Paclitaxel Suppressed N6-Methyladenosine of PUM1 offsets cetuximab resistance in colorectal cancer

Abstract

Abstract Background We have previously demonstrated that RNA-binding protein Pumilio-1 (PUM1) is ubiquitous in cetuximab-resistant colorectal cancer (CRC) cells. The role of the N6-methyladenosine modification of PUM1 influenced by paclitaxel (PTX) in modulating cetuximab-resistance in CRC cells was investigated in the current work. Methods PUM1 mRNA expression in CRC tissues and cells was measured by quantitative real-time PCR (qRT-PCR), and PUM1 protein expression was measured by immunohistochemistry (IHC). The involvement of PUM1 expression in CRC prognosis was evaluated by survival analysis. RNA immunoprecipitation (RIP) was performed to evaluate combination capability of PUM1 and YTHDF1. RNA m6A dot blot assays were conducted to investigate variations in the degree of the METTL3-induced N6-methyladenosine modification of PUM1. Patient derived tumor xenograft (PDX) models to investigate the regulatory role of paclitaxel inhibited N6-demethyladenosine modification of PUM1 and diminished PUM1 mRNA level. Results The N6-methyladenosine content of PUM1 was increased in CRC by cetuximab treatment and increased PUM1 mRNA stability (P < 0.05). PUM1 induced monocyte-to-macrophage differentiation of CRC cells during in vitro functional assays and activated the WNT axis by enhancing DDX5 expression (P < 0.05). Paclitaxel increased PUM1 N6-demethyladenosine levels and decreased PUM1 expression (P < 0.05), resulting in suppression of the WNT pathway. Paclitaxel also reduced PUM1-induced cetuximab-resistance in CRC cells. CRC xenografts from human patients had enhanced m6A modification of PUM1 in paclitaxel-treated samples (P < 0.05) shown by ex vivo studies. In clinic, high PUM1 levels in CRC patients correlated with elevated TAM content and poor survival (P < 0.05). Conclusion Inhibition of N6-methyladenosine modification of PUM1 is proposed as a novel therapeutic target to overcome cetuximab-resistance in CRC.