Investigating the Role of ADP‐Ribosylation Factor 6 in Tumor Cell Invasion and Extracellular Signal‐Regulated Kinase Activation

Hoover, Holly; Muralidharan‐Chari, Vandhana; Tague, Sarah E.; D’Souza‐Schorey, Crislyn

doi:10.1016/s0076-6879(05)04014-0

Cited by 19 publications

(22 citation statements)

References 21 publications

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“…The gelatin degradation assay was as previously described[45]. Alternatively, isolated microvesicles were seeded and incubated on gelatin-coated coverslips for 6–8 hours.…”

Section: Methodsmentioning

confidence: 99%

ARF6-Regulated Shedding of Tumor Cell-Derived Plasma Membrane Microvesicles

et al. 2009

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Background Increased MAPK signaling, small GTPase activation, cytoskeletal rearrangements and the directed targeting of proteases to sites of extracellular matrix degradation, all accompany the process of tumor cell invasion. Several studies have implicated the small GTP-binding protein, ARF6, in tumor cell invasion although the molecular basis by which ARF6 facilitates this process is unclear. Results We show that the ARF6 GTP/GDP cycle regulates the release of protease-loaded plasma membrane-derived microvesicles from tumor cells into the surrounding environment. To enable microvesicle shedding, ARF6-GTP dependent activation of phospholipase D promotes the recruitment of the extracellular signal-regulated kinase (ERK) to the plasma membrane where in turn, ERK phosphorylates and activates myosin light chain kinase (MLCK). MLCK-mediated MLC phosphorylation is required for microvesicle release. Inhibition of ARF6 activation is accompanied by PKC-mediated phosphorylation of MLC, which blocks microvesicle shedding. Protein cargo appears to be selectively sorted into microvesicles and adhesion to the ECM is facilitated by microvesicle-associated integrin receptors. Conclusions Microvesicle shedding in tumor cells occurs via an actomyosin-based membrane abscission mechanism that is regulated by nucleotide cycling on ARF6. Microvesicle shedding appears to release selected cellular components, particularly those involved in cell adhesion and motility, into the surrounding environment. These findings suggest that ARF6 activation and the proteolytic activities of microvesicles both of which are thought to correlate directly with tumor progression, could potentially serve as biomarkers for disease.

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“…The gelatin degradation assay was as previously described[45]. Alternatively, isolated microvesicles were seeded and incubated on gelatin-coated coverslips for 6–8 hours.…”

Section: Methodsmentioning

confidence: 99%

ARF6-Regulated Shedding of Tumor Cell-Derived Plasma Membrane Microvesicles

et al. 2009

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“…LOX cells were cultured on glass coverslips, fixed, and processed as described (6, 11). Fluorescence was visualized using a Bio-Rad or Leica laser confocal scanning system.…”

Section: Methodsmentioning

confidence: 99%

“…The cell invasion assay was performed as previously described (6, 11). Briefly, cells were seeded on fluorescent gelatincoated coverslips.…”

Section: Methodsmentioning

confidence: 99%

ADP-Ribosylation Factor 6 Regulates Tumorigenic and Invasive Properties In vivo

Muralidharan‐Chari¹,

Hoover²,

Clancy³

et al. 2009

Cancer Research

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102

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This study shows that the small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is an important regulator of tumor growth and metastasis. Using spontaneous melanoma tumor growth assays and experimental metastasis assays in nude mice, we show that sustained activation of ARF6 reduces tumor mass growth but significantly enhances the invasive capacity of tumor cells. In contrast, mice injected with tumor cells expressing a dominantly inhibitory ARF6 mutant exhibited a lower incidence and degree of invasion and lung metastasis compared with control animals. Effects on tumor growth correlate with reduced cell proliferation capacity and are linked at least in part to alterations in mitotic progression induced by defective ARF6 cycling. Furthermore, phospho-ERK levels in subcultured cells from ARF6(GTP) and ARF6(GDP) tumor explants correlate with invasive capacity. ARF6-induced extracellular signal-regulated kinase (ERK) signaling leads to Rac1 activation to promote invadopodia formation and cell invasion. These findings document an intricate role for ARF6 and the regulation of ERK activation in orchestrating mechanisms underlying melanoma growth, invasion, and metastases.

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“…Gelatin degradation assays were performed as previously described (Hoover et al, 2005). Briefly, coverslips were coated with a thin layer of 2% gelatin in PBS and dried overnight at 4°C.…”

Section: Gelatin Degradation Assaymentioning

confidence: 99%

“…The requirements for SNAP23, syntaxin-13 and VAMP3 function in degradation of an extracellular matrix were examined using an established gelatin degradation assay (Hoover et al, 2005;Itoh et al, 2001;Tague et al, 2004). For these studies, HT-1080 cells were transfected with the indicated constructs, incubated for appropriate periods, plated on coverslips coated with fluorescently labeled (Texas red) gelatin, and incubated for 24 hours.…”

Section: Mutant Snare Constructs Inhibit Degradation Of a Gelatin Matrixmentioning

confidence: 99%

VAMP3, syntaxin-13 and SNAP23 are involved in secretion of matrix metalloproteinases, degradation of the extracellular matrix and cell invasion

Kean

Williams

Skalski

et al. 2009

Journal of Cell Science

101

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evidence that SNAREs contribute to trafficking of MMPs. Syntaxin-4 (Miyata et al., 2004) and Ti-VAMP (tetanus-insensitive vesicleassociated membrane protein, also known as VAMP7) (Steffen et al., 2008) have been shown to be involved in cellular invasion and trafficking of MT1-MMP. SNAP25 (synaptosome-associated protein of 25 kDa) has been found to partly colocalize with MMP7 in epithelial cells (Gorodeski, 2007). There is also recent evidence that directly links SNARE-mediated intracellular membrane traffic to cell-ECM interactions (Al-Awar et al., 2000;Roberts et al., 2001). We (Gonon et al., 2005;Skalski and Coppolino, 2005;Tayeb et al., 2005), along with others (Proux-Gillardeaux et al., 2005), have reported that SNARE-mediated trafficking, including that mediated by the plasma membrane SNARE SNAP23 and the endosomal SNARE VAMP3, is required for cell adhesion and migration in cultured mammalian cell systems.In the present study, we examined the roles that SNAP23, VAMP3 and syntaxin-13 play in the secretion of MMPs and the degradation of ECM by HT-1080 fibrosarcoma cells. We report that SNAP23 and VAMP3 are required for secretion of MMP2 and MMP9, and that these SNAREs and syntaxin-13 are involved in trafficking of the membrane-bound MT1-MMP to the cell surface. Inhibiting the function of SNAP23, VAMP3 or syntaxin-13 using dominantnegative mutants of the SNAREs, RNA interference (RNAi) or tetanus toxin impaired the in situ degradation of gelatin by the cells. We present data that are the first to show that VAMP3, syntaxin-13 and SNAP23 are required for efficient invasion by HT-1080 cells in vitro. The impaired ECM degradation and reduced invasive capacity observed in cells as a consequence of inhibiting SNARE function suggests an important role for SNARE-mediated traffic in tumor progression. Results GFP-tagged SNAREs partly overlap with endogenous MMP2 and MMP9 in HT-1080 cellsMMP2 and MMP9 are secreted proteinases that have been shown to contribute to tumor-cell invasion and metastasis. Having identified roles for SNAP23 (Gonon et al., 2005) and VAMP3 (Proux-Gillardeaux et al., 2005;Skalski and Coppolino, 2005;Tayeb et al., 2005) in the non-invasive motility of cells, we sought to investigate the involvement of these SNAREs in the trafficking and secretion of MMPs during cell invasion. SNAP23 is a plasma membrane SNARE found in many cell types, and VAMP3 is a known binding partner of SNAP23 (Hepp et al., 1999). To study MMP activity during cell invasion, we used the human cell line HT-1080, which is derived from a highly invasive fibrosarcoma and is known to express MMP2 and MMP9 along with MT1-MMP (Ginestra et al., 1997;Stanton et al., 1998). The subcellular distributions of endogenous MMP2 and MMP9 were compared with those of GFP-SNAP23 and GFP-VAMP3, and it was observed that SNAP23 ( Fig. 1A) and VAMP3 (Fig. 1B) partly colocalized with both MMP2 and MMP9 at the periphery of HT-1080 cells in response to treatment of the cells with the tumor promoter PMA (phorbol 12-myristate 13-acetate). PMA was added to...

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Investigating the Role of ADP‐Ribosylation Factor 6 in Tumor Cell Invasion and Extracellular Signal‐Regulated Kinase Activation

Cited by 19 publications

References 21 publications

ARF6-Regulated Shedding of Tumor Cell-Derived Plasma Membrane Microvesicles

ARF6-Regulated Shedding of Tumor Cell-Derived Plasma Membrane Microvesicles

ADP-Ribosylation Factor 6 Regulates Tumorigenic and Invasive Properties In vivo

VAMP3, syntaxin-13 and SNAP23 are involved in secretion of matrix metalloproteinases, degradation of the extracellular matrix and cell invasion

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