RhoB loss induces Rac1-dependent mesenchymal cell invasion in lung cells through PP2A inhibition.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which is mainly due to its high risk of metastatic dissemination. One critical point of this process is the ability of cancer cells to detach from the primary tumor and migrate through the extracellular matrix; however, the underlying molecular mechanisms are not yet fully understood. In the present study, we identified the small GTPase RhoB as a key regulator of bronchial cell morphology in a three-dimensional (3D) matrix. RhoB loss, which is frequently observed during lung cancer progression, induced an epithelial-mesenchymal transition (EMT) characterized by an increased proportion of invasive elongated cells in 3D. The process was mediated by Slug induction and E-cadherin repression. In addition, downregulation of RhoB induced Akt1 activation, which in turn activated Rac1 through the guanine-exchange factor Trio to control cell shape rearrangement. Further, we provide evidence that RhoB interacted with and positively regulates phosphatase PP2A through the recruitment of its regulatory subunit B55, which was found to be crucial for Akt dephosphorylation. B55 inhibition completely suppressed RhoB-mediated PP2A regulation. Finally, we show that PP2A inactivation, by targeting either its catalytic or its regulatory B55 subunit, completely reversed RhoB-dependent morphological changes and also fully prevented the ability of RhoB to decrease the invasiveness of bronchial cells. Altogether, these results highlight a novel signaling axis and describe new molecular mechanisms that could explain the tumor suppressor role of RhoB in lung cancer. Therefore, we propose that RhoB could be responsible for early metastatic prevention by inhibiting the EMT-derived invasiveness of lung cells through the control of PP2A activity.

Neuron-derived orphan receptor-1 (NOR-1) is induced by thrombin and mediates vascular endothelial cell growth.

BACKGROUND AND AIM: Neuron-derived orphan receptor-1 (NOR-1) is a transcription factor overexpressed in human atherosclerotic plaques that is involved in vascular smooth muscle cell (VSMC) proliferation. The aim of this study was to analyze the role of NOR-1 in thrombin-induced endothelial cell growth. RESULTS: Thrombin induced an early and transient up-regulation of NOR-1 in human umbilical vein endothelial cells (HUVEC). NOR-1 up-regulation by thrombin is dependent on multiple pathways, including cytosolic Ca(2+), activation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) pathways [both extracellular-regulated kinase (ERK) and p38 MAPK], and downstream activation of cAMP response element binding protein (CREB). The critical role of CREB in the induction of NOR-1 by thrombin was demonstrated using a dominant-negative of CREB. By site-direct mutagenesis we identified two CRE sites present at -79 and -53 bp in the NOR-1 promoter involved in the up-regulation of NOR-1 by thrombin. Inhibition of thrombin receptor PAR-1 abolished CREB activation, NOR-1 up-regulation and DNA synthesis (used as an index of cell proliferation). TRAP-6 mimicked both NOR-1 up-regulation and CREB activation induced by thrombin, while PPACK (an irreversible thrombin inhibitor) prevented such an effect. Direct inhibition of thrombin-induced NOR-1 up-regulation, using antisense oligonucleotides or siRNA against NOR-1, reduced DNA synthesis and endothelial cell re-growth after injury in an in vitro model of wound repair. CONCLUSIONS: These results indicate that NOR-1 up-regulation plays a key role in thrombin-induced endothelial cell growth. Strategies aimed to block NOR-1 could be useful to prevent vascular effects triggered by thrombin.