REVIEW ARTICLE

The Role of CXCR4 in Highly Malignant Human Gliomas Biology: Current Knowledge and Future Directions Filippo Gagliardi,1 Ashwin Narayanan,2 Michele Reni,3 Alberto Franzin,1 Elena Mazza,3 Nicola Boari,1 Michele Bailo,1 Paola Zordan,2 and Pietro Mortini1 Given the extensive histomorphological heterogeneity of high-grade gliomas, in terms of extent of invasiveness, angiogenesis, and necrosis and the poor prognosis for patients despite the advancements made in therapeutic management. The identification of genes associated with these phenotypes will permit a better definition of glioma heterogeneity, which may ultimately lead to better treatment strategies. CXCR4, a cell surface chemokine receptor, is implicated in the growth, invasion, angiogenesis and metastasis in a wide range of malignant tumors, including gliomas. It is overexpressed in glioma cells according to tumor grade and in glioma tumor initiating cells. There have been various reports suggesting that CXCR4 is required for tumor proliferation, invasion, angiogenesis, and modulation of the immune response. It may also serve as a prognostic factor in characterizing subsets of glioblastoma multiforme, as patients with CXCR4-positive gliomas seem to have poorer prognosis after surgery. Aim of this review was to analyze the current literature on biological effects of CXCR4 activity and its role in glioma pathogenesis. A better understanding of CXCR4 pathway in glioma will lead to further investigation of CXCR4 as a novel putative therapeutic target. GLIA 2014;00:000–000

Key words: chemokine receptor, brain tumor, cancer stem cells

Introduction

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alignant gliomas account for more than 50% of primary tumors developing within the central nervous system (Bian et al., 2007). They are characterized by highly invasive behavior and aberrant neovascularization (Bian et al., 2007; Lakka et al., 2004). Despite the advancements made in therapeutic management of high grade lesions, with rare exceptions such as anaplastic oligodendrogliomas, the median survival period of patients harboring malignant gliomas remains less than 1 year, and the 3-year survival rate after diagnosis is estimated at less than 2% of patients (Bian et al., 2007; Kleihues et al., 1995; Scott et al., 1998; Stupp et al., 2005). The aggressiveness and the refractoriness to current therapies of these tumors are mainly due to their highly invasive behavior, which makes current therapeutic options ineffective.

The identification of genes associated to glioblastoma multiforme (GBM) heterogeneity may provide the identification of different tumor subsets and the consequent development of innovative treatment strategies (Bian et al., 2007; Charles et al., 2012; Deorah et al., 2006; Misra-Press et al., 1992; von Deimling et al., 1995; Yamada et al., 1999; Zhang et al., 2003). A number of cytokines and growth factors, originally identified as mediators of leukocyte and normal stem cells trafficking and homing (Bian et al., 2007; Suratt et al., 2004), were found to play a role in tumor progression and metastasis in many types of malignant tumors (Balkwill, 2004; Bian et al., 2007; Furusato et al., 2010; Wang et al., 1998). They demonstrated a role in cell growth and angiogenesis, to negatively modulate anti-cancer immune response and to induce directional cell migration (Salmaggi et al., 2004).

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22669 Published online Month 00, 2014 in Wiley Online Library (wileyonlinelibrary.com). Received Nov 8, 2013, Accepted for publication Mar 21, 2014. Address correspondence to Filippo Gagliardi, Department of Neurosurgery and Gamma Knife Radiosurgery, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy. E-mail: [email protected] From the 1Department of Neurosurgery, San Raffaele Scientific Institute, Vita-Salute University, Milan, Italy; 2Division of Regenerative Medicine, Stem Cells and Gene Therapy, Neural Stem Cell Biology Unit, San Raffaele Scientific Institute, Milan, Italy; 3Department of Oncology, Medical Oncology Unit, San Raffaele Scientific Institute, Milan, Italy.

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Based on the number and spacing of the first two conserved cysteine residues in the N-terminus, chemokines have been divided into four subfamilies (CXC, CC, CX3C, and C). Each of these bind to specific receptors, which correspond to the four distinct subfamilies of chemokines they bind (CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors) (Murdoch and Finn, 2000). Chemokine receptors are found at the cellular surface and are constituted of about 350 aminoacids with a molecular weight of around 40 kDa (Mellado et al., 2001; Murdoch and Finn, 2000; Rossi and Zlotnik, 2000). Each receptor has a seven-transmembrane structure and coupled to G-protein (Murdoch and Finn, 2000). In case of CXC chemokines, they are distinguished into two subgroups based on the presence or absence, in the NH2-terminal domain, of three amino acid residues (GluLeu-Arg) named ELR motif; chemokines containing ELR motif were found to be associated with potent angiogenic capability (Belperio et al., 2000; Salmaggi et al., 2004). One of the major exceptions to this trait is SDF1 (CXCL12), a CXC ELR-negative chemokine. It is a known lymphocyte chemoattractant and a regulator of immune response surveillance during inflammation (Bleul et al., 1996; Nagasawa et al., 1996; Salmaggi et al., 2004; Suzuki et al., 2001). It has been demonstrated to play a critical role in tumor progression, angiogenesis, and metastasis in various tumors (Furusato et al., 2010). The receptor for SDF1, CXCR4, has been reported to promote the directional metastasis in melanoma, prostate cancer, and neuroblastoma (Bian et al., 2007; Cardones et al., 2003; Geminder et al., 2001). It is also highly expressed in human breast cancer cell lines, and injection of anti-CXCR4 antibody or silencing of this receptor significantly reduced the metastasis in the murine in vivo model (Liang et al., 2004, 2005, 2007; Muller et al., 2001). GBM cell lines have also been reported to express CXCR4, which may transduce growth signals in response to SDF1 (Bian et al., 2007; Salmaggi et al., 2004; Zhou et al., 2002). Receptor and ligand were mostly detected in tumor cells closer to the necrotic regions. Moreover, in animal xenograft experiments, a CXCR4 antagonist (AMD3100/Plerixafor) significantly inhibited the tumorigenicity and the growth of tumors (Bian et al., 2007; Rubin et al., 2003). These results suggest that CXCR4 may play a critical role in promoting the progression of human gliomas. Purpose of this study is to clarify the CXCR4 mechanisms in glioma biology by reviewing current knowledge about the role played by the receptor in human glioma cell survival, migration, invasiveness, proliferation, angiogenesis, and immunomodulation, events which are crucial for tumor 2

progression and spread, strongly limiting patients prognosis (Fig. 1). Promising molecular-targeted therapeutic approaches are systematically reviewed (Bian et al., 2007).

CXCR4 CXCR4 is a seven transmembrane G protein-coupled cell surface chemokine receptor. Among the currently known 22 chemokine receptors, it is one of the thoroughly studied, due to its role as a co-receptor for HIV entry161 and its ability to mediate metastatic process in different cancers (Burger and Kipps, 2006; Kucia et al., 2005; Sun et al., 2000, 2003, 2005, 2008; Zlotnik, 2004, 2006a,b). CXCR4 is a 352-amino acid rhodopsin-like G protein coupled receptor (GPCR). It selectively binds to the CXC chemokine stromal cell-derived factor 1 (SDF1), which is also known as CXCL12 (Fredriksson et al., 2003; Murphy et al., 2000). As a classical chemokine receptor, it plays an essential role in physiological processes such as hematopoiesis, organogenesis, and vascularization (Kucia et al., 2005; Nagasawa et al., 1996; Tachibana et al., 1998). In animal models, deficiency either of CXCR4 or of its ligand SDF-1 phenotypically results in a lethal defect in B cell lymphopoiesis, bone marrow colonization and cardiac septa development (Nagasawa et al., 1996; Tachibana et al., 1998; Zou et al., 1998). Recently, a novel receptor for SDF-1, called CXCR7, has been identified (Balabanian et al., 2005; Burns et al., 2006). It was found to be expressed on tumor-associated blood vessels and on distinct malignant cells (Burns et al., 2006; Hattermann et al., 2010; Meijer et al., 2008; Miao et al., 2007; Wang et al., 2008). In gliomas, Hattermann et al. showed that CXCR7 is much higher and broadly expressed in astrocytoma and GBM cells especially within the differentiated glioma cells when compared with CXCR4 which is present within the glioma stem like cell population. It has been demonstrated to be functionally active in glioma cell lines and it inhibits apoptosis induced by temozolomide (Hattermann et al., 2010). Further studies are needed to establish putative interactions and function of these two receptors in glioma biology.

CXCR4 Pathway SDF1 bound-CXCR4 acts via Gai, to activate the phosphoinositol-3-kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling pathways (Ganju et al., 1998; Neurath et al., 2006; Vicente-Manzanares et al., 1999). The CXCR4/SDF1 complex is incorporated into lipid rafts and may associate with members of the Src family of kinases (Zaman et al., 2008). Activated CXCR4 increases intracellular calcium mobilization and induces phosphorylation of focal adhesion components such as FAK and Pyk2 (Fernandis et al., 2004; Phillips et al., 2003, 2005). Volume 00, No. 00

Gagliardi et al.: Role of CXCR4 in Highly Malignant Glioma

FIGURE 1: Current knowledge and future directions on CXCR4 pathway in high grade gliomas.

The PI3K/AKT and MAPK signal transduction pathways contribute to chemotaxis, cell migration, and secretion of matrix metalloproteinases (MMPs) including MMP-2 and MMP-9 (Fernandis et al., 2004; Ganju et al., 1998; Janowska-Wieczorek et al., 2000; Libura et al., 2002). They also induce expression of cell surface integrins such as VLA-4 and VLA-538–40 and secretion of the angiopoietic factor, VEGF (Liang et al., 2004, 2005, 2007). Ultimately, through these pathways, SDF1-bound CXCR4 can induce cytoskeletal rearrangement, adhesion to endothelial cells, polarized migration of cells to specific organs and the secretion of angiopoietic factors (Alsayed et al., 2007; Cho et al., 2006; Hillyer et al., 2003; Kucia et al., 2005).

Histological Localization The distribution of CXCR4-positive cells in a single tumor is often extremely heterogeneous (Bian et al., 2007; Spano et al., 2004). CXCR4 and SDF1 positive cells have been observed either in regions close to necrotic areas, or in regions tightly related to neovessels (Rempel et al., 2000). Month 2014

It has been observed that SDF1 is highly expressed by the endothelium, whereas CXCR4 is mostly concentrated on the membrane of cells surrounding the neovessels. This finding strongly suggests the hypothesis, that receptor axis may play a role in promoting angiogenesis by directly inducing endothelial cells migration (Rempel et al., 2000).

CXCR4 and Tumor Grade SDF1 and CXCR4 expression was found to directly correlate to brain developmental. The two proteins were expressed at a low level in normal adult astrocytes, neurons, and microglial cells (Ohtani et al., 1998; Rempel et al., 2000; Shirozu et al., 1995). In contrast, they were observed to be highly expressed in brains of developing embryos, mainly localized in neurons of fetal cerebellum (Ma et al., 1998; Rempel et al., 2000). Low-grade tumors (Grade I and II according to WHO classification) demonstrate intermediate level of expression of SDF1 and CXCR4, whereas the highest expression levels were detected in GBMs. Nevertheless, even in GBMs, not all 3

cells of the tumor bulk are CXCR4 positive, suggesting that positive cells may represent a subpopulation of actively growing cells, which may promote invasion and metastasis (Bian et al., 2007).

potential use of this molecule in the clinical assessment of disease extent and prognosis (Ehtesham et al., 2013; Stevenson et al., 2008).

CXCR4 and Immune System Modulation Expression Profile in GBM Cancer Stem Cell Derived Lines Over the year’s majority of studies carried out on GBM involved work on immortalized GBM cell lines cultured in serum (Bian et al., 2007; Lee et al., 2006). Studies on the expression of CXCR4 on these lines have shown that they lack it entirely or when present are very lowly expressed (Bian et al., 2007; Schulte et al., 2011). Over the last decade various groups, ours included, reported that long-term proliferating cancer stem cells (CSCs) could be isolated from post-surgery specimens of human GBM by exposure to specific mitogens such as epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) (Galli et al., 2004; Lee et al., 2006; Salmaggi et al., 2006). These GBM CSC lines can be grown in vitro extensively, mostly composed of “bona fide” CSCs. Most notably, when implanted intracranially into immune-compromised mice, GBM CSC give rise to experimental tumors that very closely reproduce the major feature of human GBM, i.e., its invasive nature, as opposed to xenografts induced by serum-dependent traditional human glioma cell lines (U87, U373, etc.) (Galli et al., 2004; Lee et al., 2006). The few studies that have looked into the role of CXCR4 in CSCs, indicate that CXCR4pos cells have the capability of not only developing tumor spheres in serum free medium supplemented with growth factors, but also the other cardinal features required for a CSC population, i.e., multilineage differentiation and development of diffused tumor xenografts in intracranially injected mice (Schulte et al., 2011; Zheng et al., 2011). A study by Schulte et al., has demonstrated that CXCR4 expression both at the transcript and protein level can be found in a subfraction of glioma CSCs cultured by them. They also see a subset of these CXCR4 positive cells express the CSC marker CD133 (Schulte et al., 2011).

CXCR4 and Tumor Imaging Patterns Hyperintensity in T2-weighted MR images is strongly associated to the extent of disseminated disease burden. Ethesham et al. demonstrated a significant correlation between CXCR4 expression levels in human high grade gliomas and increased extent and intensity of T2-weighted perilesional MRI signal abnormalities. They concluded that, clinically CXCR4 level correlates with increased tumor grade and consequently with a worse prognosis, emphasizing the importance of CXCR4 as a mediator of the biologic behavior of these tumors and the 4

The highest expression of SDF1 is detected in regions involved in necrotic and perivascular areas, in which tumor integrity is compromised by damaged blood-brain barrier or leaky vessels. This finding suggests that the expression of both SDF1 and CXCR4 may be increased in areas, where the tumor is vulnerable to the immune surveillance (Rempel et al., 2000). SDF1 has been found to modulate negatively the immune response by its interactions with CXCR4, which is present on both macrophages and CD8 T cells membranes (Ameisen, 1998; Herbein et al., 1998; Rempel et al., 2000). The binding between the ligand and the receptor induces macrophages to secrete surface TNF and CD8 T cells to express TNF-R. The consequent mutual binding between macrophage and T cells through TNF/TNF-R interaction, transduces a pro-apoptotic signal in the T cell, suppressing the cytotoxic immune activity. Moreover the activation of TNF-R may lead to protection of tumor cells from immune surveillance by inducing an attack of macrophages and microglial cells toward T lymphocytes (Rempel et al., 2000; Tanabe et al., 1997).

CXCR4 and Proliferative Activity It has been established that CXCR4 is a mediator of neural progenitor cell migration in the developing brain and it was hypothesized that it may play a role in mediating the proliferation and self-renewal of CSCs trough the activation of different intracellular pathways (Ehtesham et al., 2013). SDF1 binding to CXCR4 induces the hydrolysis of phosphatidylinositol 4,5-bisphosphate yielding inositol 1,4,5trisphosphate, which releases Ca21 from intracellular stores, and diacylglycerol, which consequently activates protein kinase C cascade, required for sustaining proliferative activity (Haribabu et al., 1997; Sehgal et al., 1998). CXCR4dependent aberrant proliferative behavior of glioma cells is sustained by an autocrine/paracrine loop, which involves SDF-1 secretion followed by the binding and further activation of CXCR4 (Haribabu et al., 1997; Sehgal et al., 1998). Additionally, CXCR4 cytoplasmic tail contains a number of phosphorylation sites, some of which are believed to be involved in receptor desensitization. These include sites for protein kinase C, calmodulin-dependent protein kinases, and tyrosine protein kinases (Haribabu et al., 1997; Sehgal et al., 1998). Receptor desensitization can also occur in a phosphorylation independent manner (Haribabu et al., 1997; Sehgal et al., 1998). Several other members of the GPR family are known to be involved in the process of cell transformation and Volume 00, No. 00

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proliferation, cross-acting with CXCR4 axis. These include MAS (neuronal angiotensin sensitive receptor), serotonin 1c receptor, muscarinic acetylcholine receptors m1, m3, and m5, and a1B-adrenergic receptor (Gutkind et al., 1991; Hen et al., 1989; Jackson et al., 1988; Julius et al., 1989; Sehgal et al., 1998). As further confirmation, the overexpression of the CXCR4 receptor in GBM demonstrated an increase in cell proliferation (Ehtesham et al., 2013). However, antibody and antisense mediated inhibition of CXCR4 strongly negated cell proliferation suggesting an important role for CXCR4 in maintaining the neoplastic phenotype of GBM cells (Ehtesham et al., 2013).

express CXCR4 both at the transcriptional and protein level, which were found to 25- to 89-fold higher than that found in noninvasive tumor cells. Various reports demonstrate that GBM cells secrete SDF1 in local microenvironment improving their invasive capability toward a SDF1 gradient in vitro, through autocrine ligand-mediated receptor activation (Ehtesham et al., 2006, 2013). Furthermore, neutralization of CXCR4, using specific antibodies significantly impairs the in vitro invasive capacity of malignant glial cells (Ehtesham et al., 2006, 2013). Schulte et al. (2011) also observed the inhibition of highly invasive growth pattern in glioma CSC xenografts in vivo, using the specific antagonist AMD3100.

CXCR4 and Angiogenesis

CXCR4 and Survival

Glioma angiogenesis is activated through different pathways, mostly in correlation to the hypoxic microenvironment within the tumor (Ehtesham et al., 2013; Mellinghoff et al., 2011; Pen et al., 2007; Wuestefeld et al., 2012). CXCR4 has been identified as an activator of HIF-1 (Ehtesham et al., 2013; Staller et al., 2003), specifically in areas of hypoxia (Ehtesham et al., 2013; Rempel et al., 2000; Zagzag et al., 2000, 2006). It was demonstrated that CXCR4 levels correlate with HIF-1 levels in tumor cells and both are concurrently increased in tumor neovasculature in vivo. VEGF has been found to upregulate CXCR4 expression in human brain microvascular endothelial cells (HBMECs) (Ehtesham et al., 2013; Zagzag et al., 2000, 2006). In contrast, activation of CXCR4 in glioma cells was found to markedly increase the production of VEGF through calciumdependent mechanisms (Bian et al., 2004, 2007; Yang et al., 2005). This finding is supported by the observation that microvessel HBMECs highly express CXCR4 in highly malignant human gliomas and that glioma cells secrete SDF1 (CXCL12) through an autocrine and paracrine mechanism (Bian et al., 2007; Salmaggi et al., 2004). This observation suggests that HBMECs may sense its ligand in the tumor microenvironment and consequently increase their recruitment, proliferation, and infiltrative capability to facilitate the angiogenic processes (Bian et al., 2007). In gliomas, VEGF also increases microvascular permeability, which ultimately facilitates dissemination of malignant tumor cells (Carmeliet and Collen, 2000; Yang et al., 2005).

Similar to the phenomenon observed in ovarian cancer cells, in some human glioma cells lines, CXCR4 may act as a survival factor in vitro, through MAP kinase and Akt pathways, and by transactivating the receptor for epidermal growth factor (Bian et al., 2007; Porcile et al., 2004). Particularly, Akt signaling has been linked in many studies to increased cell survival and cancer progression (Guichet et al., 2013; Nicholson and Anderson, 2002; Zou et al., 1998). It was also shown that SDF1 isoform alpha protected against apoptosis resulting from serum-deprivation, suggesting that it could substitute for deprivation of growth factors, when fetal serum is withdrawn from the culture medium (Zou et al., 1998).

CXCR4 and Invasiveness CXCR4 pathway in glioma cells has been identified as a mediator of glioma cell invasiveness (Bian et al., 2007; Ehtesham et al., 2006, 2013). By sorting invasive tumor cell subpopulations and non-migratory neoplastic cells from the tumor core, it was found, that invasive populations over Month 2014

CXCR4 and Prognosis Recent studies on clinical data of patients harboring CXCR4positive gliomas versus those with CXCR4-negative gliomas, it was observed that CXCR4-positive patients exhibited poorer postoperative life expectancy (Bian et al., 2007). It was also observed that the positivity of CXCR4 correlates with glioma size, but not with the age or gender of the patients (Bian et al., 2007). These data on CXCR4 suggests that in patients with CXCR4-postive gliomas, it could have a significant role as a prognostic marker.

Future Therapeutic Directions Accumulating evidence on the role of CXCR4 in cancer biology and particularly in glioma biology has led to an increasing call for a therapeutic strategy against it. It has been reported that both, anti-CXCR4 antibodies and a synthetic inhibitor of CXCR4 were capable of abrogating metastasis in a number of human cancers on animal models (Bian et al., 2007; Liang et al., 2004, 2005, 2007; Muller et al., 2001). Combination of AMD 3100 and alkylating agents may cause tumor regression in vivo (Ehtesham et al., 2013). The 5

CXCR4 antagonist AMD3100 has been recently approved for clinical use in combination with granulocyte colonystimulating factor (G-CSF) for hematopoietic stem cell mobilization and collection in patients with non-Hodgkin lymphoma and multiple myeloma (Bilgin et al., 2013; Maziarz et al., 2013; Russell et al., 2013; Shaughnessy et al., 2013; Smith et al., 2013). It was also demonstrated that systemic administration of the CXCR4 antagonist AMD3100 inhibited the growth of intracranial glioblastoma xenografts by increasing apoptosis and decreasing proliferation of tumor cells (Ehtesham et al., 2013; Rubin et al., 2003). Another compound, called Nordy, which acts as a lipoxygenase inhibitor, demonstrated a decrease in CXCR4 expression and thereby inhibited the tumorigenicity and growth of human glioma cells in xenograft model (Bian et al., 2004, 2007; Ehtesham et al., 2013). It was found that it was capable of suppressing the expression of CXCR4 in malignant human glioma cells, reducing tumor cell chemotaxis, VEGF production and intracellular calcium flux (Bian et al., 2007). More importantly, Nordy markedly inhibited the growth of tumors, with no toxicity to the animals (Bian et al., 2004, 2007). A new frontier that is being explored is the application of RNA interference (RNAi) for targeting CXCR4 in cancer treatment (Ehtesham et al., 2013; Ramachandran and Ignacimuthu, 2012). RNAi-mediated knockdown of CXCR4 reduced VEGF production by glioma stem-like cells in vitro with a consequent limitation of the growth and angiogenesis of tumor xenografts in vivo (Ehtesham et al., 2013; Ping et al., 2011; Yamada et al., 1999). The preclinical studies on glioma have set a basis supporting a therapeutic rationale for targeting the CXCR4 pathway in patients. So far, no clinical studies on glioma patients have been published. An ongoing Phase I clinical trial on the use of Plerixafor in combination with bevacizumab in recurrent high-grade glioma will prove if this drug combination can be given safely (clinicaltrial.gov: NCT01339039). Additionally, this ongoing trial will give information about the ability of Plerixafor to cross the blood-brain barrier and its availability into human brain tumors. Potential limitations of the use of CXCR4 antagonist may be related to the multitude of variables influencing gliomas (invasiveness, angiogenesis, stem/progenitor function, therapeutic sensitivity), which makes it difficult to quantify the exact contribution of inhibiting CXCR4 in these different pathways. Another concern is the potential immunosuppressive effects of blocking a known mediator of immune cell trafficking (Ehtesham et al., 2013). To date, there are no reports on the use of CXCR4 antagonists as antineoplastic agent on non-hematopoietic, solid tumors. 6

Acknowledgment The authors acknowledge Rossella Galli for her essential intellectual contribution in writing this review.

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