SMAD2 linker phosphorylation impacts overall survival, proliferation, TGFβ1-dependent gene expression and pluripotency-related proteins in NSCLC.

BACKGROUND: We investigated the impact of SMAD2 linker phosphorylation (pSMAD2L) on overall and disease-free survival, signal transduction, as well as cancer-related processes in non-small cell lung cancer (NSCLC). METHODS: We generated A549 cells constitutively lacking pSMAD2L (A549L(sub)) to gain mechanistic insights and stimulated NSCLC cell lines with inhibitors against cell cycle-associated kinases or TGFβ1. In addition, we analysed SMAD2 and pSMAD2L in alveolar epithelial cells type 2 from tumour-free lung tissue as well as in benign and malignant T cells by Western blotting. Furthermore, pSMAD2L-positive tumours and immune cells were analysed in an NSCLC patient cohort (n = 316) using multiplex immunofluorescence. RESULTS: In NSCLC cell lines and benign T cells, pSMAD2L was expressed in a mitosis-dependent manner. Loss of pSMAD2L (A549L(sub)) had an anti-proliferative effect, slowed migration, and increased alternatively spliced short SMAD2 (SMAD2(ΔE3)). The gene signature in A549L(sub) was associated with developmental and morphogenetic processes and redirected canonical TGFβ1-dependent signalling. By contrast, SMAD2(ΔE3) was absent in benign T cells but present in malignant T lymphoblasts. NSCLC patients with low pSMAD2L(+) tumour cell density had a poorer prognosis, whereas low pSMAD2L(+) immune cell density favoured overall and disease-free survival. CONCLUSIONS: pSMAD2L antagonises anti-proliferative canonical TGFβ-signalling in NSCLC and redirects TGFβ1-dependent gene expression, whereas loss of pSMAD2L enhances SMAD2(ΔE3) and affects pluripotency-associated proteins in vitro. In NSCLC patients, pSMAD2L cell density influences disease-free and overall survival in a spatially distinct manner.