Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility.

The tumor suppressor VHL (von Hippel-Lindau protein) serves as a negative regulator of hypoxia-inducible factor-alpha subunits. However, accumulated evidence indicates that VHL may play additional roles in other cellular functions. We report here a novel hypoxia-inducible factor-independent function of VHL in cell motility control via regulation of fibroblast growth factor receptor 1 (FGFR1) endocytosis. In VHL null tumor cells or VHL knock-down cells, FGFR1 internalization is defective, leading to surface accumulation and abnormal activation of FGFR1. The enhanced FGFR1 activity directly correlates with increased cell migration. VHL disease mutants, in two of the mutation hot spots favoring development of renal cell carcinoma, failed to rescue the above phenotype. Interestingly, surface accumulation of the chemotactic receptor appears to be selective in VHL mutant cells, since other surface proteins such as epidermal growth factor receptor, platelet-derived growth factor receptor, IGFR1, and c-Met are not affected. We demonstrate that 1) FGFR1 endocytosis is defective in the VHL mutant and is rescued by reexpression of wild-type VHL, 2) VHL is recruited to FGFR1-containing, but not EGFR-containing, endosomal vesicles, 3) VHL exhibits a functional relationship with Rab5a and dynamin 2 in FGFR1 internalization, and 4) the endocytic function of VHL is mediated through the metastasis suppressor Nm23, a protein known to regulate dynamin-dependent endocytosis.

RNA-dependent integrin alpha3 protein localization regulated by the Muscleblind-like protein MLP1.

We show that localized expression of the integrin alpha3 protein is regulated at the level of RNA localization by the human homologue of Drosophila Muscleblind, MLP1/MBLL/MBNL2, a unique Cys3His zinc-finger protein. This is supported by the following observations: MLP1 knockdown abolishes localization of integrin alpha3 to the adhesion complexes; MLP1 is localized in adhesion plaques that contain phospho-focal adhesion kinase; this localization is microtubule-dependent; integrin alpha3 transcripts colocalize with MLP1 in distinct cytoplasmic loci; integrin alpha3 transcripts are physically associated with MLP1 in cells and MLP1 binds to a specific ACACCC motif in the integrin alpha3 3' untranslated region (UTR) in vitro; and a green fluorescent protein (GFP) open reading frame-integrin alpha3 3' UTR chimeric gene directs GFP protein localization to distinct cytoplasmic loci near the cell periphery, which is dependent on MLP1 and is mediated by the ACACCC motif but is independent of the integrin alpha3 signal peptide.

Caspase-1 is a direct target gene of ETS1 and plays a role in ETS1-induced apoptosis.

ETS1, the founding member of Ets transcriptional factor family, plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. Previous work has shown that ETS1 represses tumorigenicity of colon carcinoma cells in vivo, and that the p42-ETS1 protein bypasses a defect in apoptosis in colon carcinoma cells through the up-regulation of caspase-1 expression. In this report, we show that expression of p42-ETS1 inhibits tumorigenicity of colon cancer DLD-1 cells through induction of apoptosis in vivo. In support of the hypothesis that caspase-1 might be a target involved in the sensitization of DLD-1 cells to Fas-induced apoptosis by ETS1, overexpression of caspase-1 bypasses Fas-induced apoptosis in these cells as well. Furthermore, ETS1-mediated apoptosis was observed in MOP8 cells, a transformed mouse NIH3T3 cell line. To determine whether ETS1 activates the transcription of caspase-1, luciferase reporters driven by the wild-type and mutant caspase-1 promoters were generated. Both p51-ETS1 and p42-ETS1 transactivated the caspase-1 transcription and a functional Ets binding site is identified in the caspase-1 promoter. Wild-type caspase-1 promoter (pGL3-ICE) was strongly transactivated by ETS1 and this transactivation was dramatically diminished by the mutation of the potential Ets binding site (-525 bp). In addition, electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed complex formation between this binding site and ETS1 proteins. Taken together, ETS1 transcriptionally induces the expression of caspase-1; as such, the regulatory control of caspase-1 expression by ETS1 may underlie the apoptotic susceptibility modulated by ETS1 in specific tumor cells.

Suppression of Neu-induced mammary tumor growth in cyclin D1 deficient mice is compensated for by cyclin E.

Amplification and/or overexpression of the receptor tyrosine kinase HER2/Neu and the cell cycle regulatory gene cyclin D1 are frequently associated with human breast cancer. We studied the functional significance of cyclin D1 in Neu-induced mammary oncogenesis by developing mice overexpressing either wild-type or mutant Neu in a cyclin D1 deficient background. The absence of cyclin D1 suppresses mammary tumor formation induced by the wild-type or activated mutant form of Neu, which promote multi- and single-step progression of tumorigenesis, respectively. These data indicate that cyclin D1 is preferentially required for Neu-mediated signal transduction pathways in mammary oncogenesis. Significantly, 35% of mutant Neu/cyclin D1(-/-) mice regained mammary tumor potential due to compensation by cyclin E. Thus, shared targets of cyclins D1 and E are important in modulating Neu function in mammary tumorigenesis. Our results imply that the combinatorial inhibition of cyclins D1 and E might be useful in the treatment of malignancies induced by Neu.