RESUMO
Inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5) has been identified as a metastasis suppressor gene in pancreatic cancer. Here, we analyzed ITIH5 promoter methylation and protein expression in The Cancer Genome Atlas (TCGA) dataset and three tissue microarray cohorts (n = 618), respectively. Cellular effects, including cell migration, focal adhesion formation and protein tyrosine kinase activity, induced by forced ITIH5 expression in pancreatic cancer cell lines were studied in stable transfectants. ITIH5 promoter hypermethylation was associated with unfavorable prognosis, while immunohistochemistry demonstrated loss of ITIH5 in the metastatic setting and worsened overall survival. Gain-of-function models showed a significant reduction in migration capacity, but no alteration in proliferation. Focal adhesions in cells re-expressing ITIH5 exhibited a smaller and more rounded phenotype, typical for slow-moving cells. An impressive increase of acetylated alpha-tubulin was observed in ITIH5-positive cells, indicating more stable microtubules. In addition, we found significantly decreased activities of kinases related to focal adhesion. Our results indicate that loss of ITIH5 in pancreatic cancer profoundly affects its molecular profile: ITIH5 potentially interferes with a variety of oncogenic signaling pathways, including the PI3K/AKT pathway. This may lead to altered cell migration and focal adhesion formation. These cellular alterations may contribute to the metastasis-inhibiting properties of ITIH5 in pancreatic cancer.
Assuntos
Adesão Celular , Movimento Celular , Neoplasias Pancreáticas , Transdução de Sinais , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Movimento Celular/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Adesões Focais/metabolismo , Adesões Focais/genética , Metilação de DNA/genética , Regiões Promotoras Genéticas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Secretadas Inibidoras de ProteinasesRESUMO
In pancreatic cancer treatment, tumor stage-dependent chemotherapies are used to prolong overall survival. By measuring DNA promoter hypermethylation in the plasma of patients with stage IV pancreatic cancer, it was recently shown that promoter DNA methylation of the tumor suppressor gene SFRP1 has a high value for predicting failure of drug treatment with gemcitabine. In this study, we therefore aimed to identify as precisely as possible the region in the SFRP1 promoter that is frequently hypermethylated in pancreatic cancer tissue. First, we used the TCGA data set to define CpG-rich regions flanking the SFRP1 transcription start site that were significantly more methylated in pancreatic cancer compared to normal pancreatic acinar tissue. A core CpG island was identified that exhibited abundant tumor DNA methylation and anti-correlation of SFRP1 mRNA expression. To validate our in silico results, we performed bisulfide conversion followed by DNA pyrosequencing of 28 matched formalin-fixed, paraffin-embedded (FFPE) pancreatic cancer cases and six pancreatic cancer cell lines. A defined block of seven CpG sites within the core CpG island was identified, which confirmed our in silico results by showing significantly higher SFRP1 methylation in pancreatic cancer specimens than in normal pancreatic tissue. By selecting this core CpG island, we were able to determine a median overall survival benefit for the low SFRP1 methylation group compared to the high SFRP1 methylation group (702 versus 517 days, p = 0.01) in the TCGA pancreatic cancer cohort. We propose a compact pyrosequencing assay that can be used in the future to further investigate the prognostic value of SFRP1 promoter hypermethylation in predicting pancreatic cancer chemoresistance. Therefore, instead of DNA analysis from blood (liquid biopsy), DNA easily extractable from cancer tissue blocks (FFPE material) could be used.