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

Abstract

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.

RETRACTED: The Pseudogene DUXAP8 Promotes Non-small-cell Lung Cancer Cell Proliferation and Invasion by Epigenetically Silencing EGR1 and RHOB

“…40 Mazieres et al 41 found that the level of the RhoB protein dramatically decreased over the course of lung cancer progression and was lost in 96% of invasive tumors, whereas the ectopic expression of RHOB in the lung cancer cell line A549 suppressed cell proliferation both in vitro and in vivo. Moreover, RhoB loss could induce Rac1-dependent mesenchymal cell invasion in lung cells through the control of PP2A activity, 29 and promote the invasion of human bronchial cells via the activation of AKT1. 42 Furthermore, Mazieres et al 43 also reported that the loss of RhoB expression was induced via epigenetic regulation by histone deacetylation in lung cancer, whereas we found that LSD1 recruited by DUXAP8-mediated histone demethylation also contributed to RHOB loss of expression in NSCLC cells.…”

“…EGR1 and RHOB are newly identified tumor suppressors, and their loss is frequently observed during lung cancer development and progression. 29,30 Therefore, we assumed that DUXAP8 contributes to NSCLC by repressing EGR1 and RHOB expression. Accordingly, we used western blot analysis to examine their protein levels in H1299 and H1975 cells transfected with DUXAP8 or control siRNA.…”

RASSF1A Suppresses the Invasion and Metastatic Potential of Human Non–Small Cell Lung Cancer Cells by Inhibiting YAP Activation through the GEF-H1/RhoB Pathway

“…12), tumorigenic epithelial cell lines BEAS-2B, cancer-derived cell lines A549 and H1975 from ATCC, and BEAS-2B-KRasV12 from Dr. Pradines (9,13) were grown in supplemented appropriate media (Supplementary Table S1), transfected at 30% confluence, using Lipofectamine RNAiMAX (Invitrogen) with siRNA, plasmid DNA, or control mimics (Dharmacon; Supplementary Tables S2 and S3). HBEC-3 and HBEC-3-KRasG12V were authenticated using standard karyotyping techniques as previously described (12).…”

“…S8E), and we hypothesized that such phosphatase regulation could influence GEFH1 phosphorylation state. Recently, siRNA-mediated depletion of RhoB was reported to downregulate PP2A activity (13). By immunoprecipitating PP2ACa from cells lysates derived from BEAS2B cells expressing RASSF1A, we detected both GEFH1 and RhoB with specific antibodies (Supplementary Fig.…”

“…Cells were lysed in chilled immunoprecipitation buffer and the cleared lysate (500 mg) incubated with 3 mg of the indicated antibody and 30 mL of protein-A agarose beads (Repligen) in 1 mL of IP buffer (13). Beads were resuspended in 30 mL of 2Â Laemmli buffer and subjected to Western blotting.…”