Signal transducer and activator of transcription-3 drives the high-fat diet-associated prostate cancer growth

Abstract

Prostate cancer (PCa) is the second leading cause of cancer death in men. PCa progression can be associated with obesity. Signal transducer and activator of transcription-3 (STAT3) plays a crucial role in PCa growth. However, whether STAT3 plays a role in high-fat diet (HFD)-associated PCa growth is unknown. Our data show that HFD feeding increases tumor size, STAT3 phosphorylation, and palmitic acid (PA) level in the xenograft tissues of the PCa-bearing xenograft mouse model. In vitro studies show that PA increases STAT3 expression and phosphorylation (STAT3-Y705) in PCa. Computational modeling suggests strong and stable binding between PA and unphosphorylated STAT3 at R593 and N538. The binding changes STAT3 structure and activity. Functional studies show that both STAT3 mutants (R583A and N538A) and STAT3 dominant negative significantly reduce PA-enhanced STAT3 phosphorylation, PA-increased PCa cell proliferation, migration, and invasion. In the xenograft mouse models, the HFD-increased tumor growth and STAT3 phosphorylation in tumors are reversed by STAT3 inhibition. Our study not only demonstrates the regulatory role of PA/STAT3 axis in HFD-associated PCa growth but also suggests a novel mechanism of how STAT3 is activated by PA. Our data suggest STAT3 as a therapeutic target for the treatment of HFD-associated PCa.

Fig. 1. Signal transducer and activator of transcription-3 (STAT3) phosphorylation is increased in the xenograft tissues of the prostate cancer (PCa)-bearing xenograft mouse model after high-fat diet (HFD) feeding.

a Body weight and b tumor weight of the DU145-bearing xenograft mouse models after feeding the HFD or matched control diet (CD) for 14 days. c Expression of Ki67 in the xenograft tissues. d Western blots and e immunohistochemistry staining of Stat3 and phosphorylated Stat3 (p-Stat3) at Y705 or Y727 in the xenograft tissues of the HFD-feeding and CD-feeding xenograft mouse models. Shown is the mean ± SEM, 4–10 mice in each group. *P value < 0.05

Fig. 2. High-fat diet (FD) feeding increases palmitic acid (PA) level in the xenograft tissues of the prostate cancer (PCa)-bearing xenograft mouse model and PA increases signal transducer and activator of transcription-3 (STAT3) phosphorylation (p-STAT3-Y705) in PCa.

a Principal component analysis and b relative total free fatty acid levels of the xenograft tissues of the mice. Shown is the mean ± SEM, 10 mice in each group. *P value <0.05. c Principal component analysis of the lipids in the serum of the DU145-bearing xenograft mouse models fed by HFD or control diet (CD), 5–6 mice in each group. Oil red-O staining of the lipids in d PC3 and e DU145 cells after culturing with the serum of the HFD-feeding or CD-feeding mice. f Oil red-O quantification of these cancer cells. CD culture, cells cultured with serum of CD-feeding mice; HFD culture, cells cultured with serum of HFD-feeding mice. g Western blots showing the levels of Stat3 and pStat3 at Y705 and Y727 in PCa cells upon PA challenge for 24 h. h Western blots showing the levels of p-Stat3 (Y705) in the cytoplasm and the nucleus of DU145 cells upon PA challenge. i Stat3-luciferase reporter activity upon PA challenge and j upon stearic acid challenge in DU145 and PC3 cells, respectively. Shown is the mean ± SEM, n = 3 independent experiments. *P value < 0.05 and **p value < 0.01

Fig. 3. Palmitic acid (PA) increases signal transducer and activator of transcription-3 (STAT3) transcription.

Relative messenger RNA (mRNA) levels of the oncogenic genes in a DU145 and in b PC3 cells upon PA challenge (12 µM) for 24 h. Cyclin D1, cluster of differentiation 36 (CD36), p53, nuclear factor-κB (NF-κB), GATA-binding protein 2 (GATA2), β-catenin, sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-γ (PPAR-γ), Stat3, nuclear respiratory factor-1 (NRF-1), N-myc, and c-Myc. b The relative Stat3 mRNA levels in TRAMP-C1 cells upon PA challenge. Shown is the mean ± SEM, n = 3 independent experiments. *P value < 0.05 and **p value < 0.01

Fig. 4. Palmitic acid (PA) has strong and stable binding with unphosphorylated signal transducer and activator of transcription-3 (USTAT3), which changes the protein structure of the USTAT3.

a–e Molecular simulations showing the interaction of USTAT3 with PA or stattic (STA), a specific Stat3 inhibitor. a Three-dimensional ribbon model of PA in complex with USTAT3. b Three-dimensional ribbon model of STA in complex with USTAT3. c Plots of root mean square deviation (RMSD) of heavy atoms of USTAT3-PA (red) and USTAT3-STA complex (blue). d Surface presentation of the USTAT3-PA complex crystal structure at 0 and 50 ns. e Surface presentation of the USTAT3-STA complex crystal structure at 0 and 50 ns. f Surface presentation of the homology model of human STAT3 dimer at 0 and 50 ns. g Binding mode of subunit A’s transactivation domain with subunit B’s SH2 domains (up) and subunit A’s SH2 domains with subunit B’s transactivation domain (down). h Plots of RMSD of heavy atoms of human STAT3 dimer. i Western blot showing the expressions of phosphorylated (p)-Stat3 (Y705) in DU145 cells, or DU145 cells overexpressed with Stat3-R6938A, Stat3-N538A, Stat3-R6938A/N538A, or Stat3-Y705F after treating with PA for 24 h

Fig. 5. Palmitic acid (PA) increases prostate cancer (PCa) cell proliferation which is mediated by signal transducer and activator of transcription-3 (STAT3) activity.

Proliferation of a DU145, c PC3 cells upon PA challenge at the indicated concentrations for 24 h. Proliferation of b DU145 and d PC3 cells after PA treatment for 24 h in the presence of orlistat (Orl, 1 µM). Proliferation of PC3 cells overexpressed with Stat3-Y705F treated by PA at the indicated concentrations for 24 h e in the absence or f presence of Orl (1 µM). Proliferation of PC3 cells overexpressed with Stat3-R593A treated by PA at the indicated concentrations for 24 h g in the absence or h presence of Orl (1 µM). Proliferation of PC3 cells overexpressed with STAT3-N538A treated by PA at the indicated concentrations for 24 h i in the absence or j presence of Orl (1 µM). Proliferation of PC3 cells overexpressed with Stat3-R6938A/N538A treated by PA at the indicated concentrations for 24 h k in the absence or l presence of Orl (1 µM). Proliferation of DU145 cells overexpressed with Stat3-R6938A/N538A treated by PA at the indicated concentrations for 24 h m in the absence or n presence of Orl (1 µM). Proliferation of o DU145 and p PC3 cells under PA challenge in the presence of stattic. q Proliferation of DU145 cells in the presence of stattic at the indicated concentrations. r Proliferation of DU145, PC3, and TRAMP-C1 cells after culturing with the serum of the HFD-feeding or control diet (CD)-feeding mice. CD culture, cells cultured with serum of CD-feeding mice; HFD culture, cells cultured with serum of HFD-feeding mice. Shown is the mean ± SEM, n = 3 independent experiments. *p value <0.05. s Tumor weight of the DU145-bearing xenograft mouse model fed by match CD or HFD with or without stattic treatment (2.5 mg/kg). t Western blots of phosphorylated (p)-STAT3 (Y705) in the xenograft tissues of the HFD-feeding xenograft mouse model with or without stattic treatments (2.5 mg/kg). Shown is the mean ± SEM, n = 3–4 mice in each group

Fig. 6. Palmitic acid (PA) increases prostate cancer (PCa) cell migration and invasion which are mediated by signal transducer and activator of transcription-3 (STAT3) activity.

a Twist-1 luciferase reporter activity in DU145 cells upon PA challenge. b Relative messenger RNA (mRNA) level of Twist-1 in DU145 cells upon PA challenge. c Scratches in confluent monolayer of DU145 cells in the presence or absence of 12 µM PA at 0 and 16 h. Transwell invasion assay showing the invasive capacity of DU145 cells upon PA challenge d without or f with 5 µM stattic. Transwell invasive capacity of PC3 cells upon PA challenge e with or g without 5 µM stattic. Scratches in confluent monolayer of DU145 cells overexpressed with h Stat3-Y705F and i Stat3-R6938A/N538A in the presence or absence of 12 µM PA at 0 and 16 h. Transwell invasion assay showing the invasive capacity of DU145 cells overexpressed with j Stat3-Y705F and k Stat3-R6938A/N538A upon PA challenge. Shown is the mean ± SEM, n = 3 independent experiments. *P value < 0.05