Non-canonical HIPPO-MST1/2 promotes hyper-proliferation of pulmonary vascular cells through CDC20

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Olapoju , View ORCID Profile Andressa Pena , Theodore Avolio , Derek Lin , Dmitry Goncharov , View ORCID Profile John R. Greenland , View ORCID Profile Paul J. Wolters , View ORCID Profile Horace DeLisser , View ORCID Profile Soni Savai Pullamsetti , View ORCID Profile Tatiana V. Kudryashova , View ORCID Profile Elena A. Goncharova doi: https://doi.org/10.1101/2025.04.03.646845 Tapan Dey 1 University of California at Davis , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tapan Dey Iryna Zhyvylo 1 University of California at Davis , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Iryna Zhyvylo Lifeng Jiang 1 University of California at Davis , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lifeng Jiang Samuel O. Olapoju 2 Max Planck Institute for Heart and Lung Research , Bad Nauheim, Germany 3 Department of Internal Medicine, Justus Liebig University, Institute for Lung Health (ILH), Member of German centre for Lung research (DZL) , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andressa Pena 4 University of Pittsburgh , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andressa Pena Theodore Avolio 4 University of Pittsburgh , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Derek Lin 1 University of California at Davis , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dmitry Goncharov 1 University of California at Davis , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site John R. Greenland 5 University of California, San Francisco School of Medicine 6 San Francisco Veterans Affairs Health Care System , San Francisco, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for John R. Greenland Paul J. Wolters 5 University of California, San Francisco School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paul J. Wolters Horace DeLisser 7 University of Pennsylvania School of Medicine , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Horace DeLisser Soni Savai Pullamsetti 2 Max Planck Institute for Heart and Lung Research , Bad Nauheim, Germany 3 Department of Internal Medicine, Justus Liebig University, Institute for Lung Health (ILH), Member of German centre for Lung research (DZL) , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Soni Savai Pullamsetti Tatiana V. Kudryashova 4 University of Pittsburgh , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tatiana V. Kudryashova Elena A. Goncharova 1 University of California at Davis , Davis, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elena A. Goncharova For correspondence: eagoncharova{at}ucdavis.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract HIPPO components mammalian Ste20-like protein kinases 1 and 2 (MST1/2) are well described growth suppressors. However, in pulmonary arterial hypertension (PAH), MST1/2 switch their roles and become pro-proliferative and pro-survival molecules, supporting hyper-proliferation of pulmonary artery (PA) smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs), remodeling of small PAs, and pulmonary hypertension. Here, we report that MST1/2 promotes hyper-proliferation and apoptosis resistance of human PAH PASMCs and PAAFs by up-regulating cell division cycle protein 20 (CDC20), establishing novel link between HIPPO-MST1/2 and cell cycle regulation in PAH. Authors Contributions conception and design of the work (EAG, SSP, TVK); acquisition, analysis, and interpretation of data (TD, IZ, LJ, SOO, AP, TA, DL, DG, JRG, PJW, HD, TK); drafting and editing the manuscript (EAG, SSP, TVK, JRG, PJW). To the Editor Hyper-proliferation and resistance to apoptosis of pulmonary vascular cells in small muscular pulmonary arteries (PA) are important components of pulmonary vascular remodeling, a major irreversible feature of pulmonary arterial hypertension (PAH) ( 1 ). Components of HIPPO signaling mammalian Ste20-like protein kinases 1 and 2 (MST1/2) (also known as serine/threonine-protein kinases 4/3) act as growth suppressors in somatic mammalian cells ( 2 ). However, in PAH pulmonary vasculature, MST1/2 promote hyper-proliferation and apoptosis resistance of pulmonary artery (PA) smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs) through up-regulating Budding Uninhibited By Benzimidazoles 3 Homolog (BUB3) and down-regulating Forkhead Box 3 (FOXO3), respectively ( 3 ). However, it is not clear whether PASMCs and PAAFs in PAH share any functional downstream effectors of MST1/2, which could be viewed as potential molecular targets for therapeutic intervention. The cell division cycle protein 20 (CDC20) is a pro-proliferative protein acting as an oncogene in several human cancers ( 4 ). STRING functional protein association network database predicted that CDC20 connects BUB3, STK4 (MST1), STK3 (MST2), and FOXO3, ( Figure 1A ), suggesting its potential role as a downstream effector of MST1/2 that is shared between PASMCs and PAAFs. Analysis of publicly available databases GSE117261, GSE144274, and GSE144932 ( 5 - 7 ) demonstrated that CDC20 expression is increased in the lungs and isolated PASMCs and PAAFs from patients with PAH compared to non-diseased controls ( Figure 1B-D ) and identified CDC20 as a most significantly up-regulated gene in human PAH PASMCs ( 5 ). Download figure Open in new tab Figure 1. MST1/2 promotes hyper-proliferation and survival of human PAH PASMCs and PAAFs via CDC20 A : Predicted interactions among STK4 (MST1), STK3 (MST2), BUB3, FOXO3, and CDC20 by the STRING functional protein association network database. B : CDC20 gene expression in human lung tissue from non-diseased and PAH subjects. ****p<0.0001 by Mann Whitney U. Data are derived from publicly available dataset GSE117261. C, D: Volcano plots of differentially expressed genes (DEGs) in PASMCs ( C ) and PAAFs ( D ) from PAH vs control subjects. Data are derived from the publicly available datasets GSE144274 and GSE144932. E, F : Human control and PAH PASMCs ( E ) and PAAFs ( F ) were treated with diluent (−) or 5μM MST1/2 inhibitor XMU-MP1 for 48 hours followed by immunoblot analysis to detect indicated proteins. Data are means±SE; n=5 subjects/group. *p<0.05, **p<0.01 by Kruskal Wallis (Dunn post-hoc correction). G-J : Human PAH PASMCs ( G, H ) or PAAFs ( I, J ) were transfected with siRNA MST1, siRNA MST2, or control scrambled siRNA (−) for 48 hours followed by immunoblot analysis to detect indicated proteins. Representative images and data presented as means±SE from n=5 subjects/group. *p<0.05, **p<0.01 by Kruskal Wallis (Dunn post-hoc correction). K-R : Human PAH PASMCs ( K-N ) and PAAFs ( O-R ) were transfected with pCMV3-CDC20-GFPSpark or control pCMV3-GFPSpark plasmid (−) and treated with 5μM XMU-MP1 or diluent. 48 hours later, BrdU incorporation ( K, O ), cell counts ( L, P ), cell viability (% of dead cells) ( M, Q ) were performed; apoptosis was examined using In situ cell death detection assay ( N, R ). Data are means±SE from n=5 subjects/group. *p<0.05, **p<0.01, ***p<0.001 by Kruskal Wallis (Dunn post-hoc correction). S : MST1/2 promotes hyper-proliferation and survival of PASMCs and PAAFs via up-regulating CDC20. Further studies are needed to establish whether MST1/2 regulate CDC20 via BUB3 and FOXO3. To test whether CDC20 acts downstream of MST1/2, we used pharmacological and molecular approaches. First, we treated early-passage human PAH and non-diseased (control) PASMCs and PAAFs with the selective MST1/2 inhibitor XMU-MP1. PASMCs and PAAFs from PAH patients had higher protein levels of CDC20 than respective control cells, which was significantly reduced by XMU-MP1 ( Figure 1E, F ). Supporting inhibitor-based findings, transfection of human PAH PASMCs and PAAFs with siRNA MST1 or siRNA MST2 dramatically reduced CDC20 protein content compared to cells transfected with control siRNA ( Figure 1G-J ). Taken together, these data show that CDC20 is overexpressed and acts as a downstream positive effector of MST1/2 in PASMCs and PAAFs from patients with PAH. To explore potential role of CDC20 in MST1/2-induced hyper-proliferation and survival of PAH pulmonary vascular cells, we used a “rescue” approach. We transfected human PAH PASMCs and PAAFs with mammalian vector expressing human CDC20 (pCMV3-CDC20-GFPSpark) or empty pCMV3-GFPSpark plasmid (control) in the presence or absence of MST1/2 inhibitor XMU-MP1 and tested cell proliferation and survival using BrdU incorporation, cell count and viability assays, and In Situ Cell Death Detection Kit ( 3 , 8 ). In line with published findings ( 3 ), XMU-MP1 significantly reduced proliferation and induced apoptosis in PAH PASMCs and PAAFs transfected with control plasmid ( Figure 1K-R ). In contrast, transfection with pCMV3-CDC20-GFPSpark protected cells from these XMU-MP1 effects ( Figure 1K-R ), suggesting that MST1/2 promote proliferation and survival of human PAH PASMCs and PAAFs via CDC20. In conclusion, our data identified CDC20 as a novel downstream effector of non-canonical HIPPO-MST1/2 signaling, which is required for MST1/2-dependent hyper-proliferation and apoptosis resistance of PASMCs and PAAFs from human PAH lungs ( Figure 1S ), providing novel link between HIPPO cassette and cell cycle regulation. Further studies are needed to determine the relationship between CDC20 and previously identified PAH-specific MST1/2 downstream effectors BUB3 and FOXO3. It is also important to note that dysregulation of cell cycle components cyclin-dependent kinases (CDKs) has been recently reported as an important driver of pulmonary vascular hyperproliferation, remodeling, and PAH ( 9 ). CDKs and CDC20 reciprocally regulate each other to drive cell cycle in physiological conditions ( 10 ). Our findings raise the possibility that MST1/2-induced CDC20 up-regulation could disturb physiological CDC20-CDKs coordination, further supporting unstimulated pulmonary vascular hyper-proliferation and remodeling in PAH. Our findings strongly suggest that further studies are needed to determine the mechanisms of regulation and function of CDC20 in healthy and diseased pulmonary vasculature and to evaluate a potential attractiveness of targeting CDC20 as a remodeling-focused strategy to treat PAH. Footnotes Sources of funding: R01 HL130261 (EAG), R01 HL166932 (TVK), German Research Foundation (DFG), Collaborative Research Center (CRC) 1213 projects A01 (S.S.P. and S.O.O.) References 1. ↵ Johnson S , Sommer N , Cox-Flaherty K , Weissmann N , Ventetuolo CE , Maron BA . Pulmonary Hypertension: A Contemporary Review . Am J Respir Crit Care Med 2023 ; 208 : 528 – 548 . OpenUrl CrossRef PubMed 2. ↵ Dey A , Varelas X , Guan KL . 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Olapoju , Andressa Pena , Theodore Avolio , Derek Lin , Dmitry Goncharov , John R. Greenland , Paul J. Wolters , Horace DeLisser , Soni Savai Pullamsetti , Tatiana V. Kudryashova , Elena A. Goncharova bioRxiv 2025.04.03.646845; doi: https://doi.org/10.1101/2025.04.03.646845 Share This Article: Copy Citation Tools Non-canonical HIPPO-MST1/2 promotes hyper-proliferation of pulmonary vascular cells through CDC20 Tapan Dey , Iryna Zhyvylo , Lifeng Jiang , Samuel O. Olapoju , Andressa Pena , Theodore Avolio , Derek Lin , Dmitry Goncharov , John R. Greenland , Paul J. Wolters , Horace DeLisser , Soni Savai Pullamsetti , Tatiana V. Kudryashova , Elena A. 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