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Kindler syndrome (KS) is a rare genodermatosis resulting from loss-of-function mutations in FERMT1, the gene that encodes Kindlin-1. KS patients have a high propensity to develop aggressive and metastatic cutaneous squamous cell carcinoma (cSCC). Here we show in non-KS-associated patients that elevation of FERMT1 expression is increased in actinic keratoses compared to normal skin, with a further increase in cSCC supporting a pro-tumorigenic role in this population. In contrast, we show that loss of Kindlin-1 leads to increased SCC tumor growth in vivo and in 3D spheroids, which was associated with the development of a hypoxic tumor environment and increased glycolysis. The metalloproteinase Mmp13 was upregulated in Kindlin-1-depleted tumors, and increased expression of MMP13 was responsible for driving increased invasion of the Kindlin-1-depleted SCC cells. These results provide evidence that Kindlin-1 loss in SCC can promote invasion through the upregulation of MMP13, and offer novel insights into how Kindlin-1 loss leads to the development of a hypoxic environment that is permissive for tumor growth.
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Introduction: The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Methods: Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry. Results: We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and we defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. Discussion: These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC.
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Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes, play key roles in fundamental cellular processes, including the sensing and transduction of mechanical cues. Here, we investigate systems-level changes in the integrin adhesome in patient-derived cutaneous squamous cell carcinoma cells and identify the actin regulatory protein Mena as a key node in the adhesion complex network. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We uncover an unexpected role for Mena at the nuclear membrane, where it controls nuclear architecture, chromatin repositioning and gene expression. Our findings identify an adhesion protein that regulates gene transcription via direct signalling across the nuclear envelope.
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Carcinoma de Células Escamosas , Neoplasias Cutâneas , Humanos , Actinas/genética , Actinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Núcleo Celular/metabolismo , Expressão Gênica , Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Membrana Nuclear/metabolismo , Lâmina Nuclear/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
OBJECTIVE: Immunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack of response. Here, we aimed to further investigate the immunoregulatory function of focal adhesion kinase (FAK) in PDAC, with specific emphasis on regulation of the type-II interferon response that is critical in promoting T-cell tumour recognition and effective immunosurveillance. DESIGN: We combined CRISPR, proteogenomics and transcriptomics with mechanistic experiments using a KrasG12Dp53R172H mouse model of pancreatic cancer and validated findings using proteomic analysis of human patient-derived PDAC cell lines and analysis of publicly available human PDAC transcriptomics datasets. RESULTS: Loss of PDAC cell-intrinsic FAK signalling promotes expression of the immunoproteasome and Major Histocompatibility Complex class-I (MHC-I), resulting in increased antigen diversity and antigen presentation by FAK-/- PDAC cells. Regulation of the immunoproteasome by FAK is a critical determinant of this response, optimising the physicochemical properties of the peptide repertoire for high affinity binding to MHC-I. Expression of these pathways can be further amplified in a STAT1-dependent manner via co-depletion of FAK and STAT3, resulting in extensive infiltration of tumour-reactive CD8 T-cells and further restraint of tumour growth. FAK-dependent regulation of antigen processing and presentation is conserved between mouse and human PDAC, but is lost in cells/tumours with an extreme squamous phenotype. CONCLUSION: Therapies aimed at FAK degradation may unlock additional therapeutic benefit for the treatment of PDAC through increasing antigen diversity and promoting antigen presentation.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Humanos , Animais , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Apresentação de Antígeno , Evasão da Resposta Imune , Proteômica , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Microambiente Tumoral , Linhagem Celular TumoralRESUMO
Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell-matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.
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Neoplasias Ósseas , Endoglina/metabolismo , Sarcoma de Ewing , Neoplasias Ósseas/genética , Endoglina/genética , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Proteômica , Receptores de Fatores de Crescimento , Sarcoma de Ewing/patologia , Transdução de SinaisRESUMO
In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here define a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions. Adding to nuclear FAK's known roles in regulating transcription, we now show that nuclear FAK regulates expression of many adhesion-related proteins that localise to the nucleus and that nuclear FAK binds to the adhesome component and nuclear protein Hic-5. FAK and Hic-5 work together in the nucleus, co-regulating a subset of genes transcriptionally. We demonstrate the principle that there are subcomplexes of nuclear adhesion proteins that cooperate to control transcription.
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Núcleo Celular , Proteoma , Adesão Celular , Núcleo Celular/metabolismo , Proteoma/metabolismo , Transdução de SinaisRESUMO
Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways.
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SRC is a nonreceptor tyrosine kinase with key roles in breast cancer development and progression. Despite this, SRC tyrosine kinase inhibitors have so far failed to live up to their promise in clinical trials, with poor overall response rates. We aimed to identify possible synergistic gene-drug interactions to discover new rational combination therapies for SRC inhibitors. An unbiased genome-wide CRISPR-Cas9 knockout screen in a model of triple-negative breast cancer revealed that loss of integrin-linked kinase (ILK) and its binding partners α-Parvin and PINCH-1 sensitizes cells to bosutinib, a clinically approved SRC/ABL kinase inhibitor. Sensitivity to bosutinib did not correlate with ABL dependency; instead, bosutinib likely induces these effects by acting as a SRC tyrosine kinase inhibitor. Furthermore, in vitro and in vivo models showed that loss of ILK enhanced sensitivity to eCF506, a novel and highly selective inhibitor of SRC with a unique mode of action. Whole-genome RNA sequencing following bosutinib treatment in ILK knockout cells identified broad changes in the expression of genes regulating cell adhesion and cell-extracellular matrix. Increased sensitivity to SRC inhibition in ILK knockout cells was associated with defective adhesion, resulting in reduced cell number as well as increased G1 arrest and apoptosis. These findings support the potential of ILK loss as an exploitable therapeutic vulnerability in breast cancer, enhancing the effectiveness of clinical SRC inhibitors. SIGNIFICANCE: A CRISPR-Cas9 screen reveals that loss of integrin-linked kinase synergizes with SRC inhibition, providing a new opportunity for enhancing the clinical effectiveness of SRC inhibitors in breast cancer.
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Neoplasias da Mama/genética , Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Quinases da Família src/antagonistas & inibidores , Compostos de Anilina/farmacologia , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos Knockout , Nitrilas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Quinases da Família src/metabolismoRESUMO
Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.
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Neoplasias Encefálicas/imunologia , Epigênese Genética , Glioblastoma/imunologia , Evasão da Resposta Imune/imunologia , Células Mieloides/imunologia , Células-Tronco Neoplásicas/imunologia , Microambiente Tumoral/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Metilação de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Mieloides/metabolismo , Células Mieloides/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.
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Adesões Focais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Aminoácidos/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 (Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression.
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Cromatina/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Interleucina-33/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Motivos de Aminoácidos , Comunicação Celular , Núcleo Celular/metabolismo , Humanos , Ligação ProteicaRESUMO
Cell-surface adhesion receptors mediate interactions with the extracellular matrix (ECM) to control many fundamental aspects of cell behavior, including cell migration, survival, and proliferation. Integrin adhesion receptors recruit structural and signaling proteins to form multimolecular adhesion complexes that link the plasma membrane to the actomyosin cytoskeleton. The assembly and turnover of adhesion complexes are tightly regulated, governed in part by the networks of physical protein interactions and functional signaling associations between components of the adhesome. Proteomic profiling of adhesion complexes has begun to reveal their molecular complexity and diversity. To interrogate the composition of cell-ECM adhesions, we detail herein an approach for the network analysis of adhesion complex proteomes. Integration of these proteomic data with adhesome databases in the context of predicted protein interactions enables the mapping of experimentally defined adhesion complex networks. Computational analysis of resultant network models can identify subnetworks of putative functionally linked adhesion protein communities. This approach provides a framework to predict functional adhesion protein relationships and generate new mechanistic hypotheses for further experimental testing.
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Biologia Computacional/métodos , Integrinas/metabolismo , Complexos Multiproteicos/metabolismo , Redes Neurais de Computação , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Movimento Celular , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Integrinas/genética , Complexos Multiproteicos/genética , Ligação Proteica , Proteoma/genética , SoftwareRESUMO
Reverse-phase protein array (RPPA) technology uses panels of high-specificity antibodies to measure proteins and protein post-translational modifications in cells and tissues. The approach offers sensitive and precise quantification of large numbers of samples and has thus found applications in the analysis of clinical and pre-clinical samples. For effective integration into drug development and clinical practice, robust assays with consistent results are essential. Leveraging a collaborative RPPA model, we set out to assess the variability between three different RPPA platforms using distinct instrument set-ups and workflows. Employing multiple RPPA-based approaches operated across distinct laboratories, we characterised a range of human breast cancer cells and their protein-level responses to two clinically relevant cancer drugs. We integrated multi-platform RPPA data and used unsupervised learning to identify protein expression and phosphorylation signatures that were not dependent on RPPA platform and analysis workflow. Our findings indicate that proteomic analyses of cancer cell lines using different RPPA platforms can identify concordant profiles of response to pharmacological inhibition, including when using different antibodies to measure the same target antigens. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of disease or response to therapy.
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Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Análise Serial de Proteínas/métodos , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Análise de Componente PrincipalRESUMO
Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.
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Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Membranas/química , Multimerização Proteica , Animais , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Humanos , Membranas/enzimologia , Relação Estrutura-AtividadeRESUMO
Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Angiopoietina-1/biossíntese , Angiopoietina-1/genética , Linhagem Celular , Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Cadeias alfa de Integrinas/biossíntese , Cadeias alfa de Integrinas/genética , Cadeias beta de Integrinas/biossíntese , Cadeias beta de Integrinas/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/biossíntese , Fator de Crescimento Transformador beta3/genéticaRESUMO
BACKGROUND: Triple-negative breast cancer is an aggressive type of breast cancer with high risk of recurrence. It is still poorly understood and lacks any targeted therapy, which makes it difficult to treat. Thus, it is important to understand the underlying mechanisms and pathways that are dysregulated in triple-negative breast cancer. METHODS: To investigate the role of mitochondria in triple-negative breast cancer progression, we analysed previously reported gene expression data from triple-negative breast cancer cybrids with SUM-159 as the nuclear donor cell and SUM-159 or A1N4 (c-SUM-159, c-A1N4) as the mitochondrial donor cells and with 143B as the nuclear donor cell and MCF-10A or MDA-MB-231 (c-MCF-10A, c-MDA-MB-231) as the mitochondrial donor cells. The role of potential biomarkers in cell proliferation and migration was examined in SUM-159 and MDA-MB-231 cells using sulforhodamine B and wound healing assays. RESULTS: Rank product analysis of cybrid gene expression data identified 149 genes which were significantly up-regulated in the cybrids with mitochondria from the cancer cell line. Analysis of previously reported breast tumour gene expression datasets confirmed 9 of the 149 genes were amplified, up-regulated, or down-regulated in more than 10% of the patients. The genes included NDRG1, PVT1, and EXT1, which are co-located in cytoband 8q24, which is frequently amplified in breast cancer. NDRG1 showed the largest down-regulation in the cybrids with benign mitochondria and was associated with poor prognosis in a breast cancer clinical dataset. Knockdown of NDRG1 expression significantly decreased proliferation of SUM-159 triple-negative breast cancer cells. CONCLUSIONS: These results indicate that mitochondria-regulated nuclear gene expression helps breast cancer cells survive and proliferate, consistent with previous work focusing on an Src gene signature which is mitochondria regulated and drives malignancy in breast cancer cybrids. This is the first study to show that mitochondria in triple-negative breast cancer mediate significant up-regulation of a number of genes, and silencing of NDRG1 leads to significant reduction in proliferation.
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PRK1 and PRK2 are two closely related AGC-family serine/threonine protein kinases. Here we demonstrate novel roles for them at cilia and in cancer biology. In both instances serum withdrawal leads to increased activating PRK1 and PRK2 phosphorylation (pPRK1/pPRK2) and their depletion results in reduced spheroid growth. pPRK1/pPRK2 localise to the transition zone of cilia and their co-depletion results in reduced cilia size, impaired planer polarity and impaired cilia associated signalling. High PRK2 (but not PRK1) expression correlates with poor outcome in patients with basal-like/Triple Negative (TN) Breast Cancer (BC) where there is also higher expression relative to other BC tumour subtypes. In agreement, depletion of PRK1 and PRK2 in mouse TNBC cells, or CRISPR/Cas9 mediated deletion of PRK2 alone, significantly reduces cell proliferation and spheroid growth. Finally proteomic analysis to identify PRK2 binding partners in mouse TNBC cells revealed proteins that are important for both cilia and BC biology. Taken together these data demonstrate novel roles for PRK1 and PRK2 at cilia and in BC biology and in the case of PRK2 in particular, identifies it as a novel TNBC therapeutic target.
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Cílios/enzimologia , Proteína Quinase C/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Cells have evolved mechanisms to sense the composition of their adhesive microenvironment. Although much is known about general mechanisms employed by adhesion receptors to relay signals between the extracellular environment and the cytoskeleton, the nuances of ligand-specific signalling remain undefined. Here, we investigated how glomerular podocytes, and four other basement membrane-associated cell types, respond morphologically to different basement membrane ligands. We defined the composition of the respective adhesion complexes using mass spectrometry-based proteomics. On type IV collagen, all epithelial cell types adopted a round morphology, with a single lamellipodium and large adhesion complexes rich in actin-binding proteins. On laminin (511 or 521), all cell types attached to a similar degree but were polygonal in shape with small adhesion complexes enriched in endocytic and microtubule-binding proteins. Consistent with their distinctive morphologies, cells on type IV collagen exhibited high Rac1 activity, while those on laminin had elevated PKCα. Perturbation of PKCα was able to interchange morphology consistent with a key role for this pathway in matrix ligand-specific signalling. Therefore, this study defines the switchable basement membrane adhesome and highlights two key signalling pathways within the systems that determine distinct cell morphologies. Proteomic data are availableviaProteomeXchange with identifier PXD017913.
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Membrana Basal/metabolismo , Gânglios Espinais/citologia , Laminina/farmacologia , Proteômica/métodos , Animais , Linhagem Celular , Forma Celular/efeitos dos fármacos , Colágeno Tipo IV/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Integrina alfa3/metabolismo , Ligantes , Espectrometria de Massas , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Proteína Quinase C-alfa/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
We mutated the focal adhesion kinase (FAK) catalytic domain to inhibit binding of the chaperone Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We reexpressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a subset of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair FAK-associated nuclear YAP in sensitive cancer cell lines. Here, we report the discovery of a new, clinically actionable, synergistic combination between FAK and HDAC inhibitors.
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Proteína-Tirosina Quinases de Adesão Focal/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Proliferação de Células , Sinergismo Farmacológico , Humanos , Camundongos , Transdução de SinaisRESUMO
Reverse-phase protein array (RPPA) technology is a high-throughput antibody- and microarray-based approach for the rapid profiling of levels of proteins and protein posttranslational modifications in biological specimens. The technology consumes small amounts of samples, can sensitively detect low-abundance proteins and posttranslational modifications, enables measurements of multiple signaling pathways in parallel, has the capacity to analyze large sample numbers, and offers robust interexperimental reproducibility. These features of RPPA experiments have motivated and enabled the use of RPPA technology in various biomedical, translational, and clinical applications, including the delineation of molecular mechanisms of disease, profiling of druggable signaling pathway activation, and search for new prognostic markers. Owing to the complexity of many of these applications, such as developing multiplex protein assays for diagnostic laboratories or integrating posttranslational modification-level data using large-scale proteogenomic approaches, robust and well-validated data are essential. There are many distinct components of an RPPA workflow, and numerous possible technical setups and analysis parameter options exist. The differences between RPPA platform setups around the world offer opportunities to assess and minimize interplatform variation. Crossplatform validation may also aid in the evaluation of robust, platform-independent protein markers of disease and response to therapy.