Breast cancer is the most frequently diagnosed cancer, with metastasis accounting for the majority of cancer-related deaths. The mechanisms of early-stage breast cancer metastasis to regional immune sites like lymph nodes remain elusive. Here, we performed an in-depth proteomic and phosphoproteomic analysis of a substantial series of breast cancer samples, alongside genomic and transcriptomic evaluations. This cohort encompasses 195 specimens: 65 primary breast tumors, their corresponding normal tissues, and metastatic axillary lymph nodes. We offer an overview of the molecular alterations at the transcriptomic, proteomic, and phosphoproteomic levels during lymph node metastasis. Notably, the findings indicate that regional lymph node metastasis is primarily influenced by proteomic and phosphoproteomic alterations, rather than genomic or transcriptomic changes. We found the ANGPTL4 and HMGB1 could serve as the biomarker of lymph node metastasis. Data analysis and cell experiments involving silencing of the alternative splicing factor HNRNPU demonstrated that alternative splicing plays a significant role in modulating protein expression, phosphorylation profiles and cell proliferation. The key phosphorylation sites, including MARCKSL1-S104 and FKBP15-S320, as well as the upstream kinase PRKCB, were identified as playing crucial roles in breast cancer lymph node metastasis. Targeted intervention of the kinase PRKCB resulted in effectively suppressing the proliferation and metastasis of breast cancer tumor cells. Immune profiling analysis and experimental validation of breast cancer cell cocultured with CD8+ T cell reveals correlations between phosphorylation of MARCKSL1-S104 and FKBP15-S320 with immune checkpoint PD-L1 expression, and their impact on tumor cell apoptosis, suggesting a potential mechanism of immune evasion in metastasis. This study systematically characterizes the molecular landscape and features of primary breast tumors and their matched metastatic lymph nodes. These insights enhance our understanding of early-stage breast cancer metastasis and may pave the way for improved diagnostic tools and targeted therapeutic strategies.
{"title":"Multi-omics Analysis Reveals Comprehensive Aberrant Protein and Phosphorylation Characteristics in Breast Cancer and Paired Metastatic Lymph Nodes.","authors":"Linhui Zhai,Cui-Cui Liu,Lei Zhao,Le-Wei Zheng,Chengyu Chu,Hu Hong,Yu-Wen Cai,Lie Chen,Yi-Ming Liu,Yiou Wang,Wensi Zhao,Yuqi Huang,Shiyu Duan,Zhi-Ming Shao,Yiting Jin,Minjia Tan,Ke-Da Yu","doi":"10.1093/procel/pwag002","DOIUrl":"https://doi.org/10.1093/procel/pwag002","url":null,"abstract":"Breast cancer is the most frequently diagnosed cancer, with metastasis accounting for the majority of cancer-related deaths. The mechanisms of early-stage breast cancer metastasis to regional immune sites like lymph nodes remain elusive. Here, we performed an in-depth proteomic and phosphoproteomic analysis of a substantial series of breast cancer samples, alongside genomic and transcriptomic evaluations. This cohort encompasses 195 specimens: 65 primary breast tumors, their corresponding normal tissues, and metastatic axillary lymph nodes. We offer an overview of the molecular alterations at the transcriptomic, proteomic, and phosphoproteomic levels during lymph node metastasis. Notably, the findings indicate that regional lymph node metastasis is primarily influenced by proteomic and phosphoproteomic alterations, rather than genomic or transcriptomic changes. We found the ANGPTL4 and HMGB1 could serve as the biomarker of lymph node metastasis. Data analysis and cell experiments involving silencing of the alternative splicing factor HNRNPU demonstrated that alternative splicing plays a significant role in modulating protein expression, phosphorylation profiles and cell proliferation. The key phosphorylation sites, including MARCKSL1-S104 and FKBP15-S320, as well as the upstream kinase PRKCB, were identified as playing crucial roles in breast cancer lymph node metastasis. Targeted intervention of the kinase PRKCB resulted in effectively suppressing the proliferation and metastasis of breast cancer tumor cells. Immune profiling analysis and experimental validation of breast cancer cell cocultured with CD8+ T cell reveals correlations between phosphorylation of MARCKSL1-S104 and FKBP15-S320 with immune checkpoint PD-L1 expression, and their impact on tumor cell apoptosis, suggesting a potential mechanism of immune evasion in metastasis. This study systematically characterizes the molecular landscape and features of primary breast tumors and their matched metastatic lymph nodes. These insights enhance our understanding of early-stage breast cancer metastasis and may pave the way for improved diagnostic tools and targeted therapeutic strategies.","PeriodicalId":20790,"journal":{"name":"Protein & Cell","volume":"29 1","pages":""},"PeriodicalIF":21.1,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145956196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Poly (ADP-ribose) polymerase 1 (PARP1) inhibition represents promising targeted therapy for BRCA deficient cancer patients based on synthetic lethality theory. Recent evidence shows that efficacy of DNA damage drugs depends on two aspects, DNA repair signaling and immune response. Applying a functional proteomics approach, we find that the function of spliceosome is perturbed by PARP inhibitors via enhancing interaction between PARP1 and SF3B1, a key factor of spliceosome. We demonstrate that differential alternative spliced mRNA and accumulation of double strand RNA (dsRNA) are induced by perturbation of spliceosome upon PARP inhibitors treatment, resulting in triggering dsRNA antiviral mimicry innate immune response. Moreover, we identify a novel function of BRCA1, through which BRCA1 regulates innate immune response leading to compromising of the innate immune signaling by downregulation of IRF3 in BRCA1 deficient breast cancer cells, which reduces the sensitivity to PARP inhibitors and causes intrinsic resistance. Polyinosinic-polycytidylic acid (poly(I: C)) is a dsRNA synthetic analog sensitizing PARP inhibitors through further triggering dsRNA signaling. Finally, we show that the combination of PARP inhibitors and poly(I: C) enhances antitumor efficiency in vivo. Overall, our study reveals BRCA1 deficiency impedes tumor cell intrinsic innate immune response, inducing intrinsic resistance to PARP inhibitors that can be overcome when poly(I: C) is combined.
{"title":"Tumor cell intrinsic dsRNA innate immune response triggered by PARP inhibitor is compromised in BRCA1 deficient breast cancer by repressing IRF3.","authors":"Cuiting Zhang,Jing-Bo Zhou,Josh Haipeng Lei,Irene Ling Ang,Kai Miao,Xiaoling Xu,Terence Chuen Wai Poon,Edwin Cheung,Chu-Xia Deng","doi":"10.1093/procel/pwaf104","DOIUrl":"https://doi.org/10.1093/procel/pwaf104","url":null,"abstract":"Poly (ADP-ribose) polymerase 1 (PARP1) inhibition represents promising targeted therapy for BRCA deficient cancer patients based on synthetic lethality theory. Recent evidence shows that efficacy of DNA damage drugs depends on two aspects, DNA repair signaling and immune response. Applying a functional proteomics approach, we find that the function of spliceosome is perturbed by PARP inhibitors via enhancing interaction between PARP1 and SF3B1, a key factor of spliceosome. We demonstrate that differential alternative spliced mRNA and accumulation of double strand RNA (dsRNA) are induced by perturbation of spliceosome upon PARP inhibitors treatment, resulting in triggering dsRNA antiviral mimicry innate immune response. Moreover, we identify a novel function of BRCA1, through which BRCA1 regulates innate immune response leading to compromising of the innate immune signaling by downregulation of IRF3 in BRCA1 deficient breast cancer cells, which reduces the sensitivity to PARP inhibitors and causes intrinsic resistance. Polyinosinic-polycytidylic acid (poly(I: C)) is a dsRNA synthetic analog sensitizing PARP inhibitors through further triggering dsRNA signaling. Finally, we show that the combination of PARP inhibitors and poly(I: C) enhances antitumor efficiency in vivo. Overall, our study reveals BRCA1 deficiency impedes tumor cell intrinsic innate immune response, inducing intrinsic resistance to PARP inhibitors that can be overcome when poly(I: C) is combined.","PeriodicalId":20790,"journal":{"name":"Protein & Cell","volume":"386 1","pages":""},"PeriodicalIF":21.1,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145947299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The TCR/CD3s complex translates antigenic cues into graded immune responses. Xu and colleagues (Molecular Cell, 2025) revealed a lipid-electrostatic mechanism that governs sequential phosphorylation of the CD3ζ chain. Using NMR in a membrane-mimetic system, they uncovered a gradient of membrane insertion across its ITAMs, establishing a structural basis for tunable signaling. Under chronic stimulation, ATP depletion preferentially impairs distal ITAM phosphorylation, revealing an intrinsic energy-sensitive pathway driving T cell exhaustion. These findings provide a compelling model for TCR regulation and suggest strategies to enhance immunotherapy via CD3ζ engineering or metabolic restoration.
{"title":"Lipid-Gated \"Phosphorylation Code\" for TCR Graded Signaling and T-cell exhaustion.","authors":"Hui Chen,Jizhong Lou,Wei Chen","doi":"10.1093/procel/pwaf110","DOIUrl":"https://doi.org/10.1093/procel/pwaf110","url":null,"abstract":"The TCR/CD3s complex translates antigenic cues into graded immune responses. Xu and colleagues (Molecular Cell, 2025) revealed a lipid-electrostatic mechanism that governs sequential phosphorylation of the CD3ζ chain. Using NMR in a membrane-mimetic system, they uncovered a gradient of membrane insertion across its ITAMs, establishing a structural basis for tunable signaling. Under chronic stimulation, ATP depletion preferentially impairs distal ITAM phosphorylation, revealing an intrinsic energy-sensitive pathway driving T cell exhaustion. These findings provide a compelling model for TCR regulation and suggest strategies to enhance immunotherapy via CD3ζ engineering or metabolic restoration.","PeriodicalId":20790,"journal":{"name":"Protein & Cell","volume":"20 1","pages":""},"PeriodicalIF":21.1,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145823990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
18F-fluorodeoxyglucose (18F-FDG) is the most widely used radiotracer for positron emission tomography (PET) imaging in clinical oncology, owing to the elevated glycolytic activity of tumor cells. However, transient post-radiotherapy (RT) "metabolic flares" of 18F-FDG uptake are frequently observed in patients and are traditionally attributed to localized inflammatory responses. Whether these flares are linked to immune cell dynamics, particularly tumor-infiltrating T cells, and the mechanisms involved remain poorly understood. Here, we demonstrate that RT markedly upregulates intracellular adhesion molecule-1 (ICAM-1) expression and promotes T cell infiltration in tumors, as observed in both patients and mouse models. Genetic ablation of ICAM-1 significantly attenuates RT-induced metabolic flares in irradiated tumors, primarily due to reduced 18F-FDG uptake by tumor-infiltrating T cells rather than myeloid cells. Mechanistically, ICAM-1 engages with lymphocyte function-associated antigen 1 (LFA-1) to facilitate T cell clustering, thereby promoting their intratumoral accumulation and activating glycolysis and the tricarboxylic acid (TCA) cycle via the PI3K-AKT-mTOR signaling pathway. These findings identify ICAM-1 as a critical regulator of T cell metabolic reprogramming and tumor infiltration following RT, offering a mechanistic explanation for 18F-FDG PET flares. Clinical monitoring of post-RT tumor ICAM-1 expression may enhance PET interpretation and aid in distinguishing pseudoprogression from true tumor progression.
{"title":"ICAM-1 promotes T cell glycolytic reprogramming and tumor infiltration to drive 18F-FDG PET flares following radiotherapy.","authors":"Rui Song,Meixin Zhao,Ting Zhang,Yining Zhang,Fuxin Guo,Huiying He,Haoyi Zhou,Kui Li,Jianze Wang,Jinhong Du,Feng Wang,Shixin Zhou,Hua Zhu,Jiadong Wang,Weifang Zhang,Zhi Yang,Zhaofei Liu","doi":"10.1093/procel/pwaf111","DOIUrl":"https://doi.org/10.1093/procel/pwaf111","url":null,"abstract":"18F-fluorodeoxyglucose (18F-FDG) is the most widely used radiotracer for positron emission tomography (PET) imaging in clinical oncology, owing to the elevated glycolytic activity of tumor cells. However, transient post-radiotherapy (RT) \"metabolic flares\" of 18F-FDG uptake are frequently observed in patients and are traditionally attributed to localized inflammatory responses. Whether these flares are linked to immune cell dynamics, particularly tumor-infiltrating T cells, and the mechanisms involved remain poorly understood. Here, we demonstrate that RT markedly upregulates intracellular adhesion molecule-1 (ICAM-1) expression and promotes T cell infiltration in tumors, as observed in both patients and mouse models. Genetic ablation of ICAM-1 significantly attenuates RT-induced metabolic flares in irradiated tumors, primarily due to reduced 18F-FDG uptake by tumor-infiltrating T cells rather than myeloid cells. Mechanistically, ICAM-1 engages with lymphocyte function-associated antigen 1 (LFA-1) to facilitate T cell clustering, thereby promoting their intratumoral accumulation and activating glycolysis and the tricarboxylic acid (TCA) cycle via the PI3K-AKT-mTOR signaling pathway. These findings identify ICAM-1 as a critical regulator of T cell metabolic reprogramming and tumor infiltration following RT, offering a mechanistic explanation for 18F-FDG PET flares. Clinical monitoring of post-RT tumor ICAM-1 expression may enhance PET interpretation and aid in distinguishing pseudoprogression from true tumor progression.","PeriodicalId":20790,"journal":{"name":"Protein & Cell","volume":"116 1","pages":""},"PeriodicalIF":21.1,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145786218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
PROTACs (proteolysis targeting chimeras) offer a revolutionary strategy to degrade proteins previously considered "undruggable." While the importance of the target protein ligand and linker is well-established, the strategic selection of an E3 ubiquitin ligase and its corresponding ligand is an equally critical but underexplored determinant of PROTAC efficacy and selectivity. This perspective systematically analyzes how E3 ligase-ligand pairing dictates degradation outcomes across diverse biological contexts. Our analysis, incorporating head-to-head comparisons, demonstrates that no single E3 ligand is universally superior. Instead, degradation efficiency is profoundly modulated by ternary complex cooperativity, cell-type specificity, and tissue distribution. CRBN-based degraders frequently excel in hematologic malignancies, while VHL-based PROTACs show advantages in certain solid tumors. We further highlight emerging E3 ligands (e.g., from IAP, DCAF families) as promising tools to overcome resistance and expand the degradable proteome. The perspective also explores innovative frontiers, including the potential for targeting non-protein substrates and the application of PROTACs as versatile chemical knockdown tools in research. Ultimately, this paper underscores the central paradigm that "context dictates strategy" in E3 ligase selection, providing a critical framework for optimizing PROTAC design and broadening their therapeutic and research applications.
{"title":"Context Matters: E3 Ligase-Ligand Pairing Strategies for Optimized PROTAC Performance.","authors":"Luyao Yin,Pengcheng Shu,Xiaozhong Peng","doi":"10.1093/procel/pwaf107","DOIUrl":"https://doi.org/10.1093/procel/pwaf107","url":null,"abstract":"PROTACs (proteolysis targeting chimeras) offer a revolutionary strategy to degrade proteins previously considered \"undruggable.\" While the importance of the target protein ligand and linker is well-established, the strategic selection of an E3 ubiquitin ligase and its corresponding ligand is an equally critical but underexplored determinant of PROTAC efficacy and selectivity. This perspective systematically analyzes how E3 ligase-ligand pairing dictates degradation outcomes across diverse biological contexts. Our analysis, incorporating head-to-head comparisons, demonstrates that no single E3 ligand is universally superior. Instead, degradation efficiency is profoundly modulated by ternary complex cooperativity, cell-type specificity, and tissue distribution. CRBN-based degraders frequently excel in hematologic malignancies, while VHL-based PROTACs show advantages in certain solid tumors. We further highlight emerging E3 ligands (e.g., from IAP, DCAF families) as promising tools to overcome resistance and expand the degradable proteome. The perspective also explores innovative frontiers, including the potential for targeting non-protein substrates and the application of PROTACs as versatile chemical knockdown tools in research. Ultimately, this paper underscores the central paradigm that \"context dictates strategy\" in E3 ligase selection, providing a critical framework for optimizing PROTAC design and broadening their therapeutic and research applications.","PeriodicalId":20790,"journal":{"name":"Protein & Cell","volume":"31 1","pages":""},"PeriodicalIF":21.1,"publicationDate":"2025-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145710782","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mitochondrial calcium fluxes serve as pivotal regulators of optimal organellar function and cellular viability, yet the spatiotemporal regulation of nanodomain Ca2+ transients at mitochondria-ER contact sites (MERCS) and their integration into adaptive mitochondrial stress signaling remain unresolved. In this study, we employed custom-built high temporal-spatial resolution GI/3D-SIM imaging techniques to achieve nanoscale resolution of calcium transients. We identify that MERCS-localized calcium oscillations gate retrograde stress signaling. Mechanistically, we demonstrate that augmented mitochondria-associated ER membrane (MAMs) connectivity unexpectedly attenuated global mitochondrial Ca2+ efflux, which triggering ATF5 shuttling-mediated transcriptional licensing and calcium-sensitive epigenetic reprogramming that synergistically activating stress-resilience programs. Quantitative protein expression and transcriptome analyses confirm that CsA-mediated calcium retention mimics MAMs induction preserves mitochondrial integrity and protecting cells from apoptosis in Aβ1-42-challenged neurons through synchronized UPRmt activation. Our findings reveal a novel mechanism by which MERCS decode proteotoxic stress into transcriptional and epigenetic adaptations, offering therapeutic potential for neurodegenerative diseases.
{"title":"Spatiotemporal Ca2+ nanodomain remodeling at MERCS regulates mitochondrial proteostasis.","authors":"Yanan Lv,Xuejing Zhao,Di Li,Zhaoqi Hao,Yue Zhao,Yuhang Zhou,Yujing Zhang,Han Chen,Zhongbing Lu,Dong Li,Yuting Guo","doi":"10.1093/procel/pwaf109","DOIUrl":"https://doi.org/10.1093/procel/pwaf109","url":null,"abstract":"Mitochondrial calcium fluxes serve as pivotal regulators of optimal organellar function and cellular viability, yet the spatiotemporal regulation of nanodomain Ca2+ transients at mitochondria-ER contact sites (MERCS) and their integration into adaptive mitochondrial stress signaling remain unresolved. In this study, we employed custom-built high temporal-spatial resolution GI/3D-SIM imaging techniques to achieve nanoscale resolution of calcium transients. We identify that MERCS-localized calcium oscillations gate retrograde stress signaling. Mechanistically, we demonstrate that augmented mitochondria-associated ER membrane (MAMs) connectivity unexpectedly attenuated global mitochondrial Ca2+ efflux, which triggering ATF5 shuttling-mediated transcriptional licensing and calcium-sensitive epigenetic reprogramming that synergistically activating stress-resilience programs. Quantitative protein expression and transcriptome analyses confirm that CsA-mediated calcium retention mimics MAMs induction preserves mitochondrial integrity and protecting cells from apoptosis in Aβ1-42-challenged neurons through synchronized UPRmt activation. Our findings reveal a novel mechanism by which MERCS decode proteotoxic stress into transcriptional and epigenetic adaptations, offering therapeutic potential for neurodegenerative diseases.","PeriodicalId":20790,"journal":{"name":"Protein & Cell","volume":"5 1","pages":""},"PeriodicalIF":21.1,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145704403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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