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. 2016 Oct 4;7(40):65403-65417.
doi: 10.18632/oncotarget.11395.

Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

Yeong-Chin Jou  1 , Yuh-Shyan Tsai  2 , Chang-Te Lin  1 , Chun-Liang Tung  3 , Cheng-Huang Shen  1 , Hsin-Tzu Tsai  2 , Wen-Horng Yang  2 , Hung-I Chang  2 , Syue-Yi Chen  4 , Tzong-Shin Tzai  5   6
Affiliations

Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

Yeong-Chin Jou et al. Oncotarget. .

Abstract

Hypoxia-inducible factor-1α (HIF-1α) can control a transcriptional factor forkhead box P3 (Foxp3) protein expression in T lymphocyte differentiation through proteasome-mediated degradation. In this study, we unveil a reverse regulatory mechanism contributing to bladder cancer progression; Foxp3 expression attenuates HIF-1α degradation. We first demonstrated that Foxp3 expression positively correlates with the metastatic potential in T24 cells and can increase the expression of HIF-1α-target genes, such as vascular endothelial growth factor (VEGF) and glucose transporter (GLUT). Foxp3 protein can bind with HIF-1α, particularly under hypoxia. In vivo ubiquination assay demonstrated that Foxp3 can decrease HIF-1α degradation in a dose-dependent manner. Knocking-down of Foxp3 expression blocks in vivo tumor growth in mice and prolongs mice's survival, which is associated with von Willebrand factor expression. Thirty-three of 145 (22.8 %) bladder tumors exhibit Foxp3 expression. Foxp3 expression is an independent predictor for disease progression in superficial bladder cancer patients (p = 0.032), associated with less number of intratumoral CD8+ lymphocyte. The metaanalysis from 2 published datasets showed Foxp3 expression is positively associated with GLUT-4,-9, and VEGF-A, B-, D expression. This reverse post-translational regulation of HIF-1α protein by Foxp3 provides a new potential target for developing new therapeutic strategy for bladder cancer.

Keywords: Foxp3; bladder neoplasms; glycolysis; immunohistochemistry; prognosis.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no financial disclosure.

Figures

Figure 1
Figure 1. Foxp3 expression is associated with aerobic glycolysis and VEGF, glucose transporter expression
A. Foxp3 expression is examined with western blotting assay in three T24 bladder cancer sublines (T24-P, T24-L, and T24-B). B. Assays for glucose, lactate, and ATP production in three T24 sublines. C. VEGF mRNA and D. GLUT-1~5 mRNA expression is examined in three T24 sublines using qRT-PCR assay. E. Foxp3 mRNA expression is examined in Foxp3-knocking down T24-B transfectants and its control. F. Assays for glucose and lactate production, G. VEGF mRNA expression, and H. GLUT-1~5 as assayed in Foxp3-knocking down transfectant #25 and its control. I. Foxp3 expression is examined with western blotting assay after transient transfection of Foxp3 plasmid into T24-P bladder cancer cells. J. Assays for glucose and lactate production, K. VEGF mRNA expression, and L. GLUT-1~5 as assayed in pooled Foxp3-overexpressing T24-P transfectants and its control. T24-P, T24 parental subline; T24-L, metastatic lung T24 subline; T24-B, metastatic bone T24 subline; ATP, Adenosine triphosphate; GLUT, glucose transporter; EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor; NS, not significant; *, p<0.05; **, p<0.01, ***, p<0.001. Data are represented as mean ± SD.
Figure 2
Figure 2. Foxp3 can bind with HIF-1α in hypoxia condition
A. T24-B cell were stained with anti-Foxp3, anti-HIF-1α and DAPI for confocal microscopy studies in normoxic and hypoxic circumstance as indicated times. Scale bars, 10 μm. B. T24-B cell were maintained in normoxic and hypoxic circumstance as indicated times. Cell lysates were immunoprecipitated with anti-Foxp3 antibody and subjected to Foxp3 and HIF-1α western blot.
Figure 3
Figure 3. Foxp3 expression decreased ubiquitin-proteasomal degradation of HIF-1α protein
A. HIF-1α mRNA expression were examined in T24-B Foxp3 knocking down cells and its control both in normoxic and hypoxic circumstances using qRT-PCR assays. B. Foxp3-knocking down T24B cells and its control were maintained in normoxic and hypoxic circumstance and cell lysates for harvested for western blot. C. After transfection, cells were maintained in normoxic and hypoxic circumstance and treated with MG132 (10μM) for 24 hours before harvesting for western blotting assay. D. Foxp3 and HIF-1α mRNA expression were examined in Foxp3-overexpressing T24-P cells and its control in normoxic circumstance using qRT-PCR assays. E. T24-P cells were transfected with Foxp3 cDNA-containing plasmids or control as indicated conditions with or without MG132 treatment. Cell lysates were immunoprecipitated with anti-Foxp3 or anti-ubiquitin antibody and subjected to Foxp3, HIF-1α or ubiquitin western blot. F. In vivo ubiquitination assay. T24-P cells were transfected with Foxp3 cDNA-carrying plasmid in a dose-dependent manner, as well as histdine-tagged ubiquitin cDNA. Cell lysates were harvested and Histiidine-containing protein complex were pulled down using Ni++-NTA magnetic bead for subquentent immunoblotting assay. The expression of β-actin used as a loading control. NS, not significant; *, p<0.05; **, p<0.01, ***, p<0.001. Data are represented as mean ± SD.
Figure 4
Figure 4. Effect of Foxp3 expression on bladder tumor growth in mice
Female NOD-SCID mice were subcutaneously injected with 1×106 Foxp3 knocking-down T24-B in 100 ml serum-free medium or its control. In vivo growth A. and survival curve B. were record and analyzed. C. The growing tumor were harvested at day 58 for immunostaining of von Willebrand factor, Magnification × 200. Scale bars, 50 μ. D. The integrated density were calculated using Imaging-Pro Plus software and compared with unpaired t-test.
Figure 5
Figure 5. Foxp3 protein expression and CD8+ lymphocyte in human bladder cancer
A. Negative Foxp3 expression (× 200; scale bar, 50 μm), B. Positive Foxp3 expression (× 200; scale bar, 50 μm), C. Foxp3+ lymphocytes within the bladder tumor (× 400; scale bar, 50 μm). D. More than 8 CD8+ lymphocyte within bladder tumors, E. 1≤ CD8+ lymphocyte number ≤8, and F. none of CD8+ lymphocytes based on the average number determined from 10 random 0.0328-mm2 digital images captured under high power field (× 320; scale bar, 20 μm). G. Distribution and mean number of CD8+TILs from 96 bladder cancer specimen according to Foxp3 expression. H. The comparison of average CD8+ TILs number between Foxp3-expressing tumors or not is analyzed using unpaired t-test. Foxp3-expressing lymphocytes (arrow).
Figure 6
Figure 6. Effect of Foxp3 expression on disease recurrence or progression in 115 patients with superficial bladder cancer
A. Proportion of recurrence-free survival according to the Foxp3 expression. B. Proportion of progression-free survival according to the Foxp3 expression. RFS, recurrence-free survival; PFS, progression-free survival.
Figure 7
Figure 7. Correlation of Foxp3 expression with the Glucose transporter and VEGF
Two human urothelial carcinoma dataset GSE32548 (n=131) and GSE48075 (n=142) were selected for the metaanalysis due to the used same platform (GPL6947, Illumina HumanHT-12 V3.0 expression bead chip) and larger number of patients (more than 100). A. GLUT3, B. GLUT-4, C. VEGF-A, and D. VEGF-B.
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References

    1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics 2012. CA Cancer J Clin. 2015;65:87–108. - PubMed
    1. Wu XR. Urothelial tumorigenesis: a tale of divergent pathways. Nat Rev Cancer. 2005;5:713–25. - PubMed
    1. Roupret M, Zigeuner R, Palou J, Boehle A, Kaasinen E, Sylvester R, Babjuk M, Oosterlinck W. European guidelines for the diagnosis and management of upper urinary tract urothelial cell carcinomas: 2011 update. Eur Urol. 2011;59:584–594. - PubMed
    1. Stenzl A, Cowan NC, De Santis M, Kuczyk MA, Merseburger AS, Ribal MJ, Sherif A, Witjes J A. Treatment of muscle-invasive and metastatic bladder cancer: update of the EAU guidelines. Eur Urol. 2011;59:1009–1018. - PubMed
    1. Babjuk M, Oosterlinck W, Sylvester R, Kaasinen E, Bohle A, Palou-Redorta J, Roupret M. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder the 2011 update. Eur Urol. 2011;59:997–1008. - PubMed

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