The DIAPH3/RPL6 axis regulates the cGAS-STING pathway in pancreatic cancer

The DIAPH3/RPL6 axis regulates the cGAS-STING pathway in pancreatic cancer | 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 Research Article The DIAPH3/RPL6 axis regulates the cGAS-STING pathway in pancreatic cancer Haoyang Huang, Chao Lin, Cheng Zhou, Lingling Cui, Yefei Rong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7509327/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Nov, 2025 Read the published version in European Journal of Medical Research → Version 1 posted 10 You are reading this latest preprint version Abstract The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is essential for regulating immune responses in tumours. The molecular processes regulating its activation in pancreatic cancer are still not fully understood. We demonstrate that ribosomal protein L6 (RPL6) directly interacts with cGAS, hence initiating cGAS-STING signalling in malignant pancreatic cells. Data mining from open-access sources demonstrated a strong positive connection between RPL6 abundance and immune cell infiltration. The forced expression of RPL6 greatly boosted the production of interferon-β in these cells. Additionally, diaphanous-related formin 3 (DIAPH3) reduced RPL6 protein levels by disrupting its interaction with the deubiquitinase OTUD4. Our results demonstrate that RPL6-mediated stimulation of cGAS-STING signalling profoundly influences immune control in pancreatic cancer, highlighting this pathway as a potential therapeutic target. RPL6 DIAPH3 OTUD4 cGAS tumor immune microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Pancreatic cancer remains one of the most lethal malignancies globally, with five-year survival rates never above 10%[ 1 ]. Most patients are identified at later stages, making surgical excision an uncommon curative option[ 1 , 2 ]. Standard chemotherapy regimens provide only marginal enhancements in survival rates and are often linked to significant systemic toxicity[ 1 , 2 ]. Due to its very aggressive biology and ambiguous initial clinical presentations, over 80% of patients are identified with distant metastases, significantly limiting effective treatment measures[ 1 – 3 ]. Immune checkpoint inhibition has significantly improved treatment results in other malignancies, but in pancreatic carcinoma, its effectiveness is considerably diminished owing to the highly immunosuppressive tumour microenvironment[ 3 , 4 ]. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is very important for detecting DNA in the cytosol and starting immune responses against tumours[ 5 ]. The enzyme cGAS finds DNA pieces in the cytosol that come from genomic instability or damage caused by radiation or chemotherapy[ 5 , 6 ]. It speeds up the production of cyclic GMP-AMP (cGAMP), which binds to the STING adaptor protein on the endoplasmic reticulum[ 5 , 6 ]. When STING is activated, it starts the TBK1-IRF3 signalling cascade, which causes the production of type I interferons (IFN-α/β) and pro-inflammatory cytokines[ 6 ]. These mediators augment dendritic cell recruitment and the activation of cytotoxic CD8⁺ T cells, therefore fortifying antitumour immunity[ 7 ]. Still, pancreatic tumours are usually “cold” in terms of the immune system, and their milieu actively inhibits immune activation. This makes targeted manipulation of cGAS-STING signalling a promising but difficult way to treat them[ 8 ]. Diaphanous-related formin 3 (DIAPH3) is an important controller of how the cytoskeleton changes shape and how the mitochondria stay in balance[ 9 ]. This formin protein helps create actin filaments and keeps microtubules stable, which makes it easier for cells to move, change shape, and make pseudopodia[ 9 , 10 ]. In pancreatic cancer models, DIAPH3 overexpression enhances cell invasion and motility, whereas its depletion leads to mitochondrial fragmentation, functional decline, increased accumulation of reactive oxygen species (ROS), and structural instability[ 11 ]. Clinically, elevated DIAPH3 expression in pancreatic cancer tissues is associated with increased proliferation, anchorage-independent growth, and greater invasiveness[ 11 , 12 ]. Besides its role in the cytoskeleton, DIAPH3 has been linked to selenium incorporation by direct interaction with ribosomal protein L6 (RPL6), a crucial element in selenoamino acid metabolism[ 11 ]. The precise chemical mechanisms by which DIAPH3 affects RPL6 stability are insufficiently defined. Additionally, the function of RPL6 in cancer-associated signalling pathways, particularly the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) axis, is not well understood. Addressing these inadequacies is imperative, given the established association between cGAS-STING signalling and antitumor immune responses. Understanding DIAPH3’s function in RPL6 stability and RPL6’s participation in cGAS-STING-mediated pathways might lead to new methods to stop immune evasion in pancreatic cancer. This study aims to (i) explore the molecular mechanism by which DIAPH3 regulates RPL6 protein stability and (ii) clarify the role of RPL6 in modulating cGAS-STING signalling in pancreatic cancer cells. The findings from this study may inform the development of novel therapeutic strategies that enhance immune response and improve treatment efficacy for this very lethal malignancy. Materials and Methods Cell Culture The Cell Bank of the Chinese Academy of Sciences (Shanghai, China) provided human pancreatic cancer cell lines MiaPaCa-2 and Panc01, as well as human embryonic kidney 293T (HEK293T) cells. Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL) was used to grow these cells. The cultures were kept in a humidified incubator at 37°C with 5% CO₂. For transient transfection tests, plasmid constructs were delivered into cells using Lipofectamine 8000 reagent (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s protocol. Cell Transduction The pLKO.1 backbone carried short hairpin RNA (shRNA) sequences designed to specifically silence DIAPH3, OTUD4, and RPL6 genes, as detailed in Table S1. 293T cells were used to make lentiviral particles by co-transfecting shRNA plasmids with the helper vectors PxpAX and pMD2. G. The viral suspensions that were made were collected, condensed, and utilised to infect pancreatic cancer cell lines. Infected cultures underwent puromycin selection for about 14 days, after which the effectiveness of gene silencing was assessed using Western blot analysis. Co-immunoprecipitation (co-IP) Assay 48 hours after the transfection operations were finished, the cells were broken up in ice-cold immunoprecipitation (IP) buffer containing 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl. We cleared the lysates by spinning them at 12,000 × g for 10 minutes at 4°C and then collecting the supernatants. The whole protein was incubated overnight at 4°C with moderate rotation using either anti-Flag magnetic beads (Sigma, USA; Cat# A2220) or anti-HA magnetic beads (Thermon Fisher Scientific, USA; Cat# 88836). After that, the beads were washed three times using a solution with 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl in it. By boiling the beads in 1× SDS loading buffer at 95°C for 5 minutes, the bound proteins were released. The eluates were then collected and analysed by western blotting. GST Pull-down Assay The process utilised to make cell lysates was the same as the one used for the co-immunoprecipitation investigations. After centrifugation, each sample was split in half and treated overnight at 4°C with 5 µg of either GST alone or a GST-RPL6 fusion protein. The next day, 50 µL of glutathione-conjugated beads were added to catch the GST-fusion proteins. The reaction mixtures were left at 4°C overnight with moderate rotation. We used a centrifuge to get the beads back and then put them back in 50 µL of 1× SDS loading buffer. We then heated them at 100°C for 5 minutes. After a second centrifugation step, the supernatants were put through western blot examination. Western Blotting Cells underwent two successive washing with ice-cold phosphate-buffered saline (PBS) before being lysed in RIPA buffer supplemented with a combination of protease and phosphatase inhibitors. The lysates were cleared by centrifugation, and the BCA Protein Quantification Kit (Pierce) was used to measure the protein levels in the supernatants. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate equal amount of protein from each sample. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were exposed overnight at 4°C to the following primary antibodies: anti-RPL6 (Abcam, ab126100, dilution 1:1000), anti-tubulin (Santa Cruz Biotechnology, sc-5286, 1:4000), anti-Flag (Proteintech, 66008-4-Ig, 1:1000), anti-GAPDH (Proteintech, 60004-1-Ig, 1:1000), anti-HA (Proteintech, 81290-1-RR, 1:1000), anti-OTUD4 (Proteintech, 25070-1-AP, 1:100), anti-DIAPH3 (Proteintech, 14342-1-AP, 1:100), and anti-cGAS (Proteintech, 26416-1-AP, 1:100). On the same day, the membranes were then treated for 1 to 2 hours at room temperature with secondary antibodies that were linked to horseradish peroxidase. A chemiluminescent reagent (Millipore, WBKLS0050) was used to find immunoreactive protein bands, and the Image Lab software platform was used to evaluate them quantitatively. Quantitative PCR (qPCR) Using TRIzol reagent (Invitrogen) and following standard procedures, we got total RNA from cells that had been grown. The PrimeScript™ RT Kit (Takara) was employed in accordance with the protocol provided by the manufacturer. Each sample had 1 µg of RNA turned into complementary DNA (cDNA) using reverse transcription. We used the SYBR Green detection method on a CFX96 Real-Time PCR equipment (Bio-Rad, California, USA) to do quantitative real-time PCR. Using the 2^−ΔΔCt approach, we figured out how many the levels of transcripts were determined, and β-actin was used as the internal control. Statistical Analysis We used SPSS version 23.0 (IBM, USA) and GraphPad Prism version 8.0.2 (GraphPad Software, La Jolla, CA, USA) to perform all the statistical tests. The log-rank test was used to compare the survival curves. A two-sided chi-squared (χ²) test was used to look at relationships between categorical variables, and a two-sided Student’s t-test was used to look at differences in mean values across groups. A p-value of less than 0.05 was used to show statistical significance. Results DIAPH3 interacts with RPL6 Our previous mass spectrometric analyses have previously identified ribosomal protein L6 (RPL6) as a potential binding partner of DIAPH3[ 11 ]. To confirm this observation, the vectors coding Flag-DIAPH3 and HA-RPL6 were co-transfected into the MiaPaCa-2 and Panc01 cell lines. Immunoprecipitation of these epitope-tagged proteins showed that DIAPH3 and RPL6 form a stable molecular complex (Fig. 1 A). Using glutathione S-transferase (GST) pull-down experiments, we found that a GST-RPL6 fusion protein could effectively bind endogenous DIAPH3 (Fig. 1 B). Additionally, immunoprecipitation assays using untagged proteins confirmed that the endogenous DIAPH3 and RPL6 interact under the growth conditions (Fig. 1 C). Overexpression or knockdown of DIAPH3 changed the level of RPL6 protein: overexpression of DIAPH3 lowered the levels of RPL6 (Fig. 1 D), whereas RNA interference-induced DIAPH3 knockdown upregulated RPL6 (Fig. 1 E). In summary, our findings show that DIAPH3 interacts with RPL6 in a way that makes its protein less stable. RPL6 interacts with OTUD4. Subsequent protein interaction studies revealed OTUD4 as an additional potential partner of DIAPH3[ 11 ]. This finding prompted the concept that DIAPH3 may influence RPL6 levels by altering its interaction with OTUD4. Therefore, we first examined the interaction between RPL6 and OTUD4. Experiments with MiaPaCa-2 and Panc01 cells provided definitive proof of complex formation between Flag-OTUD4 and HA-RPL6 upon their simultaneous introduction (Fig. 2 A). In the GST-pull down assay, the interaction between GST-RPL6 and OTUD4 was observed (Fig. 2 B). In line with this, both endogenous RPL6 and a GST-RPL6 recombinant fusion effectively caught native OTUD4 (Fig. 2 C). Functional studies demonstrated that OTUD4 expression directly affected RPL6 protein stability: elevated OTUD4 expression enhanced RPL6 levels (Fig. 2 D), whereas RNAi-mediated OTUD4 knockdown reduced them (Fig. 2 E). These results show that OTUD4 keeps RPL6 stable by directly interacting with it. Interestingly, higher levels of DIAPH3 expression weakened the link between OTUD4 and RPL6 (Fig. 3 A), which made it harder for OTUD4 to remove ubiquitin from RPL6 and stabilize RPL6 (Fig. 3 B-C). RPL6 interacts with cGAS and promotes activation of the cGAS–STING pathway The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is very important for controlling how the immune system works in the tumour microenvironment. Experiments in MiaPaCa-2 and Panc01 cell lines that looked at the co-expression of Flag-RPL6 and HA-cGAS showed that these two molecules did indeed form a protein complex (Fig. 4 A), and the endogenously expressed RPL6 and cGAS interacted with each other (Fig. 4 B). Glutathione S-transferase pull-down tests further confirmed that RPL6 interacts with both recombinant GST-cGAS protein (Fig. 4 C). The examination of the Human Protein Atlas (HPA) clinical dataset indicated that reduced RPL6 expression correlates with worse overall survival in pancreatic cancer patients (Fig. 4 D). Functional experiments demonstrated that the knockdown of RPL6 in pancreatic cancer cells inhibited interferon-β (IFNβ) production, whereas ectopic RPL6 expression elevated IFNβ mRNA levels (Fig. 5 A-D). Likewise, an analysis of publicly accessible transcriptome and clinical information revealed a favourable correlation between RPL6 abundance and the infiltration of cytotoxic CD8⁺ T cells (Fig. 5 E). Additionally, DIAPH3 levels exhibited an inverse correlation with the infiltration of T follicular helper (Tfh) cells, T helper 17 (Th17) cells, and neutrophils (Fig. 5 F). All these data support the idea that RPL6 is a positive regulator of the cGAS–STING pathway. Discussion Prior studies have shown that DIAPH3 is significantly elevated in pancreatic cancer and facilitates increased selenium absorption, a process partially linked to its physical interaction with the selenoprotein RPL6[ 11 ]. Even with these discoveries, the exact mechanisms that explain this link and the effects of changing RPL6 levels are still not well understood. In this work, we demonstrate that DIAPH3 functions as an inhibitor of RPL6 protein stability by obstructing OTUD4-mediated ubiquitin chain removal. Moreover, our results demonstrate direct interaction between RPL6 and cGAS, thereby promoting the activation of the cGAS-STING signalling cascade and establishing RPL6 as an active contributor to tumour-associated immune modulation. RPL6 is a vital component of the 60S ribosomal subunit, crucial for precise ribosome assembly and the translation of mRNA into functional proteins[ 13 ]. RPL6 has a huge effect on protein synthesis across the world since it is so important for ribosomal function[ 13 ]. In addition to its structural roles, RPL6 helps maintain the redox balance in cells by taking part in metabolic pathways that rely on selenium[ 11 ]. The combination of structural and regulatory roles shows how important it is in cancer development and backs up its promise as a treatment target[ 14 , 15 ]. In this study, we demonstrate that RPL6 physically interacts with cGAS and that increased RPL6 expression enhances interferon-β transcription. Examination of publicly available transcriptome data reveals a significant positive association between RPL6 abundance and the infiltration of immune effector cells in pancreatic tumours, therefore substantiating its function as an immunomodulatory element within the tumour microenvironment. Ribosomopathy, or defective ribosomal activity, is known to cause strong activation of the innate immune system[ 16 ]. Disruptions in ribosome biogenesis, whether caused by conventional cytotoxic drugs or specific inhibitors like CX-5461, may liberate ribosomal proteins, including RPL11 and RPL5, from the nucleolus[ 17 , 18 ]. These released proteins bind to the oncogenic E3 ubiquitin ligase MDM2, which stabilises and activates the tumour suppressor p53[ 19 ]. Displaced ribosomal proteins may also serve as endogenous danger-associated molecular patterns (DAMPs), therefore activating immunological sensors, including cGAS–STING and TLR4–MyD88 signalling networks[ 20 , 21 ]. RPL3 and RPL7 directly interact with toll-like receptor 4 (TLR4), which activates NF-κB via MyD88 and makes cells release more pro-inflammatory cytokines, such as IL-6 and TNF-α[ 22 ]. This kind of immunological activation makes macrophages better at killing tumours[ 22 ]. As a result, drugs that interfere with ribosome function may change tumours from an immunologically “cold” state to a “hot” phenotype by causing immunogenic stress[ 23 ]. This would make the tumours more sensitive to immune checkpoint blocking, such as PD-1 inhibition[ 23 ]. Preclinical evidence indicates that the integration of ribosome-targeted therapies with immunotherapeutic strategies might significantly diminish metastatic spread in malignancies like melanoma and triple-negative breast carcinoma[ 24 ], offering a robust method to counteract tumour immune evasion. In summary, our study elucidates the molecular determinants influencing RPL6 stability and elucidates its immunological function in pancreatic cancer. These findings highlight critical molecular targets for this extremely aggressive cancer. Abbreviations cGAS GMP-AMP synthase STING stimulator of interferon genes RPL6 ribosomal protein L6 DIAPH3 diaphanous-related formin 3 Declarations Data availability statement Data will be provided upon request. Ethics approval and consent to participate The animal procedures were authorized by the ethics committee of Fudan University (B2025-272), and were compliance with all relevant ethical regulations. Author Contribution RYF and CLL designed this study and drafted the manuscript. HHY ,LC, and ZC helped collect the data and performed statistical analysis. HHY and LC contributed to the conception, design, data interpretation, and supervision of the study. All the authors read and approved the manuscript. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Natural Science Foundation of China (82173116). References Dreyer SB, Beer P, Hingorani SR, Biankin AV: Improving outcomes of patients with pancreatic cancer . Nat Rev Clin Oncol 2025, 22 (6):439-456. Kotsiliti E: Therapy affects tumour microenvironment in pancreatic cancer . Nat Rev Gastroenterol Hepatol 2024, 21 (11):746. Kotsiliti E: Metastatic pancreatic cancer and the liver . Nat Rev Gastroenterol Hepatol 2024, 21 (9):606. Laheru D, Jaffee EM: Immunotherapy for pancreatic cancer - science driving clinical progress . Nat Rev Cancer 2005, 5 (6):459-467. Hopfner KP, Hornung V: Molecular mechanisms and cellular functions of cGAS-STING signalling . Nat Rev Mol Cell Biol 2020, 21 (9):501-521. Strzyz P: YAP/TAZ get a STING in the tail . Nat Rev Mol Cell Biol 2022, 23 (9):581. Cao LL, Kagan JC: Targeting innate immune pathways for cancer immunotherapy . 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Elhamamsy AR, Metge BJ, Alsheikh HA, Shevde LA, Samant RS: Ribosome Biogenesis: A Central Player in Cancer Metastasis and Therapeutic Resistance . Cancer Res 2022, 82 (13):2344-2353. Additional Declarations No competing interests reported. Supplementary Files TableS1.docx Cite Share Download PDF Status: Published Journal Publication published 29 Nov, 2025 Read the published version in European Journal of Medical Research → Version 1 posted Editorial decision: Revision requested 24 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 20 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers invited by journal 10 Sep, 2025 Editor assigned by journal 09 Sep, 2025 Submission checks completed at journal 05 Sep, 2025 First submitted to journal 01 Sep, 2025 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. 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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-7509327","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515522914,"identity":"563818cb-5068-477e-a18b-8ba9ceb4d3a7","order_by":0,"name":"Haoyang Huang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Haoyang","middleName":"","lastName":"Huang","suffix":""},{"id":515522915,"identity":"a6fed54e-7474-4e5a-91a5-07bbcd3011a5","order_by":1,"name":"Chao Lin","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Lin","suffix":""},{"id":515522918,"identity":"74119716-db32-474b-80f9-1b658c48cc75","order_by":2,"name":"Cheng Zhou","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zhou","suffix":""},{"id":515522921,"identity":"30ce1860-c69d-464e-a1c2-adb7655ab3a3","order_by":3,"name":"Lingling Cui","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Lingling","middleName":"","lastName":"Cui","suffix":""},{"id":515522922,"identity":"21341334-eec8-4a6c-9834-ca6adf092d60","order_by":4,"name":"Yefei Rong","email":"data:image/png;base64,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","orcid":"","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Yefei","middleName":"","lastName":"Rong","suffix":""}],"badges":[],"createdAt":"2025-09-01 14:08:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7509327/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7509327/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40001-025-03525-z","type":"published","date":"2025-11-29T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91509921,"identity":"b8c58293-435b-49e6-9363-ef46f5ea7756","added_by":"auto","created_at":"2025-09-17 08:42:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":775672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction between DIAPH3 and RPL6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Co-immunoprecipitation (co-IP) demonstrating binding between overexpressed DIAPH3 and RPL6 proteins. (B) GST pull-down illustrating the association of purified GST–RPL6 fusion protein with endogenous DIAPH3. (C) Co-IP confirming interaction between endogenous DIAPH3 and RPL6 proteins. (D–E) Immunoblot analysis showing changes in RPL6 protein abundance following DIAPH3 overexpression (D) or silencing (E).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7509327/v1/b5a08a5e8ad8fb8907cfa3d2.jpg"},{"id":91511446,"identity":"90a59b1e-75e0-456c-a8c4-c77901dc1089","added_by":"auto","created_at":"2025-09-17 08:50:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":741449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOTUD4 and RPL6 form a complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Co-immunoprecipitation (co-IP) demonstrates interaction between overexpressed OTUD4 and RPL6 proteins. (B) GST pull-down confirming association of purified GST–RPL6 fusion protein with endogenous OTUD4. (C) Co-IP verifying binding between endogenous OTUD4 and RPL6 proteins. (D–E) Immunoblot analysis depicting alterations in RPL6 protein abundance following OTUD4 overexpression (D) or silencing (E).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7509327/v1/65fdb47d05331e0b1ec768b1.jpg"},{"id":91513721,"identity":"12a5fb48-d2ed-4136-b5d6-49d98baabf5b","added_by":"auto","created_at":"2025-09-17 09:06:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":542988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDIAPH3 inhibits the interaction between OTUD4 and RPL6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Co-immunoprecipitation (co-IP) illustrates how DIAPH3 expression influences the binding between OTUD4 and RPL6. (B) Immunoblot analysis showing the impact of DIAPH3 expression on OTUD4-dependent modulation of RPL6 protein abundance. (C) Ubiquitination assay was performed to examine the deubiquitination of RPL6 by OTUD4.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7509327/v1/89ad9ce6b89fb1122a752d34.jpg"},{"id":91509932,"identity":"82cda77f-5b11-47ae-934d-1872bd9304ea","added_by":"auto","created_at":"2025-09-17 08:42:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":498698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRPL6 interacts with cGAS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Co-immunoprecipitation (co-IP) demonstrates binding between overexpressed cGAS and RPL6 proteins. (B) Co-IP verifying interaction between endogenous cGAS and RPL6 proteins. \u0026nbsp;(C) GST pull-down confirming association of purified GST-cGAS fusion protein with endogenous RPL6. (D) Correlation analysis of RPL6 expression and patient survival in pancreatic cancer based on HPA dataset.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7509327/v1/4e98dcf35687a1d21b7aa57e.jpg"},{"id":91509925,"identity":"bd7750ba-9d9a-4934-8d87-0bda82337e76","added_by":"auto","created_at":"2025-09-17 08:42:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1353106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRPL6 activates the cGAS–STING signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Immunoblot analyses showing the overexpression and knockdown of RPL6 in MiaPaCa-2 and Panc01 cells. (C, D) Quantification of IFNβ mRNA in cells overexpressing or depleted of RPL6. (E) Correlation between RPL6 expression and activated CD8⁺ T cell abundance in pancreatic cancer datasets. (F) Correlation between DIAPH3 expression and the infiltration of activated Tfh cells, neutrophils, and Th17 cells in pancreatic cancer datasets. **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7509327/v1/9bd72a798a4fa82424c21f07.jpg"},{"id":97178737,"identity":"c3ffa83c-9629-480b-b2d3-e1c9fb045f4e","added_by":"auto","created_at":"2025-12-01 16:13:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5473962,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7509327/v1/4f1fbc9f-113b-44dc-8131-7e1c0db630a7.pdf"},{"id":91509923,"identity":"9516dd54-886a-4490-a531-a1003c56fb2e","added_by":"auto","created_at":"2025-09-17 08:42:16","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16295,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7509327/v1/c9b0a7a47b8f266dcaad68a2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The DIAPH3/RPL6 axis regulates the cGAS-STING pathway in pancreatic cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer remains one of the most lethal malignancies globally, with five-year survival rates never above 10%[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Most patients are identified at later stages, making surgical excision an uncommon curative option[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Standard chemotherapy regimens provide only marginal enhancements in survival rates and are often linked to significant systemic toxicity[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to its very aggressive biology and ambiguous initial clinical presentations, over 80% of patients are identified with distant metastases, significantly limiting effective treatment measures[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImmune checkpoint inhibition has significantly improved treatment results in other malignancies, but in pancreatic carcinoma, its effectiveness is considerably diminished owing to the highly immunosuppressive tumour microenvironment[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is very important for detecting DNA in the cytosol and starting immune responses against tumours[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The enzyme cGAS finds DNA pieces in the cytosol that come from genomic instability or damage caused by radiation or chemotherapy[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It speeds up the production of cyclic GMP-AMP (cGAMP), which binds to the STING adaptor protein on the endoplasmic reticulum[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. When STING is activated, it starts the TBK1-IRF3 signalling cascade, which causes the production of type I interferons (IFN-α/β) and pro-inflammatory cytokines[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These mediators augment dendritic cell recruitment and the activation of cytotoxic CD8⁺ T cells, therefore fortifying antitumour immunity[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Still, pancreatic tumours are usually \u0026ldquo;cold\u0026rdquo; in terms of the immune system, and their milieu actively inhibits immune activation. This makes targeted manipulation of cGAS-STING signalling a promising but difficult way to treat them[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDiaphanous-related formin 3 (DIAPH3) is an important controller of how the cytoskeleton changes shape and how the mitochondria stay in balance[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This formin protein helps create actin filaments and keeps microtubules stable, which makes it easier for cells to move, change shape, and make pseudopodia[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In pancreatic cancer models, DIAPH3 overexpression enhances cell invasion and motility, whereas its depletion leads to mitochondrial fragmentation, functional decline, increased accumulation of reactive oxygen species (ROS), and structural instability[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Clinically, elevated DIAPH3 expression in pancreatic cancer tissues is associated with increased proliferation, anchorage-independent growth, and greater invasiveness[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Besides its role in the cytoskeleton, DIAPH3 has been linked to selenium incorporation by direct interaction with ribosomal protein L6 (RPL6), a crucial element in selenoamino acid metabolism[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The precise chemical mechanisms by which DIAPH3 affects RPL6 stability are insufficiently defined. Additionally, the function of RPL6 in cancer-associated signalling pathways, particularly the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) axis, is not well understood. Addressing these inadequacies is imperative, given the established association between cGAS-STING signalling and antitumor immune responses. Understanding DIAPH3\u0026rsquo;s function in RPL6 stability and RPL6\u0026rsquo;s participation in cGAS-STING-mediated pathways might lead to new methods to stop immune evasion in pancreatic cancer. This study aims to (i) explore the molecular mechanism by which DIAPH3 regulates RPL6 protein stability and (ii) clarify the role of RPL6 in modulating cGAS-STING signalling in pancreatic cancer cells. The findings from this study may inform the development of novel therapeutic strategies that enhance immune response and improve treatment efficacy for this very lethal malignancy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eThe Cell Bank of the Chinese Academy of Sciences (Shanghai, China) provided human pancreatic cancer cell lines MiaPaCa-2 and Panc01, as well as human embryonic kidney 293T (HEK293T) cells. Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) with 10% foetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 \u0026micro;g/mL) was used to grow these cells. The cultures were kept in a humidified incubator at 37\u0026deg;C with 5% CO₂. For transient transfection tests, plasmid constructs were delivered into cells using Lipofectamine 8000 reagent (Thermo Fisher Scientific, USA) in accordance with the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell Transduction\u003c/h3\u003e\n\u003cp\u003eThe pLKO.1 backbone carried short hairpin RNA (shRNA) sequences designed to specifically silence DIAPH3, OTUD4, and RPL6 genes, as detailed in Table S1. 293T cells were used to make lentiviral particles by co-transfecting shRNA plasmids with the helper vectors PxpAX and pMD2. G. The viral suspensions that were made were collected, condensed, and utilised to infect pancreatic cancer cell lines. Infected cultures underwent puromycin selection for about 14 days, after which the effectiveness of gene silencing was assessed using Western blot analysis.\u003c/p\u003e\n\u003ch3\u003eCo-immunoprecipitation (co-IP) Assay\u003c/h3\u003e\n\u003cp\u003e48 hours after the transfection operations were finished, the cells were broken up in ice-cold immunoprecipitation (IP) buffer containing 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl. We cleared the lysates by spinning them at 12,000 \u0026times; g for 10 minutes at 4\u0026deg;C and then collecting the supernatants. The whole protein was incubated overnight at 4\u0026deg;C with moderate rotation using either anti-Flag magnetic beads (Sigma, USA; Cat# A2220) or anti-HA magnetic beads (Thermon Fisher Scientific, USA; Cat# 88836). After that, the beads were washed three times using a solution with 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl in it. By boiling the beads in 1\u0026times; SDS loading buffer at 95\u0026deg;C for 5 minutes, the bound proteins were released. The eluates were then collected and analysed by western blotting.\u003c/p\u003e\n\u003ch3\u003eGST Pull-down Assay\u003c/h3\u003e\n\u003cp\u003eThe process utilised to make cell lysates was the same as the one used for the co-immunoprecipitation investigations. After centrifugation, each sample was split in half and treated overnight at 4\u0026deg;C with 5 \u0026micro;g of either GST alone or a GST-RPL6 fusion protein. The next day, 50 \u0026micro;L of glutathione-conjugated beads were added to catch the GST-fusion proteins. The reaction mixtures were left at 4\u0026deg;C overnight with moderate rotation. We used a centrifuge to get the beads back and then put them back in 50 \u0026micro;L of 1\u0026times; SDS loading buffer. We then heated them at 100\u0026deg;C for 5 minutes. After a second centrifugation step, the supernatants were put through western blot examination.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eCells underwent two successive washing with ice-cold phosphate-buffered saline (PBS) before being lysed in RIPA buffer supplemented with a combination of protease and phosphatase inhibitors. The lysates were cleared by centrifugation, and the BCA Protein Quantification Kit (Pierce) was used to measure the protein levels in the supernatants. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate equal amount of protein from each sample. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were exposed overnight at 4\u0026deg;C to the following primary antibodies: anti-RPL6 (Abcam, ab126100, dilution 1:1000), anti-tubulin (Santa Cruz Biotechnology, sc-5286, 1:4000), anti-Flag (Proteintech, 66008-4-Ig, 1:1000), anti-GAPDH (Proteintech, 60004-1-Ig, 1:1000), anti-HA (Proteintech, 81290-1-RR, 1:1000), anti-OTUD4 (Proteintech, 25070-1-AP, 1:100), anti-DIAPH3 (Proteintech, 14342-1-AP, 1:100), and anti-cGAS (Proteintech, 26416-1-AP, 1:100). On the same day, the membranes were then treated for 1 to 2 hours at room temperature with secondary antibodies that were linked to horseradish peroxidase. A chemiluminescent reagent (Millipore, WBKLS0050) was used to find immunoreactive protein bands, and the Image Lab software platform was used to evaluate them quantitatively.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative PCR (qPCR)\u003c/h2\u003e\u003cp\u003eUsing TRIzol reagent (Invitrogen) and following standard procedures, we got total RNA from cells that had been grown. The PrimeScript\u0026trade; RT Kit (Takara) was employed in accordance with the protocol provided by the manufacturer. Each sample had 1 \u0026micro;g of RNA turned into complementary DNA (cDNA) using reverse transcription. We used the SYBR Green detection method on a CFX96 Real-Time PCR equipment (Bio-Rad, California, USA) to do quantitative real-time PCR. Using the 2^\u0026minus;ΔΔCt approach, we figured out how many the levels of transcripts were determined, and β-actin was used as the internal control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eWe used SPSS version 23.0 (IBM, USA) and GraphPad Prism version 8.0.2 (GraphPad Software, La Jolla, CA, USA) to perform all the statistical tests. The log-rank test was used to compare the survival curves. A two-sided chi-squared (χ\u0026sup2;) test was used to look at relationships between categorical variables, and a two-sided Student\u0026rsquo;s t-test was used to look at differences in mean values across groups. A p-value of less than 0.05 was used to show statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDIAPH3 interacts with RPL6\u003c/h2\u003e\u003cp\u003eOur previous mass spectrometric analyses have previously identified ribosomal protein L6 (RPL6) as a potential binding partner of DIAPH3[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To confirm this observation, the vectors coding Flag-DIAPH3 and HA-RPL6 were co-transfected into the MiaPaCa-2 and Panc01 cell lines. Immunoprecipitation of these epitope-tagged proteins showed that DIAPH3 and RPL6 form a stable molecular complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using glutathione S-transferase (GST) pull-down experiments, we found that a GST-RPL6 fusion protein could effectively bind endogenous DIAPH3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, immunoprecipitation assays using untagged proteins confirmed that the endogenous DIAPH3 and RPL6 interact under the growth conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Overexpression or knockdown of DIAPH3 changed the level of RPL6 protein: overexpression of DIAPH3 lowered the levels of RPL6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), whereas RNA interference-induced DIAPH3 knockdown upregulated RPL6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In summary, our findings show that DIAPH3 interacts with RPL6 in a way that makes its protein less stable.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRPL6 interacts with OTUD4.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSubsequent protein interaction studies revealed OTUD4 as an additional potential partner of DIAPH3[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This finding prompted the concept that DIAPH3 may influence RPL6 levels by altering its interaction with OTUD4. Therefore, we first examined the interaction between RPL6 and OTUD4. Experiments with MiaPaCa-2 and Panc01 cells provided definitive proof of complex formation between Flag-OTUD4 and HA-RPL6 upon their simultaneous introduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the GST-pull down assay, the interaction between GST-RPL6 and OTUD4 was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In line with this, both endogenous RPL6 and a GST-RPL6 recombinant fusion effectively caught native OTUD4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Functional studies demonstrated that OTUD4 expression directly affected RPL6 protein stability: elevated OTUD4 expression enhanced RPL6 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), whereas RNAi-mediated OTUD4 knockdown reduced them (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results show that OTUD4 keeps RPL6 stable by directly interacting with it. Interestingly, higher levels of DIAPH3 expression weakened the link between OTUD4 and RPL6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which made it harder for OTUD4 to remove ubiquitin from RPL6 and stabilize RPL6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRPL6 interacts with cGAS and promotes activation of the cGAS\u0026ndash;STING pathway\u003c/h2\u003e\u003cp\u003eThe cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is very important for controlling how the immune system works in the tumour microenvironment. Experiments in MiaPaCa-2 and Panc01 cell lines that looked at the co-expression of Flag-RPL6 and HA-cGAS showed that these two molecules did indeed form a protein complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and the endogenously expressed RPL6 and cGAS interacted with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Glutathione S-transferase pull-down tests further confirmed that RPL6 interacts with both recombinant GST-cGAS protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The examination of the Human Protein Atlas (HPA) clinical dataset indicated that reduced RPL6 expression correlates with worse overall survival in pancreatic cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFunctional experiments demonstrated that the knockdown of RPL6 in pancreatic cancer cells inhibited interferon-β (IFNβ) production, whereas ectopic RPL6 expression elevated IFNβ mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Likewise, an analysis of publicly accessible transcriptome and clinical information revealed a favourable correlation between RPL6 abundance and the infiltration of cytotoxic CD8⁺ T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Additionally, DIAPH3 levels exhibited an inverse correlation with the infiltration of T follicular helper (Tfh) cells, T helper 17 (Th17) cells, and neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). All these data support the idea that RPL6 is a positive regulator of the cGAS\u0026ndash;STING pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrior studies have shown that DIAPH3 is significantly elevated in pancreatic cancer and facilitates increased selenium absorption, a process partially linked to its physical interaction with the selenoprotein RPL6[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Even with these discoveries, the exact mechanisms that explain this link and the effects of changing RPL6 levels are still not well understood. In this work, we demonstrate that DIAPH3 functions as an inhibitor of RPL6 protein stability by obstructing OTUD4-mediated ubiquitin chain removal. Moreover, our results demonstrate direct interaction between RPL6 and cGAS, thereby promoting the activation of the cGAS-STING signalling cascade and establishing RPL6 as an active contributor to tumour-associated immune modulation.\u003c/p\u003e\u003cp\u003eRPL6 is a vital component of the 60S ribosomal subunit, crucial for precise ribosome assembly and the translation of mRNA into functional proteins[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. RPL6 has a huge effect on protein synthesis across the world since it is so important for ribosomal function[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition to its structural roles, RPL6 helps maintain the redox balance in cells by taking part in metabolic pathways that rely on selenium[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The combination of structural and regulatory roles shows how important it is in cancer development and backs up its promise as a treatment target[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this study, we demonstrate that RPL6 physically interacts with cGAS and that increased RPL6 expression enhances interferon-β transcription. Examination of publicly available transcriptome data reveals a significant positive association between RPL6 abundance and the infiltration of immune effector cells in pancreatic tumours, therefore substantiating its function as an immunomodulatory element within the tumour microenvironment.\u003c/p\u003e\u003cp\u003eRibosomopathy, or defective ribosomal activity, is known to cause strong activation of the innate immune system[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Disruptions in ribosome biogenesis, whether caused by conventional cytotoxic drugs or specific inhibitors like CX-5461, may liberate ribosomal proteins, including RPL11 and RPL5, from the nucleolus[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These released proteins bind to the oncogenic E3 ubiquitin ligase MDM2, which stabilises and activates the tumour suppressor p53[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Displaced ribosomal proteins may also serve as endogenous danger-associated molecular patterns (DAMPs), therefore activating immunological sensors, including cGAS\u0026ndash;STING and TLR4\u0026ndash;MyD88 signalling networks[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. RPL3 and RPL7 directly interact with toll-like receptor 4 (TLR4), which activates NF-κB via MyD88 and makes cells release more pro-inflammatory cytokines, such as IL-6 and TNF-α[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This kind of immunological activation makes macrophages better at killing tumours[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As a result, drugs that interfere with ribosome function may change tumours from an immunologically \u0026ldquo;cold\u0026rdquo; state to a \u0026ldquo;hot\u0026rdquo; phenotype by causing immunogenic stress[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This would make the tumours more sensitive to immune checkpoint blocking, such as PD-1 inhibition[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Preclinical evidence indicates that the integration of ribosome-targeted therapies with immunotherapeutic strategies might significantly diminish metastatic spread in malignancies like melanoma and triple-negative breast carcinoma[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], offering a robust method to counteract tumour immune evasion.\u003c/p\u003e\u003cp\u003eIn summary, our study elucidates the molecular determinants influencing RPL6 stability and elucidates its immunological function in pancreatic cancer. These findings highlight critical molecular targets for this extremely aggressive cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ecGAS \u0026nbsp; \u0026nbsp; \u0026nbsp; GMP-AMP synthase\u003c/p\u003e\n\u003cp\u003eSTING \u0026nbsp; \u0026nbsp; stimulator of interferon genes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRPL6 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;ribosomal protein L6\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDIAPH3 \u0026nbsp; diaphanous-related formin 3\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eavailability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be provided upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal procedures were authorized by the ethics committee of\u0026nbsp;Fudan University\u0026nbsp;(B2025-272), and were compliance with all relevant ethical regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRYF and CLL designed this study and drafted the manuscript. HHY ,LC, and ZC helped collect the data and performed statistical analysis. HHY and LC contributed to the conception, design, data interpretation, and supervision of the study. All the authors read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of China (82173116).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDreyer SB, Beer P, Hingorani SR, Biankin AV: \u003cstrong\u003eImproving outcomes of patients with pancreatic cancer\u003c/strong\u003e. \u003cem\u003eNat Rev Clin Oncol \u003c/em\u003e2025, \u003cstrong\u003e22\u003c/strong\u003e(6):439-456.\u003c/li\u003e\n\u003cli\u003eKotsiliti E: \u003cstrong\u003eTherapy affects tumour microenvironment in pancreatic cancer\u003c/strong\u003e. \u003cem\u003eNat Rev Gastroenterol Hepatol \u003c/em\u003e2024, 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\u003cstrong\u003e7\u003c/strong\u003e(1):60.\u003c/li\u003e\n\u003cli\u003eHu XN, Li CF, Huang SM, Nie CL, Pang R: \u003cstrong\u003ePescadillo ribosomal biogenesis factor 1 and programmed death-ligand 1 in gastric and head and neck squamous cell carcinoma\u003c/strong\u003e. \u003cem\u003eWorld J Gastroenterol \u003c/em\u003e2025, \u003cstrong\u003e31\u003c/strong\u003e(19):106644.\u003c/li\u003e\n\u003cli\u003eElhamamsy AR, Metge BJ, Alsheikh HA, Shevde LA, Samant RS: \u003cstrong\u003eRibosome Biogenesis: A Central Player in Cancer Metastasis and Therapeutic Resistance\u003c/strong\u003e. \u003cem\u003eCancer Res \u003c/em\u003e2022, \u003cstrong\u003e82\u003c/strong\u003e(13):2344-2353.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RPL6, DIAPH3, OTUD4, cGAS, tumor immune microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-7509327/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7509327/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is essential for regulating immune responses in tumours. The molecular processes regulating its activation in pancreatic cancer are still not fully understood. We demonstrate that ribosomal protein L6 (RPL6) directly interacts with cGAS, hence initiating cGAS-STING signalling in malignant pancreatic cells. Data mining from open-access sources demonstrated a strong positive connection between RPL6 abundance and immune cell infiltration. The forced expression of RPL6 greatly boosted the production of interferon-β in these cells. Additionally, diaphanous-related formin 3 (DIAPH3) reduced RPL6 protein levels by disrupting its interaction with the deubiquitinase OTUD4. Our results demonstrate that RPL6-mediated stimulation of cGAS-STING signalling profoundly influences immune control in pancreatic cancer, highlighting this pathway as a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"The DIAPH3/RPL6 axis regulates the cGAS-STING pathway in pancreatic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 08:42:12","doi":"10.21203/rs.3.rs-7509327/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-24T15:16:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T14:26:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-21T02:02:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251193799724755234420013553015439929607","date":"2025-09-15T19:01:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158952961871655745618285795993041288560","date":"2025-09-12T08:18:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299350145102894039215944229718462000576","date":"2025-09-12T05:04:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-10T10:47:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-09T22:00:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-05T08:04:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Medical Research","date":"2025-09-01T14:06:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5274e32-209f-442f-9da5-500bce080379","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:06:12+00:00","versionOfRecord":{"articleIdentity":"rs-7509327","link":"https://doi.org/10.1186/s40001-025-03525-z","journal":{"identity":"european-journal-of-medical-research","isVorOnly":false,"title":"European Journal of Medical Research"},"publishedOn":"2025-11-29 15:57:45","publishedOnDateReadable":"November 29th, 2025"},"versionCreatedAt":"2025-09-17 08:42:12","video":"","vorDoi":"10.1186/s40001-025-03525-z","vorDoiUrl":"https://doi.org/10.1186/s40001-025-03525-z","workflowStages":[]},"version":"v1","identity":"rs-7509327","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7509327","identity":"rs-7509327","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}