PIM inhibition increases LKB1-dependent phosphorylation of AMPK
To investigate in more detail whether PIM kinases negatively regulate AMPK phosphorylation and activation, we used a pharmacological approach to inhibit PIM activity in PC3 prostate cancer, HeLa cervical cancer and MCF7 breast cancer cell lines. Cells were treated with either DMSO or 10 µM concentrations of two structurally distinct small molecule pan-PIM inhibitors, DHPCC9 or AZD1208, which inhibit the catalytic activity of all three PIM family members[33, 38]. The relative phosphorylation level of AMPK was determined 24 h later by Western blotting with antibodies against AMPK or its phosphorylated Thr172 residue. As shown in (Fig. 1A), AMPK was expressed at a similar level in all three cell lines, but it was more prominently phosphorylated in PC3 cells than in the others. When PIM activity was inhibited by either DHPCC9 or AZD1208, AMPK phosphorylation was significantly enhanced in both PC3 and MCF7 cells, but not in HeLa cells.
These results could be explained by the observed expression of the AMPK upstream kinase LKB1 in PC3 and MCF7 cells, but not in HeLa cells. To confirm this conclusion, we used the CRISPR/Cas9-based genomic editing technique to knock out LKB1 from both PC3 and MCF7 cells (Additional File 2, Figure S1A). As demonstrated by DNA gel electrophoresis (Additional File 2, Figure S2) and Western blotting (Fig. 1B), there was no LKB1 expression in the knock-out cells. When AMPK phosphorylation levels were analysed, no significant differences were observed between wild-type and LKB1-deficient cells that had been treated with DMSO (Fig. 1B). By contrast, treatment with the PIM inhibitor DHPCC9 induced a profound increase in AMPK phosphorylation in wild-type, but not knock-out cells. Furthermore, transient expression of FLAG-tagged LKB1 in LKB1-deficient PC3 or MCF7 cells restored the response to DHPCC9 (Fig. 1C). Altogether, these data indicate that LKB1 is necessary for the PIM inhibition-induced increase in AMPK phosphorylation.
AMPK phosphorylation levels are inversely correlated with PIM expression levels
In order to analyse the respective contribution of the different PIM family members in regulating AMPK phosphorylation and activity, we used the CRISPR/Cas9 technique to generate both individual and combined PIM knock-out cells (Additional File 2, Figure S1B-D). As confirmed by DNA gel electrophoresis (Additional File 2, Figure S2) and Western blotting (Fig. 2A), single (KO) as well as triple (TKO) knock-out lines were successfully produced from both PC3 and MCF7 cells. Lack of any single PIM protein did not result in notable changes in AMPK phosphorylation in either cell line (Fig. 2B). By contrast, significantly elevated levels of phosphorylation were observed in the two independent PC3 and MCF7 TKO cell clones (Fig. 2C), while transient expression of His-tagged PIM1 in these cells restored AMPK phosphorylation back to its basal level (Fig. 2D). Furthermore, FDCP1 myeloid cells stably overexpressing PIM1 (FD/PIM1) exhibited significantly lower levels of AMPK phosphorylation than the corresponding control cells (FD/Neo) (Fig. 2E). Taken together, our data indicate that either pharmacological inactivation or knock-out of all three PIM family members results in increased AMPK phosphorylation.
LKB1 is a novel substrate for PIM kinases
As LKB1 was indispensable for AMPK phosphorylation in both PC3 and MCF7 cells, this raised the question of whether PIM kinases downregulate LKB1 activity by directly phosphorylating it. To address this question, we subjected GST-tagged PIM family members, LKB1 or their combinations to radioactive in vitro kinase assays. Visualisation of 32P-labeled phosphoproteins by autoradiography revealed that all three PIM kinases phosphorylate LKB1 in vitro, and that LKB1 does not undergo autophosphorylation (Fig. 3A). Thus, our data indicate that LKB1 indeed is a novel substrate targeted by all three PIM kinases. These data were further confirmed by a non-radioactive in vitro kinase assay (Fig. 3B), where phosphoproteins were visualised by Western blotting with the phospho-Akt substrate (PAS) antibody. This antibody recognises not only the AKT-targeted sequence RXXS/T, but also the PIM-targeted consensus sequence RXRHXS/T (Fig. 3C).
For LKB1, multiple phosphorylation sites have been identified[40, 41]. However, only a few of them, including Ser334 and Ser428, resemble PIM-target sites that can be recognised by the PAS antibody. To determine whether one or both of them are PIM target sites, we mutated them separately to alanine residues and subjected the mutant proteins to in vitro kinase assays. When His-tagged wild-type (WT) or mutant proteins were incubated in the presence of GST-PIM1, there was a 40% decrease in the intensity of the 32P-labeled signal for the S334A mutant as compared to the WT protein, while no significant changes were observed for the S428A mutant (Fig. 3D), indicating that Ser334 is a prominent PIM target site. This was confirmed by non-radioactive in vitro kinase assays followed by Western blotting with the PAS antibody (Fig. 3E). However, as the S334A mutation did not completely remove the residual signals in either assay, it remains possible that PIM kinases also target other sites in LKB1.
Having established PIM proteins as upstream kinases of LKB1, we examined their intracellular interactions. In co-immunoprecipitation assays, His-tagged PIM1 could be captured by FLAG-tagged LKB1 from both cell lines (Fig. 3F). In FLIM (fluorescence-lifetime imaging microscopy) analysis, significantly reduced GFP lifetimes were observed when GFP-tagged LKB1 and RFP-tagged PIM1 were co-expressed in either MCF7 or PC3 cells (Fig. 3G), suggesting that they physically interacted with each other.
PIM kinases target Ser334 in LKB1 to regulate AMPK phosphorylation
To verify that PIM kinases phosphorylate LKB1 in cells, we transiently expressed FLAG-tagged LKB1 in both PC3 and MCF7 cells. At 24 h after transfection, cells were treated with DMSO or 10 µM DHPCC9 for another 24 h, after which cells were lysed, FLAG-LKB1 proteins were pulled down with the FLAG antibody and their phosphorylation levels were analysed by Western blotting with the PAS antibody. As shown in Fig. 4A, the relative phosphorylation levels of LKB1 were significantly reduced by PIM inhibition in both types of cells. In addition to the pharmacological approach, we analysed LKB1 phosphorylation in WT and TKO MCF7 cells transiently expressing either FLAG-tagged LKB1 or the corresponding S334A mutant. In line with our data on PIM inhibition, LKB1 phosphorylation was dramatically decreased in TKO cells lacking all three PIM kinases (Fig. 4B). Notably, there was no significant difference between the phosphorylation level of the S334A mutant in WT and TKO cells, suggesting that Ser334 is a prominent PIM target site in LKB1.
To explore the impact of Ser334 phosphorylation of LKB1 on AMPK phosphorylation, FLAG-tagged LKB1 or the corresponding S334A mutant were transiently expressed in LKB1 KO derivatives of PC3 and MCF7 cells, and the cells were treated with either DMSO or 10 µM DHPCC9. As expected, DHPCC9 treatment did not trigger any considerable increase in AMPK phosphorylation in either type of FLAG-transfected cells lacking LKB1 (Fig. 4C). By contrast, reintroduction of WT LKB1, but not the phosphorylation-deficient S334A mutant restored the response of cells to DHPCC-9, resulting in a significant increase in AMPK phosphorylation. These data suggest that phosphorylation of LKB1 at Ser334 is involved in the regulation of AMPK phosphorylation by PIM kinases and their inhibitors.
AKT has been reported to phosphorylate LKB1 at Ser334, resulting in its nuclear sequestration by the 14-3-3 protein. To determine whether PIM-dependent phosphorylation of this site has similar consequences in PC3 or MCF7 cells, we fractionated LKB1KO cells transiently expressing FLAG-tagged WT LKB1 or the phosphodeficient S334A mutant. According to our analyses, both WT and mutant proteins were mainly localised in the nuclear fractions of both PC3 and MCF7 derivatives (Additional File 2: Figure S3A). By contrast, the endogenous LKB1 in parental cells was mostly localised in the cytosolic fractions, and this was not influenced by pharmacological PIM inhibition (Additional File 2: Figure S3B) or by knocking out of all three PIM kinase members (Additional File 2: Figure S3C). However, the level of AMPK phosphorylation in the cytoplasmic fraction was increased in both cases. To confirm that there is no compensatory activation of AKT in the PIM TKO cells, we analysed AKT Ser473 phosphorylation levels from them, but did not observe any major changes as compared to WT cells (Additional File 2: Figure S3D).
Combined knock-out of LKB1 and PIM kinases impairs cell proliferation and tumor growth
We next performed an in silico analysis of mRNA expression levels in patient-derived samples and observed that in prostate carcinomas and certain breast carcinomas, PIM expression was elevated and LKB1/STK11 expression was reduced (Additional File 2: Figure S4). However, in the breast medullary carcinoma dataset, both PIM and LKB1 expression levels were highly upregulated. As LKB1 expression and LKB1-dependent AMPK activation are often associated with cell growth suppression, this prompted us to evaluate the proliferation rates for WT PC3 or MCF7 cells or their LKB1-deficient derivatives in response to treatment with DMSO or 10 µM DHPCC9. Proliferation was followed for 5 days by measuring cell confluence with the IncuCyte live cell imaging system, where the two independent LKB1 KO clones behaved similarly to the WT cells (Fig. 5A). All the DMSO-treated cells proliferated well with sigmoidal growth curves, while the DHPCC9 treatment retarded cell growth.
The proliferation rates of TKO cells lacking all PIM kinases were reduced (Fig. 5B), which was in line with what we observed following the DHPCC9 treatment of WT cells. Surprisingly, cells lacking both LKB1 and PIM kinases (TKOLKB1 KO) grew even slower than TKO clones. As shown in (Fig. 5C), increased AMPK phosphorylation levels in TKO clones correlated well with their slower proliferation rates as compared to their WT counterparts. On the other hand, due to the absence of LKB1, the phosphorylation levels of AMPK in TKOLKB1 KO clones were lower than in TKO clones, yet the proliferation rates of TKOLKB1 KO clones were slower. These data suggest that under the conditions of PIM inhibition, changes in AMPK phosphorylation levels are not directly connected to the proliferation properties of LKB1 KO cells.
We next employed the chick embryo chorioallantoic membrane (CAM) xenograft model to investigate the in vivo behaviour of the knock-out cells lacking both PIM and LKB1. WT MCF7 and PC3 cells or their KO and/or TKO derivatives were implanted onto the CAM of eggs on day 7 of incubation to allow the development of tumors. On day 14, tumors were excised from the CAM and weighed. Interestingly, there was a significant increase in the mass of tumors derived from LKB1 KO clones in PC3 but not MCF7 cells (Fig. 5D), but there was no significant difference between WT and TKO clones in either cell type. However, the mass of TKOLKB1KO MCF7 tumor cells was significantly lower than that of WT or LKB1KO samples. Intriguingly, while loss of LKB1 alone in PC3 cells increased tumor load, this effect was abolished, when combined with the loss of PIM kinases. Conversely, transient over-expression of PIM1 triggered further increases in tumor mass in two independent PC3 LKB1KO clone xenografts but not in WT samples (Fig. 5E). These data indicate that LKB1 and PIM kinases cooperate in the regulation of tumorigenic growth.