Abstract
Using a cell-based high-throughput screen designed to detect small chemical compounds that inhibit cell growth and survival, we identified three structurally related compounds, 21A8, 21H7, and 65D4, with differential activity on cancer versus normal cells. Introduction of structural modifications yielded compound M-110, which inhibits the proliferation of prostate cancer cell lines with IC(50)s of 0.6 to 0.9 μmol/L, with no activity on normal human peripheral blood mononuclear cells up to 40 μmol/L. Screening of 261 recombinant kinases and subsequent analysis revealed that M-110 is a selective inhibitor of the PIM kinase family, with preference for PIM-3. The prostate cancer cell line DU-145 and the pancreatic cancer cell line MiaPaCa2 constitutively express activated STAT3 (pSTAT3(Tyr705)). Treatment of DU-145 cells with M-110 or with a structurally unrelated PIM inhibitor, SGI-1776, significantly reduces pSTAT3(Tyr705) expression without affecting the expression of STAT3. Furthermore, treatment of DU-145 cells with M-110 attenuates the interleukin-6-induced increase in pSTAT3(Tyr705). To determine which of the three PIM kinases is most likely to inhibit expression of pSTAT3(Tyr705), we used PIM-1-, PIM-2-, or PIM-3-specific siRNA and showed that knockdown of PIM-3, but not of PIM-1 or PIM-2, in DU-145 cells results in a significant downregulation of pSTAT3(Tyr705). The phosphorylation of STAT5 on Tyr694 in 22Rv1 cells is not affected by M-110 or SGI-1776, suggesting specificity for pSTAT3(Tyr705). These results identify a novel role for PIM-3 kinase as a positive regulator of STAT3 signaling and suggest that PIM-3 inhibitors cause growth inhibition of cancer cells by downregulating the expression of pSTAT3(Tyr705).
Highlights
The PIM family of oncogenic serine/threonine kinases consists of three members, PIM-1, PIM-2, and PIM-3
The pim-1 proto-oncogene was first identified as a locus frequently activated by proviral integration in Moloney murine leukemia virus–induced mouse T-cell lymphomas [1, 2], and pim-2 was identified as a gene frequently activated in secondary transplants of virus-induced lymphomas
To determine whether these compounds were active on epithelial tumor–derived cells, we tested the effect of 65D4 on the survival of the human prostate cancer cell line DU-145
Summary
The PIM family of oncogenic serine/threonine kinases consists of three members, PIM-1, PIM-2, and PIM-3. The pim-1 proto-oncogene was first identified as a locus frequently activated by proviral integration in Moloney murine leukemia virus–induced mouse T-cell lymphomas [1, 2], and pim-2 was identified as a gene frequently activated in secondary transplants of virus-induced lymphomas. The oncogenic nature of PIM-1 and PIM-2 was confirmed by the observation that transgenic mice overexpressing these kinases in the lymphoid system develop lymphomas. Simultaneous overexpression of c-myc further increases the frequency of lymphoma-. Authors' Affiliations: 1Genetics and Development Division, Toronto Western Research Institute, University Health Network; 2Department of Immunology, University of Toronto, Toronto, Ontario, Canada; and 3State Key Laboratory of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P.R. China
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