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Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is an aggressive subtype of this disease. Targeted treatment has improved outcome, but there is still a need for new therapeutic strategies as some patients respond poorly to treatment. Our aim was to identify compounds that substantially affect viability in HER2+ breast cancer cells in response to combinatorial treatment. We performed a high-throughput drug screen of 278 compounds in combination with trastuzumab and lapatinib using two HER2+ breast cancer cell lines (KPL4 and SUM190PT). The most promising drugs were validated in vitro and in vivo, and downstream molecular changes of the treatments were analyzed. The screen revealed multiple drugs that could be used in combination with lapatinib and/or trastuzumab. The Src-inhibitor dasatinib showed the largest combinatorial effect together with lapatinib in the KPL4 cell line compared to treatment with dasatinib alone (p < 0.01). In vivo, only lapatinib significantly reduced tumor growth (p < 0.05), whereas dasatinib alone, or in combination with lapatinib, did not show significant effects. Protein analyses of the treated xenografts showed significant alterations in protein levels compared to untreated controls, suggesting that all drugs reached the tumor and exerted a measurable effect. In silico analyses suggested activation of apoptosis and reduced activity of survival pathways by all treatments, but the opposite pattern was observed for the combinatorial treatment compared to lapatinib alone.
Assuntos
Antineoplásicos , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Lapatinib/farmacologia , Lapatinib/uso terapêutico , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral , Trastuzumab/uso terapêuticoRESUMO
Most cancer alterations occur in the noncoding portion of the human genome, where regulatory regions control gene expression. The discovery of noncoding mutations altering the cells' regulatory programs has been limited to few examples with high recurrence or high functional impact. Here, we show that transcription factor binding sites (TFBSs) have similar mutation loads to those in protein-coding exons. By combining cancer somatic mutations in TFBSs and expression data for protein-coding and miRNA genes, we evaluate the combined effects of transcriptional and post-transcriptional alterations on the regulatory programs in cancers. The analysis of seven TCGA cohorts culminates with the identification of protein-coding and miRNA genes linked to mutations at TFBSs that are associated with a cascading trans-effect deregulation on the cells' regulatory programs. Our analyses of cis-regulatory mutations associated with miRNAs recurrently predict 12 mature miRNAs (derived from 7 precursors) associated with the deregulation of their target gene networks. The predictions are enriched for cancer-associated protein-coding and miRNA genes and highlight cis-regulatory mutations associated with the dysregulation of key pathways associated with carcinogenesis. By combining transcriptional and post-transcriptional regulation of gene expression, our method predicts cis-regulatory mutations related to the dysregulation of key gene regulatory networks in cancer patients.
Assuntos
MicroRNAs , Neoplasias , Humanos , Regulação da Expressão Gênica , Neoplasias/genética , Mutação , MicroRNAs/fisiologia , Redes Reguladoras de GenesRESUMO
Long non-coding RNAs (lncRNAs) are involved in breast cancer pathogenesis through chromatin remodeling, transcriptional and post-transcriptional gene regulation. We report robust associations between lncRNA expression and breast cancer clinicopathological features in two population-based cohorts: SCAN-B and TCGA. Using co-expression analysis of lncRNAs with protein coding genes, we discovered three distinct clusters of lncRNAs. In silico cell type deconvolution coupled with single-cell RNA-seq analyses revealed that these three clusters were driven by cell type specific expression of lncRNAs. In one cluster lncRNAs were expressed by cancer cells and were mostly associated with the estrogen signaling pathways. In the two other clusters, lncRNAs were expressed either by immune cells or fibroblasts of the tumor microenvironment. To further investigate the cis-regulatory regions driving lncRNA expression in breast cancer, we identified subtype-specific transcription factor (TF) occupancy at lncRNA promoters. We also integrated lncRNA expression with DNA methylation data to identify long-range regulatory regions for lncRNA which were validated using ChiA-Pet-Pol2 loops. lncRNAs play an important role in shaping the gene regulatory landscape in breast cancer. We provide a detailed subtype and cell type-specific expression of lncRNA, which improves the understanding of underlying transcriptional regulation in breast cancer.
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Neoplasias da Mama , RNA Longo não Codificante , Neoplasias da Mama/patologia , Metilação de DNA , Feminino , Regulação da Expressão Gênica , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Microambiente TumoralRESUMO
Many breast cancer patients are diagnosed with small, well-differentiated, hormone receptor-positive tumors. Risk of relapse is not easily identified in these patients, resulting in overtreatment. To identify metastasis-related gene expression patterns, we compared the transcriptomes of the non-metastatic 67NR and metastatic 66cl4 cell lines from the murine 4T1 mammary tumor model. The transcription factor nuclear factor, erythroid 2-like 2 (NRF2, encoded by NFE2L2) was constitutively activated in the metastatic cells and tumors, and correspondingly a subset of established NRF2-regulated genes was also upregulated. Depletion of NRF2 increased basal levels of reactive oxygen species (ROS) and severely reduced ability to form primary tumors and lung metastases. Consistently, a set of NRF2-controlled genes was elevated in breast cancer biopsies. Sixteen of these were combined into a gene expression signature that significantly improves the PAM50 ROR score, and is an independent, strong predictor of prognosis, even in hormone receptor-positive tumors.
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Neoplasias da Mama , Fator 2 Relacionado a NF-E2 , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Recidiva Local de Neoplasia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aberrant DNA methylation is an early event in breast carcinogenesis and plays a critical role in regulating gene expression. Here, we perform genome-wide expression-methylation Quantitative Trait Loci (emQTL) analysis through the integration of DNA methylation and gene expression to identify disease-driving pathways under epigenetic control. By grouping the emQTLs using biclustering we identify associations representing important biological processes associated with breast cancer pathogenesis including regulation of proliferation and tumor-infiltrating fibroblasts. We report genome-wide loss of enhancer methylation at binding sites of proliferation-driving transcription factors including CEBP-ß, FOSL1, and FOSL2 with concomitant high expression of proliferation-related genes in aggressive breast tumors as we confirm with scRNA-seq. The identified emQTL-CpGs and genes were found connected through chromatin loops, indicating that proliferation in breast tumors is under epigenetic regulation by DNA methylation. Interestingly, the associations between enhancer methylation and proliferation-related gene expression were also observed within known subtypes of breast cancer, suggesting a common role of epigenetic regulation of proliferation. Taken together, we show that proliferation in breast cancer is linked to loss of methylation at specific enhancers and transcription factor binding and gene activation through chromatin looping.
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Purpose: Human epidermal growth factor receptor 2 positive (HER2+) breast cancers responding poorly to targeted therapy need improved treatment options. miR-101-5p has shown tumor-suppressive properties in multiple cancer forms, and we assessed the effect and mechanism of action of this miRNA in HER2+ breast cancer. Methods: Expression levels of miR-101-5p in two clinical datasets, TCGA and METABRIC, were compared between tumor and normal adjacent samples, and across molecular subtypes and HER2 status. The ability of miR-101-5p to sensitize for treatment with lapatinib, tucatinib and trastuzumab was explored in HER2+ breast cancer cells responding poorly to such targeted drugs. Proliferation and apoptosis assays and downstream protein analysis were performed. Results: Expression levels of miR-101-5p were significantly lower in tumor compared to normal adjacent tissue (p < 0.001), and particularly low in HER2+ tumors, both the HER2-enriched subtype (p ≤ 0.037) and clinical HER2-status (p < 0.001). In a HER2+ cell line (KPL4) responding poorly to targeted drugs, miR-101-5p overexpression inhibited proliferation (p < 0.001), and combinatorial treatment with lapatinib and trastuzumab significantly further decreased this inhibition (p = 0.004). Proteomic data and in silico analyses revealed PI3K/Akt- and HER2-pathways among the predicted regulated pathways. miR-101-5p alone (p = 0.018) and in combination with lapatinib and trastuzumab (p < 0.001) induced apoptosis, while the targeted drugs alone did not exert any significant effect neither on proliferation nor apoptosis. Conclusion: miR-101-5p acts as a tumor suppressor by inducing apoptosis in HER2+ breast cancer and sensitizes cells with initially poor response to lapatinib and trastuzumab.
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HER2-positive (HER2 +) breast cancer patients that do not respond to targeted treatment have a poor prognosis. The effects of targeted treatment on endogenous microRNA (miRNA) expression levels are unclear. We report that responsive HER2 + breast cancer cell lines had a higher number of miRNAs with altered expression after treatment with trastuzumab and lapatinib compared to poorly responsive cell lines. To evaluate whether miRNAs can sensitize HER2 + cells to treatment, we performed a high-throughput screen of 1626 miRNA mimics and inhibitors in combination with trastuzumab and lapatinib in HER2 + breast cancer cells. We identified eight miRNA mimics sensitizing cells to targeted treatment, miR-101-5p, mir-518a-5p, miR-19b-2-5p, miR-1237-3p, miR-29a-3p, miR-29c-3p, miR-106a-5p, and miR-744-3p. A higher expression of miR-101-5p predicted better prognosis in patients with HER2 + breast cancer (OS: p = 0.039; BCSS: p = 0.012), supporting the tumor-suppressing role of this miRNA. In conclusion, we have identified miRNAs that sensitize HER2 + breast cancer cells to targeted therapy. This indicates the potential of combining targeted drugs with miRNAs to improve current treatments for HER2 + breast cancers.
Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica/métodos , Lapatinib/farmacologia , MicroRNAs/genética , Trastuzumab/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Prognóstico , Análise de Sobrevida , Regulação para CimaRESUMO
BACKGROUND: Abnormal DNA methylation is observed as an early event in breast carcinogenesis. However, how such alterations arise is still poorly understood. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play key roles in various biological processes. Here, we integrate miRNA expression and DNA methylation at CpGs to study how miRNAs may affect the breast cancer methylome and how DNA methylation may regulate miRNA expression. METHODS: miRNA expression and DNA methylation data from two breast cancer cohorts, Oslo2 (n = 297) and The Cancer Genome Atlas (n = 439), were integrated through a correlation approach that we term miRNA-methylation Quantitative Trait Loci (mimQTL) analysis. Hierarchical clustering was used to identify clusters of miRNAs and CpGs that were further characterized through analysis of mRNA/protein expression, clinicopathological features, in silico deconvolution, chromatin state and accessibility, transcription factor binding, and long-range interaction data. RESULTS: Clustering of the significant mimQTLs identified distinct groups of miRNAs and CpGs that reflect important biological processes associated with breast cancer pathogenesis. Notably, two major miRNA clusters were related to immune or fibroblast infiltration, hence identifying miRNAs associated with cells of the tumor microenvironment, while another large cluster was related to estrogen receptor (ER) signaling. Studying the chromatin landscape surrounding CpGs associated with the estrogen signaling cluster, we found that miRNAs from this cluster are likely to be regulated through DNA methylation of enhancers bound by FOXA1, GATA2, and ER-alpha. Further, at the hub of the estrogen cluster, we identified hsa-miR-29c-5p as negatively correlated with the mRNA and protein expression of DNA methyltransferase DNMT3A, a key enzyme regulating DNA methylation. We found deregulation of hsa-miR-29c-5p already present in pre-invasive breast lesions and postulate that hsa-miR-29c-5p may trigger early event abnormal DNA methylation in ER-positive breast cancer. CONCLUSIONS: We describe how miRNA expression and DNA methylation interact and associate with distinct breast cancer phenotypes.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Hormônios/farmacologia , MicroRNAs/genética , Cromatina/metabolismo , Ilhas de CpG/genética , DNA Metiltransferase 3A/metabolismo , Elementos Facilitadores Genéticos/genética , Feminino , Redes Reguladoras de Genes , Humanos , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Família Multigênica , Fenótipo , Locos de Características Quantitativas/genéticaRESUMO
Different miRNA profiling protocols and technologies introduce differences in the resulting quantitative expression profiles. These include differences in the presence (and measurability) of certain miRNAs. We present and examine a method based on quantile normalization, Adjusted Quantile Normalization (AQuN), to combine miRNA expression data from multiple studies in breast cancer into a single joint dataset for integrative analysis. By pooling multiple datasets, we obtain increased statistical power, surfacing patterns that do not emerge as statistically significant when separately analyzing these datasets. To merge several datasets, as we do here, one needs to overcome both technical and batch differences between these datasets. We compare several approaches for merging and jointly analyzing miRNA datasets. We investigate the statistical confidence for known results and highlight potential new findings that resulted from the joint analysis using AQuN. In particular, we detect several miRNAs to be differentially expressed in estrogen receptor (ER) positive versus ER negative samples. In addition, we identify new potential biomarkers and therapeutic targets for both clinical groups. As a specific example, using the AQuN-derived dataset we detect hsa-miR-193b-5p to have a statistically significant over-expression in the ER positive group, a phenomenon that was not previously reported. Furthermore, as demonstrated by functional assays in breast cancer cell lines, overexpression of hsa-miR-193b-5p in breast cancer cell lines resulted in decreased cell viability in addition to inducing apoptosis. Together, these observations suggest a novel functional role for this miRNA in breast cancer. Packages implementing AQuN are provided for Python and Matlab: https://github.com/YakhiniGroup/PyAQN.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Algoritmos , Biomarcadores/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Simulação por Computador , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Análise de Sequência com Séries de Oligonucleotídeos , Linguagens de Programação , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Previously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER-), cytokeratin 5/6 positivity and basal-like features of breast cancer. METHODS: We investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed. RESULTS: CISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b. CONCLUSIONS: Our study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER- tumours.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Macrófagos/imunologia , MicroRNAs/genética , Células Estromais/metabolismo , Biomarcadores Tumorais/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Estudos de Coortes , Bases de Dados Genéticas/estatística & dados numéricos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Células Estromais/imunologia , Células Estromais/patologiaRESUMO
The long non-coding RNA NEAT1 locus is transcribed into two overlapping isoforms, NEAT1_1 and NEAT1_2, of which the latter is essential for the assembly of nuclear paraspeckles. NEAT1 is abnormally expressed in a wide variety of human cancers. Emerging evidence suggests that the two isoforms have distinct functions in gene expression regulation, and recently it was shown that NEAT1_2, but not NEAT1_1, expression predicts poor clinical outcome in cancer. Here, we report that NEAT1_2 expression correlates with HER2-positive breast cancers and high-grade disease. We provide evidence that NEAT1_1 and NEAT1_2 have distinct expression pattern among different intrinsic breast cancer subtypes. Finally, we show that NEAT1_2 expression and paraspeckle formation increase upon lactation in humans, confirming what has previously been demonstrated in mice.
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Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7RESUMO
Genomic amplification of 3q26.2 locus leads to the increased expression of microRNA 551b-3p (miR551b-3p) in triple-negative breast cancer (TNBC). Our results demonstrate that miR551b-3p translocates to the nucleus with the aid of importin-8 (IPO8) and activates STAT3 transcription. As a consequence, miR551b upregulates the expression of oncostatin M receptor (OSMR) and interleukin-31 receptor-α (IL-31RA) as well as their ligands OSM and IL-31 through STAT3 transcription. We defined this set of genes induced by miR551b-3p as the "oncostatin signaling module," which provides oncogenic addictions in cancer cells. Notably, OSM is highly expressed in TNBC, and the elevated expression of OSM associates with poor outcome in estrogen-receptor-negative breast cancer patients. Conversely, targeting miR551b with anti-miR551b-3p reduced the expression of the OSM signaling module and reduced tumor growth, as well as migration and invasion of breast cancer cells.
Assuntos
Progressão da Doença , MicroRNAs/metabolismo , Oncostatina M/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos Nus , MicroRNAs/genética , Terapia de Alvo Molecular , Invasividade Neoplásica , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica , Ativação Transcricional/genética , Regulação para Cima/genética , beta Carioferinas/metabolismoRESUMO
BACKGROUND: Breast cancer is a heterogeneous disease at the clinical and molecular level. In this study we integrate classifications extracted from five different molecular levels in order to identify integrated subtypes. METHODS: Tumor tissue from 425 patients with primary breast cancer from the Oslo2 study was cut and blended, and divided into fractions for DNA, RNA and protein isolation and metabolomics, allowing the acquisition of representative and comparable molecular data. Patients were stratified into groups based on their tumor characteristics from five different molecular levels, using various clustering methods. Finally, all previously identified and newly determined subgroups were combined in a multilevel classification using a "cluster-of-clusters" approach with consensus clustering. RESULTS: Based on DNA copy number data, tumors were categorized into three groups according to the complex arm aberration index. mRNA expression profiles divided tumors into five molecular subgroups according to PAM50 subtyping, and clustering based on microRNA expression revealed four subgroups. Reverse-phase protein array data divided tumors into five subgroups. Hierarchical clustering of tumor metabolic profiles revealed three clusters. Combining DNA copy number and mRNA expression classified tumors into seven clusters based on pathway activity levels, and tumors were classified into ten subtypes using integrative clustering. The final consensus clustering that incorporated all aforementioned subtypes revealed six major groups. Five corresponded well with the mRNA subtypes, while a sixth group resulted from a split of the luminal A subtype; these tumors belonged to distinct microRNA clusters. Gain-of-function studies using MCF-7 cells showed that microRNAs differentially expressed between the luminal A clusters were important for cancer cell survival. These microRNAs were used to validate the split in luminal A tumors in four independent breast cancer cohorts. In two cohorts the microRNAs divided tumors into subgroups with significantly different outcomes, and in another a trend was observed. CONCLUSIONS: The six integrated subtypes identified confirm the heterogeneity of breast cancer and show that finer subdivisions of subtypes are evident. Increasing knowledge of the heterogeneity of the luminal A subtype may add pivotal information to guide therapeutic choices, evidently bringing us closer to improved treatment for this largest subgroup of breast cancer.
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Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Análise por Conglomerados , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/mortalidade , Variações do Número de Cópias de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Redes e Vias Metabólicas , Metabolômica/métodos , MicroRNAs/genética , Noruega/epidemiologia , Prognóstico , RNA Mensageiro/genéticaRESUMO
Breast cancer patients with Luminal A disease generally have a good prognosis, but among this patient group are patients with good prognosis that are currently overtreated with adjuvant chemotherapy, and also patients that have a bad prognosis and should be given more aggressive treatment. There is no available method for subclassification of this patient group. Here we present a DNA methylation signature (SAM40) that segregates Luminal A patients based on prognosis, and identify one good prognosis group and one bad prognosis group. The prognostic impact of SAM40 was validated in four independent patient cohorts. Being able to subdivide the Luminal A patients may give the two-sided benefit of identifying one subgroup that may benefit from a more aggressive treatment than what is given today, and importantly, identifying a subgroup that may benefit from less treatment.
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Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Metilação de DNA , Transcriptoma , Neoplasias da Mama/patologia , Análise por Conglomerados , Epigênese Genética , Epigenômica/métodos , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , PrognósticoRESUMO
Breast cancer consists of at least five main molecular "intrinsic" subtypes that are reflected in both pre-invasive and invasive disease. Although previous studies have suggested that many of the molecular features of invasive breast cancer are established early, it is unclear what mechanisms drive progression and whether the mechanisms of progression are dependent or independent of subtype. We have generated mRNA, miRNA, and DNA copy-number profiles from a total of 59 in situ lesions and 85 invasive tumors in order to comprehensively identify those genes, signaling pathways, processes, and cell types that are involved in breast cancer progression. Our work provides evidence that there are molecular features associated with disease progression that are unique to the intrinsic subtypes. We additionally establish subtype-specific signatures that are able to identify a small proportion of pre-invasive tumors with expression profiles that resemble invasive carcinoma, indicating a higher likelihood of future disease progression.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , RNA Mensageiro/genética , Transdução de Sinais/genéticaRESUMO
BACKGROUND: The role played by microRNAs in the deregulation of protein expression in breast cancer is only partly understood. To gain insight, the combined effect of microRNA and mRNA expression on protein expression was investigated in three independent data sets. METHODS: Protein expression was modeled as a multilinear function of powers of mRNA and microRNA expression. The model was first applied to mRNA and protein expression for 105 selected cancer-associated genes and to genome-wide microRNA expression from 283 breast tumors. The model considered both the effect of one microRNA at a time and all microRNAs combined. In the latter case the Lasso penalized regression method was applied to detect the simultaneous effect of multiple microRNAs. RESULTS: An interactome map for breast cancer representing all direct and indirect associations between the expression of microRNAs and proteins was derived. A pattern of extensive coordination between microRNA and protein expression in breast cancer emerges, with multiple clusters of microRNAs being associated with multiple clusters of proteins. Results were subsequently validated in two independent breast cancer data sets. A number of the microRNA-protein associations were functionally validated in a breast cancer cell line. CONCLUSIONS: A comprehensive map is derived for the co-expression in breast cancer of microRNAs and 105 proteins with known roles in cancer, after filtering out the in-cis effect of mRNA expression. The analysis suggests that group action by several microRNAs to deregulate the expression of proteins is a common modus operandi in breast cancer.
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The let-7 microRNA (miRNA) family has been implicated in the regulation of diverse cellular processes and disease pathogenesis. In cancer, loss-of-function of let-7 miRNAs has been linked to tumorigenesis via increased expression of target oncogenes. Excessive proliferation rate of tumor cells is often associated with deregulation of mitotic proteins. Here, we show that let-7b contributes to the maintenance of genomic balance via targeting Aurora B kinase, a key regulator of the spindle assembly checkpoint (SAC). Our results indicate that let-7b binds to Aurora B kinase 3'UTR reducing mRNA and protein expression of the kinase. In cells, excess let-7b induced mitotic defects characteristic to Aurora B perturbation including increased rate of polyploidy and multipolarity, and premature SAC inactivation that leads to forced exit from chemically induced mitotic arrest. Moreover, the frequency of aneuploid HCT-116 cells was significantly increased upon let-7b overexpression compared to controls. Interestingly, together with a chemical Aurora B inhibitor, let-7b had an additive effect on polyploidy induction in HeLa cells. In breast cancer patients, reduced let-7b expression was found to be associated with increased Aurora B expression in grade 3 tumors. Furthermore, let-7b was found downregulated in the most aggressive forms of breast cancer determined by clinicopathological parameters. Together, our findings suggest that let-7b contributes to the fidelity of cell division via regulation of Aurora B. Moreover, the loss of let-7b in aggressive tumors may drive tumorigenesis by up-regulation of Aurora B and other targets of the miRNA, which further supports the role of let-7b in tumor suppression.
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Aurora Quinase B/metabolismo , Neoplasias da Mama/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Poliploidia , RNA Neoplásico/metabolismo , Regiões 3' não Traduzidas , Aurora Quinase B/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Células HeLa , Humanos , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Neoplásico/genéticaRESUMO
A large number of breast cancers are characterized by amplification and overexpression of the chromosome segment surrounding the HER2 (ERBB2) oncogene. As the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to trastuzumab resistance. We integrated array comparative genomic hybridization (aCGH) data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes with several phenotypic endpoints in a panel of trastuzumab responding and non-responding HER2 positive breast cancer cells. In this Data in Brief we give a detailed description of the experimental procedures and the data analysis methods used in the study (1).
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BACKGROUND: The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. RESULTS: We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. CONCLUSIONS: Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.
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Neoplasias da Mama/genética , Variações do Número de Cópias de DNA , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Replicação do DNA , Feminino , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismoRESUMO
Genomic copy number alterations are common in cancer. Finding the genes causally implicated in oncogenesis is challenging because the gain or loss of a chromosomal region may affect a few key driver genes and many passengers. Integrative analyses have opened new vistas for addressing this issue. One approach is to identify genes with frequent copy number alterations and corresponding changes in expression. Several methods also analyse effects of transcriptional changes on known pathways. Here, we propose a method that analyses in-cis correlated genes for evidence of in-trans association to biological processes, with no bias towards processes of a particular type or function. The method aims to identify cis-regulated genes for which the expression correlation to other genes provides further evidence of a network-perturbing role in cancer. The proposed unsupervised approach involves a sequence of statistical tests to systematically narrow down the list of relevant genes, based on integrative analysis of copy number and gene expression data. A novel adjustment method handles confounding effects of co-occurring copy number aberrations, potentially a large source of false positives in such studies. Applying the method to whole-genome copy number and expression data from 100 primary breast carcinomas, 6373 genes were identified as commonly aberrant, 578 were highly in-cis correlated, and 56 were in addition associated in-trans to biological processes. Among these in-trans process associated and cis-correlated (iPAC) genes, 28% have previously been reported as breast cancer associated, and 64% as cancer associated. By combining statistical evidence from three separate subanalyses that focus respectively on copy number, gene expression and the combination of the two, the proposed method identifies several known and novel cancer driver candidates. Validation in an independent data set supports the conclusion that the method identifies genes implicated in cancer.