Comparative proteomic analysis implicates eEF2 as a novel target of PI3Kgamma in the MDA-MB-231 metastatic breast cancer cell line

Cancer cell migration is fundamentally required for breast tumour invasion and metastasis. The insulin-like growth factor 1 tyrosine kinase receptor (IGF-1R) and the chemokine G-protein coupled receptor, CXCR4 have been shown to play an important role in breast cancer metastasis. Our previous study has shown that IGF-1R can transactivate CXCR4 via a physical association in the human MDA-MB-231 metastatic breast cancer cell line and that this plays a key role in IGF-I-induced migration of these cells. In the present study we used pharmacological inhibition and RNAi to identify PI3Kγ as an important migration signalling molecule downstream of receptor transactivation in MDA-MB-231 cells. To identify PI3K?-regulated proteins upon transactivation of CXCR4 by IGF-I, we undertook a comparative proteomics approach using 2-D- Fluorescence Difference Gel Electrophoresis (DIGE) and identified the proteins by mass spectrometry.Meizhi Niu, Manuela Klingler-Hoffmann, Julie A Brazzatti, Briony Forbes, Chareeporn Akekawatchai, Peter Hoffmann and Shaun R McCol

CXCR4/CXCL12 Participate in Extravasation of Metastasizing Breast Cancer Cells within the Liver in a Rat Model

INTRODUCTION: Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer. METHODS: Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4. RESULTS: In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p,0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p < 0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. CONCLUSION: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.Claudia Wendel, André Hemping-Bovenkerk, Julia Krasnyanska, Sören Torge Mees, Marina Kochetkova, Sandra Stoeppeler and Jörg Haie

Chemokines and chemokine receptors in cancer progression

Directed cell migration is a fundamental component of numerous biological systems and is critical to the pathology of many diseases. Although the importance of chemokines in providing navigational cues to migrating cells bearing specific receptors is well-established, how chemokine function is regulated is not so well understood and may be of key importance to the design of new therapeutics for numerous human diseases, particularly for the control of cancer growth and metastasis, diseases in which chemokines have recently been implicated. In this review, we discuss the general views on the role of specific chemokines in these pathological processes. In addition, we discuss two novel aspects of chemokine cancer biology; cross-talk between chemokine and growth factor receptors, and refractory chemokine receptors.Chareeporn Akekawatchai, Marina Kochetkova, Jane Holland and Shaun R McCol

Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells

The definitive version can be found at http://www.jbc.org/In the multimolecular environment in tissues and organs, crosstalk between growth factor and G protein-coupled receptors is likely to play an important role in both normal and pathological responses. In this report, we demonstrate transactivation of the chemokine receptor CXCR4 by the growth factor insulin-like growth factor (IGF)-1 is required for IGF-1-induced cell migration in metastatic MDA-MB-231 cells. The induction of chemotaxis in MDAMB- 231 cells by IGF-1 was inhibited by pretreatment of the cells with pertussis toxin (PTX) and by RNAi-mediated knockdown of CXCR4. Transactivation of the CXCR4 pathway by IGF-1 occurred independently of CXCL12, the chemokine ligand of CXCR4. Neither CXCR4 knockdown nor PTX had any effect on the ability of IGF-1 to activate IGF-1R, suggesting thatCXCR4andGproteins are activated subsequent to, or independently of, phosphorylation of IGF-1R by IGF-1. Coprecipitation studies revealed the presence of a constitutive complex containing IGF-1R, CXCR4, and theGprotein subunits, Giα2 and Gß, and stimulation of MDA-MB-231 cells with IGF-1 led to the release of Giα2 and Gß from CXCR4. Based on our findings, we propose that CXCR4 constitutively forms a complex with IGF-1R in MDA-MB-231 cells, and that this interaction allows IGF-1 to activate migrational signaling pathways through CXCR4, Giα2 and Gß.Chareeporn Akekawatchai, Jane D. Holland, Marina Kochetkova, John C. Wallace and Shaun R. McCol

Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells

© 2006 American Association for Cancer ResearchCXCR4 is a G protein–coupled receptor of considerable biological significance, and among its numerous functions, it is suggested to play a critical role in cancer metastasis. We have investigated the expression and function of CXCR4 in a range of breast cancer cell lines covering a spectrum of invasive phenotypes and found that, while surface levels of CXCR4 were uniform across the entire panel, only highly invasive cells that are metastatic in immunocompromised mice expressed functional receptors. CXCL12/SDF-1 induced cellular responses such as calcium mobilization, actin polymerization, and chemotaxis in metastatic cells, whereas noninvasive cells were unresponsive. Moreover, CXCL12 activated multiple signaling pathways downstream of G proteins in highly invasive cells but failed to activate any of the examined kinase cascades in noninvasive cell lines. This blockade in nonmetastatic cell lines seems to be due to the inability of G protein A and B subunits to form a heterotrimeric complex with CXCR4. GA and GB were able to bind to CXCR4 independently in all cell lines, but the association of G protein AB; heterotrimers with the receptor, a prerequisite for signal transduction downstream from G protein–coupled receptors, was only observed in the highly invasive cell lines. Our findings show, for the first time, that CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein–receptor complex formation, and this regulation may play a role in the transition from nonmetastatic to malignant tumors.Jane D. Holland, Marina Kochetkova, Chareeporn Akekawatchai, Mara Dottore, Angel Lopez and Shaun R. McCol

Chemokine receptors CXCR4 and CCR7 promote metastasis by preventing anoikis in cancer cells

Chemokine receptors are essential mediators of the metastatic spread in various cancer types; however their precise function in the development of secondary tumors remains poorly understood. We report here a novel property of the chemokine receptors CXCR4 and CCR7 in inhibiting detachment-induced cell death – anoikis, which is believed to be one of the major blocks in the metastatic spread of various neoplasms. Activation of these chemokine receptors by their respective ligands, CXCL12 and CCL21 specifically reduced the sensitivity of metastatic breast cancer cells to anoikis by a distinct mechanism of selective regulation of pro-apoptotic Bmf and anti-apoptotic Bcl-xL proteins. Consequently, functional CXCR4 and CCR7 increased cell survival in the absence of correct ECM attachment both in vitro and in vivo. We also demonstrated that preventing chemokine-induced reduction in Bmf levels significantly attenuated breast cancer metastasis in an experimental mouse model. These results provide evidence for a previously unknown axis in malignant tumors, which connects chemokine receptors with deregulated apoptosis in the absence of the appropriate cell – ECM interaction and may offer novel targets for therapeutic intervention for the treatment of metastatic breast and potentially other tumors.M Kochetkova, S Kumar and S R McCol

Chemokine receptor oligomerization to tweak chemotactic responses

Chemokine receptors guide cell migration by responding to local chemokine gradients during immune surveillance and inflammation. Similar to other G protein-coupled receptors, chemokine receptors can form oligomeric complexes that might have distinct pharmacological and biochemical properties as compared to their individual constituents. The majority of evidence for chemokine receptor oligomers came from transfected cells using tagged receptors to monitor their close proximity or physical association. However, translation of these observations to (patho)-physiological consequences is puzzling for the majority of chemokine receptor oligomers due to experimental limitations and challenges to distinguish oligomer- from downstream signaling-mediated crosstalk. Recent methodological advances allow in situ validation of chemokine receptor oligomers in native cells, disruption of oligomers, and detection of oligomer-mediated signaling. Chemokine receptor oligomerization modulates cell migration in (patho)-physiology and consequently offers novel therapeutic targets