Marion De Toledo

Loss of p53 promotes RhoA–ROCK-dependent cell migration and invasion in 3D matrices

In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA–ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.

Characterization of TCL, a New GTPase of the Rho Family related to TC10 and Cdc42

GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis. We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively. This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14. TCL mRNA is 2.5 kilobases long and is mainly expressed in heart. In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10. Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP. Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane. Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.

Pro-metastatic splicing of Ron proto-oncogene mRNA can be reversed: Therapeutic potential of bifunctional oligonucleotides and indole derivatives

Alternative splicing is a key molecular mechanism for increasing the complexity of the human transcriptome. Nearly all human genes are regulated by alternative splicing and the deregulation of this process has a causative role in various human diseases, including cancer. The discovery that alternatively spliced isoforms of several genes are expressed selectively in tumor cells opened the exciting possibility that pharmacological treatment of aberrant splicing could lead to new anti-cancer therapeutic approaches. An alternatively spliced isoform of a scatter factor receptor and proto-oncogene, Ron, accumulates during tumor progression of epithelial tissues and is able to confer an invasive phenotype to the expressing cells. This isoform, called ΔRon, originates from skipping of exon 11, and this specific splicing event is controlled by the expression level of the splicing factor and proto-oncogene SF2/ASF. Over-expression of SF2/ASF, which occurs frequently in various human tumors, induces the production of ΔRon and activates the epithelial to mesenchymal transition (EMT), leading to increased cell motility. In this paper, we have used targeted oligonucleotide enhancers of splicing (TOES) to recruit positive splicing factors to Ron exon 11 and thereby stimulate its inclusion. As an alternative approach, we have used selected indole derivatives that target ASF/SF2 splicing activity. Both treatments correct aberrant ΔRon splicing, restoring the incorporation of Ron exon 11. Notably, indole derivatives are also able to affect the invasive phenotype of the cells. Thus, these treatments may have therapeutic applications for anti-cancer purposes.

The gene for a new brain specific RhoA exchange factor maps to the highly unstable chromosomal region 1p36.2-1p36.3.

Guanine nucleotide exchange factors from the Dbl family are proto-oncogenic proteins that activate small GTPases of the Rho family. Here we report the characterization of GEF720, a novel Dbl-like protein related to p115Rho-GEF. GEF720 activated RhoA both in our recently developed Yeast Exchange Assay and in biochemical in vitro exchange assays. GEF720 induced RhoA dependent assembly of actin stress fibers in REF52 fibroblastic cells. In NIH3T3 cells this Dbl-like protein elicited formation of transformation foci with a morphology similar to RhoA-V14 induced foci. In the PC12 neuron-like cell line, expression of GEF720, whose mRNA is brain specific, inhibited NGF-induced neurite outgrowth. Finally, GEF720 gene is located on human chromosome 1 on band 1p36, between Tumor Protein 73 and Tumor Necrosis Factor Receptor 12, two genes rearranged in many neuroblastoma cell lines. Together, these results show that this new Dbl related protein, GEF720, is an exchange factor that can directly activate RhoA in vivo and is potentially involved in the control of neuronal cell differentiation. GEF720 is also a new candidate gene involved in the progression of neuroblastoma and developmental abnormalities associated with rearrangements in the 1p36 chromosomal region.

TNFα induces sequential activation of Cdc42- and p38/p53-dependent pathways that antagonistically regulate filopodia formation

Cell migration is an essential function in various physiological processes, including tissue repair and tumour invasion. Repair of tissue damage requires the recruitment of fibroblasts to sites of tissue injury, which is mediated in part by the cytokine tumour necrosis factor α (TNFα). As dynamic rearrangements of actin cytoskeleton control cell locomotion, this implicates that TNFα is a potent coordinator of cellular actin changes. We have investigated the role of TNFα in regulating the cortical actin-containing structures essential for cell locomotion called filopodia. Kinetic analysis of TNFα-treated mouse embryonic fibroblasts (MEFs) revealed a dual effect on filopodia formation: a rapid and transient induction mediated by Cdc42 GTPase that is then counteracted by a subsequent sustained inhibition requiring activation of the mitogen-activated protein kinase p38 but not Cdc42 activity. This inhibition also involves the tumour suppressor p53, given that it is activated in response to TNFα following the same time course as the decrease of filopodia formation. This functional activation of p53, measured by transcription induction of its target p21WAF1(p21), is also associated with p38 kinase-dependent phosphorylation of p53 at serine 18. Furthermore, TNFα did not inhibit filopodia formation in MEFs treated with the transcription inhibitor actinomycin D, in p53-deficient MEFs, or MEFs expressing p53 mutants H273 or H175, which supports a role for the transcriptional activity of p53 in mediating TNFα-dependent filopodia inhibition. Our data delineate a novel inhibitory pathway in which TNFα prevents filopodia formation and cell migration through the activation of the mitogen-activated protein kinase (MAPK) p38, which in turn activates p53. This shows that TNFα on its own initiates antagonistic signals that modulate events linked to cell migration.

Cooperative anti-invasive effect of Cdc42/Rac1 activation and ROCK inhibition in SW620 colorectal cancer cells with elevated blebbing activity.

Rho GTPases are key regulators of tumour cell invasion and therefore constitute attractive targets for the design of anticancer agents. Several strategies have been developed to modulate their increased activities during cancer progression. Interestingly, none of these approaches took into account the existence of the well-known antagonistic relationship between RhoA and Rac1. In this study, we first compared the invasiveness of a collection of colorectal cancer cell lines with their RhoA, Rac1 and Cdc42 activities. A marked decrease of active Cdc42 and Rac1 correlated with the high invasive potential of the cell lines established from metastatic sites of colorectal adenocarcinoma (LoVo, SKCo1, SW620 and CoLo205). Conversely, no correlation between RhoA activity and invasiveness was detected, whereas the activity of its kinase effector ROCK was higher in cancer cell lines with a more invasive phenotype. In addition, invasiveness in these colon cancer cell lines was correlated with a typical round and blebbing morphology. We then tested whether treatment with PDGF to restore Cdc42 and Rac1 activities and/or with Y27632, a chemical inhibitor of ROCK, could decrease the invasiveness of SW620 cells. The association of both treatments substantially decreased the invasive potential of SW620 cells and this effect was accompanied by loss of membrane blebbing, restoration of a more elongated cell morphology and re-establishment of E-cadherin-dependent adherens junctions. This study paves the road to the development of therapeutic strategies in which different Rho GTPase modulators are combined to modulate the cross-talk between Rho GTPases and their specific input in metastatic progression.

Antagonistic functions of LMNA isoforms in energy expenditure and lifespan

Alternative RNA processing of LMNA pre‐mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson‐Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin‐expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS‐like mice, whereas lamin C‐only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C‐only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.

TP53 drives invasion through expression of its Δ133p53β variant

TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53β promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53β increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53β is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53β depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53β induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression. DOI: http://dx.doi.org/10.7554/eLife.14734.001

Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

Matching sets of human primary fibroblasts cocultured with placenta explants are used to compare tissue capacities to support trophoblast invasion. Substituting endometrium with dermis dramatically reduces EVCT interstitial invasion, a phenomenon related to the ECM fibronectin content, FN alternative splicing, and expression of the SR protein SRSF1.