SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLNTRY INFORTION OI:.8/ncb7 Torrano, Valcarcel et al. Supplementary Figure Log mrn RT Log mrn H9 p=.767 p=.8 p=.96 p=.98 p=. p=.789 p=.7 p=.67 Log mrn NOR Log mrn NRIP Log mrn PG H H9 KT H NRIP NOR RT PG NO RT NO NO KT H7 H NOR R SIRT H RT H p=e- p=. p=e-8 p=.6 p=.86 p=e- p=.8 p=6e- p=e- p=.9 p=.8 p=.67 p=. p=. p=. p=. p=. P=. p=.8 p=.7 p=. p=.9 p=. p=.6 p=.67 p=.68 p=.9 p=.889 p=.667 p=.89 p=.9 Log mrn H p=.9 p=.999 p=.86 p=. Log mrn H p=.9 p= p= 9e- p=.6979 Log mrn H7 Log mrn PG p=.8 p=. p=.7 Lapointe et al. Varambally et al. Tomlins et al. p=.8 PG expression (a.u.)..... P H Lapointe et al. Varambally et al. Tomlins et al. Log mrn PG p=.8 a n c e r F Log mrn PG (edian centered) p=.9 p=.7 Supplementary Figure, xpression of metabolic co-regulators in Taylor dataset (N: normal; Pa: prostate cancer)., xpression of 7 metabolic co-regulators from figure a in four additional prostate cancer datasets (N: normal; Pa: prostate cancer). In Varambally dataset gene expression levels are presented in Log. In Tomlins, Grasso and Lapointe datasets gene expression levels are presented in median centred Log. -, ssociation of PG expression with Gleason score in TG provisional data 6,7 () and Taylor datasets ()., nalysis of PG expression in benign prostatic hyperplasia (PH) and Pa specimens from asurto University Hospital cohort (qrtpr, PH n= patient specimens and ancer n=6 patient specimens). F, PG expression in normal prostate (N), primary tumour (PT) and metastatic (et) specimens in Grasso dataset. Points outlined by circles indicate statistical outliers (,, and F). rror bars represent minimum and maximum values (,,, and F) or median with interquartile range (). Statistic test: two-tailed Student T test (, ), two-tailed ann Whitney U test () and NOV (, and F). 6 acmillan Publishers Limited. ll rights reserved

2 SUPPLNTRY INFORTION Pten expression (a.u.) S c o r e... W t (n=-7) P t e n K O Pgca expression (a.u.) Glandular inflitration..... P T N K O S c o r e (n=7-8) K O Stromal Infiltration ge (months; ave ± stdv) ann n Whitney p-val Pten wt Pgca wt. ±.8. Pten pc+/+ ; Pgca pc-/-. ±. 9 Pten pc+/- ; Pgca pc+/+. ± Pten pc+/- ; Pgca pc-/-. ±. Pten pc-/- ; Pgca pc+/+.7 ±. 7.9 Pten pc-/- ; Pgca pc-/-.±.9 Vegfa expression (a.u.) Torrano, Valcarcel et al. Supplementary Figure... F V / f i e l d % of mice with vascular invasion Pten pc-/- ; Pgca pc +/+ 8 6 Pten pc-/- ; Pgca pc-/- Pten KO KO µm µm. G P t e n K O K O Ptenpc-/- ; Pgca pc-/- P t e n K O K O P t e n K O K O I Pten pc-/- ; Pgca pc+/+ Pten pc-/- ; Pgca pc-/- P t e n K O K O J Pten pc-/- ; Pgca pc-/- µm PanK µm PanK µm R µm H K L HGPIN HGPIN + focal invasion Invasive cancer % of mice with PanK positive cells in LN 8 6 Pten pc-/- ; Pgca pc+/+ (n = 6) % of mice with dissemination 8 6 Pten KO KO % of mice 8 6 Pten pc-/- ; Pgca pc+/- (n = ) % of mice with vascular invasion 8 6 Pten pc-/- ; Pgca pc+/- (n = ) N % of mice with metastasis 8 6 LN Liver Lung Pten pc-/- ; Pgca pc+/- (n = ) O PTN mrn TG PG mrn R=.9 p=.e-6 P G m R N s i g n a l 8 6 iploid TG Shallow eep deletions deletions P T N s t a t u s Supplementary Figure, nalysis of Pten and Pgca gene expression in Gs of the indicated genotype (Pten wt Pgca wt n= mice; Pten pc-/- Pgca pc+/+ n=7 mice; Pten pc+/+ Pgca pc-/- n=6 mice; Pten pc-/- Pgca pc-/- n= mice; data is normalized to Gapdh expression)., ge comparison between experimental cohorts (n as in Figure d)., Quantification of prostate tissue with histological vascular invasion signs in Pten KO ( mice) and KO mice (9 mice) (limited to mice with invasive signs)., Histological analysis of inflammatory signs (stromal and glandular infiltration) in Pten KO and KO mice (Pten KO, n=7 mice; KO, n= mice). Quantification of Vefga mrn expression in Pten KO and KO mice (Pten KO, n=7 mice; KO, n=6 mice; data is normalized to Gapdh expression). F, Quantification of microvessel density (V) (dot plot, left panels) and representative images of immunodetection (right panels) in Pten KO and KO mice (Pten KO, n= mice; KO, n= mice). G, Representative haematoxylin and eosin staining depicting liver metastasis in KO (bar=µm). H, Incidence of small groups of Pan-cytokeratin (Pan-K) positive cells in the lymph nodes of Pten KO mice (6 mice). I-J, representative immunohistochemical detection (X) of Pan-K positive cells in the bone marrow () of Pten KO and KO (I) and androgen receptor (R) in the bone marrow of KO (J) (bar=µm). Pink arrows indicate immunoreactive cells. K, Quantification of dissemination frequency (Pten KO, 6 mice; KO, 8 mice). L-, Histopathological characterization of the prostate tissue (L) and frequency of vascular invasion signs (, only in mice with invasion signs) in Pten pc-/-, Pgca pc+/- mice ( mice). N, Frequency of metastatic lesions in lymph nodes (LN), liver and lung of Pten pc-/-, Pgca pc+/- mice ( mice). O, orrelation between PG and PTN gene expression in prostate cancer specimens (left panel) and the association of PTN genomic loss to PG gene expression (right panel), in TG provisional dataset. pc, prostate-specific allelic changes; +, Wildtype allele; -, deleted allele; wt: any given genotype resulting in the lack of deletion of Pgca and Pten alleles. Pten KO = Pten pc-/-, Pgca pc+/+ ; KO = Pten pc-/-, Pgca pc-/-. Stdv: standard deviation of the mean. ll error bars represent median with interquartile range. p = p-value. a.u: arbitrary units. Statistic tests: one-tailed ann-whitney U test (, ), two-tailed ann-whitney U test (, and F); NOV (O, right panel); Pearson s coefficient (O, left panel). p <., p < acmillan Publishers Limited. ll rights reserved

3 SUPPLNTRY INFORTION shs Gene expression (a.u.) shpg ean sd N PT 6.6,.7 et 6..7 ell lines 6..8 N vs ell line: p=.9e- PT vs ell line: p=.9 et vs ell line: p=.97 PGα protein level (a.u) Gene expression (a.u.) O P U LnaP S Soft garf SF rug H Fold change... Soft agar Fold change rdu G S G/ I J Pgcα GPH 7 % of cells 8 6 % Ki67 positive cells 8 6 K Gene expression (a.u.) Relative cell number P P ays SK Xeno qpr O HH L VGF expression (a.u.) SL VGF P VGF expression (a.u.) VGF xenon SN V xeno Xenos V/field Xenos µm µm Supplementary Figure, mrn expression of PG, O and HH by qrtpr in P cells transduced with scramble shrn (shs) or PGtargeting shrn (shpg) (n=)., PG expression in normal (N, n=9), primary tumour (PT, n=), metastasis (et, n=9) specimens and metastatic cell lines. ata is shown as Log mrn expression., ensitometry of PGα protein expression in ewo (endogenous) and P TRIPZ-H-Pgcα (ectopic) cell lines, relative to β-ctin (n=, independent experiments)., ffect of Pgcα induction (+ox) on O mrn expression in P (n=, independent experiments), U (n=7, independent experiments) and LnaP cells (n=, independent experiments). -F, ffect of Pgcα expression on anchorage-independent growth (, n=, independent experiments) and rdu incorporation (F, n=, independent experiments) in U cells. G, ffect of Pgcα expression on cell cycle progression in P cells (n=, independent experiments). H, ffect of doxycycline treatment (.µg/ml) on cell growth of non-transduced P cells (n=, independent experiments). I-J, Pgcα protein expression and cell proliferation by Ki67 immunoreactivity in xenograft samples from Fig. f ( n= tumours, n=6 tumours). K, mrn expression of O and HH in xenograft samples from Fig. f. ( n=9 tumours, n=6 tumours). L-, nalysis of VFG mrn expression upon Pgcα induction in P cells (L, n=, independent experiments) and xenograft samples (, n=9 tumours and n=6 tumours). N, Quantification of microvessel density (V) in xenograft samples ( n=9 tumours and n=7 tumours). Right panels show representative staining micrographs. rror bars indicate s.e.m (,,,, F, G, H, L) and median with interquartile range (J, K,, N). Statistic tests: two-tailed Student T test (,,,,, F, G, H, L) and one-tailed ann-whitney U test (J, K,, N). p <., p <., p < acmillan Publishers Limited. ll rights reserved

4 SUPPLNTRY INFORTION Fold change relative to p=.6 G O T T P I H F H L Y L P T P T P I N P Fold change relative to G O T T P I H Fold change.... P G I H I S U S H Supplementary Figure -, Validation of the microarray by qrtpr in U (n=, independent experiments) and LnaP (n=, independent experiments) TRIPZ-H-Pgca cells. Gene expression values relative to cells are represented (reference gene expression values are indicated with a dotted line), mrn expression of PGα target genes in doxycycline-treated (.µg/ml) non-transduced P cells (n=, independent experiments). rror bars represent s.e.m. Statistic test: One tail Student T test. p <., p <., p < acmillan Publishers Limited. ll rights reserved

5 SUPPLNTRY INFORTION etabolite abundance (a.u) : : 8 : 6 6 : : O H β-oxidation (a.u.) U Pgcα β-oxidation (a.u.)... P F G itrate abundance (a.u) U Pgcα OR to TP (pol/min) 8 6 P Pgcα OR to TP (pol/min) 8 6 U Pgcα TP signal (a.u) 8 6 U Pgcα H P signal (a.u) KO.... Pten KO I P signal (a.u).... KO Pten KO P Pgcα U Pgcα Xenografts Gs P Pgcα U Pgcα Xenografts Gs J P a l m i t a te ( U % f r o m N o o x ) 8 6 P Pgcα K Lactate production (a.u) P Pgcα L Lactate production (a.u).... P OR (pol/min) P Supplementary Figure, nalysis of differential abundance in metabolites involved in fatty acid catabolism by untargeted L-HRS in U TRIPZ-H-Pgcα cells (n=, independent experiments). - valuation of the dehydrogenation of H-palmitate (readout of β-oxidation) in U cells upon Pgcα expression (, n=, independent experiments) and, in doxycycline-treated (.µg/ml) non-transduced P cells (, n=, independent experiments). Values relative to cells are presented., ffect of Pgcα expression on citrate abundance measured by L-HRS metabolomics in U cells (n=, independent experiments). -F, TPproducing OR (upon complex V inhibition by oligomycin injection) in P (, n=, independent experiments) and U (F, n=, independent experiments) cells upon Pgcα expression. G, asal mitochondrial TP production in U cells upon Pgcα expression (n=, independent experiments). H-I, L-HRS quantification of P (H) and P (I) abundance in P Pgcα (n=, independent experiments), U Pgcα (n=, independent experiments), xenografts ( n=8 tumours; n= tumours) and Gs (Pten KO n= mice; KO n= mice). J, Quantification of area under the curve (U, relative to ) of Palmitate labelling from -U 6 -Glucose in P TRIPZ-H-Pgcα cells (data related to Fig. j, n=, independent experiments). K, etermination of extracellular lactate in P TRIPZ-H-Pgcα cells (n=, independent experiments). L-, Lactate production (L) and OR () in doxycycline-treated (.µg/ml) nontransduced P cells (n=, independent experiments). rror bars represent s.e.m., except xenograft and G data in H-I, that represent median with interquartile range. Statistic tests: two tailed Student T test (,,,,, F, G, H (P and U), I (P and U), J, K, L, ) and one tailed ann Whitney U test (H (Xenografts and Gs), I (Xenografts and Gs)). p <., p <., p < acmillan Publishers Limited. ll rights reserved

6 SUPPLNTRY INFORTION Single object volume (µm ) itochondria volume (µm ) 8 6 P TRIPZ-H-Pgca 7 Torrano, Valcarcel et al. Supplementary Figure 6 PG WT PG LL µg/ml PGα b-ctin TP signal (x.) shs shrrα shs shrrα $ WT WT LL LL ito TP 7 sh-scr shrrα PGα RRα GPH mrn fold from scr - dox $ p=.6 F mrn fold from ox Veh J L Fluorescence signal x - (a.u) 6 $ p=. T P I H T P Pgcα $ p=.9 Fluorescence signal x - (a.u) itosox F itosox F G Relative cell number 8 6 U Pgcα I H T T P I S U G O T nmol /mg tissue 8 6 Xenografts ontrol m N m N µ ntp growth H Relative OR (a.u).... K asal OR I TP signal (x.) Lipid peroxidation $ ito TP + Veh + Veh + XT 79 + XT 79 Fold change ays ays ays ays Supplementary Figure 6, nalysis of mitochondrial morphology (mitochondrial volume) in P cells upon Pgcα expression (n=, independent experiments)., xpression of PGαWT and PGαLL in P cells after treatment with. μg/ml doxycycline (ox) (a representative experiment with technical replicates is presented, similar results were obtained in three independent experiments)., asal mitochondrial TP production in PGαWT and PGαLL P cells (n=, independent experiments)., xpression of Pgcα and RRα in doxycycline-treated P TRIPZ-H-Pgcα cells transduced with shscramble (shs) or shrrα (a representative experiment with technical replicates is presented, similar results were obtained in three independent experiments)., mrn expression of PGα target genes in doxycyclinetreated P TRIPZ-H-Pgcα cells transduced with shs or shrrα (n= for T and n= for IH, TP, ISU, GOT and ; independent experiments). F-I, mrn expression of PGα target genes (F, n= for T and n= for TP and IH; independent experiments), cell number (G, n=, independent experiments), basal oxygen consumption (H, n=, independent experiments) and basal mitochondrial TP production (I, n= 7 for + XT79; n=8 the rest; independent experiments) in vehicle (Veh) or XT79-treated Pgcα-inducible P cells. J, valuation of cellular (F) and mitochondrial-specific (itosox) ROS production in Pgcα-expressing P (left panel; n=, independent experiments) and U (right panel; n=6, independent experiments) cells. K, valuation of lipid peroxidation in xenograft tissues from Fig. f ( n= tumours; n= tumours). L, ffect of the indicated antioxidant treatments on cell number (relative to day ) of Pgcα-expressing P cells (n=, independent experiments). F:,7 -dichlorodihydrofluorescein. : not significant. rror bars represent s.e.m. (,,, F, G, H, I, J, L) or median with interquartile range (K). Statistic tests: two tailed Student T test (,,, F, G, H, I, J, L) or one tailed Student T test (comparison between conditions in,, F, G, H, I) and two tailed ann-whitney U test (K). /$ p <., / p <., /$ p <.. sterisks indicate statistic between and conditions (unless represented otherwise) and dollar symbol between either, vehicle (Veh) and XT79-treated Pgcαexpressing cells, shs and shrra-transduced Pgcα-expressing cells or PgcαWT and PgcαLL acmillan Publishers Limited. ll rights reserved

7 SUPPLNTRY INFORTION v e r a g e s i g n a l N O V p <. ## ##. v e r a g e s i g n a l N O V p <. Torrano, Valcarcel et al. Supplementary Figure 7 v e ra g e s ig n a l N O V p =. # v e r a g e s i g n a l N O V p <. ## N P T e t N P T e t N P T e t N P T e t I S U S U L S L L H G S T T P L G e n e e x p r e s s i o n ( a. u. ) Log mrn (median centered) Log mrn SL NG NTXR RHGF6 TP GST p=.e-6 p=.e-7 p=. p=.e- p=.9 p=.7 p=. p=.7 p=.e-6 p=.7e- p=.e-7 p=.8e- p=. p=.e-7 p=.87 p=.6e-6 Lapointe et al. Log mrn (median centered) Log mrn (median centered) p=.66 p=.7e-8 p=. p=. p=. p=. p=.e- ISU L LH NNT PPI RNS SO SUL p=.e-7 p=.e-6 p=.e-9 p=.e- p=e-6 p=.7e- p=. p=.9e- p=. TPINP p=8.7e- P=e- p=.9e- p=.e- p=.e- p=6.8e-6 p=.7e-7 p=.e-8 p=. Lapointe et al. Log mrn Log mrn (median centered) p=. p=.8 p=. p=e-7 p=. p=. p=. p=.9 hi sq. p<. hi sq. p<. F r e q u e n c y ( % ) F r e q u e n c y ( % ) S i g n a t u r e n e g a t i v e S i g n a t u r e p o s i t i v e N P T e t N P T e t Supplementary Figure 7 -, Representation of the average signal of genes within the PGα-upregulated gene set () (Fig. 7b, blue circle) and within the PGα-dependent RRα-upregulated gene set () (Fig. 7b, yellow circle, Table S6) in the indicated datasets,,6,7, in normal (N; Taylor n=9 and Grasso n=), primary tumours (PT; Taylor n= and Grasso n=9) and metastasis (ets; Taylor n=9 and Grasso n=7)., qrtpr mrn expression analysis of PGα target genes from, in benign prostatic hyperplasia (PH) and Pa specimens from asurto University Hospital cohort (PH n= patient specimens; Prostate cancer n=6 patient specimens)., xpression of the indicated genes (from Supplementary Table 7) in different disease states (N: normal, Lapointe n=9, Taylor n=9 and Grasso n=; PT: primary tumour, Lapointe n=, Taylor n= and Grasso n=9; et: metastasis, Lapointe n=, Taylor n=9 and Grasso n=7) in three Pa datasets,,., Representation of PGα-RRα Q signature frequency within different tumour types (N: normal; PT: primary tumour; et: metastasis) in two datasets, (Taylor: N, n=9; PT, n= ; et, n=9; Grasso: N, n=; PT, n=9; et, n=7). rror bars represent s.e.m. (, ), median with interquartile range () and maximum and minimum (). Statistic tests: Statistic tests: NOV (,, ); two tailed Student T test (, ), one tailed ann Whitney U test (), hi Square (). sterisks in, indicates statistics between normal and metastasis and hash between primary tumours and metastasis. p: p-value. /# p <., /## p <., p < acmillan Publishers Limited. ll rights reserved

8 SUPPLNTRY INFORTION PGα 7 Hsp9 : arker (Precision Plus Protein ual olor Standards, Ref #67) PGα 7 7 PGα 7 GPH RRα GPH 7 b-ctin PGα 7 : arker (Pink Prestained protein ladder, Nippon Genetics, at.no. WP) Supplementary Figure 8 Unprocessed blots., Western blot corresponding to Figure a., Western blot corresponding to Figure b., Western blot corresponding to Supplementary Figure I., Western blot corresponding to Supplementary Figure 6., Western blot corresponding to Supplementary Figure 6. Precision Plus Protein ual olor Standards (Ref #67) markers was used in -. Pink Pre-stained protein ladder, Nippon Genetics, at.no. WP, was used in acmillan Publishers Limited. ll rights reserved

9 SUPPLNTRY INFORTION Table titles and legends Supplementary Table Gene expression profiling in P TRIPZ-H-Pgcα cells (oxycycline vs. No oxycycline, (.µg/ml). Supplementary Table Untargeted L-HRS metabolomic profiling in P TRIPZ-H-Pgcα cells (oxycycline vs. No oxycycline, (.µg/ml). Supplementary Table Untargeted L-HRS metabolomic profiling in U TRIPZ-H-Pgcα cells (oxycycline vs. No oxycycline, (.µg/ml). Supplementary Table Untargeted L-HRS metabolomic profiling in xenograft-derived tissues (from P TRIPZ-H-Pgcα cells) upon induction of Pgcα expression (oxycycline diet vs. chow). Supplementary Table Untargeted L-HRS metabolomic profiling in G-derived prostate tissues (Pten pc-/-, Pgca pc-/- vs. Pten pc-/-, Pgca pc+/+ ). Supplementary Table 6 efinition of RRα signature within the PGα gene list. Genes included in the TGTY_V$RR_Q dataset or identified in the study by Stein et al (STIN_SRR_TRGTS 8 ) were considered as RRα targets. Supplementary Table 7 List of Pgcα-regulated genes in P (Supplementary Table ) that show significant and consistent correlation with PG in human prostate cancer datasets (R>.; p<.) in at least three out five datasets. Supplementary Table 8 List of primers and probes (Universal Probe Library, Roche) used in qrtpr. Supplementary Table 9 Statistics source data for animal experiments reported in Fig. k, and Fig. 6e, h. ll data are organized into different sheets and named based on the corresponding figure/panel numbers. References: Taylor,. S. et al. Integrative genomic profiling of human prostate cancer. ancer ell 8, -, doi:.6/j.ccr...6 (). Varambally, S. et al. Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression. ancer ell 8, 9-6, doi:.6/j.ccr... (). Tomlins, S.. et al. Integrative molecular concept modeling of prostate cancer progression. Nat Genet 9, -, doi:.8/ng9 (7). Grasso,. S. et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 87, 9-, doi:.8/nature (). Lapointe, J. et al. Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl cad Sci U S, 8-86, doi:.7/pnas.6 (). 6 erami,. et al. The cio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. ancer iscov, -, doi:.8/ (). 7 Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cioportal. Sci Signal 6, pl, doi:.6/scisignal.88 (). 8 Stein, R.. et al. strogen-related receptor alpha is critical for the growth of estrogen receptor-negative breast cancer. ancer Res 68, 88-88, doi:.8/8-7.n-8-9 (8) acmillan Publishers Limited. ll rights reserved