Targeting LIF With Cyclovirobuxine D to Suppress Tumor Progression via LIF/p38MAPK/p62-Modulated Mitophagy in Hepatocellular Carcinoma

Abstract

Leukemia inhibitory factor (LIF) exerts an oncogenic function in several types of cancer, including hepatocellular carcinoma (HCC). However, small-molecule inhibitors of LIF haven't been established. Here, we identified that LIF was remarkably overexpressed in HCC by multi-omics approaches, indicating that inhibition of LIF would be a promising therapeutic strategy. Inhibiting LIF could suppress proliferation and metastasis by activating p38MAPK/p62-modulated mitophagy. Interestingly, we found that the natural small-molecule Cyclovirobuxine-D (CVB-D), was a new inhibitor of cytoplasmic LIF in HCC. We further validated LIF as a potential target of CVB-D through biotin-modified CVB-D-Probe utilizing mass spectrometry. Mechanistically, we showed that CVB-D could bind to LIF at Val145, thereby inducing mitophagy, accompanied by cell cycle arrest and inhibition of invasion and migration. Moreover, we demonstrated that CVB-D had a therapeutic potential by targeting LIF-modulated mitophagy in patient-derived xenograft (PDX) models, which would elucidate LIF as a druggable target and regulatory mechanisms and exploit CVB-D as the novel small-molecule inhibitor of LIF for future HCC drug discovery.

Keywords

cyclovirobuxine D (CVB-D) / hepatocellular carcinoma (HCC) / leukemia inhibitory factor (LIF) / mitophagy / small-molecule inhibitor

References

[1] R. L. Siegel, K. D. Miller, N. S. Wagle, and A. Jemal, “Cancer Statistics, 2023,” CA: A Cancer Journal for Clinicians 73 (2023): 17-48.

[2] B. Sangro, P. Sarobe, S. Hervás-Stubbs, and I. Melero, “Advances in Immunotherapy for Hepatocellular Carcinoma,” Nature Reviews Gastroenterology & Hepatology 18 (2021): 525-543.

[3] J. M. Llovet, M. Kudo, P. Merle, et al., “Lenvatinib Plus Pembrolizumab Versus Lenvatinib Plus Placebo for Advanced Hepatocellular Carcinoma (LEAP-002): A Randomised, Double-Blind, Phase 3 Trial,” Lancet Oncology 24 (2023): 1399-1410.

[4] D. Q. Huang, A. G. Singal, F. Kanwal, et al., “Hepatocellular Carcinoma Surveillance—Utilization, Barriers and the Impact of Changing Aetiology,” Nature Reviews Gastroenterology & Hepatology 20 (2023): 797-809.

[5] L. L. Chan and S. L. Chan, “Drug Development for Hepatocellular Carcinoma,” Lancet Oncology 24 (2023): 1292-1294.

[6] H. N. Nguyen, E. H. Noss, F. Mizoguchi, et al., “Autocrine Loop Involving IL-6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators,” Immunity 46 (2017): 220-232.

[7] J. Hall, G. Guo, J. Wray, et al., “Oct4 and LIF/Stat3 Additively Induce Krüppel Factors to Sustain Embryonic Stem Cell Self-Renewal,” Cell Stem Cell 5 (2009): 597-609.

[8] R. Hallett, E. Bonfill-Teixidor, R. Iurlaro, et al., “Therapeutic Targeting of LIF Overcomes Macrophage Mediated Immunosuppression of the Local Tumor Microenvironment,” Clinical Cancer Research 29, no. 4 (2022): 791-804.

[9] B. P. Haines, R. B. Voyle, T. A. Pelton, R. Forrest, and P. D. Rathjen, “Complex Conserved Organization of the Mammalian Leukemia Inhibitory Factor Gene: Regulated Expression of Intracellular and Extracellular Cytokines,” Journal of Immunology 162 (1999): 4637-4646.

[10] S.-C. Liu, T. Hsu, Y.-S. Chang, et al., “Cytoplasmic LIF Reprograms Invasive Mode to Enhance NPC Dissemination Through Modulating YAP1-FAK/PXN Signaling,” Nature Communications 9 (2018): 5105.

[11] Y. Shi, W. Gao, N. K. Lytle, et al., “Targeting LIF-mediated Paracrine Interaction for Pancreatic Cancer Therapy and Monitoring,” Nature 569 (2019): 131-135.

[12] C. Zhang, J. Liu, J. Wang, W. Hu, and Z. Feng, “The Emerging Role of Leukemia Inhibitory Factor in Cancer and Therapy,” Pharmacology & Therapeutics 221 (2021): 107754.

[13] S. A. Jones and B. J. Jenkins, “Recent Insights Into Targeting the IL-6 Cytokine family in Inflammatory Diseases and Cancer,” Nature Reviews Immunology 18 (2018): 773-789.

[14] M. Pascual-García, E. Bonfill-Teixidor, E. Planas-Rigol, et al., “LIF Regulates CXCL9 in Tumor-Associated Macrophages and Prevents CD8⁺ T Cell Tumor-Infiltration Impairing Anti-PD1 Therapy,” Nature Communications 10 (2019): 2416.

[15] C. Hu, R. Xia, X. Zhang, et al., “circFARP1 Enables Cancer-Associated Fibroblasts to Promote Gemcitabine Resistance in Pancreatic Cancer via the LIF/STAT3 Axis,” Molecular Cancer 21 (2022): 24.

[16] X. Zhang, B. Hu, Y. Sun, et al., “Arsenic Trioxide Induces Differentiation of Cancer Stem Cells in Hepatocellular Carcinoma Through Inhibition of LIF/JAK1/STAT3 and NF-kB Signaling Pathways Synergistically,” Clinical and Translational Medicine 11 (2021): e335.

[17] J. Zhang, M. He, W. Fu, et al., “Primate-Specific microRNA-637 Inhibits Tumorigenesis in Hepatocellular Carcinoma by Disrupting Signal Transducer and Activator of Transcription 3 Signaling,” Hepatology 54 (2011): 2137-2148.

[18] B. P. Haines, R. B. Voyle, and P. D. Rathjen, “Intracellular and Extracellular Leukemia Inhibitory Factor Proteins Have Different Cellular Activities That Are Mediated by Distinct Protein Motifs,” MBoC 11 (2000): 1369-1383.

[19] T. Hisaka, A. Desmoulière, J.-L. Taupin, et al., “Expression of Leukemia Inhibitory Factor (LIF) and Its Receptor gp190 in Human Liver and in Cultured Human Liver Myofibroblasts. Cloning of New Isoforms of LIF mRNA,” Comparative Hepatology 3 (2004): 10.

[20] L. Zhang, R. Fu, D. Duan, et al., “Cyclovirobuxine D Induces Apoptosis and Mitochondrial Damage in Glioblastoma Cells through ROS-Mediated Mitochondrial Translocation of Cofilin,” Frontiers in Oncology 11 (2021): 656184.

[21] L. Zhou, H. Tang, F. Wang, et al., “Cyclovirobuxine D Inhibits Cell Proliferation and Migration and Induces Apoptosis in Human Glioblastoma Multiforme and Low‑Grade Glioma,” Oncology Reports 43, no. 3 (2020): 807-816.

[22] F. Jiang, Y. Chen, S. Ren, et al., “Cyclovirobuxine D Inhibits Colorectal Cancer Tumorigenesis via the CTHRC1‑AKT/ERK‑Snail Signaling Pathway,” International Journal of Oncology 57, no. 1 (2020): 183-196.

[23] J. Zhang, Y. Chen, J. Lin, et al., “Cyclovirobuxine D Exerts Anticancer Effects by Suppressing the EGFR-FAK-AKT/ERK1/2-Slug Signaling Pathway in Human Hepatocellular Carcinoma,” DNA and Cell Biology 39 (2020): 355-367.

[24] S. Zhang, J. Zhang, Y. An, et al., “Multi-Omics Approaches Identify SF3B3 and SIRT3 as Candidate Autophagic Regulators and Druggable Targets in Invasive Breast Carcinoma,” Acta Pharmaceutica Sinica B 11 (2021): 1227-1245.

[25] T. Lamark, S. Svenning, and T. Johansen, “Regulation of Selective Autophagy: The p62/SQSTM1 Paradigm,” Essays in Biochemistry 61 (2017): 609-624.

[26] H.-U. Simon, R. Friis, S. W. G. Tait, and K. M. Ryan, “Retrograde Signaling From Autophagy Modulates Stress Responses,” Science Signaling 10 (2017): eaag2791.

[27] A. Cassidy-Stone, J. E. Chipuk, E. Ingerman, et al., “Chemical Inhibition of the Mitochondrial Division Dynamin Reveals Its Role in Bax/Bak-Dependent Mitochondrial Outer Membrane Permeabilization,” Developmental Cell 14 (2008): 193-204.

[28] S. T. Luk, K. Ng, L. Zhou, et al., “Deficiency in Embryonic Stem Cell Marker Reduced Expression 1 Activates Mitogen-Activated Protein Kinase Kinase 6-Dependent p38 Mitogen-Activated Protein Kinase Signaling to Drive Hepatocarcinogenesis,” Hepatology 72 (2020): 183-197.

[29] A. Tomás-Loba, E. Manieri, B. González-Terán, et al., “p38γ is Essential for Cell Cycle Progression and Liver Tumorigenesis,” Nature 568 (2019): 557-560.

[30] Z. Huang, L. Chu, J. Liang, et al., “H19 Promotes HCC Bone Metastasis Through Reducing Osteoprotegerin Expression in a Protein Phosphatase 1 Catalytic Subunit Alpha/p38 Mitogen-Activated Protein Kinase-Dependent Manner and Sponging microRNA 200b-3p,” Hepatology 74 (2021): 214-232.

[31] Y.-Y. Zhang, Z.-Q. Mei, J.-W. Wu, and Z.-X. Wang, “Enzymatic Activity and Substrate Specificity of Mitogen-Activated Protein Kinase p38α in Different Phosphorylation States,” Journal of Biological Chemistry 283 (2008): 26591-26601.

[32] T. Pantsar, P. D. Kaiser, M. Kudolo, M. Forster, U. Rothbauer, and S. A. Laufer, “Decisive Role of Water and Protein Dynamics in Residence Time of p38α MAP Kinase Inhibitors,” Nature Communications 13 (2022): 569.

[33] A. J. Burton, M. Haugbro, L. A. Gates, J. D. Bagert, C. D. Allis, and T. W. Muir, “In Situ Chromatin Interactomics Using a Chemical Bait and Trap Approach,” Nature Chemistry 12 (2020): 520-527.

[34] L. P. Conway, A. M. Jadhav, R. A. Homan, et al., “Evaluation of Fully-functionalized Diazirine Tags for Chemical Proteomic Applications,” Chemical Science 12 (2021): 7839-7847.

[35] S. Viswanadhapalli, K. V. Dileep, K. Y. J. Zhang, H. B. Nair, and R. K. Vadlamudi, “Targeting LIF/LIFR Signaling in Cancer,” Genes & Diseases 9, no. 4 (2021): 973-980.

[36] Y. Loriot, A. Marabelle, J. P. Guégan, et al., “Plasma Proteomics Identifies Leukemia Inhibitory Factor (LIF) as a Novel Predictive Biomarker of Immune-Checkpoint Blockade Resistance,” Annals of Oncology 32 (2021): 1381-1390.

[37] M.-T. Wang, N. Fer, J. Galeas, et al., “Blockade of Leukemia Inhibitory Factor as a Therapeutic Approach to KRAS Driven Pancreatic Cancer,” Nature Communications 10 (2019): 3055.

[38] X. Bao, Y. Liu, J. Huang, et al., “Stachydrine Hydrochloride Inhibits Hepatocellular Carcinoma Progression via LIF/AMPK Axis,” Phytomedicine 100 (2022): 154066.

[39] S. Yin, W. Jin, Y. Qiu, L. Fu, T. Wang, and H. Yu, “Solamargine Induces Hepatocellular Carcinoma Cell Apoptosis and Autophagy via Inhibiting LIF/miR-192-5p/CYR61/Akt Signaling Pathways and Eliciting Immunostimulatory Tumor Microenvironment,” Journal of Hematology & Oncology 15 (2022): 32.

[40] Y.-N. Liu, S. Niu, W.-Y. Chen, et al., “Leukemia Inhibitory Factor Promotes Castration-Resistant Prostate Cancer and Neuroendocrine Differentiation by Activated ZBTB46,” Clinical Cancer Research 25 (2019): 4128-4140.

[41] Y. Shao, W. Pu, R. Su, et al., “Autocrine and Paracrine LIF Signals to Collaborate Sorafenib-resistance in Hepatocellular Carcinoma and Effects of Kanglaite Injection,” Phytomedicine 136 (2024): 156262.

[42] J. S. Fitzgerald, T. G. Poehlmann, E. Schleussner, and U. R. Markert, “Trophoblast Invasion: The Role of Intracellular Cytokine Signalling via Signal Transducer and Activator of Transcription 3 (STAT3),” Human Reproduction Update 14 (2008): 335-344.

[43] X. Zheng, M. Huang, L. Xing, et al., “The circRNA circSEPT9 Mediated by E2F1 and EIF4A3 Facilitates the Carcinogenesis and Development of Triple-Negative Breast Cancer,” Molecular Cancer 19 (2020): 73.

[44] W.-H. Lin, Y.-W. Chang, M.-X. Hong, et al., “STAT3 Phosphorylation at Ser727 and Tyr705 Differentially Regulates the EMT-MET Switch and Cancer Metastasis,” Oncogene 40 (2021): 791-805.

[45] H. Yu, X. Yue, Y. Zhao, et al., “LIF Negatively Regulates Tumour-Suppressor p53 Through Stat3/ID1/MDM2 in Colorectal Cancers,” Nature Communications 5 (2014): 5218.

[46] Q. Zhang, S. Cao, F. Qiu, and N. Kang, “Incomplete Autophagy: Trouble Is a Friend,” Medicinal Research Reviews 42 (2022): 1545-1587.

[47] J. Lu, D. Sun, S. Gao, Y. Gao, J. Ye, and P. Liu, “Cyclovirobuxine D Induces Autophagy-Associated Cell Death via the Akt/mTOR Pathway in MCF-7 Human Breast Cancer Cells,” Journal of Pharmacological Sciences 125 (2014): 74-82.

[48] J. Wu, Z. Tan, J. Chen, and C. Dong, “Cyclovirobuxine D Inhibits Cell Proliferation and Induces Mitochondria-Mediated Apoptosis in Human Gastric Cancer Cells,” Molecules 20 (2015): 20659-20668.

[49] C. Zeng, T. Zou, J. Qu, X. Chen, S. Zhang, and Z. Lin, “Cyclovirobuxine D Induced-Mitophagy Through the p65/BNIP3/LC3 Axis Potentiates Its Apoptosis-Inducing Effects in Lung Cancer Cells,” IJMS 22 (2021): 5820.

[50] H. Tanton, T. Sewastianik, H.-S. Seo, et al., “A Novel β-catenin/BCL9 Complex Inhibitor Blocks Oncogenic Wnt Signaling and Disrupts Cholesterol Homeostasis in Colorectal Cancer,” Science Advances 8 (2022): eabm3108.

[51] Y. Mo, Y. Wang, Y. Wang, et al., “Circular RNA circPVT1 Promotes Nasopharyngeal Carcinoma Metastasis via the β-TrCP/c-Myc/SRSF1 Positive Feedback Loop,” Molecular Cancer 21 (2022): 192.

[52] J. Ju, A. Chen, Y. Deng, et al., “NatD Promotes Lung Cancer Progression by Preventing Histone H4 Serine Phosphorylation to Activate Slug Expression,” Nature Communications 8 (2017): 928.

[53] L. Wang, C.-M. Cheng, J. Qin, et al., “Small-Molecule Inhibitor Targeting Orphan Nuclear Receptor COUP-TFII for Prostate Cancer Treatment,” Science Advances 6 (2020): eaaz8031.

[54] R. J. Chai, H. Werner, P. Y. Li, et al., “Disrupting the LINC Complex by AAV Mediated Gene Transduction Prevents Progression of Lamin Induced Cardiomyopathy,” Nature Communications 12 (2021): 4722.

[55] M. D. Wilkerson and D. N. Hayes, “ConsensusClusterPlus: A Class Discovery Tool With Confidence Assessments and Item Tracking,” Bioinformatics 26 (2010): 1572-1573.

[56] P. Langfelder and S. Horvath, “WGCNA: An R Package for Weighted Correlation Network Analysis,” BMC Bioinformatics 9 (2008): 559.

[57] J. Reimand, R. Isserlin, V. Voisin, et al., “Pathway Enrichment Analysis and Visualization of Omics Data Using G:Profiler, GSEA, Cytoscape and EnrichmentMap,” Nature Protocols 14 (2019): 482-517.

[58] P. Shannon, A. Markiel, O. Ozier, et al., “Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks,” Genome Research 13 (2003): 2498-2504.

[59] M. I. Love, W. Huber, and S. Anders, “Moderated Estimation of Fold Change and Dispersion for RNA-seq Data With DESeq2,” Genome Biology 15 (2014): 550.

[60] A. Zhu, J. G. Ibrahim, and M. I. Love, “Heavy-Tailed Prior Distributions for Sequence Count Data: Removing the Noise and Preserving Large Differences,” Bioinformatics 35 (2019): 2084-2092.

[61] G. Yu, L.-G. Wang, Y. Han, and Q.-Y. He, “clusterProfiler: An R Package for Comparing Biological Themes among Gene Clusters,” OMICS: A Journal of Integrative Biology 16 (2012): 284-287.

[62] C. Ginestet, “ggplot2: Elegant Graphics for Data Analysis: Book Reviews,” Journal of the Royal Statistical Society: Series A (Statistics in Society) 174 (2011): 245-246.

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