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RNA-seq analysis of FACS-sorted pre-metastatic lung endothelial cells reveal transcriptional programs regulated by cisplatin and FAK in the pre-metastatic lung

ABSTRACT: This study investigates the mechanisms underlying DNA-damage driven vascular endothelial-cell senescence regulated by EC-FAK and its role in lung cancer metastasis. RNA-seq analysis has been performed FACS-sorted pre-metastatic lung endothelial cells to identify FAK-regulated transcriptomic changes due to DNA- damaging therapy in tumour burdened and non-tumour burdened mice (comparisons between Cre-negative ECFAK WT mice with Cre-positive ECFAK KO mice in Cisplatin treated samples.

ORGANISM(S): Mus musculus

PROVIDER: GSE328693 | GEO | 2026/09/09

REPOSITORIES: GEO

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Phospho- and total- proteomic analysis of FAK knockout in primary mouse lung endothelial cells

Project description:We assessed the impact of Endothelial Focal Adhesion kinase (ECFAK) knockout, using primary lung endothelial cells isolated (at P10-P14) from a FAK flox/flox mice crossed with mice expressing a tamoxifen-inducible Cre(iCreERT2) that is driven by endothelial cell (EC)-selective platelet-derived growth factor subunit B (Pdgfb) promoter (Pdgfb-iCreERT2) (Tavora et al., 2010). To induce EC restricted FAK deletion, we injected 4-hydroxytamoxifen (4-OHT) from postnatal day 1 (P1) to P2. Molecular signaling events modulated by ECFAK loss, with or without ex vivo VEGF stimulation, were then examined by phospho and total quantitative proteomics analysis using 6plex isobaric tandem mass tagging multiplexing (TMT). In total, three experiments were performed (a an initial pilot experiment not included in this study, followed by two main biological replicates included - i.e. experiments 2, and 3). The following 4 TMT channels were utilised in each experiment: TMT-126: WT-mock(PBS) stimulated TMT-127: WT-Vegf stimulated TMT128: FAK KO-mock (PBS)stimulated TMT 129:FAK KO-Vegf stimulated

2022-04-13 | PXD030824 | Pride

Cancer Associated Fibroblast-FAK regulates malignant cell metabolism

Project description:Breast and pancreatic cancers are characterised by profound metabolic changes1,2. Until recently, these changes have been ascribed to their intrinsic genetic profiles2-4. However, the contribution of cancer-associated fibroblasts (CAFs) to cell metabolism has not been fully investigated5. Here, we provide clinical evidence that reduction in stromal Focal Adhesion Kinase (FAK) correlates with reduced overall survival in human breast and pancreatic cancer patients. To model this, we developed FSP-Cre+;FAKfl/fl mice where FAK was deleted in a subpopulation of CAFs. We establish that loss of CAF-FAK is sufficient to enhance tumour growth without affecting desmoplasia in orthotopically injected breast and pancreatic carcinomas. Additionally, deletion of CAF-FAK enhances disease progression in the MMTV-PyMT spontaneous model of breast cancer. Furthermore, despite this pro-tumourigenic effect, a significant reduction in tumour angiogenesis was observed in late-stage tumours, but not early-stage, size-matched tumours in FSP-Cre+;FAKfl/fl mice. This indicates that loss of CAF-FAK is sufficient to reduce malignant cell dependency on blood vessel support suggesting acquired metabolic alterations in cancer cells. Indeed, 18F-FDG-PET imaging and glucose flux analysis revealed enhanced glucose catabolism in early-stage, size-matched tumours in FSP-Cre+;FAKfl/fl mice in vivo. Mechanistically, we demonstrate that conditioned medium from FAK-null CAFs enhances glycolysis and glycolytic capacity in malignant cells. Proteomics and phosphoproteomics analysis reveal enriched cytokine-mediated signalling pathways in FAK-null CAFs and subsequent enrichment of associated phosphopeptides in malignant cells, respectively. Overall, our data uncover a novel mechanism by which cytokines, regulated by CAF-FAK, can promote cancer growth and progression, and identify a new set of CAF-derived targets to interfere with cancer metabolism.

2020-02-18 | PXD008276 | Pride

Low endothelial cell FAK expression reduces pancreatic ductal adenocarcinoma liver metastasis after gemcitabine treatment

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2023-03-10 | PXD021542 | Pride

Elucidating the role of the kinase activity of endothelial cell focal adhesion kinase in angiocrine signalling and tumour growth

Project description:A common limitation of cancer treatments is chemotherapy resistance. We have previously identified a molecular mechanism where chemotherapy resistance is regulated by endothelial cells. Endothelial cell specific knockout of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. Our current study addresses the kinase dependent component of endothelial cell FAK sensitisation to the DNA damaging chemotherapeutic drug doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that specific inactivation of the FAK kinase domain in endothelial cells of blood vessels in established subcutaneous B16F0 tumours sensitises melanoma cells to doxorubicin. Tumour growth was reduced in response to doxorubicin treatment in FAK kinase-dead but not in wild-type mice. Furthermore, we demonstrate that doxorubicin reduces perivascular proliferation, enhances apoptosis and DNA-damage in endothelial cell FAK kinase dead tumours. When we treated human pulmonary microvascular endothelial cells with the FAK kinase inhibitors defactinib, PF-562,271 and PF-573,228 in combination with doxorubicin, we observed a reduction in cytokine levels, implying a possible mechanism for FAK kinase domain in chemosensitisation. Together, these results confirm the role of the kinase domain of EC-FAK in chemosensitising tumour cells to doxorubicin.

2021-11-02 | PXD019728 | Pride

RNA-seq analysis of FACS-sorted pre-metastatic lung endothelial cells reveal transcriptional programs regulated by cisplatin and FAK in the pre-metastatic lung

Project description:RNA-seq analysis of FACS-sorted pre-metastatic lung endothelial cells reveal transcriptional programs regulated by cisplatin and FAK in the pre-metastatic lung

| PRJNA1456142 | ENA

RNA-seq expression profiling of metastasis-associated endothelial cells from murine bone marrow

Project description:Breast tumour cells were found to remodel the murine bone marrow vascular microenvironment to support metastatic expansion. To gain insight into the host vasculature in response to tumor colonization, cells from the isogenic murine mammary tumour cell line 4T1.2 were intracardially injected into immunocompetent BALB/c mice. We analysed the transcriptional profiles of endothelial cells extracted from heavily infiltrated (Metastatic) bone marrow stroma and the adjacent tumor-free bone marrow stroma (Non-metastatic), as well as from healthy bones (Control). Methods: Immunocompetent BALB/c mice were intracardically injected with 4T1.2 tumor cells. The bones of these tumor-burdened mice were examined under fluorescence stereomicroscope to microdissect heavily-infiltrated (Metastatic) and the adjacent tumour-free (Non-metastatic) areas. Bones from healthy mice were used as controls. The dissected bone material from both burdened and control mice was processed to isolate EMCNhi/CD31hi endothelial cells using flow cytometry. RNA was extracted for 75bp paired-end RNA-seq profiling using an Illumina NextSeq 500 sequencer.

2021-09-16 | GSE160100 | GEO

ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning

Project description:Precise vascular patterning is critical for normal growth and development. The ERG transcription factor drives Delta like ligand 4 (DLL4)/Notch signalling and is thought to act as pivotal regulators of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC specific Focal Adhesion Kinase (FAK)-knockout (KO) and point-mutant FAK-knockin mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins identified that endothelial nuclear-FAK interacts with the de-ubiquitinase USP9x and the ubiquitin ligase TRIM25 enzymes. Further in silico analysis corroborates that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.

2022-07-05 | PXD033447 | Pride

Tumor FAK Orchestrates Immunosuppression in Ovarian Cancer via the CD155/TIGIT axis

Project description:An immune profiling of cells recovered from the peritoneal cavity of mice seeded with KMF ovarian cancer cells with FAK knockout reconstituted with FAK or a kinase dead FAK An immune profiling of cells recovered from the peritoneal cavity of mice seeded with KMF ovarian cancer cells with FAK kockout reconstituted with FAK, then treated with FAKi VS4716 (100mg/kg) or vehicle by gavage twice daily.

2022-06-15 | GSE182726 | GEO

Role of FAK, dominant-negative FAK, FAK-CD and FAKsiRNA in MCF-7 human breast cancer cell tumorigenesis

Project description:Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in proliferation, motility, adhesion, invasion, angiogenesis, and survival signaling. Focal adhesion kinase has been shown to be overexpressed in many types of tumors, including breast cancer at early stages of tumorigenesis. To study the biological role of FAK in breast tumorigenesis, we used FAKsiRNA to down-regulate FAK in MCF-7 cell lines.

2009-08-17 | GSE11581 | GEO

FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy [KMF parental, KMF clone KT13 (FAK-/-), KT13 + GFP-FAK, KT13 + GFP-FAK R454, and KT13 + deltaGSK beta-catenin]

Project description:Gene copy number changes, cancer stem cell (CSC) increases, and platinum chemotherapy resistance contribute to poor prognosis in patients with recurrent high grade serous ovarian cancer (HGSOC). CSC phenotypes involving Wnt-b-catenin and aldehyde dehydrogenase activities, platinum resistance, and tumor initiating frequency are here associated with spontaneous genetic gains, including genes encoding KRAS, MYC and FAK, in a new murine model of ovarian cancer (KMF). Noncanonical FAK signaling was sufficient to sustain human and KMF tumorsphere proliferation, CSC survival, and platinum resistance. Increased FAK tyrosine phosphorylation occurred in HGSOC patient tumors surviving neo-adjuvant platinum and paclitaxel chemotherapy and platinum resistant tumorspheres acquired FAK dependence for growth. Importantly, combining a pharmacologic FAK inhibitor with platinum overcame chemoresistance and triggered apoptosis in vitro and in vivo. Knockout, rescue, genomic and transcriptomic analyses collectively identified more than 400 genes regulated along a FAK/b-catenin/Myc axis impacting stemness and DNA repair in HGSOC, with 66 genes gained in a majority of Cancer Genome Atlas samples. Together, these results support combinatorial testing of FAK inhibitors for the treatment of recurrent ovarian cancer.

2019-07-01 | GSE129098 | GEO

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