Zhixiang Wang

ERBB Receptor-mediated Cell Signaling, Breast Cancer, Drug Resistance and Novel Therapies

There are more than 90 known protein tyrosine kinase genes in the human genome; 58 encode transmembrane protein receptor tyrosine kinases (RTKs) distributed into 20 subfamilies. Among them, ErbB receptor family, also known as EGF Receptor family or type I receptor family, include epidermal growth factor (EGF) receptor (EGFR) or ErbB1/Her1, ErbB2/Her2, ErbB3/Her3 and ErbB4/Her4. Among all RTKs, EGFR is first identified and first linked to cancer. Thus, EGFR is also the mostly intensively studied among all RTKs.

ERBB receptors were first implicated in human cancer approximately three decades ago when the avian erythroblastosis tumor virus was found to encode an aberrant form of the human epidermal growth factor (EGF) receptor (EGFR). Scientific communities have since developed substantial understanding of the cell signaling mediated by ErbB receptors and the biology underlying the dependence of cancers on aberrant ErbB receptor signaling.

Breast cancer is the most common type of cancer in women with more than one million reported new cases diagnosed per year worldwide. Among these cases, 20-30% present with metastatic or locally advanced disease, and another 30% will develop recurrent or metastatic disease. Metastatic breast cancer (MBC) is a heterogeneous disease and among the leading causes of cancer mortality. Based on the expression of ErbB2 and/or hormone receptors as well as unique gene expression patterns, breast cancer was categorized into at least five subtypes: luminal like subtypes A and B (expression of hormone receptors and luminal cytokeratins 8 and 18), basal-like (expression cytokeratings 5 and 17 and typically no expression of hormone or ErbB2 receptors), ErbB2-positive, and normal-like. Chemotherapy is often used to treat breast cancer.

The central theme of my research is to understand how the activation of ERBB receptors regulate cell signalling, how the signalling is terminated through receptor endocytosis/trafficking/degradation and through inhibitory antibodies including trastuzumab and pertuzumab, how the breakdown of this regulation contributes to cancer development, and how an intervention can be provided.

Currently there are several projects ongoing in the lab:

1. Heterogeneity of ERBB receptor-mediated cell signaling
   ErbB receptors are activated after homo- or hetero-dimerization. The ErbB family is unique among various groups of RTKs in that ErbB3 has impaired kinase activity, while ErbB2 does not have a direct ligand. Therefore, heterodimerization is an important mechanism that allows the activation of all ErbB receptors in response to ligand stimulation. The activated ErbB receptors bind to many signaling proteins and stimulate the activation of many signaling pathways including RAS/RAF/MEK/ERK, PI3K/AKT/TOR, Src kinases, Phospholipase C-γ1, and STAT transcription factors. The specificity and potency of intracellular signaling cascades are determined by the expression of positive and negative regulators, the specific composition of activating ligand(s), receptor dimer constituents, and the array of proteins that associate with the tyrosine phosphorylated C-terminal domain of the ErbB receptors. Through the controlling of these diverse signaling networks, ErbB receptors regulate many critical cellular processes, such as cell proliferation, cell differentiation, cell survival, cell metabolism, cell migration, and cell cycle.
   In spite of significant advances in our understanding of ERBBR signaling and trafficking, some critical knowledge is still lacking. To date, the study of receptor signaling and endocytosis has largely been done assuming cellular homogeneity. The heterogeneity of receptor signaling and endocytosis has been rarely explored. In the last 20 years our research has been focused on the heterogeneity of ERBB receptor signalling. We have been studying the heterogeneity of EGFR signaling from different subcellular locations (cell organelles) along its endocytic trafficking pathway, the heterogeneity of EGFR signaling in response to ligand intensity and duration, the heterogeneity of EGFR signaling among various phases of cell cycle, and the heterogeneity through the formation of various homo- and hetero-dimers.
2. Phosphorylation of Rac1 and the function of nuclear Rac1
   We have studied the role of PLC-γ1 in EGF-induced cell signaling for many years. During our research, we noticed that PLC-γ1 interacts with Rac1 in response to EGF. Following this lead, we began our research on the regulation of Rac1. We demonstrate that PLC-γ1 SH3 domain is a Rac1 GEF in vitro. Our data for the first time established a direct cross talk between PLC-γ1 and Rac1 pathways in the control of EGF-induced cytoskeleton reorganization and cell migration. Moreover, we recently showed that Rac1 T108 is phosphorylated by ERK in response to EGF. Most interestingly, we showed that T108 phosphorylation targets Rac1 to the nucleus. Translocation of Rac1 to nucleus hinders Rac1's role in cell migration. We also showed that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus.
   Our preliminary data further showed that nuclear Rac1 localized to specific nuclear domain, likely nuclear speckle, and a phosphomimetic mutant Rac1T108E binds to many hnRNP isoforms that also localized to nuclear speckles and control RNA splicing. These data support a novel function of Rac1 in nucleus. We are currently exploring the function of nuclear Rac1.
3. Mechanism underlying the action and synergism of trastuzumab and pertuzumab in breast cancer
   Overexpression of EGFR, HER2 and HER3 occurs in 30-40%, 20-30% and ~20% of breast cancer cases, respectively. Targeting HER2 has proven to be an effective therapeutic strategy for HER2-positive breast cancer. Following its approval by FDA in 1998, trastuzumab, an antibody targeting HER2, has changed the paradigm for the treatment of HER2-positive breast cancer. However, despite the positive outcome initially, acquired resistance to trastuzumab has gradually developed, which posts a challenge that needs to be overcome. The later approval of pertuzumab by FDA to be used in combination with trastuzumab and docetaxel to treat metastatic HER2-positive breast cancer significantly improved the outcome of the breast cancer patients. It is shown recently that when added to adjuvant trastuzumab and chemotherapy, pertuzumab significantly improves the outcomes among patients with HER2-positive early breast cancer. However, the lack of a good understanding of the mechanisms underlying the action and synergism of trastuzumab and pertuzumab could severely limit its application and efficacy. Indeed, despite these achievements, the persisting high toll of deaths resulting from HER2-positive breast cancer calls for newer therapies and combinations. A thorough understanding of the mechanisms underlying the function of trastuzumab, pertuzumab and the synergism of these two antibodies is essential for the development of better therapies to combat HER2-positve breast cancer.
   We are currently study the effects of trastuzumab and pertuzumab on the homodimerization and activation of ERBB2 in CHO cells stably expressing human ERBB2. We are also examining the effects of these two antibodies on the cell signaling, proliferation and survival of breast cancer cells with different expression patterns of ERBB receptors. Furthermore, we are exploring the novel mechanisms underlying the action of trastuzumab.
4. Taxane resistance and novel therapy to overcome taxane resistance in breast cancer
   Breast cancer is among the leading causes of cancer mortality in Canada. Despite advances in treatment, disease recurrence and progression remains a major obstacle to therapy. Frontline chemotherapy regimens for treating breast cancer typically include a taxane, alone or in combination with other chemotherapy. Despite an initial response, metastatic disease will eventually progress due to the development of drug resistance. Subsequent chemotherapeutic agents used clinically for taxane-resistant MBC include anthracyclines, antimetabolites, and vinca alkaloids, however, with limited progressive success. Thus, development of novel therapy against taxane resistance becomes an urgent issue in BC treatment.
   We have been study the molecular mechanisms underlying taxane resistance. We showed that in addition to high expression ABCB1 and ABCC1, the distinct expression profiles of β-tubulin isotypes, also contributes to taxane resistance. To date, microtubule targeting agents (MTAs) remain amongst the most reliable antimitotic drugs used in cancer chemotherapy. Current MTAs all bind to β-tubulin and are divided into two categories: microtubule-stabilizing agents including taxanes, and microtubule-destabilizing agents including vinca alkaloid, colchicine-site binding agents (CSBAs), and recently developed Eribulin. We showed recently that two taxane-resistant MCF-7 cells, MCF-7TXT and MCF-7TAX cells are also cross-resistant to vinca alkaloid, however, they are more sensitive to CSBAs including colchicine, 2MeOE2, ABT-751 and phosphorylated combretastatin A-4 (CA-4P) than the parental non-resistant MCF-7CC cells. This suggests that CSBAs could be an effective therapy for taxane-resistant BC patients. Moreover, previous research suggests that CSBAs may not be sensitive to ABCB1 export and they have different binding preference to various β-tubulin isotypes compared to taxanes and vinca alkaloids. Indeed, both MCF-7TXT and MCF-7TAX cells overexpress ABCB1 and ABCC1, and they show distinctive profile of various β-tubulin isotypes.
   Currently we are collaborating with other labs to generate and developing novel CSBAs to overcome taxane resistance in breast cancer. One promising novel agent we are test CR42-024, we are able to demonstrate that the taxane-resistant MCF-7TXT and MCF-7TAX cells are more sensitive to CR42-024 than the non-resistant MCF-7CC cells in terms of microtubule polymerization and cell toxicity. Moreover, CR42-024 has higher killing capabilities, as indicated by a lower IC50 value (concentration required to kill 50% of cells in culture) when compared with existing CSBAs. We plan to test CR42-024 in cell, mouse and primary cells isolated from breast cancer patients.

Selected Publications:

A. Books:

Wang, Z. (2017) Editor for "ErbB Receptor Signaling: Methods and Protocols." ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.

B. Book Chapters:

1. Brandwein, D. Tong, J., Li, L., Ballermann, B., and Wang, Z. (2018) An in vitro kinase assay to assess Rac1 phosphorylation by ERK. In Rho GTPases: Methods and Protocols, Second Edition, Ed. Rivero, F., eBook. Serial Editor JM Walker, "Methods in Molecular Biology". Publisher: Springer Nature. ©Springer Science+Business Media LL In Press.
2. Wang, Z (2017) ErbB Receptor and Cancer. Page 3-35. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.
3. Maadi H, Nami B, Wang Z. (2017). Dimerization Assessment of Epithelial Growth Factor Family of Receptor Tyrosine Kinases by Using Cross-Linking Reagent. Page 101-108. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.
4. Nami B, Wang Z. (2017). Application of Immunofluorescence Staining to Study ErbB Family of Receptor Tyrosine Kinases. Page 109-116. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.
5. Wang Y, Billing S, Wang Z. (2017). Activation of Endosome-Associated Inert EGF Receptor Following Internalization. Page 117-126. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.
6. Pennock S, Billing S, Wang Z, Wang Y. (2017) Two-Pulse Endosomal Stimulation of Receptor Tyrosine Kinases Induces Cell Proliferation. Page 127-133. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.
7. Wang Q. and Wang Z. (2017). Study of EGFR Signaling/Endocytosis by Site-Directed Mutagenesis. Page 135-143. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.
8. Tong J. and Wang Z. (2017) Analysis of Epidermal Growth Factor Receptor-Induced Cell Motility by Wound Healing Assay. Page 159-163. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.
9. Wee, P. and Wang, Z. (2017) Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation. Page 167-181. In ErbB Receptor Signaling: Methods and Protocols. Ed. Wang, Z. ISBN 978-1-4939-7218-0, Hard cover, 303 pages, ISBN 978-1-4939-7219-7, eBook. Serial Editor JM Walker, "Methods in Molecular Biology" 1652. Publisher: Springer Nature. ©Springer Science+Business Media LL.

C. Refereed Papers:

1. Nami, B. and Wang, Z. (2018) Genetics and expression profile of tubulin gene superfamily in breast cancer subtypes and its relation to taxane resistance. Cancers (Accepted)
2. Nguyen, A.H., Abdelrasoul, G.N., Lin, D., Maadi, H., Tong, J., Chen, G., Wang, R., Shoute, Lian, Chen, J., and Wang, Z. (2018) Polyethylenimine-coated iron oxide magnetic nanoparticles for high efficient gene delivery. Applied Nanoscience 8(4) 811-821, https://doi.org/10.1007/s13204-018-0775-z
3. Maadi, H., Nami, B., Tong, J., Li, G., and Wang, Z. (2018). The effects of trastuzumab on HER2-mediated intracellular signaling in CHO cells expressing human HER2. BMC Cancer 18(1):238. doi:10.1186/s12885-018-4143-x
4. Brandwein, D. and Wang, Z. (2017) Interaction between Rho GTPases and 14-3-3 proteins. Int J Mol Sci. 18(10). pii: E2148. doi: 10.3390/ijms18102148
5. Wang, R., Chen, X., Parissenti, A., Joy, A.A., Tuszynski, J., Brindley, D.N., and Wang, Z. (2017) Sensitivity of docetaxel-resistant MCF-7 breast cancer cells to microtubule-destabilizing agents including vinca alkaloids and colchicine-site binding agents. PloS one, 12(8):e0182400
6. Wang, R., Wang, H., and Wang, Z. (2017) Live imaging to study microtubule dynamic instability in taxane-resistant breast cancers. J. Vis. Exp. (120), e55027, doi:10.3791/55027
7. Nami, B. and Wang, Z. (2017). HER2 in breast cancer stemness: A negative feedback loop towards trastuzumab resistance. Cancers. 9(5): E40
8. Wee, P and Wang, Z. (2017) Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways. Cancers. 9(5): E52
9. Kourouniotis, G., Wang, Y., Pennock, S., Chen, X., and Wang, Z. (2016) Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor. Int J Mol Sci. 2016 Jul 25;17(8). pii: E1200. doi: 10.3390/ijms17081200
10. Wang, Z. (2016) Transactivation of Epidermal Growth Factor Receptor by G Protein-coupled Receptors: Recent Progress, Challenges and Future Research. Int. J. Mol. Sci. 2016, 17(1), 95
11. Tong, J., Li, L., Ballermann, B. and Wang, Z. (2016) Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation. PLOS One 27;11(1):e0147103. doi: 10.1371
12. Tavasoli, M., Li, L., Al-Momany, A., Zhu, L., Adam, B.A., Wang, Z. and Ballermann, B. (2016) CLIC5A stimulates Rac1-dependent Pak and ERM protein activation, protecting against hypertension-induced glomerular injury. Kidney International 89(4):833-47
13. Wang, Z. (2015) Personalized medicine for HER2-positive breast cancer. Breast Cancer Management, 4(5), 237-240 , doi: 10.2217/bmt.15.16
14. Wee, P., Shi, H., Jiang, J., Wang, Y., and Wang, Z. (2015) EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis. Cellular Signalling 27:638-51
15. Wang, Q., Chen, X. and Wang, Z. (2015) Dimerization drives EGF receptor endocytosis through two sets of compatible endocytic codes. Journal of Cell Science 128:935-950
16. Wang, Z. (2014) Taxane resistance in Breast Cancer. (review) Cancer Cell & Microenvironment 1 (3): 57-65 doi: 10.14800/ccm.126
17. Wang H., Vo T., Hajar A., Li S., Chen X., Parissenti A. M., Brindley D.N., Wang Z. (2014): Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxel-resistant MCF-7 breast cancer cells. BMC Cancer, 14:37.
18. 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.
19. 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 33(33):4253-64.