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Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells

ABSTRACT: Integrins provide an essential bridge between cancer cells and the extracellular matrix, playing a central role in every stage of disease progression. Despite the recognized significance of integrin phosphorylation in several biological processes, the regulatory mechanisms and their relevance remained elusive. To address this gap, we engineered a fluorescence resonance energy transfer biosensor for integrin β1 phosphorylation, screening 96 protein tyrosine phosphatases and identifying Shp2 and PTP-PEST as negative regulators. Mutation of the integrin NPxY(783/795) sites revealed the importance of integrin phosphorylation for efficient cancer cell invasion, further supported by inhibition of the identified integrin phosphorylation regulators Shp2 and Src kinase. Using proteomics approaches, we uncovered Cofilin as a component of the phosphorylated integrin-Dok1 complex and linked this axis to effective invadopodia formation, a process supporting breast cancer invasion. These data further implicate dynamic modulation of integrin β1 phosphorylation at NPxY sites at different stages of metastatic dissemination.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

SUBMITTER: James Conway

LAB HEAD: Prof. Johanna

PROVIDER: PXD048646 | Pride | 2025-02-05

REPOSITORIES: pride

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			Action	DRS
	190612_100ng_BiCAP_1.raw	Raw
	190612_100ng_BiCAP_2.raw	Raw
	190612_100ng_BiCAP_3.raw	Raw
	190612_100ng_Venus_1.raw	Raw
	190612_100ng_Venus_2.raw	Raw

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Inhibition of ß1 integrin induces its association with MT1-MMP and decreases MT1- MMP internalization and cellular invasiveness

Project description:Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of β1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of β1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and β1 integrin were sequestered at the cell surface when β1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analysis. Sequestration of β1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and β1 integrin ultimately led to a loss of invasive behaviour.

2022-12-22 | PXD024000 | Pride

TMTC1 promotes invasiveness of ovarian cancer

Project description:Ovarian cancer is the most lethal gynecological malignancy and is characterized by peritoneal disseminated metastasis. Although O-mannosyltransferase TMTC1 is highly expressed by ovarian cancer, its pathophysiologic role in ovarian cancer remains unclear. Here, immunohistochemistry showed that TMTC1 was overexpressed in ovarian cancer tissues compared with the adjacent non-tumor ovarian tissues and high TMTC1 expression was associated with poor prognosis in patients with ovarian cancer. Silencing TMTC1 reduced ovarian cancer cell viability, migration, and invasion in vitro as well as suppressed peritoneal tumor growth and metastasis in vivo. Moreover, TMTC1 knockdown reduced cell-laminin adhesion, which was associated with the decreased phosphorylation of FAK at pY397. Conversely, TMTC1 overexpression promoted these malignant properties in ovarian cancer cells. Glycoproteomic analysis and Concanavalin A (ConA) pull-down assays showed that integrins β1 and β4 were novel O-mannosylated protein substrates of TMTC1. Furthermore, TMTC1-mediated cell migration and invasion were significantly reversed by siRNA-mediated knockdown of integrin β1 or β4. Collectively, these results suggest that TMTC1-mediated invasive behaviors are primarily through integrins β1 and β4 and that TMTC1 is a potential therapeutic target for ovarian cancer.

2023-10-24 | PXD037907 | Pride

PTEN deficiency exposes a requirement for an ARF GTPase module in integrin-dependent invasion in ovarian cancer

Project description:Dysregulation of the PI3K/AKT pathway is a common occurrence in ovarian carcinomas. Loss of the tumour suppressor PTEN in high-grade serous ovarian carcinoma (HGSOC) is associated with a patient subgroup with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We utilise long-term time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency does not affect proliferation but rather induces PI(3,4,5)P3-rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor β1-integrin as key ARF6 interactors regulating the PTEN loss-associated invasion phenotype. ARF6 functions to promote invasion by controlling the recycling of internalised, active β1-integrin complexes to maintain invasive activity into the ECM. The expression of the ARF6-centred complex in HGSOC patients is inversely associated with outcome, allowing identification of patient groups with improved versus poor outcome. ARF6 may represent a new therapeutic vulnerability in PTEN-depleted HGSOC tumours.

2023-07-11 | PXD038305 | Pride

Expression profiling of 5 novel breast cancer cell lines

Project description:The Fra-1 transcription factor promotes tumor cell growth, invasion and metastasis. While characterizing five breast cancer cell lines derived from primary human breast tumors, we identified BRC-31 as a novel basal-like cell model that expresses elevated Fra-1 levels. BRC-31 cells display elevated FAK, SRC and ERK2 phosphorylation relative to luminal breast cancer models. Inhibition of this signaling axis, through the use of pharmacological inhibitors, reduces the phosphorylation and stabilization of Fra-1. Elevated integrin αVβ3 expression in these cells suggested that integrin receptors might activate this FAK-SRC-ERK2 signaling axis to enhance Fra-1 phosphorylation. These cells also express high levels of uPAR, a GPI-anchored receptor that has been shown to enhance integrin-mediated signaling initiated by Vitronectin engagement. Transient knockdown of uPAR in BRC31 cells grown on Vitronectin reduces Fra-1 phosphorylation and stabilization and uPAR and Fra-1 are required for Vitronectin-induced cell invasion. In clinical samples, a molecular component signature consisting of Vitronectin-uPAR-uPA-Fra-1 predicts poor overall survival in patients with breast cancer and correlates with a Fra-1 transcriptional signature. Taken together, we have identified a novel-signaling axis that leads to phosphorylation and stabilization of Fra-1, a transcription factor that is emerging as an important modulator of breast cancer progression and metastasis.

2017-08-03 | GSE69915 | GEO

Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells

Project description:Our work highlights a new role of SHP2 in regulating breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, this work enforces the rational for targeting SHP2 in breast cancer in order to reduce local invasion and block the metastatic potential of neoplastic cells.

2022-06-23 | PXD017219 | Pride

Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing

Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged. To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.

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Transcription profiling of human MDAMB231 breast cancer cells by Parvin-beta in 2D vs 3D culture conditions

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SILAC-based proteomic approach to delineate molecular alterations associated with erlotinib resistance in head and neck squamous cell carcinoma

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