HFD enhances PCa progression, adipocyte infiltration, and adipolysis in the tumor microenvironment in vivo
The PCa model were generated by intraperitoneally inoculating PC-3M-luc-C6 cells with stable luciferase expression, and randomly divided the mice into HFD and control diet (CD) groups fed with a HFD or CE-2 diet, respectively. Tumor progression was evaluated using an IVIS imaging system four weeks after inoculation of the cells. The extent of tumor burden, as evaluated by luciferase activity, was significantly higher in the HFD group than in the CD group (Fig. 1a). The Ki67 positivity was significantly higher in the HFD group than in the CD group by immunohistochemistry (28.3 ± 5.3% vs 13.9 ± 2.1%, *P < 0.05; Fig. 1b). In addition, the hematoxylin and eosin staining of the mouse peritoneum tumor demonstrated a higher transmigration of PC-3M-luc-C6 PCa cells into the peritoneum stroma in the HFD group than in the CD group (arrow in the middle panel, Fig. 1c). Interestingly, a HFD markedly stimulated adipocyte infiltration to the xenograft tumor microenvironment (Fig. 1c, right). A recent report has shown that the adipocytes surrounding tumor cells may provide many types of nutrients, such as fatty acids (FAs), for growing tumor cells by increasing adipolysis . In line with this observation, we investigated the role of adipocytes in the tumor microenvironment by measuring lipase activity in both the xenograft tumor extract and the serum with a lipase activity assay kit. The lipase activity was markedly higher in the HFD group than the CD group (P < 0.05 and P < 0.05, respectively; Fig. 1d and 1e). In addition, we measured the level of acid FFAs in the xenograft extracts and serum using a FFA quantification kit. We demonstrated that the FFA level was significantly higher in the HFD group than in the CD group (*P < 0.05 and **P < 0.01, respectively; Fig. 1f and 1 g). These findings suggest that a HFD influences PCa progression and enhances adipocyte infiltration and lipolysis in the PCa microenvironment.
HFD and exogenous FFAs enhance the expression and secretion of MIC-1 in vivo and in vitro.
MIC-1 is a divergent member of the transforming growth factor-β family , and has been shown to be expressed in both PCa cells and in prostate stromal fibroblasts [15, 16]. Therefore, we investigated the expression of MIC-1 and glial-derived neurotrophic factor receptor alpha-like (GFRAL), the cognate receptor of MIC-1, semi-quantitatively by immunohistochemistry using the mouse xenograft tumor sample. We demonstrated that although the expression level of MIC-1 was significantly higher in the HFD group than in the CD group (P < 0.05, Fig. 2a), there was no statistical difference in the expression of GFRAL between the two groups (Fig. 2a). In addition, the mean serum level of MIC-1 was significantly higher in the HFD group than in the CD group (3506.0 ± 1888.4 and 2618.8 ± 710.4 pg/ml, P < 0.05; Fig. 2b). Considering the accumulating evidence suggesting that palmitic acid (PA) stimulates MIC-1 expression in many types of cancer cells [7, 25], we examined the role of increased FFAs on MIC-1 expression under a HFD condition. The mRNA expression level of MIC-1, but not GFRAL and TGF-beta, was significantly higher in PCa LNCaP, PC3, and DU145 cells after treatment with 0.125 mmol/l of PA, 0.25 mmol/l of oleic acid (OA), or 0.15 mmol/l of linoleic acid (LA) (Figure S1 and Fig. 2c). In addition, the expression of monomeric and dimeric MIC-1 (molecular weight, ∼14 kDa and ∼28 kDa, respectively) was markedly increased in the conditioned medium (CM) of PC-3 cells treated with FFAs compared with that of the PC-3 cells cultured with 2% BSA as control (Fig. 2d). The proliferation rate was significantly higher in the PC-3 and PC-3M-luc-C6 cells following treatment with 50 ng/ml rMIC-1, and the effect was attenuated by pretreatment with 50 nM GFRAL siRNA for 12 h (Figure S2a). Similarly, the invasive capacity of PC-3 and PC-3M-luc-C6 cells was significantly increased by treatment with rMIC-1, and the effect was abrogated by pretreatment with GFRAL siRNA (Figure S2b). These findings suggest that enhanced FFA release in the tumor microenvironment through upregulated adipolysis stimulated the expression and secretion of MIC-1 in tumor cells under the HFD condition, and that the MIC-1-GFRAL signaling stimulated PCa progression.
Activation and cytokine secretion in PCa stromal fibroblasts by upregulated MIC-1 in the HFD condition
The expression level of αSMA, a activation marker of stromal fibroblasts was significantly higher in the xenograft tumor of HFD group than in the CD group (P < 0.05; Fig. 3a), We measured the mouse serum cytokine levels using a human cytokine cytometric bead array for IL-8, IL-1β, IL-6, IL-10, TNFα, and IL-12p70, and the mean IL-8 level was significantly higher in the HFD group than in the CD group (1358.9 ± 528.5 and 773.2 ± 210.7 pg/ml, respectively, P < 0.05; Fig. 3b). Next, we evaluated the relationship between the increased cytokine secretion in prostate stromal cells (PrSC) and the upregulated MIC-1 in PCa cells. The IL-8 and IL-6 levels were significantly increased by 3.4- and 3.2-fold, respectively in the CM of PrSC cells treated with 50 ng/ml rMIC-1 compared to untreated PrSC, and were increased by 4.7- and 5.4-fold in the CM of PrSC cells co-cultured with PC-3 cells. However, the effect was markedly abrogated by pretreatment of the PC-3 cells with 50 nM MIC-1 siRNA (siMIC-1) for 12 h (Fig. 3c). Similarly, the IL-8 and IL-6 levels were significantly higher in the CM of the PrSC cells co-cultured with DU145 cells compared to those cultured alone or co-cultured with DU145 cells pretreated with siMIC-1 for 12 h (Figure S3). In addition, the expression of αSMA was significantly upregulated in PrSC cells by treatment with 50 ng/ml rMIC-1, and the phospho-ERK1/2 (pERK1/2) expression was also upregulated in PrSC cells in response to treatment with rMIC-1 (Fig. 3d). The mRNA expression levels of IL-8 and IL-6 were markedly increased in PrSC cells by treatment with 50 ng/ml rMIC-1, and the effect was attenuated by pretreatment with 1 µM U0126, an ERK kinase inhibitor (Fig. 3e). These findings suggest that upregulated MIC-1 in PCa cells stimulates the surrounding stromal fibroblasts, resulting in increased secretion of protumorigenic cytokines such as IL-8 and IL-6 through activation of the ERK signaling pathway in the PCa microenvironment under a HFD condition.
Periprostatic adipocytes directly stimulated MIC-1 secretion in PCa cells and subsequently increased IL-8 secretion in PrSC through upregulated adipolysis and FFA release in a direct co-culture system
To address the functional role of periprostatic adipocytes on MIC-1 and cytokine expression in the PCa stromal microenvironment, a co-culture assay was performed by direct incubation of periprostatic adipocytes (PPACs) with PC-3 cells and/or PrSC cells. The isolated PPACs from the radical prostatectomies of PCa patients (n = 13) were directly incubated in 6-well plates previously seeded 2 × 105 PC-3 cells pretreated with or without 50 nM siMIC-1, and further in the presence or absence of 4 × 104 PrSC cells in DMEM containing for 48 h. The levels of adipolysis and FFA release were significantly higher in the CM harvested from the co-culture of PPAC with PC-3 cells in the CM from the PPAC culture alone, regardless of in the presence or absence of PrSC cells (*P < 0.05 and **P < 0.01; Fig. 4a and 4b). Interestingly, the levels of adipolysis and FFA release were also increased in the co-culture of PPAC and PC-3 cells pretreated with siMIC-1 (Fig. 4a and 4b). In addition, the MIC-1 level was significantly higher in the CM of the co-culture of PC-3 cells and PPAC, regardless of the presence, or absence of PrSC cells (5178.0 ± 801.8 vs 1883.6 ± 143.1 pg/ml, and 4597.7 ± 723.0 vs 1883.6 ± 143.1, respectively). On the contrary, the MIC-1 level was significantly decreased by pretreatment of PC-3 with siMIC-1 in co-culture with PPAC (**P < 0.01 and **P < 0.01, Fig. 4c). The IL-8 level was significantly higher in the CM of the co-culture of PrSC cells and PPAC, and further significantly increased in the co-presence of PC-3 cells than that of in the CM of PrSC cells, PC-3 cells or PPAC single culture, or PrSC cells and PPAC co-culture, respectively (6697.7 ± 1673.5 vs 1266.7 ± 147.9 pg/ml, and 6697.7 ± 1673.5 vs 2688.4 ± 401.6, respectively). Furthermore, IL-8 was markedly decreased in the CM of the co-culture of PrSC cells and PC-3 cells when the PC-3 cells were pretreated with siMIC-1 compared to the co-culture of native PC-3 cells, PPAC, and PrSC cells (*P < 0.05, Fig. 4d). These findings strongly suggest that the periprostatic adipocytes in the tumor microenvironment have bidirectional crosstalk with PCa cells and the surrounding stromal cells, and enhance PCa progression through the upregulation of MIC-1 in PCa cells. This in turn, is thought to enhance IL-8 production from PrSC cells, especially under conditions of a HFD and/or HFD-mediated obesity.
Overexpression and secretion of MIC-1 was correlated with cancer stroma activation, high serum level of proinflammatory cytokines, high body mass index and serum lipase activity, and advanced human PCa progression
To further determine the role of MIC-1 in human PCa progression, we performed MIC-1 and αSMA immunohistochemistry in specimens of 67 PCa patients treated by radical prostatectomy. MIC-1 was highly expressed in PCa cells and was also expressed in the surrounding tumor stromal cells (Figure S4). αSMA was predominantly expressed in the PCa stroma, and the staining level of αSMA in PCa specimens was calculated according to the proportion of αSMA-positive cells (Fig. 5a). In addition, we measured the serum MIC-1 in the PCa patient and divided the samples into two groups (high and low) according to the median serum level of MIC-1 (MIC-1-low group, 735.0 ± 67.1 pg/ml, n = 33; and MIC-1-high group, 1206.9 ± 274.3 pg/ml, n = 34). The αSMA staining levels and serum levels of IL-8 and IL-6 were compared between the two groups. In subjects, with the low serum MIC-1-level (MIC-1-low group), 51.5%, 36.4%, and 12.1% were classified as low, moderate, and high αSMA staining, respectively, while 29.4%, 38.2%, and 32.4% of subjects with high MIC-1 levels (MIC-1-high group) were classified as low, moderate, and high αSMA staining (P = 0.011, Fig. 5b). In addition, the mean serum level of IL-8 and IL-6 was significantly higher in the MIC-1-high group than the MIC-1-low group (IL-8: 230.1 ± 395.0 and 61.4 ± 145.9 pg/ml, P = 0.035; and IL-6: 10.0 ± 17.5 and 3.0 ± 3.5 pg/ml P = 0.044, respectively, Fig. 5c and 5d). In addition, the serum MIC-1 levels were compared between the patients with a low PSA (< 10 ng/dl, n = 45) level and those with a high PSA (≥ 10 ng/dl, n = 22) level, and patients with a low Gleason score (GS, < 7, n = 43) and those with a high GS (≥ 7, n = 24). The serum MIC-1 levels were significantly higher in the high PSA group than in the low PSA group (1080.1 ± 297.9 and 897.7 ± 238.8 pg/ml, respectively, P = 0.009, Fig. 5e), and also higher in the high GS group than in the low GS group (996.1 ± 241.7 and 887.8 ± 200.6 pg/ml, respectively, P = 0.074, Fig. 5f). In addition, when the patients were divided into two groups according to median body mass index (BMI), the mean serum MIC-1 level was significantly higher in the high BMI group than in the low BMI group (984.2 ± 254.1, n = 33, and 863.3 ± 179.2 pg/ml, n = 34, respectively, P = 0.037, Fig. 5g). When the patients were divided into two groups according to median serum lipase activity, the serum MIC-1 levels were significantly higher in the high lipase activity group than in the low lipase activity group (1064.8 ± 329.1, n = 27, and 845.2 ± 207.7 pg/ml, n = 40, respectively; P = 0.001; Fig. 5h). These findings strongly suggest that the overexpression and secretion of MIC-1 may play an important role in PCa progression by enhanced secretion of proinflammatory cytokines through the activation of stromal fibroblasts.
GFRAL is expressed in PCa cells and surrounding stromal fibroblasts, and expression is affected by androgen deprivation therapy and chemotherapy
To determine the role of GFRAL, a MIC-1 functional receptor, on PCa progression, we performed GFRAL immunohistochemistry in specimens from PCa patients who underwent radical prostatectomy. GFRAL was predominantly expressed in the cytoplasm and membrane of PCa cells, and also in the surrounding stromal fibroblasts (Fig. 6a). The staining level of GFRAL tended to be lower in the specimens from patients who received neoadjuvant therapy, but the difference was not significant (None, n = 10 and NEO, n = 9, P = 0.208, Fig. 6b). Furthermore, the GFRAL staining level was significantly lower in the stromal fibroblasts of patients who received neoadjuvant chemohormonal therapy than in those who did not (None, n = 10 and NEO, n = 9, P = 0.017, Fig. 6b). In addition, as shown in Figure S5, the mRNA level of IL-8 and IL-6 in PrSC cells that was upregulated by rMIC-1 was significantly abrogated by pretreatment of 50 nM siGFRAL. Thus, GFRAL expression may be essential for stimulation of cytokine expression in stromal fibroblasts, as well as for the secretion of MIC-1 from PCa cells. These findings also suggest that MIC-1-GFRAL signaling in the tumor microenvironment plays a critical role in PCa progression by affecting the interaction between PCa cells and stromal fibroblasts.