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
Abstract 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. LncR...
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2025-07-01
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| Series: | Journal of Experimental & Clinical Cancer Research |
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| Online Access: | https://doi.org/10.1186/s13046-025-03465-w |
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| author | Lei Zhao Ke Si Shenjian Luo Lantian Zhang Shuai Mao Wenliang Zhang |
| author_facet | Lei Zhao Ke Si Shenjian Luo Lantian Zhang Shuai Mao Wenliang Zhang |
| author_sort | Lei Zhao |
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| description | Abstract 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. |
| format | Article |
| id | doaj-art-4c26c639eea744f692b5f01ad1646600 |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-07-01 |
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| spelling | doaj-art-4c26c639eea744f692b5f01ad16466002025-08-20T04:02:42ZengBMCJournal of Experimental & Clinical Cancer Research1756-99662025-07-0144112110.1186/s13046-025-03465-wNon-canonical activation of MAPK signaling by the lncRNA ASH1L-AS1-encoded microprotein APPLE through inhibition of PP1/PP2A-mediated ERK1/2 dephosphorylation in hepatocellular carcinomaLei Zhao0Ke Si1Shenjian Luo2Lantian Zhang3Shuai Mao4Wenliang Zhang5The Key Laboratory of Advanced Interdisciplinary Studies, The First Affiliated Hospital of Guangzhou Medical University, GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macao Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou Medical UniversityThe Key Laboratory of Advanced Interdisciplinary Studies, The First Affiliated Hospital of Guangzhou Medical University, GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macao Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou Medical UniversityDepartment of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical UniversityThe Key Laboratory of Advanced Interdisciplinary Studies, The First Affiliated Hospital of Guangzhou Medical University, GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macao Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou Medical UniversityDepartment of Hepatic Surgery, the First Affiliated Hospital, Sun Yat-sen UniversityThe Key Laboratory of Advanced Interdisciplinary Studies, The First Affiliated Hospital of Guangzhou Medical University, GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macao Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou Medical UniversityAbstract 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.https://doi.org/10.1186/s13046-025-03465-wASH1L-AS1Liver cancerLncRNA encoded-microproteinMAPK signalingPP1/PP2A |
| spellingShingle | Lei Zhao Ke Si Shenjian Luo Lantian Zhang Shuai Mao Wenliang Zhang 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 Journal of Experimental & Clinical Cancer Research ASH1L-AS1 Liver cancer LncRNA encoded-microprotein MAPK signaling PP1/PP2A |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | ASH1L-AS1 Liver cancer LncRNA encoded-microprotein MAPK signaling PP1/PP2A |
| url | https://doi.org/10.1186/s13046-025-03465-w |
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