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.

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BackgroundCD24, a mucin-like membrane glycoprotein, plays a critical role in carcinogenesis, but its role in human gastric cancer and the underlying mechanism remains undefined.MethodsThe contents of CD24 and epidermal growth factor receptor (EGFR) in gastric cancer cells (SGC-7901 and BGC-823) and non-malignant gastric epithelial cells (GES-1) were evaluated by Western blotting assay. Cellular EGFR staining was examined by immunofluorescence assay. Cell migration rate was measured by wound healing assay. The effects of depletion/overexperssion of CD24 on EGFR expression and activation of EGF/EGFR singaling pathways were evaluated by immunofluorescence, qPCR, Western blotting and flow cytometry techniques. RhoA activity was assessed by pulldown assay. CD24 and EGFR expression patterns in human gastric tumor samples were also investigated by immunohistochemistry staining.ResultsCD24 was overexpressed in human gastric cancer cells. Ectopic expression of CD24 in gastric epithelial cells augmented the expression of EGFR, while knockdown of CD24 in gastric cancer cells decreased the level of EGFR and cell migration velocity. To further explore the mechanisms, we investigated the effect of CD24 expression on EGF/EGFR signaling. We noticed that this effect of CD24 on EGFR expression was dependent on promoting EGFR internalization and degradation. Lower ERK and Akt phosphorylations in response to EGF stimulation were observed in CD24-depleted cells. In addition, we noticed that the effect of CD24 on EGFR stability was mediated by RhoA activity in SGC-7901 gastric cancer cells. Analysis of gastric cancer specimens revealed a positive correlation between CD24 and EGFR levels and an association between CD24 expression and worse prognosis.ConclusionThus, these findings suggest for the first time that CD24 regulates EGFR signaling by inhibiting EGFR internalization and degradation in a RhoA-dependent manner in gastric cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0787-y) contains supplementary material, which is available to authorized users.

Aims and Hypothesis: This study aims to investigate the mechanism involved in intracellular regulation of EGFR degradation induced by EGF.Methods: Phosphorylation of proteins related to EGFR signaling was examined by western blot analysis. Activation, connection between Rab35 and folliculin (FLCN) were assessed by pulldown, coimmunoprecipitation assays separately. The relationship between FLCN and cell growth was detected using gene overexpression and knock-down techniques.Results: Here, we demonstrate that interfering with FLCN, a tumor suppressor, reduces the rate of EGF-induced EGFR degradation, resulting in prolonged activation of downstream signaling. Rab35 is also involved in these processes. Moreover, C-terminal of FLCN binds to and activates Rab35. Of special interest is the observation that erlotinib, a selective EGFR inhibitor, not only obstructs the EGFR-mediated cellular signaling, but also abolishes EGF-stimulated EGFR degradation. Further results reveal that EGF facilitates the activation of Rab35, and FLCN modulates EGF-dependent Rab35 activation and cell growth.Conclusions: Taken together, our study proposes a negative-feedback regulation model in which FLCN mediates EGF-induced Rab35 activation, thereby increasing EGFR degradation and attenuating EGFR signaling.

Hypoxia-inducible factor 2α (HIF2α) plays critical roles in cancer progression. Although the mechanisms of HIF2α translation and degradation have been well studied, the mechanism for HIF2α regulation at transcriptional level is still not fully understood. Here, we present evidence that DNA methylation in promoter contributes to transcription of EPAS1 coding HIF2α. Methylated CpG binding protein 3 (MBD3) contributes to the intricate regulatory mechanism. We showed that MBD3 bound to the EPAS1 promoter in breast cancer cells and amplified EPAS1 transcription through demethylating CpG located around transcriptional start site in MDA-MB-468 cells. This enabled MDA-MB-468 cells to activate HIF2α-mediated angiogenesis. However, in 7860 cells, the demethylation function of MBD3 on EPAS1 was not observed because of the poor methylated-CpG promoter. Nevertheless, depletion of MBD3 induced by shRNA decreased EPAS1 transcription and therefore decreased HIF2α-mediated cellular response in both MDA-MB-468 and 7860 cancer cells. These results indicated that the endogenous MBD3 was involved in regulating the transcription and therefore the transcriptional activities of HIF2α, suggesting that MBD3 may be a potential therapeutic target of tumor.