RESUMO
Ameloblastoma (AB) is an odontogenic tumor that arises from ameloblast-lineage cells. Although relatively uncommon and rarely metastatic, AB tumors are locally invasive and destructive to the jawbone and surrounding structures. Standard-of-care surgical resection often leads to disfigurement, and many tumors will locally recur, necessitating increasingly challenging surgeries. Recent genomic studies of AB have uncovered oncogenic driver mutations, including in the mitogen-activated protein kinase (MAPK) and Hedgehog signaling pathways. Medical therapies targeting those drivers would be a highly desirable alternative or addition to surgery; however, a paucity of existing AB cell lines has stymied clinical translation. To bridge this gap, here we report the establishment of 6 new AB cell lines-generated by "conditional reprogramming"-and their genomic characterization that reveals driver mutations in FGFR2, KRAS, NRAS, BRAF, PIK3CA, and SMO. Furthermore, in proof-of-principle studies, we use the new cell lines to investigate AB oncogene dependency and drug sensitivity. Among our findings, AB cells with KRAS or NRAS mutation (MAPK pathway) are exquisitely sensitive to MEK inhibition, which propels ameloblast differentiation. AB cells with activating SMO-L412F mutation (Hedgehog pathway) are insensitive to vismodegib; however, a distinct small-molecule SMO inhibitor, BMS-833923, significantly reduces both downstream Hedgehog signaling and tumor cell viability. The novel cell line resource enables preclinical studies and promises to speed the translation of new molecularly targeted therapies for the management of ameloblastoma and related odontogenic neoplasms.
Assuntos
Ameloblastoma , Tumores Odontogênicos , Humanos , Ameloblastoma/tratamento farmacológico , Ameloblastoma/genética , Proteínas Hedgehog , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Recidiva Local de Neoplasia , Tumores Odontogênicos/genética , Classe I de Fosfatidilinositol 3-Quinases/uso terapêutico , Proteínas Quinases Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Linhagem CelularRESUMO
Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.
Assuntos
Cisteína Endopeptidases/genética , Entamoeba histolytica/enzimologia , Entamoeba histolytica/patogenicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Amplificação de Genes , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , VirulênciaRESUMO
Extracellular neutral cysteine proteinases are an important virulence factor of E. histolytica. Experimental evidence supporting its role in invasion includes the ability to degrade components of the extracellular matrix and activate complement by specifically cleaving C3. We had previously reported the isolation of fragments encoding cysteine proteinase genes from HM-1 (ACP1) and a nonpathogenic strain (REF291, ACP2) by PCR using consensus sequences based on conserved structural motifs of eukaryotic cysteine proteinases. Using similar techniques, we have now identified a third gene encoding a cysteine proteinase which is present in both pathogenic and nonpathogenic strains and have correlated cysteine proteinase specific-mRNA levels with enhanced proteolytic activity and cytopathic effect on a fibroblast cell monolayer, a quantitative assay of virulence.
Assuntos
Cisteína Endopeptidases/genética , Entamoeba histolytica/genética , Genes de Protozoários , Isoenzimas/genética , Proteínas de Protozoários/genética , Animais , Células Cultivadas , Cisteína Endopeptidases/fisiologia , Entamoeba histolytica/enzimologia , Entamoeba histolytica/patogenicidade , Fibroblastos , Isoenzimas/metabolismo , Proteínas de Protozoários/metabolismo , RNA Mensageiro/análise , RNA de Protozoário/análise , Virulência/genéticaRESUMO
As experience with implants has grown, the aesthetic expectations of both dentists and patients has increased. Controlled placement techniques can benefit the aesthetic result, but anatomic constraints and procedural limitations may prevent ideal implant placement. This article reviews guidelines to proper placement of implants and presents surgical and restorative approaches to correction of improper positioning. The key to a successful, aesthetically pleasing reconstruction lies in careful case planning and communication.