Pharmacologic Inhibition of MLK3 Kinase Activity Blocks the In Vitro Migratory Capacity of Breast Cancer Cells but Has No Effect on Breast Cancer Brain Metastasis in a Mouse Xenograft Model

Rhoo, Kun Hyoe; Granger, Megan; Sur, Joynita; Feng, Changyong; Gelbard, Harris A.; Dewhurst, Stephen; Polesskaya, Oksana

doi:10.1371/journal.pone.0108487

Cited by 10 publications

(6 citation statements)

References 25 publications

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“…On the other hand, the mouse model showed a difference of adhesion between the two cancer cell lines where MDA-MB-231 cells interacted in a larger amount compared to MCF-7. This result using the mouse in vitro BBB model is consistent with the results obtained with animal studies [ 29 ]. Hence, associated to in vivo experiments, the mouse model could represent a relevant BBB model to study brain metastases at a cellular and molecular level but is not suitable for human mechanistic studies.…”

Section: Discussionsupporting

confidence: 91%

Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines

et al. 2016

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Around 7–17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities.

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Section: Discussionsupporting

confidence: 91%

Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines

et al. 2016

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“…C57BL/6J male mice were randomly assigned to eight groups: Sham, TAC, TAC + U-099, Sham + AAV NC , TAC + AAV NC , TAC + AAV MLK3− , TAC + atagomir, and TAC + agomir. TAC + U-099 mice were generated by intraperitoneal (i. p.) injection of URMC-099, an inhibitor of MLK3, has been reported to play an important role in a variety of diseases including anti-inflammatory and cognitive decline 34,37 and as a useful tool to investigate the role of MLK3 in other diseases is common [38][39][40] (10 mg/kg, dissolved in 10% DMSO, 40% PEG300, and 50% saline, every 12 h, MedChemExpress, Shanghai, China) 7 days before TAC surgery. Sham and TAC mice received corresponding isotype i.p.…”

Section: Animalmentioning

confidence: 99%

Pyroptosis and ferroptosis induced by mixed lineage kinase 3 (MLK3) signaling in cardiomyocytes are essential for myocardial fibrosis in response to pressure overload

et al. 2020

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Chronic heart failure (CHF) is the final outcome of many cardiovascular diseases, and is a severe health issue faced by the elderly population. Mixed lineage kinase 3 (MLK3), a member of MAP3K family, is associated with aging, inflammation, oxidative stress, and related diseases, such as CHF. MLK3 has also been reported to play an important role in protecting against cardiomyocyte injury; however, its function in myocardial fibrosis is unknown. To investigate the role of MLK3 in myocardial fibrosis, we inhibited the expression of MLK3, and examined cardiac function and remodeling in TAC mice. In addition, we assessed the expression of MLK3 protein in ventricular cells and its downstream associated protein. We found that MLK3 mainly regulates NF-κB/NLRP3 signaling pathway-mediated inflammation and that pyroptosis causes myocardial fibrosis in the early stages of CHF. Similarly, MLK3 mainly regulates the JNK/p53 signaling pathway-mediated oxidative stress and that ferroptosis causes myocardial fibrosis in the advanced stages of CHF. We also found that promoting the expression of miR-351 can inhibit the expression of MLK3, and significantly improve cardiac function in mice subjected to TAC. These results suggest the pyroptosis and ferroptosis induced by MLK3 signaling in cardiomyocytes are essential for adverse myocardial fibrosis, in response to pressure overload. Furthermore, miR-351, which has a protective effect on ventricular remodeling in heart failure caused by pressure overload, may be a key target for the regulation of MLK3.

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“…We treated HIV-1 ADA strain-infected (HIV-1 ADA -infected) MDMs with 400 ng/ml URMC-099, followed by subtherapeutic concentrations (1 μM) of nanoformulated ATV (nanoATV) at 1, 3, and 5 days after infection at a multiplicity of 0.1 infectious virions per cell. The concentration of URMC-099 used elicited a maximal therapeutic efficacy that included drug particle retentions, amyloid β clearance, cellular vitality, and robust neuroprotective and antiinflammatory responses (11,(16)(17)(18)(19)(20). As concentrations of nanoATV of 100 μM or greater suppress viral replication, we performed URMC-099 dose-response determinations using subtherapeutic nanoATV concentrations for the measurement of viral growth.…”

Section: Urmc-099 Facilitates Nanoart Activitymentioning

confidence: 99%

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

Gnanadhas¹,

Dash²,

Sillman³

et al. 2017

Journal of Clinical Investigation

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Long-acting anti-HIV products can substantively change the standard of care for patients with HIV/AIDS. To this end, hydrophobic antiretroviral drugs (ARVs) were recently developed for parenteral administration at monthly or longer intervals. While shorter-acting hydrophilic drugs can be made into nanocarrier-encased prodrugs, the nanocarrier encasement must be boosted to establish long-acting ARV depots. The mixed-lineage kinase 3 (MLK-3) inhibitor URMC-099 provides this function by affecting autophagy. Here, we have shown that URMC-099 facilitates ARV sequestration and its antiretroviral responses by promoting the nuclear translocation of the transcription factor EB (TFEB). In monocyte-derived macrophages, URMC-099 induction of autophagy led to retention of nanoparticles containing the antiretroviral protease inhibitor atazanavir. These nanoparticles were localized within macrophage autophagosomes, leading to a 4-fold enhancement of mitochondrial and cell vitality. In rodents, URMC-099 activation of autophagy led to 50-fold increases in the plasma drug concentration of the viral integrase inhibitor dolutegravir. These data paralleled URMC-099-mediated induction of autophagy and the previously reported antiretroviral responses in HIV-1-infected humanized mice. We conclude that pharmacologic induction of autophagy provides a means to extend the action of a long-acting, slow, effective release of antiretroviral therapy. Center for Neural Development and Disease, University of Rochester Medical Center (URMC), Rochester, New York, USA. Conflict of interest:H.A. Gelbard and H.E. Gendelman are members of the scientific advisory board for WavoDyne Therapeutics Inc., which is developing URMC-099 as a firstin-class MLK-3 inhibitor for clinical trials. URMC-099 is owned by URMC (patent nos. US 8,846,909 B2;8,877,772;and 9,181,247 and associated international patents). tion of TFEB mRNA by approximately 4-to 8-fold under different treatment conditions ( Figure 2F). With HIV-1 infection, TFEB mRNA levels were reduced by 2-to 3-fold compared with levels in uninfected controls, and URMC-099 could recover the phenotype. Increased TFEB translocation was delayed and did not reach significant levels until day 7 after URMC-099 treatment (Supplemental Figure 2B). These data confirm that URMC-099 regulates TFEB nuclear translocation in an mTORC1-dependent manner. URMC-099 stimulates autophagy in human MDMs. On the basis of the preceding data sets, we theorized that nuclear translocation of TFEB induces autophagy and lysosomal biogenesis. TFEB serves as the master regulator of both autophagy and lysosomal biogenesis (26), and such regulation could have a profound role in nanoART intracellular sequestration in MDMs. Thus, we examined the autophagosome markers microtubule-associated protein 1 light chain 3 β (LC3B, also known as MAP1LC3B) and beclin 1 (BECN1). Using Western blot assays, we found that each marker was significantly upregulated by URMC-099 ( Figure 3, A-C and Supplemental Figure 3A). URMC-099 increased the expression ...

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Pharmacologic Inhibition of MLK3 Kinase Activity Blocks the In Vitro Migratory Capacity of Breast Cancer Cells but Has No Effect on Breast Cancer Brain Metastasis in a Mouse Xenograft Model

Cited by 10 publications

References 25 publications

Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines

Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines

Pyroptosis and ferroptosis induced by mixed lineage kinase 3 (MLK3) signaling in cardiomyocytes are essential for myocardial fibrosis in response to pressure overload

Autophagy facilitates macrophage depots of sustained-release nanoformulated antiretroviral drugs

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