CXCR4-Targeted Library

INTRODUCTION

Chemokines (chemotactic/chemoattractant cytokines) are highly basic small secreted proteins consisting on average of 70-125 amino acids with molecular masses ranging from 6 to 14 kDa which mediate their effects through binding to seven transmembrane domain (7-TMS) of the specific family of G-protein-coupled receptors (GPCR) located on target cell membrane. Initially, chemokines were recognized as chemo-attractants and activators of specific types of leucocytes in a variety of immune and inflammatory responses. Notable, over the past few years there has been a “chemokine revolution” in the realm of anti-cancer drug therapy and the great majority of authoritative scientists and clinicians in oncology-related field of science are now aware of their crucial role at all stages of neoplastic transformation and progression [1]. Thus, tumor cells extensively express functional chemokine receptors, which can sustain proliferation, angiogenesis, survival and promote organ specific localization of distant cancer metastases [2].

The chemokine receptor CXCR4 possesses multiple fundamental functions in both normal and pathologic physiology. CXCR4 is a GPCR receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1, previously SDF1-α). The interaction between CXCL12 and CXCR4 plays a critical role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This interaction is also known to be involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA), asthma and pulmonary fibrosis.

1. The pivotal role of CXCR4 chemokine receptor in cancer pathology

Unlike other chemokine receptors, CXCR4 is expressed in many normal tissues, including those of the central nervous system, while it is also commonly expressed by over 25 different tumor cells including cancers of epithelial, mesenchymal, haematopoeitic origin, etc. [3]. For example, tumor cells from breast, prostate, pancreatic, lung and ovarian carcinomas, neuroblastoma and glioblastoma, all express CXCR4 [4-6]. This receptor was also found in acute lymphoblastic and myeloblastic leukemia, in non-Hodgkin`s lymphoma, in tumors derived from kidney, as well as in melanoma and rhabdomyosarcoma [6,7]. In other cancer cells studied, CXCR4 may be co-expressed with other CC or CXC chemokine receptors or less commonly, other receptors are present without expression of CXCR4. Several lines of evidence show that the CXCL12-CXCR4 chemokine system may also be involved in promoting tumor cell survival and growth. For instance, in cells from adult glioblastoma and pediatric medulloblastoma, CXCR4-CXCL12 signalling network induced chemotaxis and enhanced proliferation and survival [8]. In several types of cancer, including glioma, melanoma, NSCLC, renal and thyroid, CXCL12 can stimulate tumor proliferation and/or survival of CXCR4-expressing tumor cells [9]. Production of CXCL12, both at mRNA and at protein level, has been detected in several CXCR4-expressing tumors, thus suggesting a possible autocrine or paracrine loop of growth. It was recently reported that CXCR4 is frequently expressed in human pancreatic cancer cells and that CXCL12, in addition to enhancing motility and invasion, promotes their proliferation and excites anti-apoptotic effects [10]. Furthermore, Wang and Ma have suggested that β-catenin is a vital key intracellular factor in the CXCR4-CXCL12 axis promoting metastatic events of pancreatic cancer [11]. It was recently reported that CXCR4 is expressed in several types of malignant brain tumors [12]. Human breast cancer cells express CXCR4 and CCR7 [13]. Especially, CXCR4 repression was found to effectively inhibit metastasis of breast cancer cells in experimental animal models [6]. The specific ligands for these receptors CXCL12 and CCL21 are found at elevated levels in lymph nodes, lung, liver and bone marrow, organs to which breast tumours generally metastasize. In addition, as communicated in several previous studies, melanoma cells generally express CCR7 and CCR10 chemokine receptors [13] they also significantly co-express CXCR4 and CXCR3 which play a critical roole in tumor growth, metastasis and tissue invasion [14]. Leukaemic and lymphoma cells also express a wide variety of chemokine receptors, including CXCR4. Recently, CXCR4 have reported to be a key effector in the formation of peritoneal carcinomatosis in gastric cancer in proliferation, migration and invasion of epithelial ovarian cancer cells [15,16]. Furthermore, there is growing in vitro and in vivo evidence that CXCR4 expression by leukaemia cells allows for homing and their retention within the marrow. As such, leukaemia cells appear to utilise CXCR4 to access niches that are normally restricted to progenitor cells, and thereby reside in a microenvironment that favours their growth and survival [17]. It was also recently reported that CXCR4-CXCL12 signaling activates phosphorylation of extracellular- signal regulated kinase 1/2 (ERK1/2) and stimulates meningioma cell proliferation [18]. Considering the really high diversity and infinite complexity of chemokine related signal propagation we discuss here the basic features of chemokine CXCR4-CXCL12 signslling network and disclose the main principles of how this pair works in the field of cancer growth and progression.

It should be noted that CXCR4 is not a tumor specific marker and not all cancers express this receptor. Both receptor and its specific ligand are widely expressed in normal tissues and play a fundamental role in a variety of physiological processes including fetal development, joint mobilization of haemopoietic stem cells and specific trafficking of a majority of leukocyte types [19,20].

2. Signaling via CXCR4-CXCL12 chemokine system

The chemokine receptor CXCR4 belongs to the large superfamily of GPCR receptors, and is directly involved in a number of biological processes including organogenesis, hematopoiesis, and immune response. Several recent reports highlight the high complexity of intra/extracellular signal transduction initiated by chemokine receptors, especially by CXCR4 [21,22]. In general, chemokines activate 7-TM GPCR chemokine receptors which are coupled to heterotrimeric Gαβγ protein subunit. Heterotrimeric G- proteins associate with the intracellular domains of GPCRs when in their inactive, or guanosine diphosphate (GDP)-bound, state. Upon chemokine ligand binding, the GDP is readily exchanged for guanosine triphosphate (GTP) resulting in instantaneous activation of corresponding G-protein. The active G-protein subsequently initiates dissociation of Gαβγ into its Gα and Gβγ subunits to stimulate many intracellular mediators. In contrast to other chemokine receptors, stimulation of CXCR4 can lead to prolonged activation of both mentioned subunits [23]. Signaling via CXCR4 also enhances tyrosine phosphorylation, association of components of focal adhesion complexes such as paxillin and NF-κB activity in nuclear extracts [24]. In a metastatic breast cancer cell line, NF-κB directly regulates the CXCR4 promoter and can upregulate expression of CXCR4, facilitating increased responses to CXCL12 [25]. In breast cancer cell lines, CXCL12 also induces phosphorylation of FAK, Pyk2, the cytoskeletal proteins paxillin and Crk, the tyrosine phosphatase SHP2 and the adaptor protein Cbl [26]. There is one interesting report of cross talk between the BCR/ABL oncogenic tyrosine kinase and CXCR4 signalling [27]. In CML, BCR/ABL kinase phosphorylates, activates and disregulates proliferation and survival pathways of progenitor cells in the bone marrow. Immature leukaemic cells leave the marrow and are found in large numbers in the blood and spleen. BCR/ABL strongly activates a CXCR4-dependent signalling component through the Src family tyrosine kinase, Lyn. Cross talk between BCR/ABL and CXCR4 signalling may allow the oncoprotein to couple to PI3-kinase, MAPK cascades and ‘take over’ the chemokine pathway. This could lead to disruption of chemotaxis and hence release of the transformed cells into the periphery. Undoubtly, the precise signaling mechanisms by which all chemokine receptors regulate cellular function will become evident in time, but it has proven to be difficult to link specific CCR or CXCR signalling pathways right through to a biological response such as chemotaxis, cell growth and progression.

3. The complicity of CXCR4 in cancer metastasis, angiogenesis, adhesion and invasion

Cancer metastasis results from a non-random process, in which organ selectivity by the tumor cells is highly dependent on interactions between tumour and host stromal cells as well as between cancer cell and several essential molecular factors expressed at the remote organs that eventually turn into preferred sites of metastasis formation [28,29]. In aggregate, these factors support the consecutive steps required for metastasis formation, including tumor cell adhesion to microvessel walls, extravasation into target tissue and migration. For instance, chemokine CXCL12, lysophosphatidic acid (LPA) and thrombin promote the migration and invasion of cancer cells through their cognate receptors, CXCR4, LPA1 and PAR1, respectively, enabling the cancer cells to escape from the location of the primary tumour [30]. Notable, metastasis is a major cause of morbidity and mortality in breast cancer patients. To prevent these lethal outcomes, improved strategies to treat metastatic neoplasms are strongly needed. Blood flow and other mechanical factors influence the delivery of cancer cells to specific organs, whereas molecular interactions between the cancer cells and the new organ influence the probability that the cells will grow there. Inhibition of the growth of metastases in secondary sites offers a promising approach for effective cancer therapy. Metastases arise following the spread of cancer from a primary site and the formation of new tumor nidus in distant organs. When cancer is detected at an early stage, before it has spread, it can often be treated successfully by small molecule inhibitors of tumor growth or surgery/local irradiation, and the patient will be cured. However, when cancer is detected after it is known to have metastasized, treatments are much less successful.

Many chemokines play multiple roles in tumor growth, invasion and metastasis by inducing cellular transformation, angiogenesis, secretion of proteinases, and organ specific metastasis [31]. As mentioned above, chemokines control the directional migration of leukocytes and it seems that mechanisms utilised for leukocyte trafficking may also used by tumor cells. Studies on the contribution of chemokine receptors to organ specific metastasis are providing important clues about why some cancers metastasize to specific organs. Recent elegant studies have shown that tumour cells express patterns of chemokine receptors, including CXCR4, that ‘match’ chemokines that are specifically expressed in organs to which these cancers commonly metastasize [32].

Cancer cells migrate towards the chemoattractant gradient until reaching the site for secondary colonization. For chemokine receptor expression by a cancer cell to be advantageous a chemokine gradient is required/needs to be established and in breast, prostate and ovarian cancer, neuroblastoma, melanoma and some forms of leukaemia, the respective ligand is strongly expressed at sites of tumor spread. Tumour cell migration in response to CXCR4 stimulation requires the polarization of intracellular signalling molecules that results in a leading edge that protrudes outward, coupled with contractile forces at the back and sides of the cell to propel the cell towards a chemoattractant. For example, melanoma cells express functional CCR7, CCR10 (and lower levels of CXCR4, not shown) (Fig. (1)). Melanoma cells also express specific ligands for these receptors at the two major sites of metastasis, skin and lymph nodes [13]. Both breast cancer cells and primary breast tumours were found to express the chemokine receptors CXCR4 and CCR7 at high levels. The specific soluble ligands for these receptors CXCL12 and CCL21 are found at elevated levels in lymph nodes, lung, liver and bone marrow - organs to which breast tumours often metastasize, whereas skin tissue expresses high levels of CCL27, a soluble ligand for the CCR10 receptor [33,34]. Therefore, breast cancer cells that are taken to the lung by the blood flow would find a strong chemokine-receptor ‘match’, which would lead to chemokine mediated signal activation. By contrast, breast cancer cells taken to skin would not find such a match. Melanoma cells, however, taken to skin by the circulation (or by local invasion) would find a CCL27-CCR10 chemokine-receptor ‘match’ that would lead to the activation of chemokine-mediated pathways. These results found further support in experimental tumor models: transduction of tumor cells with CCR7 conferred improved ability to metastasize to regional lymph nodes [35], while CXCR4-transfected cells preferentially migrated to the lung [36].

Cyclooxygenase 2 (COX2) expressed in tumour and stromal cells generates prostaglandin E2 (PGE2), which binds to EP2 (pro-angiogenic factor) receptors on cancer cells and promotes tumour cell proliferation and extracellular matrix (ECM) degradation through the expression of matrix metalloproteinase 2 (MMP2) and MMP9 [37], a response also elicited by thrombin and CXCL12. Stimulation of mentioned GPCR receptors (CXCR4, LPA1, PAR1 and EP2) also causes increased release of vascular endothelial growth factor (VEGF), thereby promoting vascular permeability, which is important for tumour cell extravasation and tumour angiogenesis. Specifically for solid tumours, as they grow, the hypoxic condition in the tumour microenvironment results in the stabilization of hypoxia- inducible factor-1 (HIF1), which upregulates CXCL12 and VEGF. Cancer cells also produce several CC and CXC chemokines, such as CCL2, CCL5, CXCL8 (interleukin 8 (IL8)) to recruit tumor associated macrophages (TAMs) and leukocytes to the tumour. These immune cells then help to promote blood vessel growth by releasing VEGF and other angiogenic factors (AF). Concomitantly, tumour or stromal inflammatory mediators that act on GPCRs, such as IL8, prostaglandin E2 (PGE2) and sphingosine-1- phosphate (S1P), can also regulate the activity of MMPs that degrade the ECM, which clears a path, at the same time as endothelial cell chemotaxis, often involving the coordinated activation of a network of small GTPases such as Rho and Rac and their downstream targets by Gα13 or Gβγ when released from Gαi, paves the way for new blood vessel growth. Finally, S1P is released following the activation of sphingosine kinase activity, and functions in an autocrine and paracrine manner to cause tumour and endothelial cell proliferation and migration. Inflammatory cytokines that accumulate in the tumour milieu also stimulate the nuclear factor B (NFB)-dependent increased expression and release of IL8 from stromal and cancer cells, which promotes endothelial cell migration towards the growing tumour. Ultimately, pro- angiogenic GPCRs activate a network of small GTPases, Akt and mitogen-activated protein kinase (MAPK) signalling that promotes the migration, survival and growth of endothelial cells. Several other important mediators are also implicated in cancer groth, metastasis, invasion and angiogenesis, these include HIF1α, hypoxia-inducible factor-1; IL8, interleukin 8; NFB, nuclear factor B, ect.