Background: The mutations of oncogenic epidermal growth factor receptor (EGFR) is an important cause of lung adenocarcinoma (LUAD) malignance. It has been knowm that metabolic reprogramming is an important hallmark of malignant tumors, and purine metabolism is a key metabolic pathway for tumor progression and drug resistance, but its relationship with the EGFR-mutant LUAD is unclear.
Methods: Metabolic reprogramming was studied through capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolic profiling analysis. Cell proliferation in vitro was evaluated by EdU staining and cell cycle assay. Tumorigenicity in vivo was tested by subcutaneous tumor formation experiment in nude mice. The binding of hypoxia-inducible factor-1 alpha (HIF-1α) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was detected by DNA pull‑down assay and Chromatin immunoprecipitation (ChIP) assays. HIF-1α, HPRT1, DNA damage and cell apoptosis related genes were examined by western blot. In addition, RNA sequencing, mass spectrometry and bioinformatics analysis were performed.
Results: We found that mutated EGFR (muEGFR) upregulates HPRT1 to promote purine metabolism and tumorigenesis of EGFR-mutant LUAD. Mechanistically, muEGFR increases HIF-1α expression through protein stability. Meanwhile, up-regulated HIF-1α bound to the promoter of HPRT1 and transcriptionally activates HPRT1 expression, enhancing purine metabolism to maintain rapid tumor cell proliferation in EGFR-mutant LUAD. Further, gefitinib inhibited the synthesis of purine nucleotides, and HPRT1 inhibition increased the sensitivity of gefitinib to EGFR-mutant LUAD.
Conclusions: Our study reveals that muEGFR-HIF-1α-HPRT1 axis plays a key role in EGFR-mutant LUAD and provides a new strategy-inhibiting purine metabolism for treating EGFR-mutant LUAD.
背景:致癌表皮生长因子受体(EGFR)突变是肺腺癌(LUAD)恶性化的重要原因。人们已经知道,代谢重编程是恶性肿瘤的一个重要标志,而嘌呤代谢是肿瘤进展和耐药性的一个关键代谢途径,但其与表皮生长因子受体突变型肺腺癌(LUAD)的关系尚不清楚:方法:通过基于毛细管电泳-飞行时间质谱(CE-TOF/MS)的代谢谱分析研究了代谢重编程。体外细胞增殖通过EdU染色和细胞周期测定进行评估。体内致瘤性通过裸鼠皮下肿瘤形成实验进行检测。缺氧诱导因子-1α(HIF-1α)和次黄嘌呤磷酸核糖基转移酶1(HPRT1)的结合通过DNA牵引试验和染色质免疫沉淀(ChIP)试验进行检测。通过 Western 印迹检测了 HIF-1α、HPRT1、DNA 损伤和细胞凋亡相关基因。此外,还进行了 RNA 测序、质谱分析和生物信息学分析:结果:我们发现,突变的表皮生长因子受体(muEGFR)上调HPRT1,促进表皮生长因子受体突变的LUAD的嘌呤代谢和肿瘤发生。从机理上讲,muEGFR通过蛋白稳定性增加HIF-1α的表达。同时,上调的HIF-1α与HPRT1的启动子结合,转录激活HPRT1的表达,促进嘌呤代谢,从而维持EGFR突变型LUAD肿瘤细胞的快速增殖。此外,吉非替尼抑制了嘌呤核苷酸的合成,HPRT1抑制增加了吉非替尼对表皮生长因子受体突变型LUAD的敏感性:我们的研究揭示了muEGFR-HIF-1α-HPRT1轴在表皮生长因子受体突变型LUAD中的关键作用,并为抑制嘌呤代谢治疗表皮生长因子受体突变型LUAD提供了新策略。
{"title":"HIF-1α-HPRT1 axis promotes tumorigenesis and gefitinib resistance by enhancing purine metabolism in EGFR-mutant lung adenocarcinoma.","authors":"Pengyu Geng, Fei Ye, Peng Dou, Chunxiu Hu, Jiarui He, Jinhui Zhao, Qi Li, Miao Bao, Xiangnan Li, Xinyu Liu, Guowang Xu","doi":"10.1186/s13046-024-03184-8","DOIUrl":"10.1186/s13046-024-03184-8","url":null,"abstract":"<p><strong>Background: </strong>The mutations of oncogenic epidermal growth factor receptor (EGFR) is an important cause of lung adenocarcinoma (LUAD) malignance. It has been knowm that metabolic reprogramming is an important hallmark of malignant tumors, and purine metabolism is a key metabolic pathway for tumor progression and drug resistance, but its relationship with the EGFR-mutant LUAD is unclear.</p><p><strong>Methods: </strong>Metabolic reprogramming was studied through capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolic profiling analysis. Cell proliferation in vitro was evaluated by EdU staining and cell cycle assay. Tumorigenicity in vivo was tested by subcutaneous tumor formation experiment in nude mice. The binding of hypoxia-inducible factor-1 alpha (HIF-1α) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was detected by DNA pull‑down assay and Chromatin immunoprecipitation (ChIP) assays. HIF-1α, HPRT1, DNA damage and cell apoptosis related genes were examined by western blot. In addition, RNA sequencing, mass spectrometry and bioinformatics analysis were performed.</p><p><strong>Results: </strong>We found that mutated EGFR (muEGFR) upregulates HPRT1 to promote purine metabolism and tumorigenesis of EGFR-mutant LUAD. Mechanistically, muEGFR increases HIF-1α expression through protein stability. Meanwhile, up-regulated HIF-1α bound to the promoter of HPRT1 and transcriptionally activates HPRT1 expression, enhancing purine metabolism to maintain rapid tumor cell proliferation in EGFR-mutant LUAD. Further, gefitinib inhibited the synthesis of purine nucleotides, and HPRT1 inhibition increased the sensitivity of gefitinib to EGFR-mutant LUAD.</p><p><strong>Conclusions: </strong>Our study reveals that muEGFR-HIF-1α-HPRT1 axis plays a key role in EGFR-mutant LUAD and provides a new strategy-inhibiting purine metabolism for treating EGFR-mutant LUAD.</p>","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"43 1","pages":"269"},"PeriodicalIF":11.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11441087/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-30DOI: 10.1186/s13046-024-03193-7
Zaozao Wang, Haibo Han, Chenghai Zhang, Chenxin Wu, Jiabo Di, Pu Xing, Xiaowen Qiao, Kai Weng, Hao Hao, Xinying Yang, Yifan Hou, Beihai Jiang, Xiangqian Su
Background: Somatic copy number alterations (SCNAs) are pivotal in cancer progression and patient prognosis. Dysregulated long non-coding RNAs (lncRNAs), modulated by SCNAs, significantly impact tumorigenesis, including colorectal cancer (CRC). Nonetheless, the functional significance of lncRNAs induced by SCNAs in CRC remains largely unexplored.
Methods: The dysregulated lncRNA LOC101927668, induced by copy number amplification, was identified through comprehensive bioinformatic analyses utilizing multidimensional data. Subsequent in situ hybridization was employed to ascertain the subcellular localization of LOC101927668, and gain- and loss-of-function experiments were conducted to elucidate its role in CRC progression. The downstream targets and signaling pathway influenced by LOC101927668 were identified and validated through a comprehensive approach, encompassing RNA sequencing, RT-qPCR, Western blot analysis, dual-luciferase reporter assay, evaluation of mRNA and protein degradation, and rescue experiments. Analysis of AU-rich elements (AREs) within the mRNA 3' untranslated region (UTR) of the downstream target, along with exploration of putative ARE-binding proteins, was conducted. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays were employed to elucidate potential interacting proteins of LOC101927668 and further delineate the regulatory mechanism between LOC101927668 and its downstream target. Moreover, subcutaneous xenograft and orthotopic liver xenograft tumor models were utilized to evaluate the in vivo impact of LOC101927668 on CRC cells and investigate its correlation with downstream targets.
Results: Significantly overexpressed LOC101927668, driven by chr7p22.3-p14.3 amplification, was markedly correlated with unfavorable clinical outcomes in our CRC patient cohort, as well as in TCGA and GEO datasets. Moreover, we demonstrated that enforced expression of LOC101927668 significantly enhanced cell proliferation, migration, and invasion, while its depletion impeded these processes in a p53-dependent manner. Mechanistically, nucleus-localized LOC101927668 recruited hnRNPD and translocated to the cytoplasm, accelerating the destabilization of RBM47 mRNA, a transcription factor of p53. As a nucleocytoplasmic shuttling protein, hnRNPD mediated RBM47 destabilization by binding to the ARE motif within RBM47 3'UTR, thereby suppressing the p53 signaling pathway and facilitating CRC progression.
Conclusions: The overexpression of LOC101927668, driven by SCNAs, facilitates CRC proliferation and metastasis by recruiting hnRNPD, thus perturbing the RBM47/p53/p21 signaling pathway. These findings underscore the pivotal roles of LOC101927668 and highlight its therapeutic potential in anti-CRC interventions.
{"title":"Copy number amplification-induced overexpression of lncRNA LOC101927668 facilitates colorectal cancer progression by recruiting hnRNPD to disrupt RBM47/p53/p21 signaling.","authors":"Zaozao Wang, Haibo Han, Chenghai Zhang, Chenxin Wu, Jiabo Di, Pu Xing, Xiaowen Qiao, Kai Weng, Hao Hao, Xinying Yang, Yifan Hou, Beihai Jiang, Xiangqian Su","doi":"10.1186/s13046-024-03193-7","DOIUrl":"10.1186/s13046-024-03193-7","url":null,"abstract":"<p><strong>Background: </strong>Somatic copy number alterations (SCNAs) are pivotal in cancer progression and patient prognosis. Dysregulated long non-coding RNAs (lncRNAs), modulated by SCNAs, significantly impact tumorigenesis, including colorectal cancer (CRC). Nonetheless, the functional significance of lncRNAs induced by SCNAs in CRC remains largely unexplored.</p><p><strong>Methods: </strong>The dysregulated lncRNA LOC101927668, induced by copy number amplification, was identified through comprehensive bioinformatic analyses utilizing multidimensional data. Subsequent in situ hybridization was employed to ascertain the subcellular localization of LOC101927668, and gain- and loss-of-function experiments were conducted to elucidate its role in CRC progression. The downstream targets and signaling pathway influenced by LOC101927668 were identified and validated through a comprehensive approach, encompassing RNA sequencing, RT-qPCR, Western blot analysis, dual-luciferase reporter assay, evaluation of mRNA and protein degradation, and rescue experiments. Analysis of AU-rich elements (AREs) within the mRNA 3' untranslated region (UTR) of the downstream target, along with exploration of putative ARE-binding proteins, was conducted. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays were employed to elucidate potential interacting proteins of LOC101927668 and further delineate the regulatory mechanism between LOC101927668 and its downstream target. Moreover, subcutaneous xenograft and orthotopic liver xenograft tumor models were utilized to evaluate the in vivo impact of LOC101927668 on CRC cells and investigate its correlation with downstream targets.</p><p><strong>Results: </strong>Significantly overexpressed LOC101927668, driven by chr7p22.3-p14.3 amplification, was markedly correlated with unfavorable clinical outcomes in our CRC patient cohort, as well as in TCGA and GEO datasets. Moreover, we demonstrated that enforced expression of LOC101927668 significantly enhanced cell proliferation, migration, and invasion, while its depletion impeded these processes in a p53-dependent manner. Mechanistically, nucleus-localized LOC101927668 recruited hnRNPD and translocated to the cytoplasm, accelerating the destabilization of RBM47 mRNA, a transcription factor of p53. As a nucleocytoplasmic shuttling protein, hnRNPD mediated RBM47 destabilization by binding to the ARE motif within RBM47 3'UTR, thereby suppressing the p53 signaling pathway and facilitating CRC progression.</p><p><strong>Conclusions: </strong>The overexpression of LOC101927668, driven by SCNAs, facilitates CRC proliferation and metastasis by recruiting hnRNPD, thus perturbing the RBM47/p53/p21 signaling pathway. These findings underscore the pivotal roles of LOC101927668 and highlight its therapeutic potential in anti-CRC interventions.</p>","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"43 1","pages":"274"},"PeriodicalIF":11.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11440719/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vigorous CD8+ T cells play a crucial role in recognizing tumor cells and combating solid tumors. How T cells efficiently recognize and target tumor antigens, and how they maintain the activity in the "rejection" of solid tumor microenvironment, are major concerns. Recent advances in understanding of the immunological trajectory and lifespan of CD8+ T cells have provided guidance for the design of more optimal anti-tumor immunotherapy regimens. Here, we review the newly discovered methods to enhance the function of CD8+ T cells against solid tumors, focusing on optimizing T cell receptor (TCR) expression, improving antigen recognition by engineered T cells, enhancing signal transduction of the TCR-CD3 complex, inducing the homing of polyclonal functional T cells to tumors, reversing T cell exhaustion under chronic antigen stimulation, and reprogramming the energy and metabolic pathways of T cells. We also discuss how to participate in the epigenetic changes of CD8+ T cells to regulate two key indicators of anti-tumor responses, namely effectiveness and persistence.
活跃的 CD8+ T 细胞在识别肿瘤细胞和抗击实体瘤方面发挥着至关重要的作用。T 细胞如何有效识别和靶向肿瘤抗原,以及如何在实体瘤微环境的 "排斥 "中保持活性,是人们关注的主要问题。最近,人们对 CD8+ T 细胞的免疫学轨迹和寿命的认识取得了进展,这为设计更优化的抗肿瘤免疫疗法方案提供了指导。在此,我们回顾了新发现的增强 CD8+ T 细胞抗实体瘤功能的方法,重点是优化 T 细胞受体(TCR)表达、改善工程 T 细胞的抗原识别、增强 TCR-CD3 复合物的信号转导、诱导多克隆功能 T 细胞向肿瘤归巢、逆转 T 细胞在慢性抗原刺激下的衰竭以及重编程 T 细胞的能量和代谢途径。我们还讨论了如何参与 CD8+ T 细胞的表观遗传变化,以调节抗肿瘤反应的两个关键指标,即有效性和持久性。
{"title":"Focusing on CD8<sup>+</sup> T-cell phenotypes: improving solid tumor therapy.","authors":"Zhouchi Yao, Yayun Zeng, Cheng Liu, Huimin Jin, Hong Wang, Yue Zhang, Chengming Ding, Guodong Chen, Daichao Wu","doi":"10.1186/s13046-024-03195-5","DOIUrl":"https://doi.org/10.1186/s13046-024-03195-5","url":null,"abstract":"<p><p>Vigorous CD8<sup>+</sup> T cells play a crucial role in recognizing tumor cells and combating solid tumors. How T cells efficiently recognize and target tumor antigens, and how they maintain the activity in the \"rejection\" of solid tumor microenvironment, are major concerns. Recent advances in understanding of the immunological trajectory and lifespan of CD8<sup>+</sup> T cells have provided guidance for the design of more optimal anti-tumor immunotherapy regimens. Here, we review the newly discovered methods to enhance the function of CD8<sup>+</sup> T cells against solid tumors, focusing on optimizing T cell receptor (TCR) expression, improving antigen recognition by engineered T cells, enhancing signal transduction of the TCR-CD3 complex, inducing the homing of polyclonal functional T cells to tumors, reversing T cell exhaustion under chronic antigen stimulation, and reprogramming the energy and metabolic pathways of T cells. We also discuss how to participate in the epigenetic changes of CD8<sup>+</sup> T cells to regulate two key indicators of anti-tumor responses, namely effectiveness and persistence.</p>","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"43 1","pages":"266"},"PeriodicalIF":11.4,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11437975/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-28DOI: 10.1186/s13046-024-03189-3
Silvia Sozzi, Isabella Manni, Cristiana Ercolani, Maria Grazia Diodoro, Armando Bartolazzi, Francesco Spallotta, Giulia Piaggio, Laura Monteonofrio, Silvia Soddu, Cinzia Rinaldo, Davide Valente
Background: Pancreatic ductal adenocarcinoma (PDAC) features KRAS mutations in approximately 90% of human cases and excessive stromal response, termed desmoplastic reaction. Oncogenic KRAS drives pancreatic carcinogenesis by acting on both epithelial cells and tumor microenvironment (TME). We have previously shown that Homeodomain-Interacting Protein Kinase 2 (HIPK2) cooperates with KRAS in sustaining ERK1/2 phosphorylation in human colorectal cancers. Here, we investigated whether HIPK2 contributes to oncogenic KRAS-driven tumorigenesis in vivo, in the onset of pancreatic cancer.
Methods: We employed an extensively characterized model of KRASG12D-dependent preinvasive PDAC, the Pdx1-Cre;LSL-KRasG12D/+ (KC) mice. In these mice, HIPK2 was inhibited by genetic knockout in the pancreatic epithelial cells (KCH-/-) or by pharmacologic inactivation with the small molecule 5-IodoTubercidin (5-ITu). The development of preneoplastic acinar-to-ductal metaplasia (ADM), intraepithelial neoplasia (PanIN), and their associated desmoplastic reaction were analyzed.
Results: In Hipk2-KO mice (KCH-/-), ERK phosphorylation was lowered, the appearance of ADM was slowed down, and both the number and pathologic grade of PanIN were reduced compared to Hipk2-WT KC mice. The pancreatic lesion phenotype in KCH-/- mice was characterized by abundant collagen fibers and reduced number of αSMA+ and pSTAT3+ desmoplastic cells. These features were reminiscent of the recently described human "deserted" sub-TME, poor in cells, rich in matrix, and associated with tumor differentiation. In contrast, the desmoplastic reaction of KC mice resembled the "reactive" sub-TME, rich in stromal cells and associated with tumor progression. These observations were confirmed by the pharmacologic inhibition of HIPK2 in KC mice.
Conclusion: This study demonstrates that HIPK2 inhibition weakens oncogenic KRAS activity and pancreatic tumorigenesis providing a rationale for testing HIPK2 inhibitors to mitigate the incidence of PDAC development in high-risk individuals.
{"title":"Inactivation of HIPK2 attenuates KRAS<sup>G12D</sup> activity and prevents pancreatic tumorigenesis.","authors":"Silvia Sozzi, Isabella Manni, Cristiana Ercolani, Maria Grazia Diodoro, Armando Bartolazzi, Francesco Spallotta, Giulia Piaggio, Laura Monteonofrio, Silvia Soddu, Cinzia Rinaldo, Davide Valente","doi":"10.1186/s13046-024-03189-3","DOIUrl":"10.1186/s13046-024-03189-3","url":null,"abstract":"<p><strong>Background: </strong>Pancreatic ductal adenocarcinoma (PDAC) features KRAS mutations in approximately 90% of human cases and excessive stromal response, termed desmoplastic reaction. Oncogenic KRAS drives pancreatic carcinogenesis by acting on both epithelial cells and tumor microenvironment (TME). We have previously shown that Homeodomain-Interacting Protein Kinase 2 (HIPK2) cooperates with KRAS in sustaining ERK1/2 phosphorylation in human colorectal cancers. Here, we investigated whether HIPK2 contributes to oncogenic KRAS-driven tumorigenesis in vivo, in the onset of pancreatic cancer.</p><p><strong>Methods: </strong>We employed an extensively characterized model of KRAS<sup>G12D</sup>-dependent preinvasive PDAC, the Pdx1-Cre;LSL-KRas<sup>G12D/+</sup> (KC) mice. In these mice, HIPK2 was inhibited by genetic knockout in the pancreatic epithelial cells (KCH<sup>-/-</sup>) or by pharmacologic inactivation with the small molecule 5-IodoTubercidin (5-ITu). The development of preneoplastic acinar-to-ductal metaplasia (ADM), intraepithelial neoplasia (PanIN), and their associated desmoplastic reaction were analyzed.</p><p><strong>Results: </strong>In Hipk2-KO mice (KCH<sup>-/-</sup>), ERK phosphorylation was lowered, the appearance of ADM was slowed down, and both the number and pathologic grade of PanIN were reduced compared to Hipk2-WT KC mice. The pancreatic lesion phenotype in KCH<sup>-/-</sup> mice was characterized by abundant collagen fibers and reduced number of αSMA<sup>+</sup> and pSTAT3<sup>+</sup> desmoplastic cells. These features were reminiscent of the recently described human \"deserted\" sub-TME, poor in cells, rich in matrix, and associated with tumor differentiation. In contrast, the desmoplastic reaction of KC mice resembled the \"reactive\" sub-TME, rich in stromal cells and associated with tumor progression. These observations were confirmed by the pharmacologic inhibition of HIPK2 in KC mice.</p><p><strong>Conclusion: </strong>This study demonstrates that HIPK2 inhibition weakens oncogenic KRAS activity and pancreatic tumorigenesis providing a rationale for testing HIPK2 inhibitors to mitigate the incidence of PDAC development in high-risk individuals.</p>","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"43 1","pages":"265"},"PeriodicalIF":11.4,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11437985/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-20DOI: 10.1186/s13046-024-03158-w
Carlos Casas-Arozamena, Ana Vilar, Juan Cueva, Efigenia Arias, Victoria Sampayo, Eva Diaz, Sara S Oltra, Cristian Pablo Moiola, Silvia Cabrera, Alexandra Cortegoso, Teresa Curiel, Alicia Abalo, Mónica Pamies Serrano, Santiago Domingo, Pablo Padilla-Iserte, Marta Arnaez de la Cruz, Alicia Hernández, Virginia García-Pineda, Juan Ruiz-Bañobre, Rafael López, Xavier Matias-Guiu, Eva Colás, Antonio Gil-Moreno, Miguel Abal, Gema Moreno-Bueno, Laura Muinelo-Romay
Background: There has been a rise in endometrial cancer (EC) incidence leading to increased mortality. To counter this trend, improving the stratification of post-surgery recurrence risk and anticipating disease relapse and treatment resistance is essential. Liquid biopsy analyses offer a promising tool for these clinical challenges, though the best strategy for applying them in EC must be defined. This study was designed to determine the value of cfDNA/ctDNA monitoring in improving the clinical management of patients with localized and recurrent disease.
Methods: Plasma samples and uterine aspirates (UA) from 198 EC patients were collected at surgery and over time. The genetic landscape of UAs was characterized using targeted sequencing. Total cfDNA was analyzed for ctDNA presence based on the UA mutational profile.
Results: High cfDNA levels and detectable ctDNA at baseline correlated with poor prognosis for DFS (p-value < 0.0001; HR = 9.25) and DSS (p-value < 0.0001; HR = 11.20). This remained clinically significant when stratifying tumors by histopathological risk factors. Of note, cfDNA/ctDNA analyses discriminated patients with early post-surgery relapse and the ctDNA kinetics served to identify patients undergoing relapse before any clinical evidence emerged.
Conclusions: This is the most comprehensive study on cfDNA/ctDNA characterization in EC, demonstrating its value in improving risk stratification and anticipating disease relapse in patients with localized disease. CtDNA kinetics assessment complements current strategies to monitor the disease evolution and the treatment response. Therefore, implementing cfDNA/ctDNA monitoring in clinical routines offers a unique opportunity to improve EC management.
Translational relevance: The study demonstrates that high levels of cfDNA and detectable ctDNA at baseline are strong indicators of poor prognosis. This enables more accurate risk stratification beyond traditional histopathological factors, allowing clinicians to identify high-risk patients who may benefit from more aggressive treatment and closer monitoring. Moreover, longitudinal analysis of cfDNA/ctDNA can detect disease recurrence months before clinical symptoms or imaging evidence appear. This early warning system offers a significant advantage in clinical practice, providing a window of opportunity for early intervention and potentially improving patient outcomes.
{"title":"Role of cfDNA and ctDNA to improve the risk stratification and the disease follow-up in patients with endometrial cancer: towards the clinical application.","authors":"Carlos Casas-Arozamena, Ana Vilar, Juan Cueva, Efigenia Arias, Victoria Sampayo, Eva Diaz, Sara S Oltra, Cristian Pablo Moiola, Silvia Cabrera, Alexandra Cortegoso, Teresa Curiel, Alicia Abalo, Mónica Pamies Serrano, Santiago Domingo, Pablo Padilla-Iserte, Marta Arnaez de la Cruz, Alicia Hernández, Virginia García-Pineda, Juan Ruiz-Bañobre, Rafael López, Xavier Matias-Guiu, Eva Colás, Antonio Gil-Moreno, Miguel Abal, Gema Moreno-Bueno, Laura Muinelo-Romay","doi":"10.1186/s13046-024-03158-w","DOIUrl":"https://doi.org/10.1186/s13046-024-03158-w","url":null,"abstract":"<p><strong>Background: </strong>There has been a rise in endometrial cancer (EC) incidence leading to increased mortality. To counter this trend, improving the stratification of post-surgery recurrence risk and anticipating disease relapse and treatment resistance is essential. Liquid biopsy analyses offer a promising tool for these clinical challenges, though the best strategy for applying them in EC must be defined. This study was designed to determine the value of cfDNA/ctDNA monitoring in improving the clinical management of patients with localized and recurrent disease.</p><p><strong>Methods: </strong>Plasma samples and uterine aspirates (UA) from 198 EC patients were collected at surgery and over time. The genetic landscape of UAs was characterized using targeted sequencing. Total cfDNA was analyzed for ctDNA presence based on the UA mutational profile.</p><p><strong>Results: </strong>High cfDNA levels and detectable ctDNA at baseline correlated with poor prognosis for DFS (p-value < 0.0001; HR = 9.25) and DSS (p-value < 0.0001; HR = 11.20). This remained clinically significant when stratifying tumors by histopathological risk factors. Of note, cfDNA/ctDNA analyses discriminated patients with early post-surgery relapse and the ctDNA kinetics served to identify patients undergoing relapse before any clinical evidence emerged.</p><p><strong>Conclusions: </strong>This is the most comprehensive study on cfDNA/ctDNA characterization in EC, demonstrating its value in improving risk stratification and anticipating disease relapse in patients with localized disease. CtDNA kinetics assessment complements current strategies to monitor the disease evolution and the treatment response. Therefore, implementing cfDNA/ctDNA monitoring in clinical routines offers a unique opportunity to improve EC management.</p><p><strong>Translational relevance: </strong>The study demonstrates that high levels of cfDNA and detectable ctDNA at baseline are strong indicators of poor prognosis. This enables more accurate risk stratification beyond traditional histopathological factors, allowing clinicians to identify high-risk patients who may benefit from more aggressive treatment and closer monitoring. Moreover, longitudinal analysis of cfDNA/ctDNA can detect disease recurrence months before clinical symptoms or imaging evidence appear. This early warning system offers a significant advantage in clinical practice, providing a window of opportunity for early intervention and potentially improving patient outcomes.</p>","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"43 1","pages":"264"},"PeriodicalIF":11.4,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11414036/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142299857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1186/s13046-024-03179-5
Adrien Krug, Aymen Saidane, Chiara Martinello, Floriane Fusil, Alexander Michels, Christian J. Buchholz, Jean-Ehrland Ricci, Els Verhoeyen
For angioimmunoblastic T cell lymphoma (AITL), a rare cancer, no specific treatments are available and survival outcome is poor. We previously developed a murine model for AITL that mimics closely human disease and allows to evaluate new treatments. As in human AITL, the murine CD4+ follicular helper T (Tfh) cells are drivers of the malignancy. Therefore, chimeric antigen receptor (CAR) T cell therapy might represent a new therapeutic option. To prevent fratricide among CAR T cells when delivering an CD4-specific CAR, we used a lentiviral vector (LV) encoding an anti-CD4 CAR, allowing exclusive entry into CD8 T cells. These anti-CD4CAR CD8-targeted LVs achieved in murine AITL biopsies high CAR-expression levels in CD8 T cells. Malignant CD4 Tfh cells were eliminated from the mAITL lymphoma, while the CAR + CD8 T cells expanded upon encounter with the CD4 receptor and were shaped into functional cytotoxic cells. Finally, in vivo injection of the CAR + CD8-LVs into our preclinical AITL mouse model carrying lymphomas, significantly prolonged mice survival. Moreover, the in vivo generated functional CAR + CD8 T cells efficiently reduced neoplastic T cell numbers in the mAITL tumors. This is the first description of in vivo generated CAR T cells for therapy of a T cell lymphoma. The strategy described offers a new therapeutic concept for patients suffering from CD4-driven T cell lymphomas.
血管免疫母细胞T细胞淋巴瘤(AITL)是一种罕见的癌症,目前尚无特效治疗方法,生存率很低。我们之前开发了一种血管免疫母细胞淋巴瘤小鼠模型,该模型与人类疾病非常相似,可用于评估新疗法。与人类 AITL 一样,小鼠 CD4+ 滤泡辅助性 T(Tfh)细胞是恶性肿瘤的驱动因素。因此,嵌合抗原受体(CAR)T细胞疗法可能是一种新的治疗选择。为了在递送 CD4 特异性 CAR 时防止 CAR T 细胞自相残杀,我们使用了编码抗 CD4 CAR 的慢病毒载体 (LV),使其能独家进入 CD8 T 细胞。在小鼠 AITL 活检中,这些抗 CD4CAR CD8 靶向 LV 在 CD8 T 细胞中实现了高 CAR 表达水平。恶性 CD4 Tfh 细胞从 mAITL 淋巴瘤中被清除,而 CAR + CD8 T 细胞在与 CD4 受体相遇后得到扩增,并形成功能性细胞毒性细胞。最后,将 CAR + CD8-LVs 体内注射到携带淋巴瘤的临床前 AITL 小鼠模型中,可显著延长小鼠的存活时间。此外,体内生成的功能性 CAR + CD8 T 细胞有效减少了 mAITL 肿瘤中的肿瘤性 T 细胞数量。这是首次描述体内生成的 CAR T 细胞用于治疗 T 细胞淋巴瘤。所述策略为 CD4 驱动的 T 细胞淋巴瘤患者提供了一种新的治疗理念。
{"title":"In vivo CAR T cell therapy against angioimmunoblastic T cell lymphoma","authors":"Adrien Krug, Aymen Saidane, Chiara Martinello, Floriane Fusil, Alexander Michels, Christian J. Buchholz, Jean-Ehrland Ricci, Els Verhoeyen","doi":"10.1186/s13046-024-03179-5","DOIUrl":"https://doi.org/10.1186/s13046-024-03179-5","url":null,"abstract":"For angioimmunoblastic T cell lymphoma (AITL), a rare cancer, no specific treatments are available and survival outcome is poor. We previously developed a murine model for AITL that mimics closely human disease and allows to evaluate new treatments. As in human AITL, the murine CD4+ follicular helper T (Tfh) cells are drivers of the malignancy. Therefore, chimeric antigen receptor (CAR) T cell therapy might represent a new therapeutic option. To prevent fratricide among CAR T cells when delivering an CD4-specific CAR, we used a lentiviral vector (LV) encoding an anti-CD4 CAR, allowing exclusive entry into CD8 T cells. These anti-CD4CAR CD8-targeted LVs achieved in murine AITL biopsies high CAR-expression levels in CD8 T cells. Malignant CD4 Tfh cells were eliminated from the mAITL lymphoma, while the CAR + CD8 T cells expanded upon encounter with the CD4 receptor and were shaped into functional cytotoxic cells. Finally, in vivo injection of the CAR + CD8-LVs into our preclinical AITL mouse model carrying lymphomas, significantly prolonged mice survival. Moreover, the in vivo generated functional CAR + CD8 T cells efficiently reduced neoplastic T cell numbers in the mAITL tumors. This is the first description of in vivo generated CAR T cells for therapy of a T cell lymphoma. The strategy described offers a new therapeutic concept for patients suffering from CD4-driven T cell lymphomas.","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"53 60 1","pages":""},"PeriodicalIF":11.3,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1186/s13046-024-03182-w
Satish kumar Reddy Noonepalle, Maria Gracia-Hernandez, Nima Aghdam, Michael Berrigan, Hawa Coulibaly, Xintang Li, Christian Zevallos-Delgado, Andrew Pletcher, Bryan Weselman, Erica Palmer, Tessa Knox, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra
Macrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies. In this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT. Macrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME. In summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.
{"title":"Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma","authors":"Satish kumar Reddy Noonepalle, Maria Gracia-Hernandez, Nima Aghdam, Michael Berrigan, Hawa Coulibaly, Xintang Li, Christian Zevallos-Delgado, Andrew Pletcher, Bryan Weselman, Erica Palmer, Tessa Knox, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra","doi":"10.1186/s13046-024-03182-w","DOIUrl":"https://doi.org/10.1186/s13046-024-03182-w","url":null,"abstract":"Macrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies. In this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT. Macrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME. In summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"49 1","pages":""},"PeriodicalIF":11.3,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249795","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1186/s13046-024-03176-8
Yang Chen, Hengyang Shen, Zhenling Wang, Changzhi Huang, Hongqiang Zhang, Yu Shao, Ying Tong, Lei Xu, Yunfei Lu, Zan Fu
Long-term accumulation of misfolded proteins leads to endoplasmic reticulum (ER) stress in colorectal cancer (CRC). However, the precise pathways controlling the decision between survival and apoptosis in CRC are unclear. Therefore, in this study, we investigated the function and molecular mechanism of glucosidase I (GCS1) in regulating ER stress in CRC. A public database was used to confirm the expression level of GCS1 in CRC and normal tissues. Clinical samples from our center were used to confirm the mRNA and protein expression levels of GCS1. Cell proliferation, migration, invasion, and apoptosis assays revealed the biological role of GCS1. Immunohistochemical techniques were used to evaluate the expression of key proteins in subcutaneous implanted tumors in nude mice, which provided further evidence for the biological function of GCS1 in promoting cancer in vivo. The results of coimmunoprecipitation-mass spectrometry analysis and immunofluorescence colocalization analysis the interaction between GCS1 and GRP78. In addition, the mechanism of action of USP10, GRP78, and GCS1 at the post- translational level was investigated. Finally, a tissue microarray was used to examine the connection between GCS1 and GRP78 expression and intracellular localization of these proteins using immunohistochemistry and immunofluorescence. The experimental results revealed that GCS1 was substantially expressed in CRC, with higher expression indicating a worse prognosis. Thus, GCS1 can enhance the proliferation and metastasis while inhibiting the apoptosis of CRC cells both in vivo and in vitro. Mechanistically, GCS1 binds to GRP78, recruits USP10 for deubiquitination of GRP78 to promote its degradation, and decreases ER stress-mediated apoptosis, increasing CRC cell proliferation and metastasis. In summary, GCS1 stimulates CRC growth and migration and reduces ER stress-mediated apoptosis via USP10-mediated deubiquitination of GRP78. Our findings identify a possible therapeutic target for CRC.
{"title":"Recruitment of USP10 by GCS1 to deubiquitinate GRP78 promotes the progression of colorectal cancer via alleviating endoplasmic reticulum stress","authors":"Yang Chen, Hengyang Shen, Zhenling Wang, Changzhi Huang, Hongqiang Zhang, Yu Shao, Ying Tong, Lei Xu, Yunfei Lu, Zan Fu","doi":"10.1186/s13046-024-03176-8","DOIUrl":"https://doi.org/10.1186/s13046-024-03176-8","url":null,"abstract":"Long-term accumulation of misfolded proteins leads to endoplasmic reticulum (ER) stress in colorectal cancer (CRC). However, the precise pathways controlling the decision between survival and apoptosis in CRC are unclear. Therefore, in this study, we investigated the function and molecular mechanism of glucosidase I (GCS1) in regulating ER stress in CRC. A public database was used to confirm the expression level of GCS1 in CRC and normal tissues. Clinical samples from our center were used to confirm the mRNA and protein expression levels of GCS1. Cell proliferation, migration, invasion, and apoptosis assays revealed the biological role of GCS1. Immunohistochemical techniques were used to evaluate the expression of key proteins in subcutaneous implanted tumors in nude mice, which provided further evidence for the biological function of GCS1 in promoting cancer in vivo. The results of coimmunoprecipitation-mass spectrometry analysis and immunofluorescence colocalization analysis the interaction between GCS1 and GRP78. In addition, the mechanism of action of USP10, GRP78, and GCS1 at the post- translational level was investigated. Finally, a tissue microarray was used to examine the connection between GCS1 and GRP78 expression and intracellular localization of these proteins using immunohistochemistry and immunofluorescence. The experimental results revealed that GCS1 was substantially expressed in CRC, with higher expression indicating a worse prognosis. Thus, GCS1 can enhance the proliferation and metastasis while inhibiting the apoptosis of CRC cells both in vivo and in vitro. Mechanistically, GCS1 binds to GRP78, recruits USP10 for deubiquitination of GRP78 to promote its degradation, and decreases ER stress-mediated apoptosis, increasing CRC cell proliferation and metastasis. In summary, GCS1 stimulates CRC growth and migration and reduces ER stress-mediated apoptosis via USP10-mediated deubiquitination of GRP78. Our findings identify a possible therapeutic target for CRC.","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"36 1","pages":""},"PeriodicalIF":11.3,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142208600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified. We confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclear‒cytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model. This study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration. These findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA.
胆管癌(CCA)是一种高度恶性的肿瘤,其特点是缺乏有效的靶向治疗策略。蛋白质 UHRF1 在保护 DNA 甲基化方面发挥着关键作用,并与 DNMT1 协同作用。转录后修饰(PTM),如泛素化,在促进这一过程中发挥着不可或缺的作用。然而,CCA中调控UHRF1的特定PTM仍未确定。我们通过质谱分析证实了 STUB1 和 UHRF1 之间的相互作用。此外,我们还通过共IP实验、变性IP泛素化实验、核-胞质分离和免疫荧光实验研究了STUB1-UHRF1/DNMT1轴的内在机制。通过 RNA-seq 鉴定了受 STUB1-UHRF1/DNMT1 轴调控的下游 PLA2G2A 基因。通过亚硫酸氢盐测序 PCR(BSP)实验评估启动子甲基化的变化,探索了 PLA2G2A 的负调控机制。我们使用 CCK-8 试验、集落形成试验、Transwell 试验、伤口愈合试验和异种移植小鼠模型评估了 PLA2G2A 和 STUB1 在 CCA 细胞增殖、侵袭和迁移中的作用。我们利用原代 CCA 小鼠模型评估了 STUB1/UHRF1 对胆管癌的影响。研究发现,STUB1 与 UHRF1 相互作用,导致 UHRF1 的 K63 链接泛素化增加。因此,这促进了 UHRF1 的核转位,并增强了其与 DNMT1 的结合亲和力。STUB1-UHRF1/DNMT1 轴导致 PLA2G2A 启动子的 DNA 甲基化增加,进而抑制其表达。STUB1 在 CCA 中的表达增加与肿瘤进展和总生存期成反比。相反,PLA2G2A 在 CCA 中通过抑制细胞增殖、侵袭和迁移发挥抑瘤作用。这些研究结果表明,STUB1 介导的 UHRF1 泛素化通过表观遗传沉默 PLA2G2A,在肿瘤进展过程中发挥了关键作用,这凸显了 STUB1 作为 CCA 预后生物标志物和治疗靶点的潜力。
{"title":"STUB1-mediated K63-linked ubiquitination of UHRF1 promotes the progression of cholangiocarcinoma by maintaining DNA hypermethylation of PLA2G2A","authors":"Junsheng Chen, Da Wang, Guanhua Wu, Fei Xiong, Wenzheng Liu, Qi Wang, Yiyang Kuai, Wenhua Huang, Yongqiang Qi, Bing Wang, Yongjun Chen","doi":"10.1186/s13046-024-03186-6","DOIUrl":"https://doi.org/10.1186/s13046-024-03186-6","url":null,"abstract":"Cholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified. We confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclear‒cytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model. This study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration. These findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA. ","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"8 1","pages":""},"PeriodicalIF":11.3,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142208603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC. Microarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings. Increased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8+ T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8+ T-cell exhaustion. Blocking Nectin2 improved CD8+ T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2+ TANs and OLR1+ TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs. TANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC.
肿瘤相关中性粒细胞(TANs)是肿瘤浸润免疫细胞中的一个重要组成部分,最近已成为胰腺导管腺癌(PDAC)进展过程中的一个关键角色。本研究旨在阐明TAN的促瘤机制,并为有效的PDAC免疫疗法找到新的靶点。研究人员进行了微阵列和细胞因子阵列分析,以确定TANs的功能机制。人和小鼠的TAN分别来自分化的HL-60细胞和正位移植的PDAC肿瘤。通过体外共培养和体内正位或皮下模型评估了TANs与癌细胞和细胞毒性T细胞的相互作用。对PDAC患者的单细胞转录组进行了分析,以验证细胞研究结果。肿瘤微环境中中性粒细胞浸润的增加与PDAC患者的生存率低下有关。TANs分泌大量趋化因子配体5(CCL5),从而增强了癌细胞的迁移和侵袭。TANs亚群与PDAC的细胞毒性CD8+ T细胞浸润呈负相关,并促进T细胞功能障碍。TANs上调Nectin2的膜表达,导致CD8+ T细胞衰竭。阻断Nectin2可改善CD8+ T细胞功能,抑制小鼠模型的肿瘤进展。人类 PDAC 的单细胞分析显示了两种免疫抑制 TANs 表型:Nectin2+ TANs和OLR1+ TANs。内质网应激调节TANs的原瘤活性。TANs通过分泌CCL5和上调Nectin2促进PDAC的进展。靶向免疫检查点Nectin2可能是提高PDAC免疫疗法疗效的一种新策略。
{"title":"Tumor-associated neutrophils upregulate Nectin2 expression, creating the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma","authors":"Haizhen Luo, Naoki Ikenaga, Kohei Nakata, Nobuhiro Higashijima, Pingshan Zhong, Akihiro Kubo, Chenyi Wu, Chikanori Tsutsumi, Yuki Shimada, Masataka Hayashi, Koki Oyama, Satomi Date, Toshiya Abe, Noboru Ideno, Chika Iwamoto, Koji Shindo, Kenoki Ohuchida, Yoshinao Oda, Masafumi Nakamura","doi":"10.1186/s13046-024-03178-6","DOIUrl":"https://doi.org/10.1186/s13046-024-03178-6","url":null,"abstract":"Tumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC. Microarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings. Increased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8+ T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8+ T-cell exhaustion. Blocking Nectin2 improved CD8+ T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2+ TANs and OLR1+ TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs. TANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC.","PeriodicalId":50199,"journal":{"name":"Journal of Experimental & Clinical Cancer Research","volume":"58 1","pages":""},"PeriodicalIF":11.3,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142208601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}