Interleukin-17 promotes development of castration-resistant prostate cancer potentially through creating an immunotolerant and pro-angiogenic tumor microenvironment

Abstract

BACKGROUND

Interleukin-17 (IL-17) has been demonstrated to promote formation and growth of hormone-naïve prostate adenocarcinoma in mice. IL-17’s role in development of castration-resistant prostate cancer is unknown. In the present study, we investigated IL-17’s role in castration-resistant prostate cancer in a mouse model.

METHODS

IL-17 receptor C (IL-17RC) deficient mice were interbred with Pten conditional mutant mice to produce RC⁺ mice that maintained IL-17RC expression and RC⁻ mice that were IL-17RC deficient. Male RC⁺ and RC⁻ mice were Pten-null and were castrated at 16 weeks of age when invasive prostate cancer had already formed. At 30 weeks of age, all male mice were analyzed for the prostate phenotypes.

RESULTS

RC⁻ mice displayed prostates that were smaller than RC⁺ mice. Approximately 23% of prostatic glands in RC⁻ mice, in contrast to 65% of prostatic glands in RC⁺ mice, developed invasive adenocarcinomas. Compared to castrate RC⁺ mice, castrate RC⁻ mouse prostate had lower rates of cellular proliferation and higher rates of apoptosis as well as lower levels of MMP7, YBX1, MTA1, and UBE2C proteins. In addition, castrate RC⁻ mouse prostate had less angiogenesis, which was associated with decreased levels of COX-2 and VEGF. Moreover, castrate RC⁻ mouse prostate had fewer inflammatory cells including lymphocytes, myeloid-derived suppressor cells, and macrophages.

CONCLUSIONS

Taken together, our findings suggest that IL-17 promotes development of invasive prostate adenocarcinomas under castrate conditions, potentially through creating an immunotolerant and pro-angiogenic tumor microenvironment.

INTRODUCTION

Locally confined prostate cancer is treated by surgery or radiation. At the advanced stage when metastases occur, prostate cancer is treated with androgen deprivation therapy. However, Castration-induced regression of tumor is typically followed by re-growth with castrate levels of androgens, a status known as castration-resistant prostate cancer (CRPC) [1]. The mechanisms of how hormone-sensitive prostate cancer develops into CRPC remain to be defined. Alterations of androgen receptor (AR) signaling pathways, such as AR gene amplification, increase in AR expression, and AR gene mutations [2], may cause hypersensitivity of AR to low levels of both endocrine and intracrine androgens [3]. AR splicing variants may constitutively activate AR signaling in ligand-independent manners [4]. AR signaling may also be activated by growth factors in the absence of androgens [5]. Activation of HER-2/neu and Ras/mitogen-activated protein kinase pathways causes androgen-independent AR activities [6]. Transcriptional coactivators may lead to ligand-independent AR activation [7]. Focal neuroendocrine differentiation seems to be a common feature of prostate cancer. By secretion of a number of growth factor-like molecules (such as bombesin, calcitonin, and parathyroid hormone-related peptide), neuroendocrine cells can support the growth and progression of surrounding prostate cancer cells towards the castration-resistant state [8]. Androgen ablation up-regulates expression of the anti-apoptotic Bcl-2 gene [9] and clusterin gene [10], whereas the pro-apoptotic p53 gene is often mutated [11]. Decreased PTEN or increased Akt activities are linked to castration-resistant progression of prostate cancer [12,13]. Expression of TMPRSS2-ERG (transmembrane protease, serine 2 - E-twenty six related gene) fusion protein [14,15] and some microRNAs [16,17] has also been associated with CRPC.

Interleukin-6 (IL-6) and IL-8 have been found to play a role in development of CRPC [18,19]. Both IL-6 and IL-8 are downstream targets of IL-17, a cytokine that is produced by T_H17 cells, γδ T cells, and other immune cells [20]. IL-17 acts through a heterodimer of receptors IL-17RA and IL-17RC [21–23], thus, either Il17ra knockout (KO) or Il17rc KO completely abolishes IL-17 signaling [24,25]. We have previously reported that IL-17RC protein expression as detected by the anti-IL-17RC intracellular domain antibodies is significantly increased in CRPC, compared to hormone-sensitive prostate cancer [26,27]. Recently, we cross-bred Il17rc KO (Il17rc^−/−) mice with Pten conditional KO mice (Pten^L/L;Cre⁺) and found that, in Pten-deficient context, Il17rc KO mice developed significantly smaller prostate tumors compared to Il17rc wild-type mice [28]. Our findings suggest that IL-17 promotes formation and growth of hormone-naïve prostate adenocarcinoma. However, it is unknown whether IL-17 plays any role in the development of CRPC. In the present study, we castrated the mice at 16 weeks of age and examined them at 30 weeks of age. We found that Il17rc KO mice developed significantly smaller prostates compared to Il17rc wild-type mice under castrate conditions.

MATERIALS AND METHODS

Mice

Animal protocol was approved by the Animal Care and Use Committee of Tulane University. The breeding strategy and genotyping protocols have been described previously, using Pten^loxp/loxp (Pten^L/L) mice, PB-Cre4 mice, and Il17rc^−/− mice [28]. Male RC⁺ (n = 9) and RC⁻ (n = 9) mice at 16 weeks of age were castrated. This age was chosen because a majority of RC⁺ and RC⁻ mice had already developed invasive adenocarcinomas by this age [28,29]. The castration procedures were as the following: mice were anesthetized with 4% isoflurane; the skin over the scrotum was disinfected by 70% ethanol and Betadine solution; a 0.5-cm incision was made over the scrotum; the testes were exposed by pulling the adipose tissue; a hemostat was applied to curtail blood flow followed by silk ligation of blood vessels; the ductus deferentes were ligated and cut; the testes were excised; and the skin incision was closed with #5-0 nylon suture that was removed 7 days later. All instruments used were sterile. To alleviate pain, Carprofen (2 mg/kg) was injected subcutaneously at the end of surgical procedure and then every 12 hours up to 48 hours.

Histopathology

Mice were euthanized and weighed at 30 weeks of age. The genitourinary (GU) blocs were photographed, weighed with an empty bladder, and fixed en bloc as described previously [28,30]. Twenty-eight consecutive 4-μm sections of each prostate were cut and 4 sections (from every seventh section) were stained with hematoxylin and eosin (H&E) for histopathologic assessment in a blinded fashion according to the Bar Harbor Classification [30]. The prostatic glands were assessed under low- and high-power magnifications, and approximately 27 to 94 prostatic glands in each prostate were counted, with a total of over 500 prostatic glands in 9 mouse prostates per genotype. The number of inflammatory cells in the connective tissue space between the prostatic glands was counted in ten high-power fields (x400 magnification) of each dorsal prostatic lobe; the average number of inflammatory cells per high-power field in 9 mouse prostates per genotype was compared.

Immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining

Immunohistochemical staining and double immunofluorescent staining were performed as described previously [28]. The antibodies used were: rabbit anti-p-Akt (1:100), mouse anti-PTEN (26H9, 1:50), rabbit anti-YB1 (D299, 1:50), and rabbit anti-MTA1 (D40D1, 1:25) from Cell Signaling Technology, Inc., Danvers, MA, USA; rabbit anti-Ki-67 (1:100, EMD Millipore, Billerica, MA, USA); rabbit anti-VEGF (A-20, sc-152, 1:200), goat anti-HIF-1α (Y-15, sc-12542, 1: 50), rabbit anti-NOS2 (iNOS, N-20, sc-651, 1:100), rabbit anti-Integrin αM (CD11b, H-61, sc-28664, 1:100), goat anti-Ly6C (P-12, sc-23080, 1:100), goat anti-Ly6G (Y-11, sc-103603, 1:100), goat anti-arginase I (V-20, sc-18345, 1:100), and goat anti-COX-2 (C-20, sc-1745, 1:200) from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; rabbit anti-CD31 (Ab28364, 1:50, Abcam, PLC., Cambridge, MA, USA); rabbit anti-laminin (1:100, Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-α-smooth muscle actin (1:200, Pierce Biotechnology, Rockford, IL, USA); goat anti-MMP7 (1:200, R&D systems, Minneapolis, MN, USA); rabbit anti-UbcH10/UBE2C (1:200, Boston Biochem, Cambridge, MA, USA); and Cy3-conjugated anti-goat IgG and DyLight 488-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using TACS.XL^® Blue Label In Situ Apoptosis Detection Kits (Trevigen, Inc., Gaithersburg, MD, USA) according to the manufacturer’s instructions [28]. To quantify Ki-67-positive and TUNEL-positive cells, three animals from each genotype group were randomly selected; three representative prostate sections from each animal were stained; approximately 200 cells per field of ten high-power fields (x400 magnification) of each prostate lobe were counted; and the percentages of positive cells were calculated as the number of positive cells divided by the total number of cells. The density of microvessels was evaluated by counting the CD31-positive microvessels in ten high-power fields per lobe; the average number of CD31-positive microvessels per high-power field in three random mouse prostates per genotype was compared. The myeloid-derived suppressor cells (MDSCs) were defined as CD11b/Gr-1 (CD11b/Ly6C and CD11b/Ly6G) double positive cells. The M1 and M2 macrophages were defined as iNOS-positive and arginase I-positive cells, respectively. The numbers of MDSCs, M1 macrophages, and M2 macrophages in the connective tissue space between the prostatic glands were counted in ten high-power fields (x400 magnification) in each dorsal prostatic lobe; the average numbers of MDSCs, M1 and M2 macrophages per high-power field in three random mouse prostates per genotype were compared.

Statistical analysis

Comparisons of the GU-bloc weights were analyzed using Student’s t test. Kruskal -Wallis test was used to compare the incidences of normal, PIN and invasive adenocarcinoma. Student’s t test was used to analyze the remaining data.

RESULTS

Castrate RC⁻ mice developed smaller prostate glands than castrate RC⁺ mice

Previously we found that there were no significant differences in the expression of Il17rc mRNA and protein, GU-bloc weight, and histopathology between Il17rc^+/+;Pten^L/L;Cre⁺ and Il17rc^+/−;Pten^L/L;Cre⁺ mice [28]. Therefore, we put Il17rc^+/+;Pten^L/L;Cre⁺ mice and Il17rc^+/−;Pten^L/L;Cre⁺ mice into one group, named RC⁺ mice that expressed IL-17RC receptor. Likewise, Il17rc^−/−;Pten^L/L;Cre⁺ mice were named RC⁻ mice that did not express IL-17RC receptor. Both RC⁺ and RC⁻ mice had Pten gene conditionally knocked out in the prostatic epithelium due to probasin promoter-driven Cre recombinase [28]. We reported that the GU blocs, including the prostatic glands, were clearly larger in the non-castrate (or intact) RC⁺ mice than in the intact RC⁻ mice at 30 weeks of age (Fig. 1A and 1B) [28]. In the present study, we found that the GU blocs also appeared larger in the castrate RC⁺ mice than in the castrate RC⁻ mice at 30 weeks of age (Fig. 1C and 1D). The average GU bloc weight was 946.3 mg in the intact RC⁺ mice compared to 644.7 mg in the intact RC⁻ mice (P < 0.01, Fig. 1E). Castration at 16 weeks of age significantly reduced the GU bloc weight at 30 weeks of age in both RC⁺ and RC⁻ mice to an average of 224.1 mg and 164.3 mg, respectively (P < 0.01, Fig. 1E). Yet, the GU bloc weight of castrate RC⁺ mice was still significantly heavier than that of castrate RC⁻ mice (P < 0.05, Fig. 1E).

Fig. 1.

Castrate RC⁻ mice developed smaller prostate glands than castrate RC⁺ mice. A–D: Representative photographs of the GU blocs; arrows indicate urinary bladders and arrowheads indicate the ventral prostatic lobes for orientation of the view. E: The GU bloc weights at 30 weeks of age; n = 20 for intact RC⁺ mice, n = 12 for intact RC⁻ mice, n = 9 for castrate RC⁺ mice, and n = 9 for castrate RC⁻ mice.

Castrate RC⁻ mice developed fewer invasive adenocarcinomas than castrate RC⁺ mice

It has been reported that intact RC⁺ mice developed invasive adenocarcinomas, that is, the neoplastic cells have invaded through the basement membrane and into the stroma, at 30 weeks of age with 100% penetrance [28,29], whereas intact RC⁻ mice developed invasive adenocarcinomas in about 70% of prostates at 30 weeks of age [28]. In the castrate RC⁺ mice, we found that approximately 65% of prostatic glands presented with invasive adenocarcinomas (Fig. 2A), 33% of prostatic glands had prostatic intraepithelial neoplasia (PIN), and 2% of prostatic glands appeared “normal” as they presented with a single layer of luminal epithelial cells. In contrast, the castrate RC⁻ mice showed approximately 23% of prostatic glands with invasive adenocarcinomas, 64% of prostatic glands with PIN, and 12% of prostatic glands with normal epithelia (Fig. 2B). Under higher magnification, the invasive adenocarcinoma cells presented with atypical hyperchromatic nuclei and invaded into the surrounding stroma (Fig. 2C and 2D). The rate of invasive adenocarcinomas was 42% lower in RC⁻ mice than in RC⁺ mice (P < 0.001, Fig. 2E).

Fig. 2.

Castrate RC⁻ mice developed fewer invasive adenocarcinomas than castrate RC⁺ mice. AD: Representatives of H&E-stained dorsal prostatic lobes; arrows indicate normal glands with a single layer of luminal epithelium, arrowheads indicate PIN lesions, and rectangular frames indicate invasive adenocarcinomas; original magnification, x100 for (A–B) and x400 for (C–D). E: Percentages of normal, PIN, and invasive cancer in dorsal, lateral, and ventral prostatic lobes.

To verify our diagnosis of invasive adenocarcinoma versus PIN based on H&E-stained tissue sections, consecutive sections were stained with H&E and immunohistochemically stained with anti-laminin or anti-α-smooth muscle actin (α-SMA) antibodies. As shown in Supplementary Figure 1A, invasive adenocarinoma showed lack of staining or discontinuity of staining for laminin. In contrast, PIN lesion presented a continuous layer of laminin staining around the prostatic gland (Supplementary Fig. 1B). Similarly, α-SMA staining showed lack of continuity in invasive adenocarcinoma (Supplementary Fig. 1C), whereas a continuous layer of α-SMA staining was present in the PIN lesion and normal gland (Supplementary Fig. 1D).

Castrate RC⁻ prostate had less cellular proliferation and more apoptosis than castrate RC⁺ prostate

We found that RC⁺ mouse prostate had clearly more Ki-67-positive neoplastic cells (Fig. 3A) than RC⁻ mouse prostate (Fig. 3B). The differences between RC⁺ and RC⁻ mice were statistically significant in the dorsal prostatic lobes (DP), lateral prostatic lobes (LP), and ventral prostatic lobes (VP) (P < 0.05 or P < 0.01, Fig. 3C). On the other hand, the number of apoptotic cells as detected by TUNEL staining was fewer in the RC⁺ mouse prostatic lobes (Fig. 3D) than in the RC⁻ mouse prostatic lobes (Fig. 3E), which was statistically significant (P < 0.01, Fig. 3F).

Fig. 3.

Castrate RC⁻ prostate had less cellular proliferation and more apoptosis than castrate RC⁺ prostate. A–B: Ki-67 staining; arrows indicate the positive cells. C: Percentages of Ki-67-positive cells in dorsal (DP), lateral (LP), and ventral (VP) prostatic lobes; *P < 0.05 and **P < 0.01. D–E: TUNEL staining; arrow indicates the positive cells. F: Percentages of apoptotic cells in prostatic lobes; **P < 0.01.

Castrate RC⁻ prostate decreased expression of invasion-related proteins

Previously we reported that the intact RC⁻ mice expressed significantly less MMP7 in the prostate than the intact RC⁺ mice, which partially explained the lower rate of invasive cancer in RC⁻ mice compared to RC⁺ mice [28]. In the castrate mice, the level of MMP7 expression was also lower in the RC⁻ prostate than in the RC⁺ prostate (Fig. 4A and 4B).

Fig. 4.

Castrate RC⁻ prostate decreased expression of invasion-related proteins. A–H: Representatives of immunohistochemical staining; arrows indicate neoplastic cells; original magnification, x400.

To search for other proteins that might contribute to the different incidence rates of invasive cancer in our animal models, we tested several candidates that were reportedly involved in prostate carcinogenesis. YBX1 (also known as YB-1) is a Y-box binding protein [31], which has been demonstrated to confer invasiveness to breast cancer cells [32]. YBX1 level is elevated in human PIN and invasive adenocarcinomas [33], similar to the pattern of MMP7 expression [28]. We found that YBX1 expression was clearly decreased in the castrate RC⁻ prostate compared to the castrate RC⁺ prostate (Fig. 4C and 4D). Metastasis associated 1 (MTA1) was originally identified from rat mammary adenocarcinoma cell lines [34] and recently it has been associated with the invasiveness of human prostate cancer cells [35]. We found that MTA1 expression was discernibly decreased in the castrate RC⁻ prostate compared to the castrate RC⁺ prostate (Fig. 4E and 4F). Ubiquitin-conjugating enzyme E2C (UBE2C, also called UBCH10) is needed for degradation of mitotic cyclins [36], which has been associated with malignant transformation and aggressiveness of many tumors [37]. UBE2C level is undetectable in human normal prostate, low in hormone-sensitive prostate cancer and high in CRPC [38]. Again, we found that UBE2C level was obviously higher in the castrate RC⁺ prostate than the castrate RC⁻ prostate (Fig. 4G and 4H).

Castrate RC⁻ prostate had less angiogenesis than castrate RC⁺ prostate

IL-17 has been found to be able to promote migration and cord formation of vascular endothelial cells through induction of a variety of proangiogenic factors [39], thus IL-17 may enhance in vivo lung cancer growth via promoting angiogenesis [40]. By immunohistochemical staining of CD31, we found that there were clearly more blood vessels in the castrate RC⁺ prostate than in the castrate RC⁻ prostate (Fig. 5A and 5B), which was statistically significant in the prostatic lobes examined (P < 0.01, Fig. 5C). We and other investigators have shown that IL-17 can induce angiogenic CXC chemokines including CXCL1, CXCL5 and CXCL8 expression [28,40]. It has been reported that cyclooxygenase-2 (COX-2) is induced by IL-17 in keratinocytes [41]. In the present study, we found that COX-2 level was dramatically lower in RC⁻ prostate than RC⁺ prostate (Fig. 5D and 5E). We also found that hypoxia inducible factor 1-α (HIF1A) level was not discernibly different between RC⁺ and RC⁻ prostates (Fig. 5F and 5G). However, the level of vascular endothelial growth factor A (VEGFA) was clearly higher in RC⁺ prostate than RC⁻ prostate (Fig. 5H and 5I). It has been demonstrated that the COX-2-VEGF pathway is involved in gastric angiogenesis [42]. Our findings suggest that the COX-2-VEGF pathway plays a role in prostatic angiogenesis while HIF1A’s role may be very limited.

Fig. 5.

Castrate RC⁻ prostate had less angiogenesis than castrate RC⁺ prostate. A–B: Representatives of CD31 staining; arrowheads indicate microvessels. C: Density of microvessels in prostatic lobes; **P < 0.01. D–I: Representatives of immunohistochemical staining; arrows indicate neoplastic cells; original magnification, x400.

Castrate RC⁻ prostate had less inflammatory cell infiltration than castrate RC⁺ prostate

Previously we reported that the inflammatory cell population was mainly composed of macrophages (or myeloid cells) and lymphocytes in the intact mouse prostate and the number of inflammatory cells was significantly reduced in RC⁻ prostate compared to RC⁺ prostate [28]. In the castrate mouse prostate, the inflammatory cell population was mainly composed of lymphocytes (Fig. 6A). Consistent with our observation in the intact mice, we found that the number of inflammatory cells was much fewer in the castrate RC⁻ prostate than the castrate RC⁺ prostate (Fig. 6A and 6B), which was statistically significant (P < 0.01, Fig. 6C).

Fig. 6.

Castrate RC⁻ prostate had less inflammatory cell infiltration than castrate RC⁺ prostate. AB: H&E staining; arrows indicate lymphocytes. C: Number of inflammatory cells counted on H&E-stained sections. D–E: Double immunofluorescent staining of CD11b and Ly6C. F: Number of Ly6C MDSCs. G–H: Double immunofluorescent staining of CD11b and Ly6G. I: Number of Ly6G MDSCs. J–K: Immunofluorescent staining of iNOS-positive M1 macrophages. L: Number of M1 macrophages. M–N: Immunofluorescent staining of arginase I-positive M2 macrophages; arrowhead indicates positively stained prostatic epithelium. O: Number of M2 macrophages. Original magnification, x400; arrows indicate positive cells; *P < 0.05 and **P < 0.01.

It has been shown that IL-17 induces infiltration of myeloid-derived suppressor cells (MDSCs) to promote prostate tumor growth [43]. MDSCs are considered as immature myeloid cells that are identified as CD11b/granulocyte-differentiation antigen-1 (Gr-1) double-positive cells. Gr-1 antigen consists of two epitopes recognized by anti-Ly6G (lymphocyte antigen 6 complex, locus G) and anti-Ly6C (lymphocyte antigen 6 complex, locus C). Thus, MDSCs consist of two major subsets: cells with granulocytic phenotype marked by CD11b⁺/Ly6G⁺ and cells with monocytic phenotype marked by CD11b⁺/Ly6C⁺, both subsets having equal suppressive activities against T cell function [44]. Therefore, we examined the infiltration of the two MDSC subsets in mouse prostate. We found that the numbers of both MDSC subsets were significantly reduced in RC⁻ prostate compared to RC⁺ prostate (P < 0.05, Fig. 6D to 6I).

It has been reported that the inducible nitric oxide synthase (iNOS)-positive M1 macrophages and arginase I-positive M2 macrophages are present in the mouse prostate tumors, where M1 macrophages have anti-tumor functions while M2 macrophages have pro-tumor functions [45]. We found that there were slightly fewer M1 macrophages than M2 macrophages in both RC⁺ and RC⁻ prostates (Fig. 6J to 6O). Yet, the numbers of M1 and M2 macrophages were significantly reduced in RC⁻ prostate compared to RC⁺ prostate (Fig. 6L and 6O).

DISCUSSION

We previously reported that Il17rc knockout inhibited formation and growth of hormone-naïve prostate adenocarcinoma in the Pten conditional knockout mouse model [28]. In the present study, we used the same mouse model and castrated the animals at 16 weeks of age. Fourteen weeks after castration, we found that invasive adenocarcinomas were present in both RC⁺ and RC⁻ mouse prostates, albeit at different incidence rates. The GU bloc size, judged by the GU bloc weight, was significantly smaller in the castrate mice than the intact mice (Fig. 1). The remaining invasive adenocarcinomas in the castrate mice presumably are CRPC based on the direct evidence that cellular proliferation was still present 14 weeks after the absence of testicular androgens. Another line of indirect evidence is that UBE2C level was very high in the castrate RC⁺ mouse prostate as UBE2C was only expressed at high levels in CRPC [38].

The most significant phenotypic difference between the castrate RC⁺ and RC⁻ mice is the incidence rate of invasive adenocarcinomas. RC⁺ mice presented invasive adenocarcinomas in 65% of prostatic glands, in sharp contrast to 23% of prostatic glands in RC⁻ mice (Fig. 2E). These incidence rates are much lower than the rates in the non-castrate RC⁺ and RC⁻ mice (i.e., 100% and 70%, respectively) [28,29]. One possible reason is that the AR-positive prostatic epithelium undergoes increased apoptosis in response to castration. A 10-times increase of apoptotic cells in the Pten-null prostate cancer was found compared to the intact mouse prostate cancer, and the apoptotic rate was even higher in the prostate cancer than the normal epithelium 3 days post-castration [29]. Apoptosis may possibly lead to regression of some invasive adenocarcinomas and PIN lesions, resulting in a heterogeneous appearance of normal epithelium, PIN, and invasive adenocarcinomas.

There are several possible reasons to explain the different incidence rates of invasive adenocarcinomas between the castrate RC⁺ and RC⁻ mice. First, the cellular proliferation rate is higher in RC⁺ prostate than RC⁻ prostate while the apoptotic rate is lower in RC⁺ prostate than RC⁻ prostate. These differences confer an advantage to the tumor growth in RC⁺ mice over RC⁻ mice. Second, several proteins, namely, MMP7, YBX1, MTA1, and UBE2C, are expressed at higher levels in RC⁺ prostate than RC⁻ prostate. These proteins have been associated with the invasiveness and aggressiveness of human prostate cancer cells [28,32,33,35,38]. While we have demonstrated that MMP7 is a direct downstream target of IL-17 signaling pathway [28], it remains to be determined if YBX1, MTA1, and UBE2C are also IL-17 downstream targets. Third, angiogenesis is reduced in RC⁻ prostate compared to RC⁺ prostate. Angiogenesis is an integral hallmark of cancer and it has recently been associated with early neoplastic progression besides its well-known role in macroscopic tumors [46]. One mechanism by which IL-17 promotes lung cancer growth is through induction of angiogenesis [40]. The reduced levels of COX-2 and VEGFA in RC⁻ prostate may be responsible for the decreased angiogenesis, as the COX-2-VEGF pathway has been associated with gastric angiogenesis [42]. COX-2 is an IL-17 downstream target in keratinocytes [41]. And last, inflammatory cell infiltration is reduced in RC⁻ prostate compared to RC⁺ prostate. The number of inflammatory cells appears to be more in the prostate of the castrate mice than the intact mice. It has been reported that androgen ablation increased infiltration of CD4+ T cells and macrophages in human prostate tumors [47]. The inflammatory cell population shifts from myeloid cells/lymphocytes in the intact mice to mainly lymphocytes in the castrate mice. This finding is in line with a recent report that castration elicits infiltration of T_H1 cells followed by predominantly T_H17 cells in rat prostate [48]. The subtypes of lymphocytes in our animal models are the subjects of our ongoing studies. Nevertheless, we have shown that the numbers of two major myeloid cell types, MDSCs and macrophages, are significantly reduced in RC⁻ mice compared to RC⁺ mice. We have recently demonstrated that IL-17 is a chemoattractant for monocytes/macrophages [49]. The reduced infiltration of MDSCs and macrophages may be caused by lack of IL-17RC receptor on these cells and/or indirectly by the decreased chemokine levels in the tumor microenvironment of RC⁻ mice. Since MDSCs and M2 macrophages are pro-tumor inflammatory cells, a decrease in their numbers may partially contribute to the phenotype of reduced incidence rate of invasive adenocarcinomas in RC⁻ mice.

In summary, the present study demonstrates that IL-17 promotes development of CRPC in the Pten conditional knockout mouse model. IL-17 may affect several hallmark capabilities of cancer, including sustaining proliferation, resisting cell death, activating invasion, inducing angiogenesis, and recruiting pro-tumor inflammatory cells [46]. These findings suggest that blocking IL-17 signaling through pharmacological interventions may have potentials in the prevention and treatment of CRPC.

CONCLUSIONS

IL-17 promotes development of invasive prostate adenocarcinomas in Pten conditional knockout mice under castrate conditions, potentially through creating an immunotolerant and pro-angiogenic tumor microenvironment.

ABBREVIATIONS

AR

androgen receptor

Bcl-2

B-cell lymphoma 2

COX-2

cyclooxygenase-2

Cre

Cre recombinase

CRPC

castration-resistant prostate cancer

CXCL

C-X-C motif ligand

GU

genitourinary

Gr-1

granulocyte-differentiation antigen-1

H&E

hematoxylin and eosin

HER-2

human epidermal growth factor receptor 2

HIF

hypoxia inducible factor

IL

interleukin

IL-17RC

IL-17 receptor C

iNOS

inducible nitric oxide synthase

KO

knockout

MDSCs

myeloid-derived suppressor cells

MMP

matrix metalloproteinase

MTA1

metastasis associated 1

PIN

prostatic intraepiethelial neoplasia

Pten

phosphatase and tensin homolog

Ras

rat sarcoma gene

SMA

smooth muscle actin

T_H17

T helpher cells expressing IL-17

TMPRSS2-ERG

transmembrane protease, serine 2 - E-twenty six related gene

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

UBE2C

ubiquitin-conjugating enzyme E2C

VEGF

vascular endothelial growth factor

YBX1

Y-box binding protein 1