G protein coupled receptors (GPCRs) are cell surface receptors and members of the seven-transmembrane superfamily that initiates intracellular signaling primarily via the activation of heterotrimeric G proteins (alpha, beta, and gamma). GPCRs are activated by several ligands including neurotransmitters, chemokines, hormones, calcium ions (Ca2 +), and sensory stimuli. The processes of GPCR signaling system includes regulation of receptor desensitization, internalisation and signal termination. Chemokine receptors have important biological functions and classified into two groups: G protein-coupled chemokine receptors (GPCCR), and atypical chemokine receptors, which scavenges chemokine to reduce inflammation or shape chemokine gradients. GPCCRs are responsible in leukocytes trafficking and positioning throughout the body by activating directed cell migration, which is also referred as chemotaxis. Leukocyctes involvement is vital for immune response, but inappropriate trafficking can lead to pathological conditions such as inflammatory diseases, cancer or human immunodeficiency virus (HIV).
Chemokines are constitutes of the superfamily, and divided into four subclasses (C, CC, CX3C and CXC). The main CC and CXC classes are involved in tertiary structure that contains a flexible N-terminus, which is a region that is important for chemokine-receptor binding interactions and subsequent activation. Chemokines are also known to promote angiogenesis, proliferation, and apoptosis therefore interaction between chemokines, chemokine receptors and GPCRs also helps in human cancer metastasis.
Overexpression of CXCL12 chemokine and its receptor CXCR4 are commonly found at sites of cancer metastases including lymph node, lung, liver, and the marrow. Production of CXCL12 from marrow stromal cells is a major source of chemoattractant for lymphocytes, monocytes and its receptor. CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and breast cancer metastases. Normally, CXCR4 expression is low in normal breast tissues but high in malignant tumours, especially HER2-mediated metastasis. CXCL12 or also known as stromal-cell derived factor (SDF1), causes proliferation of B cell progenitors and regulate B cell maturation. Interactions of CXCL12-CXCR4 result in metastasis of breast cancer cells to regional lymph nodes and lungs. As a result, this interaction stimulate many intracellular signals such as increasing in calcium influx, extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation, activation of phosphatidylinositol 3-kinase (PI3K) and Akt, tyrosine phosphorylation of focal adhesion complex components such as Pyk-2 and Crk, and an increase in NF-κB activity. To incorporate calcium ions, the major regulator of its receptor known as calmodulin (CaM) is involved for physiological processes (such as cell proliferation, metabolic homeostasis or muscle contraction). High concentration of free Ca2+ in the cytosol and proliferating into the nucleus upon cell activation, causes production of mitogenic factors and other agnoists, therefore promoting GPCR signaling.
Upon the GPCR activation, it involves binding of agonists to extracellular domains of the receptor causing conformational changes in the seven transmembrane spanning domain, thus facilitating interactions with intracellular heterotrimetric G-proteins and enables signal transmission. When GPCRs are activated, they function as guanine nucleotide exchange factors (GEFs) for the Gα subunit which causes guanosine diphosphate (GDP) to convert into guanosine triphosphate (GTP), this causes dissociation of the GTP-bound Gα subunit from the Gβγ heterodimers hence allowing both subunits to mediate downstream signal transduction cascades (Figure 1). There are various G-proteins divided into four subfamilies: Gαs, Gαi, Gαq, and Gα12/13, but most chemokine signalings are activated by Gαi proteins, however some have been reported to interact with Gαq. Gαi is an inhibitory G-protein that inhibits the production of cAMP, while Gαq activates phospholipase C (PLC). In other words, when the G protein subunits are triggered it can either positively or negatively regulate downstream effector such as adenylyl cyclases and cyclic adenosine monophosphate (cAMP) production, affecting the Src kinase and CREB transcriptional activity. For example in breast cancer, when adenylyl cyclase is stimulated with binding to lutenizing hormone (ligand) this results to increased estrogen levels. As breast cancer is estrogen-mediated, consequently it enhances cancer cell growth and development. Nitric oxide (NO) synthetease is also another second messenger that function as a neurotransmitter. Cyclic guanosine monophosphate (GMP) produced by the NO-stimulated soluble guanylyl cyclase is responsible in regulating ion channel conductivity and blocking gap junction conductivity. As a consequent, steroid hormones and carcinogens promote transcription factors to initiate gene expression which in turn causes cancer invasion.
On the other hand, G𝛽𝛾 subunits are required for chemotaxis, consequently activating various signaling cascades such as GPCR kinases (GRKs), ion channels, and phospholipase C-𝛽 (PLC-𝛽). PLC-𝛽 catalyses phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to inositol trisphosphate (IP3 and diacylglycerol (DAG). These causes IP3 to release in calcium from endoplasmic reticulum (ER) stores, and DAG can activate protein kinase C (PKC), which is important in receptor regulation through phosphorylation and desensitization (uncoupling).
GRKs and arrestins are the major sources in signaling termination through phosphorylation of activated GPCRs. The mechanism involved the interactions between arrestins and GPCRs upon phosphorylation by GRKs, hence these regulatory proteins terminate GPCR intracellular signal transduction, and control G protein subunit coupling, therefore controlling second messenger signal transduction.
Arrestins function as signalosome adaptor or scaffolding proteins. According to studies, β-arrestins 1 and 2 in metastatic breast cancer regulates cell proliferation, enhance cell invasion and migration, transmit anti-apoptotic survival signals and affect other features of tumours (e.g. angiogenesis, invasion and metatstatic potential). GPCR internalisation and desensitisation are initiated by β-arrestins 1 and 2. Moreover, β-arrestin1 is directed into the nucleus to transactivate epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptors (VEGF), hence this mechanism enhances invasion and metastasis of cancer by stimulating various signaling cascades including mitogen-activated protein kinase (MAPK), ERK, Akt, and nuclear factor (NF)-κB pathways (Figure 2). When EGFR is introduced, angiogenic factors are released and result to infrastructure of new blood vessels. Consequently producing tumorigenesis during cancer progression and thus metastasis occurs.
GPCRs are known to induce different signalling pathways initiated by ligand binding. In conclusion, to treat breast cancer or reduce metastasis, specific-downstream regulatory pathways or receptors are the targets. For example, inhibiting CXCR4 must be achieved as it is a receptor, which is a metastatic potential. However, precautions are required in treatment with CXCR4 inhibitors since CXCR4 inhibition initiates progenitor or stem cell mobilization from the bone marrow. Hypoxia-inducible factor-1 (HIF-1α) triggered by hypoxia, increases CXCR4 transcription. In advanced stages of breast cancer, CXCR4 may also couple to Gα12/13 when Gα13 protein is overactivated, resulting to invasion via lymphatic vessels and site-specific metastasis in a Gα12/13-RhoA-dependent manner. This mechanism is mediated by protease-activated receptors (PARs) and lisophosphatidic acid (LPA), which may also be possible targets for metastasis prevention and treatment. Furthermore, combination of novel drug and therapies is also one of the possible therapeutics in managing tumorigenesis and thus breast cancer metastasis.