An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism

Abstract

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

ARF GTPases and Their Ubiquitous Role in Intracellular Trafficking Beyond the Golgi

“…Recently, ARF5 and IQSEC1 were also implicated in Ca 2+ -dependent disassembly of focal adhesions during cell migration (D'Souza et al, 2020). Further supporting a role for ARF5 in migration, the GTPase was shown to be recruited to vesicular structures in cell protrusions via IQSEC1 and to promote invasion and metastatic cancer by controlling phosphoinositide metabolism (Nacke et al, 2021).…”

Identification of MARCO and PCSK6 As Key Genes Promoting Hepatic Metastasis in Pancreatic Cancer via Bioinformatics Analysis

“…Reprogramming of tumor metabolism played a signi cant role in tumor metastasis [21,22]. Plenty of tumor metabolism related genes had been discovered [23][24][25][26]. Among the H-DEGs, iron metabolism related genes including GOT1 and CP [27,28], glutamine metabolism related gene GOT1 [29], pyruvate metabolism related gene PC [30], proline metabolism related gene ALDH4A1 [31], and so on were found in this study.…”

The small GTPase ARF3 controls metastasis and invasion modality by regulating N-cadherin levels

“…Method described previously 3 , briefly cells were plated on 96 well plates for 48 hours, fixed and then stained, as described in 'Fixed 3D culture and analysis…”

“…Method described previously 3 , briefly PC3 cells were incubated on 120mm plates for 48 hours at 37°C, 5% CO 2. Cells were then lysed on ice in pulldown-lysis buffer (50mM Tris, 100mM NaCl, 2mM MgCl2, 0.1% SDS, 0.5% Na-deoxycholate, 1% Triton X-100, 10% glycerol).…”

“…is presented in a line graph (PRISM 7, GraphPad) as luminescence fold change to t = 0 at 24 hour time points. P values and the statistical test used are described in Figure Legends.Invasion and Migration AssaysMethod described previously3 , briefly ImageLock plates were coated with 20μl of 10% GFRM diluted in medium overnight at 37 o C. 70,000 PC3 cells in 100μl medium were plated in each well for 4 hours at 37 o C. The resultant monolayer was wounded using a wound making tool (Satorius), washed three times with medium and overlaid with either 50μl of 25% GFRM for invasion assays or 100μl medium for migration assays. After incubation at 37 o C for an hour 100μl medium was added to each well of invasion assay and plates imaged every hour for 4-6 days using the IncuCyte ZOOM.For each experimental replicate the average Relative Wound Density (RWD) of all control samples (Scr shRNA) was calculated and each technical replicate normalized to this value.…”