Brain tumours originate from various types of cells, of which gliomas are most frequent. Recent epidemiological data in UK confirmed that glioblastoma (GB) is also the most common among glial tumours with 5–7 cases per 100.000 individuals yearly, and represents 50% of all gliomas.1 The World Health Organisation (WHO) distinguishes four grades of glioma, of which GB is the most aggressive, invasive, and lethal among all types of brain tumours. According to the standard histological classification, GB originates from neoplastic glial cells, also called astrocytes, either
There are other reasons for GB therapy resistance,
Solid tumour progression is not only relaying on the genetic and epigenetic variations of cancerous cells acquired during their evolution, but also on how their homotypic and heterotypic interactions with the stromal cells of associated microenvironment are. The “tumour microenvironment” (TME) consists not only of local, resident cells being invaded by the tumour cells, but also of infiltrating host cells, e.g. bone-marrow and blood-derived mesenchymal stem cells (MSC) and haematopoietic stem cells (HSC) and their progenitors, e.g. mature lymphocytes, macrophages, etc. Very recently, Salmon
In glioblastoma in addition to their autonomous (inter-tumour) heterogeneity11, the increasing attention is paid to their non-autonomous heterogeneity (intra-tumour heterogeneity), that is presenting an obstacle to a more informed treatment, as we still do not understand the ability of GB cells to manipulate and exploit these non-cancerous cells collectively termed “tumour stroma”. As stated by Broekman
Thus, GB cells spreading to the brain involve multiple modes of communication with stromal cells as extensively reviewed by Matias
As reviewed by Balkwill18, chemokines are chemotactic cytokines that cause directed migration of stromal cells, such as leukocytes, that are induced by inflammatory cytokines. Chemokine signalling results in transcription of target genes that are involved in cell invasion, motility, interactions with the extracellular matrix (ECM) and cell survival. Chemokine signalling can coordinate cell movement during inflammation, as well as the homeostatic transport of HSCs, MSCs, myeloids cells lymphocytes, dendritic cells and neutrophils, as well as cancer-associated fibroblasts (CAFs).19 Directed migration of cells that express the appropriate chemokine receptor, occurs along a chemical gradient of ligand - known as the chemokinereceptor axis — allowing cells to move towards high local concentrations of chemokines. More than 50 human chemokines and 20 chemokine receptors have been discovered so far. Cytokines, as pro-inflammatory mediators, when excessive, also play a role in causing chronic inflammation, for example induced by bacterial (e.g. by
The cytokine CCL5, also classified as C-C motif chemokine 5, has been initially termed RANTES (“Regulated upon Activation Normal T cell Expressed and Secreted”), and as a potent chemokine, attracting leukocytes.22 Later, CCL5 was recognised as a versatile inflammatory mediator, expressed by breast cancer cells (BC), where along with CCL2 it promoted pro-malignant activities by attracting macrophages, T-cells and granulocytes, as well as mesenchymal stem cells and enhancing angiogenesis.23 CCL5 has been suggested as potential therapeutic target to impair the disease progression. In immune cells, CCL5 was reported initially as a HIV-suppressive factor and expressed mainly by CD8+ T cells.24 It binds to its cognate receptor C-C chemokine receptor type 5 (CCR5), which is (alongside C-X-C chemokine receptor type 4 (CXCR4)), an HIV entry co-receptor into CD4+ T cells.25 The Food and Drug Administration approved the first CCR5-based entry inhibitor, now called maraviroc (MRV) in 2009, and based on this, new drugs that promote CCR5 and CXCR4 internalization, independent of canonical cellular signalling, provided clinical benefits for HIV patients with minor side effects.
The mode of CCL5 action thus comprises binding not only to its cognate and the most studied interacting partner, the CCR5 membrane receptor, but also to other members of the G-protein coupled receptors (GPCR), such as C-C chemokine receptor type 1 (CCR1) and C-C chemokine receptor type 3 (CCR3). In addition to that, non-conventional or auxiliary receptors of CCL5 are CD4426 and GPR75 (Figure 1A).27 This is not unusual, as many chemokine receptors display promiscuous ligand binding, meaning they have more than one high-affinity ligand.28 Such variety of CCL5 interactions causes the activation of multiple pathways and gives the ligand a diverse range of not only physiological, but also pathological functions, including in cancer.29 In this vein, CCL5 has been shown to be highly expressed in a plethora of cancer types, such as colorectal30, lung31, prostate32, breast28,33,34 and cervical cancer.35 In addition, tissue or plasma CCL5 also serve as a marker of poor prognosis, as it is the case in cervical35, prostate32, gastric36, breast37, pancreatic cancer38, as well as in glioblastoma.39 The cells that express and secrete CCL5 in cancer, can either be cancerous cells themselves or stromal cells.40
Chemokine CCL5 promiscuous binding to receptors. A variety of chemokine-receptors are interacting with CCL5/CCR5 in the signalling axis:
The receptor CCR5, classified as C-C chemokine receptor 5, alternatively termed also CD195, is the main receptor through which CCL5 transduces signalisation.41,42 Structurally, CCR5 is a GPCR, as are many other chemokine receptors.41 This means that it has a N-terminal extracellular tail responsible for ligand binding, seven hydrophobic transmembrane regions, the six loops that connect them, and a C-terminal cytosolic tail. This is crucial for transducing the signal caused by ligand binding after its heterotrimeric G-proteins binding or through G-protein independent pathways.41
Similar to other chemokine receptors, CCR5 also acts redundantly for signalling pathways.28 High affinity ligands that bind to CCR5 are CCL5, as well as chemokine (C-C motif) ligand 3 (CCL3) (also known as MIP-1α) and chemokine (C-C motif) ligand 4 (CCL4) (also known as MIP-1β) (Figure 1B).42,43 As already mentioned, substantial research has been dedicated to the role of CCR5 in HIV infection; M-tropic or macrophage strain HIV-1 uses CD4 as its main receptor to bind to and enter CD4+ T cells, but for this it also needs co-receptors, CCR5 and CXCR4 acting as such. Small molecular inhibitors (maraviroc [MRV]) and the humanized monoclonal antibody (leronlimab)44,45 are CCR5 antagonists that inhibit HIV-1 virus from entering the T-cells.43 MRV binds to CCR5 and acts as an allosteric inverse agonist, locking CCR5 in an inactive conformation.42 However, recent research has focused more on cancer, as similarly to CCL5, CCR5 is overexpressed in many types of cancers, including breast28,33,34,46, prostate32 and in particular glioblastoma.47,48 Both maraviroc49 and leronlimab46 have been shown to potently block cancer metastasis in murine xenografts.
The canonical (conventional) way of signalling is through a hepta-helical chemokine receptor and adjacent G proteins, more specifically their Gα subunit and Gβγ dimer.41 Upon ligand binding, CCR5 activates Gαβγ trimer by causing guanosine exchange (GDP à GTP) and the dissociation of the membrane-bound Gα subunit from the Gβγ dimer.50 Activated Gα affects adenylyl cyclase, and subsequently cellular cyclic adenosine monophosphate (cAMP) levels that activates cytosolic protein kinase A (PKA). Gα together with Gβγ affect various targets (e.g. PLCβ), resulting in the production of secondary messengers, such as inositol-1,4,5-triphosphate (IP3), diacylglycerol (DAG) and increased cytosolic calcium concentration. Both Gα and Gβγ trigger calcium influx, therefore the direct interaction of chemokine and its receptor can be confirmed by a calcium mobilisation assay.38 Influx of calcium activates calcium-dependant pathways (NF-κB), as well pathways independent of G-proteins, all favouring malignancy in one way or the other.41
The CCL5/CCR5 axis has been recently reported as a mechanism of tumour progression in pancreatic38, gastric20 and breast cancer.33, 44 Noteworthy, in cancer, the CCL5-receptors signalling can favour cancer progression directly by affecting proliferation, migration and cell survival of cancer cells, or indirectly, by affecting tumour microenvironment,
Cancer cell proliferation is regulated by many pathways, one of them, most commonly mediated by CCL5/CCR5 signalling, is mammalian target of rapamycin mTOR) pathway. This pathway is convergent with the Phosphatidylinositol 3-kinase (PI3K) pathway, as they both activate the Akt (protein kinase B) pathway (Figure 2).54
Phosphatidylinositol 3-kinase (PI3K)/pAkt-kinase pathway as a central CCL5/CCR5 signalling cascade in cancer cells. Upon CCL5 binding to its cognate receptor CCR5, primarily the PI3K/Akt pathway is triggered. This favours the phosphorylation of PIP2 to PIP3, a secondary messenger responsible for the activation of the Akt kinase, which in turn phosphorylates several downstream effectors. This causes the inhibition of pro-apoptotic effectors and the upregulation of survival genes. Another target of PI3K is the mammalian target of rapamycin complex 1 (mTORC1), which also activates the Akt kinase. However, it has also been shown that a secondary intracellular target, Focal Adhesion kinase (FAK) binding-SRC kinase complex can be activated, resulting in additional PI3K activation (in prostate cancer62).
The “mTOR is a kinase, encoded by mTOR gene
Cell migration along or through 3D extracellular matrix (ECM) is fundamental to normal tissue formation and regeneration, stem cells and immune cells trafficking, and cancer cell invasion and metastasis.57 As in various cancer cell types, migratory glioblastoma cell acquire mesenchymal type of movement58, where invasion rates are governed by the capacity of cells to induce a proteolytic cascade. This includes metalloproteases (MMPs), plasminogen and its activators as well as cathepsins59 and integrin- actomyosin mediated mechano-coupling. The process starts with cell polarisation of the actin cytoskeleton, enabling directional movement of the migrating cell. By forming frontal protrusions that activate integrin receptors, the cells are attached to the ECM integrins. Intracellularly, this triggers activity of small cytosolic GTPase proteins, RhoG, Cdc42 and Rac, which are essential in coordinating these processes58 and thereby metastasis
Early studies showed the importance of CCR5 in the invasion of breast and prostate cancer cells.61 In human lung cancer cells, CCL5/CCR5 activation augments the migratory ability by increasing their surface expression of αvβ3 integrin31 and phosphorylation of PI3K/Akt kinases. Further, the authors have shown that by PI3K/Akt inhibitors or transfection of the CCL5 treated cells by mutant PI3K and Akt, lead to a decrease in αvβ3 integrin expression and migration. CCL5/CCR5 activation of PI3K/Akt signalling also activated IKKα/β and NF-κB, again enhancing αvβ3 integrin expression and migration.31 Presumably, the major mechanisms of CCL5 and other chemokines activation involves the PI3K-γ isoform through the Gβγ dimer of the G-protein, which is coupled with the CCR5 receptor.51
Actin polymerisation as a result of CCL5 activation of CCR5 was observed also in pancreatic cancer38 and recently in breast cancer epithelial cell lines34, enhancing migration by a PI3K/Akt pathway. In GB cell lines U87 and U251, CCL5 stimulation enhanced their migratory ability.56 After treatment of U87 cells with a PI3K inhibitor and CCR5 siRNA, inhibition of Akt phosphorylation was demonstrated in CCL5-treated cells and significant inhibition of growth was observed in U87 glioma xenografts in mouse model. Finally, high CCR5 expression in MES- GB was correlated with high p-Akt expression in patients’ samples. We have shown that GB cell invasion is triggered by intracellular cathepsin B62, followed by its activation of plasminogen system63 and finally activating executive metalloproteases of which MMP-9 is directly degrading EMC, and the latter was also downregulated by downregulated CCR5-PI3K/ Akt signalisation.56 Wang
Tumour’s maintenance of cancer cell survival is a necessity for its progression. This is achieved by overexpression of DNA repair and/or by increasing the apoptotic threshold to avoid cancer cell death. CCR5 signalling promotes breast cancer cell survival in both ways34, but in glioblastoma the CCL5/CCR5 activation mostly affects apoptosis. In human breast cancer, high CCR5 expression correlates with poor outcome, as recently reported by Jiao
In GB, Pan
Tumour-induced immunosuppression involves recruitment of different cells forming tumour microenvironment, such as tumour infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSCs), innate lymphoid cells, mesenchymal stem cells (MSC), immature dendritic cells (IDC) and tumour-associated macrophages (TAM), many of these cells expressing CCR5 and its ligand CCL5.46,48,65 TAMs actually comprise as two ontogenetically distinct subsets, microglia and glioblastoma infiltrated macrophages (MDMs) derived from monocyte are representing about 30% of all cells in glioblastoma.66,67 The difference between MDMs and microglia is also reflected in cytokine gene expression.65 Microglia mediated immuno-suppression dwells also on the CCL5/CCR5 and effect of CCR5 signalling on TAM activation (polarization) and GB progression has been investigated by Laudati
CCL5-CCR5 system and microglia polarization. The pharmacological blockade of CCR5 with maraviroc prevents the activity of glioblastoma-associated anti-inflammatory microglia M2 phenotype in and induces (green arrow) the conversion to prevailing pro-inflammatory M1 microglia phenotype.
Furthermore, under conditions mimicking the late stage of glioma pathology, CCR5 blockade thus induces a prevailing M1 pro-inflammatory state. Such changes in microglia polarization profile are potentially associated with cytotoxic and anti-tumour properties, which leads to a potential reduction in tumour growth (Figure 3), as emphasised by Jiao
The concept of hierarchical tumour evolution from cancer stem cells is now widely accepted and proven also in GB.7 GSCs and their progenies, represent the final obstacles for therapy failure, as these represent the most resistant cell phenotype in the GB. These cells, although a minority of total cancer cell populations, define the functional progression of GB by expressing a panel of stemless markers and cell damage resistance genes. Besides molecular set up of these cells’ subpopulation, also the micro environmental cues contribute to their resistance by stromal cells’ protection in the so-called tumour tissue niches. We have recently shown the cellular and functional features of GB niches around a fraction of arterioles by immuno-histochemistry.70 Besides the crucial endothelial-GSCs paracrine interactions, maintained mainly by CCL12-CXCR4 axis, other interactions with resident mesenchymal stem cells (MSCs) are plausible, and may be maintained by CCL5/CCR5 axis as shown on Figure 4 (unpublished data).
CCL5 and CCR5 in Glioblastoma microenvironment
The basic question when investigating chemokine paracrine signalling is
The most commonly observed situation in CCL5/CCR5 signalling is activating host CCR5, expressed by differentiated glioblastoma cells, by stromal cells such as TAM, as discussed above and by Wang
Another way of heterotypic cellular cross-talk in glioblastoma is via secreted CCL5 by GB cells. This ligand affects infiltrating or stromal cells that express CCR5, thus affecting their intracellular signalisation that results for example in the immunosuppression of the GB microenvironment, as has been discussed above in chapter
Another hypothesis by Kouno
In MES-GB subtype, the autocrine CCL5/CCR5 activation loop has been examined as well by adding CCL5. Because no significant difference between wild-type tumours and those with additional stromal CCL5 was noticed, they concluded that CCL5 promotes survival and proliferation of the cells in a cell-autonomous and autocrine manner. Low grade gliomas seem to rely on stromal chemokine stimulation, whereas high grade gliomas (GB), establish autocrine chemokine stimulation. An interesting interpretation of this is that the ability of autocrine activation grants gliomas for relative stromal independency and this in turn causes the stromal cells to have less control over the regulation of the tumour leading in tumour malignancy.29