RNA-seq analysis evaluating cell cycle specific gene expression and the effect of KRTAP2-3 gene inactivation in SAS cells expressing Fucci.

Proliferation and migration of cancer cells are regulated by the stimuli from tumour microenvironment during its progression and metastasis. While transforming growth factor-β (TGF-β) increases migration and metastasis of various types of epithelial cancer cells via induction of epithelial-mesenchymal transition (EMT), it also decreases their proliferation by inducing G1 cell cycle arrest. However, the correlation of these tumour promoting and suppressing effects of TGF-β remains to be elusive because of the lack of analysis at a single cell level. Here, we showed that TGF-β confers the oral cancer cells residing in G1 phase with higher motility and mesenchymal phenotypes, suggesting a correlation between TGF-β-dependent cell cycle arrest and increased migration. We identified keratin-associated protein 2-3 (KRTAP2-3) as a regulator of these dual effects of TGF-β. Expression of KRTAP2-3 was associated with both TGF-β-induced cell cycle arrest and increased migration, and also was correlated with poor prognosis of head and neck cancer patients. We found that deletion of KRTAP2-3 gene decreased in vitro migration and in vivo metastasis of oral cancer cells via induction of mesenchymal-epithelial transition (MET). KRTAP2-3 also induces the expression of Regulator of G-protein signalling 4 (RGS4) and Zinc finger BED domain-containing protein 2 (ZBED2), which suppresses proliferation and increases migration of oral cancer cells, respectively. Present findings revealed novel mechanisms how TGF-β orchestrates proliferation and migration of cancer cells by inducing the expression of KRTAP2-3, and highlighted the importance of targeting the motile cancer cells under G1 cell cycle arrest to suppress metastasis. A total of 12 samples, 6 conditions (G1 and S/G2/M fractions, with/without TGF-b; KRTAP2-3 KO or control clones, no treatment), each in biological duplicate.