RhoB as a tumor suppressor: It's all about localization.

The small GTPase RhoB is distinguished from other Rho proteins by its unique subcellular localization in endosomes, multivesicular bodies, and nucleus. Despite high sequence homology with RhoA and RhoC, RhoB is mainly associated with tumor suppressive function, while RhoA and RhoC support oncogenic transformation in most malignancies. RhoB regulates the endocytic trafficking of signaling molecules and cytoskeleton remodeling, thereby controlling growth, apoptosis, stress response, immune function, and cell motility in various contexts. Some of these functions may be ascribed to RhoB's unique subcellular localization to endocytic compartments. Here we describe the pleiotropic roles of RhoB in cancer suppression in the context of its subcellular localization, and we discuss possible therapeutic avenues to pursue and highlight priorities for future research.

Studying Chemotactic Migration in Dunn Chamber: An Example Applied to Adherent Cancer Cells.

Cell migration is a vital process in the development of multicellular organisms. When deregulated, it is involved in many diseases such as inflammation and cancer metastisation. Some cancer cells could be stimulated using chemoattractant molecules, such as growth factor Heregulin ß1. They respond to the attractant or repellent gradients through a process known as chemotaxis. Indeed, chemotactic cell motility is crucial in tumour cell dissemination and invasion of distant organs. Due to the complexity of this phenomenon, the majority of available in vitro methods to study the chemotactic motility process have limitations and are mainly based on endpoint assays, such as the Boyden chamber assay. Nevertheless, in vitro time-lapse microscopy represents an interesting opportunity to study cell motility in a chemoattracting gradient, since it generates large volume image-based information, allowing the analysis of cancer cell behaviours. Here, we describe a detailed time-lapse imaging protocol, designed for tracking T47D human breast cancer cell line motility, toward a gradient of Heregulin ß1 in a Dunn chemotaxis chamber assay. The protocol described here is readily adapted to study the motility of any adherent cell line, under various conditions of chemoattractant gradients and of pharmacological drug treatments. Moreover, this protocol could be suitable to study changes in cell morphology, and in cell polarity.

NR1D1 regulation by Ran GTPase via miR4472 identifies an essential vulnerability linked to aneuploidy in ovarian cancer.

While aneuploidy is a main enabling characteristic of cancers, it also creates specific vulnerabilities. Here we demonstrate that Ran inhibition targets epithelial ovarian cancer (EOC) survival through its characteristic aneuploidy. We show that induction of aneuploidy in rare diploid EOC cell lines or normal cells renders them highly dependent on Ran. We also establish an inverse correlation between Ran and the tumor suppressor NR1D1 and reveal the critical role of Ran/NR1D1 axis in aneuploidy-associated endogenous DNA damage repair. Mechanistically, we show that Ran, through the maturation of miR4472, destabilizes the mRNA of NR1D1 impacting several DNA repair pathways. We showed that NR1D1 interacts with both PARP1 and BRCA1 leading to the inhibition of DNA repair. Concordantly, loss of Ran was associated with NR1D1 induction, accumulation of DNA damages, and lethality of aneuploid EOC cells. Our findings suggest a synthetic lethal strategy targeting aneuploid cells based on their dependency to Ran.

ARF6 controls RHOB targeting to endosomes regulating cancer cell invasion.

Endocytic trafficking has emerged as an essential mechanism to spatiotemporally coordinate signaling protein complexes that control cytoskeletal dynamics and cell motility. Our study established an unexpected regulatory mechanism whereby ADP ribosylation factors 6 (ARF6) controls the stability and endosomal localization of RAS homologous protein B (RHOB) to regulate cell invasion downstream of the oncogenic receptor tyrosine kinase, MET.

CD154 inhibits death of T cells via a Cis interaction with the α5ß1 integrin.

CD154 plays a major role in the pathogenesis of several autoimmune and inflammatory diseases. In addition to CD40, soluble CD154 (sCD154) binds to other receptors namely αIIbß3, αMß2, α5ß1 and αvß3 integrins. We have previously reported that binding of sCD154 to α5ß1 integrin expressed on several human T cell lines is capable of inhibiting Fas-induced cell death. In the current study, we show that such effect of the sCD154/α5ß1 interaction is not restricted to the cell death response induced by Fas but could also be exhibited toward other death signals such as TRAIL and TNF- α. We also demonstrate that sCD154 is capable of inhibiting Fas-mediated death of human activated T cells, more importantly of CD4+ than CD8+ T ones. Our data also show that membrane-bound CD154 and α5ß1 integrin expressed on the surface of distinct cells failed to influence cell death responses. However, when membrane-bound CD154 and α5ß1 are expressed on the surface of same cell, their interaction was capable of down regulating cell death. CD154 was shown to co-localize with the α5ß1 integrin on the surface of these cells. These data strongly suggest a cis-type of interaction between CD154 and α5ß1 when both are expressed on the same cell surface, rather than a trans-interaction which usually implicates the ligand and its receptor each expressed on the surface of a distinct cell. Taken together, these findings add to the list of roles through which CD154 is contributing to the pathogenesis of autoimmune-inflammatory diseases, i.e. by protecting T cells from death and enhancing their survival.

Real-time Three-dimensional Tracking of Endocytic Vesicles.

Endocytic trafficking and recycling are fundamental cellular processes that control essential functions such as signaling protein complexes transport and membrane identity. The small GTPase Rabs are indispensable component of the endosomal recycling machinery. The Rabs bind to effectors to mediate their functions, such as protein sorting and degradation, membrane tethering or lipid modification, and organelle motility. Due to the complex and dynamic nature of endosomal compartments and tracking route, detailed multiparametric analyses of three-dimensional data by quantitative methods are challenging. Here, we describe a detailed time-lapse imaging protocol designed for the quantitative tracking of single endosomal vesicles, using GFP-Rab4-positive recycling endosomes. This method permits automated tracking of single endocytic vesicles in three-dimensional live cell imaging, allowing the study of multiple parameters such as abundance, speed, directionality, and subcellular localization, as well as protein colocalization. This protocol can be broadly used in any kind of cellular models, under various contexts, including growth factors stimulation, gene knockdowns, drug treatments, and is suitable for high throughput screens.

Colorimetric RhoB GTPase Activity Assay.

The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton rearrangements and FA dynamics and are required for effective cell migration and invasion, as well as cell cycle progression and apoptosis. The Rho GTPases activity is regulated by conformational switching between GTP-bound (active) and GDP-bound (inactive) states. This GTP/GDP cycling is tightly controlled by the guanine nucleotide exchange factors (GEFs), which function as activators by catalyzing the exchange of GDP for GTP and by the GTPase-activating proteins (GAPs), which enable hydrolysis of GTP leading to the Rho GTPase inactivation. Here, we describe a detailed protocol to perform a RhoB G-LISA activation assay to detect the level of GTP-loaded RhoB in vitro. This is the first colorimetric assay designed to specifically measure RhoB activation. This method was developed by adapting the RhoA G-LISA Activation Assay Kit (Cytoskeleton, Inc.) and allow the precise measurement of RhoB activity in less than 3 hours. This rapid methodology can be broadly used to assess the level of GTP-loaded RhoB in any kind of cellular models, to appreciate either the role RhoB activation in physiological processes, diseases, oncogenic transformation or for drug discovery in high throughput screens.

Arf6 regulates RhoB subcellular localization to control cancer cell invasion.

The ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates endocytic recycling processes in concert with various effectors. Arf6 controls cytoskeletal organization and membrane trafficking; however, the detailed mechanisms of regulation remain poorly understood. Here, we report that Arf6 forms a complex with RhoB. The interaction between RhoB and Arf6 is mediated by the GCI (glycine, cysteine, and isoleucine) residues (188-190) of RhoB. Specific targeting of Arf6 to plasma membrane or mitochondrial membranes promotes recruitment and colocalization of RhoB to these membrane microdomains. Arf6 depletion promotes the loss of RhoB from endosomal membranes and leads to RhoB degradation through an endolysosomal pathway. This results in defective actin and focal adhesion dynamics and increased 3D cell migration upon activation of the Met receptor tyrosine kinase. Our findings identify a novel regulatory mechanism for RhoB localization and stability by Arf6 and establish the strict requirement of Arf6 for RhoB-specific subcellular targeting to endosomes and biological functions.

Ran promotes membrane targeting and stabilization of RhoA to orchestrate ovarian cancer cell invasion.

Ran is a nucleocytoplasmic shuttle protein that is involved in cell cycle regulation, nuclear-cytoplasmic transport, and cell transformation. Ran plays an important role in cancer cell survival and cancer progression. Here, we show that, in addition to the nucleocytoplasmic localization of Ran, this GTPase is specifically associated with the plasma membrane/ruffles of ovarian cancer cells. Ran depletion has a drastic effect on RhoA stability and inhibits RhoA localization to the plasma membrane/ruffles and RhoA activity. We further demonstrate that the DEDDDL domain of Ran is required for the interaction with serine 188 of RhoA, which prevents RhoA degradation by the proteasome pathway. Moreover, the knockdown of Ran leads to a reduction of ovarian cancer cell invasion by impairing RhoA signalling. Our findings provide advanced insights into the mode of action of the Ran-RhoA signalling axis and may represent a potential therapeutic avenue for drug development to prevent ovarian tumour metastasis.

CLIP-170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration.

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus-end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward-directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP-170, as well as the association of CLIP-170 to MT plus-ends. In response to HGF, entry of Met into Rab4-positive endosomes results in Golgi-localized γ-ear-containing Arf-binding protein 3 (GGA3) and CLIP-170 recruitment to an activated Met RTK complex. We conclude that CLIP-170 co-ordinates the recycling and the transport of Met-positive endocytic vesicles to plus-ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration.

COMMD5/HCaRG Hooks Endosomes on Cytoskeleton and Coordinates EGFR Trafficking.

COMMD5/HCaRG is involved in tissue repair, and its low expression is associated with tumorigenicity. Cell growth, migration, and differentiation are controlled by COMMD5. We previously reported that COMMD5 inhibited the growth of renal carcinoma cells by regulating expression or phosphorylation of ErbB members. Here, we demonstrate that COMMD5 is crucial for the stability of the cytoskeleton. Its silencing leads to a major re-organization of actin and microtubule networks. The N terminus of COMMD5 binds to the endosomal Rab5, and its C terminus, including the COMMD domain, binds to the cytoskeletal scaffolding. COMMD5 participates in long-range endosome transport, including epidermal growth factor receptor (EGFR) recycling, and provides the strength to deform and assist the scission of vesicles into sorting endosomes. This study establishes the molecular mechanism by which COMMD5 acts as an adaptor protein to coordinate endosomal trafficking and reveals its important role for EGFR transport and activity.

5'-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion.

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5'-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2-Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease.

Met receptor tyrosine kinase signals through a cortactin-Gab1 scaffold complex, to mediate invadopodia.

Invasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2-associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure-function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1-cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.

ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells.

Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDia1. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDia1, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coli) and cytoplasmic linker-associated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering.

Memo-RhoA-mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells.

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as alpha-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo-RhoA-mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.