PIM kinases inhibit AMPK activation and promote tumorigenicity by phosphorylating LKB1

Abstract

Background 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. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells. Methods We 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. Results We 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. Conclusion PIM 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.

Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

“…This effect may be associated with the AMPK inhibition by TDRD7 , identified by Subramanian et al ( 15 , 16 ). PIM1 has been identified to block AMPK activation ( 42 ).…”

Proviral Insertion in Murine Lymphomas 2 Promotes Inflammation and Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Regulating AMPK and NF-κB Signalings

“…Inhibition of PIM2 reduced protein expression of Osterix to prevent bone loss in myeloma 20) . Moreover, PIM2 functioned as a negative regulator of liver kinase B1 to reduce the activity of AMPK through decreasing AMPK phosphorylation at Thr 172 29) and increasing AMPK phosphorylation at Thr 467 18) . Our results demonstrated that protein expression of CO-L1A1, OPN and RUNX2 in lipopolysaccharide-treated PDLCs were increased by knockdown of PIM2, and silence of PIM2 increased phosphorylation of AMPK at Thr 172 in lipopolysaccharide-treated PDLCs.…”

PIM kinases phosphorylate lactate dehydrogenase A at serine 161 and suppress its nuclear ubiquitination

“…FuGENEÒ HD Transfection Reagent (Promega, Madison, WI, USA) was used for plasmid transfections according to the manufacturer's protocol. The use of CRISPR/Cas9-based genome editing to prepare knock-out derivatives of MCF7 and PC3 cells lacking all three members in the PIM family has previously been described [13]. Eukaryotic and bacterial vectors pcDNA TM 3.1/V5-His-C and pGEX-6P-1 for expression of wild-type (WT) human PIM kinases have been described previously [10].…”

“…PIM kinases are a family of serine-threonine kinases with three highly homologous family members (PIM1, PIM2 and PIM3) that are often over-expressed in haematological or solid tumours [1][2][3] and that contribute to tumorigenesis there [4][5][6]. The PIM kinases are constitutively active [7] and regulate cell survival, motility, proliferation and metabolism by phosphorylating a wide range of substrates including BAD [8], NFATC1 [9], NOTCH1 [10], NOTCH3 [11], CAPZ [12] and LKB1 [13]. Knocking out all three members of the PIM family results in LKB1-dependent AMPK activation [13,14], increased production of reactive oxygen species as well as altered expression of several metabolic enzymes involved in glycolysis, pentose phosphate pathway or oxidative phosphorylation [15,16].…”

“…The PIM kinases are constitutively active [7] and regulate cell survival, motility, proliferation and metabolism by phosphorylating a wide range of substrates including BAD [8], NFATC1 [9], NOTCH1 [10], NOTCH3 [11], CAPZ [12] and LKB1 [13]. Knocking out all three members of the PIM family results in LKB1-dependent AMPK activation [13,14], increased production of reactive oxygen species as well as altered expression of several metabolic enzymes involved in glycolysis, pentose phosphate pathway or oxidative phosphorylation [15,16]. There is also increasing evidence on the ability of PIM kinases to directly phosphorylate metabolic enzymes, such as lactate dehydrogenase (LDH) [17], enolase [12,17], pyruvate kinase M2 [18], hexokinase 2 [19] and hypoxia-inducible factor 1 [20].…”