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Triple-negative (TN) breast cancers (ER − PR − HER2 − ) are highly metastatic and associated with poor prognosis. Within this subtype, invasive, stroma-rich tumours with infiltration of inflammatory cells are even more aggressive. The effect of myeloid cells on reactive stroma formation in TN breast cancer is largely unknown. Here, we show that primary human monocytes have a survival advantage, proliferate in vivo and develop into immunosuppressive myeloid cells expressing the myeloid-derived suppressor cell marker S100A9 only in a TN breast cancer environment. This results in activation of cancer-associated fibroblasts and expression of CXCL16, which we show to be a monocyte chemoattractant. We propose that this migratory feedback loop amplifies the formation of a reactive stroma, contributing to the aggressive phenotype of TN breast tumours. These insights could help select more suitable therapies targeting the stromal component of these tumours, and could aid prediction of drug resistance. A reactive tumour stroma is associated with poor prognosis. Here, the authors show that in patients with triple negative breast cancer resident monocytes activate cancer-associated fibroblasts and induce production of CXCL16, which acts as a monocyte chemoattractant, resulting in an amplificatory feedback loop.

Quality control of cell lines used in biomedical research is essential to ensure reproducibility. Although cell line authentication has been widely recommended for many years, misidentification, including cross-contamination, remains a serious problem. We outline a multi-stakeholder, incremental approach and policy-related recommendations to facilitate change in the culture of cell line authentication.

Background Eukaryotic Initiation Factor 4E-Binding Protein ( EIF4EBP1 , 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy. Methods Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1. Results Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes. Conclusions These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

Introduction Toll-like receptors (TLRs) are a family of pattern recognition receptors that are expressed on cells of the innate immune system. The ligands can be pathogen derived (pathogen associated molecular patterns; PAMPs) or endogenous (damage associated molecular patters; DAMPs) that when bound induces activation of nuclear factor kappa B (NF-κB) and transcription of pro-inflammatory genes. TLRs have also been discovered in various malignant cell types, but with unknown function. Methods In this study we performed a detailed analysis of TLR and co-receptor expression pattern and function in breast cancer. Expression patterns were examined using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) on three estrogen receptor-positive (ER + ) and four estrogen receptor/progesterone receptor-negative (ER − /PR − ; ER/PR-negative) breast cancer cell lines, and a breast cancer cohort consisting of 144 primary breast cancer samples. The function was investigated using in vitro assays comprising PAMP/DAMP-stimulation, downstream signaling and TLR-silencing experiments. Results We found that TLR4 was expressed in a biologically active form and responded to both PAMPs and DAMPs primarily in ER/PR-negative breast cancers. Stimulation of TLR2/4 in vitro induced expression of pro-inflammatory genes and a gene expression analysis of primary breast cancers showed a strong correlation between TLR4 expression and expression of pro-inflammatory mediators. In line with this, TLR4 protein expression correlated with a decreased survival. Conclusions These findings suggest that TLR4 is expressed in a functional form in ER/PR-negative breast cancers. Studies regarding TLR4-antagonist therapies should be focusing on ER/PR-negative breast cancer particularly.

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well-characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal-like, luminal A, luminal B, HER2-enriched and normal-like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2−/Ki67low, ER+/HER2−/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal-like, triple negative and HER2-enriched tumors, and high H3K27me3 in luminal A, HER2-enriched and normal-like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal-like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease-free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer. ► Using IHC we studied the abundance of EZH2 and H3K27me3 in breast tumors. ► We found differential abundance of both markers in breast tumor subtypes. ► The expression of EZH2 was high in basal-like and HER2-enriched tumors. ► The occurrence of H3K27me3 was low in tumors classified as basal-like or luminal B. ► Survival differences were seen based on abundance of the two markers.

Purpose NSD3 has been implicated as a candidate driver oncogene from the 8p11-p12 locus, and we have previously published evidence for its amplification and overexpression in human breast cancer. This aim of this study was to further characterize the transforming function of NSD3 in vivo. Methods We generated a transgenic mouse model in which NSD3 gene expression was driven by the MMTV promoter and expressed in mammary epithelium of FVB mice. Mammary glands were fixed and whole mounts were stained with carmine to visualize gland structure. Mammary tumors were formalin-fixed, and paraffin embedded (FFPE) tumors were stained with hematoxylin and eosin. Results Pups born to transgenic females were significantly underdeveloped compared to pups born to WT females due to a lactation defect in transgenic female mice. Whole mount analysis of the mammary glands of transgenic female mice revealed a profound defect in functional differentiation of mammary gland alveoli that resulted in the lactation defect. We followed parous and virgin NSD3 transgenic and control mice to 50 weeks of age and observed that several NSD3 parous females developed mammary tumors. Whole mount analysis of the mammary glands of tumor-bearing mice revealed numerous areas of mammary hyperplasia and ductal dysplasia. Histological analysis showed that mammary tumors were high-grade ductal carcinomas, and lesions present in other mammary glands exhibited features of alveolar hyperplasia, ductal dysplasia, and carcinoma in situ. Conclusions Our results are consistent with our previous studies and demonstrate that NSD3 is a transforming breast cancer oncogene.

Background The gene desert on human chromosomal band 8q24 harbors multiple genetic variants associated with common cancers, including breast cancer. The locus, including the gene desert and its flanking genes, MYC , PVT1 and FAM84B , is also frequently amplified in human breast cancer. We generated a megadeletion (MD) mouse model lacking 430-Kb of sequence orthologous to the breast cancer-associated region in the gene desert. The goals were to examine the effect of the deletion on mammary cancer development and on transcript level regulation of the candidate genes within the locus. Methods The MD allele was engineered using the MICER system in embryonic stem cells and bred onto 3 well-characterized transgenic models for breast cancer, namely MMTV-PyVT , MMTV-neu and C3(1)-TAg. Mammary tumor growth, latency, multiplicity and metastasis were compared between homozygous MD and wild type mice carrying the transgenes. A reciprocal mammary gland transplantation assay was conducted to distinguish mammary cell-autonomous from non-mammary cell-autonomous anti-cancer effects. Gene expression analysis was done using quantitative real-time PCR. Chromatin interactions were evaluated by 3C. Gene-specific patient outcome data were analysed using the METABRIC and TCGA data sets through the cBioPortal website. Results Mice homozygous for the MD allele are viable, fertile, lactate sufficiently to nourish their pups, but maintain a 10% lower body weight mainly due to decreased adiposity. The deletion interferes with mammary tumorigenesis in mouse models for luminal and basal breast cancer. In the MMTV-PyVT model the mammary cancer-reducing effects of the allele are mammary cell-autonomous. We found organ-specific effects on transcript level regulation, with Myc and Fam84b being downregulated in mammary gland, prostate and mammary tumor samples. Through analysis using the METABRIC and TCGA datasets, we provide evidence that MYC and FAM84B are frequently co-amplified in breast cancer, but in contrast with MYC , FAM84B is frequently overexpressed in the luminal subtype, whereas MYC activity affect basal breast cancer outcomes. Conclusion Deletion of a breast cancer-associated non-protein coding region affects mammary cancer development in 3 transgenic mouse models. We propose Myc as a candidate susceptibility gene, regulated by the gene desert locus, and a potential role for Fam84b in modifying breast cancer development.

Normal breast epithelial cells require insulin and EGF for growth in serum-free media. We previously demonstrated that over expression of breast cancer oncogenes transforms MCF10A cells to an insulin-independent phenotype. Additionally, most breast cancer cell lines are insulin-independent for growth. In this study, we investigated the mechanism by which oncogene over expression transforms MCF10A cells to an insulin-independent phenotype. Analysis of the effects of various concentrations of insulin and/or IGF-I on proliferation of MCF10A cells demonstrated that some of the effects of insulin were independent from those of IGF-I, suggesting that oncogene over expression drives a true insulin-independent proliferative phenotype. To test this hypothesis, we examined metabolic functions of insulin signaling in insulin-dependent and insulin-independent cells. HER2 over expression in MCF10A cells resulted in glucose uptake in the absence of insulin at a rate equal to insulin-induced glucose uptake in non-transduced cells. We found that a diverse set of oncogenes induced the same result. To gain insight into how HER2 oncogene signaling affected increased insulin-independent glucose uptake we compared HER2-regulated gene expression signatures in MCF10A and HER2 over expressing MCF10A cells by differential analysis of time series gene expression data from cells treated with a HER2 inhibitor. This analysis identified genes specifically regulated by the HER2 oncogene, including VAMP8 and PHGDH, which have known functions in glucose uptake and processing of glycolytic intermediates, respectively. Moreover, these genes specifically implicated in HER2 oncogene-driven transformation are commonly altered in human breast cancer cells. These results highlight the diversity of oncogene effects on cell regulatory pathways and the importance of oncogene-driven metabolic transformation in breast cancer.

A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these determinants have a poor prognosis. We used cell line models of EGFR-positive/PTEN null TNBC to elucidate the signaling networks that drive the malignant features of these cells and cause resistance to EGFR inhibitors. In these cells, amphiregulin (AREG)-mediated activation of EGFR results in up-regulation of fibronectin (FN1), which is known to be a mediator of invasive capacity via interaction with integrin β1. EGFR activity in this PTEN null background also results in Wnt/beta-catenin signaling and activation of NF-κB. In addition, AKT is constitutively phosphorylated in these cells and is resistant to gefitinib. Expression profiling demonstrated that AREG-activated EGFR regulates gene expression differently than EGF-activated EGFR, and functional analysis via genome-scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are essential for survival of SUM-149 cells, but are uncoupled from EGFR signaling. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, which likely mediates resistance to EGFR inhibitors. •Activation of EGFR by AREG alters signaling and gene expression compared to EGF.•Activation of EGFR by AREG reduces mTORC1 pathway expression and phosphorylation.•EGF-positive, PTEN-null TNBC cells are poised for Wnt/beta-catenin signaling.•Wnt/beta-catenin activity occurs in a subset of cells and is enhanced in mammospheres.•Regulation of growth/survival genes is uncoupled from EGFR in PTEN-null TNBC cells.

More than 20 different PIK3CA gene mutations were identified in breast cancer with different frequencies. Whether these breast cancer associated mutations have similar biological effects is largely unknown. In this study, we established a novel cell model using the lentivirus system to express 10 different PIK3CA genes (wild type and mutant) based on the human mammary epithelial cell MCF10A. We found that nine different PIK3CA mutants harbor different abilities to promote cell proliferation and EGF independent growth. In addition, most PIK3CA mutants (except for the wild type PIK3CA , the Q60K and the K111N mutants) had the ability to change the morphogenesis of the MCF10A cell in 3D Matrigel assay. Moreover, different PIK3CA mutants have different abilities to promote colony formation and cell invasion. We further observed that most of the PIK3CA mutants could activate p-AKT and p-p70-S6K in the absence of EGF stimulation. Finally, LY294002, a PI3K inhibitor, can effectively inhibit cell growth in cell lines with different PIK3CAs. Taken together, our results support the notion that different PIK3CA mutations differentially contribute to breast cancer transformation, and exploration of the therapeutic application of these mutations will benefit breast cancer patients with the PIK3CA mutations.