Prognostic Value of CMTM6 Expression in Breast Cancer

Prognostic Value of CMTM6 Expression in Breast 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 Prognostic Value of CMTM6 Expression in Breast Cancer Lingting Jiang, Aiyu Liu, Puchao Peng, Shizhen Zhang, Chengliang Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7752340/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 Introduction CMTM6, a regulator of tumor cell PD-L1 stability via inhibiting lysosome-mediated degradation, is linked to anti-tumor immunity and tumorigenesis. However, its expression and prognostic value in breast cancer subtypes remain unclear. Methods We conducted an integrated bioinformatics analysis of CMTM6 using multiple public databases: cBioPortal for examining genetic alterations in TCGA breast cancer datasets; GEPIA for comparing expression between tumor and normal tissues; bc-GenExMiner 4.0 for assessing expression across subtypes and performing meta-analysis; Kaplan-Meier Plotter and the Human Protein Atlas for prognostic assessment. Results CMTM6 was ubiquitous in human tissues but low in normal breast (glandular/myoepithelial cells only). It was higher in breast cancer than normal tissues, lower in HER2-positive subtypes, higher in P53 wild-type tumors, and uncorrelated with lymph node status/age. Breast cancer CMTM6 gene alterations were rare (amplification: 0.56%; deep deletion: 0.14%). High CMTM6 (mRNA/protein) correlated with better overall survival, specifically in lymph node-positive and Luminal A subtypes, but not in other subtypes. Discussion CMTM6 is differentially expressed in breast cancer/subtypes and acts as a favorable prognostic indicator for lymph node-positive/Luminal A breast cancer, serving as a potential subtype-specific biomarker. CMTM6 Breast cancer Prognosis Overall survival Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key points 1. CMTM6 exists in many human tissues, is low in normal breast tissue but higher in breast cancer tissue. It is lower in HER2-positive breast cancer and higher in breast cancer with wild-type P53, and rarely has genetic changes in breast cancer. 2. High CMTM6 levels (mRNA and protein) mean better overall survival for breast cancer patients, especially those with lymph node-positive or Luminal A subtype—this doesn’t apply to other subtypes. 3. CMTM6 may be a subtype-specific biomarker to predict prognosis for some breast cancer patients, like those with lymph node-positive or Luminal A breast cancer. 1 Introduction CMTM6 is a transmembrane protein recently identified as a critical regulator of PD-L1 stability in tumor cells. Localized at the plasma membrane, CMTM6 colocalizes with PD-L1 and sustains its expression throughout the cell cycle by preventing lysosomal degradation 1 , 2 . This stabilization mechanism contributes to the suppression of T-cell activation and dampens anti-tumor immunity 1 , 2 . Depletion of CMTM6 reduces PD-L1 levels, alleviates T-cell immunosuppression, and enhances tumor-specific T-cell activity in both in vitro and in vivo models 1 . Therefore, targeting the CMTM6–PD-L1 interaction to restore anti-tumor immune responses has emerged as a promising therapeutic strategy for cancer treatment 1 , 2 . Beyond its role in immune regulation, CMTM6 has been increasingly implicated in tumorigenesis and cancer progression. According to The Cancer Genome Atlas (TCGA) data, CMTM6 mRNA is expressed across tumor samples from 30 different cancer types 1 . Its expression patterns and clinical implications, however, vary considerably among malignancies. In gliomas, elevated CMTM6 expression is associated with malignant phenotypes and co-occurs with frequent genomic alterations in key driver oncogenes 3 . In non-small cell lung cancer (NSCLC), CMTM6 is more highly expressed than in small cell lung cancer (SCLC) and shows an inverse correlation with metastatic tendency 4 . Conversely, in pancreatic adenocarcinoma, high CMTM6 mRNA levels are linked to poorer overall survival 5 , a trend also observed in colorectal cancer, where elevated CMTM6 expression predicts unfavorable patient outcomes 6 . Notably, in NSCLC, CMTM6 serves as an independent predictive biomarker for response to PD-L1 inhibitors 7 . In hepatocellular carcinoma (HCC), bioinformatics analyses and immunofluorescence staining consistently indicate a negative correlation between CMTM6 expression and prognosis 8 . Within breast cancer, CMTM6 expression is significantly higher in triple-negative breast cancer (TNBC) compared to HER2-positive subtypes, and higher levels are associated with shorter progression-free survival in TNBC patients 9 , 10 . Nevertheless, the role and prognostic value of CMTM6 in other breast cancer subtypes remain poorly characterized. This study therefore aims to integrate and expand upon existing annotations to systematically evaluate CMTM6 expression in breast cancer and assess its subtype-specific prognostic significance. 2 Materials and Methods 2.1 Bioinformatic Analysis of CMTM6 Expression 1) cBioPortal for Cancer Genomics ( http://www.cbioportal.org/ ): This platform provides tools for exploring, visualizing, and analyzing multidimensional cancer genomics data. It was used to assess CMTM6 mRNA expression, copy number alterations, and mutations in the breast cancer cohort from the TCGA database. 2) Gene Expression Profiling Interactive Analysis (GEPIA): This database was utilized to compare CMTM6 gene expression between breast cancer tissues and normal breast tissues. 3) Breast Cancer Gene-Expression Miner Version 4.0 (bc-GenExMiner 4.0): A comprehensive database containing annotated genomic data from 5,609 breast cancer patients, used to analyze CMTM6 mRNA expression across different breast cancer subtypes. 2.2 Bioinformatic Analysis of CMTM6 Prognostic Value 1) Meta-analysis with bc-GenExMiner 4.0: This database was used to integrate previously annotated genomic data and evaluate the association between CMTM6 expression and survival outcomes in breast cancer patients. 2) Kaplan-Meier Plotter ( https://kmplot.com ): A web-based tool for analyzing the correlation between gene expression levels and patient prognosis, used here to assess the prognostic value of CMTM6 mRNA in breast cancer. 3) Human Protein Atlas ( https://www.proteinatlas.org ): This database was employed to examine the CMTM6 protein expression in breast cancer patients. 3 Results 3.1 CMTM6 is ubiquitously expressed in human tissues CMTM6 is a widely expressed transmembrane protein implicated in epigenetic regulation, embryonic development, and tumorigenesis 1 , 11 . Analysis of the Human Protein Atlas (HPA) database confirmed that CMTM6 is ubiquitously expressed across human tissues with low tissue specificity (Fig. 1 A and Figure S1). At the mRNA level, CMTM6 expression was relatively high in the liver but low in skeletal muscle (Fig. 1 A). In contrast, CMTM6 protein expression was elevated in the cerebellum, endometrium, fallopian tube, rectum, and urinary bladder, while lower expression levels were observed in adipose tissue, cerebral cortex, liver, oral mucosa, ovary, salivary gland, soft tissue, and testis (Fig. 1 B). These findings suggest that CMTM6 may undergo post-transcriptional regulation in a tissue-specific manner. 3.2 CMTM6 is lowly expressed in breast tissues Single-cell RNA-seq data from the Human Protein Atlas revealed that CMTM6 mRNA is primarily expressed in specific clusters of breast glandular and myoepithelial cells. In contrast, other cell types within breast tissue—such as T cells, macrophages, fibroblasts, and adipocytes—exhibited minimal CMTM6 expression (Fig. 2 A and Figure S2). Consistent with these findings, immunohistochemical (IHC) staining of normal breast tissues confirmed that CMTM6 protein is detectable in glandular and myoepithelial cells (Fig. 2 B), but absent in adipocytes (Fig. 2 C). 3.3 CMTM6 is widely expressed in human cancers CMTM6 has previously been reported in several malignancies, including lung cancer 7 , 12 , hepatocellular carcinoma 12 , glioma 3 and TNBC 9 . Analysis of the Human Protein Atlas further demonstrated widespread CMTM6 mRNA expression across multiple human cancer cell lines and tumor types, with low cell line and cancer-type specificity (Fig. 3 A‒3B). Among cell lines, the choriocarcinoma-derived BeWo and Hodgkin’s lymphoma-derived HDLM-2 cells showed relatively high CMTM6 mRNA levels (Fig. 3 A). In tumor samples, ovarian cancer exhibited elevated CMTM6 mRNA expression (Fig. 3 B). At the protein level, IHC analysis revealed moderate to strong cytoplasmic staining in stomach, liver, pancreatic, urothelial, and ovarian cancers, while most other cancer types showed negligible expression (Figure S3A‒S3B). 3.4 CMTM6 gene shows amplification in breast cancer We examined CMTM6 genetic alterations across multiple cancer types using cBioPortal. Alterations—including mutations, fusions, amplifications, and deep deletions—were generally infrequent. The highest amplification frequency was observed in bladder cancer, the highest deep deletion rate in clear cell renal cell carcinoma, and the highest mutation frequency in uterine cancer (Fig. 4 A). In breast cancer, CMTM6 gene amplification and deep deletion accounted for 0.56% and 0.14% of copy number alterations, respectively (Fig. 4 B). 3.5 CMTM6 expression in breast cancer Using the GEPIA database 13 , we found that CMTM6 expression was significantly higher in breast cancer tissues compared to normal breast tissues (Fig. 5 A). Further analysis with bc-GenExMiner 4.0 indicated that CMTM6 mRNA levels were lower in HER2-positive subtypes compared to other subtypes (Fig. 5 B), and higher in p53 wild-type tumors than in p53 mutant cases (Fig. 5 C). No significant correlation was observed between CMTM6 expression and lymph node status or patient age (Figure S4A‒S4B). 3.6 CMTM6 overexpression is associated with better outcome in breast cancer Elevated CMTM6 expression has been linked to improved prognosis in certain cancers 7 , 14 . In triple-negative breast cancer (TNBC), 44.7% of cases showed positive CMTM6 staining by IHC 9 . Through the Human Protein Atlas, we found that 73.86% of breast cancer tissues exhibited high CMTM6 protein expression (Figure S5A), which was associated with improved survival (Fig. 6 A). Analysis of the Kaplan–Meier Plotter database further confirmed that high CMTM6 mRNA expression correlated significantly with better overall survival in breast cancer patients (Fig. 6 B). Subgroup analyses indicated that this favorable prognostic association was particularly evident in lymph node-positive and luminal A subtypes (Fig. 6 C‒6D), but not in lymph node-negative, luminal B, HER2-positive, basal-like, or p53-mutant subtypes (Figure S5B‒F). 4 Discussion In the present study, utilizing publicly available annotated databases, we demonstrated that CMTM6 is highly expressed in several human cancers, including stomach, liver, pancreatic, urothelial, and ovarian cancers. In breast cancer, CMTM6 expression was significantly elevated in tumor tissues compared to normal breast tissues. Moreover, higher expression levels of CMTM6—both at the mRNA and protein levels—were significantly associated with improved survival outcomes in breast cancer patients. The CMTM family, which includes CKLF and CMTM1–8, has been implicated in various diseases such as cardiovascular disorders 15 , autoimmune conditions 16 and tumorigenesis 17 . Previous studies have localized CMTM6 to the plasma membrane in multiple cancer types 3 , 7 , 18 , 19 . Our findings further support these observations, confirming elevated CMTM6 expression in stomach, liver, pancreatic, urothelial, and ovarian cancers (Fig. 3 ). Recent groundbreaking research has identified CMTM6 as a key regulator of PD-L1. By binding to PD-L1, CMTM6 inhibits its lysosomal degradation, thereby stabilizing PD-L1 expression. Consequently, inhibition of CMTM6 reduces PD-L1 levels and enhances tumor-specific T-cell activity 1 , 2 , 20 , 21 . Furthermore, CMTM6 expression has been linked to improved response to anti-PD-1 therapies 7 , 22 . In head and neck squamous cell carcinoma, CMTM6 expression correlates with PD-L1 levels 23 , and promotes epithelial–mesenchymal transition (EMT) and cancer stemness via activation of the Wnt/β-catenin pathway 18 . In HCC, CMTM6 upregulation enhances cancer cell proliferation through β-catenin signaling 8 , It is also positively correlated with PD-L1 expression and CD8⁺ T-cell infiltration, with high membrane CMTM6 levels associated with tumor recurrence and proliferation 24 . Additionally, CMTM6 interacts with and stabilizes vimentin, facilitating EMT and thereby promoting HCC proliferation, migration, and invasion 25 . In oral squamous cell carcinoma (OSCC), CMTM6 stabilizes membrane-bound Enolase-1, activating AKT/GSK-3β-mediated Wnt signaling and contributing to cisplatin resistance 26 . In NSCLC, CMTM6 is upregulated and colocalizes with EGFR and RAB11. High CMTM6 expression correlates with poor prognosis, and a CMTM6-targeting nanobody has been shown to disrupt CMTM6–EGFR interaction, reduce EGFR levels, and suppress proliferation in EGFR-TKI-resistant NSCLC models both in vitro and in vivo 27 . In breast cancer, EMT-induced SNAI1 upregulates CMTM6, leading to increased cell surface PD-L1 expression 19 . However, the precise biochemical mechanisms underlying CMTM6 regulation and its role in breast cancer pathogenesis remain incompletely understood and warrant further investigation. Notably, we found that elevated CMTM6 mRNA expression predicted better survival specifically in luminal A breast cancer, but not in lymph node-negative, luminal B, HER2-positive, or basal-like subtypes. This suggests a potential interplay between CMTM6 and estrogen/progesterone receptor signaling pathways during breast cancer progression. In conclusion, our study indicates that CMTM6 expression is upregulated in breast cancer compared to normal tissues and may serve as an independent prognostic factor for predicting patient survival. Declarations Funding This study was supported by grant from the National Natural Science Foundation of China Project (Grant nos. 82202838, 82473005). Conflict of interest The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Author contributions All authors contributed to the study conception and design. Data collection and analysis were performed by L. J., A. L., P. P. and S. Z.. The first draft of the manuscript was written by L. J. and A. L., and all authors commented on previous versions of the manuscript. Supervision by C. C. and Y. Z. All authors read and approved the final manuscript. 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J translational Med 19. 10.1186/s12967-021-02787-5 Mohapatra P, Shriwas O, Mohanty S, Ghosh A, Smita S, Kaushik SR, Arya R, Rath R, Majumdar D, Muduly SK et al (2021) D.K.,. CMTM6 drives cisplatin resistance by regulating Wnt signaling through the ENO-1/AKT/GSK3β axis. JCI insight 6 . 10.1172/jci.insight.143643 Xia L, Wang J, Xue H, Li H, Li Q, Qin S, Yu C, Liu Y, Gao Y, Li L et al (2025) A CMTM6 Nanobody Overcomes EGFR-TKI Resistance in Non-Small Cell Lung Cancer. Adv Sci (Weinh) 12:e2410945. 10.1002/advs.202410945 Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif Figure S1: CMTM6 is ubiquitously expressed in human tissues with low tissue specificity. FigureS2.tif Figure S2: Single cell RNA-seq analysis of breast tissues. FigureS3.tif Figure S3: CMTM6 is widely expressed in human cancers A. CMTM6 protein expression was presented in most of human cancer tissues; B. IHC staining showed that several human cancers, such as stomach, liver, pancreatic, urothelial and ovarian cancers displayed moderate to strong cytoplasmic immunoreactivity. FigureS4.tif Figure S4: CMTM6 expression in breast cancer A-B. CMTM6 mRNA expression levels did not relate to lymph nodal status (A) or age stages (B). FigureS5.tif Figure S5: CMTM6 overexpression is associated with better outcome of breast cancer A. CMTM6 was expressed highly in 73.86% of breast cancer tissues; B-F. CMTM6 mRNA expression did not indicate survival significance in lymph node negative (B), luminal B (C), HER2-positive (D), Basal-like (E), and P53-mutant (F) types of breast cancers. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":1307811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCMTM6 is ubiquitously expressed in human tissues with low tissue specificity \u003c/strong\u003eA. The mRNA expression of CMTM6 in different human tissues; B. The CMTM6 protein expression in different human tissues.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/576d2d4b2d982d16853e4f21.png"},{"id":92942721,"identity":"058dbd80-6521-4b15-a5f2-60f4a117eeaa","added_by":"auto","created_at":"2025-10-07 11:48:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5145194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCMTM6 is lowly expressed in breast tissues \u003c/strong\u003eA. Single cell RNA-seq analysis of breast tissues showed that \u003cem\u003eCMTM6\u003c/em\u003e mRNA is only expressed in specific cluster of breast glandular cells and myoepithelial cells; B-C. The IHC staining analysis of normal breast tissues showed that CMTM6 protein was only expressed in glandular cells and myoepithelial cells (B), but not in adipocytes (C).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/df53d56a36034823a2f507c9.png"},{"id":92944322,"identity":"54ec0c8f-8cc5-4828-a074-42ef9e101c97","added_by":"auto","created_at":"2025-10-07 12:04:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2014580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCMTM6 is widely expressed in human cancers \u003c/strong\u003eA. The \u003cem\u003eCMTM6\u003c/em\u003e mRNA was presented in most of human cancer cell lines; B. The \u003cem\u003eCMTM6\u003c/em\u003emRNA was presented in most of human cancer tissues.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/fba4e9a5b6f4fcb79c164669.png"},{"id":92943512,"identity":"9e7eb944-fe09-4858-bc76-82ae4513f0fa","added_by":"auto","created_at":"2025-10-07 11:56:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2122619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCMTM6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene shows amplification in breast cancer\u003c/strong\u003e A. \u003cem\u003eCMTM6\u003c/em\u003e gene alterations were existed at low frequencies in majority of human cancers, with the highest amplification frequency in bladder cancer, highest deep deletion rate in clear cell renal cell carcinoma, and highest mutation frequency in uterine cancer; B.\u003cem\u003e CMTM6\u003c/em\u003e gene amplification accounted for 0.56% of the copy number alternations, and deep deletion comprised 0.14% in breast cancer.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/c23ad932262e1fc016dea5c8.png"},{"id":92942727,"identity":"863e728b-cc30-4f77-aff6-29bb9808498b","added_by":"auto","created_at":"2025-10-07 11:48:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":672042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCMTM6 expression in breast cancer\u003c/strong\u003e A. The expression of \u003cem\u003eCMTM6 \u003c/em\u003ewere significantly increased in breast cancer tissues than that in normal tissues; B. The \u003cem\u003eCMTM6\u003c/em\u003e mRNA was expressed at a lower level in HER2-expressing subtypes than in other subtypes of breast cancer; C. The \u003cem\u003eCMTM6\u003c/em\u003e mRNA expressed at a higher level in p53 wild-type breast cancer than in p53 mutant breast cancer.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/28971e081c40c28c8354e372.png"},{"id":92943513,"identity":"50862505-85e1-48f4-a92c-26ae8240bf57","added_by":"auto","created_at":"2025-10-07 11:56:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1318427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCMTM6 overexpression is associated with better outcome of breast cancer \u003c/strong\u003eA. High expression of CMTM6 was associated with better survival of breast cancer; B. Higher \u003cem\u003eCMTM6\u003c/em\u003emRNA expression was significantly associated with better survival of breast cancer; C-D. The elevated \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression indicated better survival in lymph node positive (C) and luminal A (D) type breast cancers.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/1a47c5fbad4b4c2dd742b82b.png"},{"id":93433365,"identity":"21574015-fce8-4d0f-9004-05b66789819c","added_by":"auto","created_at":"2025-10-13 19:01:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12474821,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/403b922f-c2b7-4324-bd84-6dc8fe23bcf1.pdf"},{"id":92942728,"identity":"255d5a41-fc6b-4525-808c-dcd800151c78","added_by":"auto","created_at":"2025-10-07 11:48:15","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14420640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1: CMTM6 is ubiquitously expressed in human tissues with low tissue specificity.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/ff648972358aa98025f5e7ff.tif"},{"id":92942730,"identity":"745c2e1e-6ea4-49d1-bc07-f4e23f5ed438","added_by":"auto","created_at":"2025-10-07 11:48:15","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13293528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2: Single cell RNA-seq analysis of breast tissues.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/044d4c82674c3b98b8c3ca37.tif"},{"id":92943515,"identity":"52895631-7bd5-4581-9f56-f8799bb5ab3b","added_by":"auto","created_at":"2025-10-07 11:56:15","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12581676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3: CMTM6 is widely expressed in human cancers\u003c/strong\u003e A. CMTM6 protein expression was presented in most of human cancer tissues; B. IHC staining showed that several human cancers, such as stomach, liver, pancreatic, urothelial and ovarian cancers displayed moderate to strong cytoplasmic immunoreactivity.\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/81952b610e72fe7539c02c58.tif"},{"id":92942729,"identity":"ee2a0edf-180f-428e-8fe9-743c8faed8d1","added_by":"auto","created_at":"2025-10-07 11:48:15","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8373620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S4: CMTM6 expression in breast cancer \u003c/strong\u003eA-B. \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression levels did not relate to lymph nodal status (A) or age stages (B).\u003c/p\u003e","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/d12c5455f082fe0d709ee193.tif"},{"id":92942737,"identity":"57278968-0fd4-4a8b-97da-5339522b75c5","added_by":"auto","created_at":"2025-10-07 11:48:15","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11426512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S5: CMTM6 overexpression is associated with better outcome of breast cancer \u003c/strong\u003eA. CMTM6 was expressed highly in 73.86% of breast cancer tissues; B-F. CMTM6 mRNA expression did not indicate survival significance in lymph node negative (B), luminal B (C), HER2-positive (D), Basal-like (E), and P53-mutant (F) types of breast cancers.\u003c/p\u003e","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7752340/v1/c5a2fbf31594b521448bd2cd.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prognostic Value of CMTM6 Expression in Breast Cancer","fulltext":[{"header":"Key points","content":"\u003cp\u003e1. CMTM6 exists in many human tissues, is low in normal breast tissue but higher in breast cancer tissue. It is lower in HER2-positive breast cancer and higher in breast cancer with wild-type P53, and rarely has genetic changes in breast cancer.\u003c/p\u003e\n\u003cp\u003e2. High CMTM6 levels (mRNA and protein) mean better overall survival for breast cancer patients, especially those with lymph node-positive or Luminal A subtype\u0026mdash;this doesn\u0026rsquo;t apply to other subtypes.\u003c/p\u003e\n\u003cp\u003e3. CMTM6 may be a subtype-specific biomarker to predict prognosis for some breast cancer patients, like those with lymph node-positive or Luminal A breast cancer.\u003c/p\u003e"},{"header":"1 Introduction","content":"\u003cp\u003eCMTM6 is a transmembrane protein recently identified as a critical regulator of PD-L1 stability in tumor cells. Localized at the plasma membrane, CMTM6 colocalizes with PD-L1 and sustains its expression throughout the cell cycle by preventing lysosomal degradation \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This stabilization mechanism contributes to the suppression of T-cell activation and dampens anti-tumor immunity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Depletion of CMTM6 reduces PD-L1 levels, alleviates T-cell immunosuppression, and enhances tumor-specific T-cell activity in both in vitro and in vivo models \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Therefore, targeting the CMTM6\u0026ndash;PD-L1 interaction to restore anti-tumor immune responses has emerged as a promising therapeutic strategy for cancer treatment \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBeyond its role in immune regulation, CMTM6 has been increasingly implicated in tumorigenesis and cancer progression. According to The Cancer Genome Atlas (TCGA) data, \u003cem\u003eCMTM6\u003c/em\u003e mRNA is expressed across tumor samples from 30 different cancer types \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Its expression patterns and clinical implications, however, vary considerably among malignancies. In gliomas, elevated CMTM6 expression is associated with malignant phenotypes and co-occurs with frequent genomic alterations in key driver oncogenes \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In non-small cell lung cancer (NSCLC), CMTM6 is more highly expressed than in small cell lung cancer (SCLC) and shows an inverse correlation with metastatic tendency \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Conversely, in pancreatic adenocarcinoma, high \u003cem\u003eCMTM6\u003c/em\u003e mRNA levels are linked to poorer overall survival \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, a trend also observed in colorectal cancer, where elevated CMTM6 expression predicts unfavorable patient outcomes \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, in NSCLC, CMTM6 serves as an independent predictive biomarker for response to PD-L1 inhibitors \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In hepatocellular carcinoma (HCC), bioinformatics analyses and immunofluorescence staining consistently indicate a negative correlation between CMTM6 expression and prognosis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWithin breast cancer, CMTM6 expression is significantly higher in triple-negative breast cancer (TNBC) compared to HER2-positive subtypes, and higher levels are associated with shorter progression-free survival in TNBC patients \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the role and prognostic value of CMTM6 in other breast cancer subtypes remain poorly characterized. This study therefore aims to integrate and expand upon existing annotations to systematically evaluate CMTM6 expression in breast cancer and assess its subtype-specific prognostic significance.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003e\u003cb\u003e2.1 Bioinformatic Analysis of CMTM6 Expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003e1) cBioPortal for Cancer Genomics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"http://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e): This platform provides tools for exploring, visualizing, and analyzing multidimensional cancer genomics data. It was used to assess \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression, copy number alterations, and mutations in the breast cancer cohort from the TCGA database.\u003c/p\u003e\u003cp\u003e2) Gene Expression Profiling Interactive Analysis (GEPIA): This database was utilized to compare \u003cem\u003eCMTM6\u003c/em\u003e gene expression between breast cancer tissues and normal breast tissues.\u003c/p\u003e\u003cp\u003e3) Breast Cancer Gene-Expression Miner Version 4.0 (bc-GenExMiner 4.0): A comprehensive database containing annotated genomic data from 5,609 breast cancer patients, used to analyze \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression across different breast cancer subtypes.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.2 Bioinformatic Analysis of CMTM6 Prognostic Value\u003c/b\u003e\u003c/p\u003e\u003cp\u003e1) Meta-analysis with bc-GenExMiner 4.0: This database was used to integrate previously annotated genomic data and evaluate the association between CMTM6 expression and survival outcomes in breast cancer patients.\u003c/p\u003e\u003cp\u003e2) Kaplan-Meier Plotter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kmplot.com\u003c/span\u003e\u003cspan address=\"https://kmplot.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e): A web-based tool for analyzing the correlation between gene expression levels and patient prognosis, used here to assess the prognostic value of \u003cem\u003eCMTM6\u003c/em\u003e mRNA in breast cancer.\u003c/p\u003e\u003cp\u003e3) Human Protein Atlas (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e): This database was employed to examine the CMTM6 protein expression in breast cancer patients.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e3.1 CMTM6 is ubiquitously expressed in human tissues\u003c/h2\u003e\u003cp\u003eCMTM6 is a widely expressed transmembrane protein implicated in epigenetic regulation, embryonic development, and tumorigenesis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Analysis of the Human Protein Atlas (HPA) database confirmed that CMTM6 is ubiquitously expressed across human tissues with low tissue specificity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Figure S1). At the mRNA level, \u003cem\u003eCMTM6\u003c/em\u003e expression was relatively high in the liver but low in skeletal muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, CMTM6 protein expression was elevated in the cerebellum, endometrium, fallopian tube, rectum, and urinary bladder, while lower expression levels were observed in adipose tissue, cerebral cortex, liver, oral mucosa, ovary, salivary gland, soft tissue, and testis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These findings suggest that CMTM6 may undergo post-transcriptional regulation in a tissue-specific manner.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.2 CMTM6 is lowly expressed in breast tissues\u003c/h2\u003e\u003cp\u003eSingle-cell RNA-seq data from the Human Protein Atlas revealed that \u003cem\u003eCMTM6\u003c/em\u003e mRNA is primarily expressed in specific clusters of breast glandular and myoepithelial cells. In contrast, other cell types within breast tissue\u0026mdash;such as T cells, macrophages, fibroblasts, and adipocytes\u0026mdash;exhibited minimal CMTM6 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Figure S2). Consistent with these findings, immunohistochemical (IHC) staining of normal breast tissues confirmed that CMTM6 protein is detectable in glandular and myoepithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), but absent in adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.3 CMTM6 is widely expressed in human cancers\u003c/h2\u003e\u003cp\u003eCMTM6 has previously been reported in several malignancies, including lung cancer \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, glioma \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and TNBC \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Analysis of the Human Protein Atlas further demonstrated widespread \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression across multiple human cancer cell lines and tumor types, with low cell line and cancer-type specificity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA‒3B). Among cell lines, the choriocarcinoma-derived BeWo and Hodgkin\u0026rsquo;s lymphoma-derived HDLM-2 cells showed relatively high \u003cem\u003eCMTM6\u003c/em\u003e mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In tumor samples, ovarian cancer exhibited elevated \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At the protein level, IHC analysis revealed moderate to strong cytoplasmic staining in stomach, liver, pancreatic, urothelial, and ovarian cancers, while most other cancer types showed negligible expression (Figure S3A‒S3B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.4 \u003cem\u003eCMTM6\u003c/em\u003e gene shows amplification in breast cancer\u003c/h2\u003e\u003cp\u003eWe examined \u003cem\u003eCMTM6\u003c/em\u003e genetic alterations across multiple cancer types using cBioPortal. Alterations\u0026mdash;including mutations, fusions, amplifications, and deep deletions\u0026mdash;were generally infrequent. The highest amplification frequency was observed in bladder cancer, the highest deep deletion rate in clear cell renal cell carcinoma, and the highest mutation frequency in uterine cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In breast cancer, \u003cem\u003eCMTM6\u003c/em\u003e gene amplification and deep deletion accounted for 0.56% and 0.14% of copy number alterations, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.5 CMTM6 expression in breast cancer\u003c/h2\u003e\u003cp\u003eUsing the GEPIA database \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, we found that CMTM6 expression was significantly higher in breast cancer tissues compared to normal breast tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Further analysis with bc-GenExMiner 4.0 indicated that \u003cem\u003eCMTM6\u003c/em\u003e mRNA levels were lower in HER2-positive subtypes compared to other subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and higher in p53 wild-type tumors than in p53 mutant cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). No significant correlation was observed between CMTM6 expression and lymph node status or patient age (Figure S4A‒S4B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.6 CMTM6 overexpression is associated with better outcome in breast cancer\u003c/h2\u003e\u003cp\u003eElevated CMTM6 expression has been linked to improved prognosis in certain cancers \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In triple-negative breast cancer (TNBC), 44.7% of cases showed positive CMTM6 staining by IHC \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Through the Human Protein Atlas, we found that 73.86% of breast cancer tissues exhibited high CMTM6 protein expression (Figure S5A), which was associated with improved survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Analysis of the Kaplan\u0026ndash;Meier Plotter database further confirmed that high \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression correlated significantly with better overall survival in breast cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Subgroup analyses indicated that this favorable prognostic association was particularly evident in lymph node-positive and luminal A subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC‒6D), but not in lymph node-negative, luminal B, HER2-positive, basal-like, or p53-mutant subtypes (Figure S5B‒F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn the present study, utilizing publicly available annotated databases, we demonstrated that CMTM6 is highly expressed in several human cancers, including stomach, liver, pancreatic, urothelial, and ovarian cancers. In breast cancer, CMTM6 expression was significantly elevated in tumor tissues compared to normal breast tissues. Moreover, higher expression levels of CMTM6\u0026mdash;both at the mRNA and protein levels\u0026mdash;were significantly associated with improved survival outcomes in breast cancer patients.\u003c/p\u003e\u003cp\u003eThe CMTM family, which includes CKLF and CMTM1\u0026ndash;8, has been implicated in various diseases such as cardiovascular disorders \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, autoimmune conditions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and tumorigenesis \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Previous studies have localized CMTM6 to the plasma membrane in multiple cancer types \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Our findings further support these observations, confirming elevated CMTM6 expression in stomach, liver, pancreatic, urothelial, and ovarian cancers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Recent groundbreaking research has identified CMTM6 as a key regulator of PD-L1. By binding to PD-L1, CMTM6 inhibits its lysosomal degradation, thereby stabilizing PD-L1 expression. Consequently, inhibition of CMTM6 reduces PD-L1 levels and enhances tumor-specific T-cell activity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Furthermore, CMTM6 expression has been linked to improved response to anti-PD-1 therapies \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn head and neck squamous cell carcinoma, CMTM6 expression correlates with PD-L1 levels \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and promotes epithelial\u0026ndash;mesenchymal transition (EMT) and cancer stemness via activation of the Wnt/β-catenin pathway \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In HCC, CMTM6 upregulation enhances cancer cell proliferation through β-catenin signaling \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, It is also positively correlated with PD-L1 expression and CD8⁺ T-cell infiltration, with high membrane CMTM6 levels associated with tumor recurrence and proliferation \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Additionally, CMTM6 interacts with and stabilizes vimentin, facilitating EMT and thereby promoting HCC proliferation, migration, and invasion \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In oral squamous cell carcinoma (OSCC), CMTM6 stabilizes membrane-bound Enolase-1, activating AKT/GSK-3β-mediated Wnt signaling and contributing to cisplatin resistance \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In NSCLC, CMTM6 is upregulated and colocalizes with EGFR and RAB11. High CMTM6 expression correlates with poor prognosis, and a CMTM6-targeting nanobody has been shown to disrupt CMTM6\u0026ndash;EGFR interaction, reduce EGFR levels, and suppress proliferation in EGFR-TKI-resistant NSCLC models both in vitro and in vivo \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In breast cancer, EMT-induced SNAI1 upregulates CMTM6, leading to increased cell surface PD-L1 expression \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the precise biochemical mechanisms underlying CMTM6 regulation and its role in breast cancer pathogenesis remain incompletely understood and warrant further investigation. Notably, we found that elevated \u003cem\u003eCMTM6\u003c/em\u003e mRNA expression predicted better survival specifically in luminal A breast cancer, but not in lymph node-negative, luminal B, HER2-positive, or basal-like subtypes. This suggests a potential interplay between CMTM6 and estrogen/progesterone receptor signaling pathways during breast cancer progression.\u003c/p\u003e\u003cp\u003eIn conclusion, our study indicates that CMTM6 expression is upregulated in breast cancer compared to normal tissues and may serve as an independent prognostic factor for predicting patient survival.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grant from the National Natural Science Foundation of China Project (Grant nos. 82202838, 82473005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Data collection and analysis were performed by L. J., A. L., P. P. and S. Z.. The first draft of the manuscript was written by L. J. and A. L., and all authors commented on previous versions of the manuscript. Supervision by C. C. and Y. Z. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBurr ML, Sparbier CE, Chan YC, Williamson JC, Woods K, Beavis PA, Lam EYN, Henderson MA, Bell CC, Stolzenburg S et al (2017) CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. 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CMTM6 drives cisplatin resistance by regulating Wnt signaling through the ENO-1/AKT/GSK3β axis. JCI insight \u003cem\u003e6\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/jci.insight.143643\u003c/span\u003e\u003cspan address=\"10.1172/jci.insight.143643\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXia L, Wang J, Xue H, Li H, Li Q, Qin S, Yu C, Liu Y, Gao Y, Li L et al (2025) A CMTM6 Nanobody Overcomes EGFR-TKI Resistance in Non-Small Cell Lung Cancer. Adv Sci (Weinh) 12:e2410945. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/advs.202410945\u003c/span\u003e\u003cspan address=\"10.1002/advs.202410945\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"CMTM6, Breast cancer, Prognosis, Overall survival","lastPublishedDoi":"10.21203/rs.3.rs-7752340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7752340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e\u003cp\u003eCMTM6, a regulator of tumor cell PD-L1 stability via inhibiting lysosome-mediated degradation, is linked to anti-tumor immunity and tumorigenesis. However, its expression and prognostic value in breast cancer subtypes remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe conducted an integrated bioinformatics analysis of CMTM6 using multiple public databases: cBioPortal for examining genetic alterations in TCGA breast cancer datasets; GEPIA for comparing expression between tumor and normal tissues; bc-GenExMiner 4.0 for assessing expression across subtypes and performing meta-analysis; Kaplan-Meier Plotter and the Human Protein Atlas for prognostic assessment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCMTM6 was ubiquitous in human tissues but low in normal breast (glandular/myoepithelial cells only). It was higher in breast cancer than normal tissues, lower in HER2-positive subtypes, higher in P53 wild-type tumors, and uncorrelated with lymph node status/age. Breast cancer \u003cem\u003eCMTM6\u003c/em\u003e gene alterations were rare (amplification: 0.56%; deep deletion: 0.14%). High CMTM6 (mRNA/protein) correlated with better overall survival, specifically in lymph node-positive and Luminal A subtypes, but not in other subtypes.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e\u003cp\u003eCMTM6 is differentially expressed in breast cancer/subtypes and acts as a favorable prognostic indicator for lymph node-positive/Luminal A breast cancer, serving as a potential subtype-specific biomarker.\u003c/p\u003e","manuscriptTitle":"Prognostic Value of CMTM6 Expression in Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 11:48:10","doi":"10.21203/rs.3.rs-7752340/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":"343a7dc5-4a41-4c5d-8c59-8b06700f0806","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T18:53:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-07 11:48:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7752340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7752340","identity":"rs-7752340","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}