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.

BACKGROUND: MAPK/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. METHODS: Translatable 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. RESULTS: A 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. CONCLUSIONS: This 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.