Inflammatory Microenvironment in Prostate Carcinogenesis

Geraldine Gueron, Javier Cotignola and Elba Vazquez

Additional information is available at the end of the chapter

1. Introduction

The association between prostate cancer and inflammation was first formally addressed in the nineteen century and since then many authors have confirmed the biological and clinical evidence of this association. However, the molecular mechanism involved is yet to be deciphered.

There are two well established pathways linking inflammation and cancer: the extrinsic pathway from conditions that cause non-resolving smouldering inflammatory responses and the intrinsic pathway where the misregulation of oncogenes and tumor suppressor genes switch on the expression of inflammation-related programs.

Prostate cancer is a complex and progressive disease. Over time the cells become resistance to hormonal therapies that are designed to block the release and/or the uptake of androgens. During this stage androgen receptor (AR) mutants are able to bind promiscuous steroids, and may convert AR antagonists to agonists. Other hormones and their receptors are involved in the abnormal growth of the gland. Particularly, oestrogens and oestrogen receptors defined a subclass of prostate cancer with a very aggressive clinical phenotype (such as the TMPRSS2-ERG fusion). In addition, other signaling cascades are switched on bypassing the androgen/AR axis and favoring tumor progression. Among them, cyclooxygenase-2 (COX-2), neuroendocrine differentiation and the loss of the tumor suppressor phosphatase and tensin homolog (PTEN), with the concomitant inhibition of the PI3K/Akt, resulting in Bcl-2 overexpression and the burst of pro-inflammatory cytokines, chemokines and other growth factors production, contributing all to the progression to the hormonal-resistance disease. As in other malignancies in prostate cancer, reactive oxygen species (ROS) cause ox-


© 2013 Gueron et al., licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

open science | open minds idative damage to macromolecules in epithelial cells and can react with other cellular components initiating a free radical chain reaction, thus sustaining the prostate carcinogenic process and its progression.

The molecular mechanisms that prime the pathogenesis of cancer-related inflammation are complex and involve a delicate interplay between tumor and its microenvironment. In prostate tumors, the switch to an angiogenic phenotype is known to be critical for its progression. Unless a tumor can stimulate the formation of new blood vessels, it remains restricted to a microscopic size. Inflammation and hypoxia are widely accepted as key elements in the induction of angiogenesis.

Dissection of the diversity of cancer-related inflammation is critical for the design of innovative diagnostic and therapeutic strategies in prostate cancer.

Specifically, the following topics and molecular events are reviewed and discussed in this chapter:

• The cytokine and chemokine orchestration and the associated downstream genetic events that cause neoplastic transformation in the prostatic tissue.

• Acknowledging the oxidative stress imbalance in the tumoral niche as key mediators of signaling cascades.

• The relevance of microRNAs as oncogenes and tumor suppressor genes and how micro-RNA expression profiles can be used for markers of prostate cancer prevention and therapeutics.

• The potential of prostate tumoral cells in the inflammatory microenvironment to express an endothelial-like phenotype and mimic vasculogenic networks.

2. Body

2.1. The cytokine & chemokine orchestration in prostate cancer: Strategies, avenues and traits

Cytokines are a family of cell-signaling protein molecules that are secreted by various cell types and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins. A variety of cyto-kines are secreted by cells in the tumor microenvironment and can impact on prostate cancer growth. These cytokines can then act in a paracrine fashion on tumor cells to stimulate a variety of physiological activities including cell proliferation, invasion, migration, che-moresistance, etc.

The tumor inflammatory microenvironment is characterized by immune cell infiltration: tumor-associated macrophages, mast cells, dendritic cells, natural killer cells, neutrophils, eo-sinophils and lymphocytes. These cells produce a variety of cytotoxic mediators such as ROS and reactive nitrogen species (RNS), serine and cysteine proteases, matrix metallopro-

teinase (MMP), tumor necrosis factor a (TNFa), interleukins, interferons and enzymes, as COX-2, lipooxygenase-5 and phospholipase A2, which activate or are activated by transcription factors such as nuclear factor kB (NF-kB) and signal transducers and activators of tran-scription-3 (STAT3), activator protein 1 and hypoxia-inducible factor 1a (HIF-1a) that mediate tumor cell proliferation, transformation, metastasis, survival, invasion, angiogene-sis, chemoresistance and radioresistance.

Present discoveries highlight chemokines and their receptors as relevant factors for inflammation. The directed migration of a cell toward the source of a secreted protein signal, known as chemotaxis, has been commonly associated to the leukocyte trafficking triggered by infection and to secondary lymphoid organs. Although extensively studied as part of the immune system, chemokines have lately been investigated as mediators of tumor development. Chemokines, the executors of chemotactic signals, are constitutively expressed in destined cell types and tissues maintaining the homeostasis of the hematopoietic and the immune system. However, inflammatory chemokines, either produced by the tumor cells or by tumor-associated cells, behave differently and their expression is induced upon inflammatory stimuli promoting proliferation and angiogenesis, contributing to the malignant progression. They certainly modify the sensitivity of prostate cancer cells to environmental stresses such as hypoxia, oxidative stress, DNA damage, altering several pathways crosstalk and producing hormone-refractory aggressive tumors. In addition to the classical roles described above, their pleiotropic effects include: potentiating the production of growth factors, inducing growth signals, attenuating apoptosis, further linking the cytokine signaling to the hypothesis that inflammation and inflammatory mediators rise as the seventh hallmark of cancer [1]. In this section we will focus on some of the several cytokines implicated in the prostate cancer microenvironment given that there are too many factors to describe.

2.1.1. The chemokine family acquaintance

To date, over 50 chemokines and 20 chemokine receptors have been recollected. These are grouped into four categories, C, CC, CXC and CX3C, according to the location of the main cysteine residues near the N-terminal domain of these proteins [2]. Chemokine binding to their corresponding seven transmembrane-domain G-protein-coupled receptors causes the activation of signal transduction networks leading to chemotaxis. These receptors have been implicated in the migration of breast, prostate and lung cells to secondary sites in the bone [3]. Up to date the most relevant chemokine receptors in prostate cancer dissemination, are CXCR4, CXCR7 and CXCR6 [3].

The CXCR4/CXCL12 axis exerts multifactorial effects and has been related to both, the homing of tumor cells to specific organs and the growth of tumor cells at specific locations. CXCL12, also known as SDF-1 (stromal derived factor 1), is considered a homeostatic che-mokine which regulates the hematopoietic cell trafficking and secondary lymphoid tissue architecture. It is constitutively expressed in several organs including lung, liver, skeletal muscle, brain, kidney, heart, skin, bone marrow and its secretion is linked to tissue damage. CXCR4 is expressed in endothelial cells and pericytes of hypoxic, injured, or pathological tissues. Of note, endothelial precursor cells also express and secrete CXCL12. In turn,

CXCR4 is widely expressed on hematopoietic cells including CD34+ hematopoietic stem cells, T- and B-lymphocytes, monocytes and macrophages, neutrophils and eosinophils as well as by brain, lung, colon, heart, kidney, liver endothelial and epithelial cells, microglia, astrocytes, neuronal cells, and progenitor cells including endothelial and smooth muscle progenitors. Functional CXCR4 is expressed on embryonic pluripotent stem cells and several types of tissue-committed stem cells. These cells with functional CXCR4 expression migrate and/or invade along CXCL12 gradients. CXCR4+ pro-angiogenic cells include immature and mature hematopoietic cells, endothelial precursor cells, and smooth muscle cell progenitors, which have direct or indirect pro-angiogenic properties. Interestingly, CXCL12 plays a role in the mobilization and recruitment of these cells to the neo-angiogenic niches supporting revascularization of ischemic tissue and tumor growth [4]. This axis has been strongly implicated in prostate cancer tumorigenesis and progression [5].

2.1.2. Chemokines and their relevance in the metastatic behavior of prostate cancer

Metastases is a multistep process including: invasion of the primary tumor cells to adjacent tissue, intravasation, dissemination through the blood or lymph, extravasation and seeding, adapting to a different tissue microenvironment and finally proliferating in such distant organs. This process involves both the selection of features that favor cancer cells growth and the concomitant alteration of the stroma generating a "fertile soil" which facilitates invasion, anchoring and survival of metastatic cells [6].

Prostate neoplasms have a striking tendency to metastasize to bone. The molecular mechanisms underlying the bone homing behavior have yet to be decoded. However, such mechanisms may include signaling cascades that induce a vascular pathway, that produce the trigger of chemotactic factors by bone marrow stromal cells and the production of growth factors within the bone, reinforcing the survival and proliferation of tumoral cells. It is of common knowledge that hematopietic stem cells are directed to the bone during bone marrow transplantation and human fetal development [7, 8] and CXCL12/CXCR4 appears in this scene as key molecules in bone seeding. Metastatic prostate cancer cells may use a similar pathway to localize to the bone. Several human prostate cancer cell lines express functional CXCR4 and differential levels of its ligand alter physiological processes of these cells such as adhesion, migration and invasion, assigning a role for this axis in prostate advanced disease. It is worth mentioning some controversial reports regarding the expression of this receptor and its ligand in prostate cancer. Mochizuki et al. [9] reported that the expression of CXCR4, but not its ligand, was increased in prostate carcinoma indicating that prostate cancer cells may also be affected by exogenous SDF-1. However, other authors showed high expression of both, ligand and receptor [10].

Interestingly, the blockade of CXCR4 inhibited the expression of vascular endothelial growth factor (VEGF) and the concomitant angiogenesis and even reduced significantly bone metastasis in vivo [11]. Furthermore, CXCR4 is positively regulated by AR [12]. Andro-gen-induced CXCR4 expression was functional in TMPRSS2-ERG-positive prostate cancer cells, further indicating the relevance of this chemokine in prostate cancer metastasis [13]. The immunohistochemical pattern of CXCR4 expression in patients with metastatic prostate cancer has shown that high expression of this chemokine in tumors had poorer cancer-specific survival than patients with low expression of CXCR4. This receptor expression has proved to be a useful prognostic factor for patients with metastatic prostate cancer treated with androgen-withdrawal therapy [14].

Strikingly, regulation of CXCL12 expression in the tumor microenvironment has been poorly studied. Some reports indicate that hypoxia may induce its expression in endothelial cells and in prostate tumor cells [5]. Could CXCL12 have an additional role to its chemo-attrac-tant properties? Could it also act as a growth factor or prevent the apoptosis of tumoral cells enabling metastasis to take place? These questions still need to be answered.

CXCR7 (RDC1), a second receptor for CXCL12, regulates a spectrum of normal and pathological processes but fails to couple to G-proteins and to induce the typical chemokine receptor mediated cellular responses. It also binds to CXCL11 and dimerizes with CXCR4. This receptor with dual specificity is up-regulated in many tumors, but its function within the tumoral niche needs further clarification [15]. Studies show that CXCR7 expression provides proliferation and survival advantages and increased adhesion properties between prostate cancer cells and the host endothelial cells. It is also more highly expressed in prostate metastases (specially those to the bone) compared to primary tumors and elevated levels of CXCR7 correlate with the aggressiveness of the disease. In the vasculature, the expression of CXCR7 is elevated in endothelial cells associated with tumors [16] and this chemokine receptor has been further linked to tumor angiogenesis in vivo [17].

Other inflammatory mediators may regulate CXCR7 function. Of note, high serum levels of IL8 have been reported in patients with advanced metastatic prostate cancer. In primary prostate carcinoma tissues, IL8 strongly correlates with biochemical prostate specific antigen (PSA) recurrence and CXCR7 expression is induced by IL8 in prostate tumor cells. As survival following androgen deprivation is a critical step in the emergence of castration-resistant tumors, IL8-induced up-regulation of CXCR7 may enhance the survival and proliferation properties of those tumor cells. Thus the up-regulation of CXCR7 induced by IL8 emerges as a promoter of castration-resistant tumors survival [15]. Moreover, CXCR7-depleted tumors showed significantly reduced levels of relevant factors for prostate tumori-genesis like cyclin D1, VEGF and phosphorylated epidermal growth factor receptor [15]. There is also additional evidence for a potential role of CXCR7 as a CXCL12 scavenger, suggesting that this receptor in turn modulates the activity of CXCR4 in tumor formation and is critical for the fine-tuning of the motility of hematopoietic cells in the bone marrow and lymphoid organs.

However, the blockade of the CXCR4 and CXCR7 only partially impaired the metastatic behavior of prostate cancer in vivo, arguing that other functional chemokine/chemokine receptor pairs may be envolved in prostate cancer progression [18].

The third chemokine receptor noteworthy in prostate cancer is CXCR6, displaying high expression not only in prostate cancer cell lines but also in prostate tissues [19]. This receptor is also known as Bonzo, STRL33 or TYMSTR. In humans, Bonzo is expressed by small subsets of T cells and CD16+ cells, but not by B cells, monocytes or dendritic cells [20].

CXCL16 is one of the two known transmembrane chemokines. It is also constitutively expressed on fibroblasts, keratinocytes and cancer cells of various origin tissues [19]. CXCL16 was identified as the ligand for this receptor and was found to signal through NF-kB via heterotrimeric G proteins/PI3K/PDK-1/Akt/IKK/IKB [21]. It was also reported to signal through the Akt/mTOR pathway [22]. A variety of chemokines contain a conserved sequence motif (ELR, glutamic acid-leucine-arginine) that precedes the first cysteine residue near the amino-terminal end which is critical for the receptor binding, for the chemotactic activity and for the promotion of angiogenesis. Intriguingly, although lacking an ELR motif in the chemokine domain, CXCL16 appears as proangiogenic. CXCR6 was shown to regulate blood vessel formation by an autocrine/paracrine loop established between prostate cancer and endothelial cells and was observed that both IL8 and IL6 levels were altered in response to changes in CXCR6 expression [18]. The striking similarities between CXCL16 and CXCL12 are likely to result in additive effects [23]. Moreover, CXCL12 and CXCL16 were observed in tissues enriched with plasma cells and in cultured human bone marrow stromal cells [23]. Thus, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4 and CXCR6 [18]. CXCL16 not only attracts T cells and natural killer T cells toward dendritic cells but also supports their firm adhesion to dendritic cells [24]. Taken together, high CXCL16/CXCR6 expression may be strongly related to aggressive cancer behavior, and particularly, high-secreted ligand expression to bone metastases of prostate cancer [19].

While it is well accepted that chemokines promote tumor development, these molecules may in turn be used to the benefit of cancer patients, acting in the recruitment of dendritic cells and /or effector cells or for their angiostatic properties. However, chemokine-mediated recruitment of immature dendritic cells within tumors, due to factors produced by the tumor milieu, may induce immune tolerance. In this context, the balance between positive and negative effects should be examined when designing novel strategies to eradicate tumors based on chemokine targeting.

2.1.3. Role of IL8 and IL6 in the transition to hormone refractory prostate cancer

Prostate cancer cells and the surrounding stroma are exposed to a plethora of interleukins and chemokines, receiving their signaling stimuli, re-enforcing tumor-promoting functions. Similar to other chemokines that recognize and bind G-protein-coupled receptors, IL8 acts through CXC receptors. The expression of CXCL8 (also known as IL8), one of the best-characterized members of the chemokine family, has been described as a key effector in prostate cancer. Normal prostate epithelial cells and tissues produce low amount of IL8, whereas prostate cancer cells from primary and metastatic tumors produce progressively greater amounts [25]. High levels of CXCL8 also correlate to an elevated adherence of the prostate tumor cells to the endothelium, hence increasing angiogenesis, tumorigenicity and lymph node metastasis in vivo [26, 27]. Even more, CXCL8 is a transcriptional target of NF-kB and its expression is elevated in androgen independent prostate cancer, contributing to the transition to a castration-resistant state and to resistance to standard chemotherapeutic drugs [28]. To date, the chemotherapy strategy utilized for advanced prostate cancer disease is based on the combination of docetaxel (a cytostatic drug) with prednisone (a glucocorticoid prodrug). However, this therapeutic strategy shows a modest survival benefit over palliative care, where many patients respond initially, but eventually develop a resistance to do-cetaxel. Among other factors, increased IL8 production decreases the sensitivity of hormone-resistant cells to the cytotoxic chemotherapeutic agents and also reduces prostate cancer cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In experimental prostate cancer a naphthalimide was shown to decrease IL8 expression and to enhance taxol activity when co-administered with this compound. Thus, negative regulators of this chemokine could emerge as second line treatment for patients with docetaxel-resist-ant advanced prostate cancer [29].

One of the most interesting mediators clearly implicated in prostate cancer is IL6, a multifunctional cytokine, produced by inflammatory cells, osteoblasts and even prostate cancer cells. There are multiple lines of clinical and experimental evidence preponderantly showing that IL6 contributes to prostate cancer progression. Both, patients with prostate cancer and patients with advanced metastatic disease display high expression levels of IL6 and its soluble receptor in the circulating plasma [30]. These observations have led to study whether this axis could predict biochemical recurrence in radical prostatectomy patients [31] providing a rationale for the clinical relevance of IL6 as a prognostic factor. In particular, a phase II study assessed the efficacy of siltuximab, in men with castrate resistant prostate cancer that had been treated with one prior chemotherapy with the primary endpoint being PSA response rate (defined by a 50% reduction of PSA) [32]. This drug, also known as CNTO 328, is a human-mouse chimeric monoclonal neutralizing IL6 antibody. The response rate was small and no men with disease had a Response Evaluation Criteria in Solid Tumors (RE-CIST) response. This criterion defines a set of rules that assesses whether a patient improves ("responds"), stays the same ("stabilizes"), or worsens ("progression") during treatments. The results obtained evidenced the lack of a beneficial therapeutic effect of IL6 neutralization in patients with advanced androgen resistant disease. However, there are still some positive prospects for IL6 neutralization, providing an additional benefit to other chemotherapy regimes, especially in light of its anti-apoptotic effects [33].

In addition to the clinical observations, in vitro studies have provided evidence that IL6 modulates prostate cancer cell growth of hormone-refractory cells, but had no effect on the growth of hormone-dependent cell lines [33].

IL6 has also been implicated in other aspects of prostate cancer pathophysiology such as tu-morigenesis in the prostate microenvironment. IL6 foremost effect is the activation of Janus kinase (JAK) signaling and of signal transducers and activators of transcription (STAT) proteins, especially STAT3. Through this signaling pathway, IL6 stimulates autocrine activation of insulin-like type I growth factor receptor (IGF-IR) to confer tumorigenesis [34]. Depending on the cellular context, IL6 can also signal through MAPK and phosphatidylinositol-3 kinase (PI3K) pathways [35, 36].

This cytokine can be produced autocrinaly in castrate resistant prostate cells and can trans-activate the AR in those cells. However, the AR status as well as other interacting signaling cascades will define the role of IL6 on ligand-independent AR activation, tumor formation, and subsequent growth. Additionally, IL6 has been proposed to initiate an intracrine signaling pathway, alternative to the androgen receptor axis, affecting metabolic enzyme levels. Surprisingly, testosterone plasma levels were significantly increased when IL6 overexpress-ing prostate cancer cells were inoculated in castrated mice, showing that this cytokine regulates the expression of esteroidogenic genes in tumoral cells [5].

Overall, IL6 strongly correlates with more advanced stages of the disease, therapy resistance, poor prognosis and can be predictive of recurrence after treatment of localized cancer. Based on all the clinical and preclinical evidence, further exploration for IL6 inhibition is justified; however, its efficacy may greatly depend on the stage of disease or other individualized factors.

2.1.4. Tumor Necorsis factor: Linking inflammation to prostate cancer

TNF was named for its ability to induce rapid haemorrhagic necrosis of experimental cancers [37]. However, it soon became noticeable that this cytokine presented anti-tumoral activity and cytotoxicity against several tumoral cells [38]. Currently, TNF is considered as a relevant player in host defense and inflammation with several activities extending far beyond its original anti-tumoral action. Among its effects, TNF signaling may lead to both, cell apoptosis and necrosis, and also to tumor progression and metastasis by switching on survival genes [39].

TNF signals through TNF receptor 1 (TNF-R1) and TNF-R2. While TNF-R1 is expressed con-stitutively in most tissues, TNF-R2 is modulated and is mostly found on immune system cells. TNF binds to the death domain containing TNF-R1 to recruit TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD) and caspase-8, forming the death-inducing signaling complex [40]. Interestingly, when TNF-R1 is activated, it also recruits receptor-interacting protein (RIP) and TNF receptor-associated factor 2 (TRAF2) and activates NF-kB, involved in cell survival, proliferation, anti-apoptotic activity and highly implicated in the inflammatory response [41].

TNFa plays critical roles in cancer pathophysiology building an elaborate association between inflammation and cancer. It functions as a key regulator of the tumor microenvironment, promoting tumor progression, even in the absence of invading inflammatory cells [42]. It facilitates cancer development acting directly on neoplastic cells or indirectly through endothelial and other inflammatory cells [43]. However, the mechanisms by which TNFa enables these events are not fully described. A recent publication from Davis et al. [44] explains the dichotomy of TNFa effect on the control of apoptosis in prostate cancer cells. These authors propose a physiologic role for TNFa in prostate regression after androgen withdrawal. This factor is required for castration-induced prostate regression, but membrane-bound TNFa protein and stromal cell specific TNFa mRNA levels increase in rat prostate after castration, which is coincident with a paracrine effect of TNFa in prostate cancer regression. However, when wild-type non-castrated mice were treated with TNFa no regression of the gland was observed [44]. All these evidences showed that this cytokine acts in the context of supplemental castration-induced signals.

Summarizing, the chemokine scene displays a vast crosstalk of pathways involved in the day-to-day dialogue between the cancer cells and the inflammatory microenvironment. The challenge relies in identifying the homeostatic target/targets that govern this setting in order to successfully re-direct the therapeutic efforts against prostate cancer.

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