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MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells.

ABSTRACT: Mixed-lineage kinase 3 (MLK3), a mitogen-activated protein kinase kinase kinase (MAP3K), has critical roles in metastasis of triple-negative breast cancer (TNBC), in part by regulating paxillin phosphorylation and focal adhesion turnover. However the mechanisms and the distinct step(s) of the metastatic processes through which MLK3 exerts its influence are not fully understood. Here we report that in non-metastatic, estrogen receptor-positive breast cancer (ER+ BC) cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), which is accompanied by elevation of matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced ER+ BC cell invasion is abrogated by FRA-1 silencing, demonstrating that MLK3 drives invasion through FRA-1. Conversely, in metastatic TNBC models, high FRA-1 levels are significantly reduced upon depletion of MLK3 by either gene silencing or by the CRISPR/Cas9n editing approach. Furthermore, ablation of MLK3 or MLK inhibitor treatment decreases expression of both MMP-1 and MMP-9. Consistent with the role of tumor cell-derived MMP-1 in endothelial permeability and transendothelial migration, both of these are reduced in MLK3-depleted TNBC cells. In addition, MLK inhibitor treatment or MLK3 depletion, which downregulates MMP-9 expression, renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells derived from TNBC-bearing mice display increased levels of FRA-1 and MMP-1 compared with parental cells, supporting a role for the MLK3-FRA-1-MMP-1 signaling axis in vascular intravasation. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMPs axis suggest that MLK3 is a promising therapeutic target for treatment of TNBC.

SUBMITTER: Rattanasinchai C

PROVIDER: S-EPMC5519193 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells.

Rattanasinchai C C Llewellyn B J BJ Conrad S E SE Gallo K A KA

Oncogenesis 20170612 6

Mixed-lineage kinase 3 (MLK3), a mitogen-activated protein kinase kinase kinase (MAP3K), has critical roles in metastasis of triple-negative breast cancer (TNBC), in part by regulating paxillin phosphorylation and focal adhesion turnover. However the mechanisms and the distinct step(s) of the metastatic processes through which MLK3 exerts its influence are not fully understood. Here we report that in non-metastatic, estrogen receptor-positive breast cancer (ER+ BC) cells, induced MLK3 expression ...[more]

PMID: 28604765

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Project description:Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations. Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay. Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells. Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

Dataset Information

MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells.

Publications

MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells.

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