Dissection of CXCR4 and CXCR7 mediated signaling in breast cancer

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2013

Dissection of CXCR4 and CXCR7 mediated signaling in breast

cancer.

Supervisors: Azra Mujic-Delic (VU) and Annemiek Wilmink (AVANS)

Churnella La Cruz (2042995)

Medicinal Chemistry, Faculty of Sciences, VU

Avans University, ATGM

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Abstract

The involvement of G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) in several diseases has been widely studied and reported. Chemokines and chemokine receptors are well known to be involved in leukocyte trafficking and inflammation. Furthermore, some family members have shown to be overexpressed in cancer. CXCR4 and its ligand CXCL12 have been widely studied and are known that they play an important role in cancer. Recently, it was found that CXCL12 is able to bind another novel

chemokine receptor CXCR7. This receptor is also overexpressed in many different cancers and in addition to CXCL12, CXCR7 binds with lower affinity to the chemokine CXCL11. Specific alleles of CXCL12 are associated with and increases the risk of breast cancer, and CXCL12 has been shown to transactivate Her2/ neu (ErbB2) through its receptor CXCR4, an established oncogene in breast cancer. ErbB family receptors are RTKs from which is also known to be overexpressed in cancers. ErbB2 is overexpressed in 25% - 30% of breast cancer and is associated with poor prognosis and shorter survival. The crosstalk between CXCR7 and ErbB2 in breast cancer is not yet clarified.

The BT-474 breast cancer cell line which expresses CXCR4, CXCR7 and ErbB2, was used in this project to get a clear picture of the cross-talk occurring between these receptors. Using both 3_{H thymidine}

incorporation and cell titer blue assays, the proliferation of BT474 cells was found to be initiated not only by CXCR4 but working together with HER2 and CXCR7. There is an indication that CXCR4 and CXCR7 are involved in the phosphorylation of ErbB2 and ErbB3 kinases. CXCR4, CXCR7 ErbB2 might be involved in the proliferation and downstream signaling via p44/42 MAPK and AKT.

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1. Introduction

Chemokines are a family of small cytokines, a group of small proteins (8 - 15kDa) that bind to chemokine receptors, which are members of G-protein-coupled receptors (GPCRs). Chemokine gradient can induce a response in cells expressing the chemokine receptors that will direct these cells to move to specific locations of the chemokine gradient1_{. GPCR’s which transduce extracellular signals into intracellular effector}

pathways through the activation of heterotrimeric (α, β, γ) G proteins, include about 900 members, representing the most prominent family of validated pharmacological targets in biomedicine2_{. There are 4} subfamilies in which chemokines are divided, based on the arrangement of the first two cysteines residues these are CXC, CC, C and CX3C. The CXC and CC subfamilies contain the majority of the chemokine members, respectively 17 an 28 members3, 36_{. Chemokines bind to the chemokine receptors which are} GPCRs. These chemokines receptors are divided by the CCR, CXCR, XCR1 and CX3CR1 subfamilies. Amongst these, there are also D6, DARC and CCX-CKR which are decoy receptors because they activate traditional G-protein dependent signaling pathways upon ligand binding3_{. Many chemokines can bind} multiple receptors and most receptors are able to bind multiple chemokines (see figure 1).

Figure 1: Chemokines and the chemokine receptors. The interaction of different chemokines with different chemokine receptor and

vice versa. Some chemokine receptors can only bind one ligand3_.

Chemokines and chemokine receptors are well known to be involved in leukocyte trafficking and

inflammation. Furthermore, some family members have shown to be overexpressed in cancer. In this disease, chemokines and their receptors are important for cell trafficking into and out of the tumor

microenvironment1_{. More recently, chemokines and their receptors have been identified as mediators of} chronic inflammation, which plays an important role in the progression of cancers, amongst others breast cancer, cancers of the lung and prostate cancer3_{Earlier studies have also shown high expression of the} chemokine receptors CXCR4, CCR7, CCR9 and CCR10 in a variety of cancer cells3_{. CXCR4 receptor and} its only ligand CXCL12 have been well studied in cancer and are known to play an important role in cancer cell migration and the initiation of metastases. It was recently found that CXCL12 is able to bind another

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novel chemokine receptor CXCR7. This receptor is also overexpressed in many different cancers and inhibition of this receptor by a small molecule lead to tumor growth inhibition in animal studies4_.

As chemokine receptors belong to the family of GPCRs, it is well established that all members are able to signal through the activation of Gi protein leading to Ca2+ release and migration. CXCR7 however is an atypical chemokine receptor as it is not able to signal through G-protein activation. So far, not a lot is known about the signaling of this receptor. It has been purposed that its function is to remove chemokines from the cell surface and internalizing, thereby making them unavailable for other receptors, implying that this receptor works as a ‘decoy receptor’. The CXCR7 characteristics suggest that like CXCR4, CXCR7 plays an important role in regulating immunity, angiogenesis, stem cell trafficking, and mediating organ-specific metastasis of cancer4, 5_{. CXCR7 is able to recruit β-arrestin upon CXCL12 binding and might be responsible} for G-protein-independent signals through ERK1/2 (MAPK) phosphorylation 6,7_{. Also, in some cell lines, it} has been shown that activation of this receptor by CXCL12 leads to phosphorylation of Akt.

In this project, the aim is to find out which signaling pathways are activated by CXCL12 stimulation and which receptors are responsible for the observed responses. For this purpose we are using the breast cancer cell line BT474 that endogenously expresses both CXCR4 and CXCR7. We have also studied different other cell lines, to find one that has similar receptor expression as the BT-474 cells.

The chemokine system of CXCR4 and CXCR7, ErbB family signaling and their functionality are described in Chapter 2. Chapter 3 will define the project approach. The protocols used during this project will be described in Chapter 4, Chapter 5 summarizes the results and these will be further discussed in Chapter 6. Finally conclusive remarks are presented in Chapter 7.

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2. The Signaling Network

2.1. The Chemokine System and Signaling

Chemokines are a family of small cytokines, a group of small proteins (8 - 15kDa) that bind to chemokine receptors, which are members of G-protein-coupled receptors (GPCRs). Chemokine gradient can induce a response in cells expressing the chemokine receptors that will direct these cells to move to specific locations of the chemokine gradient1_{. Chemokines are divided into four different groups, CXC, CC, CX3C or C,} depending on the position of the conserved cysteine residues found in the N-terminus of the polypeptides1,8_. Chemokines are involved in development, normal physiology and immune responses.

The chemokine receptors are heterotrimeric seven-transmembrane receptors coupled to G proteins, found on the surface of all cells of multicellular organisms and are major mediators of intracellular signalling9, 5_.

G protein-coupled receptors (GPCRs), which transduce extracellular signals into intracellular effector pathways through the activation of heterotrimeric (α, β, γ) G proteins, include about 900 members, representing the most prominent family of validated pharmacological targets in biomedicine2_{. GPCRs are} present in the human genome, and each receptor subtype respond to hormones, neurotransmitters,

chemokines, odorants, or tastans4_{. The signals of GPCRs are transduced by GTP-binding proteins, usually} known as G proteins. The α-subunits binds GDP or GTP and has an intrinsic, slow GTPase activity. In a resting cell, the receptor-associated G proteins form a stable inactive complex containing guanosine diphosphate (GDP) bound to Gα subunits (figure 2). Binding of a ligand (chemokine) to the receptor stimulates a rapid exchange of GTP for GDP on Gα10_.

Figure 2: G-proteins and their interaction with GPCRs. The known coupling specificity of the GPCRs to their alpha subunit and the

effect of each unit11_.

The binding of GTP causes Gα to dissociate from Gβγ, activating Gα and Gβγ and making it possible for these proteins to bind various effectors. The binding of an effector functions as a switch to activate or deactivate different systems in the cell, and the signal will be passed to different second messengers. The activated Gα interacts and regulates molecules as Ca2+_{, Na}+_{-channels, adenyl cyclase, protein kinases (see} figure 2). The activated Gβγ can regulate a many effectors in addition to the K+_{-channel, also adenylyl} cyclase, PI3-kinase, and β-adrenergic receptor kinase. The essential GTPase activity of Gα leads to the alteration of bound GTP into GDP and consequently inactivating the G protein cascade37_.

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2.1.1 Chemokine Signaling

In order for the immune system and responses to be effective, cells should be recruited to the site of inflammation and then be properly activated and regulated. Growing evidence suggest that the chemokines are not only involved in chemotaxis, since chemokines have been involved in dendritic cell maturation, macrophage activation, neutrophil degranulation and B cell antibody class switching. Chemokines are involved in the recruitment, polarization, activation and differentiation of T cells53_.

Chemotaxis of inflammatory cells toward a chemokine gradient requires signals coming from the short intracytoplasmic tails of the chemokine GPCRs8_{. The binding of a chemokine to its GPCR activates a series} of downstream effectors that facilitate internalization of the receptor and signal transduction1_{. Ligand} binding to most chemokine receptors, like other 7-TM receptors, activates the receptor and leads to phosphorylation of the receptor by a G protein receptor kinase (GRK). Phosphorylation of the receptor causes recruitment of a cytosolic adapter protein, β-arrestin. The complex of ligand, receptor, and β-arrestin is then internalized, removing the receptor from the cell membrane12_.

Chemokine signaling results in the transcription of target genes that are involved in cell invasion, motility, interactions with the extracellular matrix (ECM) and survival13_{. Chemokine signaling induces the expression} of integrins on the cell surface that causes adhesion of cells to the endothelial wall at the injured site1, 8_. Polarization of the actin cytoskeleton is also one of the main responses of signal transduction which allows directional sensing, cell polarization, accumulation of small GTPases, PI3K, resulting in actin

polymerization and F-actin formation. Actin polymerization and breakdown accounts for the extension and retraction of lamellipodia that propel the cell toward the chemokine gradient1,8_{. Directed migration of cells} that express the appropriate chemokine receptor occurs along a chemical gradient of ligand -known as the chemokine gradient- allowing cells to move towards high local concentrations of chemokines11_{. Other} changes that occur after receptor activation include classical calcium fluxes within the cell and the generation of both oxygen radicals and lipid mediators8_.

2.1.2. CXCR4 axis

CXCR4 is a highly conserved seven-span transmembrane GPCR that binds the ligand CXCL12α. It is expressed in a broad range of tissues during development, including immune and central nervous system, and can mediate migration of resting leukocytes and hematopoietic progenitors in response to CXCL12 functioning in a number of physiological processes. In the immune system, CXCR4 is highly expressed in monocytes, B cells and naive T cells in the peripheral blood as well as early hematopoietic progenitor cells in bone marrow. Once CXCL12 binds to CXCR4, the receptor forms a complex with the Gαi subunit G protein, resulting in inhibition of adenylyl cyclase-mediated cyclyc adenosine monophosphate production and

mobilization of intracellular calcium. Dissociation of the Gα subunit from Gβγ leads to activation of multiple downstream targets, including MAPK, JNK and AKT effectors3, 4, 5_.

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Figure 3: CXCR4 downstream signaling pathways after binding of its only ligand CXCL12 38_.

There are four Gα subunit families namely, Gαs, Gαi, Gαq, and Gα12 and each subunit transduces the GPCR signal via different pathways. In the CXCL12/CXCR4 pathway as mentioned earlier, Gαi inhibits adenyl cyclase whereas the Gαs subunit stimulates adenyl cyclase38_{. Both Gαi and Gαq activate MAPK, while Gαq} also activates the transcription factor NFκB. The Gαq family acts via PLC to generate the two second messengers IP3 and DAG. In the meanwhile, the Gβγ subunit can trigger PLC activation and the formation of IP3 and DAG, which results in mobilization of Ca2+. 38_.

2.1.3. CXCR7 axis

The receptor CXCR7, also known as RDC-1, was originally cloned on the basis of the homology with conserved domains of GPCRs. The CXCR7 gene maps to human chromosome 2, where the genes encoding CXCR1, CXCR2 and CXCR4 are located.

In addition to CXCL12, CXCR7 binds CXCL11 with lower affinity. CXCR7 expression has been found in T lymphocytes and during CXCL12-mediated chemotaxis. CXCR7 expression is tightly regulated in B cell development and differentiation. It has also been shown that CXCR7 is elevated in endothelial cells associated with tumors. The CXCR7 characteristics suggest that like CXCR4, CXCR7 plays a role in regulating immunity, angiogenesis, stem cell trafficking, and mediating organ-specific metastasis of cancer. Ligand binding to CXCR7 results in crosstalk with CXCR4 mediated by intracellular signaling molecules. CXCR7 may heterodimerize with CXCR4 and modulate signaling pathways initiated through CXCL12-CXCR4, but some effects of CXCX7 in growth and metastasis of cancer cells and cell migration seems to be independent of CXCR412_.

Growing evidence suggest that CXCR7 functions as a decoy receptor, which does not activate Gi pathways of a chemokine receptor that would result in GTP hydrolysis or calcium mobilization3, 5,10_{. Decoy chemokine} receptors bind chemokine ligands and internalize without initiating signaling pathways characteristically associated with activated chemokine receptors12_{. Recent studies demonstrated that CXCR7 interacts with} β-arrestin in a ligand dependent manner. It signals through β-β-arrestin and act as an endogenous β- β-arrestin-biased receptor3, 4, 5_{. An earlier study showed that an exposure to chemokine ligands drives progressive} increases in association of β-arrestin 2 with CXCR7, contrasting with the rapid onset and short duration of this process for CXCR4 and most other chemokine receptors. They also established that β-arrestin 2 is necessary for the efficient uptake of chemokine ligands by CXCR754_.

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2.1.4. CXCL12, CXCR4 and CXCR7 axis in breast cancer

As observed in breast cancer cell lines, detection of CXCR4 protein does not necessarily indicate CXCR4-mediated signaling. There is enough evidence that CXCR4 interacts with several growth factor receptor tyrosine kinases. CXCL12 is produced by myofibroblasts associated with breast cancer, and increases tumor growth by malignant cell proliferation, survival and angiogenesis (figure 4). Specific alleles of CXCL12 have been associated with increased risk for breast cancer, and transactivation of ErbB2, which is an affirmed oncogene in breast cancer4_{. A function of CXCR7 in cell growth/survival was observed in} CXCR7-transfected cells, where it expanded more rapidly in culture than non-CXCR7-transfected cells. The expression of CXCR7 provides a survival advantage to cells that becomes experimentally understandable using culture conditions that are less optimal for cell growth4, 5_{. An increased CXCR7 mRNA expression was also}

observed in several studies. The expression of CXCR7 on breast cancer cells in animal models enhances the ability of these cells to seed and proliferate in lung metastasis14_{. Another study has shown that high levels of} CXCR7 in breast cancer facilitate cancer cells ability to pass through the blood-brain barrier, leading to brain metastasis15_.

Figure 4: Signalling pathways of the crosstalk between the receptors CXCR3, CXCR4 and CXCR7. The bold lines represents the

signal after ligand binding, the thin line denotes regular signals and the dotted lines stand for signals that are not yet well affirmed4_.

Cell proliferation mediated via PI3 kinase-AKT activation through both Gαi and Gβγ subunits is induced by CXCR4. The activated Gαi also induces Ras–RAF–MEK–ERK and PI3K–AKT–NFkB signaling cascades to ease survival and proliferation of tumor cells. CXCL12 induces tumor cell proliferation by activation of MEK-ERK and PI3K-AKT signaling pathways4, 40_{. This occurs by inactivating the apoptosis regulator} protein Bcl-2. Cell proliferation mediated via PI3K -AKT activation through both Gαi and Gβγ subunits is induced CXCR4. The activated Gαi also induces Ras–RAF–MEK–ERK and PI3K–AKT–NFkB signaling cascadesto ease survival and proliferation of tumor cells. The recruitment of β-arrestin by CXCR4 has been

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described as regulator of receptor trafficking and reduction of the sensitivity, via G-protein coupled receptor kinase (GRK) 4_{. A novel role of β-arrestin has been shown in CXCR7 mediated activation of ERK1/2,} inducing tumor cell proliferation and adhesion. It has been shown that CXCR7 may act as a scavenger of CXCL12, generating it’s gradient and leading to distinctive signaling of the CXCR4. Heterodimerization amongst these receptors has also been reported as they act as co-receptors41_{. It has also been suggested that} β-arrestin is constitutively recruited after dimerization instead of Gi proteins.

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2.2. The EGFR/ErbB/HER family

The epidermal growth factor receptor (EGFR) and its close relatives HER2/c-Erb-B2, Erb-B3 and Erb-B4 are type 1 transmembrane receptors tyrosine kinases

(RTK) with key roles in embryonic development, tissue renewal/repair and cancer16_{. All family} members have in common a cysteine-rich extracellular ligand-binding domain, a single membrane-spanning region and a cytoplasmic domain composed of a tyrosine kinase domain and several tyrosine residues that are phosphorylated upon receptor activation17_{. A family of ligands, the} EGF-related peptide growth factors, bind the extracellular domain of ErbB receptors leading to the formation of both homo- and heterodimers at the cell surface, followed by internalization of the dimerized receptor17, 18, 19_{. Dimerization}

consequently stimulates the intrinsic tyrosine kinase activity of the receptors and triggers

autophosphorylation of specific tyrosine residues within the cytoplasmic domain. These

phosphorylated residues serve as binding sites for the recruitment of signaling molecules involved in

the regulation of intracellular signaling cascades17, 18_{. Dysregulation of ErbB kinases occur in a variety of} diseases, including cancer, diabetes, and autoimmune, cardiovascular, inflammatory and nervous system disorders. Various soluble factors regulate the activation state of cellular receptors, which are coupled to a signal transduction network that ultimately generates signals defining the required biological response21_.

The signaling network composed of the epidermal growth factor (EGF) family of hormones and their receptors regulates the proliferation of many tissue types. Deregulation of this network is a significant factor in the genesis of progression of several human cancers, including breast cancer17_{. Overexpression of HER2,} which occurs in 25% - 30% of breast cancer, is associated with poor prognosis and shorter survival27, 39_{. Such} overexpression produces intense signal generation and activation of downstream signaling pathways,

resulting in cells that have more aggressive growth and invasiveness characteristics39_.

2.2.1. ErbB receptor signaling

The ErbB signaling system comprises a highly complex and interactive network. Signals are initiated at the cell surface, where a ligand interacts with the receptor, and the resulting receptor dimerization and activation relays and amplifies the signal through the cytoplasm via a complex system of enzymes, proteins and small molecule secondary messengers21_.

This process of signal transduction ends up in the nucleus, where gene control and protein transcription are modified, producing effects on regulatory processes of the cell, such as differentiation, adhesion, growth,

Figure 5: There four members of the Erbb family. Each receptor is

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migration and apoptosis. Dysregulation and/or disruption of receptor function of any or all downstream processes may result in cell transformation and malignancy21, 22_.

Activation of ErbB receptors is initiated by ligand binding, leading to the formation of both active homo- and heterodimeric ErbB receptor complexes and consequently ErbB stimulation. Dimerization results in

activation of the kinase domain, transphosphorylation, and the induction of intracellular signaling cascades that mediate cell growth and survival21, 43_{. In their inactive monomeric state, the tyrosine kinase domains of} the receptors are not phosphorylated. The process of dimerization activates the cytoplasmic domain of the receptor, resulting in the autophosphorylation of multiple tyrosine residues within the domain. This in turn activates downstream proteins that induce physiological responses. Signaling diversity depends not only on the presence of specific receptors, but also on the characteristics of individual ligands. Two important signaling pathways activated by the ErbB family dimers are the phosphatidylinositol-3-kinase (PI3K) pathways, which promote tumor cell survival, and the mitogen activated protein kinase (MAPK) pathway, which stimulates cell proliferation. Intracellular signal transduction is initiated by the cytoplasmic domain of the receptor21_.

2.2.2. ErbB receptors in breast cancer

EGFR receptors are found in 30– 48% of breast cancers overall, and their presence is associated with estrogen receptor negative phenotype. EGFR overexpression has been found to be a significant prognostic factor for relapse and survival in node negative23_{and early-stage breast cancer}18_{. Some of the best systems} for studying the association of ErbB2 expression with cancer are breast cancers, among other things the protein is overexpressed, due to gene amplification, in 15– 30% 25_{of invasive ductal breast cancers.}

Overexpression correlates with tumor size, spread of the tumor to lymph nodes, high grade, high percentage of S-phase cells and aneuploidy and lack of steroid hormone receptor, implying that ErbB2 confers a strong proliferative and survival advantage on tumor cells. Another important observation pertaining to ErbB hetero-dimer collaboration during tumor development is that expression of ErbB3 is seen in many of the same tumors that overexpress ErbB2, including breast, bladder and melanomas. Furthermore, many ErbB2 overexpressing breast tumors display elevated levels of phosphotyrosine on ErbB3, probably as a result of spontaneous dimerization with ErbB218, 26_{. An earlier study showed mammary tumors of transgenic mice} expressing transforming mutants of the ErbB2 gene exhibit selective upregulation of the ErbB3 expression and activity, suggesting that there might be a selectivity profit causing both receptors to co-expression18_.

2.3. Crosstalk between the chemokine axis and ErbB receptors

The ErbB network might integrate not only its own inputs but also heterologous signals, including hormones, neurotransmitters, lymphokines and stress inducers. Many of these trans-regulatory interactions are mediated by protein kinases that directly phosphorylate ErbBs, thereby affecting their kinase activity or endocytic transport. The most extensively studied mechanism involves activation of GPCRs by agonists such as lysophosphatidic acid (LPA), carbachol (which specifically activates muscarinic acetylcholine receptors) or thrombin (figure 5). Experiments done with mutants and inhibitors of ErbBs imply that the ability to start cell division of some GPCR agonists requires transactivation of ErbB proteins27_{. These agents increase} tyrosine phosphorylation of ErbB1 and ErbB2, either by increasing their intrinsic kinase activity or by inhibiting an associated phosphatase activity19_.

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An earlier study documented the involvement of the CXCR4-CXCL12 axis in the trans-activation of

members of the ErbB receptor family through cross-activation of the Src non-receptor tyrosine kinase family. The Src kinase receptor family is involved in many signaling pathways and is known to interact with

multiple tyrosine kinase receptor families, including ErbB receptors28_{. Src kinase is activated by G-protein} coupled receptors through interactions with Gβγ-subunit and can also be activated by binding to ß-arrestin29, 30_.

Figure 6: Crosstalk between the ErbB network and GPCR signaling pathways. Src phosphorylates the intracellular domains of ErbB

receptor, while GPCRs are able to transactivate Src.

Since Src family tyrosine kinases are activated downstream of many receptor tyrosine kinases,

transactivation provides an indirect mechanism of GPCR stimulated Src activation29_{. Src activated through} crosstalk is clearly important to GPCR signaling, precisely because it is important to signaling by the transactivated receptor tyrosine kinases or focal adhesion complex. In cells where transactivated EGF receptors account for GPCR-stimulated ERK1/2 activation, Src activation is vital to GPCR-stimulated cell proliferation.

As it has been thought in earlier studies, the GPCR signaling and the mitogenic/survival signaling involving the Src family tyrosine kinases are distinct, it is now clear that both kinases function as a complex of components of common signaling networks29_.

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3. Research Approach

CXCR4 receptor and its only ligand CXCL12 have been well studied in cancer and recently it was found that CXCL12 is able to bind another newly discovered chemokine receptor CXCR7, which is also overexpressed in many different cancers. Previous studies have shown that GPCRs, amongst others CXCR4, indirectly activate Src, which phosphorylates the intracellular domains of ErbBs on tyrosine residues. However it is still unclear how this novel chemokine receptor CXCR7 interacts with ErbB receptors. The aim in this project is to unravel the crosstalk between chemokine receptors CXCR4 and CXCR7 and the ErbB receptors and their role on the p44/42 MAPK and Akt pathways in breast cancer, activated by CXCL12 stimulation and which receptors are responsible for the observed responses.

The human breast cancer cell line BT-474, which expresses CXCR4, CXCR7 and all the ErbB family receptors was used for the different assays. Also several new cell lines will be studied to find out if any of them show similar expression pattern as seen in BT-474 cells. A reverse phase protein array (RPPA) has been performed in collaboration with MD Anderson Cancer Center in Houston TX, to determine changes in (phosphorylation) protein levels upon stimulation of these cells with CXCL12. The results that were obtained by RPPA will be validated. The expression of these receptors will be evaluated by RealTime PCR (qPCR), ELISA and Whole cell binding assays. The phosphorylation of ErbB2 and ErbB3, MAPK and Akt will be assessed by immunoblotting. The proliferation of the cells will be studied by the 3H-Tymidine incorporation, the Celltiter-blue assay and by a Cell cycle assay, while the cells are stimulated with different inhibitors and combination of these inhibitors. CXCR4, CXCR7 and ErbB2 inhibitors will be tested to obtain a clear picture of the cross talk occurring between these receptors.

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4. Materials and Method

Cell Culturing

The human cell line BT-474 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin (Pen/Strep) and maintained as monolayers at 37°C in 5% CO2. Phosphate Buffered Saline (PBS) was used to wash the cells before trypsinization with trypsin-EDTA and resuspended in DMEM.

Western Blot

BT-474 (4.0x105_{) cells were cultured in each required well of a 6 well plate, for 24 hrs and then}

serumstarved for 36 hrs. Appropriate wells were pre-incubated 1 hour with 3.16µM of either IT1t (CXCR4 antagonist), VUF11207.3 (CXCR7 antagonist), combination of IT1t and VUF11207.3 or left untreated (DMSO control). Cells were then stimulated with 1nM CXCL12 at different time points followed by removing the media and washing the cells with ice-cold PBS, where after RPPA lysis buffer [containing

α-complete (Roche), NaVO

3, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF)] was added. Cells were scraped from the plate, sonicated and centrifuged to collect the supernatant. The protein concentration in lysates was determined using BCA Assay Kit (Pierce). Lysate samples containing equal amounts of protein (15 µg) were then added to SDS-page loading buffer and H2O and heated for 5min at 95°C. Proteins were separated using a 8% SDS–PAGE gel and electrotransferred to a PVDF membrane (GE Healthcare) at 200mA for 60 min. The membrane was immersed in 5% skim milk in a TBS-T solution [10 mM Tris base, (pH 8.0), 150 mM NaCl, 0.1% Tween-20] for 1h at room temperature and rinsed once quickly in TBS-T. After 1 hour in blocking buffer, membranes were incubated with primary antibody’s in 5% bovine serum albumin (BSA) in TBS-T overnight at 4°C. The membranes were subjected to three 15 min rinses in TBS-T and then incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody for 1 h at room temperature, and subjected to three 15 min rinses in TBS-T. The blot was developed with a SuperECL Plus kit (Applygen, Beijing, China), antibody’s were detected by chemoluminescense (BioRad Molecular Imager).

Table 1: List of antibodies

Name Company Ordernr. Dilution Host

pHer 2 (Tyr1248) Millipore 06-229 1:1000 Rabbit

pHer 3 (Tyr1289) Cell Signaling 4791S 1:1000 Rabbit

Total Her 2 Cell Signaling 4370S 1:1000 Rabbit

Total Her 3 Cell Signaling 4060P 1:1000 Rabbit

pMAPK (42/44

phospho) X Cell Signaling 4370S 1:1000 Rabbit

pAkt (S473) Cell Signaling 4060P 1:1000 Rabbit

p44/42 MAPK Cell Signaling 9102S 1:1000 Rabbit

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Tritiated Thymidine Incorporation Assay

BT-474 (4.0x104_{) cells were seeded on a poly-L-lysine coated 48-wells plate for 24 hrs, where after the} medium was removed and replaced with serum-free DMEM containing inhibitors and incubated for 48hrs. Hereafter 3_{H-Thymidine was added to the culture and incubated for 24 hrs, where after the cells were washed} twice with ice-cold PBS and fixed for 10 minutes with ice-cold methanol. The cells were washed 6 times with H2O and incubated for 3hrs at room temperature in 0.2 M NaOH 1% SDS solution on a shaker. Each well content was transferred into a 20ml scintillation tube containing 4ml scintillation fluid. The 3_{H in the} scintillation counter was measured by a beta counter.

ELISA

BT-474 cells were seeded on a poly-L-lysine 48-wells plate and after 24hrs incubation, medium was aspirated and cells fixed with 4% PFA at RT for 10 min. Cells were washed with TBS and incubated for 30min at RT with TBS/0.5% NP-40 (Boehringer) to permeabilize cells. After incubation, plate was blocked using 0.1M NaHCO3 + 1% skim milk and incubated 4hrs at RT on a shaker. After blocking, the diluted (in blocking buffer) primary antibodies against CXCR4 (IMG-537, ImGENEX), CXCR7 (C1C2,GeneTex) and HER2 (Mab 1129, R&D) were added and incubated o/n at RT on a shaker. Cells were washed 3 times with TBS, subsequently secondary antibody (anti-mouse) was added and incubated for 2hrs at RT on a shaker. After incubation, cells were washed 3 times with TBS and OPD (Sigma) substrate solution was added and stained on a shaker. Coloring was stopped by adding 1.0M H2SO4. Each well content was transferred to a 96-wells plate and the OD490 was measured.

RNA Isolation, Reverse Transcription and qPCR

Purification of total RNA from BT-474 cells was done using the RNeasy Mini Kit (QIAGEN,Venlo, The Netherlands) in accordance with the manufacturer’s recommendations. RNA quality was assessed using a 2% agarose gel. DNA was synthesized from 500ng of total RNA using the iScript cDNA synthesis kit (Bio-Rad laboratories B.V, The Netherlands). The DNase mix was made and mixed with cDNA followed by a volume adjustment with H2O to a final volume of 10µl. Sample was incubated for 30min at 37°C and subsequently stopped by adding EDTA and incubating the sample for 10min at 65°C. After cooling the samples to 42°C, the iScript mastermix was added and incubated for 60min at 42°C followed by a 5 min incubation at 85°C. Samples were cooled to 4°C and then diluted 20 times in nuclease free H2O. A mix of cDNA/dH2O, primers working solution (5 pM) and SYBR Green was made and pipetted into a 96-wells plate. The plate was measured and assessed using the 2 step SYBR Green 60°C + melting curve protocol on the MyiQ Detection system (Bio-Rad).

Cell Titer Blue Proliferation Assay

BT-474 (2.0x105_{) cells were seeded in a white 96-wells plate and after 24hrs incubation, medium was} replaced with serum free medium and 10% FBS medium containing the inhibitors. After incubation for 72 hours, Cell Titer-Blue dye (Promega, USA) diluted 1:1 in DMEM was added. After 3 hours incubation fluorescence was measured using Victor 2 (Wallac, 1420 multilabel counter).

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Immunofluorescence

BT-474 cells were seeded on a poly-L-lysine coated coverslip in a 12-wells plate. After 24hrs cells were fixed with 4% formaldehyde/PBS for 10min and washed 3 times with PBS for 5min. Cells were incubated in 5% skim milk for 30 min and for permeabilized cells 5% skim milk + 0.15% TritonX100 was used. Cover slips were then incubated with primary antibody against CXCR4 (12G5) and CXCR7 (C1C2) dissolved in blocking buffer on parafilm for 1 hour. After washing 3 times with PBS for 5min, cover slips were incubated with green Alexafluor488 secondary antibody in blocking buffer on parafilm for 1 hour. Cover slips were sequentially stained with Alexafluor594 red membrane marker and DAPI blue nucleus marker and then loaded on to glass plates with mounting medium and incubated overnight in the fridge. The slides were viewed the day after using a fluorescent microscope.

Guava Cell Cycle

BT-474 cells (5.0x105_{) were seeded on a 6 well plate for 24hrs, and here after serum starved for 24hrs.} Medium was aspirated and cells were stimulated for 72hrs with different inhibitors. The cells were harvested, diluted to 1.0x106_{cells/ml and added to a 96-well round bottom plate. After rinsing with PBS, 70% ethanol} was added to the cell pellet and refrigerated o/n. The cells were centrifuged the day after and supernatant was removed. The pellet was rinsed with PBS, supernatant discarded and Cell Cycle Staining Reagent (EMD Millipore Corporation, Hayward CA) was added to each well. Plate was incubated at RT shielding away from light for 30 min before acquiring the samples on the Guava PCA-96 system.

Whole Cell Binding

Cells (4.0x104_{) were seeded on a poly-L-lysine coated 48 well plate for 24hrs, where after the medium was} aspirated and HEPES binding buffer (HBB) with 0,5% BSA with or without CXCL11 and CXCL12 cold ligands was added. The radiolabeled ligand (125_{I CXCL12, Perkin-Elmer) was then added to the plate and} incubated for 3 hrs at 4°C. The cells were washed with HBB wash buffer, harvested with RIPA buffer and transferred into counting tubes. Bound 125_{I CXCL12 radioactivity was measured with Compugamma CS} Universal Gamma Counter.

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5. Results

5.1 Evaluation of the expression of CXCR4, CXCR7 and other project related receptors.

The gene expression of CXCR4, CXCR7 and the HER2 and other study related genes were evaluated in the BT474 breast cancer cell line by qPCR and the endogenous protein expression was

evaluated by ELISA. We were interested in the expression of CXCR3, CXCL11 and CXCL12 since it’s well known that ligand CXCL11 and CXCL12 both bind with high affinity to CXCR7. Also it is know that CXCR3 interacts with ligand CXCL11. To exclude the presence and influence of CXCR3 and CXCL11 on the results of future experiments, their expression was evaluated31_{. β-arrestin play an essential role in the} interrelated processes of homologous desensitization and GPCR sequestration, which leads to the termination of G protein activation. β-arrestin binding to GPCRs both uncouples receptors from heterotrimeric G proteins and targets them to clathrin-coated pits for endocytosis32_{. Also recent studies} demonstrated that CXCR7 interacts with β-arrestin in a ligand dependent manner5_{. Earlier studies have} revealed crosstalks between the chemokine and ErbB families, making chemokine receptors a potential target in HER2 positive breast cancers. Because of this knowledge, we studied the expression of

β-arrestin1/2 (data not shown) and HER family receptors in BT474 cells. In addition to BT-474 cells, new cell lines (see table 5.1) were studied in trying to find one that shows similarities in receptor expression. By doing this we aimed to find a cell line that could be used in the other assays, and in this way compare the behavior of each cell while being treated under the same conditions.

Table 5.1: Different cell lines used and their origin.

Name Origin

PC-3 Prostate cancer

OE Head & Neck cancer

U373 Glioblastoma

HepG2 Liver hepatocellular carcinoma

Cado-Es1 Human Ewing’s sarcoma

Figure 5.1.1 A (left): mRNA expression levels in BT474 breast cancer cell line. B (left): mRNA expression levels of CXCR4,

CXCR7 and HER2 in OE, PC-3 and U373 cell lines.

On mRNA level we observed the expression of CXCR4, CXCR7 and HER2. Also the expression of β-arrestin1/2 and the HER family receptors in the BT474 cells was demonstrated. The qPCR results also made

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it clear that CXCR3 and CXCL11 are not expressed in these cells, which is a very pleasing result knowing that the results in other experiments will not be influenced by this receptors and ligand. The OE cell line expresses CXCR4, CXCR7 and HER2 while the expression observed in the PC-3 and U373 cell lines were not significant for this study. Certainly the fact that PC-3 cells express CXCL11 (data not shown) is a disadvantage in this study.

We continued studying the endogenous expression of HER2 in the OE cells since they showed more similarities with BT-474 compared to the other cell lines and some other new cell lines, by comparing them with the BT-474 cell line. The expression was studied on the cell membrane and intracellular by

permeabilizing the cells. We also studied the OE’s expression of CXCR4, CXCR7 and HER2 on protein level by western blot.

Figure 5.1.2 A(left): HER2 expression in various cell lines. The assay was performed on permeabilized(Perm) and

non-permeabilized (NP) cells, in order to detect antigens on cell membrane and intracellular. A control (cntr) was taken for both condition separately. B (right): CXCR4, CXCR7 and HER2 protein expression in various cell lines. BT-474 cells were used as a control for these receptors, while OE cells and BT-474 cherry stained(cry) were tested for the presence of these receptor proteins.

Compared to the BT-474 cells, the expression of HER2 in the other cell lines is negligible. These cells do express HER2, but their expression is not similar to BT-474. This was also the case for the HER2 protein expression in the OE cell line. Only CXCR7 was similar in both BT-474 and OE cells on protein level and this similarity was earlier observed in the Ct values obtained from the qPCR results.

The results obtained from the ELISA for the expression of CXCR4 and CXCR7 in the HepG2 and Cado-Es cell lines clearly show the expression of CXCR7 on the cell surface and intracellular. There is no significant expression of CXCR4 observed in these cell lines.

Figure 5.1.3 A(left): CXCR4 expression in BT-474, HepG2 and Cado-Es cell lines. B (right): CXCR7 expression in BT-474, HepG2

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The whole cell binding assay uses cold ligands CXCL11 and CXCL12 for the chemokine receptors while competing with 125_{I radiolabeled CXCL12 (figure 5.1.4). The chemokine ligand CXCL12 was specific} for CXCR4, while CXCL11 and CXCL12 both target CXCR7. As observed from the whole cell binding, it is clear that CXCR7 is expressed in BT-474 due to significant displacement of CXCR7 specific ligand

CXCL11 by the radioligand. These results also suggest that these cells also express in a lower quantity the CXCR4 receptor. These results confirm the results from the other assays earlier mentioned, where there could be observed that CXCR7 has a higher expression in BT-474 cells.

Figure 5.1.4: Whole cell binding different cell lines compared to . The cells were incubated with CXCR4 and CXCR7 specific cold

ligands that followed by displaced with 125_{I-radiolabeled CXCL12.}

Cado-Es cells are those showing more similarities with BT-474 cells. The HepG2 and OE cell lines have a different expression pattern compared to BT-474 cells.

5.2 CXCR4 and CXCR7 dependent transactivation of ErbB receptor phosphorylation.

To establish the effect of CXCL12 ligand binding to CXCR4 and CXCR7 on the phosphorylation of ErbB2 and ErbB3, BT-474 cells were treated with CXCL12 at different time points. Western blotting was used for the identification and quantification of specific proteins; in this project it is Tyr1248 (major

autophosphorylation site in ErbB2) and Tyr1289 (phosphorylation site in ErbB3).

Figure 5.2.1: The phosphorylation of ErbB2 (A) and ErbB3 (B) in BT-474 cells. Cells were treated with 1nM CXCL12 at different

time points after 36hrs of serum starvation. Figure A shows the blot incubated with the antibody against the phosphorylation site Tyr1248 at ErbB/HER2 and figure B shows the blot incubated with the antibody against the phosphorylation site Tyr1289 at ErbB/ HER3.

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A previous report demonstrates that HER2 enhances the expression of CXCR433_{, while a novel} mechanism of HER2-neu transactivation induced by CXCL12/CXCR4 interactions in HER2- and CXCR4-expressing breast cancer cells through, G protein–dependent signal transduction mechanism has been

studied28_{. It has been proposed that ErbB3 might be a partner for ErbB2 in promoting cellular transformation} and proliferation in breast cancer26_{. Due to this knowledge, the phosphorylation of both receptors in BT-474} became interesting to study.

After quantification and normalization done by the Molecular Imager (BioRad), a time-dependent

stimulation of the phosphorylation of Tyr1248 and Tyr1289 after binding with CXCL12 was observed. The phosphorylation reached the maximum after 10 min stimulation. These results could not be reproduced after trying several times, we were not able to observe any CXCL12 induced phosphorylation (see figure 5.2.2). To distinguish between the effect of CXCR4 and CXCR7 individually on the transactivation of ErbB2, BT-474 cells were either pre-incubated for 1hr with the CXCR4 inhibitor It1t, the CXCR7 functional

antagonist VUF, and also Herceptin the monoclonal antibody that interferes with ErbB2.

Figure 5.2.2: The phosphorylation of ErbB2 in BT-474 cells untreated or treated with Herceptin, IT1t or VUF11207.3 and stimulated

at different time points with CXCL12. Total MAPK was taken as loading control.

As mentioned earlier, stimulation of the cells with CXCL12 didn’t exhibit a time dependent

phosphorylation of ErbB2. Inhibition of ErbB2with Herceptin decreased the ErbB2 signal in the absence of CXCL12 stimulation. This can be an indication of the interference of Herceptin with ErbB2 phosphorylation. The phosphorylation increases after 2min stimulation with CXCL12, and decreases around 60min to basal level. This is a clear indication that ErbB2 can be transactivated even though the receptor has been interfered by Herceptin. It was further observed that IT1t was not able to decrease the CXCL12 induced

phosphorylation of ErbB2, suggesting that CXCR4 is not or partially involved in the transactivation of ErbB2. However, VUF was able to decrease the CXCL12 induced phosphorylation of ErbB2 after 10min, implying that also CXCR7 might be involved in the transactivation of ErbB2.

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5.3 Assessment of CXCL12 (SDF-1) activation of p44/42 MAPK and Akt pathways in BT474

cells.

The activation of both CXCR4 and CXCR7 signaling may affect several major signaling pathways involved in cell survival, proliferation and migration (see Figure 4). Earlier studies have shown that CXCL12 activates PI3K/Akt, IP3, and MAPK pathways via CXCR4, thus regulating cell survival, proliferation and

chemotaxis7, 28_{. Less is known about signaling via CXCR7 after CXCL12 activation, making also this} receptor interesting to study. A novel role of β-arrestin has been shown in CXCR7 mediated activation of ERK1/2, inducing tumor cell proliferation and adhesion. It has been shown that CXCR7 may act as a scavenger of CXCL12, generating it’s gradient and leading to distinctive signaling of the CXCR441_{. An} earlier study studied CXCR7-positive glioma cells (CXCR4- and CXCR3-negative) after stimulation by CXCL12, and they observed a transient induction of the p44/42 MAPK (Erk1/2) phosphorylation42_. To distinguish between the effect of ErbB2, CXCR4 and CXCR7 on the phosphorylation of p44/42 MAPK and Akt (Ser473), BT-474 cells were either pre-incubated for 1hr with the CXCR4 inhibitor It1t, the CXCR7 functional antagonist VUF, and also Herceptin the antibody that interferes with ErbB2.

Figure 5.3.1: The phosphorylation of p44/42 MAPK in BT-474 cells untreated or treated with Herceptin, IT1t or VUF11207.3 and

stimulated at different time points with CXCL12. Total AKT was taken as loading control.

As seen from figure 5.3.1, CXCL12 was not able to increase the phosphorylation in the untreated cells and those treated with Herceptin and IT1t. Remarkable is that the basal signal observed in cells treated with Herceptin is higher than the basal level of untreated cells. In the meanwhile, the opposite can be observed in cells treated with IT1t. A CXCL12 time dependent induction of MAPK phosphorylation was seen in cells treated with VUF.

When it comes to the phosphorylation of Akt Ser473, as was also observed for phospho p44/42 MAPK CXCL12 was not able increase the phosphorylation in the untreated cells and those treated with Herceptin and IT1t. Inhibiting CXCR7 and afterward stimulation with CXCL12 seems to increase phosphorylation of Akt (see figure 5.3.2).

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Figure 5.3.2: The phosphorylation of Akt (S473) in BT-474 cells untreated or treated with Herceptin, IT1t or VUF11207.3 and

stimulated at different time points with CXCL12. Total MAPK was taken as a loading control.

5.4 Assessment of BT474 proliferation

To assess the function of the receptors of interest on the proliferation of BT-474 cells, the cells were treated with inhibitors for the different receptors. The 3_{H Thymidine assay was used to study the proliferation of the} cells after being treated with the inhibitors for 48hrs. The thymidine incorporation assay, utilizes a strategy wherein a radioactive nucleoside, 3_{H-thymidine, is incorporated into new strands of chromosomal DNA} during mitotic cell division. Following the monotreatment assays, Herceptin (HER2 antagonist),

VUF11207.3 (CXCR7 antagonist) and IT1t (CXCR4 antagonist) were subsequently assessed in combination with each other to determine whether synergistic inhibitory effects could be achieved, as shown in Figure 5.4.1.

Figure 5.4.1: Assessment of BT-474 cells proliferation after mono- and combination therapy with several inhibitors. Figure A (left)

shows the results of the 3H-Tymidine assay. BT-474 cells were treated with mono- and combination therapy of the different inhibitors. Figure B (right) shows the validated data of BT-474 cells proliferation after monotherapy with different inhibitors, obtained by the CTB proliferation assay.

To validate the results collected from the 3_{H Thymidine assay, a cell CellTiter-Blue Proliferation Assay was} done.Lapatinib (EGFR/HER2 inhibitor) was also used to study the proliferation of the cells, since we expected to see moreinhibition on the proliferation by Herceptin.

VUF, IT1t and Herceptin were not able to decrease proliferation in monotherapy in BT-474 cells. VUF was neither able to decrease proliferation in combination with Herceptin. There was 45% of inhibition observed

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This could be an indication that CXCR4 alone does not initiate proliferation but works together with HER2 and CXCR7 to promote proliferation in the BT-474 cells. Lapatinib shows a significant decrease in

proliferation showing the contribution of HER2 in the proliferation of these cells. Also some apoptosis was observed on microscopic level in cells treated with Lapatinib(3,16uM) and after testing several

concentrations of Lapatinib(not shown), we observed that the cells don’t undergo apoptosis when treated with a concentration < 1.0 uM. These results state that both proliferation and apoptosis was being measured. The activation of signaling pathways as MAPK and Akt that are involved in cell proliferation and survival, and the knowledge that CXCR4, CXCR7 and ErbB2 might induce these pathways makes it

interesting to also study the cell cycle as a validation for the proliferation assays. During the G0/G1, cells are in the resting phase where cells contain two copies of each chromosome. Cells start cycling and DNA synthesis starts in the S phase, while the entire DNA has doubled in the G2/M phase. Proliferating cells show a higher level of cells in the S and G2/M phase. To determine the percentage of cells at various phases of cell cycle, serum starved cells were treated with 1.0µM Herceptin, IT1t and VUF and 0.1µM Lapatinib for 72 hrs, untreated control (serum starved and growing in 10%FBS) and were analyzed.

Figure 5.4.2: Assessment of BT-474 cell cycle classifications, after treating the cells with different factors. Cells were treated 72hr

while being starved. (1.0µM Herceptin, IT1t and VUF was used and 0.1µM Lapatinib)

We expected treated cells to induce the G0/G1 cycle arrest > 72.4% (0% FBS control), since the cells were treated under starvation conditions. As observed in figure 5.4.2 and table 5.4, the treated cells have a higher percentage of the S and G2/M phase compared to the control. This is an indication that the cells are still proliferating after being treated. Cells treated with IT1t have a similar classification as the control, as this was also observed in the other proliferation assays.

Table 5.4: Cell Cycle classification in percentage

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6. Discussion

6.1 Assessment of the receptors expressed in BT-474 cells.

Earlier studies have reported that most HER2-positive patients tend to be insensitive to Herceptin

treatment or acquire drug resistance34_._{GPCRs have an important role in many physiological functions and in} several diseases, including the development of cancer and metastasis, making them interesting as a target for development of new drugs. We examined the BT-474 cells for the expression of different receptors on RNA level, endogenous and protein level. Identifying the receptors of interest on mRNA level, the expression of both CXCR4 and CXCR7 was demonstrated. The absence of CXCR3 and CXCL11 was very pleasing, knowing that this ligand will not be present to bind to CXCR7 causing a different downstream reaction that could be confused with those by CXCL12 binding to CXCR7. Observations from every expression assay that was done, indicated that CXCR7 showed much higher expression compared to CXCR4. The expression of every HER family member receptor was studied, since it has been reported that ligand binding to these receptors can cause them to hetero- or homodimerize and in turn induce tyrosine kinase phosphorylation and signaling cascades in the cell17, 18, 21, 43_{. BT-474 cells brings every HER family member receptor to}

expression.

6.2

ErbB2 phosphorylation induced through activation of GPCR’s

A previous report demonstrates that HER2 enhances the expression of CXCR433_{, while a novel} mechanism of HER2-neu transactivation induced by CXCL12/CXCR4 interactions in HER2- and CXCR4-expressing breast cancer cells through, G protein–dependent signal transduction mechanism has been studied28_{. The result suggests that PI-3K/Akt s473/mTOR pathway might be involved in the HER2-induced} CXCR4 expression. Stimulation with CXCL12 increased the phosphorylation of HER2-neu MDA-MB-361 breast cancer cells. CXCL12-induced transactivation of both HER2-neu and EGFR receptor tyrosine kinases was shown in SKBR3 cells28_.

It has been proposed that ErbB3 might be a partner for ErbB2 in promoting cellular transformation and proliferation in breast cancer26_{. On the one hand, overexpressed active ErbB2 might function on it’s own; on} the other hand, ErbB2 might still need to dimerize with another ErbB receptor. On the basis of the results of an earlier study, they propose that in cancer cells, ErbB2 cannot act alone but requires ErbB3 for its full signaling potential26_{. In addition the cell lines studied expressed also ErbB1, while BT-474 also expressed} ErbB4, but to drive proliferation, neither ErbB1 nor ErbB4 was able to replace ErbB3 as ErbB2’s dimer partner. These suggest that ErbB3 is the most biologically relevant partner for overexpressed ErbB226, 52,_. As shown in figure 5.2.1, we observed a time-dependent increase in phosphorylation just as Cabioglu et al.26 did, after stimulating cells with CXCL12. We were not able to repeat these results for ErbB2 as can also be seen in figure 5.2.2. Inhibition of ErbB2 by Herceptin decreased the basal activity of ErbB2 phosphorylation and after stimulation with CXCL12, the signal increases and goes back to basal level after 10min. Inhibition of CXCR7 shows a decrease in the phosphorylation after 2 min. These results suggest that both CXCR4 and CXCR7 are involved in the transactivation of ErbB2. It could be that these receptors dimerize or even signal through the b-arrestin pathways that can be activated by CXCR7 without ligand binding. These could be taking place in the cells since it’s known that CXCR7 constitutively interacts with Gαi proteins and undergoes CXCL12-mediated conformational changes41_.

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6.3

CXCR4, CXCR7 and HER2 play a role in signaling cascades through MAPK and AKT.

Two pre-clinical studies addressed the role of Herceptin in abolishing the PI3K/Akt signaling pathway, and found that treatment of HER2-gene-amplified causes growth inhibition through PTEN upregulation and downregulation of the PI3K activity and Akt funtion43_{. Inhibition of the HER2 pathways by Herceptin might} result in the activation of other signaling pathways such as the HER3 and IGF1R pathways. It has been demonstrated that the HER3 pathways can in turn activate the PI3K/Akt pathway. An earlier report that studied the effects of Herceptin in different breast cancer cell lines, reported that after treating BT-474 cells with Herceptin the pMAPK and pAkt-ser473 in BT474 was inhibited 8-12hrs after treatment44_{. It has also} been reported that activation of MAPK is one of the consequences of the GPCR-induced activation of Src family kinases46_{. In this study we treated B-474 cells for 1hr with Herceptin subsequently stimulated at} different time points with CXCL12. We observed an increase in the pMAPK signal in those cells treated with Herceptin, compared to the untreated cells. These observations might be a result of the activation of other CXCR4 or CXCR7 pathways in the cells or transactivation via the Src tyrosine kinases. It could be possible that the cells should be treated longer in order to completely inhibit the pMAPK signaling. The pAkt signal was lower compared to the untreated cells, but the signal was not completely inhibited. For both pMAPK and pAkt, we didn’t observe a time-dependent increase or decrease in the phosphorylation levels.

The CXCL12-CXCR4 pathway has been widely studied and reported to be involved in many biological effects in normal and cancer cells. Some of the most important signaling pathways from activated CXCR4 are calcium flux activation, the MAPK p44/42-ELK-1 and PI3K-Akt-NF-kB axis. Some studies observed strong phosphorylation of MAPK p44/42 and serine-threonine kinase Akt short after CXCR4 activation38, 47,48_{. The phosphorylation of Akt and MAPK seem to be both involved in CXCL12 mediated cell motility} and chemotaxis. A study showed the induction of Akt-ser473 phosphorylation after CXCL12 stimulation in MDA-MB-361 cells that highly express CXCR4 and HER2, and the phosphorylation of p44/42 MAPK in SKBR3 cells that express moderate levels of CXCR4 and HER2 compared to the MDA-MB-361 cells28_. When it comes to our observations, BT-474 cells treated with IT1t and afterwards stimulated with CXCL12 showed inhibition of the phopho p44/42 MAPK. This might be a strong indication that CXCR4 is involved in activation of the p44/42 MAPK signaling pathway, as has also been observed in previous studies. We didn’t observe any inhibition when it comes to the phosphorylation of Akt. In an other study done at our lab, by treating BT-474 cells for 2hrs with IT1t, we observed no inhibition. Stimulating the cells in this current study with CXCL12 didn’t show any significant Akt phosphorylation.

Stimulation of CXCR7 with CXCL12 has been proposed to be involved in the activation of p44/42 MAPK, after a study done in CXCR7-positive glioma cells (CXCR4- and CXCR3-negative) 49_._{A previous} study also reported the signaling of activated CXCR7 upon CXCL12 stimulation, through the activation of Akt in prostate cancer cells50_{. In this study we observed a time dependent increase in both p44/42 MAPK} and Akt-ser473 phosphorylation, when treating cells with VUF and subsequently stimulating them with CXCL12. This is an indication that phosphorylation of these pathways still takes place even after CXCR7 has been inhibited.

A previous study proposed based on their data and some other reports, that CXCR7 expression in T lymphocytes might have 2 ways of regulating chemotaxis. First the sequestration of CXCL12 by CXCR7, thereby modifying the extracellular chemokine concentration, and independent on CXCL12/CXCR7

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interactions, CXCR7 is able to heterodimerize with CXCR4 thereby altering CXCR4- mediated activation of G proteins49_{. It has been suggested that CXCR4/CXCR7 heterodimers can regulate CXCR4 functions} through an allosteric mechanism and affect ligand binding and/or signalling41, 49_{. This process explains the} observation previously seen in a study, where the co-expression of CXCR7 with CXCR4 decreased early ERK activation on CXCL12 exposure51_.

6.4

Induction of proliferation in BT-474 cells might be dependent of the crosstalk amongst the

receptors.

Studying the proliferation of the BT-474 cells by treating them with different inhibitors, we observed

that Herceptin didn’t show significant decrease in the proliferation as was expected, knowing that it has been approved for the treatment of breast cancer patients with Her2 protein overexpression and HER2 gene amplification. Earlier studies have reported that most HER2-positive patients tend to be insensitive to Herceptin treatment or acquire drug resistance34_{. This might also be an explanation for the observed lack of} decrease in proliferation in BT-474 breast cancer cells by Herceptin. It is also known that many patients do not respond to monotherapy, requiring the use of Herceptin as combination therapy35_{. In this study a higher} decrease in proliferation was observed in cells treated with Herceptin in combination with IT1t. This could be an indication that CXCR4 alone does not initiate proliferation but, works together with HER2 to promote proliferation in the BT-474 cells.An earlier report studied the regulation of CXCR4 in HER2 tumor

metastasis and found that HER2 enhances the expression of CXCR4. Down regulation of CXCR4 by Herceptin was also observed in BT-474 cells33_{. Therefore, these results indicate that inhibiting HER2 and} CXCR4 could be more effective in reduction of the proliferation in BT-474 cells. CXCR4 expression level in BT-474 cells is lower compared to CXCR7 and HER2, and may be indicative for the reason why no effect can be seen on the CXCR4 inhibition. This observation was seen in every proliferation assay done in this study.There was also a decrease in proliferation observed in cells treated with a combination of VUF and IT1t, suggesting that CXCR4 works also together with CXCR7 to promote proliferation in BT-474 cells. CXCR4 and CXCR7 can also form heterodimers, whereby CXCR7 changes the conformation of the

CXCR4/G-protein complexes and abrogates its signalling7, 41_{. Data obtained in a previous study done on the} CXCL12-CXCR4-CXCR7 axis indicate that CXCR7 and CXCR4 form constitutive homodimers and heterodimers when they are co-expressed in the same cell41_.

It has also been reported that Lapatinib is a more efficient treatment in Herceptin-resistant, Her2-amplified tumors based on its distinct mechanism of anti-HER2 action from Herceptin35_{. Lapatinib is known to be the} tyrosine kinase inhibitor that exhibits the highest selectivity and has a dual inhibitory effect on EGFR/ HER234_{. While Herceptin reduces the HER2 mediated signaling, it doesn’t interfere with signaling mediated} from other HER family dimers45_{. This might suggest a combination therapy for different HER family} receptors.

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7. Conclusions

In conclusion, it seems that CXCR4 and CXCR7 are involved in the transactivation of ErbB2, but to what extend they are involved should be further assessed. CXCR7 might signal differently to CXCL12 responses either regulating CXCR4 signaling through dimerization, or through it’s own pathway. How each of these receptors signal individually or by their dimers should be further studied, especially since less is known about the signaling mechanisms of CXCR7 in these cells. CXCR4, CXCR7 and ErbB2 seem to be involved in the proliferation and downstream signaling via p44/42 MAPK and Akt. This is an indication that

pharmacological compounds that trigger a combination of receptors should be taken into account, since combination therapies have been giving the best results in cancer cells studied.

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Dissection of CXCR4 and CXCR7 mediated signaling in breast cancer

2013