Secreted spermidine synthase reveals a paracrine role for PGC1α-induced growth suppression in prostate cancer

Abstract

Prostate cancer is the fifth cause of death by cancer worldwide, second in incidence in the male population. The definition of the molecular basis of its development and the oncogenic signals driving lethality continue to be important objectives in prostate cancer research. Prior work from others and us has demonstrated that loss of PGC1α expression results in a metabolic, signaling and transcriptional reprogramming that supports the development of metastatic disease. However, we do not fully understand the spectrum of tumor suppressive effects regulated by this co-regulator. Here we show that PGC1α governs non-cell autonomous paracrine tumor suppression in prostate cancer. A systematic analysis of the transcriptional landscapes associated to PGC1α loss of expression revealed that PGC1α alters the expression of genes encoding for secreted proteins. Cell secretome studies corroborated that PGC1α-dependent ERRα regulation in prostate cancer cells suppresses the growth of tumor cells exposed to their conditioned media, independently of androgen receptor status. The integration of in vitro and in vivo secretomics data and genetic perturbation assays revealed spermidine synthase as a transcriptional target of PGC1α and mediator of the paracrine metabolic growth suppressive effect. Moreover, the activity of the regulatory axis PGC1α-ERRα-SRM was reflected in patients and had prognostic value. Altogether, this work provides unprecedented evidence of the non-cell autonomous suppressive role of PGC1α, which broadens the view of this co-regulator as a multifactorial tumor suppressor in prostate cancer.

Fig. 1. PGC1α-driven conditioned media reduced 2D-cell proliferation of aggressive prostate cancer cells.

A Schematic representation of the experimental approach for condition media production. B Quantification of 2D-cell proliferation in AR negative, PC3 (n = 7) and DU145 (n = 5) cells, and AR positive 22Rv1 (n = 4) and LnCaP (n = 4) cells grown with differential conditioned media produced by PGC1α non-expressing (CM (-D)) and expressing (CM (+D)) PC3 cells. A representative image of the crystal violet staining is included below the quantifications. C Schematic representation of the experimental approach for production and combination of conditioned medias. D Dose-dependent effect of PGC1α-expressing PC3 cells´ conditioned media (CM (+D)). Different percentages of PGC1α-expressing and non-expressing PC3 conditioned media were used to grow and monitor 2D cell proliferation of recipient PC3 cells for 7 days (n = 3). E. Quantification of proliferation rate of PC3 TRIPZ cells co-cultured with PGC1α-expressing PC3 cells for 7 and 12 (7 + 5) days (n = 3). In B, D, E, data are normalized to the -Dox (non-PGC1α expressing) conditions, depicted by a black dotted line. R: recipient cells. CM conditioned media, D or Dox doxycycline, FC fold change. Statistics: one sample t-test with reference value 1 (B, D), ordinary one-way ANOVA (D, depicted with a dollar symbol). *p.value < 0.05; **p.value < 0.01; ***/$$$ p.value < 0.001. Error bars indicate s.e.m.

Fig. 2. PGC1α- non-cell autonomous anti-proliferative effect is dependent on ERRα and restricted to the soluble fraction of the conditioned media.

A Quantification of 2D-cell proliferation (crystal violet) of PC3 (n = 3) grown with differential conditioned media produced by PGC1α non-expressing and expressing PC3 cells with or without deletion of ERRα. A representative image of the crystal violet staining is included the quantifications. B Quantification of 2D-cell proliferation (crystal violet) of PC3 (n = 3) grown with the heavy fraction of conditioned media (>10 kDa) produced by PGC1α non-expressing and expressing PC3 cells with or without deletion of ERRα. C Effect of EVs produced by PGC1α non-expressing and expressing PC3 cells on the 2D-cell proliferation (crystal violet) of PC3 cells during 5 (left panel, n = 4) and 7 days (right panel, n = 5). D Effect of EVs-depleted fraction produced by PGC1α non-expressing and expressing PC3 cells on the 2D-cell proliferation (crystal violet) of PC3 cells during 5 (left panel, n = 4) and 7 days (right panel, n = 4). All data are normalized to the CM - Dox (non-PGC1α expressing) condition, depicted by a black dotted line. R: recipient cells. CM conditioned media. SFs soluble factors, EVs extracellular vesicles. D or Dox: doxycyline. Statistics: one sample t-test with reference value 1 (A–D); paired-t-test (A, B). */$ p.value 0.05; **/$$ p.value < 0.01; ***/$$$ p.value 0.001. Asterisks indicate statistical difference between No Dox and Dox conditions and dollar symbols indicate statistical difference between Control Dox and sgERRα#1/sgERRα#2 Dox. Error bars indicate s.e.m.

Fig. 3. PGC1α regulates spermidine synthase expression and secretion in prostate cancer cells.

A Volcano plot representing label-free LC/MS data of proteins differentially secreted by PGC1α expressing and non-expressing PC3 cells. B Volcano plot representing label-free LC/MS data of proteins differentially detected in the tumor interstitial liquid (TIL) isolated from Pten and Pten/Pgc1a KO prostate tumors. C Venn Diagram (left panel) showing the common secreted proteins differentially detected by LC/MS in vitro (CM) and in vivo (TIL) and histogram (right panel) showing the degree of change in the detection of SRM. Effect of PGC1α re-expression on SRM in PC3 cells (D, RT-qPCR, n = 3; E one representative Western blot out of 4, quantifications are shown below). F Effect of ERRα deletion on the PGC1α-driven transcriptional deregulation of SRM (RT-qPCR, n = 3). G Chromatin immunoprecipitation of exogenous PGC1α on SRM promoter in PC3-PGC1α expressing cells after induction with 0.5 mg/mL doxycycline (n = 3). Final data were normalized to IgG (negative immunoprecipitation control). CM conditioned media, D or Dox: doxycycline, FC fold change. Statistics: one sample t-test with reference value 1 (D, F, G); paired-t-test (F). */$ p.value 0.05; **/$$ p.value < 0.01; ***/$$$ p.value < 0.001. Asterisks indicate statistical difference between No Dox and Dox conditions and dollar symbols indicate statistical difference between Control Dox and sgERRα#1/sgERRα#2 Dox. Error bars indicate s.e.m.

Fig. 4. Differential secretion of spermidine synthase contributes to the non-cell autonomous anti-proliferative effect of PGC1α in prostate cancer.

A Incorporation of ¹³C from U-13C5-L-Methionine (2 h pulse) into spermidine and spermine metabolites after 3 day-treatment of PC3 cells grown with the indicated CM. B Validation of SRM overexpression in PC3 cells with inducible expression of PGC1α (Western blot, one representative image out of 4). C Quantification of 2D-cell proliferation (crystal violet) of PC3 grown with differential CM produced by: PGC1α non-expressing and expressing PC3 cells with or without overexpression of SRM (n = 4). D Quantification of PC3-luc cells co-injected in nude mice together with PGC1α non-expressing (No Dox) and expressing (Dox) PC3 cells with or without overexpression of SRM, at day 6 (left panel) and 16 (right panel). (n = 10 tumors per group; 2 injections per mice). E Confirmation of SRM silencing in PC3 cells using two independent short hairpin RNAs (Western blot, one representative image out of 3). F Quantification of 2D-cell proliferation (crystal violet) of PC3 grown with differential CM produced by PC3 cells in which the expression of SRM was silenced (n = 3). In C, E, a representative image of the crystal violet staining is included beside and below the quantification, respectively. In C, F, data are normalized to the CM -Dox (non-PGC1α expressing) condition (C) or to the CM shScr (F), depicted by a black dotted line. P producer cells, R recipient cells, CM conditioned media, D or Dox doxycycline, FC fold change. n.s not significant. Statistics: one sample t-test with reference value 1 (C, F), unpaired-t-test (A, C), Mann Whitney test (D). Asterisks and dollar symbols indicate statistical differences between the experimental groups. */$ p.value < 0.05; **p.value < 0.01; *** p.value < 0.001. Error bars indicate s.e.m.

Fig. 5. PGC1α expression and activity inversely correlate with SRM in prostate cancer patients and has prognostic value.

A Analysis of SRM mRNA in PCa patients stratified according to the mean expression of PGC1α mRNA (PPARGC1A). B Correlation analysis between PPARGC1A and SRM mRNA expression in primary tumor specimens of different prostate cancer datasets. C Association of the combined expression of PPARGC1A and SRM mRNA with disease-free survival (DFS) in human PCa specimens. Patients were grouped according to the average mRNA expression of both genes. H: high, above average. L: low, below average. Four groups were generated: PGC1α H - SRM L, PGC1α L - SRM H, PGC1α H - SRM H and PGC1α L - SRM L. Sample sizes: Grasso, n = 45; Taylor, n = 131; Glinsky, n = 79 and TCGA provisional, n = 497. Statistics: Mann Whitney test (A) Spearman correlation Rho/ρ (B), Log-rank (Mantel–Cox) test (C). *p.value < 0.05; **p.value < 0.01; ***p.value < 0.001. Error bars indicate s.e.m. HR harzard ratio, p.v p-value.