Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma and metastatic colorectal cancer. Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximabresistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin, heparinbinding EGF and b-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and resensitization to cetuximab. In addition, expression of a nuclear localization sequence-tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab.

The proto-oncogene c-Src (Src) is a nonreceptor tyrosine kinase whose expression and activity is correlated with advanced malignancy and poor prognosis in a variety of human cancers. Nine additional enzymes with homology to Src have been identified and collectively are referred to as the Src family kinases (SFKs). Together, SFKs represent the largest family of nonreceptor tyrosine kinases and interact directly with receptor tyrosine kinases, G-protein-coupled receptors, steroid receptors, signal transducers and activators of transcription and molecules involved in cell adhesion and migration. These interactions lead to a diverse array of biological functions including proliferation, cell growth, differentiation, cell shape, motility, migration, angiogenesis, and survival. Studies investigating mutational activation of Src in human cancers suggest this may be a rare event and wild-type Src is weakly oncogenic. Thus, the role of Src in the development and progression of human cancer has remained unclear. Recently, it has been suggested that increased SFK protein levels and, more importantly, SFK tyrosine kinase activity is linked to cancer progression and metastatic disease by facilitating the action of other signaling proteins. This accumulating body of evidence indicates that SFKs may represent a promising therapeutic target for the treatment of solid tumors. This review discusses the role of SFKs in solid tumors and the recent therapeutic advances aimed at targeting this family of tyrosine kinases in cancer.

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical problem. In this study we show that overexpression of the oncogenic receptor kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft assays, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL targeting drugs to treat cetuximab-resistant cancers.

The epidermal growth factor receptor (eGFR) is a receptor tyrosine kinase belonging to the HeR family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the Pi3K/AKT, RAs/RAF/MeK/ eRK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. increased activation by gene amplification, protein overexpression or mutations of the eGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NsCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the eGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five eGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKis) and two monoclonal antibodies have gained FdA approval for use in oncology. Both approaches to targeting the eGFR have shown clinical promise and the anti-eGFR antibody cetuximab is used to treat HNsCC and CRC. despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. in this review we focus on the biology of the eGFR, the role of eGFR in human cancer, the development of

The epidermal growth factor receptor (EGFR) has been one of the most targeted receptors in the field of oncology. While anti-EGFR inhibitors have demonstrated clinical success in specific cancers, most patients demonstrate either intrinsic or acquired resistance within one year of treatment. Many mechanisms of resistance to EGFR inhibitors have been identified, one of these being attributed to alternatively localized EGFR from the cell membrane into the cell’s nucleus. Inside the nucleus, EGFR functions as a co-transcription factor for several genes involved in cell proliferation and angiogenesis, and as a tyrosine kinase to activate and stabilize proliferating cell nuclear antigen and DNA dependent protein kinase. Nuclear localized EGFR is highly associated with disease progression, worse overall survival in numerous cancers, and enhanced resistance to radiation, chemotherapy, and the anti-EGFR therapies gefitinib and cetuximab. In this review the current knowledge of how nuclear EGFR enhances resistance to cancer therapeutics is discussed, in addition to highlighting ways to target nuclear EGFR as an anti-cancer strategy in the future.

Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.

Purpose Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard of care treatments for HNSCC patients include surgery, radiation and chemotherapy. Additionally, the anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult to treat malignancy. Thus, identification of new molecular targets is critical. Experimental Design In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient derived xenografts (PDXs), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard of care treatment regimes used in HNSCC. Results AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases and shorter relapse free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. Additionally, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Conclusion Collectively, this study provides evidence for the role of AXL in HNSCC pathogenesis and supports further pre-clinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a major role in oncogenesis. Cetuximab is an EGFR-blocking antibody that is FDA approved for use in patients with metastatic colorectal cancer (mCRC) and head and neck squamous cell carcinoma (HNSCC). Although cetuximab has shown strong clinical benefit for a subset of cancer patients, most become refractory to cetuximab therapy. We reported that cetuximab-resistant NSCLC line NCI-H226 cells have increased steady-state expression and activity of EGFR secondary to altered trafficking/degradation and this increase in EGFR expression and activity lead to hyper-activation of HER3 and down stream signals to survival. We now present data that Src family kinases (SFKs) are highly activated in cetuximab-resistant cells and enhance EGFR activation of HER3 and PI(3)K/Akt. Studies using the Src kinase inhibitor dasatinib decreased HER3 and PI(3)K/Akt activity. In addition, cetuximab-resistant cells were resensitized to cetuximab when treated with dasatinib. These results indicate that SFKs and EGFR cooperate in acquired resistance to cetuximab and suggest a rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab.