RESUMO
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.
Assuntos
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
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.
Assuntos
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
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.
Assuntos
Núcleo Celular , Proteoma , Adesão Celular , Núcleo Celular/metabolismo , Proteoma/metabolismo , Transdução de SinaisRESUMO
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.