Molecular characterization and clinical relevance of RCAN1 in diffuse gliomas

Molecular characterization and clinical relevance of RCAN1 in diffuse gliomas | 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 Molecular characterization and clinical relevance of RCAN1 in diffuse gliomas Jianhua Han, Zheng Zhao, Shuai Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9444214/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background . RCAN1 encodes a calcineurin inhibitor conventionally viewed as a tumor suppressor, yet its RCAN1‑4 isoform is paradoxically enriched in mesenchymal glioblastoma. The precise contribution of RCAN1 to glioma biology across the full histologic and molecular spectrum, however, remains poorly defined. Methods . We performed a comprehensive pan‑glioma analysis integrating RNA‑sequencing data from two independent Chinese Glioma Genome Atlas (CGGA) cohorts (CGGA‑325, n = 325; CGGA‑693, n = 693) and a publicly available single‑cell RNA‑sequencing dataset of glioblastoma. Associations between RCAN1 expression and clinicopathological features, survival outcomes, transcriptional programs, and the immune microenvironment were systematically evaluated. Results . RCAN1 expression increased progressively with WHO grade and was selectively elevated in IDH-wildtype gliomas. Single‑cell transcriptomics revealed that RCAN1 expression was predominantly confined to mesenchymal‑like (MES‑like) and astrocytic‑like (AC‑like) malignant cell states. High RCAN1 expression independently predicted shorter overall survival in both CGGA cohorts. Gene set enrichment analysis demonstrated that RCAN1-high tumors exhibited marked enrichment of TNFα/NF‑κB signaling, hypoxia response, and interferon‑γ response pathways, and RCAN1 correlated positively with mesenchymal markers CD44 and SOD2. Immune deconvolution further linked elevated RCAN1 to an immunosuppressive microenvironment characterized by enrichment of M2 macrophages and depletion of naïve CD4⁺ T cells. Conclusions . RCAN1 upregulation in high‑grade, IDH‑wildtype gliomas reflects mesenchymal transition‑associated transcriptional reprogramming and contrasts with its canonical tumor‑suppressive role in other cancers. These findings nominate RCAN1 as a subtype‑specific prognostic biomarker and potential therapeutic target in aggressive gliomas. RCAN1 glioma mesenchymal subtype transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Diffuse gliomas are the most common primary malignant tumors of the adult central nervous system 1 . Molecular characterization has profoundly reshaped our understanding of glioma biology and supported the implementation of an integrated diagnostic classification, in which IDH mutation and 1p/19q codeletion serve as core diagnostic biomarkers 2 . These advances have improved prognostic stratification and facilitated the development of targeted therapeutic strategies. Notably, IDH-targeted therapies have recently demonstrated encouraging clinical efficacy in patients with glioma 3 . Despite substantial progress in molecular understanding, the clinical management of diffuse gliomas remains highly challenging. The standard-of-care for glioblastoma (GBM)—maximal safe surgical resection followed by concurrent radiotherapy and temozolomide—has provided only modest survival benefits over the past two decades, and therapeutic resistance ultimately develops in nearly all patients 4 . There remains an urgent need to identify novel actionable therapeutic targets and clarify the molecular mechanisms underlying glioma progression and treatment failure. Among the signaling pathways implicated in glioma pathobiology, the calcium-dependent calcineurin/NFAT axis has been linked to glioma cell proliferation, migration, apoptosis, and malignant phenotypes 5 , 6 . Regulator of calcineurin 1 (RCAN1) acts as an endogenous inhibitor of calcineurin phosphatase activity, thereby limiting NFAT dephosphorylation and nuclear translocation 7 , 8 . Consistent with its reported tumor-suppressive functions in several malignancies 9 – 11 , ectopic expression of RCAN1 has been demonstrated to markedly reduce glioma cell viability through induction of apoptosis, an effect mechanistically attributed to attenuation of NF-κB signaling 12 . Paradoxically, however, a recent study 13 showed that the RCAN1-4 isoform is highly enriched in the most aggressive mesenchymal glioblastoma and may represent an immunotherapeutic target. Despite these provocative observations, the precise contribution of RCAN1 to glioma biology across the full histologic and molecular spectrum remains poorly defined. To address this gap in knowledge, the present study undertakes a comprehensive pan‑glioma analysis to delineate the expression landscape of RCAN1, interrogate its prognostic significance across diffuse glioma subtypes, and characterize its biological behaviors. By integrating transcriptomic profiling with functional interrogation, we aim to clarify the dichotomous role of RCAN1 and assess its potential utility as both a prognostic biomarker and a subtype‑specific therapeutic vulnerability in neuro‑oncology. Materials and Methods Data Acquisition and Processing RNA‑sequencing (RNA‑seq) transcriptome profiles and corresponding clinical annotations for diffuse gliomas were obtained from the Chinese Glioma Genome Atlas (CGGA; http://www.cgga.org.cn/ ), comprising two independent cohorts: CGGA-325 (n = 325 cases) and CGGA-693 (n = 693 cases) 14 . For visualization of gene expression distributions, RCAN1 expression values were presented as log2(FPKM + 1). Public single-cell RNA-seq data from human glioblastoma were obtained from a prior publication 15 , accessible at https://singlecell.broadinstitute.org/single_cell/study/SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma . RCAN1 Expression Across Clinicopathological Subgroups To characterize the expression landscape of RCAN1, clinicopathological comparisons were performed separately in the CGGA-325 and CGGA-693 cohorts. RCAN1 expression was compared across WHO grade, IDH mutation status, MGMT promoter methylation status, integrated IDH/1p19q status in lower-grade gliomas, and primary versus recurrent disease status. Single‑Cell RNA‑Sequencing Analysis To resolve RCAN1 expression at single-cell resolution, we analyzed a public human glioblastoma scRNA-seq dataset. Original malignant-cell state annotations were used, including mesenchymal-like (MES-like), astrocytic-like (AC-like), neural progenitor-like (NPC-like), and oligodendrocyte progenitor-like (OPC-like) states. RCAN1 expression was visualized on the malignant-cell state map based on relative meta-module scores and on tSNE embeddings of all cells, and compared across these annotated states. Survival Analysis To evaluate the prognostic significance of RCAN1 in diffuse gliomas, both univariate and multivariate survival analyses were performed. Patients in each cohort were dichotomized into RCAN1-high and RCAN1-low groups according to the cohort-specific median RCAN1 expression value. Kaplan–Meier survival curves were generated for the two groups and compared using the log-rank test. Survival analysis was conducted in the entire glioma cohort as well as in subgroups stratified by IDH mutational status. To determine whether RCAN1 expression represents an independent prognostic factor, multivariable Cox proportional hazards regression models were fitted separately in the CGGA-325 and CGGA-693 cohorts, including age at diagnosis, WHO grade, IDH mutation status, 1p/19q codeletion status, MGMT promoter methylation status and RCAN1 expression group. Gene Set Enrichment Analysis To elucidate the biological pathways and processes associated with RCAN1 expression, Pearson correlation analysis was first performed between RCAN1 and all other genes in the analyzed RNA-seq cohort. Genes meeting predefined thresholds of absolute correlation coefficient and adjusted P value (|r| > 0.5 and adjusted P < 0.05) were considered significantly correlated with RCAN1 and were used for downstream heatmap visualization and over-representation analyses, with positively and negatively correlated genes examined separately where appropriate. Genes were also ranked according to their correlation coefficients with RCAN1 for Gene Set Enrichment Analysis (GSEA) using the clusterProfiler R package against the Molecular Signatures Database (MSigDB) Hallmark gene set collection (H) and the Gene Ontology Biological Process (GO:BP) collection, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway collection. A gene set was considered significantly enriched at a false discovery rate (FDR) q < 0.05. Normalized enrichment scores were calculated for each significantly enriched pathway. Pathway-Focused Co-expression Analysis Within selected representative enriched pathways identified in the CGGA-693 cohort, Pearson correlation analysis was further performed between RCAN1 and constituent genes. Genes within the selected pathway were ranked according to their correlation coefficients with RCAN1, and the direction and statistical significance of these associations were annotated for figure-based visualization. Representative pathway genes were further compared between the RCAN1-high and RCAN1-low groups defined by the cohort-specific median RCAN1 expression value. Immune Infiltration Analysis To evaluate the relationship between RCAN1 expression and the glioma immune microenvironment, CIBERSORT-derived estimates of 22 tumor-infiltrating immune cell subsets in the CGGA-693 cohort were used for downstream analysis. Pearson correlation analysis was used to assess associations between RCAN1 expression and the estimated fraction of each immune subset. Statistical Analysis All statistical analyses were performed in the R computational environment (version 4.5.3) using publicly available packages. Comparisons of continuous variables between two groups were performed using the Wilcoxon rank-sum test; for three or more groups, the Kruskal-Wallis test was applied. Pearson correlation analysis was used for gene co-expression analyses, pathway-focused correlation analyses, and associations between RCAN1 expression and immune-cell fractions. Adjusted P values were calculated using the Benjamini–Hochberg method where applicable. All reported P values are two-sided, and P < 0.05 was considered statistically significant. Results RCAN1 Expression Is Associated with Glioma Grade, Molecular Features, and Recurrence Status We first examined RCAN1 mRNA expression across clinicopathological and molecular subgroups in the two independent CGGA RNA-seq cohorts. In both CGGA-325 and CGGA-693, RCAN1 expression increased with WHO grade and was highest in WHO IV tumors (Fig. 1 A, F). Within LGGs, IDH-wildtype tumors showed the highest RCAN1 expression, whereas no significant difference was observed between IDH-mutant/1p19q-codeleted and IDH-mutant/1p19q-non-codeleted tumors (Fig. 1 B, G). Consistently, RCAN1 expression was significantly higher in IDH-wildtype than in IDH-mutant tumors in both LGG and GBM in the two cohorts (Fig. 1 C, H). MGMT promoter methylation was not associated with RCAN1 expression in LGG in either cohort; in GBM, higher RCAN1 expression was observed in the unmethylated subgroup only in CGGA-325, whereas no significant difference was detected in CGGA-693 (Fig. 1 D, I). Regarding progression status, recurrent LGG showed higher RCAN1 expression than primary LGG in both cohorts, whereas GBM showed lower recurrent expression in CGGA-325 and no significant difference in CGGA-693 (Fig. 1 E, J). RCAN1 Is Enriched in MES-like and AC-like Malignant Cell States at Single-Cell Resolution To resolve RCAN1 expression at single-cell resolution, we analyzed a public glioblastoma scRNA-seq dataset. On the relative meta-module score map, RCAN1 signal was enriched primarily in MES-like and AC-like malignant states and was weaker in NPC-like and OPC-like states (Fig. 2 A, B). Complementary t-SNE visualization further showed that RCAN1 expression was concentrated mainly in malignant cells rather than in non-malignant cell populations (Fig. 2 C, D). These findings provide single-cell support for the association between RCAN1 and the mesenchymal-like glioblastoma phenotype. High RCAN1 Expression Independently Predicts Poor Survival in Diffuse Gliomas To evaluate the prognostic relevance of RCAN1, Kaplan–Meier analyses were performed in both CGGA cohorts. In CGGA-325, high RCAN1 expression was associated with shorter overall survival in the all-grade, IDH-mutant, and IDH-wildtype groups (all P < 0.01; Fig. 3 A-C). The same pattern was confirmed in CGGA-693 (all P < 0.01; Fig. 3 D-F). Multivariable Cox regression further showed that RCAN1-high status remained independently associated with worse survival (CGGA-325: HR 2.05, P < 0.01; CGGA-693: HR 1.36, P = 0.03; Fig. 3 G, H). GSEA Implicates RCAN1 in Inflammatory and Hypoxia-Associated Pathways In the CGGA-693 cohort, GSEA revealed that higher RCAN1 expression was significantly associated with multiple biological pathways relevant to glioma pathobiology (Fig. 4 A, B). Specifically, higher RCAN1 expression was associated with enrichment of gene sets related to inflammatory and immune signaling, including those governing tumor necrosis factor alpha signaling via NF‑κB and interferon‑gamma response. In addition, significant enrichment was observed for pathways involved in apoptosis, hypoxia response, plasma membrane organization, and epithelial cell development. Within the HALLMARK_TNFA_SIGNALING_VIA_NFKB gene set, RCAN1 showed positive associations with representative genes such as CD44, PLAU, IFNGR2, GADD45A, and SOD2, whereas RCAN1 expression exhibited weak inverse correlations with BMP2 and SNN (Fig. 4 C). RCAN1 Expression Is Associated with an Immunosuppressive Tumor Microenvironment In the CGGA-693 cohort, correlation analysis of CIBERSORT-derived immune fractions indicated that RCAN1 clustered positively with gamma‑delta T cells, M0 and M2 macrophages, and activated dendritic cells. Inverse correlations were most evident for naïve CD4⁺ T cells, whereas the association with memory B cells appeared less prominent (Fig. 5 ). Discussion The present study addresses a persistent conundrum in neuro‑oncology regarding the observation that RCAN1, an established endogenous inhibitor of the pro‑tumorigenic calcineurin/NFAT and NF‑κB signaling pathways, is paradoxically upregulated in the most aggressive mesenchymal subtype of glioblastoma. By integrating pan‑glioma transcriptomic profiling, single‑cell resolution analysis, and functional pathway interrogation, our findings provide transcriptomic evidence supporting a context‑dependent association of RCAN1 with aggressive glioma phenotypes. Our clinicopathological analysis demonstrates that RCAN1 expression increases progressively with WHO grade and is selectively enriched in IDH-wildtype gliomas, with further elevation observed in recurrent LGG tumors. Single-cell transcriptomic analysis further refined these findings, revealing that RCAN1 expression is predominantly enriched in AC-like and MES-like malignant cell states, with particularly prominent signal in the MES-like compartment. This cell‑state‑specific enrichment aligns with prior studies 13 characterizing RCAN1—specifically its RCAN1‑4 isoform— as a hallmark of MES‑like GBM cells and reinforces its tight association with mesenchymal transition. This stepwise upregulation, together with its independent prediction of shortened overall survival in multivariate models, establishes RCAN1 as a robust biomarker of aggressive disease and poor clinical outcome. Notably, this expression pattern diverges markedly from that reported in the majority of other solid malignancies, where RCAN1 is frequently downregulated relative to normal tissues and reduced expression portends advanced disease and inferior survival—consistent with its function as a calcineurin-dependent tumor suppressor. In stark contrast, our findings indicate that RCAN1 is not diminished but rather progressively elevated in high-grade gliomas, reaching maximal levels in IDH-wildtype GBM, with additional elevation observed in recurrent LGG, whereas recurrent GBM showed a cohort-dependent pattern. This discrepancy suggests that the regulatory logic governing RCAN1 expression may be rewired in the glial lineage, likely during mesenchymal transition 16 . Rather than fulfilling a tumor‑suppressive role, RCAN1 in glioma may be transcriptionally co‑opted—potentially via isoform switching to the mesenchymal‑specific RCAN1‑4 variant 13 —to support a pro‑invasive, therapy‑resistant, and immunosuppressive phenotype. Thus, the paradoxical upregulation of RCAN1 in gliomas not only distinguishes it from the prevailing expression paradigm in extracranial malignancies but also warrants further functional and isoform-specific validation. Functional genomic analyses further indicate that elevated RCAN1 expression was associated with transcriptomic programs linked to malignant behavior in gliomas. Enrichment of TNFα/NF-κB signaling and hypoxia response gene sets in RCAN1-high tumors aligns with transcriptional programs driving proliferation, invasion, stem-like properties, and mesenchymal transition 17 – 19 . Positive correlations between RCAN1 and mesenchymal markers CD44 and SOD2 further support its association with the mesenchymal subtype 20 . Notably, CD44 has been implicated in glioma stemness, invasion, and immunosuppressive microenvironmental features 21 , 22 , whereas SOD2 enhances survival under oxidative and hypoxic stress 20 . Activation of complement and interferon-γ response pathways additionally implicates RCAN1 in shaping a pro-inflammatory, immunosuppressive microenvironment 23 . Collectively, these findings suggest that RCAN1 operates within a pro-oncogenic network that potentiates invasion, stemness, and adaptive resistance. These co-expression patterns argue against a tumor-suppressive role and instead support transcriptional co-option of the RCAN1 locus during mesenchymal transition to reinforce the aggressive biology of high-grade gliomas. Immune deconvolution analysis revealed that RCAN1 expression correlates positively with gamma‑delta T cells, M0 and M2 macrophages, and activated dendritic cells, and negatively with naïve CD4⁺ T cells. This profile—enrichment of immunosuppressive myeloid subsets coupled with depletion of naïve T cells—mirrors the immunologically “cold” microenvironment characteristic of mesenchymal glioblastoma 24 , 25 . The association with M2 macrophages, in particular, aligns with their established roles in promoting immune tolerance and therapeutic resistance 26 . These findings reinforce the notion that elevated RCAN1 expression demarcates a glioma subset characterized by profound immunosuppression, thereby highlighting the therapeutic imperative of RCAN1‑targeted strategies capable of circumventing local immune dysfunction. In summary, our study reframes the role of RCAN1 in glioma biology. Rather than functioning as a conventional tumor suppressor, its elevated expression in high-grade, IDH-wildtype gliomas reflects the transcriptional reprogramming and isoform diversion that accompany mesenchymal transition. The association of RCAN1 with a pro-invasive network, therapy-resistant pathways, and an immunosuppressive microenvironment underscores its centrality to the aggressive biology of glioblastoma. Importantly, these findings nominate RCAN1 as candidate biomarkers and potential subtype-selective therapeutic targets. Declarations Author Contribution S. L., Z. Z. conceptualized the project. Z. Z., J. H. and S. L. analyzed and interpreted the sequencing data; Z. Z., J. H. and S. L. wrote the manuscript and prepared all the figures. All authors reviewed the manuscript. Acknowledgments This study was supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (2025ZD0552017), the National Natural Science Foundation of China (82472841), the Beijing Municipal Health Commission Fund (11000023T000002044300-5). Data Availability The data used in this study were obtained from the publicly accessible Chinese Glioma Genome Atlas (CGGA) database. 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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-9444214","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633537469,"identity":"78a4f3cf-40a3-4867-9d9a-d4fb519ff3d2","order_by":0,"name":"Jianhua Han","email":"","orcid":"","institution":"Beijing Tian Tan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jianhua","middleName":"","lastName":"Han","suffix":""},{"id":633537470,"identity":"1864e9a2-8d08-4940-b172-37ed4611f073","order_by":1,"name":"Zheng Zhao","email":"","orcid":"","institution":"Beijing Neurosurgical Institute","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Zhao","suffix":""},{"id":633537471,"identity":"aa6bb1de-9dfe-48a7-b1ce-23a26d8a8f95","order_by":2,"name":"Shuai Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYFAC5oYDDBUHmBkYeIjWwgjUcuYAMw8bKVoYGNsOMBCvRb6BsfEw77w77PbyvQcYPu6pJazF4ABjw2Hebc+ADuNLYJzx7DgRWhjAWg6D/GLAzHPgGFEOA2qZQ4oWBrDDGuBaaohw2GHGhoNzjgH9cizH4OCMAweIcFh78+EPb2ruJLM3nzF88OFAHREOY4ZQyWBHMjAcJkILFNhBaWJsGQWjYBSMgpEGAJnUOdXLL/3rAAAAAElFTkSuQmCC","orcid":"","institution":"Beijing Tian Tan Hospital","correspondingAuthor":true,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-04-17 05:09:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9444214/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9444214/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108429349,"identity":"f0e298bb-1e5e-4a51-8a86-2ff4d62180ef","added_by":"auto","created_at":"2026-05-04 14:22:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4245101,"visible":true,"origin":"","legend":"\u003cp\u003eRCAN1 expression across clinicopathological and molecular subgroups in two CGGA RNA-seq cohorts. (A–E) CGGA-325 and (F–J) CGGA-693. RCAN1 expression was compared according to WHO grade, integrated IDH/1p19q status in lower-grade glioma (LGG), IDH status, MGMT promoter methylation status, and primary/recurrent status. GBM, glioblastoma; Mut-Co, IDH-mutant with 1p/19q codeletion; Mut-nCo, IDH-mutant without 1p/19q codeletion; WT, IDH wild type; Mut, IDH mutation, M, methylated; uM, unmethylated; Pri, primary; Rec, recurrent. P values are shown in the panels.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9444214/v1/f3af17393d37b785383da499.png"},{"id":108493487,"identity":"f609ddc2-f6a8-4da0-b425-e0dcf509ffe5","added_by":"auto","created_at":"2026-05-05 10:00:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1009719,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-cell distribution of RCAN1 expression in glioblastoma. (A) Relative meta-module score map showing OPC-like, NPC-like, AC-like, and MES-like malignant states. (B) RCAN1 expression projected onto the malignant-cell state map. (C) t-SNE plot of cell assignments. (D) RCAN1 expression projected onto the t-SNE map. RCAN1 expression is enriched mainly in MES-like/AC-like malignant cells.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9444214/v1/4c277a3a25f5b4b79c16ccb5.png"},{"id":108429350,"identity":"b7228113-f5d9-4b82-b881-8c3d13c87c14","added_by":"auto","created_at":"2026-05-04 14:22:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3370812,"visible":true,"origin":"","legend":"\u003cp\u003ePrognostic significance of RCAN1 in diffuse gliomas. Kaplan-Meier curves for all-grade, IDH-mutant, and IDH-wildtype gliomas in CGGA-325 (A–C) and CGGA-693 (D–F), stratified by median RCAN1 expression. (G, H) Multivariable Cox models for the two cohorts.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9444214/v1/a6d802809540fd6f112c4270.png"},{"id":108493395,"identity":"f3805240-3ea6-4256-b1b2-48bb06699798","added_by":"auto","created_at":"2026-05-05 10:00:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3233744,"visible":true,"origin":"","legend":"\u003cp\u003eBiological programs associated with RCAN1 expression in the CGGA-693 cohort. (A) Heatmap of RCAN1-correlated genes and top enriched GO biological process and Hallmark terms. (B) GSEA plots for selected positively enriched pathways. (C) Correlation ranking and representative genes within HALLMARK_TNFA_SIGNALING_VIA_NFKB. Positive and negative associations are shown at opposite ends of the ranked list.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9444214/v1/5da9416311e5d38e21436dfa.png"},{"id":108429353,"identity":"46605f90-e8fe-4e6c-bf00-93cabacc882b","added_by":"auto","created_at":"2026-05-04 14:22:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4321088,"visible":true,"origin":"","legend":"\u003cp\u003eImmune-cell correlations associated with RCAN1 expression in the CGGA-693 cohort. Correlation matrix showing the relationship between RCAN1 and CIBERSORT-estimated immune-cell fractions. Warm colors indicate positive correlations and cool colors indicate negative correlations.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9444214/v1/005040e7288bf1cecafd3762.png"},{"id":108803665,"identity":"7be2121e-d631-4c0e-8399-41d85a7c335c","added_by":"auto","created_at":"2026-05-08 15:02:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16019473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9444214/v1/a6f983df-b0f9-422c-82ba-fea52d64a588.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular characterization and clinical relevance of RCAN1 in diffuse gliomas","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiffuse gliomas are the most common primary malignant tumors of the adult central nervous system\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Molecular characterization has profoundly reshaped our understanding of glioma biology and supported the implementation of an integrated diagnostic classification, in which IDH mutation and 1p/19q codeletion serve as core diagnostic biomarkers\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These advances have improved prognostic stratification and facilitated the development of targeted therapeutic strategies. Notably, IDH-targeted therapies have recently demonstrated encouraging clinical efficacy in patients with glioma\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite substantial progress in molecular understanding, the clinical management of diffuse gliomas remains highly challenging. The standard-of-care for glioblastoma (GBM)\u0026mdash;maximal safe surgical resection followed by concurrent radiotherapy and temozolomide\u0026mdash;has provided only modest survival benefits over the past two decades, and therapeutic resistance ultimately develops in nearly all patients\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. There remains an urgent need to identify novel actionable therapeutic targets and clarify the molecular mechanisms underlying glioma progression and treatment failure.\u003c/p\u003e \u003cp\u003eAmong the signaling pathways implicated in glioma pathobiology, the calcium-dependent calcineurin/NFAT axis has been linked to glioma cell proliferation, migration, apoptosis, and malignant phenotypes\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Regulator of calcineurin 1 (RCAN1) acts as an endogenous inhibitor of calcineurin phosphatase activity, thereby limiting NFAT dephosphorylation and nuclear translocation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Consistent with its reported tumor-suppressive functions in several malignancies\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, ectopic expression of RCAN1 has been demonstrated to markedly reduce glioma cell viability through induction of apoptosis, an effect mechanistically attributed to attenuation of NF-κB signaling\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Paradoxically, however, a recent study\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e showed that the RCAN1-4 isoform is highly enriched in the most aggressive mesenchymal glioblastoma and may represent an immunotherapeutic target.\u003c/p\u003e \u003cp\u003eDespite these provocative observations, the precise contribution of RCAN1 to glioma biology across the full histologic and molecular spectrum remains poorly defined. To address this gap in knowledge, the present study undertakes a comprehensive pan‑glioma analysis to delineate the expression landscape of RCAN1, interrogate its prognostic significance across diffuse glioma subtypes, and characterize its biological behaviors. By integrating transcriptomic profiling with functional interrogation, we aim to clarify the dichotomous role of RCAN1 and assess its potential utility as both a prognostic biomarker and a subtype‑specific therapeutic vulnerability in neuro‑oncology.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData Acquisition and Processing\u003c/h2\u003e \u003cp\u003eRNA‑sequencing (RNA‑seq) transcriptome profiles and corresponding clinical annotations for diffuse gliomas were obtained from the Chinese Glioma Genome Atlas (CGGA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cgga.org.cn/\u003c/span\u003e\u003cspan address=\"http://www.cgga.org.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), comprising two independent cohorts: CGGA-325 (n\u0026thinsp;=\u0026thinsp;325 cases) and CGGA-693 (n\u0026thinsp;=\u0026thinsp;693 cases)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. For visualization of gene expression distributions, RCAN1 expression values were presented as log2(FPKM\u0026thinsp;+\u0026thinsp;1). Public single-cell RNA-seq data from human glioblastoma were obtained from a prior publication\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://singlecell.broadinstitute.org/single_cell/study/SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma\u003c/span\u003e\u003cspan address=\"https://singlecell.broadinstitute.org/single_cell/study/SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRCAN1 Expression Across Clinicopathological Subgroups\u003c/h3\u003e\n\u003cp\u003eTo characterize the expression landscape of RCAN1, clinicopathological comparisons were performed separately in the CGGA-325 and CGGA-693 cohorts. RCAN1 expression was compared across WHO grade, IDH mutation status, MGMT promoter methylation status, integrated IDH/1p19q status in lower-grade gliomas, and primary versus recurrent disease status.\u003c/p\u003e\n\u003ch3\u003eSingle‑Cell RNA‑Sequencing Analysis\u003c/h3\u003e\n\u003cp\u003eTo resolve RCAN1 expression at single-cell resolution, we analyzed a public human glioblastoma scRNA-seq dataset. Original malignant-cell state annotations were used, including mesenchymal-like (MES-like), astrocytic-like (AC-like), neural progenitor-like (NPC-like), and oligodendrocyte progenitor-like (OPC-like) states. RCAN1 expression was visualized on the malignant-cell state map based on relative meta-module scores and on tSNE embeddings of all cells, and compared across these annotated states.\u003c/p\u003e\n\u003ch3\u003eSurvival Analysis\u003c/h3\u003e\n\u003cp\u003eTo evaluate the prognostic significance of RCAN1 in diffuse gliomas, both univariate and multivariate survival analyses were performed. Patients in each cohort were dichotomized into RCAN1-high and RCAN1-low groups according to the cohort-specific median RCAN1 expression value. Kaplan\u0026ndash;Meier survival curves were generated for the two groups and compared using the log-rank test. Survival analysis was conducted in the entire glioma cohort as well as in subgroups stratified by IDH mutational status. To determine whether RCAN1 expression represents an independent prognostic factor, multivariable Cox proportional hazards regression models were fitted separately in the CGGA-325 and CGGA-693 cohorts, including age at diagnosis, WHO grade, IDH mutation status, 1p/19q codeletion status, MGMT promoter methylation status and RCAN1 expression group.\u003c/p\u003e\n\u003ch3\u003eGene Set Enrichment Analysis\u003c/h3\u003e\n\u003cp\u003eTo elucidate the biological pathways and processes associated with RCAN1 expression, Pearson correlation analysis was first performed between RCAN1 and all other genes in the analyzed RNA-seq cohort. Genes meeting predefined thresholds of absolute correlation coefficient and adjusted P value (|r| \u0026gt; 0.5 and adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considered significantly correlated with RCAN1 and were used for downstream heatmap visualization and over-representation analyses, with positively and negatively correlated genes examined separately where appropriate. Genes were also ranked according to their correlation coefficients with RCAN1 for Gene Set Enrichment Analysis (GSEA) using the clusterProfiler R package against the Molecular Signatures Database (MSigDB) Hallmark gene set collection (H) and the Gene Ontology Biological Process (GO:BP) collection, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway collection. A gene set was considered significantly enriched at a false discovery rate (FDR) q\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Normalized enrichment scores were calculated for each significantly enriched pathway.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePathway-Focused Co-expression Analysis\u003c/h2\u003e \u003cp\u003eWithin selected representative enriched pathways identified in the CGGA-693 cohort, Pearson correlation analysis was further performed between RCAN1 and constituent genes. Genes within the selected pathway were ranked according to their correlation coefficients with RCAN1, and the direction and statistical significance of these associations were annotated for figure-based visualization. Representative pathway genes were further compared between the RCAN1-high and RCAN1-low groups defined by the cohort-specific median RCAN1 expression value.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmune Infiltration Analysis\u003c/h3\u003e\n\u003cp\u003eTo evaluate the relationship between RCAN1 expression and the glioma immune microenvironment, CIBERSORT-derived estimates of 22 tumor-infiltrating immune cell subsets in the CGGA-693 cohort were used for downstream analysis. Pearson correlation analysis was used to assess associations between RCAN1 expression and the estimated fraction of each immune subset.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed in the R computational environment (version 4.5.3) using publicly available packages. Comparisons of continuous variables between two groups were performed using the Wilcoxon rank-sum test; for three or more groups, the Kruskal-Wallis test was applied. Pearson correlation analysis was used for gene co-expression analyses, pathway-focused correlation analyses, and associations between RCAN1 expression and immune-cell fractions. Adjusted P values were calculated using the Benjamini\u0026ndash;Hochberg method where applicable. All reported P values are two-sided, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRCAN1 Expression Is Associated with Glioma Grade, Molecular Features, and Recurrence Status\u003c/h2\u003e \u003cp\u003eWe first examined RCAN1 mRNA expression across clinicopathological and molecular subgroups in the two independent CGGA RNA-seq cohorts. In both CGGA-325 and CGGA-693, RCAN1 expression increased with WHO grade and was highest in WHO IV tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, F). Within LGGs, IDH-wildtype tumors showed the highest RCAN1 expression, whereas no significant difference was observed between IDH-mutant/1p19q-codeleted and IDH-mutant/1p19q-non-codeleted tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, G). Consistently, RCAN1 expression was significantly higher in IDH-wildtype than in IDH-mutant tumors in both LGG and GBM in the two cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, H). MGMT promoter methylation was not associated with RCAN1 expression in LGG in either cohort; in GBM, higher RCAN1 expression was observed in the unmethylated subgroup only in CGGA-325, whereas no significant difference was detected in CGGA-693 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, I). Regarding progression status, recurrent LGG showed higher RCAN1 expression than primary LGG in both cohorts, whereas GBM showed lower recurrent expression in CGGA-325 and no significant difference in CGGA-693 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRCAN1 Is Enriched in MES-like and AC-like Malignant Cell States at Single-Cell Resolution\u003c/h2\u003e \u003cp\u003eTo resolve RCAN1 expression at single-cell resolution, we analyzed a public glioblastoma scRNA-seq dataset. On the relative meta-module score map, RCAN1 signal was enriched primarily in MES-like and AC-like malignant states and was weaker in NPC-like and OPC-like states (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Complementary t-SNE visualization further showed that RCAN1 expression was concentrated mainly in malignant cells rather than in non-malignant cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). These findings provide single-cell support for the association between RCAN1 and the mesenchymal-like glioblastoma phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHigh RCAN1 Expression Independently Predicts Poor Survival in Diffuse Gliomas\u003c/h2\u003e \u003cp\u003eTo evaluate the prognostic relevance of RCAN1, Kaplan\u0026ndash;Meier analyses were performed in both CGGA cohorts. In CGGA-325, high RCAN1 expression was associated with shorter overall survival in the all-grade, IDH-mutant, and IDH-wildtype groups (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). The same pattern was confirmed in CGGA-693 (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Multivariable Cox regression further showed that RCAN1-high status remained independently associated with worse survival (CGGA-325: HR 2.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CGGA-693: HR 1.36, P\u0026thinsp;=\u0026thinsp;0.03; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eGSEA Implicates RCAN1 in Inflammatory and Hypoxia-Associated Pathways\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn the CGGA-693 cohort, GSEA revealed that higher RCAN1 expression was significantly associated with multiple biological pathways relevant to glioma pathobiology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Specifically, higher RCAN1 expression was associated with enrichment of gene sets related to inflammatory and immune signaling, including those governing tumor necrosis factor alpha signaling via NF‑κB and interferon‑gamma response. In addition, significant enrichment was observed for pathways involved in apoptosis, hypoxia response, plasma membrane organization, and epithelial cell development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWithin the HALLMARK_TNFA_SIGNALING_VIA_NFKB gene set, RCAN1 showed positive associations with representative genes such as CD44, PLAU, IFNGR2, GADD45A, and SOD2, whereas RCAN1 expression exhibited weak inverse correlations with BMP2 and SNN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRCAN1 Expression Is Associated with an Immunosuppressive Tumor Microenvironment\u003c/h2\u003e \u003cp\u003eIn the CGGA-693 cohort, correlation analysis of CIBERSORT-derived immune fractions indicated that RCAN1 clustered positively with gamma‑delta T cells, M0 and M2 macrophages, and activated dendritic cells. Inverse correlations were most evident for na\u0026iuml;ve CD4⁺ T cells, whereas the association with memory B cells appeared less prominent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study addresses a persistent conundrum in neuro‑oncology regarding the observation that RCAN1, an established endogenous inhibitor of the pro‑tumorigenic calcineurin/NFAT and NF‑κB signaling pathways, is paradoxically upregulated in the most aggressive mesenchymal subtype of glioblastoma. By integrating pan‑glioma transcriptomic profiling, single‑cell resolution analysis, and functional pathway interrogation, our findings provide transcriptomic evidence supporting a context‑dependent association of RCAN1 with aggressive glioma phenotypes.\u003c/p\u003e \u003cp\u003eOur clinicopathological analysis demonstrates that RCAN1 expression increases progressively with WHO grade and is selectively enriched in IDH-wildtype gliomas, with further elevation observed in recurrent LGG tumors. Single-cell transcriptomic analysis further refined these findings, revealing that RCAN1 expression is predominantly enriched in AC-like and MES-like malignant cell states, with particularly prominent signal in the MES-like compartment.\u003c/p\u003e \u003cp\u003eThis cell‑state‑specific enrichment aligns with prior studies\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e characterizing RCAN1\u0026mdash;specifically its RCAN1‑4 isoform\u0026mdash; as a hallmark of MES‑like GBM cells and reinforces its tight association with mesenchymal transition. This stepwise upregulation, together with its independent prediction of shortened overall survival in multivariate models, establishes RCAN1 as a robust biomarker of aggressive disease and poor clinical outcome. Notably, this expression pattern diverges markedly from that reported in the majority of other solid malignancies, where RCAN1 is frequently downregulated relative to normal tissues and reduced expression portends advanced disease and inferior survival\u0026mdash;consistent with its function as a calcineurin-dependent tumor suppressor. In stark contrast, our findings indicate that RCAN1 is not diminished but rather progressively elevated in high-grade gliomas, reaching maximal levels in IDH-wildtype GBM, with additional elevation observed in recurrent LGG, whereas recurrent GBM showed a cohort-dependent pattern. This discrepancy suggests that the regulatory logic governing RCAN1 expression may be rewired in the glial lineage, likely during mesenchymal transition\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Rather than fulfilling a tumor‑suppressive role, RCAN1 in glioma may be transcriptionally co‑opted\u0026mdash;potentially via isoform switching to the mesenchymal‑specific RCAN1‑4 variant\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u0026mdash;to support a pro‑invasive, therapy‑resistant, and immunosuppressive phenotype. Thus, the paradoxical upregulation of RCAN1 in gliomas not only distinguishes it from the prevailing expression paradigm in extracranial malignancies but also warrants further functional and isoform-specific validation.\u003c/p\u003e \u003cp\u003eFunctional genomic analyses further indicate that elevated RCAN1 expression was associated with transcriptomic programs linked to malignant behavior in gliomas. Enrichment of TNFα/NF-κB signaling and hypoxia response gene sets in RCAN1-high tumors aligns with transcriptional programs driving proliferation, invasion, stem-like properties, and mesenchymal transition\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Positive correlations between RCAN1 and mesenchymal markers CD44 and SOD2 further support its association with the mesenchymal subtype\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Notably, CD44 has been implicated in glioma stemness, invasion, and immunosuppressive microenvironmental features\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, whereas SOD2 enhances survival under oxidative and hypoxic stress\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Activation of complement and interferon-γ response pathways additionally implicates RCAN1 in shaping a pro-inflammatory, immunosuppressive microenvironment\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Collectively, these findings suggest that RCAN1 operates within a pro-oncogenic network that potentiates invasion, stemness, and adaptive resistance. These co-expression patterns argue against a tumor-suppressive role and instead support transcriptional co-option of the RCAN1 locus during mesenchymal transition to reinforce the aggressive biology of high-grade gliomas.\u003c/p\u003e \u003cp\u003eImmune deconvolution analysis revealed that RCAN1 expression correlates positively with gamma‑delta T cells, M0 and M2 macrophages, and activated dendritic cells, and negatively with na\u0026iuml;ve CD4⁺ T cells. This profile\u0026mdash;enrichment of immunosuppressive myeloid subsets coupled with depletion of na\u0026iuml;ve T cells\u0026mdash;mirrors the immunologically \u0026ldquo;cold\u0026rdquo; microenvironment characteristic of mesenchymal glioblastoma\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The association with M2 macrophages, in particular, aligns with their established roles in promoting immune tolerance and therapeutic resistance\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These findings reinforce the notion that elevated RCAN1 expression demarcates a glioma subset characterized by profound immunosuppression, thereby highlighting the therapeutic imperative of RCAN1‑targeted strategies capable of circumventing local immune dysfunction.\u003c/p\u003e \u003cp\u003eIn summary, our study reframes the role of RCAN1 in glioma biology. Rather than functioning as a conventional tumor suppressor, its elevated expression in high-grade, IDH-wildtype gliomas reflects the transcriptional reprogramming and isoform diversion that accompany mesenchymal transition. The association of RCAN1 with a pro-invasive network, therapy-resistant pathways, and an immunosuppressive microenvironment underscores its centrality to the aggressive biology of glioblastoma. Importantly, these findings nominate RCAN1 as candidate biomarkers and potential subtype-selective therapeutic targets.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS. L., Z. Z. conceptualized the project. Z. Z., J. H. and S. L. analyzed and interpreted the sequencing data; Z. Z., J. H. and S. L. wrote the manuscript and prepared all the figures. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (2025ZD0552017), the National Natural Science Foundation of China (82472841), the Beijing Municipal Health Commission Fund (11000023T000002044300-5).\u003c/p\u003e \u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data used in this study were obtained from the publicly accessible Chinese Glioma Genome Atlas (CGGA) database.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeller M, Wen PY, Chang SM, et al. Glioma. \u003cem\u003eNat Rev Dis Primers.\u003c/em\u003e 2024; 10(1):33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLouis DN, Perry A, Wesseling P, et al. 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New insights into M1/M2 macrophages: key modulators in cancer progression. \u003cem\u003eCancer cell international.\u003c/em\u003e 2021; 21(1):389.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RCAN1, glioma, mesenchymal subtype, transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-9444214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9444214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e. RCAN1 encodes a calcineurin inhibitor conventionally viewed as a tumor suppressor, yet its RCAN1‑4 isoform is paradoxically enriched in mesenchymal glioblastoma. The precise contribution of RCAN1 to glioma biology across the full histologic and molecular spectrum, however, remains poorly defined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e. We performed a comprehensive pan‑glioma analysis integrating RNA‑sequencing data from two independent Chinese Glioma Genome Atlas (CGGA) cohorts (CGGA‑325, n = 325; CGGA‑693, n = 693) and a publicly available single‑cell RNA‑sequencing dataset of glioblastoma. Associations between RCAN1 expression and clinicopathological features, survival outcomes, transcriptional programs, and the immune microenvironment were systematically evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e. RCAN1 expression increased progressively with WHO grade and was selectively elevated in IDH-wildtype gliomas. Single‑cell transcriptomics revealed that RCAN1 expression was predominantly confined to mesenchymal‑like (MES‑like) and astrocytic‑like (AC‑like) malignant cell states. High RCAN1 expression independently predicted shorter overall survival in both CGGA cohorts. Gene set enrichment analysis demonstrated that RCAN1-high tumors exhibited marked enrichment of TNFα/NF‑κB signaling, hypoxia response, and interferon‑γ response pathways, and RCAN1 correlated positively with mesenchymal markers CD44 and SOD2. Immune deconvolution further linked elevated RCAN1 to an immunosuppressive microenvironment characterized by enrichment of M2 macrophages and depletion of naïve CD4⁺ T cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e. RCAN1 upregulation in high‑grade, IDH‑wildtype gliomas reflects mesenchymal transition‑associated transcriptional reprogramming and contrasts with its canonical tumor‑suppressive role in other cancers. These findings nominate RCAN1 as a subtype‑specific prognostic biomarker and potential therapeutic target in aggressive gliomas.\u003c/p\u003e","manuscriptTitle":"Molecular characterization and clinical relevance of RCAN1 in diffuse gliomas","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 14:22:34","doi":"10.21203/rs.3.rs-9444214/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c365210e-231e-447f-8326-aaec1784c4f2","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T14:22:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 14:22:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9444214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9444214","identity":"rs-9444214","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}