ABSTRACT
In recent years, comprehensive cancer genomics platforms, such as The Cancer Genome Atlas (TCGA), provide access to an enormous amount of high throughput genomic datasets for each patient, including gene expression, DNA copy number alterations, DNA methylation, and somatic mutation. While the integration of these multi-omics datasets has the potential to provide novel insights that can lead to personalized medicine, most existing approaches only focus on gene-level analysis and lack the ability to facilitate biological findings at the pathway-level. In this article, we propose Bayes-InGRiD (Bayesian Integrative Genomics Robust iDentification of cancer subgroups), a novel pathway-guided Bayesian sparse latent factor model for the simultaneous identification of cancer patient subgroups (clustering) and key molecular features (variable selection) within a unified framework, based on the joint analysis of continuous, binary, and count data. By utilizing pathway (gene set) information, Bayes-InGRiD does not only enhance the accuracy and robustness of cancer patient subgroup and key molecular feature identification, but also promotes biological understanding and interpretation. Finally, to facilitate an efficient posterior sampling, an alternative Gibbs sampler for logistic and negative binomial models is proposed using Pólya-Gamma mixtures of normal to represent latent variables for binary and count data, which yields a conditionally Gaussian representation of the posterior. The R package "INGRID" implementing the proposed approach is currently available in our research group GitHub webpage (https://dongjunchung.github.io/INGRID/).
Subject(s)
Genomics , Neoplasms , Humans , Bayes Theorem , Neoplasms/genetics , Models, Statistical , DNA MethylationABSTRACT
Several years ago, the SUM panel of human breast cancer cell lines was developed, and these cell lines have been distributed to hundreds of labs worldwide. Our lab and others have developed extensive omics data sets from these cells. More recently, we performed genome-scale shRNA essentiality screens on the entire SUM line panel, as well as on MCF10A cells, MCF-7 cells, and MCF-7LTED cells. These gene essentiality data sets allowed us to perform orthogonal analyses that functionalize the otherwise descriptive genomic data obtained from traditional genomics platforms. To make these omics data sets available to users of the SUM lines, and to allow users to mine these data sets, we developed the SUM Breast Cancer Cell Line Knowledge Base. This knowledge base provides information on the derivation of each cell line, provides protocols for the proper maintenance of the cells, and provides a series of data mining tools that allow rapid identification of the oncogene signatures for each line, the enrichment of KEGG pathways with screen hit and gene expression data, an analysis of protein and phospho-protein expression for the cell lines, as well as a gene search tool and a functional-druggable signature tool. Recently, we expanded our database to include genomic data for an additional 27 commonly used breast cancer cell lines. Thus, the SLKBase provides users with deep insights into the biology of human breast cancer cell lines that can be used to develop strategies for the reverse engineering of individual breast cancer cell lines.
ABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/metabolism , Oncogenes , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Cycle Checkpoints , Cell Cycle Proteins , Cell Proliferation , Chromosomes, Human, Pair 8/genetics , Disease-Free Survival , Everolimus/pharmacology , Female , Gene Amplification , Gene Knockdown Techniques , Humans , Kaplan-Meier Estimate , MCF-7 Cells , Phosphoproteins/genetics , Phosphorylation , Prognosis , Receptors, Estrogen , Recurrence , TOR Serine-Threonine Kinases/antagonists & inhibitors , TransfectionABSTRACT
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.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Genes, myc/genetics , Mammary Neoplasms, Experimental/genetics , Neoplasm Proteins/genetics , Animals , Female , Gene Knockout Techniques , Membrane Proteins , Mice , Mice, TransgenicABSTRACT
Cellular immunity established via immunotherapy holds the potential to eliminate solid tumors. Yet, cancer vaccines have failed to induce tumor-reactive T cells of sufficient quality to control disease. The inducible T cell costimulator (ICOS) pathway has been implicated in both the selective induction of immunity over tolerance as well as licensing of IL-17-polarized cellular immunity. Herein, we evaluated the ability of ICOS ligand (ICOSL) to augment the immunogenicity of adenoviral-based vaccination targeting the unglycosylated MUC1 peptide antigen. Vaccination disrupted immunotolerance in a transgenic mouse model recognizing human MUC1 as a self-antigen, inducing robust MUC1-specific immunity. Augmenting vaccination with ICOSL induced a bipolar Th17/Th1 effector profile, marked by increased MUC1-specific IL-17A production and RORγt expression in CD4+ but not CD8+ T cells which predominantly expressed IFNγ/IL-2 and T-bet. The polarization and maintenance of Th17 cells established following ICOSL augmented vaccination was highly durable, with elevated IL-17A and RORγt levels detected in CD4+ T cells up to 10â¯months after initial immunization. Furthermore, provision of ICOSL significantly enhanced MUC1-specific IgG antibody in response to immunization. ICOSL signaling dramatically influenced CD4+ T cell phenotype, altering gene expression of transcription factors and regulators of effector function following immunization. Interestingly, ICOSL augmentation failed to alter the transcriptional profile of CD8+ T cells following immunization, affecting the magnitude, but not distribution, of gene expression. Collectively, ICOSL supports the induction of durable, antigen-specific Th17/Th1-mediated immunity in vivo, establishing a vaccination platform to enhance CD4+ T cell-mediated antitumor immunity and providing a crucial component of an effective cancer vaccine.
Subject(s)
Mucin-1/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Vaccination/methods , Adenoviridae/genetics , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunity, Cellular/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal TransductionABSTRACT
Aberrant activation of phosphatidylinosito-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) signaling in cancer has led to pursuit of inhibitors for targeting this pathway. However, inhibitors of PI3K and AKT have failed to yield efficacious results without adverse effects. Here, we screened a library containing 441 authenticated traditional chinese medicine (TCM) plant extracts by examining their effect on cell viability of a human mammary epithelial cell line HMEC-PIK3CAH1047R, which expresses mutant PIK3CAH1047R and has constitutively active AKT signaling. We found that Oridonin, an extract from Rabdosia rubescens, reduced cell viability to the greatest extent. Oridonin binds to AKT1 and potentially functions as an ATP-competitive AKT inhibitor. Importantly, Oridonin selectively impaired tumor growth of human breast cancer cells with hyperactivation of PI3K/AKT signaling. Moreover, Oridonin prevented the initiation of mouse mammary tumors driven by PIK3CAH1047R. Our results suggest that Oridonin may serve as a potent and durable therapeutic agent for the treatment of breast cancers with hyperactivation of PI3K/AKT signaling.
ABSTRACT
There is mounting evidence that cancer stem-like cells (CSC) are selectively enriched in residual tumors after anticancer therapies, which may account for tumor recurrence and metastasis by regenerating new tumors. Thus, there is a critical need to develop new therapeutic agents that can effectively eliminate drug-resistant CSCs and improve the efficacy of cancer therapy. Here, we report that Triptolide (C1572), a small-molecule natural product, selectively depletes CSCs in a dose-dependent fashion in human triple-negative breast cancer (TNBC) cell lines. Nanomolar concentrations of C1572 markedly reduced c-MYC (MYC) protein levels via a proteasome-dependent mechanism. Silencing MYC expression phenocopied the CSC depletion effects of C1572 and induced senescence in TNBC cells. Limited dilution assays revealed that ex vivo treatment of TNBC cells with C1572 reduced CSC levels by 28-fold. In mouse xenograft models of human TNBC, administration of C1572 suppressed tumor growth and depleted CSCs in a manner correlated with diminished MYC expression in residual tumor tissues. Together, these new findings provide a preclinical proof of concept defining C1572 as a promising therapeutic agent to eradicate CSCs for drug-resistant TNBC treatment. Cancer Res; 77(23); 6641-50. ©2017 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , Diterpenes/pharmacology , Neoplasm Recurrence, Local/drug therapy , Neoplastic Stem Cells/drug effects , Phenanthrenes/pharmacology , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Animals , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Epoxy Compounds/pharmacology , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Recurrence, Local/pathology , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , RNA, Small Interfering/genetics , Spheroids, Cellular , Triple Negative Breast Neoplasms/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Carcinoma, Ductal, Breast/pathology , Cell Transformation, Neoplastic/pathology , Histone-Lysine N-Methyltransferase/genetics , Mammary Neoplasms, Experimental/pathology , Nuclear Proteins/genetics , Animals , Carcinoma, Ductal, Breast/genetics , Cell Transformation, Neoplastic/genetics , Female , Humans , Hyperplasia , Lactation , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Transgenic , Neoplasm Grading , Promoter Regions, GeneticABSTRACT
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.
Subject(s)
Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Chemokine CXCL16/metabolism , Chemotactic Factors/pharmacology , Monocytes/pathology , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Animals , Cancer-Associated Fibroblasts/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Coculture Techniques , Collagen/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunosuppression Therapy , Mice, Nude , Monocytes/drug effects , Monocytes/metabolism , Myeloid Cells/metabolism , Myeloid Cells/pathology , Solubility , Stromal Cells/pathology , Triple Negative Breast Neoplasms/genetics , Tumor Microenvironment/drug effects , Xenograft Model Antitumor AssaysABSTRACT
A critical first step in the personalized approach to cancer treatment is the identification of activated oncogenes that drive each tumor. The Identification of driver oncogenes on a patient-by-patient basis is complicated by the complexity of the cancer genome and the fact that a particular genetic alteration may serve as a driver event only in a subset of tumors that harbor it. In this study, we set out to identify the complete set of functional oncogenes in a small panel of breast cancer cell lines. The cell lines in this panel were chosen because they each contain a known receptor tyrosine kinase (RTK) oncogene. To identify additional drivers, we integrated functional genetic screens with copy number and mutation analysis, and cancer genome knowledge databases. The resulting functional oncogene signatures were able to predict responsiveness of cell lines to targeted inhibitors. However, as single agents, these drugs had little effect on clonogenic potential. By contrast, treatment with drug combinations that targeted multiple oncogenes in the signatures, even at very low doses, resulted in the induction of apoptosis and striking synergistic effects on clonogenicity. In particular, targeting a driver oncogene that mediates AKT phosphorylation in combination with targeting the anti-apoptotic BCL2L1 protein had profound effects on cell viability. Importantly, because the synergistic induction of cell death was achieved using low levels of each individual drug, it suggests that a therapeutic strategy based on this approach could avoid the toxicities that have been associated with the combined use of multiple-targeted agents.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , Oncogene Proteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Death/drug effects , Cell Line , Cell Line, Tumor , Drug Synergism , Female , HEK293 Cells , High-Throughput Nucleotide Sequencing/methods , Humans , Molecular Targeted Therapy/methods , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , RNA Interference , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
Basal subtype breast cancers have a particularly poor prognosis, with high invasiveness and resistance to most targeted therapies. TGFß and MYC drive central features of basal breast cancer: TGFß is an autocrine and paracrine signaling factor that drives cell invasion and metastasis, and MYC is a central regulator of cellular proliferation that is upregulated in many cancer types. We show here that genetic or pharmacologic inhibition of MYC in MCF10A basal breast cells results in increased sensitivity to TGFß-stimulated invasion and metastasis and also show that this signaling loop is dependent on activation of SRC. Analysis of human breast cancer datasets and additional experiments with breast cancer cell lines further suggest the relevance of this signaling loop in basal, but not luminal, breast cancers. Our results imply precaution should be taken when utilizing therapeutic inhibitors of MYC with basal breast cancer patients as this could lead to increased metastasis; however, simultaneous pharmacologic inhibition of SRC and MYC for these patients could facilitate the antiproliferative effects of MYC inhibition while blocking the consequent promotion of metastasis. Cancer Res; 76(12); 3520-30. ©2016 AACR.
Subject(s)
Breast Neoplasms/pathology , Proto-Oncogene Proteins c-myc/physiology , Transforming Growth Factor beta/pharmacology , Cell Line, Tumor , Female , Humans , Integrin alphaVbeta3/physiology , Neoplasm Invasiveness , Neoplasm Metastasis , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , src-Family Kinases/physiologyABSTRACT
The 8p11-p12 amplicon occurs in approximately 15% of breast cancers in aggressive luminal B-type tumors. Previously, we identified WHSC1L1 as a driving oncogene from this region. Here, we demonstrate that over-expression of WHSC1L1 is linked to over-expression of ERα in SUM-44 breast cancer cells and in primary human breast cancers. Knock-down of WHSC1L1, particularly WHSC1L1-short, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM-44 cells do not require exogenous estrogen for growth in vitro; however, they are dependent on ERα expression, as ESR1 knock-down or exposure to the selective estrogen receptor degrader fulvestrant resulted in growth inhibition. ChIP-Seq experiments utilizing ERα antibodies demonstrated extensive ERα binding to chromatin in SUM-44 cells under estrogen-free conditions. ERα bound to ERE and FOXA1 motifs under estrogen-free conditions and regulated expression of estrogen-responsive genes. Short-term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes to which ERα was bound under estrogen-free conditions. Finally, knock-down of WHSC1L1 in SUM-44 cells resulted in loss of ERα binding to chromatin under estrogen-free conditions, which was restored upon exposure to estradiol. These results indicate the SUM-44 cells are a good model of a subset of luminal B breast cancers that have the 8p11-p12 amplicon, over-express WHSC1L1, and over-express ERα, but are independent of estrogen for binding to chromatin and regulation of gene expression. Breast cancers such as these, that are dependent on ERα activity but independent of estradiol, are a major cause of breast cancer mortality.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast/pathology , Estrogen Receptor alpha/genetics , Estrogens/metabolism , Gene Expression Regulation, Neoplastic , Histone-Lysine N-Methyltransferase/genetics , Nuclear Proteins/genetics , Breast/metabolism , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Chromatin/genetics , Chromatin/metabolism , Chromatin/pathology , Estrogen Receptor alpha/metabolism , Female , Humans , Up-RegulationSubject(s)
Cell Culture Techniques/methods , Databases, Factual , Animals , Cell Line , Humans , Species SpecificityABSTRACT
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 patterns; 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.
Subject(s)
Breast Neoplasms/genetics , Gene Expression/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Toll-Like Receptor 4/genetics , Cell Line, Tumor , Female , Humans , MCF-7 Cells , NF-kappa B/genetics , Signal Transduction/genetics , Toll-Like Receptor 2/geneticsABSTRACT
Currently, there are no effective targeted therapies for triple-negative breast cancer (TNBC) indicating a critical unmet need for breast cancer patients. Tumors that fall into the triple-negative category of breast cancers do not respond to the targeted therapies currently approved for breast cancer treatment, such as endocrine therapy (tamoxifen, aromatase inhibitors) or human epidermal growth factor receptor-2 (HER2) inhibitors (trastuzumab, lapatinib), because these tumors lack the most common breast cancer markers: estrogen receptor, progesterone receptor, and HER2. While many patients with TNBC respond to chemotherapy, subsets of patients fare poorly and relapse very quickly. Studies indicate that epidermal growth factor receptor (EGFR) is frequently overrepresented in TNBC (>50%), suggesting EGFR could be used as a biomarker and target in breast cancer. While it is clear that this growth factor receptor plays an integral role in TNBC, little is known about the mechanisms of sustained EGFR activation and how to target this protein despite availability of EGFR-targeted inhibitors, suggesting that our understanding of EGFR deregulation in TNBC is incomplete.
Subject(s)
Antineoplastic Agents/therapeutic use , ErbB Receptors/metabolism , Triple Negative Breast Neoplasms/metabolism , Biomarkers, Tumor/metabolism , Female , Humans , Lapatinib , Ligands , Quinazolines/therapeutic use , Receptor, ErbB-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Trastuzumab/therapeutic use , Triple Negative Breast Neoplasms/drug therapyABSTRACT
Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Cell Survival , Chaperonin Containing TCP-1/physiology , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Cell Line, Tumor , Female , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Oncogenes , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Survival AnalysisABSTRACT
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 ß1. 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.
Subject(s)
Breast Neoplasms/metabolism , EGF Family of Proteins/metabolism , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , PTEN Phosphohydrolase , Wnt Signaling Pathway , Amphiregulin , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , EGF Family of Proteins/genetics , ErbB Receptors/genetics , Female , HumansABSTRACT
The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.
Subject(s)
Breast Neoplasms/genetics , Chromosomes, Human, Pair 8 , Histone Acetyltransferases/genetics , Oncogenes , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cluster Analysis , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Histone Acetyltransferases/metabolism , Humans , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Prognosis , Protein Binding , RNA Interference , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Spheroids, Cellular , Tumor Cells, Cultured , Tumor Stem Cell AssayABSTRACT
Epithelial-to-mesenchymal transition (EMT) has been associated with poor treatment outcomes in various malignancies and is inversely associated with miRNA145 expression. Therefore, we hypothesized that SNAI2 (Slug) may mediate 5-fluorouracil (5FU) chemotherapy resistance through inhibition of miR145 in colorectal cancer and thus represents a novel therapeutic target to enhance current colorectal cancer treatment strategies. Compared with parental DLD1 colon cancer cells, 5FU-resistant (5FUr) DLD1 cells demonstrated features of EMT, including >2-fold enhanced invasion (P < 0.001) and migration, suppressed E-cadherin expression, and 2-fold increased SNAI2 expression. DLD1 and HCT116 cells with stable expression of SNAI2 (DLD1/SNAI2; HCT116/SNAI2) also demonstrated EMT features such as the decreased E-cadherin as well as significantly decreased miR145 expression, as compared with control empty vector cells. On the basis of an miR145 luciferase promoter assay, we demonstrated that SNAI2 repressed activity of the miR145 promoter in the DLD1 and HCT116 cells. In addition, the ectopic expressing SNAI2 cell lines demonstrated decreased 5FU sensitivity, and, conversely, miR145 replacement significantly enhanced 5FU sensitivity. In the parental SW620 colon cancer cell line with high SNAI2 and low miR145 levels, inhibition of SNAI2 directly with short hairpin sequence for SNAI2 and miR145 replacement therapy both decreased vimentin expression and increased in vitro 5FU sensitivity. In pretreatment rectal cancer patient biopsy samples, low miR145 expression levels correlated with poor response to neoadjuvant 5FU-based chemoradiation. These results suggested that the SNAI2:miR145 pathway may represent a novel clinical therapeutic target in colorectal cancer and may serve as a response predictor to chemoradiation therapy.