Abstract 3960: Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation (2010)

Abstract Background: We have shown previously that mesothelin (MSLN) overexpression in pancreatic cancer (PC) cells leads to enhanced cell survival/proliferation in vitro & tumor progression in a xenograft mouse model. The objective of this study is to determine the underlying MSLN-induced growth factors/signaling that contribute to the PC cell proliferation. Methods: PC tissue/serum & cell lines were used to evaluate MSLN/IL-6 expression. MIA PaCa-2 & Panc1 cells stably over-expressing MSLN (MIA-MSLN, Panc1-MSLN) & vector control cell lines (MIA-V, Panc1-V) were generated by using retrovirus expression system. IL-6 was measured by BioPlex. Silencing of MSLN/IL-6 was done by using specific siRNAs. NF-κB activation was examined by western blot & reporter assay. Different forms of IL-6Rs were determined by western blot, real-time PCR & FACS. Cell proliferation was measured by MTT. Cell cycle analysis was performed using PI staining & apoptosis determination by caspase3 cleavage. Results and Conclusions: MSLN expression positively correlated with secreted IL-6 in a panel of human PC specimens (serum) & cell lines (supernatants). Both MIA-MSLN & Panc1-MSLN showed high IL-6 expression & siRNA silencing of MSLN significantly reduced the IL-6 level (p&lt;0.01). Constitutive NF-κB activation in MIA-MSLN cells was blocked by IKK inhibitor wedelolactone with concomitant IL-6 reduction (p&lt;0.01). MIA-MSLN cells survived/proliferated even under serum free conditions with massively increased IL-6 production, indicating its possible role in autocrine stimulation. Expectedly, treatment with IL-6 siRNA (siIL-6) reduced cell proliferation & cell cycle progression. Longer IL-6 siRNA treatment (&gt;5 day) induced apoptosis with significant decrease of c-myc/bcl-2 levels. Interestingly, exogenous rIL-6 induced proliferation of MIA-MSLN cells but not MIA-V cells. Although there were no changes in mRNA/protein levels of the IL-6R (gp80), the soluble fraction (sIL-6R) was significantly elevated in MIA-MSLN cells. Treatment with the TACE/ADAM17 inhibitor TAPI-1 significantly reduced sIL-6R levels, indicating intramembrane cleavage of IL-6R in these cells. This, along with the absence of any difference in membrane IL-6R suggested IL-6 trans-signaling operative in these cells. Consequently, blocking this IL-6/sIL-6R axis using sIL-6R specific antibody abrogated serum free survival as well as rIL-6-induced proliferation. Moreover, a rIL-6/rsIL-6R mix induced proliferation was observed even in MIA-V cells. Our data suggest MSLN-activated NF-κB induces elevated IL-6 expression, which acts as growth factor to support pancreatic cancer cell survival/proliferation through a novel auto/paracrine IL-6/sIL-6R trans-signaling. The close correlation between MSLN & IL-6 provides an opportunity for formulating a new combined therapy for effective control of MSLN-overexpressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3960.