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Wheeler Lab

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Research Team

Deric L. Wheeler, PhD


Mari Iida, PhD

Post-doc Students

Associate Research Specialist

Hannah Pearson, B.S.

Graduate Students

Nellie Black, B.S.


Rachel Orbuch

Olivia Ondracek

Undergraduate Opportunities

We are currently seeking an undergraduate student interested in molecular biology. Tasks will include creating functional viral protein expression systems using recombinant DNA approaches to test specific questions by graduate students and scientists in the lab. High work ethic and motivation necessary.

Research Themes

The focus of my laboratory centers around the epidermal growth factor receptor (EGFR) which is ubiquitously expressed receptor tyrosine kinase (RTK). Upon ligand binding, the EGFR initiates a spectrum of signaling pathways that promote cell proliferation, differentiation, migration, motility, and cellular adhesion. The EGFR is recognized as a key mediator of proliferation and progression in many human tumors and strategies to inhibit EGFR signaling have emerged as highly promising cancer therapy approaches. Following more than 20 years of preclinical development, five EGFR inhibitors, two monoclonal antibodies and three small molecule tyrosine kinase inhibitors (TKIs), have recently gained FDA approval in oncology (cetuximab, panitumumab, erlotinib, gefitinib and lapatinib). Both strategies of EGFR inhibition have demonstrated major tumor regressions in approximately 10-20% of advanced cancer patients. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to treatment. The underlying mechanisms of intrinsic and acquired resistance to EGFR inhibitors remain largely unexplored. In an effort to examine mechanisms of acquired resistance to EGFR inhibition we have developed a series of cetuximab-resistant cancer cell lines (H&NSCC1 and NCI-H226) models to elucidate molecular pathways leading to resistance to targeted therapies. The overall goal is to elucidate pathways that resistant cells have activated and aim at blocking these pathways and restoring sensitivity to the original target agents.

HER Family Members

A major class of the RTK superfamily is comprised of the ERBB/HER or epidermal growth factor (EGF) receptors and consists of EGFR (ErbB1/HER1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) (Figure 1). All family members contain an extracellular ligand-binding domain, a single membrane-spanning region, a juxtamembrane nuclear localization signal and a cytoplasmic tyrosine kinase domain (TKD). ERBB receptors are ubiquitously expressed in various cell types, but primarily include those of epithelial, mesenchymal and neuronal origin. Under homeostatic conditions, receptor activation is tightly regulated by the availability of ligands, which collectively form the EGF growth factor family (Figure 1). This family is divided into three distinct groups. The first includes EGF, transforming growth factor alpha and amphiregulin (AR), which all bind specifically to the EGFR. The second group includes betacellulin (BTC), heparin-binding EGF (HB-EGF) and epiregulin (EPR), which bind to both the EGFR and ERBB4. The third group is composed of the neuregulins (NRG1-4) that is further subdivided based on their ability to bind ERBB3 and ERBB4 (NRG1 and NRG2) or only to ERBB4 (NRG3 and NRG4). ERBB2 has no known ligand. Ligand binding leads to receptor homo or heterodimerization at the plasma membrane. This interaction activates the receptor tyrosine kinase and, thereby, causes autophosphorylation of the cytoplasmic tail. Of note, ERBB3 is the only family member that lacks intrinsic kinase activity, however, downstream signaling is achieved through heterodimerization. Phosphorylated cytoplasmic tails serve as docking sites for numerous proteins that contain Src Homology (SH2) and phosphotyrosine binding domains (PB). ERBB activation stimulates many complex intracellular signaling pathways that are tightly regulated by the presence and identity of ligand, the heterodimer composition and the availability of phosphotyrosine-binding proteins. The two primary pathways activated by ERBB ligands are the RAS/RAF/MEK/ERK and the phosphatidylinositol 3-kinase (PI3K)-Akt axes; however, SRC tyrosine kinases, PLC gamma, PKC and signal transducer and activator of transcription (STATs) activation have also been documented (Figure 1). Tumor cell proliferation, survival, invasion and angiogenesis ultimately can be promoted through these pathways.



EGFR is a member of the ErbB family of receptor tyrosine kinases named after its identification as an oncogene encoded by the erythorblastosis virus. Four ErbB members have been identified to date: EGFR (ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). EGFR is a 170kDa protein product of a gene composed of 28 exons spanning approximately 200 kb found on chromosome 7p11.2. EGFR plays a critical role in development and in neoplastic processes of cell proliferation, inhibition of apoptosis, angiogenesis, and metastatic spread. Stimulation of the receptor through ligand binding leads to receptor oligomerization at the plasma membrane. This activates the receptor tyrosine kinase and thereby promotes autophosphorylation of tyrosine residues in the cytoplasmic tail (Figure 2). These phosphorylated tyrosines serve as docking sites for various proteins which contain Src Homology domains (SH2) and phosphotyrosine binding domains (PB). These events lead to the activation of several signaling cascades most notably the MAPK, PI3K/AKT, STAT and PLCγ pathways which ultimately result in proliferative signals to the cell nucleus.

Figure 2CSTEGFR.jpg

Nuclear EGFR Signaling

Figure 1: EGFR biology A) Classical EGFR cytoplasmic signaling: A diagrammatic view of the EGFR cytoplasmic tail Ligand binding to the EGFR leads to receptor homo- or hetero- dimerization leading to transphosphorylation of the cytoplasmic tail tyrosine residues. Lysine 721 (K721) has been shown to be the critical site for ATP-binding domain and kinase activity of the EGFR. Mutation of this amino acid causes the receptor to become inactive so that it is neither internalized nor degraded 132, 133. Tyrosine phosphorylations in the C-terminal include Y974, Y992, Y1045, Y1068, Y1086, Y1148 and Y1173 (shown in orange), or Src family kinases can phosphorylate Y845 and Y1101 (shown in purple). Reported biological effects of phosphorylation of each tyrosine are noted. Phosphorylation of Y845 by SFKs stabilizes the activation loop, maintains the enzyme in an active state, and regulates signal transducer and activator of transcription 5b (STAT5b) activity134. Phospholipase Cγ (PLCγ)-mediated signaling is stimulated by PLCγ binding to phosphorylated Y992 site. The phosphorylation of Y1045 creates a docking site for CBL, which enables receptor ubiquitylation and degradation135. Tyrosines 1068 and 1086 cooperate to bind the SH2 domain of growth factor receptor-bound protein 2 (GRB2). This binding results in mitogen activated protein kinase (MAPK) activation through the Ras/Raf/Mek/Mapk pathway 136. SHP1 phosphatase can bind to the phosphorylated Y1173 domain, which leads to EGFR dephosphorylation137.

B) Nuclear and kinase independent EGFR signaling The EGFR has been consistently detected in the nuclei of cancer cells, primary tumor specimens and highly proliferative tissues 24-28. Since these initial observations it has been shown that EGFR can translocate to the nucleus where it performs both kinase dependent and independent activities. The EGFR has been shown to bind to STAT3 to increase expression of INOS33, E2F1 to increase expression of B-Myb32 and with STAT5 to increase expression Aurora A138. It has also been reported to increase the expression of cyclin D1, but no co-transcription factor has yet been identified 26. In addition, the EGFR has been shown to have kinase dependent activity within the nucleus or proliferating cells. The two main functions described have been the phosphorylation of PCNA leading to its stability and enhancing cell proliferation 81 and translocation and activation of DNA-PK 139.

Figure1 EGFR biology.png

EGFR as a Therapeutic Target

Targeting EGFR has been intensely pursued in the last decade as a cancer treatment strategy. One approach uses monoclonal antibodies (mAbs) to target the extracellular domain of the EGFR to block natural ligand binding (Mendelsohn, 2003) (Figure 3). Cetuximab (IMC-C225, Erbitux) prevents receptor activation and dimerization and ultimately induces receptor internalization and downregulation (Sunada et al., 1986). Cetuximab exhibits promising anti-tumor activity as monotherapy or in combination with chemotherapy and/or radiation (Baselga and Arteaga, 2005; Mendelsohn and Baselga, 2006). A series of clinical trials demonstrating clinical benefit led to the FDA approval of cetuximab for use in patients with head and neck cancer and in metastatic colorectal cancer (Bonner et al., 2006; Cunningham et al., 2004). A second monoclonal antibody, panitumumab, has also gained recent FDA approval for use in the metastatic colorectal cancer setting (Van Cutsem et al., 2007). A second approach involves the use of small molecule tyrosine kinase inhibitors (TKIs) that bind to the ATP-binding site in the tyrosine kinase domain (TKD) of the EGFR and HER2 (Figure 3). These agents inhibit EGFR autophosphorylation and ultimately lead to blockade of downstream signaling and cellular proliferation. Three anti-EGFR TKIs, erlotinib (OSI-774, Tarceva), gefitinib (ZD1839, Iressa) and Lapatinib (GW572016, Tykerb) are now approved by the FDA for use in oncology.

Both approaches to EGFR inhibition show considerable clinical promise. However, similar to the development of acquired resistance in patients treated with imantinib for chronic myeloid leukemia or with herceptin for breast cancer, increasing evidence suggests that patients who initially respond to EGFR inhibitors may subsequently become refractory (Pao et al., 2005). Therefore, an improved understanding of molecular mechanisms of acquired resistance to EGFR inhibitors may provide valuable leads to enhance the efficacy of this class of agents. The identification of catalytic domain EGFR mutations that predict for response to EGFR-TKIs in selected lung cancer patients represents a landmark development in the EGFR field (Sequist et al., 2007). Mutation in exon 21 of the EGFR TKD, L858R, may predict increased sensitivity to TKIs, whereas the T790M mutation in exon 20 is associated with acquired resistance to TKI therapy (Riely et al., 2006). These recent findings suggest that patient selection may be critical for successful therapies using EGFR TKIs (Arteaga and Baselga, 2004). Although EGFR TKD mutations appear to correlate with response to erlotinib and gefitinib, no such correlation exists for cetuximab response (Mukohara et al., 2005).

Her Family Summary Slide with TKisWebPage.jpg

Mechanisms of Resistance to Cetuximab

To study mechanisms of acquired resistance to cetuximab, we established a series of cetuximab-resistant clones in vitro following long-term exposure to cetuximab in NSCLC (H226) and HNSCC (SCC-1) cell lines. Following establishment of stable clones, we performed high-throughput screening to examine the activity of 42 membrane receptor tyrosine kinases (RTKs). Through comparative analysis of cetuximab-resistant versus parental lines, we identified that EGFR along with HER2, HER3 and cMET are all highly activated in the resistant clones. Further studies suggest that acquired resistance to cetuximab reflects dysregulation of EGFR internalization/degradation and subsequent EGFR-dependent activation of HER3 (Wheeler et al, Oncogene 2008).

Figure 2 mechanisms of antibody resistance.png

Figure 2: Proposed mechanisms of resistance to EGFR monoclonal antibodies

A) One reported mechanism of resistance to cetuximab therapy was the overexpression of the EGFR ligand TGFα 140. B) In addition to overexpression of the ligand overexpression of the EGFR has been implicated in the development of acquired resistance 78 C) Two reports, with opposite findings, showed that ubiquitylation is important for mechanisms of escape to cetuximab therapy. One report indicated decreased ubiquitylation of the receptor while the other reported increased ubiquitylation of the receptor78, 79. D) Modulation of the EGFR by Src Family Kinases and increased activity of SFKs in cetuximab resistant lines have been reported 79, 80. E) In cells with acquired resistance to cetuximab the activation of binding and activation of EGFR or HER2 to HER3 have been reported 78, 80. This activation allowed for prolonged signals to the PI3K/Akt pathway. F) Translocation of the EGFR to nucleus has been reported to play a role in resistance to cetuximab. It is thought that nuclear EGFR may serve as a second compartment of proliferation during challenge with cetuximab by regulating gene transcription. G) Cells that developed acquired resistance to cetuximab in vivo were shown to have increased VEGF production leading to altered angiogenesis and enhanced escape from cetuximab therapy 76, 77. H and I) Mutations in both PTEN and Ras have been implicated in impaired response to cetuximab therapy56. PTEN mutations lead to resistance to by decreased dephosphorylation of PIP3 leading to enhanced AKT recruitment and activation. Mutations in the KRAS protein, most notably in codons 12 and 13 lead to decreased binding of GTPase activation protein (GAPS). This keeps KRAS in a constant GTP bound, active state and can send signals downstream independently from RTK activation.