Abstract
SummaryHere we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5β1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5β1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo.
Highlights
Metastasizing cells need to invade the extracellular matrix (ECM) that surrounds tissues and tumors, and must survive and grow within these microenvironments (Sahai, 2005)
Chloride Intracellular Channel Protein 3 (CLIC3) Is Upregulated in a Rab25 and 3D MatrixDependent Fashion We plated A2780 cells stably expressing either Rab25 (A2780Rab25) or a control vector (A2780-DNA3) onto plastic or cellderived matrix (CDM)—a thick, pliable matrix composed mainly of fibrillar collagen and fibronectin that recapitulates aspects of the matrix found in connective tissues—and compared their mRNA profiles using Affymetrix arrays
We ranked gene expression changes primarily according to the less conservative step-up p value and secondarily according to fold changes, and this revealed CLIC3 to be the most significant Rab25-upregulated gene when cells were plated onto CDM, but not following adherence to plastic surfaces (Figure 1A)
Summary
Metastasizing cells need to invade the extracellular matrix (ECM) that surrounds tissues and tumors, and must survive and grow within these microenvironments (Sahai, 2005) As they invade, tumor cells form dynamic interactions with the ECM that provide traction force for forward motion and ECM remodeling and promote cell growth and survival (Kumar and Weaver, 2009). Tumor cells form dynamic interactions with the ECM that provide traction force for forward motion and ECM remodeling and promote cell growth and survival (Kumar and Weaver, 2009) Both the motility and the growth and survival of tumor cells are controlled by integrins: transmembrane proteins that interact extracellularly with ECM proteins, such as fibronectin and collagen, and intracellularly with the cytoskeleton and the cell’s signaling and vesicular transport machinery (Caswell et al, 2009; Hynes, 2002). Expression of p53 mutants or inhibition of avb integrin can drive recruitment of the Rab effector, Rab-coupling protein (RCP), to the cytotail of b1 integrin, which enables RCP to associate with the EGF receptor 1 (EGFR1). a5b1 and EGFR1 are coordinately recycled to the plasma membrane in a way that potentiates EGFR1 signaling to Akt, a kinase that promotes invasion (Caswell et al, 2008; Muller et al, 2009), cell growth, and survival
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