ABSTRACT
L1 cell adhesion molecule (L1CAM) overexpression is often associated with bad prognosis in various human carcinomas. Recent studies also suggest a role of L1CAM in pancreatic ductal adenocarcinomas (PDAC). To further address its contribution, we expressed functional domains of L1CAM in PT45-P1 PDAC cells. We found that L1CAM that is full length (L1-FL), but neither the soluble ectodomain (L1ecto) nor the cytoplasmic part (L1cyt), could enhance cell proliferation or tumour growth in mice. Expression of L1-FL resulted in constitutive activation of NF-kappaB, which was abolished by L1CAM knockdown. We showed that the expression of IL-1beta was selectively upregulated by L1-FL, and increased IL-1beta levels were instrumental for sustained NF-kappaB activation. IL-1beta production and NF-kappaB activation were abolished by knockdown of alpha5-integrin and integrin-linked kinase, but insensitive to depletion of L1CAM cleavage proteinases. Supporting these data, PT45-P1 cells transduced with an L1CAM mutant deficient in integrin binding (L1-RGE) did not support the described L1-FL functions. Our results suggest that membranous L1CAM interacts with RGD-binding integrins, leading to sustained NF-kappaB activation by IL-1beta production and autocrine/paracrine signalling. The unravelling of this novel mechanism sheds new light on the important role of L1CAM expression in PDAC cells.
Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Interleukin-1beta/biosynthesis , NF-kappa B/metabolism , Neural Cell Adhesion Molecule L1/metabolism , Pancreatic Neoplasms/metabolism , Animals , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Adhesion/physiology , Cell Growth Processes/physiology , Cell Line, Tumor , Drug Resistance, Neoplasm , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Mice , Mice, Inbred NOD , Mice, SCID , NF-kappa B/genetics , Neural Cell Adhesion Molecule L1/genetics , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
An elevated proteasome activity contributes to tumorigenesis, particularly by providing cancer cells with antiapoptotic protection and efficient clearance from irregular proteins. Still, the underlying mechanisms are poorly known. In this study, we report that in colon cancer patients, higher proteasome activity was detected in tumoral tissue compared with surrounding normal tissue, and also that increased levels of proteasomal subunit proteins, such as S5a/PSMD4 and alpha-5/PSMA5, could be detected. Colon tumors showed higher nuclear levels of nuclear factor E2-related factor 2 (Nrf2), a transcription factor supposed to be involved in the control of proteasomal subunit protein expression. The induction or overexpression of Nrf2 led to stronger S5a and alpha-5 expression in the human colon cancer cell lines, Colo320 and Lovo, as well as in NCM460 colonocytes along with higher proteasome activity. The small interfering RNA (siRNA)-mediated Nrf2 knockdown decreased S5a and alpha-5 expression and reduced proteasome activity. Additionally, Nrf2-dependent S5a and alpha-5 expression conferred protection from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, an effect preceded by an increased nuclear factor (NF)-kappaB activation and higher expression of antiapoptotic NF-kappaB target genes. These findings point to an important role of Nrf2 in the gain of proteasome activity, thereby contributing to colorectal carcinogenesis. Nrf2 may therefore serve as a potential target in anticancer therapy.
Subject(s)
Colorectal Neoplasms/metabolism , NF-E2-Related Factor 2/metabolism , Proteasome Endopeptidase Complex/metabolism , Blotting, Western , Cell Line, Tumor , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/pathology , Epithelial Cells/cytology , Humans , Immunohistochemistry , NF-E2-Related Factor 2/genetics , Oxidative Stress , Proteasome Endopeptidase Complex/biosynthesis , Proteasome Endopeptidase Complex/genetics , Signal Transduction , UbiquitinationABSTRACT
Pancreatic cancer exhibits profound chemoresistance resulting either from pre-existing (intrinsic) mechanisms, or from anticancer drug treatment itself (acquired chemoresistance). To identify molecular alterations leading to acquired chemoresistance, the chemosensitive pancreatic carcinoma cell line PT45-P1 was exposed to low-dose treatment with etoposide for 6 weeks. Afterwards, these cells (PT45-P1res) were much more resistant to high-dose treatment with anticancer drugs than parental cells. Among several differentially expressed genes in PT45-P1res cells, IL-1beta was most significantly upregulated, a finding in line with our previous observation that IL-1beta accounts for intrinsic chemoresistance of pancreatic carcinoma cells. Elevated IL-1beta expression in PT45-P1res cells was confirmed by real-time PCR and ELISA, and treatment with the IL-1 receptor antagonist restored drug-induced apoptosis. The increased IL-1beta secretion was accompanied by an elevated formation of nitric oxide (NO) and a NO-dependent inhibition of the etoposide-induced caspase-3/-7/-8/-9 activity. Caspase activation was restored either by the iNOS inhibitor 1400W, the reducing agent dithiothreitol or the IL-1 receptor antagonist, resulting in greater sensitivity towards anticancer drug treatment. Conversely, IL-1beta or the NO-donor SNAP decreased caspase activation and apoptosis in etoposide-treated PT45-P1 cells. These data confirm IL-1beta and NO as determinants of chemoresistance in pancreatic cancer, and indicate that the intrinsic and acquired chemoresistance rely to some extent on common molecular targets beneficial for improved therapeutical strategies.
