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. 2019 Dec 16;19(1):1216.
doi: 10.1186/s12885-019-6418-2.

Preoperative plasma fatty acid metabolites inform risk of prostate cancer progression and may be used for personalized patient stratification

Eugenio Zoni  1 , Martina Minoli  1 , Cédric Bovet  2 , Anne Wehrhan  2 , Salvatore Piscuoglio  3   4   5 , Charlotte K Y Ng  4   6 , Peter C Gray  7 , Martin Spahn  8   9 , George N Thalmann  1   10 , Marianna Kruithof-de Julio  11   12
Affiliations

Preoperative plasma fatty acid metabolites inform risk of prostate cancer progression and may be used for personalized patient stratification

Eugenio Zoni et al. BMC Cancer. .

Abstract

Background: Little is known about the relationship between the metabolite profile of plasma from pre-operative prostate cancer (PCa) patients and the risk of PCa progression. In this study we investigated the association between pre-operative plasma metabolites and risk of biochemical-, local- and metastatic-recurrence, with the aim of improving patient stratification.

Methods: We conducted a case-control study within a cohort of PCa patients recruited between 1996 and 2015. The age-matched primary cases (n = 33) were stratified in low risk, high risk without progression and high risk with progression as defined by the National Comprehensive Cancer Network. These samples were compared to metastatic (n = 9) and healthy controls (n = 10). The pre-operative plasma from primary cases and the plasma from metastatic patients and controls were assessed with untargeted metabolomics by LC-MS. The association between risk of progression and metabolite abundance was calculated using multivariate Cox proportional-hazard regression and the relationship between metabolites and outcome was calculated using median cut-off normalized values of metabolite abundance by Log-Rank test using the Kaplan Meier method.

Results: Medium-chain acylcarnitines (C6-C12) were positively associated with the risk of PSA progression (p = 0.036, median cut-off) while long-chain acylcarnitines (C14-C16) were inversely associated with local (p = 0.034) and bone progression (p = 0.0033). In primary cases, medium-chain acylcarnitines were positively associated with suberic acid, which also correlated with the risk of PSA progression (p = 0.032, Log-Rank test). In the metastatic samples, this effect was consistent for hexanoylcarnitine, L.octanoylcarnitine and decanoylcarnitine. Medium-chain acylcarnitines and suberic acid displayed the same inverse association with tryptophan, while indoleacetic acid, a breakdown product of tryptophan metabolism was strongly associated with PSA (p = 0.0081, Log-Rank test) and lymph node progression (p = 0.025, Log-Rank test). These data were consistent with the increased expression of indoleamine 2,3 dioxygenase (IDO1) in metastatic versus primary samples (p = 0.014). Finally, functional experiments revealed a synergistic effect of long chain fatty acids in combination with dihydrotestosterone administration on the transcription of androgen responsive genes.

Conclusions: This study strengthens the emerging link between fatty acid metabolism and PCa progression and suggests that measuring levels of medium- and long-chain acylcarnitines in pre-operative patient plasma may provide a basis for improving patient stratification.

Keywords: Acylcarnitines; Disease progression; Fatty acid metabolism; Metabolomics; Prostate cancer.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Characterization of disease progression in PCa cases. a Overall survival calculated by the Kaplan Meier method for high-risk with progression (HR + P, blue solid line), high-risk without progression (HR-P, red dotted line) and low-risk (LR, black dashed line) PCa cases. P-value estimated with Log-Rank test. b PSA progression free survival calculated by the Kaplan Meier ethod for HR + P, HR-P and LR PCa cases. P-value estimated with Log-Rank test. c Local progression free survival calculated by the Kaplan Meier method for HR + P, HR-P and LR PCa cases. P-value estimated by the Log-Rank test. d Lymph node progression-free survival calculated by the Kaplan Meier method for HR + P, HR-P and LR PCa cases. P-value estimated by the Log-Rank test. e Bone progression-free survival calculated by the Kaplan Meier method for HR + P, HR-P and LR PCa cases. P-value estimated with the Log-Rank test. For statistical details see Methods section
Fig. 2
Fig. 2
Sample classification by principal component analysis and overview of metabolomics profiling. a Principal component analysis (PCA) of all the m/z features measured in positive and negative ionization mode. The variation retained by PC1 (16.3%) is represented of the X axis and the variation retained by the PC2 (9.3%) is represented on the Y axes. Ellipses represent the 95% confidence interval for each group. b Heatmap generated with scale and centered normalized m/z abundances measured in positive and negative ionization mode. Euclideian distance between groups and Minkowski distance between metabolites was used and clustering was calculated with the Ward (Ward.D2) method for minimum variance between m/z values. c Principal component analysis (PCA) of the annotated m/z features in positive and negative ionization mode. The variation retained by PC1 (16.7%) is represented of the X axis and the variation retained by the PC2 (13.5%) is represented on the Y axis. Ellipses represent the 95% confidence interval for each group. d Heat map generated with scale and centered normalized m/z abundances of the annotated metabolites. Same methods used for Fig. 2b were applied. For statistical details see Methods section
Fig. 3
Fig. 3
Hazard ratio (95% confidence interval) and Kaplan Meier curves for the association of acylcarnitines with PSA progression. a Hazard ratios and 95% confidence interval (CI) for the association of the complete panel of acylcarnitines with the risk of PSA progression. Groups (lower and higher risk) were separate by median cut-off (suffix “_med”) of the normalized abundances for each molecule. C4 = butyrylcarnitine, C4_M = methylmalonylcarnitine. b PSA progression-free survival calculated by the Kaplan Meier method for acetylcarnitine (C2). Groups are defined by median-cut off of normalized abundances (vs L-carnitine) of high acetylcarnitine (C2 High, blue solid line) vs low acetylcarnitne (C2 Low, red dotted line). P-value was estimated with Log-Rank test. c PSA progression free survival calculated by the Kaplan Meier method for isovalerylcarnitine (C5). Groups are defined by median-cut off of normalized abundances (versus L-carnitine) of High isovalerylcarnitine (C5 High, red dot line) versus low isovelrylcarnitne (C5 Low, blue solid line). P-value was estimated with Log-Rank test. d PSA progression-free survival calculated by the Kaplan Meier method for hexanoylcarnitine (C6). Groups are defined by median-cut off of normalized abundances (vs L-carnitine) of high hexanoylcarnitine (C6 High, blue solid line) versus low hexanoylcarnitne (C6 Low, red dotted line). P-value was estimated with Log-Rank test. For statistical details see Methods section
Fig. 4
Fig. 4
Hazard ratio (95% confidence interval) and Kaplan Meier curves for the association of acylcarnitines with lymph node progression. a Hazard ratios and 95% confidence interval (CI) for the association of the complete panel of acylcarnitines with the risk of lymph node progression. Groups (lower and higher risk) were separate by median cut-off (suffix “_med”) of the normalized abundances for each molecule. C4 = butyrylcarnitine, C4_M = methylmalonylcarnitine. b Lymph node progression-free survival calculated by the Kaplan Meier method for L-carnitine (C0). Groups are defined by median-cut off of normalized abundances of high L-carnitine (C0 High, red dotted line) versus low L-carnitne (C0 Low, blue solid line). P-value was estimated with Log-Rank test. c Lymph node progression free survival calculated by the Kaplan Meier method for isovalerylcarnitine (C5). Groups are defined by median-cut off of normalized abundances (versus L-carnitine) of high isovalerylcarnitine (C5 High, red dotted line) vs low isovelrylcarnitne (C5 Low, blue solid line). P-value was estimated with Log-Rank test. For statistical details see Methods section
Fig. 5
Fig. 5
Correlation matrix for all metabolites identified in PCa cases and controls. Correlations are shown for the different acylcarnitines in primary PCa cases (a) and controls (b). c Insert with plot A displays the correlation among the acylcarnitines and all the annotated metabolites in primary cases. d Correlation among acylcarnitines in metastatic cases. The sizes of the circles are dependent on the Pearson correlation coefficient. Blue circles correspond to positive correlations and red circles correspond to negative correlations. Correlations that do not reach significance (p > 0.05) are indicated by an empty square box. For statistical details see methods section
Fig. 6
Fig. 6
Association between annotated metabolites and disease progression and analysis of gene expression data. PSA progression-free survival calculated by the Kaplan Meier method for (a) suberic acid and (b) indoleacetic acid and lymph node progression-free survival for indoleacetic acid is represented in (c). Groups are defined by median-cut off of normalized abundances (low abundance, blue solid line) vs high abundance (red dotted line). P-value was estimated with log-rank test. IDO1 expression data of primary and androgen ablation resistant metastasis are shown from GSE6752 (d) and GSE6919 (e-f). ACADM expression data in the same set of samples are displayed in (g-h-i). Fold change (FC) is calculated versus the normal or primary tumor samples and p-value (P) for significance between two groups estimated by t-test. For statistical details see Methods section
Fig. 7
Fig. 7
In vitro functional characterization of DHT and palmitate (PA) stimulation on AR positive and negative PCa cells. a Proliferation assessed in C4–2B by MTS assay upon PA (range 50–400 μM) or vehicle (control) stimulation for 48 h. b-d Representative bright field images of cultured cells under indicated experimental conditions. e Proliferation assessed in PC-3 M-Pro4 by MTS assay upon PA (range 50–400 μM) or vehicle (control) stimulation for 48 h. f-h Representative bright field images of cultured cells upon experimental conditions. i-l Relative expression or AR responsive genes upon dihydrotestosterone (DHT) or control (EtOH) stimulation, and palmitate (PA) or vehicle (BSA fatty acid free-FFA) or combination (PA + DHT) in AR positive C4–2 and (M-P) LNCaP cells. P-value indicated in the plots is relative to ANOVA. Multiple comparison significance between experimental condition is indicated by * (** p < 0.01, *** p < 0.001). Details related to quantification and normalization are included in the Methods section
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