The identification and characterization of PPM1G in the regulation of cancer stemness in hepatocellular carcinoma

Abstract

PPM1G, belonging to the protein phosphatase family, is designated as Mg2+/Mn2+ dependent 1G. The protein structure of PPM1G consists of three main domains: phosphatase domains located at both termini and an acidic domain situated in the center. Liver development model revealed highest expression of PPM1G in both of the embryonic stem cells stage and tumor tissues of patient with hepatocellular carcinoma (HCC). Additionally, scRNA-seq data (GSE125449) indicated that PPM1G is enriched in stem-like HCC cluster. Clinically, PPM1G was found to be elevated in HCC samples across 24 types of cancer and was prognosis factor of worse overall survival in 9 types of cancer by the TCGA database. In another dataset GSE40267, PPM1G was significantly overexpressed in HCC patients with lung metastasis compared to patients without. Furthermore, analysis of both TCGA and ICGC-JP databases demonstrated that PPM1G serves as an independent adverse prognosis indicator for both the overall survival and disease-free survival in patients with HCC.

Functionally, our in vitro experiments indicated that knockdown of PPM1G undermined stemness features like sphere formation, ALDH activity, and ATRA drug (Tretinoin) induced differentiation. Knockdown of PPM1G also impaired tumor proliferation, cell-cycle progression, Foci formation, migration/invasion, and Cisplatin-mediated apoptosis. As for in vivo experiments, Silencing of PPM1G significantly inhibited the development of subcutaneous and liver orthotopic tumors, reduced lung metastasis, and attenuated cancer stemness, as evidenced by limited dilution experiments.

To explore the underlying mechanism, gene ontology analysis revealed that genes exhibiting a strong positive correlation with PPM1G (Spearman R > 0.5) showed enrichment in mRNA related process. Through the overlapping analysis of three databases (RBP2GO, HiPredict, and GEPIA), YBX1 (an mRNA m5C reader protein) was identified. This finding was further supported by in vivo immunoprecipitation and immunofluorescence experiments. Knockdown of PPM1G inhibited the cytoplasmic to nuclear translocation of YBX1, while its expression level remained almost unaffected according to qPCR and western blot analysis. YBX1 has previously been shown to be involved in the stability of mRNA of MYC (one of the four cell reprogramming transcription factors). Interestingly, bioinformatics analysis of PPM1G-regulated genes also revealed enrichment as MYC and NSUN2 (an m5C writer) targets, and qPCR analysis demonstrated significantly downregulated MYC expression in PPM1G knockdown cell lines. The Actinomycin assay further confirmed that the stability of MYC mRNA relies on PPM1G.

In conclusion, there is evidence to suggest that through PPM1G/YBX1/MYC axis, knockdown of PPM1G hinders the translocation of YBX1 from cytoplasm to the nucleus, thereby preventing the reading of m5C modified MYC mRNA by the writer NSUN2. This leads to increased degradation of MYC mRNA, impairing HCC stemness, chemo-resistance, tumor growth, and metastasis.