Mesothelin confers pancreatic cancer cell resistance to TNF-α-induced apoptosis through Akt/PI3K/NF-κB activation and IL-6/Mcl-1 overexpression

Mol Cancer. 2011 Aug 31;10:106. doi: 10.1186/1476-4598-10-106.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects*
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism
  • Caspase 3 / metabolism
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Chromones / pharmacology
  • Coumarins / pharmacology
  • Cyclin A / metabolism
  • Drug Resistance, Neoplasm
  • Enzyme Activation
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / metabolism*
  • Gene Expression
  • Gene Knockdown Techniques
  • Genes, Reporter
  • Humans
  • I-kappa B Kinase / antagonists & inhibitors
  • I-kappa B Kinase / metabolism
  • Interleukin-6 / genetics
  • Interleukin-6 / metabolism*
  • Luciferases, Renilla / biosynthesis
  • Luciferases, Renilla / genetics
  • Morpholines / pharmacology
  • Myeloid Cell Leukemia Sequence 1 Protein
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / metabolism*
  • Pancreatic Neoplasms
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Transport
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • RNA Interference
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • S Phase / drug effects
  • Tumor Necrosis Factor-alpha / pharmacology*

Substances

  • Antineoplastic Agents
  • Apoptosis Regulatory Proteins
  • Chromones
  • Coumarins
  • Cyclin A
  • GPI-Linked Proteins
  • Interleukin-6
  • Morpholines
  • Myeloid Cell Leukemia Sequence 1 Protein
  • NF-kappa B
  • Phosphoinositide-3 Kinase Inhibitors
  • Proto-Oncogene Proteins c-bcl-2
  • Recombinant Proteins
  • Tumor Necrosis Factor-alpha
  • wedelolactone
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Luciferases, Renilla
  • Proto-Oncogene Proteins c-akt
  • I-kappa B Kinase
  • Caspase 3
  • mesothelin