Dual roles of immune cells and their factors in cancer development and progression

Traditional wisdom holds that intact immune responses, such as immune surveillance or immunoediting, are required for preventing and inhibiting tumor development; but recent evidence has also indicated that unresolved immune responses, such as chronic inflammation, can promote the growth and progression of cancer. Within the immune system, cytotoxic CD8(+) and CD4(+) Th1 T cells, along with their characteristically produced cytokine IFN-γ, function as the major anti-tumor immune effector cells, whereas tumor associated macrophages (TAM) or myeloid-derived suppressive cells (MDSC) and their derived cytokines IL-6, TNF, IL-1β and IL-23 are generally recognized as dominant tumor-promoting forces. However, the roles played by Th17 cells, CD4(+) CD25(+) Foxp3(+) regulatory T lymphocytes and immunoregulatory cytokines such as TGF-β in tumor development and survival remain elusive. These immune cells and the cellular factors produced from them, including both immunosuppressive and inflammatory cytokines, play dual roles in promoting or discouraging cancer development, and their ultimate role in cancer progression may rely heavily on the tumor microenvironment and the events leading to initial propagation of carcinogenesis.     

The resolution of inflammation and cancer

Inflammation has long been thought to contribute to the development of cancer; however there is also clear evidence that the immune system can recognize and eliminate cancer cells. Current research suggests that cancer-associated inflammation has a dual role in tumor progression; inflammatory mediators promote the malignant activity of cancer cells by acting as growth factors and also stimulate angiogenesis, however, cancer-associated inflammation is also linked with immune-suppression that allows cancer cells to evade detection by the immune system. In this review we will discuss the dual role of inflammation in cancer and how endogenous anti-inflammatory mechanisms may equally be important in carcinogenesis.     

Tumor-Produced Versican V1 Enhances hCAP18/LL-37 Expression in Macrophages through Activation of TLR2 and Vitamin D3 Signaling to Promote Ovarian Cancer Progression In Vitro

Tumor-associated macrophages have been shown to promote tumor growth. They may have an obligatory function in angiogenesis, invasion, and metastasis through release of inflammatory mediators. Their presence in ovarian cancer has been correlated with poor prognosis in these patients. The human cationic antimicrobial protein-18 (hCAP18)/LL-37 was originally identified as an effector molecule of the innate immune system. It is released by innate immune cells, such as macrophages, to combat microorganisms. Previous studies have characterized the hCAP18/LL-37 as a growth factor that has been shown to promote ovarian tumor progression. However, the role hCAP18/LL-37 has in macrophage-promoted ovarian tumor development and how its expression is controlled in this context remains poorly understood. Here, we demonstrate in co-culture experiments of macrophages and ovarian cancer cells a significant increase in the in vitro proliferation and invasiveness of the tumor cells is observed. These enhanced growth and invasion properties correlated with hCAP18/LL-37 induction. HCAP18/LL-37 expression was diminished by addition of two neutralizing antibodies, TLR2 or TLR6, as well as Cyp27B1 or VDR inhibitors. Furthermore, either the TLR2 or TLR6 antibody reduced vitamin D3 signaling and tumor cell progression in vitro. Addition of Cyp27B1 or VDR inhibitors abrogated TLR2/6 activation-induced expression of hCAP18/LL-37 in macrophages. Knockdown of tumor-produced versican V1 by RNAi in these tumor cells led to a decreased induction of hCAP18/LL-37 in macrophages. Versican V1 knockdown also inhibited TLR2 and vitamin D3 signaling, as well as growth and invasiveness of these tumor cells in the in vitro co-culture. In summary, we have found that versican V1 enhances hCAP18/LL-37 expression in macrophages through activation of TLR2 and subsequent vitamin D-dependent mechanisms which promote ovarian tumor progression in vitro.     

IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms

Interleukin(IL)-18 is a pleiotrophic cytokine with functions in immune modulation, angiogenesis and bone metabolism. In this study, the potential of IL-18 as an immunotherapy for prostate cancer (PCa) was examined using the murine model of prostate carcinoma, RM1 and a bone metastatic variant RM1(BM)/B4H7-luc. RM1 and RM1(BM)/B4H7-luc cells were stably transfected to express bioactive IL-18. These cells were implanted into syngeneic immunocompetent mice, with or without an IL-18-neutralising antibody (αIL-18, SK113AE4). IL-18 significantly inhibited the growth of both subcutaneous and orthotopic RM1 tumors and the IL-18 neutralizing antibody abrogated the tumor growth-inhibition. In vivo neutralization of interferon-gamma (IFN-γ) completely eliminated the anti-tumor effects of IL-18 confirming an essential role of IFN-γ as a down-stream mediator of the anti-tumor activity of IL-18. Tumors from mice in which IL-18 and/or IFN-γ was neutralized contained significantly fewer CD4(+) and CD8(+) T cells than those with functional IL-18. The essential role of adaptive immunity was demonstrated as tumors grew more rapidly in RAG1(-/-) mice or in mice depleted of CD4(+) and/or CD8(+) cells than in normal mice. The tumors in RAG1(-/-) mice were also significantly smaller when IL-18 was present, indicating that innate immune mechanisms are involved. IL-18 also induced an increase in tumor infiltration of macrophages and neutrophils but not NK cells. In other experiments, direct injection of recombinant IL-18 into established tumors also inhibited tumor growth, which was associated with an increase in intratumoral macrophages, but not T cells. These results suggest that local IL-18 in the tumor environment can significantly potentiate anti-tumor immunity in the prostate and clearly demonstrate that this effect is mediated by innate and adaptive immune mechanisms.     

IL-18 enhances thrombospondin-1 production in human gastric cancer via JNK pathway

IL-18 is a pleiotropic cytokine that is produced by many cancer cells. A recent report suggested that IL-18 plays a key role in regulating the immune escape of melanoma and gastric cancer cells. Thrombospondin (TSP-1) is known to inhibit angiogenesis in several cancers but some studies have reported that it stimulates angiogenesis in some cancers such as gastric cancer. IL-18 and TSP-1 are related to tumor proliferation and metastasis. This study investigated the relationship between IL-18 and TSP-1 in gastric cancer. RT-PCR and ELISA showed that after the cells had been treated with IL-18, the level of TSP-1 mRNA expression and TSP-1 protein production by IL-18 increased in both a dose- and time-dependent manner. The cells were next treated with specific inhibitors in order to determine the signal pathway involved in IL-18-enhanced TSP-1 production. IL-18-enhanced TSP-1 expression was blocked by SP600125, a c-Jun N-terminal kinase (JNK) specific inhibitor. In addition, Western blot showed that IL-18 enhanced the expression of phosphorylated JNK. Overall, these results suggest that IL-18 plays a key role in TSP-1 expression involving JNK.     

IL-22-mediated tumor growth reduction correlates with inhibition of ERK1/2 and AKT phosphorylation and induction of cell cycle arrest in the G2-M phase

IL-22 is a recently discovered cytokine of the IL-10 family that binds to a class II cytokine receptor composed of IL-22R1 and IL-10R2(c) and influences a variety of immune reactions. As IL-22 has also been shown to modulate cell cycle and proliferation mediators such as ERK1/2 and JNK, we studied the role of IL-22 in proliferation, apoptosis, and cell cycle regulation in EMT6 murine breast cancer cells in vitro and in vivo. In this study, we report that murine breast cancer cells express functional IL-22R as indicated by RT-PCR studies, immunoblotting, and STAT3 activation assays. Importantly, IL-22 exposure of EMT6 cells resulted in decreased levels of phosphorylated ERK1/2 and AKT protein kinases, indicating an inhibitory effect of IL-22 on signaling pathways promoting cell proliferation. Furthermore, IL-22 induced a cell cycle arrest of EMT6 cells in the G(2)-M phase. IL-22 reduced EMT6 cell numbers and the proliferation rate by approximately 50% as measured by [(3)H]thymidine incorporation. IL-22 treatment of EMT6 tumor-bearing mice lead to a decreased tumor size and a reduced tumor cell proliferation in vivo, as determined by 3'-deoxy-3'-fluorothymidine-positron emission tomography scans. Interestingly, IL-22 did not induce apoptosis, as determined in annexin V binding assay and caspase-3 activation assay and had no effect on angiogenesis in vivo. In conclusion, our results indicate that IL-22 reduced tumor growth by inhibiting signaling pathways such as ERK1/2 and AKT phosphorylation that promote tumor cell proliferation in EMT6 cells. Therefore, IL-22 may play a role in the control of tumor growth and tumor progression.     

Melanoma cell extravasation under flow conditions is modulated by leukocytes and endogenously produced interleukin 8

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for the migration of tumor cells out of the vascular system to establish metastases. Innate immune system processes can potentially promote tumor progression through inflammation dependant mechanisms. White blood cells, neutrophils (PMN) in particular, are being studied to better understand how the host immune system affects cancer cell adhesion and subsequent migration and metastasis. Melanoma cell interaction with the EC is distinct from PMN-EC adhesion in the circulation. We found PMN increased melanoma cell extravasation, which involved initial PMN tethering on the EC, subsequent PMN capture of melanoma cells and maintaining close proximity to the EC. LFA-1 (CD11a/CD18 integrin) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, while Mac-1 (CD11b/CD18 integrin) affected prolonged PMN-melanoma aggregation. Blocking E-selectin or ICAM-1 (intercellular adhesion molecule) on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. Results indicated a novel finding that PMN-facilitated melanoma cell arrest on the EC could be modulated by endogenously produced interleukin-8 (IL-8). Functional blocking of the IL-8 receptors (CXCR1 and CXCR2) on PMN, or neutralizing soluble IL-8 in cell suspensions, significantly decreased the level of Mac-1 up-regulation on PMN while communicating with melanoma cells and reduced melanoma extravasation. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines, and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the endothelium in the microcirculation, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.     

The immunosuppressive and pro-tumor functions of CCL18 at the tumor microenvironment

Chemokines are essential mediators of immune cell trafficking. In a tumor microenvironment context, chemotactic cytokines are known to regulate the migration, positioning and interaction of different cell subsets with both anti- and pro-tumor functions. Additionally, chemokines have critical roles regarding non-immune cells, highlighting their importance in tumor growth and progression.CCL18 is a primate-specific chemokine produced by macrophages and dendritic cells. This chemokine presents both constitutive and inducible expression. It is mainly associated with a tolerogenic response and involved in maintaining homeostasis of the immune system under physiological conditions. Recently, CCL18 has been noticed as an important component of the complex chemokine system involved in the biology of tumors. This chemokine induces T regulatory cell differentiation and recruitment to the tumor milieu, with subsequent induction of a pro-tumor (M2-like) macrophage phenotype. CCL18 is also directly involved in cancer cell-invasion, migration, epithelial-to-mesenchymal transition and angiogenesis stimulation, pinpointing an important role in the promotion of cancer progression. Interestingly, this chemokine is highly expressed in tumor tissues, particularly at the invasive front of more advanced stages (e.g. colorectal cancer), and high levels are detected in the serum of patients, correlating with poor prognosis.Despite the promising role of CCL18 as a biomarker and/or therapeutic target to hamper disease progression, its pleiotropic functions in a context of cancer are still poorly explored. The scarce knowledge concerning the receptors for this chemokine, together with the insufficient insight on the downstream signaling pathways, have impaired the selection of this molecule as an immediate target for translational research.In this Review, we will discuss recent findings concerning the role of CCL18 in cancer, integrate recently disclosed molecular mechanisms and compile data from current clinical studies.     

突变型人IL-18的肿瘤靶向性改造及真核表达

为了构建肿瘤靶向性的人突变型IL-18新基因并进行真核表达,以重组PCR技术构建EGF-IL-18融合基因,利用 Bac-to-Bac杆状病毒表达系统和Sf 9昆虫细胞株（来自秋天草地夜蛾）表达融合基因,纯化表达产物,并以IFN-γ诱导实验和EGFR竞争结合实验初步评价融合蛋白的生物活性。测序证明构建的融合基因为原设计EGF-IL-18融合基因。SDS-PAGE和Western blot证明EGF-IL-18融合基因在昆虫细胞中获得表达,表达的融合蛋白的Mr约为20 000,与理论值相符,纯化后融合蛋白具有特异的IL-18单抗结合活性。IFN-γ诱导实验和EGFR竞争结合实验显示,该融合蛋白具有肿瘤导向性和抗肿瘤活性。表明对突变型IL-18成功地进行了肿瘤导向性改造并使其在真核细胞获得表达。     

MDSCs might be “Achilles heel” for eradicating CSCs

During tumor initiation and progression, the complicated role of immune cells in the tumor immune microenvironment remains a concern. Myeloid-derived suppressor cells (MDSCs) are a group of immune cells that originate from the bone marrow and have immunosuppressive potency in various diseases, including cancer. In recent years, the key role of cancer stemness has received increasing attention in cancer development and therapy. Several studies have demonstrated the important regulatory relationship between MDSCs and cancer stem cells (CSCs). However, there is still no clear understanding regarding the complex interacting regulation of tumor malignancy, and current research progress is limited. In this review, we summarize the complicated role of MDSCs in the modulation of cancer stemness, evaluate the mechanism of the relationship between CSCs and MDSCs, and discuss potential strategies for eradicating CSCs with respect to MDSCs.     

Mathematical Modeling of Interleukin-35 Promoting Tumor Growth and Angiogenesis

Interleukin-35 (IL-35), a cytokine from the Interleukin-12 cytokine family, has been considered as an anti-inflammatory cytokine which promotes tumor progression and tumor immune evasion. It has also been demonstrated that IL-35 is secreted by regulatory T cells. Recent mouse experiments have shown that IL-35 produced by cancer cells promotes tumor growth via enhancing myeloid cell accumulation and angiogenesis, and reducing the infiltration of activated CD8 T cells into tumor microenvironment. In the present paper we develop a mathematical model based on these experimental results. We include in the model an anti-IL-35 drug as treatment. The extended model (with drug) is used to design protocols of anti-IL-35 injections for treatment of cancer. We find that with a fixed total amount of drug, continuous injection has better efficacy than intermittent injections in reducing the tumor load while the treatment is ongoing. We also find that the percentage of tumor reduction under anti-IL-35 treatment improves when the production of IL-35 by cancer is increased.     

Induction of an effective anti-tumor immune response and tumor regression by combined administration of IL-18 and Apoptin

Immunization strategies using plasmid DNA can potentially improve humoral and cellular immune responses that protect against cancer and infectious diseases. The chicken anemia virus-derived Apoptin protein exhibits remarkable specificity in its ability to induce apoptosis in tumor cells, but not in normal diploid cells. Interleukin-18 (IL-18) is a Th1-type cytokine that has demonstrated potential as a biological adjuvant in murine tumor models. In this study, we analyzed the anti-tumor potential and mechanism of action of simultaneous Apoptin and IL-18 gene transfer in C57BL/6 mice bearing Lewis lung carcinoma (LLC). Here we report that the growth of established tumors in mice immunized with pAPOPTIN in conjunction with pIL-18 was significantly inhibited compared with the growth of tumors in mice immunized with the empty vector (EV) or pAPOPTIN alone. Furthermore, the immunization of mice with pAPOPTIN in conjunction with pIL-18 elicited strong natural killer activity and LLC tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, T cells from lymph nodes of mice vaccinated with pIL-18 or pAPOPTIN + pIL-18 secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with Apoptin together with IL-18 may be a novel and powerful strategy for cancer immunotherapy.     

Adipose derived stem cells (ASCs) isolated from breast cancer tissue express IL-4, IL-10 and TGF-β1 and upregulate expression of regulatory molecules on T cells: do they protect breast cancer cells from the immune response?

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25^highFoxp3⁺ T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells.     

Immunoediting: evidence of the multifaceted role of the immune system in self-metastatic tumor growth

ABSTRACT: BACKGROUND: The role of the immune system in tumor progression has been subject to discussion for many decades. Numerous studies suggest that a low immune response might be beneficial, if not necessary, for tumor growth, and only a strong immune response can counter tumor growth and thus inhibit progression. METHODS: We implement a cellular automaton model previously described that captures the dynamical interactions between the cancer stem and non-stem cell populations of a tumor through a process of self-metastasis. By overlaying on this model the diffusion of immune reactants into the tumor from a peripheral source to target cells, we simulate the process of immune-system-induced cell kill on tumor progression. RESULTS: A low cytotoxic immune reaction continuously kills cancer cells and, although at a low rate, thereby causes the liberation of space-constrained cancer stem cells to drive self-metastatic progression and continued tumor growth. With increasing immune system strength, however, tumor growth peaks, and then eventually falls below the intrinsic tumor sizes observed without an immune response. With this increasing immune response the number and proportion of cancer stem cells monotonically increases, implicating an additional unexpected consequence, that of cancer stem cell selection, to the immune response. CONCLUSIONS: Cancer stem cells and immune cytotoxicity alone are sufficient to explain the three-step "immunoediting" concept - the modulation of tumor growth through inhibition, selection and promotion.     

Emerging role of tumor-associated macrophages as therapeutic targets in patients with metastatic renal cell carcinoma

Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruited into the renal cell carcinoma (RCC) microenvironment. In response to inflammatory stimuli, macrophages undergo M1 (classical) or M2 (alternative) activation. M1 cells produce high levels of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12, IL-23 and IL-6, while M2 cells produce anti-inflammatory cytokines, such as IL-10, thus contributing to RCC-related immune dysfunction. The presence of extensive TAM infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumor growth, and cellular migration and invasion. Moreover, TAMs are involved in epithelial–mesenchymal transition of RCC cancer cells and in the development of tumor resistance to targeted agents. Interestingly, macrophage autophagy seems to play an important role in RCC. Based on this scenario, TAMs represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAM recruitment, to deplete their number, to switch M2 TAMs into antitumor M1 phenotype and to inhibit TAM-associated molecules. In this review, we summarize current data on the essential role of TAMs in RCC angiogenesis, invasion, impaired anti-tumor immune response and development of drug resistance, thus describing the emerging TAM-centered therapies for RCC patients.     

Matricellular proteins and inflammatory cells: A task force to promote or defeat cancer?

In the last years it became clear that the tumor microenvironment plays a major role in neoplastic growth. Proteins secreted either by the malignant cells or by the tumor-associated stromal cells act as extracellular signal transductors, orchestrating tumor progression. Sentinel cells of the innate immune system patrol the different organs and have proven either to promote tumor growth or induce tumor suppression. In recent years, members of the matricellular family of extracellular proteins were shown to be involved in different aspects of the inflammatory response during tumor development, although in contradictory ways. In this review we discuss the evidence available up to date that relates matricellular proteins with the regulation of the inflammatory response and tumor progression.     

Role of IL-10 in immune suppression in cervical cancer

Cervical cancer is the second most common cancer in the women worldwide and the most frequent in developing countries, including India. Human papilloma virus (HPV) is the major etiological factor in cervical cancer patients. Host factors are also critical in regulating tumor growth and cytokines that modulate immunologic control may be of particular importance. In the present study, we investigated the correlation between the presence of HPV and type of cytokines expressed in cervical carcinomas and attempted to elucidate the possible reasons for the immune suppression. Cytokines investigated were type-1 cytokine IFN-gamma (shows immunostimulatory function and capable of limiting tumor growth) and type-2 cytokines IL-4, IL-10 and IL-6 (show immunosuppressive function and capable of stimulating tumor growth). Our data demonstrated the presence of HPV sub-types 16 and 18 in 86% and 13.8% of cervical tumor biopsies, respectively. The cervical tumor biopsies showed increased presence for mRNA for IL-10 and IL-1alpha, while none of the biopsies showed expression for IFN-gamma. A correlation was observed between the presence of HPV in cervical tumor biopsies and mRNA for IL-10. Increased percentages of CD4+CD25+ regulatory T cells (Tregs) were observed in circulation in cervical cancer patients, providing evidence for increased immune suppression. IL-10 may play a key role in maintenance of Tregs and explains the immunosuppressive state of cervical cancer patients.     

Muscle-to-tumor crosstalk: The effect of exercise-induced myokine on cancer progression

Physical exercise has gradually become a focus in cancer treatment due to its pronounced role in reducing cancer risk, enhancing therapeutic efficacy, and improving prognosis. In recent decades, skeletal muscles have been considered endocrine organs, exerting their biological functions via the endocrine, autocrine, and paracrine systems by secreting various types of myokines. The amount of myokines secreted varies depending on the intensity, type, and duration of exercise. Recent studies have shown that muscle-derived myokines are highly involved the effects of exercise on cancer. Multiple myokines, such as interleukin-6 (IL-6), oncostatin M (OSM), secreted protein acidic and rich in cysteine (SPARC), and irisin, directly mediate cancer progression by influencing the proliferation, apoptosis, stemness, drug resistance, metabolic reprogramming, and epithelial-mesenchymal transformation (EMT) of cancer cells. In addition, IL-6, interleukin-8 (IL-8), interleukin-15 (IL-15), brain-derived neurotrophic factor (BDNF), and irisin can improve obesity-induced inflammation by stimulating lipolysis of adipose tissues, promoting glucose uptake, and accelerating the browning of white fat. Furthermore, some myokines could regulate the tumor microenvironment, such as angiogenesis and the immune microenvironment. Cancer cachexia occurs in up to 80% of cancer patients and is responsible for 22%-30% of patient deaths. It is characterized by systemic inflammation and decreased muscle mass. Exercise-induced myokine production is important in regulating cancer cachexia. This review summarizes the roles and underlying mechanisms of myokines, such as IL-6, myostatin, IL-15, irisin, fibroblast growth factor 21 (FGF21) and musclin, in cancer cachexia. Through comprehensive analysis, we conclude that myokines are potential targets for inhibiting cancer progression and the associated cachexia.     

IL-8 expression in malignant melanoma: implications in growth and metastasis

This review article has described briefly studies supporting the concept that IL-8 expression and its regulation by inflammatory cytokines like IL-1 may play an important role in controlling the phenotypes associated with melanoma progression and metastasis. It is clear from the experiments presented here that IL-8 is an important autocrine multifunctional cytokine that modulates melanoma/cell proliferation, migration by induction of extracellular matrix degradation enzymes and induces neovascularization, all of which are critical for melanoma growth and metastasis. In addition, their expression in melanoma tumor specimens suggests an association between IL-8 expression and tumor aggressiveness. Further, inflammatory cytokines produced by either tumor cells or stromal cells may regulate IL-8 expression, which can control melanoma growth and enhance our current knowledge regarding melanoma progression and metastasis. Understanding these events and their significance will allow us to design novel therapeutic approaches for treatment of melanoma.