Cell Sigalling, Receptor Endocytosis, and Breast Cancer
ErbB receptors including ErbB1 (EGFR), ErbB2, ErbB3, and ErbB4 and other receptor tyrosine kinases (RTKs) lie at the head of a complex signal transduction cascade that modulates cell proliferation, survival, adhesion, migration and differentiation. While EGFR signalling is essential for many normal cell functions, the aberrant activity of EGFR has been shown to play a key role in the development of cancers. EGFR receptor is overexpressed in many cancers especially in breast cancer, ovarian cancer, small cell lung cancer and skin cancer. ErbB2 is overexpressed in 30% breast cancer patients. ErbB receptor overexpression correlates to poor prognosis, drug resistance, cancer metastasis and lower survival rate. All these make ErbB receptor the top choice as a target for developing cancer therapies. To date Herceptin, an antibody to ErbB2, is used clinically, and many other monoclonal antibodies (mAbs) and synthetic inhibitors of tyrosine kinase have taken central stage in fighting cancers.
The central theme of my research is to understand how the activation of EGFR regulate cell signalling, how the signalling is terminated through EGFR endocytosis, trafficking and degradation, how the breakdown of this regulation contributes to cancer development, and how an intervention can be provided. Currently, my research is focusing on the following three areas:
1. Breast cancer, drug resistance and novel treatment regimens
Despite recent advances in the treatment of breast cancer, disease recurrence remains a major obstacle to curative therapy. One of the main clinical issues is the development of drug resistance, which accounts for more than 90% of death in patients with metastatic breast cancer. Besides the existing therapeutic deficiencies, intrinsic properties of CSCs may predispose to tumor relapse. CSCs have a dormant or slow mitotic index making them less susceptible to chemotherapeutics that target rapidly dividing cells. CSCs may contain considerable variation in genetic materials due to genomic instability, which allow them to survive the various treatment regimens and to develop broad resistance.
Breast cancer cell lines selected for drug resistance have been used as a model system to reveal the mechanisms of drug resistance. Using this model system we have shown recently that multiple mechanisms are behind the resistance of selected MCF-7 cells. Given the recent findings in understanding drug resistance and the development of CSC concept, we propose that by carefully designing and monitoring the selection of drug resistant cells under various drug treatment regimens and from existing breast cancer cell lines, we will achieve following objectives:1) to determine which current breast cancer treatment regimens and administration strategies are less likely to cause drug resistance; 2) to characterize the selected drug-resistant cells to reveal the resistance mechanisms; and 3) to develop drug treatment regimens that are more effective to treat both the primary cancer and the recurrent cancer.
On the other hand, targeted therapy with trastuzumab has become a mainstay for ErbB2-positive breast cancer without a clear understanding of the mechanism of its action. While several possibilities have been suggested, the molecular mechanisms behind these possibilities have been insufficiently studied and poorly defined. Moreover, to achieve good response, trastuzumab treatment always needs to be combined with other cancer drugs such as doxorubicin and paclitaxel. However, the rationales behind the combination are not clear and it is not known what the best combination is. Furthermore, the majority of patients with metastatic breast cancer who initially respond to trastuzumab demonstrate disease progression within one year. Despite the increased importance of trastuzumab resistance in treating ErbB2-positive breast cancers, little is known regarding the molecular mechanisms of the resistance.
The lack of understanding of combined trastuzumab treatment and trastuzumab resistance, in large part, is due to the lack of understanding of the mechanisms behind the trastuzumab therapy at first place. Thus, to design more effective trastuzumab-based therapy for ErbB2-positive breast cancer, to overcome trastuzumab-resistance, it is essential to understand the molecular mechanisms of trastuzumab therapy. Our objective of this research is to reveal the mechanisms underlying the function of trastuzumab and to design better strategy for applying trastuzumab.
2. Regulation of EGFR-mediated cell signaling and endocytosis during M-phase and its implication in Cancer
It is well documented that the alteration of epidermal growth factor (EGF) receptor (EGFR)-mediated cell signaling through endocytosis and other means results in cancer development. Growth factors (GF) including EGF stimulate cell proliferation by driving the cell cycle. In the presence of serum, cultured cells move through four phases of the cell cycle: G1, S, G2 and M. GF must be present until the restriction (R) point in the G1 phase to stimulate the cell cycle. After the R point, GFs are not required for the progression of cell cycle. Mitotic (M) phase is the most dramatic period of the cell cycle, involving a major reorganization of virtually all cell components. A major difference between cancer cells and normal cells is that the cancer cells are much more mitogenic and frequently enter mitosis for proliferation. Therefore, most cancer drugs are designed to specifically target the mitotic cells. However, the role of EGF-induced cell signaling at M phase is very rarely and poorly studied. No research has been done regarding the endocytosis of EGFR at M phase.
Recently, we studied EGF-induced EGFR signaling and endocytosis during M phase and got some interesting results. We showed that at M phase EGFR is expressed and activated at the same level as in the interphase. We further showed that at M phase the activated EGFR only selectively stimulate some of the signaling pathways activated in interphase. On the other hand, we found that at M phase EGF-induced EGFR endocytosis is not ceased. This result is interesting and surprising as the current dogma is that all endocytosis is ceased at M phase. We further showed that EGF-induced EGFR endocytosis at M phase is regulated differently from interphase. EGFR endocytosis at M phase is delayed and slow compared with interphase. Moreover, EGF-induced EGFR endocytosis is dependent on EGFR kinase activation at M phase, but is largely kinase-independent at interphase. This leads us to propose that at M phase, EGF stimulates both EGFR endocytosis and EGFR-mediated cell signaling. However, both EGFR endocytosis and EGFR-mediated cell signaling are regulated differently from interphase specifically to serve the special needs of the cell at M phase. We are currently testing this hypothesis.
3. Regulation of Rho family small GTPases by phosphorylation and its implication in cancer
Accumulating evidence has implicated Rho GTPases in many aspects of cancer development, especially in metastasis. The regulatory cycle of Rho GTPases is normally controlled by three distinct families of proteins, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). However, recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the 106PNTP109 motif is likely an Erk phosphorylation site and Rac1 also has an Erk docking site 183KKRKRKCLLL192 at C-terminus. Indeed, our preliminary data showed that Rac1 T108 is phosphorylated in response to EGF. Moreover, we recently identified that PLC-g1 is a Rac1 GEF. The interaction between PLC-g1 and Rac1 is mediated by PLC-g1 SH3 domain and Rac1 proline rich motif 106PNTP109.
This leads us to form the following hypothesis: in response to EGF, PLC-g1 activates membrane-associated Rac1 and Erk phosphorylates cytosolic Rac1 at T108. Phosphorylated Rac1 is preferentially targeted to the plasma membrane and cannot be activated by PLC-g1. Thus, Rac1 T108 phosphorylation by Erk serves as a negative feed back control for Rac1 activation by PLC-g1 and serves to reduce the cell migration and the metastasis of cancers. We are currently testing our hypothesis with the following specific aims: 1) To determine whether Rac1 is phosphorylated at T108 by Erk. 2). To determine the Effects of T108 phosphorylation on Rac1 activation and cancer cell metastasis. 3) To determine the spatio-temporal control of Rac1 activation by both PLC-gamma1 and Erk.
Wang H, Vo T, Hajar A, Li S, Chen X, Parissenti AM, Brindley DN, Wang Z: Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxel-resistant MCF-7 breast cancer cells. BMC Cancer 2014, 14:37.
Tong, J., Li, L., Ballermann, B. and Wang, Z. (2013) Phosphorylation of Rac1 T108 by ERK in response to EGF: A novel mechanism to regulate Rac1 function. Mol. Cell. Biol. 33:4538-4551.
Li, L., Chakraborty, S., Yang, C., Hatanpaa, K.J., Cipher, D.J., Puliyappadamba1, V.T., Madden, A.C., Raisanen, J., Burma, S., Saha, D., Wang, Z., Pingle, S.C., Kesari, S., Boothman, D.A. and Habib, A.A. (2013) An EGFR wild type-EGFRvIII-HB-EGF feed forward loop regulates the activation of EGFRvIII. Oncogene. 2013 Sep 30. doi: 10.1038/onc.2013.400. [Epub ahead of print]
Wu, P. Wee, P., Jiang, J., Chen, X. and Wang, Z. (2012) Differential regulation of transcription factors by location-specific activation of EGF receptor signalling via a spatio-temporal interplay of Erk activation. PLOS ONE / Volume 7 / Issue 9 / e41354: 1-18
Wang, Z. (2012) Mutual Regulation of Receptor-Mediated Cell Signalling and Endocytosis: EGF Receptor System as an Example. In Molecular Regulation of Endocytosis. Ed. Ceresa, B. ISBN 978-953-51-0662-3, Hard cover, 456 pages, Publisher: InTech, Published: July 06, 2012 under CC BY 3.0 license, in subject Biochemistry, Genetics and Molecular Biology. DOI:10.5772/298950 Chen C., Uludag H., Wang Z., Jiang H. (2012) Noggin suppression decreases BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells in vitro. J Cell Biochem. 113(12):3672-80. doi: 10.1002/jcb.24240
Chen C., Uludag H., Wang Z., Rezansoff A., Jiang H. (2012) inhibit migration, metabolic activity and osteogenic differentiation of human mesenchymal stem cells in vitro. Cells Tissues Organs 195(6):473-83.
Liu, L., Shi, H., Chen, X. and Wang, Z. (2011) Regulation of EGF Receptor Endocytosis during Mitosis. Traffic 12: 201-217.
Pahara, J., Shi, H., Chen, X. and Wang, Z. (2010) Dimerization Drives PDGF Receptor Endocytosis through a C-terminal Hydrophobic Motif Shared by EGF Receptor. Exp. Cell Res 15: 2237-50.
Li, S, Wang, Q, Wang,Y. Chen, X. and Wang, Z. (2009) PLC-γ1 and Rac1 co-regulates EGF-induced cytoskeleton remodelling and cell migration. Molecular Endocrinology 23: 901-913.
Wang, Q., Wu, F., and Wang, Z. (2007) Identification of EGF Receptor C-terminal Sequences 1005-1017 and Di-leucine Motif 1010LL1011 as Essential in EGF Receptor Endocytosis. Exp. Cell Res. 313: 3349-3363.
Wang, Y., Wu, J. and Wang, Z. (2006) Akt binds to and phosphorylates phospholipase C-gamma1 in response to epidermal growth factor. Mol. Cell. Biol. 17: 2267-2277.
Wang, Q., Villeneuve, G., and Wang, Z. (2005) Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation. EMBO Rep., 6: 942-948
Wang, Y., Pennock, S., Chen, X., Kazlauskas, A. and Wang, Z. (2004) PDGF receptor-mediated signal transduction from endosomes. J. Biol. Chem., 279: 8038-8046
Pennock, S. and Wang, Z. (2003) Stimulation of cell proliferation by endosomal epidermal growth factor receptor as revealed through two distinct phases of signaling. Mol. Cell. Biol. 23: 5803-5815
Wang, Y. and Wang, Z. (2003) Regulation of EGF-induced phospholipase C-g1 translocation and activation by its SH2 and PH domains. Traffic 4: 618-630
Wang Y, Pennock S, Chen X. and Wang Z.. Internalization of inactive EGF receptor into endosomes and the subsequent activation of endosome-associated EGF receptors. Sci. STKE 2002(161): PL17.
Wang, Y., Pennock, S, Chen, X. and Wang, Z. (2002) Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival. Mol. Cell. Biol. 22 (20): 7279-7290 [Cover illustration on MCB 22 (22)]. Science's STKE highlighted this paper in "This Week in Signal Transduction" for the October 1 issue.
Wang, Z. and Moran, M. (2002) Phospholipase C-g1: a Phospholipase and Guanine Nucleotide Exchange Factor. Molecular Intervention 2: 352-355
Chen, X. and Wang, Z. (2001) Regulation of epidermal growth factor receptor endocytosis by wortmannin through activation of Rab5 rather than inhibition of phosphatidylinositol 3-kinase. EMBO Reports 2: 842-849.
Dankort, D., Maslikowski, B., Warner, N., Kanno N. Kim, H., Wang, Z., Moran, M.F., Oshima, R.G., Cardiff, R. D. and Muller, W. (2001) Grb2 and Shc adapter proteins play distinct roles in Neu (ErbB-2)-induced mammary tumorigenesis: Implications for human breast cancer. Mol. Cell. Biol. 21: 1540-1551.
Chen, X. and Wang, Z. (2001) Regulation of EGF receptor intracellular trafficking by Rab5 in the absence of Phosphatidylinosito 3-kinase activity. EMBO Reports 2:68-74. (Cover Illustration)
Chen, X., Yeung, K. T. and Wang, Z. (2000) Enhanced drug resistance in cells coexpressing ErbB2 with EGFR or ErbB3. Biochem. Biophys. Res. Commun. 277: 757-763.
Wang, Z., Zhang, L., Yeung, K. T. and Chen, X (1999) Endocytosis deficiency of EGF receptor-ErbB2 heterodimers in response to EGF stimulation. Mol. Biol. Cell 10: 1621-1636.
Yeung, K.T., Germond, C., Chen, X. and Wang, Z. (1999) Mode of action of Taxol: apoptosis at low concentration and necrosis at high concentration. Biochem. Biophys. Res. Commun. 263: 398-404.
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Dankort, D.L., Wang, Z., Blackmore, V., Moran, M. and Muller, W.J. (1997) Distinct tryrosine autophosphorylation sites negatively and positively modulate Neu-mediated transformation. Mol. Cell. Biol. 17, 5410-5425
Fam, N.P., Fan, W.-T., Wang, Z., Zhang, L.-J., Chen, H. and Moran, M. (1997) Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras. Mol. Cell. Biol. 17, 1396-1406.
Wang, Z. and Moran, M. (1996) Requirement for the adaptoer protein Grb2 in EGF receptor endocytosis. Science 272, 1935-1938.
Wang, Z., Tung, P. S. and Moran, M. (1996) Association of P120 ras GAP with endocytic components and co-localization with EGF receptor in response to EGF stimulation. Cell Growth & Differentiation 7, 123-133.
Babak Nami Mollalou