**3. Cytokines and chemokine intracellular signaling pathways**

Physiologically, inflammation is a mechanism of tissue reaction for elimination, neutralization, and destruction of the cause of aggression, as injurious stimuli such as microbial pathogens, irritants, or toxic cellular components [64]. Cells of the immune system including monocytes, macrophages, neutrophils, basophils, DCs, mast cells, T cells, and B cells play a role in this process [64]. These events are in turn controlled by a host of extracellular molecular regulators, including members of the cytokine and chemokine families that mediate both immune cell recruitment and complex intracellular signaling control mechanisms; as a result, cells assemble and disassemble a complex array of signaling pathways as they move from inactive to dedicated roles within the inflammatory response site. Disruption of these pathways triggers inflammatory disorders that could contribute for the development of some diseases as kidney cancer and other types of cancers [65].

Key pro-inflammatory cytokines in kidney cancer include interleukin-1 beta (IL-1β), IL-6, and TNFα, all of which signal via the type I cytokine receptors that are structurally divergent from other cytokine receptor types [66, 67]. IL-1 signaling starts through its binding to its receptor composed of two subunits, interleukin 1 receptor type I (IL-1RI) and interleukin 1 receptor accessory protein (IL1RAP) [68, 69]. Signaling proceeds with TIR adaptor and MyD88 by recruitment of IL-1R-associated kinases (IRAKs), which promote TNFR-associated factor 6 (TRAF6) polyubiquitination via lysine 63 linkages. Subsequently, TRAF6 interacts with the TAK1/TAB1/TAB2 complex that allows NFκB nuclear translocation (p65/p50) resulting in proinflammatory gene expression [69]. Also, TAK1/TAB1/TAB2 complex triggers the activation of the mitogen-activated protein kinases (MAPKs), c-JunN-terminal kinase (JNK), and p38, which induce the expression of pro-inflammatory genes. Similarly, TNFα binding to TNFR1 results in NFκB nuclear translocation, MAPKs, JNK, and p38, but signaling is coordinated by complex I (TRADD/TRAF2/RIP) [67, 70]. Importantly, TNFα signaling following receptor internalization is thought to be pro-apoptotic, via the formation of complex II (TRADD/FADD/ Pro-Caspase-8) [67]. On the other hand, IL-6 binds to the FIII domains of the IL-6R chains, unleashing its signal via the gp130 proteins [71, 72]. Consequently, Janus kinases (JAKs) are recruited to the receptor, phosphorylating it and themselves, triggering STAT3 activation and transcription of pro-inflammatory genes and intracellular adhesion molecules [64].

lymphocytes [53, 54]. IL1-beta is one of the major cytokines involved in the pro-inflammatory response, which is synthesized by several immune cells, such as monocytes, macrophages, DCs, B lymphocytes, NK cells, among others. It has similar activities as described for TNFα, favoring tumor invasion and the angiogenic process, as well as favoring vascular permeability and facilitating the recruitment of immune system cells to the tumor microenvironment [55]. However, TNFα is the main mediator of the acute inflammatory response, being secreted primarily by macrophages and T cells. TNFα causes vascular endothelial cells to increase the expression of leukocyte integrins inducing chemotaxis. In addition, TNFα also acts on phagocytic cells, which characterizes an autocrine effect, since macrophages, apart from secreting TNFα, may respond to the stimulus itself, releasing IL1-beta [56, 57]. IL12 is secreted primarily by macrophages, DCs, monocytes, and neutrophils. It has action in the activation of cytotoxic NK cells and TCD8 lymphocytes, but its main function in the antitumor activity is involved in the activation and proliferation of T lymphocytes and NK cells, which induces the production of IFN-gamma. Moreover, IL12 and INF-gamma together are able to differentiate T-helper cells into Th1 cells [58, 59]. IL6 is synthesized by mononuclear phagocytes, such as macrophages and also by some activated T cells and by other cell types that are not part of the immune system in response to microorganisms or IL1-beta and TNFα stimuli [60]. IL10 is a cytokine known to be anti-inflammatory, synthesized in the form of monomers of 18–20 kDa, being functional in the form of homodimers. This cytokine can be produced by Th2 lymphocytes, monocytes, and epithelial cells. Its main action is to suppress the synthesis of several inflammatory cytokines such as IL1-beta, TNFα, IL-6, IL-8, and IL-12, as well as hematopoietic growth factors (GM-CSF, G-CFS) and macrophage colony-stimulating factor (M-CFS). In addition, IL-10 can inhibit the synthesis of nitric oxide, gelatinase, and collagenase, avoiding tissue injury [61]. Although its role in the tumor context remains unclear, IL-17 is a pro-inflammatory cytokine secreted by Th17 lymphocytes, which regulates NFkB and MAPK activities. It is constantly involved in the acute phase of inflammatory diseases, such as autoimmune diseases, and it is associated with poor prognosis in patients with RCC [62, 63]. Taken all these studies together, the dual role of natural and adaptive immunity in RCC is

**3. Cytokines and chemokine intracellular signaling pathways**

diseases as kidney cancer and other types of cancers [65].

Physiologically, inflammation is a mechanism of tissue reaction for elimination, neutralization, and destruction of the cause of aggression, as injurious stimuli such as microbial pathogens, irritants, or toxic cellular components [64]. Cells of the immune system including monocytes, macrophages, neutrophils, basophils, DCs, mast cells, T cells, and B cells play a role in this process [64]. These events are in turn controlled by a host of extracellular molecular regulators, including members of the cytokine and chemokine families that mediate both immune cell recruitment and complex intracellular signaling control mechanisms; as a result, cells assemble and disassemble a complex array of signaling pathways as they move from inactive to dedicated roles within the inflammatory response site. Disruption of these pathways triggers inflammatory disorders that could contribute for the development of some

evident (**Figure 1**).

24 Evolving Trends in Kidney Cancer

Currently, it is not fully understood how the signaling triggered by these cytokines contributes to tumor progression, but high serum levels of these pro-inflammatory cytokines are associated with advanced disease [73]. Some evidence has arisen, as follows. It is well known that angiogenesis is stimulated by inflammatory mediators in the tumor microenvironment, such as those expressed by TAMs [74]. Interestingly, TAMs isolated from RCC tumors express high levels of IL-1β, TNFa, and IL-6 [75]. In addition, mouse models have demonstrated that the inhibition of IL-1β signaling reduced tumor blood vessel formation [76] and IL-1β mediates metalloproteinase-dependent RCC tumor cell invasion through the activation of cytosine-cytosine-adenosineadenosine-thymidine (CCAAT) enhancer binding protein b [67]. Regarding TNFα signaling, many studies associated it to chemokine overexpression in tumor and nontumor cells [77, 78]. Moreover, TNFα plays an important role in the progression of RCC by inducing epithelial to mesenchymal transition and CD44 expression, which may be involved in the resistance to the sunitinib treatment [66]. There is no direct correlation between IL-6R and RCC development; however, RCC cells express high levels of IL-6, and its signaling activity seems necessary for carcinogenesis, tumor progression, and tumor evasion of the immune system. STAT3 activation by IL-6 promotes tumorigenesis by preventing apoptosis while enhancing proliferation, angiogenesis, invasiveness, and immune evasion [79]. For example, activated STAT3 induces HIF-1α-mediated VEGF expression in human RCC cell [79].

Besides these pro-inflammatory cytokines, other mediators act as crucial players in RCC. Chemotactic cytokines or chemokines are responsible for the recruitment of cells from both the innate and adaptive immune systems to the site of injury or infection [64]. Chemokines induce integrin expression, such as the β2-integrin lymphocyte function-associated antigen (LFA-1), in target leukocytes, thus acting in the arrest of these cells and favoring diapedesis through the endothelium [71]. Despite this, primary chemotaxis action, chemokines, and their receptors are physiological relevant in many biological process, such as the initiation of adaptive immune responses, immune surveillance and the migration, proliferation, and survival signals in multiple cell types [64]. Chemokine signals are transduced through binding to members of the seven-transmembrane, G-protein-coupled receptor (GPCR) superfamily [80]. GPCRs exist as a heterotrimer containing three subunits: α, β, and γ. In its inactive form, the G protein is complexed in α, β, and γ, with guanosine diphosphate (GDP) fixed to the α subunit. Once stimulated by a receptor activated by its ligand, the α subunit exchanges its GDP for Guanosine-5'-triphosphate (GTP) [81]. This causes the dissociation of α which separate β and γ subunits by interacting with an effector protein or ion channel in order to stimulate or inhibit secondary intracellular messengers [81]. CXCR4 is well known for its role in the homing of progenitor cells into the bone marrow and, recently, associated with poor RCC prognosis, and it is mainly coupled to the Gαi subunit, which, after dissociation of the Gαβγ complex upon CXCR4 stimulation (**Figure 2**), is traditionally been regarded as the major signaling subunit, inhibits adenylyl cyclase activity, and triggers MAPK and phosphatidylinositol-3-kinase (PI3K) pathway activation [82]. The Gβγ subunits, in turn, lead to the activation of phospholipase C (PLC), causing the hydrolysis of the phospholipid membrane phosphatidylinositol 4,5-bisphosphate (PIP2) in inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) [13, 82]. IP3 can bind to channels of the endoplasmic reticulum, inducing the mobilization of Ca2+ ions [83, 84]. This could also be considered a downstream effect of Gαi activity, since the inhibition of Gαi activity by its potent inhibitor pertussis toxin has been reported to lead to a decreased Ca2+ mobilization from intracellular stores [85]. CXCR4 can also act by interaction with other Gα subunits, that is, Gαq or Gα12, each of which has been associated with different intracellular signaling cascades [86]. Indeed, chemokine receptors also activate signaling pathways independent of G proteins, including p38MAPK and JAK/ Stat to regulate cellular processes such as migration and gene transcription [87, 88].

Regarding the signals triggered by pro-inflammatory mediators in RCC, CXCL12 chemokine and its receptors CXCR4 and CXCR7 have gained prominence, since this pathway, which is associated with chronic inflammation, is upregulated in RCC [65, 89, 90]. In RCC cells and in other tumor cells, these chemokines activate the PI3K/Akt pathway; consequently, many downstream elements of the Akt pathway are regulated, leading to tumor cell survival [13, 91, 92]. Frequently, NFκB nuclear translocation is observed following transcription of various apoptosis inhibitors and cell-cycle-promoting genes [93], but can be activated through other pathways, such as PKC [94]. Other downstream targets of Akt include procaspase-9 and the pro-apoptotic Bcl-2 family member, BAD (Bcl-2/Bcl-XL-antagonist, causing cell death), both of which are inhibited upon phosphorylation. Other consequence of Akt activation is the inhibition of members of the FKHR (forkhead in rhabdomyosarcoma) family of transcription factors, which induce the transcription of numerous apoptotic genes [95, 96]. Besides, Akt signaling could induce p53 degradation and inhibition of GSK-3β (glycogen synthase kinase-3β), leading to stabilization of β-catenin, resulting in the downstream inhibition of negative regulators of cell cycle and the activation of cell-cycle-promoting genes [97]. mTOR (mammalian target of rapamycin) activation is also induced by Akt, which leads to p70S6K (p70 S6 kinase) activation and thus enhances protein translation of numerous cell-growth regulators [13]. Furthermore, intracellular events triggered by the activation of chemokines receptors lead to ERK1/2 signaling, following the inhibition of procaspase-9 and BAD [98, 99], the induction of transcription factors involved in cell-cycle regulation, and differentiation, thereby promoting cell proliferation [100]. Other MAPKs, including JNK, have also been implicated in chemokine-induced proliferation signaling [101]. Also, HIF-1α may be

induced by chemokines signaling that contributes to VEGF expression, which is known to be the inducer of CXCR4 expression [101]. It is important to mention that not all of these pathways have been studied in detail in RCC cells, but there is strong evidence of their involvement in the development of this disease. For example, Rac1 was not previously reported to be involved in RCC development, but some studies have shown its role in controlling tumor cell growth and chronic kidney disease [102]. In summary, CXCL12, CXCR4, and CXCR7 release result in the activation of transcription factors involved in antiapoptotic mechanisms, cell-cycle regulation, and growth factor production, favoring tumor growth and metastasis.

**Figure 2.** Signaling induced by CXCR4 via G protein. CXCL12 binding causes the dissociation of α which separate β and γ subunits; as a result, βγ PLC is activated leading to calcium mobilization. PI3K/Akt activation by Gα triggers a transduction signal that contributes with cell survival, proliferation, and migration, which is associated with RCC

Role of Immune System in Kidney Cancer http://dx.doi.org/10.5772/intechopen.77379 27

Also, there are some particularities regarding CXCR7 role in RCC, which are independent of GPCRs. CXCR7 plays a role as a decoy for CXCL12, promoting some CXCL12α accumulation and triggering a differential signaling by CXCR4 [103, 104]. Besides, CXCR7 interacts and

signals by β-arrestin in a ligand-dependent manner [83, 104].

disease. In contrast, the antagonist of CXCR4 blocks CXCR4 intracellular pathway.

survival signals in multiple cell types [64]. Chemokine signals are transduced through binding to members of the seven-transmembrane, G-protein-coupled receptor (GPCR) superfamily [80]. GPCRs exist as a heterotrimer containing three subunits: α, β, and γ. In its inactive form, the G protein is complexed in α, β, and γ, with guanosine diphosphate (GDP) fixed to the α subunit. Once stimulated by a receptor activated by its ligand, the α subunit exchanges its GDP for Guanosine-5'-triphosphate (GTP) [81]. This causes the dissociation of α which separate β and γ subunits by interacting with an effector protein or ion channel in order to stimulate or inhibit secondary intracellular messengers [81]. CXCR4 is well known for its role in the homing of progenitor cells into the bone marrow and, recently, associated with poor RCC prognosis, and it is mainly coupled to the Gαi subunit, which, after dissociation of the Gαβγ complex upon CXCR4 stimulation (**Figure 2**), is traditionally been regarded as the major signaling subunit, inhibits adenylyl cyclase activity, and triggers MAPK and phosphatidylinositol-3-kinase (PI3K) pathway activation [82]. The Gβγ subunits, in turn, lead to the activation of phospholipase C (PLC), causing the hydrolysis of the phospholipid membrane phosphatidylinositol 4,5-bisphosphate (PIP2) in inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) [13, 82]. IP3 can bind to channels of the endoplasmic reticulum, inducing the mobilization of Ca2+ ions [83, 84]. This could also be considered a downstream effect of Gαi activity, since the inhibition of Gαi activity by its potent inhibitor pertussis toxin has been reported to lead to a decreased Ca2+ mobilization from intracellular stores [85]. CXCR4 can also act by interaction with other Gα subunits, that is, Gαq or Gα12, each of which has been associated with different intracellular signaling cascades [86]. Indeed, chemokine receptors also activate signaling pathways independent of G proteins, including p38MAPK and JAK/

26 Evolving Trends in Kidney Cancer

Stat to regulate cellular processes such as migration and gene transcription [87, 88].

Regarding the signals triggered by pro-inflammatory mediators in RCC, CXCL12 chemokine and its receptors CXCR4 and CXCR7 have gained prominence, since this pathway, which is associated with chronic inflammation, is upregulated in RCC [65, 89, 90]. In RCC cells and in other tumor cells, these chemokines activate the PI3K/Akt pathway; consequently, many downstream elements of the Akt pathway are regulated, leading to tumor cell survival [13, 91, 92]. Frequently, NFκB nuclear translocation is observed following transcription of various apoptosis inhibitors and cell-cycle-promoting genes [93], but can be activated through other pathways, such as PKC [94]. Other downstream targets of Akt include procaspase-9 and the pro-apoptotic Bcl-2 family member, BAD (Bcl-2/Bcl-XL-antagonist, causing cell death), both of which are inhibited upon phosphorylation. Other consequence of Akt activation is the inhibition of members of the FKHR (forkhead in rhabdomyosarcoma) family of transcription factors, which induce the transcription of numerous apoptotic genes [95, 96]. Besides, Akt signaling could induce p53 degradation and inhibition of GSK-3β (glycogen synthase kinase-3β), leading to stabilization of β-catenin, resulting in the downstream inhibition of negative regulators of cell cycle and the activation of cell-cycle-promoting genes [97]. mTOR (mammalian target of rapamycin) activation is also induced by Akt, which leads to p70S6K (p70 S6 kinase) activation and thus enhances protein translation of numerous cell-growth regulators [13]. Furthermore, intracellular events triggered by the activation of chemokines receptors lead to ERK1/2 signaling, following the inhibition of procaspase-9 and BAD [98, 99], the induction of transcription factors involved in cell-cycle regulation, and differentiation, thereby promoting cell proliferation [100]. Other MAPKs, including JNK, have also been implicated in chemokine-induced proliferation signaling [101]. Also, HIF-1α may be

**Figure 2.** Signaling induced by CXCR4 via G protein. CXCL12 binding causes the dissociation of α which separate β and γ subunits; as a result, βγ PLC is activated leading to calcium mobilization. PI3K/Akt activation by Gα triggers a transduction signal that contributes with cell survival, proliferation, and migration, which is associated with RCC disease. In contrast, the antagonist of CXCR4 blocks CXCR4 intracellular pathway.

induced by chemokines signaling that contributes to VEGF expression, which is known to be the inducer of CXCR4 expression [101]. It is important to mention that not all of these pathways have been studied in detail in RCC cells, but there is strong evidence of their involvement in the development of this disease. For example, Rac1 was not previously reported to be involved in RCC development, but some studies have shown its role in controlling tumor cell growth and chronic kidney disease [102]. In summary, CXCL12, CXCR4, and CXCR7 release result in the activation of transcription factors involved in antiapoptotic mechanisms, cell-cycle regulation, and growth factor production, favoring tumor growth and metastasis.

Also, there are some particularities regarding CXCR7 role in RCC, which are independent of GPCRs. CXCR7 plays a role as a decoy for CXCL12, promoting some CXCL12α accumulation and triggering a differential signaling by CXCR4 [103, 104]. Besides, CXCR7 interacts and signals by β-arrestin in a ligand-dependent manner [83, 104].

Obviously, here we emphasize intracellular events in tumor cells. However, while some processes take place within the tumor cells, others would occur in the stroma: it is the synergism between these responses that contributes to the progression of the disease. RCC and other tumors interact with the surrounding tumor stroma through a variety of cytokines, chemokines, and growth factors [84]. The tumor chemotactic environment recruits inflammatory cells including neutrophils, macrophages, and lymphocytes. Although initially these cells may have a protective antitumoral role, as displayed by neutrophils, which have a higher cytotoxic activity against poorly metastatic cells, secondarily neutrophils could contribute to cancer progression. Leukocytes can produce cytokines, growth factors, and MMPs that enhance growth, proliferation, and angiogenesis, as exemplified by the TAMs, which release growth and angiogenic factors (e.g., VEGF) and basic fibroblast growth factor [105]. Thus, cellular communication by paracrine and autocrine chemokine/cytokine signaling contributes for the survival and growth of metastatic cells. In other words, stroma cells may support tumors at the same time as that tumor cells in turn modulate the microenvironment within which they reside.

TNF, has been considered for RCC treatment [115]. However, treatments with anti-TNFα monoclonal antibody showed varying results in independent studies, probably due to reactions given by this cytokine to different conditions, environmental and genetic factors and/ or other unknown or unexplained factors [116]. Besides anti-TNFα strategies, targeting IL-1β

Role of Immune System in Kidney Cancer http://dx.doi.org/10.5772/intechopen.77379 29

Promising candidates for RCC treatment have also been designed to specifically target chemokines and their receptors. One of the most widely studied compounds is AMD3100 which is thought to specifically block CXCR4 signaling [118] and that acts directly in RCC tumor [91] cells as well as local antitumor immune response, by impairing Tregs function [119], know to suppress a whole range of immune cells including B cells, NK cells, NKT cells, CD4+ or CD8+ T cells, monocytes, and DCs [119]. Also, many other chemokine antagonists have also shown potential for clinical application in cancer treatment and could be useful for RCC treatment in the future. For example, anti-CXCR7-12G8 and CCX77, CXCR7 inhibitors or CTCE-9908, which is a peptide analog of CXCL12 and an active inhibitor of the ligand, has shown promising results as well as tolerated drug that stabilized disease in early clinical trials for late-stage cancer patients [12, 120]. Recently, antibodies that inhibit T-cell coinhibitory receptors have emerged as therapeutic promises not only in the treatment of RCC but also in other tumors by inhibiting T-cell regulatory activity and increasing the antitumor immune response [14, 121]. Nivolumab and pembrolizumab (anti-PD-1) [14, 122], avelumab, and durvalumab (anti-PD-L1) are in latestage clinical development for a number of indications [14, 123], besides the first in its class, ipilimumab (anti-CTLA-4), already approved for use in a number of indications [14, 124].

Finally, the close relationship between cancer and immune system has suggested that current drug therapies used to treat inflammatory diseases or particular types of cancers could function as inhibitors of chemokine signaling and could therefore be redirected toward the treatment of other cancers [12, 116]. This hypothesis needs to be tested by further preclinical and clinical investigation, which elucidates how these drugs would act at molecular and systemic levels.

The role of immune system in kidney cancer is becoming more clear, whereas new findings that arise from clinical trials and identification of additional predictive biomarkers increase our understanding of the tumor microenvironment. Looking to the future based on the knowledge we have today, the perspective is a better understanding of immune system in

Regarding RCC therapy, a promising option is the combination therapy based on targeted agents (inhibition of mTOR or VEGF pathways associated with immunotherapies) or immunotherapy + immunotherapy, which would overcome tumor resistance, as well as to restore functional immune system cancer surveillance and response. Currently, there are many clinical trials investigating combination therapy: nivolumab (anti-PD-1) + ipilimumab (anti-CTLA-4) [125], pembrolizumab (anti-PD-1) + ipilimumab (anti-CTLA-4) [126], pidilizumab (anti-PD-1) + vaccine (DC/RCC fusion cell vaccine) [127], atezolizumab (anti-PD-L1) + bevacizumab (anti-VEGF) [127], nivolumab (anti-PD-1) + bevacizumab (anti-VEGF) [128], pembroli-

zumab (anti-PD-1) + pazopanib (TKI) [129], nivolumab (anti-PD-1) + sunitinib [130].

**5. Role of immune system in kidney cancer and the future**

tumor stroma as well as in various steps in cancer growth and metastasis.

has also been reported as possible therapy for RCC [117].
