PMEPA1 has an oncogenic role in hepatocellular carcinoma in the context of TGF signaling: data from single cell RNAseq and transgenic models

Background: Transforming growth factor β (TGFβ) acts as a tumor suppressor in early stages of carcinogenesis, but promotes invasiveness, angiogenesis and immunosuppression in advanced stages. Prostate Transmembrane Protein Androgen Induced 1 (PMEPA1) negatively regulates TGFβ signaling by interacting with SMADs, and promotes TGFβ oncogenic effects in other cancers. Here, we explored the role of PMEPA1 in hepatocellular carcinoma (HCC). Methods: We analyzed transcriptomic, genomic, epigenomic and clinicopathological data of a discovery cohort of 228 HCCs and a validation cohort of 361 HCCs. PMEPA1 levels were quantified by qPCR in the discovery cohort, and PMEPA1 overexpression was validated in 6 other publicly available cohorts (n = 916) with microarray or RNAseq data. We analyzed PMEPA1 expression at the single-cell level using a publicly available single-cell RNAseq dataset including 16 HCCs. We inferred cell-cell interactions using CellPhoneDB. Genetically engineered mouse models were generated by hydrodynamic tail-vein injection of equal amounts of plasmids overexpressing MYC and MYC+PMEPA1. Tumors were analyzed by RNAseq and histopathological stainings. Results: PMEPA1 was overexpressed in 18% of human HCCs [FC>2; n = 203/1144], a feature associated with TGFβ signaling (p < 0.05). PMEPA1 overexpression was associated with gene body hypermethylation (p < 0.0001). HCCs displaying both Wnt/TGFβ signaling and high PMEPA1 (11% of cases) were significantly enriched in signatures of immune exhaustion, late TGFβ activation, tumor microenvironment (TME) response to TGFβ and active stroma (p < 0.05) as compared with HCCs with Wnt/TGFβ signalling alone (8% of cases) or PMEPA1 overexpression alone (11% of cases). Analyses of PMEPA1 expression at the single-cell level revealed that it is not only expressed by tumoral cells but also by stromal cells such as endothelial cells (ECs), fibroblasts and pericytes CellPhoneDB analysis revealed increased interactions between PMEPA1-expressing fibroblasts/tumor cells and immune suppressive cell subtypes. in vivo, overexpression of MYC +PMEPA1 led to HCC development in 56% of the mice (n = 9/16) and was associated with a significantly reduced survival when compared to mice overexpressing MYC alone (p = 0.014), which did not develop tumors (n = 0/ 7). Overexpression of PMEPA1 was confirmed at the mRNA level. The transcriptomically profiled MYC+PMEPA1 tumors (n = 4) presented a significant enrichment in late TGFβ activation and proliferation signatures when compared to healthy livers (n = 6) and MYC-lucOS;CTNNB1 tumors (n = 5). Conclusion: PMEPA1 upregulation is linked to TGFβ activation and an aggressive phenotype in human HCC. Overexpression of PMEPA1 in combination with MYC in vivo led to HCC development, showing for the first time the oncogenic role of PMEPA1 in this cancer type. No conflict of interest.