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BACKGROUND: Particular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor-stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs. METHODS: MS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue samples and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival. RESULTS: Phosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated 'omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 amplification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and amplification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target. CONCLUSIONS: This work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.
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Neoplasias da Mama/tratamento farmacológico , Modelos Biológicos , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Terapia de Alvo Molecular , Mutação , Organoides/efeitos dos fármacos , Organoides/metabolismo , Fosforilação , Medicina de Precisão , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In prostate cancer, cancer-associated fibroblasts (CAF) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPF) and promote tumorigenesis. Resolving intercellular signaling pathways between CAF and prostate tumor epithelium may offer novel opportunities for research translation. To this end, the proteome and phosphoproteome of four pairs of patient-matched CAF and NPF were characterized to identify discriminating proteomic signatures. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a hyper reaction monitoring data-independent acquisition (HRM-DIA) workflow. Proteins that exhibited a significant increase in CAF versus NPF were enriched for the functional categories "cell adhesion" and the "extracellular matrix." The CAF phosphoproteome exhibited enhanced phosphorylation of proteins associated with the "spliceosome" and "actin binding." STRING analysis of the CAF proteome revealed a prominent interaction hub associated with collagen synthesis, modification, and signaling. It contained multiple collagens, including the fibrillar types COL1A1/2 and COL5A1; the receptor tyrosine kinase discoidin domain-containing receptor 2 (DDR2), a receptor for fibrillar collagens; and lysyl oxidase-like 2 (LOXL2), an enzyme that promotes collagen crosslinking. Increased activity and/or expression of LOXL2 and DDR2 in CAF were confirmed by enzymatic assays and Western blotting analyses. Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization and decreased CAF migration in a wound healing assay. Further, it significantly impaired the motility of co-cultured RWPE-2 prostate tumor epithelial cells. These results indicate that CAF-derived LOXL2 is an important mediator of intercellular communication within the prostate tumor microenvironment and is a potential therapeutic target.
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Aminoácido Oxirredutases/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Neoplasias da Próstata/metabolismo , Proteômica , Microambiente Tumoral , Comunicação Autócrina , Linhagem Celular Tumoral , Movimento Celular , Células Epiteliais/patologia , Matriz Extracelular/metabolismo , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Comunicação Parácrina , Fosfoproteínas/metabolismo , Fosforilação , Próstata/metabolismo , Próstata/patologia , Proteoma/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
ABSTRACT: The authors present 3 unique cases of complex fistula formations because of orbital fracture repair with a Teflon (polytetrafluroethylene) implant. A 26-year-old man presented with dacryocystitis and a cutaneous fistula 8 years after left orbital floor and medial wall fracture repair with a Teflon implant. A 46-year-old woman suffered orbital trauma after a motor vehicle accident as a teenager and the fracture was repaired with Teflon implant. Thirty-two years later, she presented with lower eyelid fistula, ectropion, and retraction. A 65-year-old woman also previously had Teflon implants for the repair of her left inferior and lateral orbital rim after a motor vehicle accident. Twenty-five years later, she presented with chronic infections involving the repaired areas, as well as left lower lid ectropion and fistula formation. The woven material nature of Teflon acted as a nidus for infection, inflammation, and led to complex cutaneous fistula formations in these patients.
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Doenças Orbitárias , Fraturas Orbitárias , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Órbita , Fraturas Orbitárias/etiologia , Fraturas Orbitárias/cirurgia , Politetrafluoretileno/efeitos adversos , Próteses e ImplantesRESUMO
BACKGROUND: Triple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target. METHODS: MS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1-3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival. RESULTS: High FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare. CONCLUSIONS: These results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion. Video abstract.
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Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/fisiopatologiaRESUMO
Inhibition of the heat shock protein 90 (Hsp90) chaperone is a promising therapeutic strategy to target expression of the androgen receptor (AR) and other oncogenic drivers in prostate cancer cells. However, identification of clinically-relevant responses and predictive biomarkers is essential to maximize efficacy and treatment personalization. Here, we combined mass spectrometry (MS)-based proteomic analyses with a unique patient-derived explant (PDE) model that retains the complex microenvironment of primary prostate tumors. Independent discovery and validation cohorts of PDEs (n = 16 and 30, respectively) were cultured in the absence or presence of Hsp90 inhibitors AUY922 or 17-AAG. PDEs were analyzed by LC-MS/MS with a hyper-reaction monitoring data independent acquisition (HRM-DIA) workflow, and differentially expressed proteins identified using repeated measure analysis of variance (ANOVA; raw p value <0.01). Using gene set enrichment, we found striking conservation of the most significantly AUY922-altered gene pathways between the discovery and validation cohorts, indicating that our experimental and analysis workflows were robust. Eight proteins were selectively altered across both cohorts by the most potent inhibitor, AUY922, including TIMP1, SERPINA3 and CYP51A (adjusted p < 0.01). The AUY922-mediated decrease in secretory TIMP1 was validated by ELISA of the PDE culture medium. We next exploited the heterogeneous response of PDEs to 17-AAG in order to detect predictive biomarkers of response and identified PCBP3 as a marker with increased expression in PDEs that had no response or increased in proliferation. Also, 17-AAG treatment led to increased expression of DNAJA1 in PDEs that exhibited a cytostatic response, revealing potential drug resistance mechanisms. This selective regulation of DNAJA1 was validated by Western blot analysis. Our study establishes "proof-of-principle" that proteomic profiling of drug-treated PDEs represents an effective and clinically-relevant strategy for identification of biomarkers that associate with certain tumor-specific responses.
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Biomarcadores Tumorais/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias da Próstata/metabolismo , Proteômica/métodos , Benzoquinonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Isoxazóis/farmacologia , Lactamas Macrocíclicas/farmacologia , Masculino , Proteínas de Neoplasias/metabolismo , Análise de Componente Principal , Neoplasias da Próstata/patologia , Proteoma/metabolismo , Reprodutibilidade dos Testes , Resorcinóis/farmacologiaRESUMO
Platinum-resistance is a major limitation to effective chemotherapy regimens in high-grade serous ovarian cancer (HGSOC). To better understand the mechanisms involved we characterized the proteome and phosphoproteome in cisplatin sensitive and resistant HGSOC primary cells using a mass spectrometry-based proteomic strategy. PCA analysis identified a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines. The most phosphorylated protein in cisplatin resistant cells was sequestosome-1 (p62/SQSTM1). Changes in expression of apoptosis and autophagy related proteins Caspase-3 and SQSTM1, respectively, were validated by Western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while SQSTM1 revealed increased expression in the resistant cell line relative to sensitive cell line. Furthermore, site-specific phosphorylation on 20 amino acid residues of SQSTM1 was detected indicating a hyper-phosphorylation phenotype. This elevated hyper-phosphorylation of SQSTM1 in resistant HGSOC cell lines was validated with Western blot analysis. Immunofluoresence staining of s28-pSQSTM1 showed inducible localization to autophagosomes upon cisplatin treatment in the sensitive cell line while being constitutively expressed to autophagosomes in the resistant cell. Furthermore, SQSTM1 expression was localized in cancer cells of clinical high-grade serous tumors. Here, we propose hyper-phosphorylation of SQSTM1 as a marker and a key proteomic change in cisplatin resistance development in ovarian cancers by activating the autophagy pathway and influencing down-regulation of apoptosis.
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Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Sequestossoma-1/metabolismo , Autofagossomos/metabolismo , Caspase 1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Espectrometria de Massas , Gradação de Tumores , Fosforilação , Estudos Prospectivos , Proteômica/métodos , Proteína Sequestossoma-1/químicaRESUMO
The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.
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Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Transdução de Sinais , Trichoderma/metabolismo , Sítios de Ligação , Celulases/metabolismo , Cromatografia Líquida , Glucanos/metabolismo , Glicólise , Glicosídeo Hidrolases/metabolismo , Hidrólise , Fosforilação , Sorbitol/metabolismo , Espectrometria de Massas em TandemRESUMO
The lung is an important reservoir of human immunodeficiency virus (HIV). Individuals infected with HIV are more prone to pulmonary infections and chronic lung disorders. We hypothesized that comprehensively profiling the proteomic landscape of bronchoalveolar lavage fluid (BALF) in patients with HIV would provide insights into how this virus alters the lung milieu and contributes to pathogenesis of HIV-related lung diseases. BALF was obtained from five HIV-negative (HIV(-)) and six asymptomatic HIV-positive (HIV(+)) subjects not on antiretroviral therapy. Each sample underwent shotgun proteomic analysis based on HPLC-tandem mass spectrometry. Differentially expressed proteins between the groups were identified using statistical methods based on spectral counting. Mechanisms of disease were explored using functional annotation to identify overlapping and distinct pathways enriched between the BALF proteome of HIV(+) and HIV(-) subjects. We identified a total of 318 unique proteins in BALF of HIV(-) and HIV(+) subjects. Of these, 87 were differentially up- or downregulated between the two groups. Many of these differentially expressed proteins are known to interact with key HIV proteins. Functional analysis of differentially regulated proteins implicated downregulation of immune responses in lungs of HIV(+) patients. Combining shotgun proteomic analysis with computational methods demonstrated that the BALF proteome is significantly altered during HIV infection. We found that immunity-related pathways are underrepresented in HIV(+) patients. These findings implicate mechanisms whereby HIV invokes local immunosuppression in the lung and increases the susceptibility of HIV(+) patients to develop a wide range of infectious and noninfectious pulmonary diseases.
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Infecções por HIV/metabolismo , HIV-1 , Proteoma/metabolismo , Adulto , Líquido da Lavagem Broncoalveolar , Estudos de Casos e Controles , Citocinas/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Humanos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Different molecular classifications for gastric cancer (GC) have been proposed based on multi-omics platforms with the long-term goal of improved precision treatment. However, the GC (phospho)proteome remains incompletely characterized, particularly at the level of tyrosine phosphorylation. In addition, previous multiomics-based stratification of patient cohorts has lacked identification of corresponding cell line models and comprehensive validation of broad or subgroup-selective therapeutic targets. To address these knowledge gaps, we applied a reverse approach, undertaking the most comprehensive (phospho)proteomic analysis of GC cell lines to date and cross-validating this using publicly available data. Mass spectrometry (MS)-based (phospho)proteomic and tyrosine phosphorylation datasets were subjected to individual or integrated clustering to identify subgroups that were subsequently characterized in terms of enriched molecular processes and pathways. Significant congruence was detected between cell line proteomic and specific patient-derived transcriptomic subclassifications. Many protein kinases exhibiting 'outlier' expression or phosphorylation in the cell line dataset exhibited genomic aberrations in patient samples and association with poor prognosis, with casein kinase I isoform delta/epsilon (CSNK1D/E) being experimentally validated as potential therapeutic targets. Src family kinases were predicted to be commonly hyperactivated in GC cell lines, consistent with broad sensitivity to the next-generation Src inhibitor eCF506. In addition, phosphoproteomic and integrative clustering segregated the cell lines into two subtypes, with epithelial-mesenchyme transition (EMT) and proliferation-associated processes enriched in one, designated the EMT subtype, and metabolic pathways, cell-cell junctions, and the immune response dominating the features of the other, designated the metabolism subtype. Application of kinase activity prediction algorithms and interrogation of gene dependency and drug sensitivity databases predicted that the mechanistic target of rapamycin kinase (mTOR) and dual specificity mitogen-activated protein kinase kinase 2 (MAP2K2) represented potential therapeutic targets for the EMT and metabolism subtypes, respectively, and this was confirmed using selective inhibitors. Overall, our study provides novel, in-depth insights into GC proteomics, kinomics, and molecular taxonomy and reveals potential therapeutic targets that could provide the basis for precision treatments.
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Proteoma , Neoplasias Gástricas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/classificação , Humanos , Proteoma/metabolismo , Linhagem Celular Tumoral , Proteômica/métodos , Fosforilação , Terapia de Alvo MolecularRESUMO
The PEAK family of pseudokinases, comprising PEAK1-3, are signalling scaffolds that play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screened for PEAK1 effectors by affinity purification and mass spectrometry, identifying calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promoted CAMK2D/G activation in TNBC cells via a novel feed-forward mechanism involving PEAK1/PLCγ1/Ca 2+ signalling and direct binding via a consensus CAMK2 interaction motif in the PEAK1 N-terminus. In turn, CAMK2 phosphorylated PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurposed RA306, a second generation CAMK2 inhibitor under pre-clinical development for treatment of cardiovascular disease. RA306 demonstrated on-target activity against CAMK2 in TNBC cells and inhibited PEAK1-enhanced migration and invasion in vitro . Moreover, RA306 significantly attenuated TNBC xenograft growth and blocked metastasis in a manner mirrored by CRISPR-mediated PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus, identify a novel mechanism for regulation of Ca 2+ signalling and its integration with tyrosine kinase signals, and identify CAMK2 as a therapeutically 'actionable' target downstream of PEAK1.
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Precision medicine has emerged as an important paradigm in oncology, driven by the significant heterogeneity of individual patients' tumour. A key prerequisite for effective implementation of precision oncology is the development of companion biomarkers that can predict response to anti-cancer therapies and guide patient selection for clinical trials and/or treatment. However, reliable predictive biomarkers are currently lacking for many anti-cancer therapies, hampering their clinical application. Here, we developed a novel machine learning-based framework to derive predictive multi-gene biomarker panels and associated expression signatures that accurately predict cancer drug sensitivity. We demonstrated the power of the approach by applying it to identify response biomarker panels for an Hsp90-based therapy in prostate cancer, using proteomic data profiled from prostate cancer patient-derived explants. Our approach employs a rational feature section strategy to maximise model performance, and innovatively utilizes Boolean algebra methods to derive specific expression signatures of the marker proteins. Given suitable data for model training, the approach is also applicable to other cancer drug agents in different tumour settings.
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Prostate cancer is the second most common cause of cancer death in males. A greater understanding of cell signalling events that occur within the prostate cancer tumour microenvironment (TME), for example, between cancer-associated fibroblasts (CAFs) and prostate epithelial or cancer cells, may identify novel biomarkers and more effective therapeutic strategies for this disease. To address this, we used cell-type-specific labelling with amino acid precursors (CTAP) to define cell-type-specific (phospho)proteomic changes that occur when prostate epithelial cells are co-cultured with normal patient-derived prostate fibroblasts (NPFs) versus matched CAFs. We report significant differences in the response of BPH-1 benign prostate epithelial cells to CAF versus NPF co-culture. Pathway analysis of proteomic changes identified significant upregulation of focal adhesion and cytoskeleton networks, and downregulation of metabolism pathways, in BPH-1 cells cultured with CAFs. In addition, co-cultured CAFs exhibited alterations in stress, DNA damage, and cytoskeletal networks. Functional validation of one of the top differentially-regulated proteins in BPH-1 cells upon CAF co-culture, transglutaminase-2 (TGM2), demonstrated that knockdown of this protein significantly reduced the proliferation and migration of prostate epithelial cells. Overall, this study provides novel insights into intercellular communication in the prostate cancer TME that may be exploited to improve patient management.
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We have determined that expression of the pseudokinase NRBP1 positively associates with poor prognosis in triple negative breast cancer (TNBC) and is required for efficient migration, invasion and proliferation of TNBC cells in culture as well as growth of TNBC orthotopic xenografts and experimental metastasis. Application of BioID/MS profiling identified P-Rex1, a known guanine nucleotide exchange factor for Rac1, as a NRBP1 binding partner. Importantly, NRBP1 overexpression enhanced levels of GTP-bound Rac1 and Cdc42 in a P-Rex1-dependent manner, while NRBP1 knockdown reduced their activation. In addition, NRBP1 associated with P-Rex1, Rac1 and Cdc42, suggesting a scaffolding function for this pseudokinase. NRBP1-mediated promotion of cell migration and invasion was P-Rex1-dependent, while constitutively-active Rac1 rescued the effect of NRBP1 knockdown on cell proliferation and invasion. Generation of reactive oxygen species via a NRBP1/P-Rex1 pathway was implicated in these oncogenic roles of NRBP1. Overall, these findings define a new function for NRBP1 and a novel oncogenic signalling pathway in TNBC that may be amenable to therapeutic intervention.
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Neoplasias de Mama Triplo Negativas , Humanos , Proteínas rac1 de Ligação ao GTP/metabolismo , Transdução de Sinais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Movimento Celular , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
PURPOSE: To examine the relation between asymmetric eyebrow elevation and ocular dominance. METHODS: A retrospective cohort study was performed in patients who underwent bilateral upper eyelid blepharoplasty from July 2007 to May 2009. Patients with pre- and postoperative involuntary asymmetric eyebrow elevation were selected from a surgical log database for inclusion in this study. Brow asymmetry was determined by examination of preoperative and postoperative digital photographs and was defined as a measured eyebrow-height difference in conjunction with the asymmetric recruitment of the frontalis muscle. Ocular dominance was determined by using a modified Porta test. Statistical analysis was performed by using the Fisher Exact test. RESULTS: Forty-seven patients met inclusion criteria. Sixty-six percent (31 of 47) involuntarily, asymmetrically elevated the right eyebrow, and 34% (16 of 47), the left. Of the 31 patients with right eyebrow elevation, 87% (27 of 31) were right-eye dominant, and 13% (4 of 31) were left-eye dominant (p < 0.001). Among patients with asymmetric left eyebrow elevation, 62.5% (10 of 16) were left-eye dominant, whereas only 37.5% (6 of 16) patients were right-eye dominant (p < 0.001). CONCLUSION: We report for the first time that involuntary asymmetric eyebrow elevation and ocular dominance are significantly associated. The assessment of ocular dominance should be included in the pre- and postoperative evaluation of patients with asymmetric brow elevation undergoing blepharoplasty. This asymmetry and its etiology must be recognized, so that an attempt to create symmetry does not result in the overcorrection of the contralateral eyelid that may lead to complications such as lagophthalmos and exposure keratopathy.
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Dominância Ocular/fisiologia , Sobrancelhas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Blefaroplastia , Sobrancelhas/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Período Pré-Operatório , Estudos Retrospectivos , Acuidade Visual/fisiologiaRESUMO
PURPOSE: To investigate a possible relationship between cranial nerve seven (CN VII) palsy and meibomian gland dysfunction. METHODS: A prospective case series of 16 patients with unilateral CN VII palsy was evaluated for meibomian gland function. Main outcome measures included the Sunnybrook Facial Grading System for CN VII palsy and a modified version of a noncontact, noninvasive meibomian gland evaluation by Arita et al., Unaffected contralateral eyes were used as the control group for comparison. RESULTS: Sixteen affected eyes were evaluated. The mean Sunnybrook Facial Grading System composite score was 34.19 ± 24 (score = -15 to 100; maximum function = 100). A paired samples t test between affected and unaffected eyes demonstrated a significant difference between mean values for tear break-up time, eyelid abnormality, meiboscore for upper eyelid, meiboscore for lower eyelid, combined meiboscore, and digital pressure. No significant difference was found for superficial punctate keratopathy nor Schirmer's test. A Pearson product-moment correlation coefficient found a significant negative correlation between the Sunnybrook Facial Grading System composite score and all aspects of meibomian gland function, except for the upper eyelid meiboscore, which only approached significance (p = 0.059). CONCLUSION: These findings suggest that a strong relationship exists between cranial nerve seven palsy and meibomian gland dysfunction. Clinical optimization of meibomian gland function may benefit cranial nerve seven palsy patients.
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Doenças Palpebrais/fisiopatologia , Doenças do Nervo Facial/fisiopatologia , Glândulas Tarsais/fisiopatologia , Fluoresceína , Corantes Fluorescentes , Fluorofotometria , Humanos , Estudos Prospectivos , Lágrimas/fisiologiaRESUMO
BACKGROUND: Asthma is a heterogeneous disease characterized by abnormal airway pathophysiology and susceptibility to different stimuli, as exemplified by a subset of patients with exercise-induced bronchoconstriction. Induced sputum provides a noninvasive method to sample airway biofluids that are enriched in proteins. OBJECTIVE: We hypothesized that novel mechanisms in the pathogenesis of asthma might be revealed by studying the patterns of protein expression in induced sputum. METHODS: We used shotgun proteomics to analyze induced sputum from 5 healthy subjects and 10 asthmatic patients, including 5 with exercise-induced bronchoconstriction. Differential protein expression among asthmatic patients, asthma subphenotypes, and control subjects was determined by using spectral counting and computational methods. RESULTS: Using Gene Ontology analysis, we defined the functional landscape of the induced sputum proteome and applied network analysis to construct a protein interaction map for this airway compartment. Shotgun proteomics analysis identified a number of proteins the differential enrichment or depletion of which robustly distinguished asthmatic patients from healthy control subjects and captured the effects of exercise on induced sputum proteome. Functional and network analysis identified key processes, including proteolytic activity, that are known contributors to airway remodeling. Importantly, this approach highlighted previously unrecognized roles for differentially expressed proteins in pathways implicated in asthma, such as modulation of phospholipase A(2) by secretoglobin, a putative role for S100A8/9 in human asthma, and selective upregulation of complement component 3a in response to exercise in asthmatic patients. CONCLUSION: Computationally intensive analysis of induced sputum proteome is a powerful approach to understanding the pathophysiology of asthma and a promising methodology to investigating other diseases of the airways.
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Asma/metabolismo , Mapas de Interação de Proteínas , Proteoma/análise , Proteoma/metabolismo , Escarro/química , Adolescente , Adulto , Western Blotting , Cromatografia Líquida , Análise por Conglomerados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Escarro/metabolismo , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
BACKGROUND: In prostate cancer, the tumour microenvironment (TME) represents an important regulator of disease progression and response to treatment. In the TME, cancer-associated fibroblasts (CAFs) play a key role in tumour progression, however the mechanisms underpinning fibroblast-cancer cell interactions are incompletely resolved. Here, we address this by applying cell type-specific labelling with amino acid precursors (CTAP) and mass spectrometry (MS)-based (phospho)proteomics to prostate cancer for the first time. METHODS: Reciprocal interactions between PC3 prostate cancer cells co-cultured with WPMY-1 prostatic fibroblasts were characterised using CTAP-MS. Signalling network changes were determined using Metascape and Enrichr and visualised using Cytoscape. Thymosin ß4 (TMSB4X) overexpression was achieved via retroviral transduction and assayed by ELISA. Cell motility was determined using Transwell and random cell migration assays and expression of CAF markers by indirect immunofluorescence. RESULTS: WPMY-1 cells co-cultured with PC3s demonstrated a CAF-like phenotype, characterised by enhanced PDGFRB expression and alterations in signalling pathways regulating epithelial-mesenchymal transition, cytoskeletal organisation and cell polarisation. In contrast, co-cultured PC3 cells exhibited more modest network changes, with alterations in mTORC1 signalling and regulation of the actin cytoskeleton. The expression of the actin binding protein TMSB4X was significantly decreased in co-cultured WPMY-1 fibroblasts, and overexpression of TMSB4X in fibroblasts decreased migration of co-cultured PC3 cells, reduced fibroblast motility, and protected the fibroblasts from being educated to a CAF-like phenotype by prostate cancer cells. CONCLUSIONS: This study highlights the potential of CTAP-MS to characterise intercellular communication within the prostate TME and identify regulators of cellular crosstalk such as TMSB4X.
Assuntos
Neoplasias da Próstata , Proteômica , Humanos , Masculino , Movimento Celular/genética , Neoplasias da Próstata/patologia , Fibroblastos/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
The pseudokinase scaffolds PEAK1 and PEAK2 are implicated in cancer cell migration and metastasis. We characterized the regulation and role of the third family member PEAK3 in cell signaling. Similar to PEAK1 and PEAK2, PEAK3 formed both homotypic and heterotypic complexes. In addition, like PEAK1, it bound to the adaptors Grb2 and CrkII. However, unlike PEAK1 and PEAK2, homodimerized PEAK3 also interacted with the ARF GTPase-activating protein ASAP1, the E3 ubiquitin ligase Cbl, and the kinase PYK2. Dimerization and subsequent phosphorylation on Tyr24, likely by a Src family kinase, were required for the binding of PEAK3 to Grb2 and ASAP1. Interactions with Grb2, CrkII, ASAP1, Cbl, and PYK2 exhibited contrasting dynamics upon cell stimulation with epidermal growth factor (EGF), in part due to PEAK3 dephosphorylation mediated by the phosphatase PTPN12. Overexpressing PEAK3 in mesenchymal-like MDA-MB-231 breast cancer cells enhanced cell elongation in a manner dependent on PEAK3 dimerization, and manipulation of PEAK3 expression demonstrated a positive role for this scaffold in regulating cell migration. Overexpressing PEAK3 in PEAK1/2 double-knockout MCF-10A breast epithelial cells enhanced acinar growth, impaired basement membrane integrity, and promoted invasion in three-dimensional cultures, with the latter two effects dependent on the binding of PEAK3 to Grb2 and ASAP1. PEAK1 and PEAK2 quantitatively and temporally influenced PEAK3 function. These findings characterize PEAK3 as an integral, signal-diversifying member of the PEAK family with scaffolding roles that promote cell proliferation, migration, and invasion.
Assuntos
Proteínas Tirosina Quinases , Transdução de Sinais , Movimento Celular , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismoRESUMO
Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in â¼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.
Assuntos
Endocitose , Proteína Quinase C-delta/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteólise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Mitógenos/farmacologia , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Ubiquitinação/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA-mutant ER+ breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA-mutant ER+ breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3ß lysosomal degradation and activation of Wnt/ß-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER+ breast cancer via PI(3,4)P2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/ß-catenin therapies.