PIM1 drives lipid droplet accumulation to promote proliferation and survival in prostate cancer

Abstract

Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer. Mechanistically, PIM1 inactivates glycogen synthase kinase 3 beta (GSK3β) via serine 9 phosphorylation. GSK3β inhibition stabilizes PPARα and enhances the transcription of genes linked to peroxisomal biogenesis (PEX3 and PEX5) and LD growth (Tip47). The effects of PIM1 on LD accumulation are abrogated with GW6471, a specific inhibitor for PPARα. Notably, LD accumulation downstream of PIM1 provides a significant survival advantage for prostate cancer cells during nutrient stress, such as glucose depletion. Inhibiting PIM reduces LD accumulation in vivo alongside slow tumor growth and proliferation. Furthermore, TKO mice, lacking PIM isoforms, exhibit suppression in circulating triglycerides. Overall, our findings establish PIM1 as an important regulator of LD accumulation through GSK3β-PPARα signaling axis to promote cell proliferation and survival during nutrient stress.

Fig. 1. PIM1 kinase is essential for LD accumulation in prostate cancer.

A Representative images of LDs in PC3TripzPIM1 cells following doxycycline treatment (50 ng/ml, 24 h). B Western blot of PC3TripzPIM1 cells following doxycycline treatment (50 ng/ml, 24 h). C Quantification of LD size and number per nuclei (n > 30 cells/group). D Representative images of basal levels of LDs, and (E) western blotting for PIM1 expression in the indicated cell lines. F Quantification of LD size and number per nuclei (n > 30 cells/group). G Representative images of LDs in tumor sections from PC3 and PC3-PIM1 xenograft tumors; LDs are in red (Oil Red O) and nuclei in blue (Hematoxylin). H Quantification of oil red o staining as % stained cells. At least 30 cells from 6 random fields were analyzed per treatment group. n = 3, mean ± SEM. ***p ≤ 0.001. For staining in (A) and (D), LDs in green (LipidSpot488) and nuclei in blue (Dapi). Scale bars, 50 µm. n = 3, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 2. PIM1 induces LD accumulation through GSK3β inhibition.

A Representative images of LDs in PC3TripzPIM1 cells following doxycycline treatment (50 ng/ml, 24 h) alone, and in combination with PIM inhibition (PIM447, 3 µM, 24 h). B Western blotting of PC3Tripz-PIM1 cells following doxycycline treatment (50 ng/ml, 24 h) alone, and in combination with PIM inhibition (PIM447, 3 µM, 24 h). C Quantification of LD size and number per nuclei (n > 30 cells/group). D Representative images of LDs and (E) western blotting of PC3TripzPIM1 cells following doxycycline treatment (50 ng/ml, 24 h) expressing vector or GSK3β (S9A) mutant. F Quantification of LD size and number per nuclei (n > 30 cells/group). For staining in (A) and (D), LDs in green (LipidSpot488) and nuclei in blue (Dapi). Scale bars, 50 µm. n = 3, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 3. GSK3β inhibition enhances peroxisomal biogenesis through PPARα to induce LD accumulation.

A Relative normalized expression of indicated genes relating to PPAR signaling (PPARα, β, γ), peroxisomal biogenesis (PEX3, PEX5, and PEX7), and Lipid droplet growth (TIP47) in PC3LN4 and PC3LN4crisprPIM1 cells. B Western blotting of TIP47 expression and GSK3β S9 phosphorylation in the indicated cell lines. C Representative images of peroxisomal marker catalase (CAT) in PC3TripzPIM1 cells following doxycycline treatment (50 ng/ml, 24 h). D Quantification of CAT puncta size and number per nuclei (n > 30 cells/group). E Representative images of basal CAT staining in the indicated cell lines. F Quantification of CAT puncta size and number per nuclei (n > 30 cells/group). G Representative images of CAT in PC3 cells following CHIR treatment (50 nM, 24 h). H Quantification of CAT puncta size and number per nuclei (n > 30 cells/group). For (C), (E), and (G), CAT is in green, and nuclei in blue (Dapi). Scale bars, 50 µm. n = 3, mean ± SEM. *p ≤ 0.05, ***p ≤ 0.001.

Fig. 4. PPARα inhibition abrogates the effect of PIM1 LD accumulation and prostate cancer proliferation.

A Representative images of LDs in PC3LN4pCIP (basal) and PC3LN4hPIM1 (basal and with PPARα inhibitor, GW6471 treatment, 4 µM, 24 h) cells. LDs are in red (Oil Red O) and nuclei in blue (Hematoxylin). Scale bars, 50 µm. B Quantification of LD size and number per nuclei (n > 27 cells/group). n = 3, mean ± SEM. ***p ≤ 0.001. C Representative images of crystal violet staining of PC3LN4pCIP and PC3LN4hPIM1 cells treated with DMSO or PPARα inhibitor (GW6471, 4 µM, 72 h). D Quantification of crystal violet staining representing fold change in cell viability. n = 3, mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001.

Fig. 5. Analysis of lipid composition with PIM1 induction in prostate cancer.

PC3Tripz-PIM1 cells were treated with DMSO or doxycycline (50 ng/ml, 24 h) and lipids were extracted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A Levels of various lipids in PC3Tripz-PIM1 cells. Raw values were normalized, and fold change vs DMSO was calculated to generate relative normalized ion intensity values. n = 3, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. B Relative normalized ion intensity values for most abundant TG species detected. Raw values were normalized, and fold change vs DMSO was calculated to generate relative normalized ion intensity values. n = 3, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. C Relative normalized ion intensity values for most abundant PC species detected. Raw values were normalized, and fold change as vs DMSO was calculated to generate relative normalized ion intensity values. n = 3, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. D Relative normalized ion intensity values for most abundant LPC species detected. Raw values were normalized, and fold change as vs DMSO was calculated to generate relative normalized ion intensity values. n = 3, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01.

Fig. 6. PIM1 promotes cell survival during nutrient stress.

A Quantitative analysis of SyTox fluorescence intensity indicating relative cell death in the indiated cell lines maintained in complete media (CM, RPMI1640, 10% dialyzed FBS, 25 mM Glucose) or depleted media (DM, RPMI1640, 10% dialyzed FBS) for 24 h. B Representative images of LDs in PC3LN4pCIP and PC3LN4hPIM1 cells maintained in complete or depleted media for 48 h. LDs in green (LipidSpot488) and nuclei in blue (Dapi). Scale bars, 50 µm. C Quantification of LD size and number per nuclei from (n > 30/treatment group). D Quantitative analysis of SyTox fluorescence intensity indicating relative cell death in the indicated cell lines maintained in complete or depleted media and treated with DMSO or Etomoxir (ETO, 100 µM, 48 h). E Representative images of crystal violet staining of PC3LN4pCIP, PC3LN4hPIM1, DU145pCIP, and DU145hPIM1 cells maintained in complete or depleted media and treated with DMSO or ETO. F Quantification of crystal violet staining. n = 3, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 7. PIM inhibition reduces LD accumulation and tumor growth in vivo.

A Representative images of LDs in PC3LN4 xenograft tumors following indicated treatment. LDs are in red (Oil Red O) and nuclei in blue (Hematoxylin). Scale bars, 50 µm. B Quantification of average LD size and number. n = 3, mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001. C Tumor volume (mm³) determined over time. n = 8, mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. D Representative images of tumor sections (4 micrometers) examined by H&E and immunohistochemical analysis. Scale bars, 50 µm. E Quantification of immunohistochemistry. F Western blot analysis of tumor samples alongside indicated cells exhibiting similar patterns in nitro and in vivo following PIM inhibition. G Heat map showing downregulation of DG and TG levels in serum samples isolated from indicated mice. H working model describing the role of PIM1 kinase in regulating LD accumulation through GSK3β-PPARα axis to promote prostate cancer survival and proliferation.