Angiopoietin-like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase-phosphoinositide 3-kinase-dependent anti-apoptotic signaling.
Bottom Line: Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells.Expression of anti-apoptotic BCL-2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells.Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase-nuclear factor of activated T cells (Syk-NFAT) pathway.
Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.Show MeSH
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Mentions: To examine signaling downstream of ANGPTL2 that might induce anti-apoptotic genes, we carried out RNA sequencing analysis of SW480/ANGPTL2-1 and SW480/Ctrl cells (Fig. 3a). We found that levels of 10 transcripts, including ANGPTL2 itself, were markedly increased in SW480/ANGPTL2-1 compared with SW480/Ctrl cells. As BCL-2 and BCL-XL were induced more robustly in SW480/ANGPTL2 compared to SW480/Ctr cells (Fig. 2), we checked their expression by RNA sequencing but did not observe significant induction of either gene using this method. However, we observed upregulation of Syk, which plays a role in cell survival,(10,12,13) in SW480/ANGPTL2 cells. Real-time PCR analysis validated that Syk expression levels in SW480/ANGPTL2-1 and -2 cells increased compared with levels seen in control SW480/Ctrl cells (Fig. 3b). Tyrosine phosphorylation is required to activate Syk kinase activity and function.(21) Thus, we examined Syk activity in SW480/ANGPTL2-1 and -2 versus control cells by Western blotting with a phospho-Syk antibody. Syk expression and phosphorylation was markedly increased in SW480/ANGPTL2-1 and -2 cells compared with SW480/Ctrl cells (Fig. 3c), suggesting that the enhanced cell survival activity seen in both lines is due to Syk upregulation and activation. Next, we asked whether Syk knockdown by siRNA in ANGPTL2-expressing SW480 cells would reduce cell viability or induce cell apoptosis following 5-FU treatment. We confirmed Syk knockdown levels in SW480/ANGPTL2-1 cells by Western blot analysis. Total Syk and phospho-Syk protein were concomitantly reduced by knockdown (Fig. 3d). We observed no significant difference in proliferation between knockdown versus scrambled control cells at 24, 48 or 72 h after plating in normal growth conditions (Fig. 3e). However, Syk knockdown cells (siRNA/A, B, and C) showed decreased viability following 5-FU treatment compared with scrambled control (siRNA Scr) cells (Fig. 3f). In addition, the proportion of apoptosis significantly increased in siRNA/A, B, and C cells compared to siRNA Scr cells (Fig. 3g). Thus, we conclude that Syk upregulation and activation enhances cell survival in ANGPTL2-overexpressing CRC cells.
Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.