Viola Hélène Lobert

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration

Although phosphatidylinositol 5‐phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3‐kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P‐binding motifs, we identified the phosphoinositide 5‐kinase PIKfyve and the phosphoinositide 3‐phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P2. The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P‐producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.

PI3K-AKT-mTOR pathway is dominant over androgen receptor signaling in prostate cancer cells

Background: Androgen receptor (AR) and the phosphatidylinositol-3 kinase (PI3K) signaling are two of the most important pathways implicated in prostate cancer. Previous work has shown that there is crosstalk between these two pathways; however, there are conflicting findings and the molecular mechanisms are not clear. Here we studied the AR–PI3K pathway crosstalk in prostate cancer cells in vitro as well as in vivo. Methods: Quantitative PCR, Western analysis, reporter assays, and proliferation analyses in vitro and in vivo were used to evaluate the effect of PI3K pathway inhibition on AR signaling and cell growth. Results: Transcriptional activity of AR was increased when the PI3K pathway was inhibited at different levels. In the androgen responsive prostate cancer cell line LNCaP, androgen and the mTOR inhibitor rapamycin synergistically activated androgen target genes. Despite increased androgen signaling, rapamycin treatment reduced LNCaP cell growth; the AR antagonist bicalutamide potentiated this effect. Furthermore, the rapamycin derivative CCI-779 reduced the growth of CWR22 prostate cancer xenografts while increasing AR target gene expression. Conclusions: These findings suggest that inhibition of the PI3K pathway activates AR signaling. Despite the increase in AR signaling which has proliferative effects, the result of PI3K pathway inhibition is antiproliferative. These findings suggest that the PI3K pathway is dominant over AR signaling in prostate cancer cells which should be considered in developing novel therapeutic strategies for prostate cancer.

The role of ESCRT proteins in attenuation of cell signalling

The ESCRT (endosomal sorting complex required for transport) machinery consists of four protein complexes that mediate sorting of ubiquitinated membrane proteins into the intraluminal vesicles of multivesicular endosomes, thereby targeting them for degradation in lysosomes. In the present paper, we review how ESCRT-mediated receptor down-regulation affects signalling downstream of Notch and growth factor receptors, and how ESCRTs may control cell proliferation, survival and cytoskeletal functions and contribute to tumour suppression.

Cell Polarity and Migration: Emerging Role for the Endosomal Sorting Machinery

The endosomal sorting complex required for transport (ESCRT) machinery has been implicated in the regulation of endosomal sorting, cell division, viral budding, autophagy, and cell signaling. Here, we review recent evidence that implicates ESCRTs in cell polarity and cell migration, and discuss the potential role of ESCRTs as tumor suppressors.

The ESCRT machinery mediates polarization of fibroblasts through regulation of myosin light chain.

Recent evidence implicates the endosomal sorting complex required for transport (ESCRT) in the regulation of epithelial polarity in Drosophila melanogaster, but the mechanisms responsible for this action remain unclear. Here we show that ESCRTs determine cell orientation during directed migration in human fibroblasts. We find that endosomal retention of α5β1 integrin and its downstream signaling effector Src in ESCRT-depleted cells is accompanied by the failure to activate myosin light chain kinase (MLCK), which thereby cannot phosphorylate myosin regulatory light chain (MRLC). Using this mechanism, ESCRT-depleted fibroblasts fail to orient their Golgi complex to undergo directional migration and show impaired focal adhesion turnover and increased spreading on fibronectin. Consistent with these findings, expression of a phosphomimetic mutant of MRLC in ESCRT-depleted cells restores normal phenotypes during cell spreading and orientation of the Golgi. These results suggest that, through their role in regulating integrin trafficking, ESCRTs regulate phosphorylation of MRLC and, subsequently, Golgi orientation and cell spreading.

PIKfyve, MTMR3 and their product PtdIns5P regulate cancer cell migration and invasion through activation of Rac1

Previously, we have shown that the phosphoinositide metabolizing enzymes PIKfyve (phosphoinositide 5-kinase, FYVE finger containing) and MTMR3 (myotubularin-related protein 3), together with their lipid product PtdIns5P, are important for migration of normal human fibroblasts. As these proteins are a kinase and a phosphatase respectively, and thereby considered druggable, we wanted to test their involvement in cancer cell migration and invasion. First, we showed that PIKfyve and MTMR3 are expressed in most cancer cells. Next, we demonstrated that depletion of PIKfyve or MTMR3 resulted in decreased velocity in three different cancer cell lines by using new software for cell tracking. Inhibition of the enzymatic activity of PIKfyve by the inhibitor YM201636 also led to a strong reduction in cell velocity. Mechanistically, we show that PIKfyve and MTMR3 regulate the activation of the Rho family GTPase Rac1. Further experiments also implicated PtdIns5P in the activation of Rac1. The results suggest a model for the activation of Rac1 in cell migration where PIKfyve and MTMR3 produce PtdIns5P on cellular membranes which may then serve to recruit effectors to activate Rac1. Finally, in an invasion assay, we demonstrate that both PIKfyve and MTMR3 are implicated in invasive behaviour of cancer cells. Thus PIKfyve and MTMR3 could represent novel therapeutic targets in metastatic cancer.

HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels

A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

Cargo-Dependent Degradation of ESCRT-I as a Feedback Mechanism to Modulate Endosomal Sorting

Ligand‐mediated lysosomal degradation of growth factor receptors, mediated by the endosomal sorting complex required for transport (ESCRT) machinery, is a mechanism that attenuates the cellular response to growth factors. In this article, we present a novel regulatory mechanism that involves ligand‐mediated degradation of a key component of the sorting machinery itself. We have investigated the endosomal localization of subunits of the four ESCRTs—Hrs (ESCRT‐0), Tsg101 (ESCRT‐I), EAP30/Vps22 (ESCRT‐II) and charged multivesicular body protein 3/Vps24 (ESCRT‐III). All the components were detected on the limiting membrane of multivesicular endosomes (MVEs). Surprisingly, however, Tsg101 and other ESCRT‐I subunits were also detected within intraluminal vesicles (ILVs) of MVEs. Tsg101 was sequestered along with cargo during endosomal sorting into ILVs and further degraded in lysosomes. Importantly, ESCRT‐mediated downregulation of two distinct cargoes, epidermal growth factor receptor (EGFR) and connexin43, mutually made cells refractory to degradation of the other cargo. Our observations indicate that the degradation of a key ESCRT component along with cargo represents a novel feedback control of endosomal sorting by preventing collateral degradation of cell surface receptors following stimulation of one specific pathway.

Ubiquitination of α-integrin cytoplasmic tails.

Recent findings have shown that ubiquitination is involved in regulating several proteins required for cell adhesion and migration. We showed that α5 integrin is ubiquitinated at its cytoplasmic lysines in response to fibronectin binding, and that this is required for its sorting to lysosomes together with fibronectin. Here we speculate whether other α integrin tails may also be ubiquitinated, and discuss the significance of ubiquitin linkages in the regulation of cell adhesion and migration.