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Role of c-Met/β1 integrin complex in the metastatic cascade in breast cancer.

ABSTRACT: Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. Although individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a complex between receptor tyrosine kinase c-Met and β1 integrin in metastases. Using cell culture and in vivo assays, we found that c-Met/β1 complex induction promoted intravasation and vessel wall adhesion in triple-negative breast cancer cells, but did not increase extravasation. These effects may have been driven by the ability of the c-Met/β1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/β1 complex induction. A β1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/β1 binding, decreased breast cancer cell invasion and mesenchymal gene expression. Bone-seeking breast cancer cells exhibited higher levels of c-Met/β1 complex than parental controls and preferentially adhered to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/β1 complex than brain metastases. Thus, the c-Met/β1 complex drove intravasation of triple-negative breast cancer cells and preferential affinity for bone-specific matrix. Pharmacological targeting of the complex may have prevented metastases, particularly osseous metastases.

SUBMITTER: Lau D

PROVIDER: S-EPMC8262466 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Role of c-Met/β1 integrin complex in the metastatic cascade in breast cancer.

Lau Darryl D Wadhwa Harsh H Sudhir Sweta S Chang Alexander Chih-Chieh AC Jain Saket S Chandra Ankush A Nguyen Alan T AT Spatz Jordan M JM Pappu Ananya A Shah Sumedh S SS Cheng Justin J Safaee Michael M MM Yagnik Garima G Jahangiri Arman A Aghi Manish K MK

JCI insight 20210622 12

Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. Although individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a complex between receptor tyrosine kinase c-Met and β1 integrin in metastases. Using cell culture and in vivo assays, we found that c-Met/β1 complex induction promote ...[more]

PMID: 34003803

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Project description:The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/β1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/β1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and β1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/β1 complex to maintain the high-affinity β1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/β1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from β1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/β1 complex whose ligand-independent cross-activation and robust affinity for FN drive invasive oncologic processes.

Project description:BackgroundTriple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Treatment options for TNBC patients are limited and further insights into disease aetiology are needed to develop better therapeutic approaches. microRNAs' ability to regulate multiple targets could hold a promising discovery approach to pathways relevant for TNBC aggressiveness. Thus, we address the role of miRNAs in controlling three signalling pathways relevant to the biology of TNBC, and their downstream phenotypes.MethodsTo identify miRNAs regulating WNT/β-catenin, c-Met, and integrin signalling pathways, we performed a high-throughput targeted proteomic approach, investigating the effect of 800 miRNAs on the expression of 62 proteins in the MDA-MB-231 TNBC cell line. We then developed a novel network analysis, Pathway Coregulatory (PC) score, to detect miRNAs regulating these three pathways. Using in vitro assays for cell growth, migration, apoptosis, and stem-cell content, we validated the function of candidate miRNAs. Bioinformatic analyses using BC patients' datasets were employed to assess expression of miRNAs as well as their pathological relevance in TNBC patients.ResultsWe identified six candidate miRNAs coordinately regulating the three signalling pathways. Quantifying cell growth of three TNBC cell lines upon miRNA gain-of-function experiments, we characterised miR-193b as a strong and consistent repressor of proliferation. Importantly, the effects of miR-193b were stronger than chemical inhibition of the individual pathways. We further demonstrated that miR-193b induced apoptosis, repressed migration, and regulated stem-cell markers in MDA-MB-231 cells. Furthermore, miR-193b expression was the lowest in patients classified as TNBC or Basal compared to other subtypes. Gene Set Enrichment Analysis showed that miR-193b expression was significantly associated with reduced activity of WNT/β-catenin and c-Met signalling pathways in TNBC patients.ConclusionsIntegrating miRNA-mediated effects and protein functions on networks, we show that miRNAs predominantly act in a coordinated fashion to activate or repress connected signalling pathways responsible for metastatic traits in TNBC. We further demonstrate that our top candidate, miR-193b, regulates these phenotypes to an extent stronger than individual pathway inhibition, thus emphasizing that its effect on TNBC aggressiveness is mediated by the coordinated repression of these functionally interconnected pathways.

Project description:After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. ?1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of ?1 integrins can be regulated by "inside-out" signals leading to extravasation from the circulation into tissues. However, a role for inside-out ?1 activation in tumor cell metastasis is uncertain. Here we show that ?1 integrin activation promotes tumor metastasis and that activated ?1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether ?1 integrin activation can influence tumor cell metastasis, the ?1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active ?1. When tumor cells expressing constitutively-active ?1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type ?1 integrins. Rescue expression with mutant ?1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to ?1 as well as ?1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type ?1, but not constitutively-active ?1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and ?1 integrin activation as well as hepatic colonization. Using an antibody that detects activated ?1 integrin, we found higher levels of activated ?1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated ?1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of ?1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of ?1 integrin signaling in neoplasia.

Dataset Information

Role of c-Met/β1 integrin complex in the metastatic cascade in breast cancer.

Publications

Role of c-Met/β1 integrin complex in the metastatic cascade in breast cancer.

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