YIPF2 is a novel Rab-GDF that enhances HCC malignant phenotypes by facilitating CD147 endocytic recycle.

An increased surface level of CIE (clathrin-independent endocytosis) proteins is a new feature of malignant neoplasms. CD147 is a CIE glycoprotein highly up-regulated in hepatocellular carcinoma (HCC). The ability to sort out the early endosome and directly target the recycling pathway confers on CD147 a prolonged surface half-life. However, current knowledge on CD147 trafficking to and from the cell-surface is limited. In this study, an MSP (membrane and secreted protein)-cDNA library was screened against EpoR/LR-F3/CD147EP-expressed cells by MAPPIT (mammalian protein-protein interaction trap). CD147 co-expressing with the new binder was investigated by GEPIA (gene expression profiling interactive analysis). The endocytosis, ER-Golgi trafficking and recycling of CD147 were measured by confocal imaging, flow cytometry, and biotin-labeled chase assays, respectively. Rab GTPase activation was checked by GST-RBD pull-down and MMP activity was measured by gelatin zymography. HCC malignant phenotypes were determined by cell adhesion, proliferation, migration, Transwell motility, and invasion assays. An ER-Golgi-resident transmembrane protein YIPF2 was identified as an intracellular binder to CD147. YIPF2 correlated and co-expressed with CD147, which is a survival predictor for HCC patients. YIPF2 is critical for CD147 glycosylation and trafficking functions in HCC cells. YIPF2 acts as a Rab-GDF (GDI-displacement factor) regulating three independent trafficking steps. First, YIPF2 recruits and activates Rab5 and Rab22a GTPases to the endomembrane structures. Second, YIPF2 modulates the endocytic recycling of CD147 through distinctive regulation on Rab5 and Rab22a. Third, YIPF2 mediates the mature processing of CD147 via the ER-Golgi trafficking route. Decreased YIPF2 expression induced a CD147 efficient delivery to the cell-surface, promoted MMP secretion, and enhanced the adhesion, motility, migration, and invasion behaviors of HCC cells. Thus, YIPF2 is a new trafficking determinant essential for CD147 glycosylation and transport. Our findings revealed a novel YIPF2-controlled ER-Golgi trafficking signature that promotes CD147-medated malignant phenotypes in HCC.

Macropinocytosis activated by oncogenic Dbl enables specific targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells.

BACKGROUND: Successful implementation of gene therapy heavily relies on efficiently delivering genetic materials and specific targeting into cells. Oncogene-driven endocytosis stimulates nutrient uptake and also develops an endocytosis-mediated defense against therapeutic agents. Cell-penetrating peptides, typically HIV-Tat, are well known for efficient delivery of nucleic acid drugs but lack targeting specificity. Various passive targeting strategies were pursued to enhance the tumor targeting efficiency; however, they are still limited by complicated cellular endocytosis routes and the heterogeneity of cancer types. METHODS: Tat/pDNA complexes were noncovalently compacted and their physiochemical properties were determined. The siRNA pool and pLV-RNAi-GFP lentivirus were used to knock down dbl oncogene (originally isolated from diffuse B-cell lymphoma) expression, and its overexpression was performed by plasmid transient transfection. The cellular uptake of fluorescent ligands was quantified by confocal imaging and flow cytometry analysis. The transgene efficiency was determined by the Luciferase expression assay. Rho GTPase activation was checked by the GST-Rho GTPase-binding domain pull-down assay. RESULTS: pGL3 plasmid DNA was noncovalently compacted with the Tat peptide into nano-size complexes at high N/P ratios. Macropinocytosis, a clathrin- and caveolin-independent endocytosis process, was shown to contribute to the uptake of middle-sized (∼600 nm) Tat/pGL3 complexes. Cell-type-specific variation in macropinocytosis was essentially controlled by the action of the Dbl oncogene. Onco-Dbl presentation constantly induced a high level of macropinocytosis activity in ovarian cancer cells. Onco-Dbl overexpression hyperstimulated macropinocytosis enhancement in cells mainly through actin cytoskeleton reorganization mediated by the PH domain and Rac1 activation. The Dbl-driven Rho GTPase signaling collectively determined the cell-type-specific macropinocytosis phenotype. CONCLUSION: Such an aspect can be exploited to selectively confer targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells. Our work provides a novel alternative for targeted delivery of cell-penetrating peptide-based nucleic acid drugs into certain tumor types if specific endocytosis pathways are used.

GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway.

Chaperone-assisted proteasome degradation of oncogenic protein acts as an upstream signal controlling tumorigenesis and progression. The understanding of the co-regulation of chaperone and oncoprotein of endocytosis pathways is extremely limited. In this study, we showed for the first time that proto-Dbl (dbl proto-oncogene product) is co-enriched with mitochondrial chaperone GRP75 in endocytosis vesicles from ovarian cancer cells. onco-Dbl, produced by oncogenic mutation/degradation of proto-Dbl, markedly enhanced cellular macropinocytosis but suppressed clathrin-mediated endocytosis and clathrin-independent endocytosis pathways, presenting a derailed endocytosis phenotype. GRP75 was associated with proto-Dbl inside cells and modulated Dbl-driven endocytosis derailed by a co-regulatory mode. In spite of not being a component of the Hsc70/Hsp90/proto-Dbl complex, the degradation of proto-Dbl was promoted by GRP75 through the CHIP-mediated ubiquitin-proteasome pathway, of which GRP75 acts as a cooperator with CHIP but also acts as a competitor to Hsc70 and Hsp90 in the multiple chaperones-assisted pro-folding/pro-degradation machinery. Knockdown or inhibition of GRP75 attenuated proto-Dbl degradation and reduced the onco-Dbl level, which differentially impaired Rho GTPases activation and therefore shifted the endocytosis-derailed phenotype. Our data uncovered a novel GRP75-Dbl endocytosis regulatory axis and provided an alternative using chaperone inhibitor to shut down the oncoprotein-driven endocytosis derailment mechanism.

Mitochondria chaperone GRP75 moonlighting as a cell cycle controller to derail endocytosis provides an opportunity for nanomicrosphere intracellular delivery.

Understanding how cancer cells regulate endocytosis during the cell cycle could lead us to capitalize this event pharmacologically. Although certain endocytosis pathways are attenuated during mitosis, the endocytosis shift and regulation during the cell cycle have not been well clarified. The conventional concept of glucose-regulated proteins (GRPs) as protein folding chaperones was updated by discoveries that translocated GRPs assume moonlighting functions that modify the immune response, regulate viral release, and control intracellular trafficking. In this study, GRP75, a mitochondria matrix chaperone, was discovered to be highly expressed in mitotic cancer cells. Using synchronized cell models and the GRP75 gene knockdown and ectopic overexpression strategy, we showed that: (1) clathrin-mediated endocytosis (CME) was inhibited whereas clathrin-independent endocytosis (CIE) was unchanged or even up-regulated in the cell cycle M-phase; (2) GRP75 inhibited CME but promoted CIE in the M-phase, which is largely due to its high expression in cancer cell mitochondria; (3) GRP75 targeting by its small molecular inhibitor MKT-077 enhanced cell cycle G1 phase-privileged CME, which provides an opportunity for intracellular delivery of nanomicrospheres sized from 40 nm to 100 nm. Together, our results revealed that GRP75 moonlights as a cell cycle controller and endocytosis regulator in cancer cells, and thus has potential as a novel interference target for nanoparticle drugs delivery into dormant cancer cells.

[Mitochondrial signal peptide guides EGFP-GRP75 fusion proteins into mitochondria].

Objective To investigate the role of mitoch-ondrial signal peptide in guiding enhanced green fluorescent protein-glucose-regulated protein 75 fusion proteins (EGFP-GRP75) into mitochondria. Methods At first, the signal peptide gene and GRP75 domain genes were spliced by overlap extension PCR. Unphosphorylatable mutant T62A/S65A, phospho-mimic mutant T62D/S65D and substrate binding-defective mutant V482F were further created through site-directed mutagenesis PCR. The fusion gene fragments were ligated into pEGFPC1 expression plasmid, respectively. The expressions of EGFP-GRP75 fusion constructs in HeLa cells were examined directly with Western blotting and laser scanning confocal microscopy. Results All the fusion proteins were highly expressed. Signal peptide remarkably reduced the expression of EGFP-GRP75 fusions compared with recombinant plasmids without signal peptide. Fluorescence was seen exclusively located in mitochondria of the cells transfected by signal peptide-contained plasmids, whereas signal peptide-absent EGFP-GRP75 fusion proteins were homogeneously distributed in the whole cell body. In addition, no changes were observed in the subcellular localization of EGFP-GRP75 fusion proteins that contained double or triple mutations at Thr 62 residues, Ser 65 residues and Val 482 residues. Conclusion Signal peptide is essential for targeting EGFP-GRP75 fusion proteins into mitochondria, and 62 threonine/65 serine/482 valine residues contribute to phosphorylation or substrate-binding characteristics are dispensable in the mitochondrial targeting function.