P-rex1 cooperates with PDGFRβ to drive cellular migration in 3D microenvironments

Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a key determinant of progression to metastasis in a number of human cancers. In accordance with this proposed role in cancer cell invasion and metastasis, we find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell line is sufficient to drive multiple migratory and invasive phenotypes. The invasive phenotype is greatly enhanced by the presence of a gradient of serum or platelet-derived growth factor, and is dependent upon the expression of functional PDGF receptor β. Consistently, the invasiveness of WM852 melanoma cells, which endogenously express P-Rex1 and PDGFRβ, is opposed by siRNA of either of these proteins. Furthermore, the current model of P-Rex1 activation is advanced through demonstration of P-Rex1 and PDGFRβ as components of the same macromolecular complex. These data suggest that P-Rex1 has an influence on physiological migratory processes, such as invasion of cancer cells, both through effects upon classical Rac1-driven motility and a novel association with RTK signalling complexes

Invasion of Normal Human Fibroblasts Induced by v-Fos Is Independent of Proliferation, Immortalization, and the Tumor Suppressors p16(INK4a) and p53

Invasion is generally perceived to be a late event during the progression of human cancer, but to date there are no consistent reports of alterations specifically associated with malignant conversion. We provide evidence that the v-Fos oncogene induces changes in gene expression that render noninvasive normal human diploid fibroblasts highly invasive, without inducing changes in growth factor requirements or anchorage dependence for proliferation. Furthermore, v-Fos-stimulated invasion is independent of the pRb/p16(INK4a) and p53 tumor suppressor pathways and telomerase. We have performed microarray analysis using Affymetrix GeneChips, and the gene expression profile of v-Fos transformed cells supports its role in the regulation of invasion, independent from proliferation. We also demonstrate that invasion, but not proliferation, is dependent on the activity of the up-regulated epidermal growth factor receptor. Taken together, these results indicate that AP-1-directed invasion could precede deregulated proliferation during tumorigenesis and that sustained activation of AP-1 could be the epigenetic event required for conversion of a benign tumor into a malignant one, thereby explaining why many malignant human tumors present without an obvious premalignant hyperproliferative dysplastic lesion

Regulation of a multigenic invasion programme by the transcription factor, AP-1: re-expression of a down-regulated gene, TSC-36, inhibits invasion

The transcription factor AP-1 (activator protein-1) is required for transformation by many oncogenes, which function upstream of it in the growth factor-ras signal transduction pathway. Previously, we proposed that one role of AP-1 in transformation is to regulate the expression of a multigenic invasion programme. As a test of this proposal we sought to identify AP-1 regulated genes based upon their differential expression in 208F rat fibroblasts transformed by FBR-v-fos (FBR), and to determine if they functioned in the invasion programme. Subtracted cDNA libraries specific for up- or down-regulated genes in FBRs compared to 208Fs were constructed and analysed. Northern analysis revealed that the cDNAs in both libraries represented differentially expressed genes. Nucleic acid sequence analysis of randomly selected cDNA clones from each library coupled with searches of nucleic acid and amino acid sequence databases determined that many of the cDNAs represented proteins that function in various aspects of the invasion process. Functional analysis of one the down-regulated genes, TSC-36/follistatin-related protein (TSC-36/Frp), which has not previously been associated with invasion, demonstrated that its expression in FBRs inhibited in vitro invasion. These results support the proposal that AP-1 in transformed cells regulates a multigenic invasion programme

Association of P-Rex1 with PDGFRβ from immortalised human fibroblasts.

<p>(A) Control vector (pLHCX) or myc-RacGEF expressing immortalised human fibroblasts were transfected with GFP-tagged PDGFRβ and plated onto glass cover slips. Cells were fixed and the myc-tagged RacGEFs were stained by immunofluorescent labelling with an antibody recognising the myc-epitope followed by AlexaFluor-555 conjugated secondary antibodies. (B, C) Myc-tagged RacGEFs were stably expressed in immortalised human fibroblasts. The cells were then grown in serum-containing medium (B) or serum-starved for 18 hr (right hand lanes in (C)) prior to lysis in a buffer containing 0.15% Tween-20. Myc-tagged RacGEFs were immunoprecipitated from lysates using an antibody recognising the myc-epitope, and immunoisolated material was probed for the presence of PDGFRβ by immunoblotting.</p

Chemical inhibition of PDGFR signalling alters the migratory behaviour of P-Rex1 expressing cells in both 2D and 3D environments.

<p>(A) Following a period of serum starvation, P-Rex1 expressing fibroblasts were stimulated with PDGF-BB, in the presence and absence of DMSO control or chemical inhibitors as indicated. Cells were the lysed and the levels of P-Rex1 and PDGFRβ were determined by Western blotting. The activity of the PI3K/Akt axis was determined by Western blotting for Akt which was phosphorylated at Thr⁴⁷³. (B) Quantification of the invasive migration of P-Rex1 expressing fibroblasts into Matrigel in the presence and absence of DMSO, PDGFRi (5 µM) or PI-103 (1 µM) as indicated (±SEM, ***<i>P</i><0.0005 Mann-Whitney rank sum test, 3 independent experiments). (C) P-Rex1 expressing fibroblasts emerging from the migratory front of scratch-wound assays in the presence and absence of DMSO PDGFRi (5 µM) or PI-103 (1 µM) were analysed using time-lapse microscopy, with images captured every 10 minutes over a period of 18 hours. Individual cells were manually tracked and the migratory path of representative cells depicted as track plots. The phase contrast images correspond to the initial frame from the supplementary movies S5–S7 as indicated. (D-F) Influence of chemical inhibition on migratory behaviour was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053982#pone-0053982-g002" target="_blank">Figure 2</a> by determination of mean cellular velocity (D), forward migratory index (E) and migratory persistence (F). (Bars 50 µm, <i>*P</i><0.05, <i>***P</i><0.0005, Mann-Whitney rank sum test).</p

PDGFRβ is required for invasive migration of P-Rex1 expressing human fibroblasts.

<p>(A) Invasion of P-Rex1 expressing human fibroblasts transfected with non-targeting siRNAs (siControl) or those targetting PDGFRα or PDGFRβ,either alone or in combination, was assayed and quantified as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053982#pone-0053982-g003" target="_blank">Figure 3</a>. (B) Embryonic fibroblasts from PDGFRα/β −/− mice were stably transfected with P-Rex1 or PDGFRβ, either alone or in combination. Invasion was determined as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053982#pone-0053982-g003" target="_blank">Figure 3</a>. (±SEM, *<i>P</i><0.05, **<i>P</i><0.005 Mann-Whitney rank sum test, 3 independent experiments).</p

Expression of P-Rex1 and PDGFRβ are required for invasive migration of WM852 melanoma cells.

<p>(A) Immunoblot showing depletion of P-Rex1 or PDGFRβ following transfection of cells with non-targeting control or siRNA oligonucleotides directed against P-Rex1 or PDGFRβ. (B,C) Invasive migration of WM852 cells into Matrigel following transfection with siRNA oligonucleotides targeting P-Rex1 or PDGFβ was determined as for Fig. 3A. In (C), panels have been appended to demonstrate the adherence of the WM852 cells to the lower surface (LS) of the transwell.(±SEM, ***<i>P<</i>0.0005 Mann-Whitney rank sum test, 3 independent experiments).</p

P-Rex1 expression promotes persistent migration on a 2D substrate.

<p>(A) Parental (Tif), and control vector (pLHCX) or RacGEF expressing fibroblasts emerging from the migratory front of scratch-wound assays were analysed using time-lapse microscopy, with images captured every 10 minutes over a period of 18 hours. Individual cells were manually tracked and the migratory path of representative cells depicted as track plots. The phase contrast images correspond to the initial frame from the supplementary movies S1–S4 as indicated. (B-D) Influence of RacGEF expression on migratory behaviour was quantified by determination of mean cellular velocity (B), forward migratory index (C) and migratory persistence (D). (Bars 50 µm, **<i>P</i><0.005, Mann-Whitney rank sum test).</p

P-Rex1 requires a factor present in serum in order to drive alterations in cell shape and invasion; PDGF can substitute for serum to drive invasion of P-Rex1 expressing cells.

<p>(A) P-Rex1 expressing fibroblasts cultured under normal growth conditions or serum-starved for 12 hours were labelled as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053982#pone-0053982-g001" target="_blank">Figure 1A</a> above. (B) High throughput imaging was used to quantify the ruffle intensity and mean cellular width/length ratio of P-Rex1 expressing fibroblasts in the presence and absence of serum. (C) The requirement for a serum gradient in the invasive migration of P-Rex1 expressing fibroblasts was determined using an inverted invasion assay as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053982#pone-0053982-g003" target="_blank">Figure 3A</a>. (D) The invasive migration of vector control (pLHCX) or P-Rex1 expressing fibroblasts into Matrigel in the absence or presence of a serum (0–10%) or PDGF-BB (0–50 ng ml⁻¹) gradient was assayed and quantified as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053982#pone-0053982-g003" target="_blank">Figure 3B</a> (±SEM, ***<i>P</i><0.0005, Mann-Whitney rank sum test, 3 independent experiments). (E) The comparative invasive migration of P-Rex1 expressing fibroblasts into Matrigel in the absence or presence of gradients of PDGF-BB or NRG1 was assayed and quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053982#pone-0053982-g003" target="_blank">Figure 3B</a> (±SEM, ***<i>P</i><0.0005 Mann-Whitney rank sum test, 3 independent experiments).</p