Abstract
BackgroundMolecules Interacting with CasL (MICAL1), a multidomain flavoprotein monoxygenase, is strongly involved in the mechanisms that promote cancer cell proliferation and survival. Activation of MICAL1 causes an up-regulation of reactive oxygen species (ROS) in HeLa cells. ROS can function as a signaling molecule that modulates protein phosphorylation, leading to malignant phenotypes of cancer cells such as invasion and metastasis. Herein, we tested whether MICAL1 could control cell migration and invasion through regulating ROS in breast cancer cell lines.MethodsThe effects of depletion/overexperssion of MICAL1 on cell invasion rate were measured by matrigel-based transwell assays. The contents of ROS in breast cancer cells were evaluated by CM2-DCFHDA staining and enhanced lucigenin chemiluminescence method. RAB35 activity was assessed by pulldown assay. The relationship of RAB35 and MICAL1 was evaluated by immunofluorescence, coimmunoprecipitation, immunoblotting and co-transfection techniques. Immunoblotting assays were also used to analyze Akt phosphorylation level.ResultsIn this study, we found that depletion of MICAL1 reduced cell migration and invasion as well as ROS generation. Phosphorylation of Akt was also attenuated by MICAL1 depletion. Likewise, the over-expression of MICAL1 augmented the generation of ROS, increased Akt phosphorylation, and favored invasive phenotype of breast cancer cells. Moreover, we investigated the effect of EGF signaling on MICAL1 function. We demonstrated that EGF increased RAB35 activation and activated form of RAB35 could bind to MICAL1. Silencing of RAB35 repressed ROS generation, prevented Akt phosphorylation and inhibited cell invasion in response to EGF.ConclusionsTaken together, our results provide evidence that MICAL1 plays an essential role in the activation of ROS/Akt signaling and cell invasive phenotype and identify a novel link between RAB35 and MICAL1 in regulating breast cancer cell invasion. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis.
Highlights
Molecules Interacting with CasL (MICAL1), a multidomain flavoprotein monoxygenase, is strongly involved in the mechanisms that promote cancer cell proliferation and survival
Molecules interacting with CasL (MICAL1) regulates breast cancer cell migration and invasion To examine the function of MICAL1 in breast cancer progression, we silenced MICAL1 expression in human breast cancer MDA-MB-231 cells with small interfering RNA (siRNA) for MICAL1
Our results provide evidence that MICAL1 plays an essential role in the activation of reactive oxygen species (ROS)/Akt signaling and cell invasive phenotype and identify a novel link between RAB35 and MICAL1 in regulating breast cancer cell invasion
Summary
Molecules Interacting with CasL (MICAL1), a multidomain flavoprotein monoxygenase, is strongly involved in the mechanisms that promote cancer cell proliferation and survival. We tested whether MICAL1 could control cell migration and invasion through regulating ROS in breast cancer cell lines. Removal of the CC domain from MICAL1 or affect the binding of CC domain to its LIM domain may cause the activation of its mono-oxygenase domain, leading to ROS production and F-actin assembly alteration. MICAL1 is a highly regulated protein, the auto-inhibition state could be relieved by the interaction occur within CC domain and other proteins such as RAB1 and Plexin under various cellular conditions [12, 13]. RABs are the largest family of small GTPases and involved in the control of intracellular membrane trafficking and cell motility through interaction with specific effector molecules [14, 15]. By a yeast two-hybrid assay, previous study has systematically screened the ability of MICAL1 binding for all the members of Rab family and found that GTPbound RAB35 was one of the few members which interacted strongly with MICAL1 [16]
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