Novel Stat3 Small Molecule Inhibitor Selectively Inhibits Mesothelin-Overexpressing Pancreatic Cancer Cell Proliferation

Introduction: Small molecules targeting signaling pathways activated in cancer cells are a promising alternative treatment option in pancreatic cancer (PC). Cell surface glycoprotein, Mesothelin (MSLN), is overexpressed in 90% of PCs. We showed MSLN overexpression activates Stat3 leading to increased cell proliferation/cell cycle progression through cyclin E/cdk2. Both Stat3 siRNA and Jak kinase inhibitor, could abrogate these effects. Tweardy's group has identified 3 novel, drug-like chemical probes that competitively target the phosphotyrosine (pY)-peptide binding site within the Src-homology 2 domain of Stat3 and block both Stat3 recruitment to activated receptors and homodimerization, while sparing Stat1, a STAT family member with anti-oncogenic function. The most active compound, C188 (4-[({3-[(carboxymethyl)thio]-4-hydroxy-1naphthyl}amino)sulfonyl]benzoic acid),is well tolerated in mice and thus could be a promising new drug to test in MSLN-overexpressing and hence Stat3-active PC cells in vitro and in vivo. Methods: MSLN-overexpressing PC cell lines CFPAC-1/BxPC3/AsPC-1 and retrovirally transduced stably MSLN-overexpressing MIA PaCa-2 (MIA-MSLN) and control cells MIA-V were used. MSLN-specific shRNA silenced AsPC (ASPC-shMSLN) and control cells (ASPC-shV) were also used. The effect of C188 on these cell lines with differential MSLN and Stat3 activation status were determined by MTT viability assay in vitro and by western blot. Based on previously reported potency of C188 in inhibiting Stat3 binding to its cognate pY-peptide ligand in surface Plasmon resonance binding (SPR) assays, in inhibiting IL-6-mediated Stat3 phosphorylation and in inhibiting nuclear translocation in a high-throughput fluorescence microscopy (HTFM) assay, we tested different doses (0.1/1/10μM) of C188 on these cells. We also examined cells incubated with JSI-124 (Cucurbitacin I), a known Jak kinase inhibitor. Results: Wefound increased p-Stat3 (Tyr705) in three naturally MSLN-high expressing cell lines (CFPAC-1, BxPC-3 & AsPC-1); C188 could effectively reduce the viability of all of these cells. In contrast, C188 had minimal effect on the viability of AsPC-shMSLN cells while strongly inhibiting AsPC-shV control cells. Furthermore, inhibition of MSLN-overexpressing PC cell proliferation by C188 treatment was dose-dependent.C188at 10 μM concentration could specifically reduce MIA-MSLN viability by almost 100% while MIA-V was unaffected by C188 treatment. Broad spectrum Jak inhibitor JSI-124 used under identical conditions proved to be equally potent against both cells irrespective of MSLN or Stat3 activation status. Conclusions: These promising results demonstrate the efficacy of a novel Stat3 inhibitor, C188, in specifically inhibiting growth of Stat3-active, MSLN-high PC cells. Our data clearly warrants further studies to determine the efficacy/specificity of C188 and its derivatives in reducing tumor burden of MSLN-overexpressing PC cells in animal models.