Adipose tissue from metabolic syndrome mice induces an aberrant miRNA signature highly relevant in prostate cancer development

Abstract

Prostate cancer (PCa) remains an important public health concern in Western countries. Metabolic syndrome (MeS) is a cluster of pathophysiological disorders with increasing prevalence in the general population that is a risk factor for PCa. Several studies have determined that a crosstalk between white adipose tissue (WAT) and solid tumors favors cancer aggressiveness. In this work, our main goal was to investigate the interaction between WAT and PCa cells through microRNAs (miRNAs), in MeS mice. We developed a MeS-like disease model using C57BL/6J mice chronically fed with high-fat diet (HFD) that were inoculated with TRAMP-C1 PCa cells. A group of five miRNAs (mmu-miR-221-3p, 27a-3p, 34a-5p, 138-5p, and 146a-5p) were increased in gonadal WAT (gWAT), tumors, and plasma of MeS mice compared to control animals. Three of these five miRNAs were detected in the media from gWAT and TRAMP-C1 cell cocultures, and significantly increased in MeS context. More importantly, hsa-miR-221-3p, 146a-5p, and 27a-3p were increased in bloodstream of PCa patients compared to healthy donors. Using miRNA microarrays, we found that 121 miRNAs were differentially released to the coculture media between HFD-gWAT and tumor cells compared to control diet-gWAT and tumor cells. Target genes for the 66 most deregulated miRNAs were involved in common pathways, mainly related to fatty acid metabolism, ER protein processing, amino acid degradation, PI3K AKT signaling, and PCa. Our findings show for the first time a signature of five miRNAs as important players involved in the interaction between WAT and PCa in MeS mice. Further research will be necessary to track these miRNAs in the interaction between these tissues as well as their role in PCa patients with MeS.

Keywords: adipose tissue; metabolic syndrome; miRNA; prostate cancer.

Fig. 1

MeS modifies the expression of miRNAs in gWAT and androgen‐sensitive prostate tumors from mice. (A) Table showing a list of miRNAs involved in adipogenesis regulation and adipocyte metabolism selected from literature. Stem‐loop RT–qPCR from gWAT (B) and TRAMP‐C1 allografts (C) obtained from CD‐ or HFD‐fed C57BL/6J mice using specific primers for the indicated miRNAs is shown (N = 8 per group). Data were normalized to geometric mean of miR‐191‐5p and miR‐103a‐3p and control and expressed as mean and SD. Statistical analysis was performed using two‐sided t‐test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2

MeS increases miRNA circulation in plasma from mice. Mouse plasma samples were analyzed by stem‐loop RT–qPCR for the indicated miRNAs and normalized to spike in cel‐39 synthetic miRNA (N = 8 per group). $2^{‐\mathrm{\Delta }{C}_{\mathrm{t}}}$ is graphed ($\mathrm{\Delta }{C}_{\mathrm{t}}\text{value}=‐C_{{\mathrm{t}}_{\text{sample}}}‐C_{{\mathrm{t}}_{\text{cel}‐\text{39}}}$) and expressed as mean and SEM. Data were analyzed by one‐sided t‐test.

Fig. 3

gWAT from MeS mice releases miRNAs impacting in prostate tumor growth. (A) Stem‐loop RT–qPCR from TRAMP‐C1 cells obtained from the coculture using specific primers for the indicated miRNAs. Data were normalized to geometric mean of miR‐191‐5p and miR‐103a‐3p and control (TRAMP‐C1 cells without the addition of gWAT) and represent mean and SD of three independent experiments with three replicates. Data were analyzed by one‐way ANOVA followed by Tukey's. (B) Stem‐loop RT–qPCR from gWAT obtained from the coculture. Data were normalized to geometric mean of miR‐191‐5p and miR‐103a‐3p and to CD group. Data were analyzed by two‐sided t‐test. (C) Stem‐loop RT–qPCR in culture medium from cocultures using primers specific for the indicated miRNAs. $2^{‐\mathrm{\Delta }{C}_{\mathrm{t}}}$ is graphed. Data were normalized to mmu‐miR‐19b ($\mathrm{\Delta }{C}_{\mathrm{t}}\text{value}=‐C_{{\mathrm{t}}_{\text{sample}}}‐C_{{\mathrm{t}}_{\text{miR-}‐\text{19b}}}$) and control (TRAMP‐C1 cells without the addition of gWAT) and represent the mean and SD of three independent experiments with three replicates. Data were analyzed by one‐way ANOVA followed by Tukey's. ND: not detectable. *P < 0.05; **P < 0.01; ***P < 0.001. White bar corresponds to the group of TRAMP‐C1 without the addition of gWAT, pink bar corresponds to the TRAMP‐C1 cell culture with gWAT from CD mice, and red bar corresponds to TRAMP‐C1 cultured with gWAT from HFD mice.

Fig. 4

Crosstalk between gWAT and prostate tumor cells by miRNAs released in a MeS environment. (A) Volcano plot representation of DE miRNAs. Colors indicate the levels of significance. The names of some of the most significant miRNAs are indicated. (B) Heat map representation of DE miRNAs. Arrows indicate the four miRNAs common to the 10 selected miRNAs based on the literature. (C) Box plots of the expression of the four selected miRNAs.

Fig. 5

Functional enrichment analysis of validated miRNA targets. (A, B) Barplot representation of the top significant KEGG pathways associated with the target genes of the downmodulated (A) and upmodulated (B) miRNAs, released to the medium of the coculture of TRAMP‐C1 cells with gWAT from HFD‐fed mice compared to CD‐fed mice. The terms common to both groups are indicated in black, and those specific to each group are highlighted in color. (C, D) Functional enrichment analysis represented as nonredundant biological terms in a functionally grouped network of miRNA targets exclusive of the downmodulated (C) and upmodulated miRNAs (D).

Fig. 6

Differential miRNA expression in tumor tissue and circulation of PCa patients. (A) Circulating miRNA expression levels of hsa‐miR‐221‐3p, 27a‐3p, 34a‐5p, 155‐5p, 138‐5p, 146a‐5p, 196a‐5p, 143‐3p, 125b‐5p, and 140‐5p in plasma of PCa and non‐PCa patient samples. Log2Intensities values are plotted. Data were analyzed by one‐way ANOVA. (B) Expression levels of hsa‐miR‐221‐3p, 27a‐3p, 34a‐5p, 155‐5p, 146a‐5p, 196aa‐5p, 143‐3p, 125b‐5p, and 140‐5p in prostate primary solid tumor and NAT. Read per millions values are graphed. Data were analyzed using paired t‐test.

Fig. 7

Hypothetical model. Upper panel: ex vivo cocultures between TRAMP‐C1 cells and adipose tissue from CD‐ or HFD‐fed mice. miRNAs induced (red arrow) or decreased (green arrow) in the cells, gWAT, and media when compared CD versus HFD groups are listed. Lower panel: miRNAs up‐ (red arrow) or downregulated (green arrow) in the postmortem adipose tissue, plasma, and allografts from CD‐ or HFD‐fed mice.