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Capping Protein Regulator and Myosin 1 Linker 3 Is Required for Tumor Metastasis

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Capping Protein Regulator and Myosin 1 Linker 3 Is Required for Tumor Metastasis Published OnlineFirst November 6, 2019; DOI: 10.1158/1541-7786.MCR-19-0722 MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS Capping Protein Regulator and Myosin 1 Linker 3 Is Required for Tumor Metastasis Huan Wang1, Chao Wang2, Guang Peng2, Doudou Yu1, Xin-Gang Cui3,4, Ying-Hao Sun2, and Xiaojing Ma1,5 ABSTRACT ◥ Metastasis accounts for 90% of deaths caused by solid tumors, but protein, that are involved in actin cytoskeletal organization, which the multitude of mechanisms underlying tumor metastasis remains is required for cell polarization and focal adhesion formation. poorly understood. CARMIL1 and 2 proteins are capping protein Moreover, molecular pathway enrichment analysis reveals that lack (CP) interactants and multidomain regulators of actin-based mobil- of CARMIL3 leads to loss of cell adhesions and low CARMIL3 ity. However, CARMIL30s function has not been explored. Through expression in breast cancer patient specimens is implicated in bioinformatic metadata analysis, we find that high CARMIL3 epithelial–mesenchymal transition. We also find that CARMIL3 expression correlates with poor survival of patients with breast and sustains adherens junction between tumor cells. This is accom- prostate cancer. Functional studies in murine and xenograft tumor plished by CARMIL3 maintaining E-cadherin transcription down- models by targeted diminution of CARMIL3 expression or forced stream of HDACs through inhibiting ZEB2 protein level, also via expression demonstrate that CARMIL3 is vitally important for protecting b-catenin from ubiquitination-mediated degradation tumor metastasis, especially for metastatic colonization. Consistent initiated by the destruction complex. with a predominantly cell-intrinsic mode of action, CARMIL3 is also crucial for tumor cell migration and invasion in vitro. Coim- Implications: This study uncovers CARMIL3 as a novel and critical munoprecipitation coupled with mass spectrometric analyses iden- regulator of metastatic progression of cancers and suggests thera- tifies a group of CARMIL3-interacting proteins, including capping peutic potentials to target CARMIL3. Introduction primary tumor and is switched on again in MET during distant colonization (11). EMT and MET take place sequentially with Metastasis is the leading cause of cancer-related death (1). morphologic changes conferring on cancer cells the metastatic During metastasis, the phenotypic transformation of tumor cells plasticity (12, 13). Thus, actin cytoskeleton remodeling, cell adhe- is closely related with epithelial-to-mesenchymal transition (EMT) sion,EMT,andMETarefinely orchestrated during tumor metas- and mesenchymal-to-epithelial transition (MET), which facilitates tasis. Despite extensive studies to elucidate the mechanisms, the tumor cells to gain the plasticity to undergo the multiple processes molecular pathways that govern these processes are still not well including local invasion, intravasation, survival in the circulation understood. system and arrest, extravasation, seeding, and colonization (2–6). There are three members in the capping protein regulator and Many of these processes require cell motility to overcome tissue myosin 1 linker (CARMIL) family in mammals. CARMIL proteins barriers, which is driven by actin cytoskeleton remodeling and is are capping protein (CP) interactants and multidomain regulators regulated by cell adhesion (7–9). E-cadherin, a major component of of actin-based mobility (14–18). Several human diseases are adherens junction (AJ), is anchored to the actin cytoskeleton via its reported to associate with CARMILs. A common CARMIL1 (also cytoplasmic tail binding with b-catenin and a-catenin (10), and it is known as LRRC16A) variant is found to be associated with downregulated in EMT during local invasion of cancer cells to leave increased susceptibility to gout (19). Mutations in CARMIL2 (also known as RLTPR) cause abrogated CD28 costimulation of T cells 1 State Key Laboratory of Microbial Metabolism, Sheng Yushou Center of Cell and decreased activation of NF-kB signaling (20, 21). In T cells Biology and Immunology, School of Life Science and Biotechnology, Shanghai fi Jiao Tong University, Shanghai, China. 2Department of Urology, Changhai isolated from patients de cient of CARMIL2, F-actin levels were Hospital, Second Military Medical University, Shanghai, China. 3Department of decreased at the leading edge and the microtubule network was Urinary Surgery, Gongli Hospital, Shanghai, China. 4Department of Urinary disorganized (22). CARMIL3 (also known as LRRC16B) was first Surgery, The Third Affiliated Hospital (Eastern Hepatobiliary Surgery Hospital), reported by Hsu and colleagues, using public information of 5 Second Military Medical University, Shanghai, China. Department of Microbi- expressed sequence tags in a systematic analysis of gene expression ology and Immunology, Weill Cornell Medicine, New York, New York. patterns, as an oncofetal protein with transforming capability and Note: Supplementary data for this article are available at Molecular Cancer reexpression in ovarian and colorectal cancer tissues (23). Our Research Online (http://mcr.aacrjournals.org/). group discovered it independently in a study of phagocytes ingest- Ã Corresponding Authors: Xiaojing Ma, Weill Cornell Medicine, 1300 York Ave- ing apoptotic cells in which LRRC16B was identified, via DNA nue, New York, NY 10021. Phone: 212-746-4404; Fax: 212-746-4423; E-mail: affinity purification coupled with mass spectrometric analysis, to be [email protected]; Xin-Gang Cui, Gongli Hospital, Second Military induced to bind to the IL12p40 promoter region promoting its Medical University, 219 Miaopu Road, Shanghai 200135, China. Phone: 8602- transcription (unpublished data). Recently, CARMIL3 was reported 1588-58730; Fax: 8602-1338-2163; E-mail: [email protected]; and Ying- Hao Sun, [email protected] to play a role in spinogenesis by localizing CP to developing synapses (24), but the involvement of CARMIL3 during cancer Mol Cancer Res 2019;XX:XX–XX progression remains virtually unknown. Here we demonstrate for doi: 10.1158/1541-7786.MCR-19-0722 the first time a critical role of CARMIL3 in tumor metastasis and Ó2019 American Association for Cancer Research. exploit the underlying molecular mechanism. AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 6, 2019; DOI: 10.1158/1541-7786.MCR-19-0722 Wang et al. Materials and Methods subtype of TCGA BRCA cohort was based on TCIA (ref. 25; https:// Bioinformatic analysis of clinical cancer patients tcia.at/home) and the clinical survival data of TCGA was retrieved The CARMIL3 CNV data of The Cancer Genome Atlas (TCGA) was according to the pipeline of Liu and colleagues (26). Survival analysis derived from Genomic Data Commons (GDC, https://gdc-portal.nci. in Fig. 1I was based on Kaplan–Meier plotter database (http://www. nih.gov/legacy-archive/) and the CARMIL3, ZEB2, CDH1 mRNA KMplot.com) and the clinical data used in Fig. 1J and Supplementary (RNA-seq), and b-catenin protein (RPPA) expression data of cancer Table S1B was retrieved from GSE42568 dataset. All the graphs were patients’ specimens was retrieved from Firehose (http://gdac.broad plotted with R version 3.4.4 (https://www.r-project.org/about.html). institute.org/), GEO (https://www.ncbi.nlm.nih.gov/geo/), or cBio- Portal (http://www.cbioportal.org/) accordingly. Kaplan–Meier anal- Cell lines ysis of cancer patients’ percent survival was grouped by median The mouse mammary tumor cell line 4T1 was a gift from Prof. CARMIL3 mRNA expression unless stated. The PAM50 luminal A Xuanming Yang of Shanghai Jiao Tong University (SJTU, Shanghai, Figure 1. High CARMIL3 expression positively correlates with human breast cancer progression. A–G, Analysis of CARMIL3 mRNA expression in breast cancer samples, in TCGA-BRCA cohort grouped by sample types (A) and tumor stages (B); in patients with TNBC of dataset GSE61723 (C and D); in dataset GSE4922 grouped by breast cancer disease grade (E); in liver metastases of dataset GSE56493 (F); and in TCGA BRCA cohort, normal tissue versus primary tumor of PAM50 luminal A subtype (G). H–J, Kaplan–Meier analysis of breast cancer patient survival, in the cohort of TCGA BRCA PAM50 luminal A subtype (H); in the lymph node positive cohort of patients with breast cancer based on Kaplan–Meier plotter dataset (I); in the cohort of GSE42568 grouped by mean CARMIL3 mRNA expression (J). OF2 Mol Cancer Res; 2019 MOLECULAR CANCER RESEARCH Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 6, 2019; DOI: 10.1158/1541-7786.MCR-19-0722 CARMIL3 Regulates Tumor Metastasis China). The 4TO7 cell line was a gift from Prof. Guohong Hu of two times independently, verifying that the targeting Carmil3 Shanghai Institute of Nutrition and Health (Shanghai, China). The genomic locus was correctly edited by one base pair loss in the mouse prostate tumor cell line TRAMP C-1 was purchased from exon, resulting in a frame shift. The identified control and Carmil3 ATCC. HEK293T and MDA-MB-231 cell lines were stored in Ma lab KO clones were used for further experiments. of SJTU (Shanghai, China). These cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 100 mg/mL peni- Mouse tumor models cillin/streptomycin (Gibco). The PC-3 and PC-3M cells were stored in All mouse studies were conducted in accordance with protocols
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