RESUMO
Pancreatic stellate cells (PSCs) constitute important cells of the pancreatic microenvironment and their close interaction with cancer cells is important in pancreatic cancer. It is currently not known whether PSCs accumulate genetic alterations that contribute to tumor biology. Our aim was to analyze genetic alterations in cancer associated PSCs. PSC DNA was matched to DNA isolated from pancreatic cancer patients' blood (n = 5) and analyzed by Next-Generation Sequencing (NGS). Bioinformatic analysis was performed using the GATK software and pathogenicity prediction scores. Sanger sequencing was carried out to verify specific genetic alterations in a larger panel of PSCs (n = 50). NGS and GATK analysis identified on average 26 single nucleotide variants in PSC DNA as compared to the matched blood DNA that could be visualized with the Integrative Genomics Viewer. The absence of PDAC driver mutations (KRAS, p53, p16/INK4a, SMAD4) confirmed that PSC isolations were not contaminated with cancer cells. After filtering the variants, using different pathogenicity scores, ten genes were identified (SERPINB2, CNTNAP4, DENND4B, DPP4, FGFBP2, MIGA2, POLE, SNRNP40, TOP2B, and ZDHHC18) in single samples and confirmed by Sanger sequencing. As a proof of concept, functional analysis using control and SERPINB2 knock-out fibroblasts revealed functional effects on growth, migration, and collagen contraction. In conclusion, PSC DNA exhibit a substantial amount of single nucleotide variants that might have functional effects potentially contributing to tumor aggressiveness.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/patologia , Genômica , Humanos , Nucleotídeos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Microambiente Tumoral/genética , Neoplasias PancreáticasRESUMO
The aim of this study was to elucidate the role of CD97 isoforms in gastric carcinoma. Out of four gastric cancer cell lines investigated, BGC-823 cells demonstrating low CD97 protein expression were stably transfected with pcDNA3.1 vector containing CD97/EGF1,2,5 or CD97/EGF1,2,3,4,5 inserts. Behavior of transfected cells was systematically investigated by employing proliferation, motility and invasive assays. As a result, we found that over-expression of CD97/EGF1,2,5 isoform correlated with increased motile and invasive ability of the clones. Furthermore, CD97/EGF1,2,5 isoform over-expression (3.8 times higher) was followed by significant decrease of CD97/EGF1,2,3,4,5 isoform (10.3 times lower). In contrast, CD97/EGF1,2,3,4,5 clones revealed significantly reduced invasive properties as compared with corresponding controls. The changes in acetylation status were one of the possible mechanisms affecting behavior of transfected cells. We concluded from the study that CD97 is closely related with advanced stages and higher invasiveness of gastric carcinoma. The study further lightened the tumor promoting role of CD97 small isoform in cancer progression and indicated the possible suppressive properties of the full length isoform of CD97.
Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Invasividade Neoplásica/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/genética , Separação Celular , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Invasividade Neoplásica/patologia , Isoformas de Proteínas , Receptores Acoplados a Proteínas G , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/patologia , TransfecçãoRESUMO
Retinoic acid (RA) acts as an anti-proliferative and redifferentiation agent in the therapy of thyroid carcinoma. Our previous studies demonstrated that pretreatment of follicular thyroid carcinoma cell lines FTC-133 and FTC-238 resulted in decreased in vitro proliferation rates and reduced tumor cell growth of xenotransplants. In addition to the previous results, we found that RA led to decreased vitality and invasiveness of FTC-133 and FTC-238 cells as they reacted with reduction of intracellular ATP levels and number of migrated cells respectively. However, the molecular mechanisms by which RA mediates these effects are not well understood. Two-dimensional (2D) screening of the proteins related to ATP metabolism and western blot analysis revealed alpha-enolase (ENO1) to be down-regulated in FTC-133 and FTC-238 cells after RA treatment. 2D gel detection and mass spectrometric analysis revealed that ENO1 existed as three separate protein spots of distinct pIs (ENO1-A1-A3). Comparative 2D difference gel electrophoresis analysis of fluorescently labeled protein samples of RA-treated and untreated FTC-133 demonstrated a selective down-regulation of ENO1-A1 which we identified as a phosphoprotein. RA caused the dephosphorylation of ENO1-A1. Both, RA-mediated and specific knock-down of ENO1/MBP-1 resulted in the reduction of MYC oncoprotein, and simultaneously decreased proliferation rates of FTC-133 and FTC-238 cell lines. In summary, the RA-mediated down-regulation of the ENO1 gene products and MYC oncoprotein provides a novel molecular mechanism facilitating the anti-proliferative effect of RA in human thyroid carcinoma cells and suggests new pathways for supportive RA therapies.