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. 2014 Aug 1;20(15):4086-95.
doi: 10.1158/1078-0432.CCR-14-0322. Epub 2014 May 19.

Prostate tumor growth is impaired by CtBP1 depletion in high-fat diet-fed mice (V体育官网入口)

Cristian P Moiola  1 , Paola De Luca  1 , Florencia Zalazar  1 , Javier Cotignola  2 , Santiago A Rodríguez-Seguí  3 , Kevin Gardner  4 , Roberto Meiss  5 , Pablo Vallecorsa  5 , Omar Pignataro  6 , Osvaldo Mazza  7 , Elba S Vazquez  2 , Adriana De Siervi  8
Affiliations

"VSports" Prostate tumor growth is impaired by CtBP1 depletion in high-fat diet-fed mice

Cristian P Moiola et al. Clin Cancer Res. .

Abstract

Purpose: Clinical and epidemiologic data suggest that obesity is associated with more aggressive forms of prostate cancer, poor prognosis, and increased mortality. C-terminal-binding protein 1 (CtBP1) is a transcription repressor of tumor suppressor genes and is activated by NADH binding VSports手机版. High calorie intake decreases intracellular NAD(+)/NADH ratio. The aim of this work was to assess the effect of high-fat diet (HFD) and CtBP1 expression modulation over prostate xenograft growth. .

Experimental design: We developed a metabolic syndrome-like disease in vivo model by feeding male nude mice with HFD during 16 weeks V体育安卓版. Control diet (CD)-fed animals were maintained at the same conditions. Mice were inoculated with PC3 cells stable transfected with shCtBP1 or control plasmids. Genome-wide expression profiles and Gene Set Enrichment Analysis (GSEA) were performed from PC3. shCtBP1 versus PC3. pGIPZ HFD-fed mice tumors. .

Results: No significant differences were observed in tumor growth on CD-fed mice; however, we found that only 60% of HFD-fed mice inoculated with CtBP1-depleted cells developed a tumor. Moreover these tumors were significantly smaller than those generated by PC3. pGIPZ control xenografts V体育ios版. We found 823 genes differentially expressed in shCtBP1 tumors from HFD-fed mice. GSEA from expression dataset showed that most of these genes correspond to cell adhesion, metabolic process, and cell cycle. .

Conclusions: Metabolic syndrome-like diseases and CtBP1 expression cooperate to induce prostate tumor growth. Hence, targeting of CtBP1 expression might be considered for prostate cancer management and therapy in the subset of patients with metabolic syndromes VSports最新版本. .

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

"VSports手机版" Figures

Figure 1.
Figure 1.
CtBP1 induces cell transformation and proliferation. A, CtBP1 IHC for radical prostatectomies from different stages of prostate cancer pathogenesis (normal gland to highly undifferentiated adenocarcinoma). An increment in positive immunostain was observed from normal to carcinomatous tissue. Similar positive nuclei stain in muscular and connective stromal fibers were seen in all tissues. ×250 magnification. B, CtBP1 RT-qPCR from prostate cancer cell lines. Fold induction was calculated normalizing data to actin β and control. Bars represent the average and SD of one representative experiment. **, P < 0.01 C, Western blot analysis from PC3.CtBP1, PC3.shCtBP1, and PC3.pGIPZ stable cells using specific CtBP1 and actin β antibodies. Quantification was obtained by normalization to actin beta and control cells as indicated under the bands. D, clonogenic assay from PC3.CtBP1, PC3.shCtBP1, and PC3.pGIPZ stable transfected cells. A representative photograph for each group, histograms for area distribution, and colony number and area analyses are shown (boxes).
Figure 2.
Figure 2.
HFD consumption for 16 weeks induced metabolic syndrome–like disease in mice. A, weekly average body weight from nu/nu mice fed with CD or HFD was plotted normalizing data to initial body weight. Box plots for (B) cholesterol and (C) total testosterone levels for CD- or HFD-fed mice are shown. Boxes represent the interquartile range, the horizontal line within each box represents the median, and the top and bottom whiskers represent the SD of one independent experiment. **, P < 0.01.
Figure 3.
Figure 3.
CtBP1 depletion decreases tumor growth in HFD-fed mice. Tumor growth curves from (A) CD or (B) HFD male nu/nu mice inoculated with PC3.pGIPZ or PC3.shCtBP1 cells after 12 weeks of diet. Each data point represents tumor volume average and SD from each group of animals. C, tumors removed were used to determine NAD+/NADH ratio. Plotted boxes represent the interquartile range, the horizontal line within each of the boxes represents the median, and the top and bottom whiskers represent the SD. *, P < 0.05. D, H&E and CtBP1 IHC staining from tumor sections generated as xenografts in CD- or HFD-fed mice (×250 magnifications). HFD PC3.shCtBP1 tumors showed irregular and hyperchromatic nuclei with several necrobiosis and leukocyte foci.
Figure 4.
Figure 4.
Microarray analysis and functional genomics of xenografts. A, functional gene ontology annotations from 823 genes differentially expressed in PC3.pGIPZ and PC3.shCtBP1 HFD xenografts using Panther software after Hugene 1.0 ST array hybridization and normalization. B, GSEA from microarray data from HFD tumor xenografts. Enrichment plot and heatmap from a selected geneset were shown. Only genes that significantly contribute to core enrichment were shown in heatmap. C, CDH1 (e-cadherin) IHC from PC3.pGIPZ and PC3.shCtBP1 xenografts HFD-fed mice (×400 magnifications). D, GSEA from microarray data from HFD tumor xenografts showing enrichment plot and heatmap from steroid hormone biosynthesis. E, RT-qPCR from the HFD-fed mice tumors using specific primers for CYP19A1. Fold induction was calculated normalizing data to actin β and control. Bars represent the average and SD from 3 mice. *, P < 0.05.
Figure 5.
Figure 5.
Differentially expressed genes identified by whole-genome expression array. A, CCND1 qRT-PCR from the HFD-fed mice tumors. Fold induction was calculated normalizing data to actin β and control. Bars represent the average and SD from 3 mice. *, P < 0.05. (B) CCND1 IHC from PC3.pGIPZ and PC3.shCtBP1 xenografts HFD-fed mice (×400 magnifications). C, GSEA from microarray data from HFD tumor xenografts. BRCA1 targets enrichment plot and heatmap geneset were shown. Only genes included in the core enrichment were shown at heatmap. D, BRCA1 IHC staining from tumor sections generated as xenografts in HFD-fed mice (×400 magnifications).
Figure 6.
Figure 6.
CtBP1 and testosterone regulate BRCA1 transcription. A, BRCA1 RT-qPCR from PC3 stable cell lines. Fold induction was calculated normalizing data to actin β and control PC3.pGIPZ cells. Bars represent the average and SD of one representative experiment. B, BRCA1 RT-qPCR from PC3 cells transiently transfected with pcDNA3 or AR5-expressing vectors, grew in charcoal FBS-medium and exposed to testosterone or vehicle for 24 hours. Fold induction was calculated normalizing data to actin β and control. Bars represent the average and SD of one representative experiment. C, CtBP1-specific ChIP scanning from PC3 cells incubated in charcoal FBS medium and exposed to testosterone or vehicle for 24 hours. Specific primers scanning BRCA1 promoter at the indicated sites were used. Enrichment was calculated normalizing data to INPUT and nonspecific IP antibody. D, BRCA1 RT-qPCR from PC3 cells, grown as indicated above and pretreated with letrozole (1 hour) and then exposed to testosterone or vehicle (24 hours). Fold induction was calculated normalizing data to actin β and control. Data represent the average and SD of one representative experiment. *, P < 0.05; **, P < 0.01.
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