Prostate-specific IL-6 transgene autonomously induce prostate neoplasm through amplifying inflammation in the prostate and peri-prostatic adipose tissue

Previously, we described that IL-6 facilitated prostate tumorigenesis and progression through autocrine IL-6 loop and re-programming oncogenic transcriptional profiles using oncogene-immortalized benign prostate epithelial cell lines [14]. Inspired by these findings, in this study, we aimed to address whether IL-6 has a causative role in de novo spontaneous prostate tumor initiation and thus generated prostate-specific IL-6 transgenic mouse lines. The encoding transgene of human IL-6 (hIL-6), which has been shown to activate downstream signaling cascade similarly in both human and mouse cells [29, 30], was expressed in the prostate of the C57BL/6 (B6) mice directed by the rat probasin (rPb) promoter [31] (Fig. 1a). We chose to express human IL-6 so that we could differentiate the expression of hIL-6 transgene from the autocrine expression of mouse IL-6 (mIL-6). The founder line that expressed the transgene IL-6 in the prostate was designated as pbIL-6. Integration of a single copy of an intact transgene was confirmed by genomic PCR against a limited template dilution standard (data not shown). Prostate-specific expression of the transgene hIL-6 was detected by RT-PCR (Fig. 1b).

To demonstrate the consequences of prostate-specific expression of hIL-6 in the adult prostate, we compared the histological characteristics of pbIL-6 transgenic mice with the wild type B57CL/6 (B6) littermates. The prostate glands from pbIL-6 transgenic mice uniformly exhibited varying degrees of prostate intraepithelial neoplasia (PIN)-like lesions at the age of 16-week old as we have examined, which were not observed in the B6 WT littermates (data not shown). By 24 weeks of age, neoplasia was evident in the pbIL-6 prostates. Specifically, the prostate exhibited increased epithelial tufting, overlapping of cells, enlarged nuclei of variable sizes and shapes with prominent nucleoli, and thickening of the stroma (Fig. 1c). As animals age, prostate glands in the pbIL-6 mice more predominantly exhibited carcinoma-like feature, such as increased proliferation, enlarged nucleoli, and disappearance of basal cells (Table 1 and Fig. 2). In two out of ten examined aged mouse (>100-week old), prostate carcinoma progressed to poorly differentiated (Additional file 1: Figure S1). Consistent with the progressive neoplasia development in pbIL-6 mice as animal ages, the rate of weight increase of prostate and genitourinary (GU) by age was significantly greater in pbIL-6 mice than in B6 WT littermates (Fig. 1d, e). Notably, unlike human, wild type mice only develop prostate intraepithelial neoplasia (PIN) but not spontaneous prostate carcinoma as they age [32]. Overall, the pbIL-6 animals presented higher mortality due to progressive tumor development in the prostate, with rare incidence in the liver (Fig. 1f).

In our previous report, we described multiple effects of IL-6 on immortalized benign human prostate epithelial cells [14]. Consistent with these findings, the prostate epithelium of the transgenic pbIL-6 mice demonstrated a focally dramatic decrease of membranous expression of E-cadherin (Fig. 2a, b). The loss of E-cadherin signifies a reduction in cell-cell adhesion and a shift of the prostate luminal cells from a clearly differentiated epithelial to a more mesenchymal phenotype and also an early stage of transformation [33, 34]. The disrupted expression of basal cell marker p63 in the prostate gland from the pbIL-6 mice also signifies the early transformation events of the prostate [35, 36] (Fig. 2a). Consistent with our previous report of the tumorigenic potential of IL-6 in epithelial cell lines [14], the prostate epithelium from pbIL-6 mice had a significant higher index of Ki-67 positivity (Fig. 2a), suggesting an active proliferation state. Moreover, in comparison to WT littermate, not only a markedly overall elevation of the oncogene β-catenin but also more significantly elevated accumulation of β-catenin in the nucleus was presented in the prostate epithelium of the pbIL-6 mice (Fig. 2a, e). Furthermore, in comparison to the B6 WT littermates, androgen receptor (AR) was not only significantly increased in the levels of expression in the prostate epithelium of the pbIL-6 mice, but also predominantly translocated to the nucleus (Fig. 2a, f), an indication of increased AR activity. Given that IL-6 has been known to increase AR activity in castration-resistant prostate cancer [37], together, these molecule features of the prostate epithelium of the pbIL-6 mice endowed its neoplasia features and tumorigenic properties, which were further substantiated by elevated expression of oncogenes, such as c-Fos and k-Ras as measured by quantitative RT-PCR (Fig. 2c, d).

In the previous report, we demonstrated that IL-6 stimulates the autocrine IL-6 loop in the immortalized benign prostate epithelial cells [14]. To investigate whether this is a bona fida effect of IL-6 signaling event during de novo tumorigenesis, we examined the expression of mouse IL-6 (mIL-6) in the prostate. Quantitative RT-PCR revealed a significant induction of mouse IL-6 expression in the prostate of pbIL-6 mice in comparison to the WT B6 littermate as we examined at 16 weeks of age (Figs. 3a, P < 0.01). By 24 weeks of age, significantly elevated serum levels of mIL-6 were detected in pbIL-6 mice (Figs. 3b, P < 0.01), presumably due to the more severe destruction of the prostate architect and release of secretory mediators. These results suggested that IL-6 in prostate tissue environment can induce bona fida IL-6 autocrine secretion. We also observed autocrine IL-6 induction in mouse prostate tumor cell lines exposed to exogenous IL-6 (Additional file 1: Figure S2).

We and the others have previously described that IL-6 signaling trans-activates the endocrine IGF signaling axis in cancer cells [14]. Activation of IGF signaling axis has been shown to play an essential role in tumor cell survival and proliferation [33]. In pbIL-6 mice, a significantly increased expression IGF-I and IGF-II was demonstrated by quantitative RT-PCR (Fig. 3c, d). Collectively, these data suggest that trans-activating IGF signaling axis may also contribute to IL-6-induced epithelium transformation.

Engagement of IL-6/IL-6Rα or IL-6/sIL-6Rα complex to the signaling adaptor molecule gp130 triggers the activation of downstream signaling cascade, most prominently the JAK2/STAT3 pathway [38]. Phosphorylation of STAT3 and translocation to the nucleus are the critical events for initiating the oncogenic transcriptional activity [39]. As shown in Fig. 4a, the prostate of pbIL-6 mice exhibited high levels of phosphorylated STAT3 (pSTAT3), whereas the prostate from WT littermates rarely presented positivity for pSTAT3. Intriguingly, pSTAT3 was present not only in the prostate epithelium cells but also in the stromal components of pbIL-6 mice. These data suggest that IL-6 signaling may pose effects on the prostate epithelium and stromal environment to induce neoplastic transformation.

We further addressed how expression of IL-6 in the prostate may impact prostate gene expression profiles using Affymetrix whole-transcript RNA array analyses. One hundred thirty genes were identified at least 2-fold upregulated in the prostate of pbIL-6 mice in comparison to the WT littermates at the significant level of P < 0.05. The most significantly regulated genes are indicated in the volcano plot of Fig. 4b and Additional file 1: Tables S1–S4. Notably, a large portion of these upregulated genes are associated with inflammation, oncogenesis, or metabolism. The most relevant upregulated genes, such as cytokine IL-6 and oncogenes c-JUN, β-catenin, and Ras have been validated by quantitative RT-PCR (Figs. 2c–e and 3a). IGF-I and the androgen signaling downstream gene TMPRSS2 were also shown in the gene expression array to be upregulated (Additional file 1: Table S3). Many of these transcriptional changes are consistent with our previous findings with in vitro IL-6 overexpression studies [14]. Together, these data indicate that expression IL-6 not only intrinsically reprograms the prostate to express pro-tumorigenic genes but also primes a pro-inflammatory tissue microenvironment whereby the combinatory effect may contribute to prostate neoplasm.

Emerging evidence suggests that chronic or recurrent inflammation may initiate and promote cancer development, including prostate cancer [40‐43]. This process involves multiple inflammatory cells as well as a broad array of inflammatory cytokines [44]. IL-6, as a major pro-inflammatory cytokine can be secreted by an array of inflammatory cell types, are not only the growth factor for epithelial cell, but also critical for the survival and proliferation of inflammatory cell types in the tissue microenvironment. This process is frequently referred as “smoldering” inflammation [40, 44‐46].

In general, an enriched infiltration of inflammatory cells was present in the prostate of the pbIL6 mice (data not shown). We thus characterized these inflammatory cell types by immunohistochemistry staining (IHC) with specific markers. The enriched infiltration of T lymphocytes (CD3⁺), macrophages (MAC-2⁺), and B lymphocytes (B220⁺) was remarkable and significantly increased in the prostate of pbIL6 mice in comparison to WT littermates (Fig. 5a, b), all of which are known to be IL-6- producing cells. Intriguingly, a rich infiltration of macrophages and T lymphocytes was also evident in the peri-prostatic adipose tissue in the pbIL6 mice, whereas infiltration is rare in the peri-prostatic adipose tissue of B6 WT littermates (Fig. 6). Together, these data suggest that enriched IL-6 expression in the prostate can amplify the inflammatory responses in the local tissue and peri-prostatic adipose tissue, which may confer as one of the pathways to induce neoplasm in the prostate.

Mice were bred and housed under specific pathogen-free conditions in the University of Washington animal facility in accordance with the institutional guidelines. All mice used in this study were on the C57BL/6 (B6) background. The rPB-IL6 expression cassette was constructed by replacing the SV40T human IL-6. The entire rPB-IL6 expression cassette was gel isolated following digestion with Hind III and was microinjected into fertilized B6 embryos at University of Washington Comparative Medicine transgenic core facility. Transgenic progeny were identified by PCR analysis of DNA extracted from tail biopsies using the forward primer specific for rPB (5′-acaagtgcatttagcctctccagta-3′) and the reverse primer specific for IL-6 (5′-tgtgtcttggtcttcatggc-3′). All experimental mice were randomly assigned to cohorts and euthanized at indicated age for evaluation of GU and the prostate.

Total RNA was extracted using TRizol (Invitrogen) followed by treatment with DNAase I (Fermentas) to exclude the genomic DNA before reversal transcription. Complementary DNA (cDNA) was synthesized using the SuperScript II kit (Invitrogen). A volume of 1 μL of cDNA was mixed with Power SYBR Green PCR MasterMix (Applied Biosystem, Carlsbad, CA, USA), and specific primer sets were added to a final concentration of 400 nM in 20 μl of reaction mixture. The reaction was performed on an ABI9700 Machine. Data were analyzed using the Lightcycler software v3.5 (Roche Applied Science, Indianapolis, IN, USA). Each sample was assayed in triplicates. Target mRNA levels were normalized against mouse GAPDH. The primers used are listed in Additional file 1: Table S5.

Total RNA of the prostates from four 24-week-old pbIL-6 transgenic male mice and wild type C57BL/6 littermates was obtained as described above. After RNA quality confirmation with a Bioanalyzer (Agilent), 300 ng of each RNA sample was used in the Affymetrix Whole-Transcript Sense Target Labeling Assay (Rev 3), followed by hybridization to a GeneChip Mouse Gene 1.0 ST Array. Eight GeneChips were used to provide biological replicates of each genotype. The Affymetrix Expression Console (v 1.1) was used to normalize data and determine signal intensity (RMA-Sketch). Analysis was performed using DAVID Bioinformatic software and R2 statistical software with bonferroni correction.

The mouse prostate tissues were fixed in 10% formaldehyde and embedded in paraffin wax. Five-micrometer sections were cut and stained with H&E for pathological evaluation. Sections were also stained with antibodies specific for: (1) E-cadherin (Santa Cruz); (2) p63 (Thermo Scientific); (3) Ki67 (Thermo Scientific); (4) β-catenin antibody (AbCAM); (5) AR (Santa Cruz); (6) pSTAT3 (Cell signaling); (7) macrophage (F4/80 or Mac-2; eBioscience); (8) B cells (B220, eBioscience); and (9) anti-CD3 (Thermo Scientific). The staining procedure has been previously described [14]. Briefly, sections were deparaffinized and incubated for 10 min in 10 mM citrate buffer (pH 6.0) at 95 °C for antigen retrieval. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol. After quenching endogenous peroxidase activity and blocking nonspecific binding, slides were incubated with specific primary antibody overnight at 4 °C followed by subsequent incubation with the appropriate biotinylated secondary antibody provided with Vectastain Elite ABC Kit. Color was developed with DAB as the perioxidase substract. All slides were counterstained with hematoxylin and mounted with Permount. Ten randomly selected fields of IHC-stained sections of the prostates from individual mice were counted for the positively stained cells and used for statistical analysis.

All results are expressed as the mean ± SEM. Differences between the mean of groups were analyzed using student’s t test with one-way ANOVA analyses. In most cases, P < 0.05 was considered as significant.