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BACKGROUND: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. METHODS: In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. RESULTS: We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. CONCLUSIONS: Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.
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[This corrects the article DOI: 10.1186/s12014-018-9197-x.].
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Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers.
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Adenocarcinoma/genética , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Quinasa 2 de Adhesión Focal/genética , Fosfoproteínas/genética , Tamoxifeno/farmacología , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenocarcinoma/terapia , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Proliferación Celular , Femenino , Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Quinasa 2 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Marcaje Isotópico , Células MCF-7 , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal , Análisis de Supervivencia , Activación Transcripcional , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The PIK3CA gene encodes for the p110 alpha isoform of PI3 kinase and is one of the most frequently mutated oncogenes in human cancers. However, the mechanisms by which PIK3CA mutations activate cell signaling are not fully understood. Here we used a phosphoproteomic approach to compare differential phosphorylation patterns between human breast epithelial cells and two isogenic somatic cell knock in derivatives, each harboring a distinct PIK3CA mutation. We demonstrated differential phosphorylation patterns between isogenic cell lines containing a PIK3CA helical domain mutation (E545K) compared to cells with a PIK3CA kinase domain mutation (H1047R). In particular, the receptor tyrosine kinase, HER3, showed increased phosphorylation at tyrosine 1328 in H1047R cells versus E545K cells. Genetic studies using shRNA demonstrated that H1047R cells have a profound decrease in growth factor independent proliferation upon HER3 knock down, but this effect was attenuated in E545K cells. In addition, HER3 knock down led to reductions in both PI3 kinase and MAP kinase pathway activation in H1047R cells, but in E545K cells only PI3 kinase pathway diminution was observed. These studies demonstrate the power of using paired isogenic cell lines for proteomic analysis to gain new insights into oncogenic signal transduction pathways.
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Neoplasias de la Mama/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteómica , Receptor ErbB-3/metabolismo , Transducción de Señal , Neoplasias de la Mama/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Estructura Terciaria de Proteína , Interferencia de ARN , Receptor ErbB-3/genéticaRESUMEN
The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis.
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Fosfatidilinositol 3-Quinasas/genética , Fosfoproteínas/genética , Proteoma/genética , Transducción de Señal/genética , Tirosina/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Humanos , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Proteoma/análisis , Proteoma/química , Proteoma/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Breast cancer is the most prevalent cancer in women worldwide. About 15-20% of all breast cancers do not express estrogen receptor, progesterone receptor or HER2 receptor and hence are collectively classified as triple negative breast cancer (TNBC). These tumors are often relatively aggressive when compared to other types of breast cancer, and this issue is compounded by the lack of effective targeted therapy. In our previous phosphoproteomic profiling effort, we identified the non-receptor tyrosine kinase TNK2 as activated in a majority of aggressive TNBC cell lines. In the current study, we show that high expression of TNK2 in breast cancer cell lines correlates with high proliferation, invasion and colony forming ability. We demonstrate that knockdown of TNK2 expression can substantially suppress the invasiveness and proliferation advantage of TNBC cells in vitro and tumor formation in xenograft mouse models. Moreover, inhibition of TNK2 with small molecule inhibitor (R)-9bMS significantly compromised TNBC proliferation.Finally, we find that high levels of TNK2 expression in high-grade basal-like breast cancers correlates significantly with poorer patient outcome. Taken together, our study suggests that TNK2 is a novel potential therapeutic target for the treatment of TNBC.
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Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Fosforilación , Pronóstico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Tasa de Supervivencia , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues.
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Proteínas de Choque Térmico HSP27/metabolismo , Isquemia/metabolismo , Fosfoproteínas/metabolismo , Fosfoserina/metabolismo , Proteómica/métodos , Animales , Biomarcadores/metabolismo , Biopsia , Línea Celular Tumoral , Humanos , Isquemia/patología , Ratones SCID , Fosforilación , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancer is the most prevalent cancer in women worldwide. About 15-20% of all breast cancers are triple negative breast cancer (TNBC) and are often highly aggressive when compared to other subtypes of breast cancers. To better characterize the biology that underlies the TNBC phenotype, we profiled the phosphotyrosine proteome of a panel of twenty-six TNBC cell lines using quantitative high resolution Fourier transform mass spectrometry. A heterogeneous pattern of tyrosine kinase activation was observed based on 1,789 tyrosine-phosphorylated peptides identified from 969 proteins. One of the tyrosine kinases, AXL, was found to be activated in a majority of aggressive TNBC cell lines and was accompanied by a higher level of AXL expression. High levels of AXL expression are correlated with a significant decrease in patient survival. Treatment of cells bearing activated AXL with a humanized AXL antibody inhibited cell proliferation and migration in vitro, and tumor growth in mice. Overall, our global phosphoproteomic analysis provided new insights into the heterogeneity in the activation status of tyrosine kinase pathways in TNBCs. Our approach presents an effective means of identifying important novel biomarkers and targets for therapy such as AXL in TNBC.
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Biomarcadores de Tumor/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteómica , Transducción de Señal , Neoplasias de la Mama Triple Negativas/enzimología , Animales , Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Activación Enzimática , Femenino , Análisis de Fourier , Humanos , Estimación de Kaplan-Meier , Espectrometría de Masas , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Fenotipo , Fosforilación , Mapas de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteómica/métodos , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapia , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing 'driver' oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets.