Leader cell myosin 10 controls adhesion dynamics, invasion, and EMT pathway activation

The epithelial-to-mesenchymal transition (EMT) relies on cytoskeletal remodeling that drives cell migration and invasion. Leader cells are a highly invasive subpopulation of cancer cells, but our knowledge of how cytoskeletal remodeling influences the EMT, and collective invasion is limited. We integrated CRISPR/Cas9 targeting of myosin 10 (MYO10), an actin-associated cytoskeletal remodeling protein, with fluorescence assisted cell sorting (FACS), western blot, Sanger sequencing, and Inference of CRISPR Edits (ICE) analysis to generate MYO10 knockout (MYO10KO), knockdown (MYO10KD) and wildtype (MYO10WT) clones. Bulk RNA sequencing and gene set enrichment analysis were used to assess differential gene expression between MYO10KO+KD and MYO10WT clones and epithelial-to-mesenchymal (EMT) pathway and target gene expression. siRNA-mediated MYO10 knockdown was also used to perturb MYO10 expression in A549 and H1975 cell lines. DNA-based tension-gauge tether (TGT) probes and reflection interference contrast and fluorescence microscopy were used to assess integrin-traction forces and cell spread area. Spheroids embedded in Matrigel were used to assess 3D invasion. Western blotting was used to assess levels focal adhesion and EMT target proteins. Two (2) MYO10KO, one (1) MYO10KD and two (2) MYO10WT clones were generated and validated. MYO10KO clones demonstrated increased expression of integrin subunits α6 and β4, and decreased integrin force generation, cell spread area, and focal adhesion kinase activity. N-cadherin expression and spheroid compaction were also reduced in MYO10KO clones while the MYO10KD clone maintained N-cadherin expression but exhibited reduced 3D invasion relative to MYO10WT clones. Paradoxically, MYO10KO and MYO10KD clones demonstrated significant activation of EMT transcriptional pathways and increased levels of the EMT transcription factor (EMT-TF) SNAIL. Loss of MYO10 influences several components of the leader cell adhesion machinery by increasing integrin subunit α6 and β4 expression and decreasing N-cadherin expression and integrin force generation while activating EMT transcriptional programs. These findings suggest a novel role for MYO10 in leader cells as a modulator of the integrin profile, traction force, spheroid organization and EMT cell state associated with collective invasion.