Human rhomboid family-1 modulates clathrin coated vesicle-dependent pro-transforming growth factor α membrane trafficking to promote breast cancer progression

Abstract

BackgroundEpidermal growth factor receptor (EGFR) signalling is critical in epithelial cancer development. Human rhomboid family-1 (RHBDF1) facilitates the secretion of TGFα, an EGFR ligand, in breast cancer; however, the underlying mechanism remains unclear. We evaluated the role for RHBDF1 in clathrin-coated vesicle (CCV)-dependent pro-TGFα membrane trafficking in breast cancer cells upon stimulation by G-protein coupled receptor (GPCR) agonists.MethodsRHBDF1 was silenced in various breast cancer cells using shRNA. TGFα levels, subcellular localization, and secretion were evaluated using ELISA, immunofluorescent staining, and coimmunoprecipitation. Phosphorylation and expression of relevant proteins were measured by western blotting. RHBDF1-dependent cell viability and invasion were measured.FindingsRHBDF1 mediates GPCR agonist-induced EGFR phosphorylation by promoting TGFα secretion in various types of breast cancer cells. RHBDF1 not only mediates ADAM17-dependent shedding of TGFα, but is essential in membrane trafficking of pro-TGFα. RHBDF1 silencing results in blocking of clathrin uncoating from CCV, a crucial step for the plasma membrane release of pro-TGFα. Interaction of RHBDF1 with auxilin-2, a CCV protein, determines the recruitment of HSC70 to CCV to facilitate clathrin uncoating. RHBDF1 function is required for the proliferation and mobility of breast cancer cells upon stimulation by Sphingosine 1 Phosphate (S1P), a GPCR agonist. We demonstrate a significant correlation between RHBDF1 overexpression and EGFR activation in breast cancer tissues.InterpretationRHBDF1 is an indispensable component of the protein trafficking machinery involved in GPCR-mediated EGFR transactivation, and is an attractive therapeutic target for cancer.FundNational Natural Science Foundation of China (81,672,740 to ZSZ, 81,272,356 and 81,330,029 to LYL).

Rhomboids and regulation of receptor tyrosine kinase ligands shedding

“…The mechanisms of action of RHBDF1 extend much further beyond what is reported in the present study of Jie Li and colleagues [ 4 ]. In colorectal cancer, RHBDF1 induces epithelial-to-mesenchymal transition (EMT) through the Wnt/β-catenin pathway [ 8 ].…”

“…In EBioMedicine, Li and colleagues [ 4 ] provide a mechanistic explanation of the role of the human rhomboid family-1 (RHBDF1) in epidermal growth factor receptor (EGFR) activation, in triple negative breast cancer (TNBC). The aberrant activation of EGFR results from increased transcriptional expression, gene amplification, or oncogenic mutations and is implicated in several human cancers.…”

“…Li et al, have now performed further research on the mechanism of action of RHBDF1 [ 4 ]. They have elegantly demonstrated that RHBDF1 promotes the secretion of the EGFR ligand, TGFα, by mediating on the one hand, ADAM17-dependent TGFα shedding, and on the other hand, clathrin-dependent plasma membrane release of pro-TGFα ( Fig.…”

“…1 ). RHBDF1 interacts with auxilin-2, a cofactor of heat shock cognate protein-70 (HSC70) that is directly involved in the uncoating process [ 4 ]. Li et al, showed that RHBDF1 inhibition decreased cell migration, proliferation, and invasion in breast cancer cells, in the presence or absence of S1P, indicating that the role of RHBDF1 may be independent of GPCR-dependent EGFR transactivation [ 4 ].…”

“…RHBDF1 interacts with auxilin-2, a cofactor of heat shock cognate protein-70 (HSC70) that is directly involved in the uncoating process [ 4 ]. Li et al, showed that RHBDF1 inhibition decreased cell migration, proliferation, and invasion in breast cancer cells, in the presence or absence of S1P, indicating that the role of RHBDF1 may be independent of GPCR-dependent EGFR transactivation [ 4 ]. Previous work from the same authors has shown that RHBDF1 facilitates the stabilization of the α -subunit of hypoxia-inducible factor-1 (HIF1α) under hypoxia in breast cancer cell lines and its expression is related with shorter progression-free survival in breast cancer patients [ 9 ].…”