DCLK1-mediated regulation of invadopodia dynamics and matrix metalloproteinase trafficking drives invasive progression in head and neck squamous cell carcinoma

IF 27.7 1区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Levi Arnold, Marrion Yap, Nathan Farrokhian, Laura Jackson, Michael Barry, Thuc Ly, Pachiappan Arjunan, Angela Kaczorowski-Worthley, Carter Tews, Avisha Pandey, Austin Morrison, Michael P. Washburn, David Standing, Juan P. Gomez, Nanda Kumar Yellapu, David Johnson, Linheng Li, Shahid Umar, Shrikant Anant, Sufi Mary Thomas
{"title":"DCLK1-mediated regulation of invadopodia dynamics and matrix metalloproteinase trafficking drives invasive progression in head and neck squamous cell carcinoma","authors":"Levi Arnold, Marrion Yap, Nathan Farrokhian, Laura Jackson, Michael Barry, Thuc Ly, Pachiappan Arjunan, Angela Kaczorowski-Worthley, Carter Tews, Avisha Pandey, Austin Morrison, Michael P. Washburn, David Standing, Juan P. Gomez, Nanda Kumar Yellapu, David Johnson, Linheng Li, Shahid Umar, Shrikant Anant, Sufi Mary Thomas","doi":"10.1186/s12943-025-02264-3","DOIUrl":null,"url":null,"abstract":"HNSCC presents a significant health challenge due to its high mortality resulting from treatment resistance and locoregional invasion into critical structures in the head and neck region. Understanding the invasion mechanisms of HNSCC has the potential to guide targeted therapies, improving patient survival. Previously, we demonstrated the involvement of doublecortin like kinase 1 (DCLK1) in regulating HNSCC cell invasion. Here, we investigated the hypothesis that DCLK1 modulates proteins within invadopodia, specialized subcellular protrusions that secrete matrix metalloproteinases to degrade the ECM. We employed tandem mass tag (TMT)-based proteomics to identify the role of DCLK1 in regulating proteins involved in HNSCC invasion and validated the findings using immunoblotting. The Cancer Genome Atlas (TCGA) database was interrogated to correlate DCLK1 expression with tumor stage, grade, and invasion-associated proteins. In vitro invasion was assessed using a Boyden chamber assay, and immunohistochemistry on patient samples determined DCLK1's distribution within tumors. Gelatin invadopodia assay was used to establish DCLK1 localization to invadopodia related gelatin degradation. Super-resolution confocal microscopy demonstrated colocalization of DCLK1 with invadopodia markers and MMP trafficking proteins. ECM degradation by MMPs in HNSCC cells with wild-type and knockdown DCLK1 was evaluated using a dye-quenched tracer, while gel zymography and MMP array identified secreted proteases. Proximity ligation assay (PLA) and co-immunoprecipitation assays were used to confirm interactions between DCLK1, MMP9, KIF16B, and RAB40B. Proteomic analysis demonstrate DCLK1's role in regulating proteins involved in cytoskeletal and ECM remodeling. Clinically, rising DCLK1 levels correlate with higher histological grade and lymph node metastasis, with heightened expression observed at the leading edge of HNSCC patient tissue. DCLK1 is localized with markers of mature invadopodia including TKS4, TKS5, cortactin, and MT1-MMP. Knockdown of DCLK1 led to reductions in invadopodia numbers and decreased in vitro invasion and ECM degradation. MMP9 colocalizes with DCLK1 within invadopodia structures and its secretion is disrupted by DCLK1 knockdown. Further, PLA and co-immunoprecipitations studies demonstrate DLCK1 complexes with KIF16B and RAB40B enabling trafficking of degradative MMP9 cargo along the invadopodia to degrade local ECM. This work unveils a novel function of DCLK1 in regulating KIF16B and RAB40B to traffic matrix degrading MMP9 cargo to the distal end of the invadopodia facilitating HNSCC invasion.","PeriodicalId":19000,"journal":{"name":"Molecular Cancer","volume":"31 1","pages":""},"PeriodicalIF":27.7000,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Cancer","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1186/s12943-025-02264-3","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

HNSCC presents a significant health challenge due to its high mortality resulting from treatment resistance and locoregional invasion into critical structures in the head and neck region. Understanding the invasion mechanisms of HNSCC has the potential to guide targeted therapies, improving patient survival. Previously, we demonstrated the involvement of doublecortin like kinase 1 (DCLK1) in regulating HNSCC cell invasion. Here, we investigated the hypothesis that DCLK1 modulates proteins within invadopodia, specialized subcellular protrusions that secrete matrix metalloproteinases to degrade the ECM. We employed tandem mass tag (TMT)-based proteomics to identify the role of DCLK1 in regulating proteins involved in HNSCC invasion and validated the findings using immunoblotting. The Cancer Genome Atlas (TCGA) database was interrogated to correlate DCLK1 expression with tumor stage, grade, and invasion-associated proteins. In vitro invasion was assessed using a Boyden chamber assay, and immunohistochemistry on patient samples determined DCLK1's distribution within tumors. Gelatin invadopodia assay was used to establish DCLK1 localization to invadopodia related gelatin degradation. Super-resolution confocal microscopy demonstrated colocalization of DCLK1 with invadopodia markers and MMP trafficking proteins. ECM degradation by MMPs in HNSCC cells with wild-type and knockdown DCLK1 was evaluated using a dye-quenched tracer, while gel zymography and MMP array identified secreted proteases. Proximity ligation assay (PLA) and co-immunoprecipitation assays were used to confirm interactions between DCLK1, MMP9, KIF16B, and RAB40B. Proteomic analysis demonstrate DCLK1's role in regulating proteins involved in cytoskeletal and ECM remodeling. Clinically, rising DCLK1 levels correlate with higher histological grade and lymph node metastasis, with heightened expression observed at the leading edge of HNSCC patient tissue. DCLK1 is localized with markers of mature invadopodia including TKS4, TKS5, cortactin, and MT1-MMP. Knockdown of DCLK1 led to reductions in invadopodia numbers and decreased in vitro invasion and ECM degradation. MMP9 colocalizes with DCLK1 within invadopodia structures and its secretion is disrupted by DCLK1 knockdown. Further, PLA and co-immunoprecipitations studies demonstrate DLCK1 complexes with KIF16B and RAB40B enabling trafficking of degradative MMP9 cargo along the invadopodia to degrade local ECM. This work unveils a novel function of DCLK1 in regulating KIF16B and RAB40B to traffic matrix degrading MMP9 cargo to the distal end of the invadopodia facilitating HNSCC invasion.
dclk1介导的浸润性动力学和基质金属蛋白酶运输调控推动头颈部鳞状细胞癌的侵袭性进展
由于治疗耐药性和局部侵袭头颈部关键结构,HNSCC的死亡率很高,因此对健康构成了重大挑战。了解HNSCC的侵袭机制有可能指导靶向治疗,提高患者生存率。先前,我们证明了双皮质素样激酶1 (DCLK1)参与调节HNSCC细胞侵袭。在这里,我们研究了DCLK1调节侵过体内的蛋白质的假设,这种特殊的亚细胞突起分泌基质金属蛋白酶来降解ECM。我们采用基于串联质量标签(TMT)的蛋白质组学方法来鉴定DCLK1在调节HNSCC侵袭相关蛋白中的作用,并使用免疫印迹法验证了这一发现。通过癌症基因组图谱(TCGA)数据库查询DCLK1表达与肿瘤分期、分级和侵袭相关蛋白的相关性。使用Boyden室法评估体外侵袭,患者样本的免疫组织化学测定DCLK1在肿瘤内的分布。明胶侵染试验建立了DCLK1对侵染虫相关明胶降解的定位。超分辨率共聚焦显微镜显示DCLK1与侵过体标记物和MMP转运蛋白共定位。使用染料猝灭示踪剂评估带有野生型和敲低型DCLK1的HNSCC细胞中MMPs对ECM的降解,而凝胶酶谱和MMP阵列鉴定分泌的蛋白酶。使用邻近结扎法(PLA)和共免疫沉淀法确认DCLK1、MMP9、KIF16B和RAB40B之间的相互作用。蛋白质组学分析表明DCLK1在调节参与细胞骨架和ECM重塑的蛋白质中发挥作用。临床上,DCLK1水平升高与更高的组织学分级和淋巴结转移相关,在HNSCC患者组织的前缘观察到表达升高。DCLK1定位于成熟侵过足的标记物,包括TKS4、TKS5、cortex和MT1-MMP。DCLK1的敲低导致侵殖细胞数量减少,体外侵袭和ECM降解减少。MMP9在侵足结构中与DCLK1共定位,DCLK1敲低会破坏其分泌。此外,PLA和共免疫沉淀研究表明,dck1与KIF16B和RAB40B的复合物可以使降解的MMP9货物沿着侵入过口运输,从而降解局部ECM。这项工作揭示了DCLK1在调节KIF16B和RAB40B到交通矩阵中的新功能,将MMP9货物降解到侵过孔的远端,促进HNSCC的侵袭。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Molecular Cancer
Molecular Cancer 医学-生化与分子生物学
CiteScore
54.90
自引率
2.70%
发文量
224
审稿时长
2 months
期刊介绍: Molecular Cancer is a platform that encourages the exchange of ideas and discoveries in the field of cancer research, particularly focusing on the molecular aspects. Our goal is to facilitate discussions and provide insights into various areas of cancer and related biomedical science. We welcome articles from basic, translational, and clinical research that contribute to the advancement of understanding, prevention, diagnosis, and treatment of cancer. The scope of topics covered in Molecular Cancer is diverse and inclusive. These include, but are not limited to, cell and tumor biology, angiogenesis, utilizing animal models, understanding metastasis, exploring cancer antigens and the immune response, investigating cellular signaling and molecular biology, examining epidemiology, genetic and molecular profiling of cancer, identifying molecular targets, studying cancer stem cells, exploring DNA damage and repair mechanisms, analyzing cell cycle regulation, investigating apoptosis, exploring molecular virology, and evaluating vaccine and antibody-based cancer therapies. Molecular Cancer serves as an important platform for sharing exciting discoveries in cancer-related research. It offers an unparalleled opportunity to communicate information to both specialists and the general public. The online presence of Molecular Cancer enables immediate publication of accepted articles and facilitates the presentation of large datasets and supplementary information. This ensures that new research is efficiently and rapidly disseminated to the scientific community.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信