Hypoxic Tumor Cell Modulates Its Microenvironment to Enhance Angiogenic and Metastatic Potential by Secretion of Proteins and Exosomes

G protein-coupled receptors stimulation and the control of cell migration.

Characterization of EHop-016, Novel Small Molecule Inhibitor of Rac GTPase

Cell biology of the movement of breast cancer cells: intracellular signalling and the actin cytoskeleton.

Rational: Patients with hepatocellular carcinoma (HCC) have a poor prognosis mostly due to intrahepatic as well as distal metastasis. Vasodilator-stimulated phosphoprotein (VASP), a regulator of actin cytoskeleton and cell migration, is overexpressed in HCC and correlated with its malignant features and poor prognosis. Very little is known about its function in HCC. Methods: qRT-PCR, Western blot and IHC were used to detect the VASP expression in tissues and cells. Transwell and wound healing assays were used to measure the migration and invasion of HCC cells. Immunoblotting and immunofluorescence were used for detection of epithelial-to-mesenchymal transition (EMT) progression in HCC cells. A lung metastasis mouse model was used to evaluate metastasis of HCC in vivo. The putative targets of miR-204 were disclosed by public databases and a dual-luciferase reporter assay. IP was used to show the interaction between VASP and CRKL. ChIP was used to analyze the binding of HIF-1α to VASP promoter region. Results: Our data involving both gain- and loss-of-function studies revealed that VASP activated AKT and ERK signaling and promoted HCC migration and invasion in vitro and in vivo by altering the EMT phenotype and expression of MMPs. We investigated the positive correlation between VASP and an adapter protein, CRKL. VASP dynamically co-localized at the SH3N domain of CRKL and mediated its function. Mechanistically, VASP overexpression at the transcriptional level was mediated by HIF-1α through direct binding to two hypoxia response elements (HRE) in the VASP promoter region. Furthermore, we identified hypoxia-induced down-regulation of miR-204, which functioned as the regulator of VASP overexpression at the post-transcriptional level. Also, hypoxia-activated p-Smad3 dependent TGF-β signaling indirectly promoted VASP expression. Conclusion: A variety of hypoxia-induced molecular mechanisms contributed to the upregulation of VASP at transcriptional and post-transcriptional levels. These mechanisms involved CRKL, HIF-1α, miR-204, and TGF-β activating the AKT and ERK signaling to promote EMT and expression of MMPs. Taken together, our results defined VASP as an oncogene of HCC pathogenesis and metastasis with the potential to serve as a prognostic biomarker.

Hypoxia-induced up-regulation of VASP promotes invasiveness and metastasis of hepatocellular carcinoma.

This study aimed to investigate the role of the cytoskeleton-associated protein vasodilator-stimulated phosphoprotein (VASP) on the migration and invasion of human breast cancer cells and its relationship to Rac1 which is a member of the Rho family and has been found to be implicated in tumorigenesis, invasion and metastasis. We detected the mRNA and protein expression levels of VASP and Rac1 of the non-invasive breast cancer cell line MCF-7 as well as the invasive cell line MDA-MB-231 by RT-PCR and Western blotting. GST pull-down assay was used to examine the activity of Rac1. Accordingly, the cell invasive migration ability was analyzed in a wound-healing assay (2D) and transwell assays (3D migration and invasion). We then used VASP-siRNA to inhibit the expression of VASP in breast cancer cells in order to study the relationship between the VASP expression level and the invasive migration ability of breast cancer cells. Rac1-siRNA was used to decrease the expression of Rac1, and observe its effect on the VASP expression level together with the migration and invasion ability of MCF-7 and MDA-MB-231 cells. Our results revealed that the invasive breast cancer cell line MDA-MB-231 showed a higher Rac1 activity and VASP expression level compared with the non-invasive MCF-7. Inhibition of Rac1 or VASP by siRNA, respectively, decreased the migration and invasion ability of breast cancer cells and the transfection of Rac1 siRNA-mediated reduction of VASP expression at mRNA and protein levels. Collectively, our data showed that the higher expression level of VASP contributed to a higher invasive migration capacity of human breast cancer cells which was controlled by the Rac1 pathway.

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Yun Wei

Papers

Positive regulation of migration and invasion by vasodilator-stimulated phosphoprotein via Rac1 pathway in human breast cancer cells