Epidermal Growth Factor/c-Met receptor signalling crosstalk drives tunneling nanotube formation in A549 lung adenocarcinoma cells

Tunneling nanotubes (TNTs) are actin-based cytoplasmic connections that can mediate intercellular transfer of various cellular cargo and have been implicated in cancer progression and chemoresistance. However, the signalling mechanisms driving their formation remain poorly understood. Given the frequent dysregulation of EGFR and c-Met signalling in non-small cell lung cancer (NSCLC), and prior evidence of TNTs in lung adenocarcinoma patient samples, we investigated the role of EGFR and c-Met receptor signalling crosstalk in TNT induction in A549 lung adenocarcinoma cells. Stimulation with EGF, HGF, or in combination induced a concentration dependent increase in the formation of TNTs. TNTs exhibited typical characteristics, including F-actin expression, non-adherence to the substratum and facilitated intercellular trafficking of lysosomes, mitochondria, and lipid vesicles. EGFR was identified as a novel component of TNTs, but had little co-localisation with the c-Met receptor. Co-stimulation with HGF and EGF did not produce consistent additive or synergistic effects on TNT formation, suggesting shared downstream signalling. Furthermore, although EGFR and c-Met inhibition blocked EGF- and HGF-induced TNTs respectively, inhibition of both receptors was required to suppress TNTs following dual HGF/EGF treatment. Interestingly, blocking the EGF receptor alongside c-Met resulted in a more potent inhibition of HGF-induced TNTs, indicating crosstalk. Furthermore, inhibition of downstream MEK and PI3K pathways reduced HGF- or EGF- induced TNT formation, but dual inhibition was required to completely block TNT formation in HGF+EGF co-stimulated cells. These findings reveal a novel convergence of EGFR and c-Met and their downstream MAPK/PI3K pathways in TNT regulation, which can have important clinical implications in combinatorial receptor and cell signalling pathway targeting in NSCLC.