Abstract
BackgroundThe oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells.MethodsWe first determined how PIM and LKB1 kinases affect AMPK phosphorylation levels. We then used in vitro kinase assays to demonstrate that LKB1 is phosphorylated by PIM kinases, and site-directed mutagenesis to identify the PIM target sites in LKB1. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9 genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate and MCF7 breast cancer cell lines. In addition to cell proliferation assays, we examined the effects of PIM and/or LKB1 loss on tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model.ResultsWe provide both genetic and pharmacological evidence to demonstrate that inhibition of PIM expression or activity increases phosphorylation of AMPK at Thr172 in both PC3 and MCF7 cells, but not in their derivatives lacking LKB1. This is explained by our observation that all three PIM family kinases can phosphorylate LKB1 at Ser334. Wild-type LKB1, but not its phosphodeficient derivative, can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. In the CAM model, loss of LKB1 enhances tumorigenicity of PC3 xenografts, while cells lacking both LKB1 and PIMs exhibit slower proliferation rates and form smaller tumors.ConclusionPIM kinases are novel negative regulators of LKB1 that affect AMPK activity in an LKB1-dependent fashion. The impairment of cell proliferation and tumor growth in cells lacking both LKB1 and PIMs indicates that these kinases possess a shared signaling role in the context of cancer. These data also suggest that PIM inhibitors may be a rational therapeutic option for LKB1-deficient tumors.6DjYH8_uPF8iBB8mWNNcxfVideo
Highlights
The oncogenic PIM kinases and the tumor-suppressive liver kinase B1 (LKB1) kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions
Cells were treated with either DMSO or 10 μM concentrations of two structurally distinct small molecule pan-PIM inhibitors, DHPCC9 or (See figure on page.) Fig. 1 AMP-activated protein kinase (AMPK) phosphorylation is enhanced by PIM inhibitors in an LKB1-dependent fashion. a PC3, Hela and MCF7 cells were treated for 24 h with DMSO (0.1%) or either DHPCC9 or AZD1208 pan-PIM inhibitor (10 μM in 0.1% DMSO) and subjected to Western blotting with antibodies against phospho-AMPK (Thr172), AMPK or LKB1
Phospho-AMPK (Thr172) versus AMPK levels were measured from WT cells in comparison to cells lacking individual b or all c PIM family members. d WT or triple knock-out (TKO) cells were transiently transfected with His or His-PIM1 plasmids, lysed 24 h later and subjected to Western blotting to determine relative AMPK phosphorylation levels. e Lysates of stably transfected FD/NEO and FD/ PIM1 cell lines were subjected to Western blotting to determine relative AMPK phosphorylation levels
Summary
The oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. PIM kinases have emerged as attractive targets for cancer therapy, especially as they possess a structurally unique ATP-binding pocket [4], and as PIM triple knock-out (TKO) mice are viable and fertile with only a mild reduction in body size [6]. This phenotype may at least partially be due to reduced cytokine responses [7] and a diminished glycolytic phenotype [8]. Inhibition of PIM expression or activity has been shown to increase AMPK phosphorylation, possibly via LKB1 [30], but the exact mechanism behind this phenomenon has remained unclear
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