Non-canonical activation of MAPK signaling by the lncRNA ASH1L-AS1-encoded microprotein APPLE through inhibition of PP1/PP2A-mediated ERK1/2 dephosphorylation in hepatocellular carcinoma.

backgroundMAPK/ERK1/2 signaling is often activated in hepatocellular carcinoma (HCC), yet classical RAS-RAF-MEK mutations are rare, indicating the involvement of non-canonical regulatory mechanisms. Long non-coding RNAs (lncRNAs) can encode microproteins that play key roles in cancer. LncRNA ASH1L-AS1 has coding potential, but its role in HCC remains unclear. Clarifying its role in MAPK signaling may uncover novel therapeutic targets for HCC.

methodsTranslatable lncRNAs associated with HCC were identified by integrating data from the TCGA-LIHC cohort and the TransLnc database. The functional role of ASH1L-AS1 and its encoded microprotein APPLE was explored through in vitro and in vivo assays, such as CCK-8, EdU incorporation, wound healing, Transwell migration and invasion, and xenograft tumor models. Mechanistic investigations were conducted to elucidate molecular mechanisms and identify potential therapeutic strategies, including co-immunoprecipitation, mass spectrometry, ChIP-qPCR, luciferase reporter assays, truncation mutation analysis, immunofluorescence, Western blot, RNA sequencing, drug sensitivity analysis etc.

resultsA total of 696 translatable lncRNAs associated with HCC were identified, with their encoded products exhibiting specific subcellular localization. Among them, ASH1L-AS1 stood out due to strong translational evidence and its significant association with disease progression, poor prognosis, immunosuppressive tumor microenvironment, and estrogen signaling. We confirmed that ASH1L-AS1 encodes a microprotein, APPLE, which is stably expressed in HCC cells and consistently upregulated in tumor tissues regardless of RAS mutation status. Functionally, APPLE promotes ERK1/2 phosphorylation, activates MAPK signaling, and enhances HCC cell proliferation, migration, invasion, and tumor growth-effects reversed by APPLE knockdown or ERK1/2 inhibition. Mechanistically, APPLE binds to ERK1/2 and phosphatases PP1/PP2A, preventing ERK1/2 dephosphorylation and sustaining MAPK pathway activation. Additionally, the transcription factor E2F1 directly binds to the ASH1L-AS1 promoter (- 300 to - 290 bp), upregulating APPLE expression and further amplifying ERK1/2 signaling. Drug sensitivity analysis identified 220 treatment combinations potentially effective against HCC subtypes driven by hyperactivation of the E2F1-ASH1L-AS1/APPLE-ERK1/2 axis.

conclusionsThis study characterized APPLE as a novel oncogenic microprotein encoded by lncRNA ASH1L-AS1, uncovering a non-canonical mechanism of MAPK activation in HCC. The identified E2F1-ASH1L-AS1/APPLE-ERK1/2 signaling axis provides new insights into HCC pathogenesis and represents a promising target for precision therapy, though further validation in clinical cohorts and preclinical studies is needed.