RESUMEN
BACKGROUND & AIMS: DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated. METHODS: A genome-wide database of 488,235 mouse coding mononucleotide repeats was established, from which a set of candidates was selected based on repeat length, gene expression, and mutation frequency. In silico prediction, in vivo immunogenicity testing, and epitope mapping was used to identify candidates for FSP vaccination. RESULTS: We identified 4 shared FSP neoantigens (Nacad [FSP-1], Maz [FSP-1], Senp6 [FSP-1], Xirp1 [FSP-1]) that induced CD4/CD8 T cell responses in naïve C57BL/6 mice. Using VCMsh2 mice, which have a conditional knockout of Msh2 in the intestinal tract and develop intestinal cancer, we showed vaccination with a combination of only 4 FSPs significantly increased FSP-specific adaptive immunity, reduced intestinal tumor burden, and prolonged overall survival. Combination of FSP vaccination with daily naproxen treatment potentiated immune response, delayed tumor growth, and prolonged survival even more effectively than FSP vaccination alone. CONCLUSIONS: Our preclinical findings support a clinical strategy of recurrent FSP neoantigen vaccination for LS cancer immunoprevention.
Asunto(s)
Antígenos de Neoplasias/farmacología , Vacunas contra el Cáncer/farmacología , Neoplasias Colorrectales Hereditarias sin Poliposis/tratamiento farmacológico , Mutación del Sistema de Lectura , Fenómenos Inmunogenéticos , Fragmentos de Péptidos/farmacología , Adyuvantes Inmunológicos/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/inmunología , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Epítopos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 2 Homóloga a MutS/genética , Naproxeno/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Carga Tumoral/efectos de los fármacos , Microambiente Tumoral , Vacunación , Eficacia de las VacunasRESUMEN
Galectins are ß-galactoside binding proteins which possess a variety of functions including modulation of apoptosis, growth and differentiation. Hence, alterations in the expression profile have been associated with loss of cellular homeostasis contributing to tumor growth and progression. Though galectin-12 is significantly downregulated in several tumor entities, including colon cancer, its impact on cellular homeostasis as well as galectin-12 specific binding partners have not been identified so far. We therefore established an experimental strategy which is based on reversible cross-link immunoprecipitation to capture the galectin-12 protein interactome in colon cancer cells. By applying this approach, we identified 10 novel candidates of galectin-12 interacting proteins including the neutral amino acid exchanger SLC1A5. Remarkably, we uncovered that binding of galectin-12 to SLC1A5 significantly reduced glutamine uptake in our model cell line. Consequently, utilization of glutamine carbon for biomass synthesis was profoundly affected, suggesting galectin-12 as a novel inhibitor of glutamine anaplerosis in colon cancer cells. More detailed analysis revealed that colon cancer cells can counteract galectin-12 mediated glutamine deprivation by induction of compensatory mechanisms which facilitate adaption to low-glutamine conditions and thus survival.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/metabolismo , Neoplasias del Colon/metabolismo , Galectinas/metabolismo , Glutamina/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Apoptosis/fisiología , Diferenciación Celular/fisiología , Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Células Tumorales CultivadasRESUMEN
DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.
Asunto(s)
Neoplasias Colorrectales , Mutación del Sistema de Lectura , Inestabilidad Genómica , Repeticiones de Microsatélite , Proteínas de Neoplasias , Degradación de ARNm Mediada por Codón sin Sentido , Fosfoproteínas , Proteínas de Unión al ARN , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Perfilación de la Expresión Génica , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteómica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Galectins, a class of lectins with specificity for ß-galactoside containing glycoconjugates, modulate several cellular processes that are involved in the control of normal cell growth, differentiation, cell-cell, and cell matrix interactions. Pathological alterations of the galectin expression pattern have been implicated in the development and progression of cancer. We therefore analyzed epigenetic mechanisms for control of galectin expression in 9 colorectal cancer (CRC) cell lines. Our data demonstrate that expression of galectins-1, -2, -7, -8, and -9 can be regulated by histone acetylation in CRC cell lines. In addition, the same set of galectins was also found to be modulated by DNA methylation. Of particular note, galectin-12 is silenced in all tested CRC cell lines but known to be re-expressed upon butyrate-induced differentiation and present in normal colonic mucosa. Loss of galectin-12 expression in undifferentiated CRC cells is associated with promoter hypermethylation and for the first time we provide detailed methylation analysis of the promoter region. In CRC tumor tissue, galectin-12 expression was downregulated in 66% of CRC tissue specimens as compared to adjacent normal tissue hinting to a possible tumor-suppressing function in CRC. © 2017 IUBMB Life, 69(12):962-970, 2017.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Epigénesis Genética , Galectinas/genética , Histonas/metabolismo , Acetilación , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Adulto , Anciano , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Estudios de Cohortes , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Metilación de ADN/efectos de los fármacos , Decitabina , Regulación hacia Abajo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Galectinas/metabolismo , Histonas/genética , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Transforming growth factor-ß (TGF-ß) signaling plays a key role in regulating normal cell growth and differentiation, and mutations affecting members of this pathway contribute to cancer development and metastasis. In DNA mismatch repair (MMR)-deficient colorectal cancers that exhibit the microsatellite instability (MSI) phenotype, biallelic frameshift mutations in the transforming growth factor ß receptor type 2 (TGFBR2) gene occur at high frequency that lead to altered signal transduction and downstream target gene expression. Although recent evidence suggests that altered TGF-ß signaling can modulate protein glycosylation patterns in MSI-high colorectal tumor cells, affected genes have not been identified. Here, we investigated in a more systematic approach, expression changes of TGFBR2-regulated genes, involved in glycosylation using a TGFBR2-reconstituted colorectal cancer cell line (HCT116-TGFBR2) and Glyco-Gene Chip analysis. Based on this oligonucleotide array of about 1000 human glycosylation-related genes, several candidates including HES1, PDGFB, JUNB and LFNG were found to be upregulated in a TGFBR2-dependent manner and subsequently validated by real-time RT-PCR analyses. Focusing on the glycosyltransferase LFNG and its target signaling protein Notch1, dual labeling with [3H]-N-acetyl-d-glucosamine ([3H]-GlcNAc) and [35S]-l-methionine revealed a significant increase in N-acetyl-d-glucosamine incorporation into immunoprecipitated Notch1 receptor upon TGFBR2 expression whereas the protein level remained unaffected. These data suggest that TGFBR2 signaling can affect Notch1 glycosylation via regulation of glycosyltransferase LFNG expression and provide a first mechanistic example for altered glycosylation in MSI colorectal tumor cells.
Asunto(s)
Acetilglucosamina/metabolismo , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Glicosiltransferasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Notch/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Western Blotting , Movimiento Celular/genética , Proliferación Celular , Células Clonales , Neoplasias Colorrectales/patología , Glicómica , Glicosilación , Glicosiltransferasas/metabolismo , Células HCT116 , Células Hep G2 , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Tipo II de Factor de Crecimiento Transformador beta , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND/AIM: The physiological compound sodium butyrate can induce differentiation in colon cancer cells in vitro. Due to the role of galectins in growth control we explored its effect on this network beyond galectins-1 and -3, with deliberate consideration of the status of microsatellite stability, for nine cell lines. MATERIALS AND METHODS: Microscopical monitoring and measurement of alkaline phosphatase activity ascertained butyrate's impact on cells. Monitoring by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting with galectin-type-specific probes characterized galectin expression. RESULTS: Controlled by expectable strong up-regulation of galectin-1 and comparatively small effects on galectin-3 regulation for galectins-4, -7, -8 and -9 were reported with no obvious association to microsatellite stability status. Neoexpression of the GAL-12 gene was observed in eight out of nine tested lines. CONCLUSION: Butyrate affects the galectin network beyond galectins-1 and -3, warranting further cell biological and histochemical studies.