RESUMO
Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.
Assuntos
Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Dicroísmo Circular , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Humanos , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Periferinas , Pichia/genética , Mutação Puntual , Estrutura Terciária de Proteína , Análise EspectralRESUMO
Opioid-binding protein/cell adhesion molecule-like (OPCML) is a tumor-suppressor gene that is frequently inactivated in ovarian cancer and many other cancers by somatic methylation. We have previously shown that OPCML exerts its suppressor function by negatively regulating a spectrum of receptor tyrosine kinases (RTK), such as ErbB2/HER2, FGFR1, and EphA2, thus attenuating their related downstream signaling. The physical interaction of OPCML with this defined group of RTKs is a prerequisite for their downregulation. Overexpression/gene amplification of EGFR and HER2 is a frequent event in multiple cancers, including ovarian and breast cancers. Molecular therapeutics against EGFR/HER2 or EGFR only, such as lapatinib and erlotinib, respectively, were developed to target these receptors, but resistance often occurs in relapsing cancers. Here we show that, though OPCML interacts only with HER2 and not with EGFR, the interaction of OPCML with HER2 disrupts the formation of the HER2-EGFR heterodimer, and this translates into a better response to both lapatinib and erlotinib in HER2-expressing ovarian and breast cancer cell lines. Also, we show that high OPCML expression is associated with better response to lapatinib therapy in breast cancer patients and better survival in HER2-overexpressing ovarian cancer patients, suggesting that OPCML co-therapy could be a valuable sensitizing approach to RTK inhibitors. Mol Cancer Ther; 16(10); 2246-56. ©2017 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular/genética , Receptores ErbB/genética , Neoplasias Ovarianas/tratamento farmacológico , Receptor ErbB-2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/administração & dosagem , Feminino , Proteínas Ligadas por GPI/genética , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinas/administração & dosagem , Receptor ErbB-2/antagonistas & inibidores , Trastuzumab/administração & dosagemRESUMO
UNLABELLED: Epithelial ovarian cancer is the leading cause of death from gynecologic malignancy, and its molecular basis is poorly understood. We previously demonstrated that opioid binding protein cell adhesion molecule (OPCML) was frequently epigenetically inactivated in epithelial ovarian cancers, with tumor suppressor function in vitro and in vivo. Here, we further show the clinical relevance of OPCML and demonstrate that OPCML functions by a novel mechanism in epithelial ovarian cancer cell lines and normal ovarian surface epithelial cells by regulating a specific repertoire of receptor tyrosine kinases: EPHA2, FGFR1, FGFR3, HER2, and HER4. OPCML negatively regulates receptor tyrosine kinases by binding their extracellular domains, altering trafficking via nonclathrin-dependent endocytosis, and promoting their degradation via a polyubiquitination-associated proteasomal mechanism leading to signaling and growth inhibition. Exogenous recombinant OPCML domain 1-3 protein inhibited the cell growth of epithelial ovarian cancers cell in vitro and in vivo in 2 murine ovarian cancer intraperitoneal models that used an identical mechanism. These findings demonstrate a novel mechanism of OPCML-mediated tumor suppression and provide a proof-of-concept for recombinant OPCML protein therapy in epithelial ovarian cancers. SIGNIFICANCE: The OPCML tumor suppressor negatively regulates a specific spectrum of receptor tyrosine kinases in ovarian cancer cells by binding to their extracellular domain and altering trafficking to a nonclathrin, caveolin-1associated endosomal pathway that results in receptor tyrosine kinase polyubiquitination and proteasomal degradation. Recombinant OPCML domain 1-3 recapitulates this mechanism and may allow for the implementation of an extracellular tumor-suppressor replacement strategy.
Assuntos
Moléculas de Adesão Celular/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Carcinoma Epitelial do Ovário , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Genes Supressores de Tumor , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Epiteliais e Glandulares/enzimologia , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Transfecção , Transplante HeterólogoRESUMO
There have been major advances in our understanding of the cellular and molecular biology of the human malignancies that are collectively referred to as ovarian cancer. At a recent Helene Harris Memorial Trust meeting, an international group of researchers considered actions that should be taken to improve the outcome for women with ovarian cancer. Nine major recommendations are outlined in this Opinion article.