Periprostatic adipose tissue promotes prostate cancer resistance to docetaxel by paracrine IGF-1 upregulation of TUBB2B beta-tubulin isoform

Abstract

Growing evidence supports the pivotal role played by periprostatic adipose tissue (PPAT) in prostate cancer (PCa) microenvironment. We investigated whether PPAT can affect response to Docetaxel (DCTX) and the mechanisms associated. Conditioned medium was collected from the in vitro differentiated adipocytes isolated from PPAT which was isolated from PCa patients, during radical prostatectomy. Drug efficacy was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide citotoxicity assay. Culture with CM of human PPAT (AdipoCM) promotes DCTX resistance in two different human prostate cancer cell lines (DU145 and PC3) and upregulated the expression of BCL-xL, BCL-2, and TUBB2B. AG1024, a well-known IGF-1 receptor inhibitor, counteracts the decreased response to DCTX observed in presence of AdipoCM and decreased TUBB2B expression, suggesting that a paracrine secretion of IGF-1 by PPAT affect DCTX response of PCa cell. Collectively, our study showed that factors secreted by PPAT elicits DCTX resistance through antiapoptotic proteins and TUBB2B upregulation in androgen independent PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte-released factors and IGF-1 axis to overcome DCTX resistance in patients with PCa.

Keywords: adipocytes; docetaxel; drug resistance; periprostatic adipose tissue; prostate cancer.

Figure 1

Adipocyte‐conditioned media effect on prostate cancer cell viability. (A) Mesenchymal stem cells from PPAT were isolated and differentiated as described in Section 2. The lysates were analyzed by immunoblotting with PPARγ and C/EBPα antibodies and autoradiography. GAPDH antibody was used for normalization. (B) DU145 and (C) PC3 (2 × 10⁴ cells) cells were plated in 96 well plates and serum starved for 16 h cells. Then, the cells were incubated with 0,25% BSA or PPAT Adipocyte‐CM from Adeno, G7(3 + 4) and G8(4 + 4) adipocytes for 48 h. Cell viability was assessed by the MTT assay. The results were reported as percentage of viable cells compared to control, considered as maximum viability (100%). Data represent the mean ± SD of triplicate samples of three independent experiments. The bars represent the mean ± SD of at least three independent experiments. *p value < .05. BSA, bovine serum albumin; C/EBPα, CCAAT/enhancer‐binding protein alpha; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; PPARγ, peroxisome proliferator‐activated receptor gamma; PPAT, periprostatic adipose tissue; SD, standard deviation

Figure 2

Docetaxel response in prostate cancer cell line upon AdipoCM treatment. DU145 and PC3 (2 × 10⁴ cells) cells were plated in 96 well plates and serum starved for 16 h cells. The cells were incubated with 0.25% BSA or PPAT Adipocyte‐CM from Adeno, G7(3 + 4) and G8(4 + 4) adipocytes for 24 h. Then, the cells were treated with docetaxel 8 nM for 48 h alone or in combination with AdipoCM. Cell viability was assessed by the MTT assay. The results were reported as percentage of viable cells compared to control, considered as maximum viability (100%). Data represent the mean ± SD of triplicate samples of three independent experiments. *p value < .05. BSA, bovine serum albumin; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; PPAT, periprostatic adipose tissue; SD, standard deviation

Figure 3

TUBB2B expression in prostate cancer cell. (A) DU145 and (B) PC3 were serum starved for 16 h cells and then incubated with 0,25% BSA or PPAT Adipocyte‐CM from G7(3 + 4) and G8(4 + 4) adipocytes for 48 h. The lysates were analyzed by immunoblotting with TUBB2B antibody and autoradiography. Actin antibody was used for normalization. The autoradiograph shown is representative of three different experiments. BSA, bovine serum albumin; PPAT, periprostatic adipose tissue; TUBB2B, β‐tubulin isoform 2B

Figure 4

Expression of IGF‐1 receptor in PCa cell lines. (A) DU145 and (B) PC3 were serum starved for 16 h cells and then incubated with 0,25% BSA or PPAT Adipocyte‐CM from adenoma and G8(4 + 4) adipocytes for 48 h. The lysates were analyzed by immunoblotting with IGF‐1 receptor antibody and autoradiography. GAPDH antibody was used for normalization. The autoradiograph shown is representative of three different experiments. BSA, bovine serum albumin; IGF‐1, insulin‐like growth factor‐1; PCa, prostate cancer; PPAT, periprostatic adipose tissue

Figure 5

Insulin‐like growth factor inhibitor AG1024 effect on docetaxel sensitivity. (A) DU145 and (B) PC3 (2 × 10⁴ cells) cells were plated in 96 well plates and serum starved for 16 h cells. Then, the cells were incubated with 0,25% BSA or PPAT Adipocyte‐CM from Adeno, G7(3 + 4) and G8(4 + 4) adipocytes for 48 h. Then, the cells were treated with docetaxel 8 nM and AG1024 8 µM alone or in combination with AdipoCM. Cell viability was assessed by the MTT assay. The results were reported as percentage of viable cells compared with control, considered as maximum viability (100%). Data represent the mean ± SD of triplicate samples of three independent experiments. *p value < .05. BSA, bovine serum albumin; MTT, 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide; PPAT, periprostatic adipose tissue; SD, standard deviation

Figure 6

AG1024 effect on TUBB2B expression in prostate cancer cell. (A) DU145 and (B) PC3 were serum starved for 16 h cells and then incubated with 0,25% BSA or PPAT Adipocyte‐CM from Adeno and G8(4 + 4) adipocytes and in combination with AG1024 8 µM. (C) DU145 and (D) PC3 cells were serum starved for 16 h, pretreated with AG1024 8 µM and then stimulated with IGF‐1 recombinant protein for 48 h. The lysates were analyzed by immunoblotting with pAKT and TUBB2B antibodies and autoradiography. GAPDH antibody was used for normalization. The autoradiograph shown is representative of three different experiments. BSA, bovine serum albumin; PPAT, periprostatic adipose tissue; TUBB2B, β‐tubulin isoform 2B

Figure 7

Schematic representation of the proposed role of PPAT in promoting DCTX resistance in PCa cell. (A) Periprostatic mature adipocytes released factors upregulated BCL‐xL, BCL‐2, and TUBB2B expression in PCa cell favoring DCTX resistance. BCL‐2, B‐cell lymphoma‐2; BCL‐xL, B‐cell lymphoma extra large; DCTX, docetaxel; PCa, prostate cancer; PPAT, periprostatic adipose tissue; TUBB2B, β‐tubulin isoform 2B