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CAPG enhances breast cancer metastasis by competing with PRMT5 to modulate STC-1 transcription.

ABSTRACT: Macrophage-capping protein (CAPG) has been shown to promote cancer cell metastasis, although the mechanism remains poorly understood. Methods: Breast cancer (BC) tissue microarray was used to test the role of CAPG in the prognosis of BC patients. Xenograft mice model was used to validate the metastasis promotion role of CAPG in vivo. Gene expression array, chromatin immunoprecipitation and luciferase report assay were performed to search for the target genes of CAPG. Protein immunoprecipitation, MS/MS analysis, tissue microarray and histone methyltransferase assay were used to explore the mechanism of CAPG regulating stanniocalcin 1 (STC-1) transcription. Results: We demonstrate a novel mechanism by which CAPG enhances BC metastasis via promoting the transcription of the pro-metastatic gene STC-1, contributing to increased metastasis in BC. Mechanistically, CAPG competes with the transcriptional repressor arginine methyltransferase 5 (PRMT5) for binding to the STC-1 promoter, leading to reduced histone H4R3 methylation and enhanced STC-1 transcription. Our study also indicates that both CAPG and PRMT5 are independent prognostic factors for BC patient survival. High CAPG level is associated with poor survival, while high PRMT5 expression favors a better prognosis in BC patients. Conclusion: Our findings identify a novel role of CAPG in the promotion of BC metastasis by epigenetically enhancing STC-1 transcription.

SUBMITTER: Huang S

PROVIDER: S-EPMC5928908 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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CAPG enhances breast cancer metastasis by competing with PRMT5 to modulate STC-1 transcription.

Huang Sheng S Chi Yayun Y Qin Yi Y Wang Ziliang Z Xiu Bingqiu B Su Yonghui Y Guo Rong R Guo Liang L Sun Hefen H Zeng Chujia C Zhou Shuling S Hu Xin X Liu Sheng S Shao Zhimin Z Wu Zhaohui Z Jin Wei W Wu Jiong J

Theranostics 20180403 9

Macrophage-capping protein (CAPG) has been shown to promote cancer cell metastasis, although the mechanism remains poorly understood. <b>Methods:</b> Breast cancer (BC) tissue microarray was used to test the role of CAPG in the prognosis of BC patients. Xenograft mice model was used to validate the metastasis promotion role of CAPG in vivo. Gene expression array, chromatin immunoprecipitation and luciferase report assay were performed to search for the target genes of CAPG. Protein immunoprecipi ...[more]

PMID: 29721098

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Project description:IntroductionAberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit.MethodsWe raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading.ResultsWith G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS).ConclusionsCapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.

Project description:BackgroundThe majority of breast cancer patients die of metastasis rather than primary tumors, whereas the molecular mechanisms orchestrating cancer metastasis remains poorly understood. Long noncoding RNAs (lncRNA) have been shown to regulate cancer occurrence and progression. However, the lncRNAs that drive metastasis in cancer patients and their underlying mechanisms are still largely unknown.MethodslncRNAs highly expressed in metastatic lymph nodes were identified by microarray. Survival analysis were made by Kaplan-Meier method. Cell proliferation, migration, and invasion assay was performed to confirm the phenotype of LINC02273. Tail vein model and mammary fat pad model were used for in vivo study. RNA pull-down and RIP assay were used to confirm the interaction of hnRNPL and LINC02273. Chromatin isolation by RNA purification followed by sequencing (ChIRP-seq), RNA-seq, ChIP-seq, and luciferase reporter assay reveal hnRNPL-LINC02273 regulates AGR2. Antisense oligonucleotides were used for in vivo treatment.ResultsWe identified a novel long noncoding RNA LINC02273, whose expression was significantly elevated in metastatic lesions compared to the primary tumors, by genetic screen of matched tumor samples. Increased LINC02273 promoted breast cancer metastasis in vitro and in vivo. We further showed that LINC02273 was stabilized by hnRNPL, a protein increased in metastatic lesions, in breast cancer cells. Mechanistically, hnRNPL-LINC02273 formed a complex which activated AGR2 transcription and promoted cancer metastasis. The recruitment of hnRNPL-LINC02273 complex to AGR2 promoter region epigenetically upregulated AGR2 by augmenting local H3K4me3 and H3K27ac levels. Combination of AGR2 and LINC02273 was an independent prognostic factor for predicting breast cancer patient survival. Moreover, our data revealed that LINC02273-targeting antisense oligonucleotides (ASO) substantially inhibited breast cancer metastasis in vivo.ConclusionsOur findings uncover a key role of LINC02273-hnRNPL-AGR2 axis in breast cancer metastasis and provide potential novel therapeutic targets for metastatic breast cancer intervention.

Project description:Background: Chemotherapy resistance is a major problem in breast cancer treatment and a leading cause of mortality in breast cancer patients. Biomarkers for chemotherapy resistance is under investigation. Methods: Paclitaxel resistant cells were established and subjected to RNA sequencing. Analysis combined with two additional RNA-seq datasets was conducted. CapG expression in patients with adjuvant chemotherapy was studied in breast cancer resection specimens using IHC and related to pathological response and disease-free survival. Paclitaxel resistance was assessed by half-maximal inhibitory concentrations (IC50) and a mouse xenograft model. Results: Increased expression of actin-binding protein CapG strongly correlated with the resistance to paclitaxel chemotherapy and decreased probability to achieve pathological complete response in breast cancer patients. Overexpressing CapG significantly enhanced paclitaxel resistance in breast cancer cells and xenograft tumors. High CapG level also significantly correlated with shorter relapse-free survival as well as hyper-activation of PI3K/Akt signaling in breast cancer patients. Mechanistically, CapG enhanced PIK3R1 expression which led to increased PI3K/Akt activation. Unexpectedly, CapG was found to bind to the variant-specific promoter of PIK3R1/P50 and directly enhance its transcription. We also identified p300/CBP as a transcriptional coregulator of CapG, which is recruited to PIK3R1 promoter through interaction with CapG, thereby increasing PIK3R1/P50 transcription by enhancing histone H3K27 acetylation. Consistently, inhibiting p300/CBP substantially decreased CapG-dependent upregulation of PIK3R1/P50 and subsequent PI3K/Akt activation, resulting in increased sensitivity to paclitaxel treatment in breast cancer cells. Conclusion: High CapG levels may predict poor paclitaxel response in breast cancer patients. Targeting CapG-mediated hyperactivation of PI3K/Akt pathway may mitigate resistance to chemotherapy in breast cancer.

Dataset Information

CAPG enhances breast cancer metastasis by competing with PRMT5 to modulate STC-1 transcription.

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CAPG enhances breast cancer metastasis by competing with PRMT5 to modulate STC-1 transcription.

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