Cyclin D1 and Dicer expression significantly correlates in luminal A and basal-like subtypes of human breast cancer. Cyclin D1 and Dicer maintain heterochromatic histone modification (Tri-m-H3K9). Cyclin D1-mediated cellular proliferation and migration is Dicer-dependent. We conclude that cyclin D1 induction of Dicer coordinates microRNA biogenesis.

Distinct Splice Variants and Pathway Enrichment in the Cell-Line Models of Aggressive Human Breast Cancer Subtypes

This study was conducted as a part of the Chromosome-Centric Human Proteome Project (C-HPP) of the Human Proteome Organization. The United States team of C-HPP is focused on characterizing the protein-coding genes in chromosome 17. Despite its small size, chromosome 17 is rich in protein-coding genes; it contains many cancer-associated genes, including BRCA1, ERBB2, (Her2/neu), and TP53. The goal of this study was to examine the splice variants expressed in three ERBB2 expressed breast cancer cell-line models of hormone-receptor-negative breast cancers by integrating RNA-Seq and proteomic mass spectrometry data. The cell lines represent distinct phenotypic variations subtype: SKBR3 (ERBB2+ (overexpression)/ER-/PR-; adenocarcinoma), SUM190 (ERBB2+ (overexpression)/ER-/PR-; inflammatory breast cancer), and SUM149 (ERBB2 (low expression) ER-/PR-; inflammatory breast cancer). We identified more than one splice variant for 1167 genes expressed in at least one of the three cancer cell lines. We found multiple variants of genes that are in the signaling pathways downstream of ERBB2 along with variants specific to one cancer cell line compared with the other two cancer cell lines and with normal mammary cells. The overall transcript profiles based on read counts indicated more similarities between SKBR3 and SUM190. The top-ranking Gene Ontology and BioCarta pathways for the cell-line specific variants pointed to distinct key mechanisms including: amino sugar metabolism, caspase activity, and endocytosis in SKBR3; different aspects of metabolism, especially of lipids in SUM190; cell-to-cell adhesion, integrin, and ERK1/ERK2 signaling; and translational control in SUM149. The analyses indicated an enrichment in the electron transport chain processes in the ERBB2 overexpressed cell line models and an association of nucleotide binding, RNA splicing, and translation processes with the IBC models, SUM190 and SUM149. Detailed experimental studies on the distinct variants identified from each of these three breast cancer cell line models that may open opportunities for drug target discovery and help unveil their specific roles in cancer progression and metastasis.

Chemical carcinogens used to induce spontaneous tumors may suppress tumor specific T-cell immunity

Meeting abstracts Many mouse models of breast cancer have been developed that closely replicate human breast cancer subtypes at the genomic level. In order to develop vaccines that could prevent a broad spectrum of breast cancer subtypes, a combination of different spontaneous tumor models are

Abstract A007: Mutant PIK3CA accelerates HER2-driven transgenic mammary tumor progression, enhances cancer stem cell features, and induces resistance to combinations of anti-HER2 therapies

Abstract The HER2 (ERBB2) oncogene is amplified in 20-25% of breast cancers and is associated with poor patient outcome. Mutational activation of PIK3CA, the gene encoding the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), occurs in ~30% of breast cancers. HER2 amplification and PIK3CA mutations often co-occur in breast cancer. Aberrant activation of the PI3K pathway has been shown to correlate with a diminished response to HER2-directed therapies. We sought to directly determine whether mutant PI3K enhances HER2-induced tumor progression and promotes resistance to HER2 inhibition in vivo. For this purpose, we generated a conditional transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared to mice expressing PIK3CA alone (p=0.0012) or HER2 alone (p&amp;lt;0.0001). HER2 and mutant PIK3CA also strongly cooperated to promote lung metastases following tail vein injection of cells derived from transgenic tumors (p&amp;lt;0.01). PIK3CA and HER2+/PIK3CA tumors were histologically diverse (i.e., adenocarcinoma, adenomyoepithelioma, adenocarcinoma with squamous metaplasia, and metaplastic carcinoma), expressed luminal and basal cytokeratins by IHC, and were associated with the claudin-low subtype of human breast cancers by microarray analysis. Accordingly, HER2+/PIK3CA tumors expressed elevated transcripts encoding EMT and stem cell markers, such as Snail, Slug, Twist, Bmi1, ITGB1 (CD29), and Procr. Further, single cells from HER2+/PIK3CA tumors formed more and larger mammospheres compared to cells from HER2-driven tumors. HER2+/PIK3CA mammospheres and tumors were completely resistant to the HER2 monocolonal antibody trastuzumab, alone and in combination with the HER2 tyrosine kinase inhibitor lapatinib or with pertuzumab, an antibody that inhibits ligand-induced HER2 dimerization. Both drug resistance and enhanced mammosphere formation were reversed by treatment with the pan-PI3K inhibitor BKM120. BKM120 treatment strongly inhibited T308 and S473 pAKT, whereas pAKT levels were unaffected by trastuzumab in combination with lapatinib or with pertuzumab. We further generated a model of resistance to the combination of trastuzumab, pertuzumab, and BKM120 by long-term treatment of HER2+/PIK3CA tumor transplants. Tumors initially responded, but a subset (3/11) resumed growth in the presence of drugs within 2 weeks of treatment initiation. Resistance was maintained following passaging in mice. We are currently performing whole-exome sequencing on resistant vs. untreated tumors in order to determine the mechanism(s) of resistance. In sum, PIK3CAH1047R accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers and generates resistance to FDA-approved combinations of anti-HER2 therapies. We propose the mouse model presented herein will be a valuable tool to investigate the underlying biology of HER2+/PI3K-mutant tumors and for the preclinical testing of therapeutic strategies against this subtype of breast cancer. Citation Format: Ariella B. Hanker, Adam Pfefferle, Justin M. Balko, Maria Gabriela Kuba, Christian D. Young, Violeta Sanchez, Cammie R. Sutton, Hailing Cheng, Charles M. Perou, Jean J. Zhao, Rebecca S. Cook, Carlos L. Arteaga. Mutant PIK3CA accelerates HER2-driven transgenic mammary tumor progression, enhances cancer stem cell features, and induces resistance to combinations of anti-HER2 therapies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A007.