Collagen hydroxylation couples NAD+/NADH dynamics to tumor dormancy and reactivation

Collagen hydroxylation couples NAD+/NADH dynamics to tumor dormancy and reactivation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Collagen hydroxylation couples NAD+/NADH dynamics to tumor dormancy and reactivation Daniela De Martino, Begoña Zapateria, Jaclyn Jaclyn Dunne, Stanislav Drapela, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6986228/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Metastasis remains the leading cause of cancer-related mortality. Disseminated tumor cells (DTCs) colonize distant organs where they enter a prolonged state of quiescence, named cellular dormancy, within collagen-rich extracellular matrix (ECM) niches. How dormant cells regulate the formation of collagen-rich niches and the mechanisms maintaining collagen proteostasis during dormancy and reactivation are not understood. Here, we identify prolyl hydroxylase P4HA2 as a key regulator of tumor dormancy through its dual role in collagen proline hydroxylation and mitochondrial function. We demonstrate that P4HA2-mediated proline hydroxylation of collagens balances the NAD+/NADH ratio, sustaining dormancy by limiting mitochondrial activity. Loss of P4HA2 disrupts collagen proteostasis, induces autophagy, and activates the proline catabolism enzyme ALDH4A1, lowering the NAD+/NADH ratio, which fuels mitochondrial energetics and triggers DTC awakening. Notably, ALDH4A1 is essential for the survival of these reactivated dormant cells, and its depletion induces apoptosis upon awakening, revealing a metabolic vulnerability in reactivated dormant cells. Our findings establish a previously unrecognized link between collagen homeostasis, NADH metabolism and tumor cell dormancy, unveiling a mechanistic framework for identifying actionable targets to eliminate DTCs and prevent metastatic relapse. Biological sciences/Cancer/Cancer microenvironment Biological sciences/Cell biology/Organelles/Mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations Yes there is potential Competing Interest. J.J.B.C is a consultant for HTL Biotechnology. P.M.A serve on the Board of Directors and are shareholders of N-zyme Scientifics. The other authors declare no competing interests. All mouse procedures were ethically approved by the Institutional Animal Care and Use Committee of the Icahn School of Medicine at Mount Sinai. Tables 1 to 3 are available in the Supplementary Files section. Supplementary Files TABLE1.xlsx Table 1. List of cell lines TABLE2.xlsx Table 2. List of shRNAs and siRNAs TABLE3.xlsx Table 3. List of antibodies Supplementallegends.docx Suppl.Figure1.jpg Suppl.Figure2.jpg Suppl.Figure3.jpg Suppl.Figure4pdf.jpg Suppl.Figure5.jpg Suppl.Figure6.jpg Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6986228","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":480193202,"identity":"dc699ecb-6352-4cb7-a864-ff4b77c29e6f","order_by":0,"name":"Daniela De Martino","email":"","orcid":"","institution":"Icahn School of Medicine at Mount Sinai","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"De Martino","suffix":""},{"id":480193203,"identity":"ce9d1fca-0364-4199-afc4-6f54366527d6","order_by":1,"name":"Begoña Zapateria","email":"","orcid":"","institution":"Albert Einstein Colelge of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Begoña","middleName":"","lastName":"Zapateria","suffix":""},{"id":480193204,"identity":"b129c361-b52c-4511-b256-4b89dbbcf534","order_by":2,"name":"Jaclyn Jaclyn Dunne","email":"","orcid":"","institution":"Medical University of South Carolina","correspondingAuthor":false,"prefix":"","firstName":"Jaclyn","middleName":"Jaclyn","lastName":"Dunne","suffix":""},{"id":480193205,"identity":"0e773308-4727-4516-9f1a-0086d12dc2de","order_by":3,"name":"Stanislav Drapela","email":"","orcid":"https://orcid.org/0000-0002-9421-9972","institution":"H. 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Lee Moffitt Cancer Center \u0026 Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Gomes","suffix":""},{"id":480193222,"identity":"cde69f31-b60b-4b92-a5ef-af3cd6dbf975","order_by":20,"name":"Peggi M. Angel","email":"","orcid":"","institution":"Protea Biosciences","correspondingAuthor":false,"prefix":"","firstName":"Peggi","middleName":"M.","lastName":"Angel","suffix":""},{"id":480193223,"identity":"323acc5a-cc59-4069-9ddc-598620c8f44e","order_by":21,"name":"Esperanza Arias","email":"","orcid":"","institution":"Albert Einstein Colelge of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Esperanza","middleName":"","lastName":"Arias","suffix":""},{"id":480193201,"identity":"1727f00b-1d70-4b67-986a-a8184ed538c4","order_by":22,"name":"Jose Javier Bravo-Cordero","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9481-1231","institution":"Icahn School of Medicine at Mount Sinai","correspondingAuthor":true,"prefix":"","firstName":"Jose","middleName":"Javier","lastName":"Bravo-Cordero","suffix":""}],"badges":[],"createdAt":"2025-06-26 20:05:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6986228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6986228/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87331697,"identity":"8a17b71d-8559-42ba-b1f9-785905c4e1df","added_by":"auto","created_at":"2025-07-22 19:08:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2364296,"visible":true,"origin":"","legend":"\u003ch1\u003eFigure 1. Collagen Proline hydroxylation is required for tumor cell dormancy\u003c/h1\u003e\n\u003cp\u003eA. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Upper panel: Representative images of CAMs 6 days (D6) after inoculation of control DMSO and 1,4 DPCA-treated D-HEp3 cells. Lower panel: Number of cells per tumor in CAM assays (n= 8 CAMs per group). D-HEp3 cells were treated for 48 hours with 1,4 DPCA at different concentrations (1, 5, 10µM) and inoculated in fertilized chicken embryos. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test.\u003c/p\u003e\n\u003cp\u003eB. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Representative multiphoton images of control DMSO and 1,4 DPCA-treated D-HEp3 cells expressing a CDK2 sensor (green) in CAMs. Second harmonic generation is shown in red (SHG) . Scale bar: 20µm. Quantification of the percentage of G0/G1 cells (with nuclear accumulation of CDK2 sensor) in D-HEp3 DMSO control (n= 6 CAMs, 14 fields) and D-HEp-3 treated with 1,4 DPCA at 5µM in CAMs (n=6 CAMs, 14 fields). Differences between groups were assessed using an unpaired twotailed t-test.\u003c/p\u003e\n\u003cp\u003eC. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Western blot for p27, phospho-histone3 (PHH3) and b-actin in D-HEp3 control and treated with 5µM of 1,4 DPCA for 48 hours.\u003c/p\u003e\n\u003cp\u003eD. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Quantification of hydroxylated collagen levels in CAM tumors from D-HEp3 control cells (DMSO) and treated with different concentrations of 1,4 DPCA (1, 5, 10µM). Concentration of hydroxyproline is represented as ng of hydroxyproline per µg of total protein content (n=5 CAMs). Group comparisons were assessed using one-way ANOVA with Dunnett’s post-hoc test to identify differences relative to the control group.\u003c/p\u003e\n\u003cp\u003eE. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;List of significantly dysregulated prolyl 4 hydroxylases (P4H) and prolyl 3 hydroxylases (P3H) genes in D-HEp3 nodules and T-HEp3 tumors grown in nude mice. Data are represented as log2 fold change D-HEp3/T-HEp3.\u003c/p\u003e\n\u003cp\u003eF. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Western blot analysis of P4HA2 expression in D-HEp3, 4T07, D2.0R and HCC1954 LCC (dormant) compared to T-HEp3, 4T1, D2.A1 and HCC1954 (proliferative) cells. b-actin was used as loading control.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/3012fd54c6f1cfd9b959b7fb.jpg"},{"id":87331914,"identity":"20ffced6-0c3d-4507-9dde-b20cb4d51ff3","added_by":"auto","created_at":"2025-07-22 19:16:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6459586,"visible":true,"origin":"","legend":"\u003ch1\u003eFigure 2. P4HA2 is required for tumor cell dormancy\u003c/h1\u003e\n\u003cp\u003eA. Representative immunofluorescence images of T-HEp3 and D-HEp3 cells stained for P4HA2 (green), ER marker PDIA3 (red) and DAPI (blue) for nuclei staining. Scale bar: 10µm.The graph shows quantification of P4HA2 intensity for D-HEp3 cells compared to T-HEp3 cells (n=74 cells/group). The two groups were statistically compared with the Mann-Whitney test.\u003c/p\u003e\n\u003cp\u003eB. Immunofluorescence images of D-HEp3 control shRNA nodules and P4HA2 KD tumors from CAMs stained for P4HA2 (green). DAPI was used for nuclei counterstaining. Scale bar: 10µm\u003c/p\u003e\n\u003cp\u003eC. Representative images of D-HEp-3 control nodules and P4HA2 KD tumors from CAMs. Number of cells from D-HEp3 control nodules (n=19 CAMs) and D-Hep3 P4HA2 knockdown tumors (shRNA1 n=11 and shRNA2 n=13 CAMs) are represented. To assess group differences, one-way ANOVA was conducted, and Dunnett’s test was applied to compare each group to the control.\u003c/p\u003e\n\u003cp\u003eD. Quantification of hydroxylated collagen levels in P4HA2 KD CAM tumors (n=6 CAMs) compared with\u003c/p\u003e\n\u003cp\u003eD-HEp3 control nodules (n=6 CAMs). Statistical significance is assessed by one-way ANOVA followed by Dunnett’s test to compare treatments to the control.\u003c/p\u003e\n\u003cp\u003eE. Representative images of D-HEp3 shRNA control nodules and D-HEp3 P4HA2 KD cells expressing a CDK2 biosensor (green). Second harmonic generation is shown in red (SHG). Scale bar: 20µm. Quantification of the percentage of G0/G1 cells per field in D-HEp3 cells shRNA control (n=15 fields, 3\u003c/p\u003e\n\u003cp\u003eCAM) and D-HEp3 P4HA2 KD (P4HA2 shRNA1 n=15 and P4HA2 shRNA3 n=15 fields, 6 CAM total) Differences between groups were analyzed by one-way ANOVA followed by Dunnett’s test to compare treatments to the control.\u003c/p\u003e\n\u003cp\u003eF. Western blot for p27 protein and PHH3 levels in P4HA2 KD D-HEp3 compared with D-HEp3 control. b-actin was used as loading control.\u003c/p\u003e\n\u003cp\u003eG. Left panels: Representative images of hematoxylin/eosin staining of mouse lungs derived from tail vein injections of D-HEp3 shRNA control and D-HEp3 P4HA2 KD. Scale bar: 5mm. Right Panels: Representative multiphoton images of solitary cells in D-HEp3 shRNA control and D-HEp3 P4HA2 KD micrometastasis in the lungs. In green, cancer cells expressing GFP. SHG signal (in red) highlights fibrillar collagens in the extracellular matrix Scale bar: 50μm.\u003c/p\u003e\n\u003cp\u003eH. Graph representing number of metastases per lung/mouse in D-HEp3 P4HA2 KD group compared with D-HEp3 control group. Statistical analysis included one-way ANOVA followed by Dunnett’s test to compare all group to the control. (shRNA control n=12, shRNA1= 13, shRNA2=12 mice)\u003c/p\u003e\n\u003cp\u003eI. Heat map showing protein levels measured by immunofluorescence in control and P4HA2-depleted primary tumors in mice. n\u0026gt;10000 cells per condition from 3 different tumors.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/2265cd0bdeeb704ad2dd8924.jpg"},{"id":87331700,"identity":"fe3d0620-ef25-4a98-92e5-ba43ecdfc0dd","added_by":"auto","created_at":"2025-07-22 19:08:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14574401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3. P4HA2 regulates the spatial distribution and abundance of proline modified collagens \u003c/strong\u003eA. Principal component analysis of human collagen peptides from control and P4HA2-depleted D-HEp3 tumors. n=3 tumors per condition.\u003c/p\u003e\n\u003cp\u003eB. Human collagen peptides showing significant differences between control and P4HA2-depleted DHEp3 tumors.\u003c/p\u003e\n\u003cp\u003eC. Representative images showing single HYP modified human collagens peptides with differential expression patterns between control and P4HA2 depleted D-HEp3 tumors.\u003c/p\u003e\n\u003cp\u003eD. Schematic representation of the experimental plan for ECM decellularization and reseeding experiment.\u003c/p\u003e\n\u003cp\u003eE. Representative ex vivo multiphoton images of CDK2 sensor (green) localization in D-HEp-3 parental cells repopulating D-HEp3 shRNA Control or D-HEp3 P4HA2 KD-derived ECM matrices. Scale bar: 50µm. The graph shows the percentage of G0/G1 cells per field. G0/G1 cells were analyzed in 6 different field per tumor.(CTR shRNA matrix: 4 CAMs, P4HA2 shRNA3 matrix: 3). Differences between groups were assessed using an unpaired two-tailed t-test.\u003c/p\u003e","description":"","filename":"Figure31.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/9e98d840c2a18aa5033896f8.jpg"},{"id":87331696,"identity":"5fa0f227-aade-4ae1-ae5f-0a9e505b47f3","added_by":"auto","created_at":"2025-07-22 19:08:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2219650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4. P4HA2 modulates autophagy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Immunoblot analysis of LC3 and p62 levels in control and P4HA2 KD D-HEp3 cells upon treatment with ammonium chloride (20 µM) and leupeptin (100 µM) for 6 hours. Ponceau staining was used as protein loading control. Quantification of autophagic flux calculated as LC3 Net flux relative to D-HEp3 shRNA control and D-HEp3 P4HA2 KD cells is shown. Quantification of p62 levels in D-HEp3 shRNA control in comparison with D-HEp3 P4HA2 KD cells. Statistical analysis was performed using an unpaired two-tailed t-test.\u003c/p\u003e\n\u003cp\u003eB. Representative immunoblot of ubiquitinated proteins in control and P4HA2 KD D-HEp3 cells following proteasome inhibition with MG132 (5µM) treatment for 6 hours.\u003c/p\u003e\n\u003cp\u003eC. Western blot and proteins levels quantification of key components of the mTOR signaling pathway, including mTOR, RAPTOR, phosphorylated 4E-BP1, and total 4E-BP1, in control and P4HA2 knockdown D-HEp3 cells. Ponceau staining is provided as a loading control. Statistical analysis was performed using an unpaired two-tailed t-test.\u003c/p\u003e\n\u003cp\u003eD. Heatmap representation of fold change in collagens accumulation upon ammonium chloride/leupeptin treatment in P4HA2 knockdown D-HEp3 cells relative to control D-HEp3 cells.\u003c/p\u003e\n\u003cp\u003eE. Representative images of COL5A1 and DAPI staining in control and P4HA2 KD cells. Quantification of COL5A1 intensity per cell is shown (n= 23 cells per condition). Scale bar: 10 µm.\u003c/p\u003e\n\u003cp\u003eF. Representative images of CAMs tumors derived from D-HEp3 cells control, P4HA2 knockdown, and P4HA2 knockdown cells transiently transfected with a small interfering RNA targeting COL5A1. The graph shows the number of cells per tumor per each group. Differences between groups were analyzed by one-way ANOVA followed by Dunnett’s test to compare treatments to the control.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/160c25d09657e062052a9c74.jpg"},{"id":87331698,"identity":"37100293-e0ca-4ec7-8431-0fc3ad40f5ab","added_by":"auto","created_at":"2025-07-22 19:08:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1052361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5. P4HA2 activity regulates mitochondria architecture and respiration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. 3D rendering of Tom20 staining and quantification of mitochondrial object number in cells expressing control shRNA (CTR shRNA) and P4HA2 shRNA (object ID color coded). Mitochondria were segmented from 3D confocal image stacks and object numbers were quantified. Scale bar: 5µm. Statistical analysis was performed using unpaired two-tailed t-tests.\u003c/p\u003e\n\u003cp\u003eB. Representative electron microscopy images of D-HEp3 KD control cells (1200x magnification, scale bar: 2 µm) and D-HEp3 P4HA2 KD cells (1200x magnification, scale bar: 2 µm). Right panel: The graph show the number of mitochondria per cell in D-HEp3 shRNA control cells versus D-HEp-3 P4HA2 knockdown cells. (n=12 cells per group). Statistical analysis was performed using an unpaired twotailed t-test.\u003c/p\u003e\n\u003cp\u003eC. Representative electron microscopy images of mitochondria structural details from D-HEp3 control (scale bar: 500nm) and D-HEp3 P4HA2 KD (scale bar: 400nm) cells.The graphs show the difference in the number of cristae, cristae width (in nm) and length (in nm) per mitochondrion between D-HEp3 shRNA Control cells (n=51 mitochondria, 231 cristae analyzed cells) and D-HEp3 P4HA2 knockdown cells (n=52 mitochondria, 310 cristae analyzed cells). Differences between groups were analyzed by one-way ANOVA followed by Dunnett’s test to compare treatments to the control.\u003c/p\u003e\n\u003cp\u003eD: Real-time measurement of oxygen consumption rate (OCR, pmol/min/cell) using the Seahorse XF Analyzer in control and P4HA2-depleted cells. Mitochondrial respiration was assessed by sequential injection of oligomycin (1 µM), FCCP (1.5 µM), and rotenone/antimycin A (0.5 µM each), as indicated. Data represent mean ± SEM of 7 technical replicates and are representative of two independent experiment.\u003c/p\u003e\n\u003cp\u003eE-K: Measurement of cellular basal respiration, maximal mitochondrial respiration rate, ATP production, spare respiratory capacity, Non-mitochondrial oxygen consumption, proton leak, and Coupling Efficiency expressed as oxygen consumption rate (pmol/min/cells) in D-HEp3 control cells compared to P4HA2 KD cells. A one-way ANOVA was performed to evaluate differences among groups, with Dunnett’s test used post hoc to compare each treatment group to the control group.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/838883d20e2a3312945b2e6a.jpg"},{"id":87331702,"identity":"b5fa93d5-9d06-4d0a-b18f-e9405cf3d069","added_by":"auto","created_at":"2025-07-22 19:08:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20051483,"visible":true,"origin":"","legend":"\u003ch1\u003eFigure 6. NAD+/NADH ratio is regulated by P4HA2 activity via ALDH4A1\u003c/h1\u003e\n\u003cp\u003eA. Cartoon Representing Mitochondrial Proline Metabolism: The diagram illustrates the key enzymatic steps in mitochondrial proline metabolism, including proline oxidation by proline dehydrogenase (PRODH) and conversion of P5C to glutamate by aldehyde dehydrogenase 4A1 (ALDH4A1), with associated production of NADH and ATP. These reactions support the tricarboxylic acid (TCA) cycle and contribute to cellular energy production.\u003c/p\u003e\n\u003cp\u003eB. Western blot analysis of ALDH4A1 (P5CDH) and PRODH protein levels in D-HEp3 shRNA control (CTR shRNA) and P4HA2 knockdown (P4HA2 shRNA) cells. β-actin was used as a loading control.\u003c/p\u003e\n\u003cp\u003eC. NAD+/NADH ratio measured in control shRNA (CTR) and P4HA2 KD D-HEp3 cells (RLU: relative luminescence units). Statistical analysis was performed using an unpaired two-tailed t-test. (n=4 measurements per group were performed).\u003c/p\u003e\n\u003cp\u003eD. NAD+/NADH ratio measured in CTR shRNA and P4HA2 knockdown cells (RLU: relative luminescence units), transfected with P5CDH or control siRNA. One-way ANOVA followed by multiple comparisons test was used to assess differences between experimental groups. (n=4 measurements per group were performed).\u003c/p\u003e\n\u003cp\u003eE. Western blot analysis of ALDH4A1 protein levels in P4HA2 KD and P4HA2 KD D-HEp3 cells transiently transfected for 48hrs with P5CDH (ALDH4A1) siRNA. b-actin was used as loading control F. Upper panel: Representative images of CAM nodules and tumors derived from DHEp3 shRNA control, D-HEp3 P4HA2 shRNA2 and D-HEp3 P4HA2 shRNA2 + siP5CDH. Lower panel: Number of cells per CAM assay (n=6 of Control, n=6 P4HA2 shRNA1, n=7 P4HA2 shRNA2, n=8 P4HA2 shRNA1+ siP5CDH, n=8 P4HA2 shRNA2+ siP5CDH CAMs). Tumors were harvested 6 days after inoculation and cells were counted. A one-way ANOVA was performed to evaluate differences among groups, with Dunnett’s test used post hoc to compare each treatment group to the control group.\u003c/p\u003e\n\u003cp\u003eG. Representative transmission electron microscopy (TEM) images of mitochondria ultrastructure (7000X) in D-HEp3 shRNA control and D-HEp-3 P4HA2 KD cells with or without silencing P5CDH (ALDH4A1). Scale bar: 500nm. The quantification categorizes mitochondria as normal, vesicular, vesicular-swollen or swollen based on morphological features and compared between different conditions. The analysis was carried out on 17 cells per each group.\u003c/p\u003e\n\u003cp\u003eH. Representative immunofluorescence images showing cleaved caspase-3 (red), F-actin (green), and DAPI for nuclei (blue) in D-HEp3 shRNA control and P4HA2 KD cells with or without silencing P5CDH (ALDH4A1). The bar graph quantifies cleaved caspase-3 staining intensity per cell (20 cells per each group). Scale bar: 25 µm. One-way ANOVA followed by multiple comparisons test was used to assess differences between experimental groups.\u003c/p\u003e","description":"","filename":"Figure6V2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/0f390bffbd45239e43395775.jpg"},{"id":87331701,"identity":"e014dcf3-4082-4961-af4c-abf954570633","added_by":"auto","created_at":"2025-07-22 19:08:37","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3249934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 7.\u003c/strong\u003e Proposed model for P4HA2 regulation of tumor dormancy. In dormant tumor cells, P4HA2 activity facilitates proper collagen folding by promoting proline hydroxylation. When P4HA2 is depleted (P4HA2 low), reduced proline hydroxylation leads to the accumulation of misfolded collagens. This triggers autophagy, which degrades the accumulated collagens and releases free proline. The recycled proline is then funneled into the mitochondria, where it is used as an energy source to support cellular awakening from dormancy.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/bf16eb3d44e4789ba528819d.jpg"},{"id":88506123,"identity":"e8b386f6-800b-46d5-8334-353cd25e8304","added_by":"auto","created_at":"2025-08-07 07:31:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":52484982,"visible":true,"origin":"","legend":"Article File","description":"","filename":"DeMartinoetal.Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1_covered_f8dada44-701d-4b8e-b0c9-7498d8fadd55.pdf"},{"id":86379179,"identity":"4b9c5fb8-30c4-4045-90f0-2ebe026a6bd8","added_by":"auto","created_at":"2025-07-10 03:48:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9770,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1. List of cell lines\u003c/p\u003e","description":"","filename":"TABLE1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/2859b9b7367849fea52f815d.xlsx"},{"id":86379180,"identity":"02b33e0d-104a-48b6-baac-9e8aca06d2fd","added_by":"auto","created_at":"2025-07-10 03:48:11","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10011,"visible":true,"origin":"","legend":"\u003cp\u003eTable 2. List of shRNAs and siRNAs\u003c/p\u003e","description":"","filename":"TABLE2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/9eb6f6b3349b3342cb2a43f4.xlsx"},{"id":86379181,"identity":"e4c84ff8-133e-4c96-a899-04b0359fc5ab","added_by":"auto","created_at":"2025-07-10 03:48:11","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10260,"visible":true,"origin":"","legend":"\u003cp\u003eTable 3. List of antibodies\u003c/p\u003e","description":"","filename":"TABLE3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/fff5eae342be27b4442d8294.xlsx"},{"id":86379186,"identity":"e05afed1-5150-4d37-9b25-ad4aebe2e1e5","added_by":"auto","created_at":"2025-07-10 03:48:12","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementallegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/f057b80bcc5c6bcb402dfcdd.docx"},{"id":87329096,"identity":"587983a0-0cae-4f95-bf3f-451a0dc07bc4","added_by":"auto","created_at":"2025-07-22 18:23:29","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5561813,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/edd2cde6973df86e89b0f059.jpg"},{"id":87329083,"identity":"8093c1b4-842b-4dd3-8c68-d972050dd42e","added_by":"auto","created_at":"2025-07-22 18:23:28","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11930094,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/507dd6bea0f566c6841a03a0.jpg"},{"id":87329082,"identity":"5d013a09-4eae-4203-a269-b575641cb005","added_by":"auto","created_at":"2025-07-22 18:23:28","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":590651,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/f011929cfec62a9de40c353e.jpg"},{"id":87329079,"identity":"17720716-c591-4a6b-b71d-7c0150bc1a1b","added_by":"auto","created_at":"2025-07-22 18:23:27","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":13407051,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure4pdf.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/7c3f513ed9d3f405874fc5b3.jpg"},{"id":87329080,"identity":"24514860-2646-4f71-af5e-c6c44c6ed6ef","added_by":"auto","created_at":"2025-07-22 18:23:28","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11072422,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/1ad76d5330b6d400844ee103.jpg"},{"id":87328717,"identity":"e8fc1c15-372c-4ff4-a891-ca0ad4d796f0","added_by":"auto","created_at":"2025-07-22 18:20:17","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":21530718,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6986228/v1/9760558fd293d565d730400c.jpg"}],"financialInterests":"\u003cp\u003e\u003cstrong\u003eYes\u003c/strong\u003e there is potential Competing Interest. J.J.B.C is a consultant for HTL Biotechnology. P.M.A serve on the Board of Directors and are shareholders of N-zyme Scientifics. The other authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eAll mouse procedures were ethically approved by the Institutional Animal Care and Use Committee of the Icahn School of Medicine at Mount Sinai.\u003c/p\u003e\n\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e","formattedTitle":"Collagen hydroxylation couples NAD+/NADH dynamics to tumor dormancy and reactivation","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6986228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6986228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Metastasis remains the leading cause of cancer-related mortality. Disseminated tumor cells (DTCs) colonize distant organs where they enter a prolonged state of quiescence, named cellular dormancy, within collagen-rich extracellular matrix (ECM) niches. How dormant cells regulate the formation of collagen-rich niches and the mechanisms maintaining collagen proteostasis during dormancy and reactivation are not understood. Here, we identify prolyl hydroxylase P4HA2 as a key regulator of tumor dormancy through its dual role in collagen proline hydroxylation and mitochondrial function. We demonstrate that P4HA2-mediated proline hydroxylation of collagens balances the NAD+/NADH ratio, sustaining dormancy by limiting mitochondrial activity. Loss of P4HA2 disrupts collagen proteostasis, induces autophagy, and activates the proline catabolism enzyme ALDH4A1, lowering the NAD+/NADH ratio, which fuels mitochondrial energetics and triggers DTC awakening. Notably, ALDH4A1 is essential for the survival of these reactivated dormant cells, and its depletion induces apoptosis upon awakening, revealing a metabolic vulnerability in reactivated dormant cells. Our findings establish a previously unrecognized link between collagen homeostasis, NADH metabolism and tumor cell dormancy, unveiling a mechanistic framework for identifying actionable targets to eliminate DTCs and prevent metastatic relapse.","manuscriptTitle":"Collagen hydroxylation couples NAD+/NADH dynamics to tumor dormancy and reactivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 03:48:07","doi":"10.21203/rs.3.rs-6986228/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3a64e909-deb3-486c-be27-6c67e4d71ad3","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50995094,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":50995095,"name":"Biological sciences/Cell biology/Organelles/Mitochondria"}],"tags":[],"updatedAt":"2025-07-24T17:10:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 03:48:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6986228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6986228","identity":"rs-6986228","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}