HGF/c-Met/β1-integrin signalling axis induces tunneling nanotubes in A549 lung adenocarcinoma cells

Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and signalling molecules. In cancer cells, TNT formation contributes to cell survival, chemoresistance, and malignancy. However, the molecular mechanisms underlying TNT formation are not well defined, especially in different cancers. TNTs are present in non-small cell lung cancer (NSCLC) patients with adenocarcinoma. In NSCLC, hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated, causing increased cancer cell growth, survival, and invasion. This study identifies c-Met, β1-integrin, and paxillin as novel components of TNTs in A549 lung adenocarcinoma cells, with paxillin localised at the protrusion site of TNTs. The HGF-induced TNTs in our study demonstrate the ability to transport lipid vesicles and mitochondria. HGF-induced TNT formation is mediated by c-Met and β1-integrin in conjunction with paxillin, followed by downstream activation of MAPK and PI3K pathways and the Arp2/3 complex. These findings demonstrate a potential novel approach to inhibit TNT formation through targeting HGF/c-Met receptor and β1-integrin signalling interactions, which has implications for multi-drug targeting in NSCLC

The role of hepatocyte growth factor and β1-integrin/cell-matrix interactions in regulating growth and tunneling nanotube formation in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases. It is associated with high mortality rates with the majority of patients diagnosed with advanced metastatic cancer. Evading programmed cell death, increasing cellular motility, suppressing immune reactivity and alterations in the interactions between tumour cells and the extracellular matrix (ECM) proteins outline the complex process of tumour invasion. Another vital understudied aspect in achieving metastasis is the cellular communication existing between cells through the formation of tunneling nanotubes (TNTs). TNTs are non-adherent F-actin-based cytoplasmic protrusions that transport organelles to propagate chemoresistance across long distances in the tumour microenvironment (TME). Moreover, different factors within the TME initiate the pro-tumourigenic processes. Hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated in NSCLC leading to aberrant signalling in NSCLC which contributes to enhanced proliferation, migration and invasion. Similarly, other pleiotropic cytokines, such as IL-6 and EGF, are also known to induce growth and invasion in NSCLC. Furthermore, remodelling of the ECM proteins such as collagen IV, fibronectin and laminin results in the propagation of growth factor signalling via β1-integrin (Yan et al. 2006). Therefore, the aim of this research was to investigate the crosstalk between HGF with other pleiotropic cytokines (e.g. IL-6 and EGF) and the ECM/β1-integrin signalling axis in inducing proliferation and TNT formation in A549 lung adenocarcinoma cells. The results of this thesis demonstrate a novel role for the combined HGF and IL-6-induced proliferation via a convergence on the MAPK pathway. Moreover, HGF mediated proliferation in A549 cells occurred via interactions with fibronectin and β1-integrin. Our results also presented a novel role for HGF and its signalling axis in TNT formation, whereby HGF/c-Met crosstalk with β1-integrin to activate Arp2/3 complex, MAPK and PI3K pathways via paxillin. C-Met, β1-integrin and paxillin were also identified as novel components of TNTs. HGF/c-Met also crosstalk with EGF/EGFR via a convergence on the MAPK and PI3K pathways to regulate TNT formation. Moreover, we discovered a subpopulation of A549 cells capable of simultaneous migration, TNT formation and cell division which highlights a novel potential mechanism of metastasis in NSCLC. To recapitulate the 3D TME, our 3D A549 tumouroid studies revealed a physical interaction with occurring between A549 tumouroids and eosinophils. Moreover, novel TNT formation was observed between A549 tumouroids upon triptolide treatment. Overall, our findings highlight the complex interactions within the TME and the importance of multi-drug targeting to fully inhibit the spectrum of protumourigenic effects induced by HGF/c-Met and its signalling partners

M1 and M2 macrophages differentially regulate colonic crypt renewal

Background: The colonic epithelium is the most rapidly renewing tissue in the body and is organized into a single cell layer of invaginations called crypts. Crypt renewal occurs through Lgr5 + gut stem cells situated at the crypt base, which divide, produce daughter cells that proliferate, migrate, differentiate into all the cells required for normal gut function, and are finally shed into the crypt lumen. In health, this rapid renewal helps maintain barrier function next to the hostile gut microbial luminal environment. Inflammation results in an influx of immune cells including inflammatory M1 macrophages into the gut mucosa next to the crypt epithelium, but the direct effect of macrophages on crypt regeneration and renewal are poorly understood. Methods: Using an in vitro macrophage-crypt coculture model, we show that homeostatic M2 macrophages and inflammatory M1 macrophages confer different effects on the crypt epithelium. Results: Both M1 and M2 increase crypt cell proliferation, with M2 macrophages requiring physical contact with the crypt epithelium, whereas M1 macrophages exert their effect through a secreted factor. Only M1 macrophages reduce goblet and Tuft cell numbers and increase Lgr5 + crypt stem cell numbers, all dependent on physical contact with the crypt epithelium. Further studies showed that M1 macrophages increase the Wnt signaling pathways cyclin D1 and LEF1 through physical contact rather than a secreted factor. Conclusions: These findings highlight the importance of understanding distinct cellular interactions and direct dialogue between cells and increase our understanding of the contribution of different immune cell subtypes on crypt cell biology during inflammation

Lineage Plasticity in SCLC Generates Non-Neuroendocrine Cells Primed for Vasculogenic Mimicry

INTRODUCTION: Vasculogenic mimicry (VM), the process of tumor cell transdifferentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including SCLC. In genetically engineered mouse models (GEMMs) of SCLC, NOTCH, and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch, and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs.METHODS: We analyzed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs, and patient biopsies. We evaluated their three-dimensional structure and defined collagen-integrin interactions.RESULTS: We found that VM vessels are present in 23/25 CDX models, 2 GEMMs, and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only NOTCH-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development, and extracellular matrix organization signatures. On Matrigel, VM-primed non-NE cells remodel extracellular matrix into hollow tubules in an integrin β1-dependent process.CONCLUSIONS: We identified VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take considerable steps toward understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve the outcomes of patients with SCLC in the future.</p