Abstract PO-040: Oral squamous carcinoma cells under TGF-β-induced cell cycle arrest represent highly motile and invasive population

Abstract

Abstract Cancer cells forming primary tumors are heterogeneous and respond in various ways to the stimuli from tumor microenvironment. Transforming growth factor-β (TGF-β) has been implicated in progression of various types of epithelial cancer. TGF-β is known to induce epithelial-mesenchymal transition (EMT) leading to increased invasiveness and thus metastasis. In addition, it has been reported that TGF-β arrests the epithelial cell in early G1 phase. While TGF-β elicits multiple effects on epithelial cancer cells, heterogeneity of tumor mass results in a different response depending on the cell state, thus it remains to be elucidated whether the cells exhibiting TGF-β-induced EMT and high motility are also under the cell cycle arrest. To examine the correlation between TGF-β-induced motility and cell cycle arrest at a single cell level, we have utilized oral squamous carcinoma cell lines, carrying fluorescent ubiquitination-based cell cycle indicator (Fucci) system. Using quantitative RT-PCR, immunoblotting and FACS analysis, we determined the expression of cell cycle-related genes and characterized the population of cells residing in certain cell cycle phases upon TGF-β treatment. Cells treated with TGF-β were also tested for their migratory ability to compare motility of cells residing in different cycle phases. Surprisingly, cells residing in G1 phase were characterized by higher motility than cells residing in S/G2/M phases suggesting a correlation between TGF-β-dependent cell cycle progression and migration. Moreover, increased motility of cells residing in G1 phase did not resulted from the progression of EMT. These results were also confirmed by our in vivo studies. In orthotropic xenograft model, cells under G1 arrest represented the majority of cells forming metastatic lymph nodules, in contrast to primary tumor mass comprising almost of equal number of cells residing in S/G2/M and G1 phases. We also performed cDNA microarray analyses of G1 and S/G2/M phase SAS-Fucci cells and identified gene whose expression was associated with both TGF-β-induced migration and cell cycle arrest. In conclusion our data suggested that cells under G1 cell cycle arrest are motile, but this increased motility is not a result of TGF-β-dependent EMT. Our data also strengthen the importance of targeting both, proliferating cancer cells and cell cycle-arrested cancer cells for the effective anti-cancer therapy. Citation Format: Katarzyna A. Inoue, Kazuki Takahashi, Maki Saito, Atsushi Kaida, Akinari Sugauchi, Toshihiro Uchihashi, Yasuhiro Yoshimatsu, Susumu Tanaka, Masahiko Miura, Mikihiko Kogo, Tetsuro Watabe. Oral squamous carcinoma cells under TGF-β-induced cell cycle arrest represent highly motile and invasive population [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-040.