title: Mesothelin confers pancreatic cancer cell resistance to TNF-α-induced apoptosis through Akt/PI3K/NF-κB activation and IL-6/Mcl-1 overexpression.
abstract: BACKGROUND
  Previous studies showed that mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. The recent success of intratumorally-injected adeno-encoded, chemo/radiation-inducible-promoter driven hTNF-α, (TNFerade) + gemcitabine in pre-clinical models of PC have renewed interest in use of TNF-α as a therapeutic component. To help find additional factors which might affect the therapy, we examined the resistance of MSLN-overexpressing pancreatic cancer cell lines to TNF-α-induced growth inhibition/apoptosis.
  
  
  METHODS
  Stable MSLN overexpressing MIA PaCa-2 cells (MIA-MSLN), stable MSLN-silenced AsPC-1 cells (AsPC-shMSLN) and other pancreatic cells (MIA-PaCa2, Panc 28, Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48) were used. NF-κB activation was examined by western blots and luciferase reporter assay. TNF-α induced growth inhibition/apoptosis was measured by MTT, TUNEL assay and caspase activation. IL-6 was measured using luminex based assay.
  
  
  RESULTS
  Compared to low endogenous MSLN-expressing MIA PaCa-2 and Panc 28 cells, high endogenous MSLN-expressing Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48 cells were resistant to TNF-α induced growth inhibition. Stable MSLN overexpressing MIA-PaCa2 cells (MIA-MSLN) were resistant to TNF-α-induced apoptosis while stable MSLN-silenced AsPC1 cells (AsPC-shMSLN) were sensitive. Interestingly, TNF-α-treated MIA-MSLN cells showed increased cell cycle progression and cyclin A induction, both of which were reversed by caspase inhibition. We further found that MIA-MSLN cells showed increased expression of anti-apoptotic Bcl-XL and Mcl-1; deactivated (p-Ser75) BAD, and activated (p-Ser70) Bcl-2. Constitutively activated NF-κB and Akt were evident in MIA-MSLN cells that could be suppressed by MSLN siRNA with a resultant increase in sensitivity of TNF-α induced apoptosis. Blocking NF-κB using IKK inhibitor wedelolactone also increased sensitivity to TNF-α-mediated cytotoxicity with concomitant decrease in Mcl-1. Blocking Akt using PI3K inhibitor also had a likewise effect presumably affecting cell cycle. MIA-MSLN cells produced increased IL-6 and were increased furthermore by TNF-α treatment. SiRNA-silencing of IL-6 increased TNF-α sensitivity of MIA-MSLN cells.
  
  
  CONCLUSIONS
  Our study delineates a MSLN-Akt-NF-κB-IL-6-Mcl-1 survival axis that may be operative in PC cells, and might help cancer cells' survival in the highly inflammatory milieu evident in PC. Further, for the success of TNFerade + gemcitabine to be successful, we feel the simultaneous inhibition of components of this axis is also essential.
authors: Michael E, DeBakey Department of Surgery, Molecular Surgeon Research Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors: Uddalak
authors: U
authors: Bharadwaj
authors: Christian
authors: C
authors: Marin-Muller
authors: Min
authors: M
authors: Li
authors: Changyi
authors: C
authors: Chen
authors: Qizhi
authors: Q
authors: Yao
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D019925:Cyclin A / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D006801:Humans; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D000090204:Mesothelin; D009025:Morpholines / Q000494:pharmacology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010190:Pancreatic Neoplasms; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D002867:Chromones; D003374:Coumarins; D019925:Cyclin A; D058851:GPI-Linked Proteins; D051550:I-kappa B Kinase; D015850:Interleukin-6; D049410:Luciferases, Renilla; D000090204:Mesothelin; D009025:Morpholines; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C000715527:MSLN protein, human; C051122:wedelolactone; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one