GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway.
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ABSTRACT:
SUBMITTER: Niu X
PROVIDER: S-EPMC6155137 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCancer-associated cachexia and muscle wasting are considered key determinants of cancer-related death and reduction in the quality of life of cancer patients. A crucial link has been established between activin signaling and skeletal muscle atrophy-hypertrophy. We previously showed that activin-?C, a novel activin-A antagonist, is a tumor modulator that abolishes the cancer-associated cachexia in a mouse genetic model of gonadal tumorigenesis, in which the normal balance of inhibin/activin signalling is disrupted by a targeted mutation in the Inha gene (inhibin ?-KO mouse). This study aimed to identify the molecular mechanism by which activin-?C increases survival and abolishes cancer-associated cachexia in ?-KO mice. We hypothesized that overexpression of activin-?C modulates the cachexia phenotype by antagonizing the activin signaling pathway and repressing muscle wasting via the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways.MethodsMale and female ActC++, ?-KO, and ?-KO/ActC++ mice and WT littermate controls were studied. Western blot analysis for the specific E3 ubiquitin ligases, atrogin-1 and MuRF1, markers of the autophagic-lysosomal pathway, Beclin-1, p62, and LC3A/B, effectors Smad-2, Smad-3 and myostatin was performed in the gastrocnemius of age-matched mice. Histopathology of the gastrocnemius and survival analysis were also conducted in animals from the same breeding cohort. Serum levels of activin-A, inflammatory cytokines, hormonal profile, and bone density were also assessed.ResultsIncreased levels of atrogin-1, MuRF-1, Beclin-1, p62, LC3A/B-I, Smad-2 and serum levels of activin-A were noted in the ?-KO mice. These mice developed gonadal cancers followed by severe weight loss, and reduced survival. Overexpression of activin- ?C antagonized the activin signaling cascade, attenuating the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways, and reduced serum levels of activin-A. ?-KO/ActC++ mice displayed a less aggressive cachectic phenotype, reduced tumor weight, and prolonged survival.ConclusionOur findings show for the first time a specific effect of activin-?C on muscle wasting and transcription factors involved in muscle protein degradation. The study indicates that activin-?C may be a novel therapy to abrogate cancer-associated weight loss and prolong survival.
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Project description:IRS4, a member of the insulin receptor substrate protein family, can induce constitutive PI3K/AKT hyperactivation and cell proliferation even in the absence of insulin or growth factors and promote tumorigenesis, but its regulation has only been explored at the transcriptional level. Methods: Scansite was used to predict the potential protein kinases that may regulate the functions of IRS4, and mass spectrometry was used to identify the E3 ligase for IRS4. The protein interaction was carried out by immunoprecipitation, and protein stability was measured by cycloheximide treatment. In vitro kinase assay was used to determine the phosphorylation of IRS4 by casein kinase 1?2 (CK1?2). Colony formation assay and xenograft-bearing mice were employed to assess the cancer cell growth in vitro and in vivo, respectively. Immunohistochemistry was performed to examine protein levels of both IRS4 and CK1?2 in osteosarcoma specimens and their relationship was evaluated by ?2 test. Two-tailed Student's t-test or the Mann-Whitney U test were used to compare the differences between subgroups. Results: IRS4 was phosphorylated at Ser859 by CK1?2 in vitro and in vivo, which promoted the polyubiquitination and degradation of IRS4 through the ubiquitin/lysosome pathway by the carboxyl terminus of Hsc70-interacting protein(CHIP). Using osteosarcoma cell lines, the ectopic nonphosphorylated mutant of IRS4 by CK1?2 triggered higher level of p-Akt and displayed faster cell proliferation and cancer growth in vitro and in nude mice. In addition, a negative correlation in protein levels between CK1?2 and IRS4 was observed in osteosarcoma cell lines and tissue samples. Conclusions: IRS4, as a new substrate of CHIP, is negatively regulated by CK1?2 at the posttranslational level, and specific CK1?2 agonists may be a potentially effective strategy for treating patients with osteosarcoma.
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