Elevated levels of circulating IL-7 and IL-15 in patients with early stage prostate cancer

Prostate cancer (PCA) is the second leading cause of cancer-related death in men with a death rate reaching 26.7% for 2001-2005 in United States[1]. Chronic inflammation has been suggested to play a major role in prostate oncogenesis[2]. Furthermore, local and systemic immunosuppression have also been reported in patients bearing PCA[3‐6]. The clarification of molecular mechanisms underlying these phenomena might provide novel insights into PCA induction and progression, of potentially high clinical relevance.

Cytokines represent key mediators of inflammation and play pivotal roles in the interaction between immune system and cancer. A number of them have been suggested to be associated with advanced stage PCA[7]. IL-4 and, in particular, IL-6 have been shown to exert antiapoptotic effects on PCA cells, whereas proangiogenic effects of IL-8 have been demonstrated[7].

A group of lymphokines including, among others, IL-2, IL-7, IL-15 and IL-21 share a receptor γ-chain[8]. Common receptor γ-chain (c- γ) cytokines regulate lymphocyte development[9‐12] and support CD4+ and CD8+ T cell homeostatic proliferation and functions[13, 14]. IL-2, IL-7, IL-15 and IL-21 are of particular interest in cancer immunotherapy[15]. Importantly, IL-2 is FDA approved for the treatment of melanoma and renal cell carcinoma, whereas IL-7 has been used with promising results in phase I/II trials in clinical oncology[16, 17]. IL-15 has not yet been used in clinical trials. However, experimental models and "in vitro" evidence suggest that it may represent a cancer treatment with high potential clinical relevance[18‐20]. Finally, IL-21 has been shown in murine models to prevent T lymphocyte exhaustion induced by chronic stimulation[21‐23]. In phase I/II clinical trials, IL-21 has also shown reproducible antitumor effects[24, 25].

Notably, however, elevated IL-7 serum levels have been detected in Hodgkin disease and in ovarian cancers [26‐29]. Furthermore, IL-7 has been shown to be produced by breast and colorectal cancer cells[30, 31]. IL-15 has also been shown to be produced by colon cancer cells and specific gene expression has been shown to be associated with distant metastases [32]. Moreover, elevated IL-15 serum levels were detected in multiple myeloma[33].

Little is known about IL-2, IL-7, IL-15 and IL-21 gene expression and protein secretion in patients bearing PCA or BPH. Indeed, IL-2 gene expression and IL-15 protein have been detected in BPH tissues[34, 35]. Furthermore, IL-7 gene has been found to be expressed in PCA tissues[36, 37] and the corresponding protein has been detected in patients' sera. However, no comparative analysis in patients with BPH and PCA has been performed so far and no data are currently available for IL-21 expression in PCA and BPH.

In order to evaluate the potential role of these IL in early phases of prostate oncogenesis, in this study we have evaluated the expression of specific genes in prostatic tissues from patients bearing either localized pT1-2c PCA or BPH and we have measured the titres of circulating proteins in their sera.

Our results indicate that in prostatic tissues from patients bearing early stage PCA, the expression of IL-7 and IL-15 genes is significantly enhanced, as compared to BPH, while no significant differences could be observed in IL-2 or IL-21 gene expression. Accordingly, titres of circulating IL-7 and IL-15 are increased in patients with localized PCA, as compared to patients with BPH. Most interestingly, IL-7 titres might help to discriminate sera from patients with PCA or BPH.

A large body of literature based on histopathological, epidemiological and molecular pathology data suggests that chronic inflammation might play an important role in prostate oncogenesis [2]. IL represent essential mediators of inflammation and titres of a number of them have been shown to be increased in patients bearing PCA[7]. Most of these studies, however, have addressed IL detection in patients with advanced stage cancers. In these conditions, high tumor burdens and mechanisms inherent with metastatic spread, e,g, osteolysis, might be involved in the induction of IL production. Furthermore, sterile inflammation primed by intratumoral ischemia and associated necrosis could also promote IL release [51]. Therefore, data from patients with advanced tumors are unlikely to provide useful information on the role eventually played by inflammation or immunosuppression in prostate oncogenesis. In order to gain insights into events occurring in early stages of prostate cancerogenesis, in this study we evaluated IL gene expression and protein production at local and systemic levels, respectively, in localized PCA, as compared with BPH.

Our data clearly indicate that the expression of a number of genes encoding pro-inflammatory and homeostatic IL, including IL-6, IL-7 and IL-15 is detectable more frequently and to a higher extent, in early stage PCA as compared to BPH tissues. Notably, the genes encoding these IL were also found to be expressed to different levels by established PCA cell lines.

To investigate the possibly systemic nature of the pro-inflammatory state detectable in PCA[37, 52], we measured IL titres in sera from patients with BPH or early stage PCA. We report here for the first time that IL-7 and IL-15 serum levels are significantly higher in patients with localized PCA than in patients with BPH. IL-6 titres were also significantly higher in patients with PCA than in patients with BPH despite the early stage of their disease [53].

IL-6 is known to be produced by multiple cell types, including activated macrophages, smooth muscle cells and PCA cells [53]. Instead, IL-7 is produced by multiple stromal cell types and epithelial cells from different districts including thymus and gut[10]. Most recently IL-7 production has been detected in normal, but not in transformed prostate epithelial cells[36]. IL-15 is typically produced by activated antigen presenting cells of the myeloid lineage, including monocytes, macrophages and dendritic cells. However, IL-15 gene has also been found to be expressed by a multiplicity of other cell types, including stromal and epithelial cells[9]. Importantly, epithelial BPH cells have been shown to express IL-15[34]. In our patients, IL serum titres were not significantly correlated with specific gene expression in the autologous prostate tissues, thereby suggesting that cells other than tumor cells might be at least in part responsible for IL production. Thus, while we here show that IL-6, IL-7 and IL-15 genes are expressed in established PCA cell lines, future studies are warranted to clarify the cellular sources of these IL within the prostate and systemically.

The nature of the mechanisms promoting IL-6, IL-7 and IL-15 gene expression and protein production in PCA is also unclear. Different microorganisms, including virus and bacteria[54, 55] have been suspected to contribute to oncogenesis in the prostate. Indeed, their presence could result in chronic inflammation. Alternatively, it has been suggested that direct damage of epithelial cells might be caused by toxic compounds contained in urines or by dietary factors[2]. Cell death might, in turn, result in the activation of the innate immune system by DAMPs [49]. Soluble factors produced by DAMP-triggered innate immune system cells, including IL-6, have repeatedly been suggested to promote prostate cancerogenesis[2]. However, here we show that at least circulating levels of HMGB1, SAA and CRP acute phase proteins are similar in patients with BPH or localized, early stage PCA.

Analysis of ROC curves indicates that IL-7, but neither IL-6 nor IL-15 titres do distinguish sera from patients with PCA from sera from patients with BPH. These findings might set the stage for larger studies also addressing the clinical relevance of IL-7 serum titres in the monitoring of PCA response to treatment and recurrences.

On the other hand, our data raise the issue of potential functional consequences of c- γ cytokine gene expression and secretion in PCA. Preliminary experiments indicate that none of the IL under investigation enhances the proliferation of cells from established PCA cell lines (data not shown). However, studies performed in breast cancer[56] indicate that IL-7 promotes the production of pro-angiogenic factors by tumor cells and proliferation of endothelial cells. IL-15 has also been shown to stimulate angiogenesis "in vivo"[57]. Furthermore, although IL-7 and IL-15 are known to promote survival and functional maturation of T cells, they have also been demonstrated to induce the expression of PD-1 "exhaustion" marker in these cells[58]. Indeed, PCA tissues have been shown to be infiltrated by T cells largely expressing PD-1, and, less frequently, PD-L1 and PD-L2[59, 60]. These data are consistent with previous reports on the phenotypic characteristics of circulating and tissue infiltrating lymphocytes in chronic viral infections[61‐64]. In this context, it is remarkable that we found that the expression of the IL-21 gene, that is of the gene encoding the cytokine preventing PD-1 mediated T cell exhaustion[21‐23] is rarely detectable in PCA.

Our work indicates that even in early stages of PCA development, genes encoding IL of pro-inflammatory significance are over-expressed in malignant tissues, as compared to BPH specimens, Importantly, corresponding proteins are detectable to higher levels in sera from patients with localized PCA as compared to patients with BPH, and, in particular, IL-7 titres might represent an additional marker of potential clinical relevance.