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

Abstract

BackgroundPrevious 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.MethodsStable 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.ResultsCompared 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.ConclusionsOur 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.

Effectiveness of mesothelin family proteins and osteopontin for malignant mesothelioma: Table 1–

“…In addition, mesothelin overexpression results in upregulation of growth/ survival pathways through autocrine production of growth factors such as interleukin (IL)-6 [13]. Mesothelin also induces an increase in nuclear factor (NF)-kB activation, which leads to resistance to tumour necrosis factor-a-induced apoptosis [14], indicating a mechanism through which mesothelin may help increase survival of tumour cells in the highly inflammatory milieu, evident in pancreatic cancer through Akt/phosphoinositide 3-kinase/NF-kB activation and IL-6 overexpression. Mesothelin overexpression results in secretion of high levels of IL-6, which could be responsible for the cells' increased viability and proliferation under serum-reduced conditions through an IL-6/soluble IL-6 receptor trans-signalling mechanism and the induction of the IL-6-STAT3 pathway [12,13].…”

Anetumab Ravtansine: A Novel Mesothelin-Targeting Antibody–Drug Conjugate Cures Tumors with Heterogeneous Target Expression Favored by Bystander Effect

“…Mesothelin may further contribute to metastasis by inducing the expression of matrix metalloproteinases 7 and 9 (13,14). In pancreatic cancer cells, mesothelin has been shown to contribute to tumorigenesis by inducing interleukin-6 expression and cell proliferation and by promoting resistance to TNF-a (15,16). Because of high expression in several cancers and a conversely restricted expression in normal tissues, mesothelin is an attractive target for anticancer therapy.…”

Evodiamine induces apoptosis in pancreatic carcinoma PANC-1 cells via NF κB inhibition

“…NF-κB is involved in the regulation of the transcription of a serial of proliferation and anti-apoptotic genes such as cyclin D1, Bcl-2 and XIAP (Ou et al, 2010;Yip-Schneider et al, 2005). Constitutive activation of NF-κB is implicated in pancreatic tumorigenesis and resistance to many chemotherapeutic agents such as TNF (Bharadwaj et al, 2011) and gemcitabine (Chen et al, 2012;Ou et al, 2010). In the light of above findings, it is important to evaluate the chemotherapeutic potential of NF-κB inhibitors.…”