Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

This study was funded by grants awarded to AGH and JB by the Agencia Estatal de Investigación (AEI) del Ministerio de Ciencia e Innovación MCIN (PID2019-104698RB-I00 and PID2022-136968OB-I00). We thank Dr. Juan Valcárcel and Dr. Elena Martin (Centre de Regulació Genètica, PRBB, Barcelona, Spain) for their advice and help with the SANJUAN splicing analyses, Prof. Dr. Daniel Navajas (Unit of Biophysics and Bioengineering, UB, Spain) for his advice on micromechanical measurements and Drs. Antoni Celià, José Yelamos, and Joaquín Arribas (IMIM, Barcelona, Spain) for providing cell lines and PDXs. We also thank Jordi Vergés and Meritxell Torrent (IMIM, Barcelona, Spain) and Carmen Escudero (IMIM, Barcelona, Spain; currently at Vall d'Hebron Institut d'Oncologia, Barcelona, Spain) for their technical support. Background: As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. Methods: We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. Results: In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain