There is presently great interest in mechanisms of acquired resistance to EGFR inhibitors that are now being used widely in the treatment of a variety of common human cancers. To investigate these mechanisms we established EGFR inhibitor resistant clones from non-small cell lung cancer cells. A comparative analysis revealed that acquired resistance to EGFR inhibitors was associated consistently with the loss of p53 and cross-resistance to radiation. To examine the role of p53, we first knocked down p53 in sensitive parental cells and found a reduction in sensitivity to both EGFR inhibitors and radiation. Conversely, restoration of functional p53 in EGFR inhibitor resistant cells was sufficient to resensitize them to EGFR inhibitors or radiation in vitro and in vivo. Further studies indicate that p53 may enhance sensitivity to EGFR inhibitors and radiation via induction of cell cycle arrest, apoptosis and DNA damage repair. Taken together, these findings suggest a central role of p53 in the development of acquired resistance to EGFR inhibitors and prompt consideration to apply p53 restoration strategies in future clinical trials that combine EGFR inhibitors and radiation.

Background

Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis. Bevacizumab is a humanized monoclonal antibody that neutralizes VEGF. We examined the impact on radiation response by blocking VEGF signaling with bevacizumab.

Methods

Human umbilical vein endothelial cell (HUVEC) growth inhibition and apoptosis were examined by crystal violet assay and flow cytometry, respectively. In vitro HUVEC tube formation and in vivo Matrigel assays were performed to assess the anti-angiogenic effect. Finally, a series of experiments of growth inhibition on head and neck (H&N) SCC1 and lung H226 tumor xenograft models were conducted to evaluate the impact of bevacizumab on radiation response in concurrent as well as sequential therapy.

Results

The anti-angiogenic effect of bevacizumab appeared to derive not only from inhibition of endothelial cell growth (40%) but also by interfering with endothelial cell function including mobility, cell-to-cell interaction and the ability to form capillaries as reflected by tube formation. In cell culture, bevacizumab induced a 2 ~ 3 fold increase in endothelial cell apoptosis following radiation. In both SCC1 and H226 xenograft models, the concurrent administration of bevacizumab and radiation reduced tumor blood vessel formation and inhibited tumor growth compared to either modality alone. We observed a siginificant tumor reduction in mice receiving the combination of bevacizumab and radiation in comparison to mice treated with bevacizumab or radiation alone. We investigated the impact of bevacizumab and radiation treatment sequence on tumor response. In the SCC1 model, tumor response was strongest with radiation followed by bevacizumab with less sequence impact observed in the H226 model.

Conclusions

Overall, these data demonstrate enhanced tumor response when bevacizumab is combined with radiation, supporting the emerging clinical investigations that are combining anti-angiogenic therapies with radiation.

Background

Motesanib is a potent inhibitor of VEGFR1, 2 and 3, PDGFR and Kit receptors. In this report we examine the interaction between motesanib and radiation in vitro and in head and neck squamous cell carcinoma (HNSCC) xenograft models.

Experimental Design

In vitro assays were performed to assess the impact of motesanib on VEGFR2 signaling pathways in human umbilical vein endothelial cells (HUVECs). HNSCC lines grown as tumor xenografts in athymic nude mice were utilized to assess the in vivo activity of motesanib alone and in combination with radiation.

Results

Motesanib inhibited VEGF-stimulated HUVEC proliferation in vitro, as well as VEGFR2 kinase activity. Additionally motesanib and fractionated radiation showed additive inhibitory effects on HUVEC proliferation. In vivo combination therapy with motesanib and radiation showed increased response compared to drug or radiation alone in UM-SCC1 (p<0.002) and SCC-1483 xenografts (p=0.001); however the combination was not significantly more efficacious than radiation alone in UM-SCC6 xenografts. Xenografts treated with motesanib demonstrated a reduction of vessel penetration into tumor parenchyma, compared to control tumors. Furthermore, triple immunohistochemical staining for vasculature, proliferation, and hypoxia demonstrated well-defined spatial relationships between these parameters in HNSCC xenografts. Motesanib significantly enhanced intratumoral hypoxia in the presence and absence of fractionated radiation.

Conclusions

These studies identify a favorable interaction when combining radiation and motesanib in HNSCC models. Data presented suggest that motesanib reduces blood vessel penetration into tumors and thereby increases intratumoral hypoxia. These findings suggest that clinical investigations examining combinations of radiation and motesanib are warranted in HNSCC.

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux®) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, sprycel®) a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src Family Kinases. We analyzed 16 CRC lines for: 1) KRAS mutation status, 2) dependence on mutant KRAS signaling, 3) expression level of EGFR and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo, and HCT116) cell lines, and two KRAS wild type cell lines (SW48 and CaCo2). In vitro, using Poly-D-Lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab whereas parental controls showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater anti-proliferative effect on KRAS mutant line as compared to either agent alone both in vitro and in vivo. To investigate potential mechanisms for this anti-proliferative response in the combinatorial therapy we performed Human Phospho-kinase Antibody Array analysis measuring the relative phosphorylation levels of phosphorylation of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the classical MAPK pathway, AKT/mTOR pathway and the family of STAT transcription factors when compared to the untreated control or monotherapy treatments. Next we analyzed tumor growth with cetuximab, dasatinib or the combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an anti-tumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation as measured by Ki67 and higher rates of apoptosis as measured by TUNEL. The data presented herein indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key-signaling pathways. Further, these results suggest that signaling via the EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. This data strengthen the rationale for clinical trials in this genetic setting combining cetuximab and dasatinib.

Purpose

In this report, we examine the interaction between panitumumab, a fully human anti-EGFR monoclonal antibody, and radiation in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines and xenografts.

Methods and Materials

HNSCC lines UM-SCC-1 and SCC-1483 as well as the NSCLC line H226 were studied. Tumor xenografts in athymic nude mice were utilized to assess the in vivo activity of panitumumab alone and in combination with radiation. In vitro assays were performed to assess the impact of panitumumab on radiation-induced cell signaling, apoptosis, and DNA damage.

Results

Panitumumab increased radiosensitivity as measured by clonogenic survival assay. Radiation-induced EGFR phosphorylation and downstream signaling through MAPK and STAT3 was inhibited by panitumumab. Panitumumab augmented radiationinduced DNA damage by 1.2–1.6-fold in each of the cell lines studied as assessed by residual γ-H2AX foci after radiation. Radiation-induced apoptosis was increased 1.4–1.9-fold by panitumumab, as evidenced by Annexin V-FITC staining and flow cytometry. In vivo, combination therapy with panitumumab and radiation was superior to panitumumab or radiation alone in H226 xenografts (p=0.01) and showed a similar trend in SCC-1483 xenografts (p=0.08). These in vivo findings correlated with immunohistochemistry examination of PCNA; panitumumab with radiation markedly reduced PCNA staining in tumor xenografts.

Conclusions

These studies identify a favorable interaction when combining radiation and panitumumab in upper aerodigestive tract tumor models, both in vitro and in vivo. These data suggest that clinical investigations examining the combination of radiation and panitumumab in the treatment of epithelial tumors warrant further pursuit.

Purpose

The epidermal growth factor receptor (EGFR) is recognized as a key mediator of proliferation and progression in many human tumors. A series of EGFR specific inhibitors have recently gained FDA approval in oncology. These strategies of EGFR inhibition have demonstrated major tumor regressions in approximately 10–20% of advanced cancer patients. However, many tumors eventually manifest resistance to treatment. Efforts to better understand the underlying mechanisms of acquired resistance to EGFR inhibitors, and potential strategies to overcome resistance, are highly needed.

Experimental Design

To develop cell lines with acquired resistance to EGFR inhibitors we utilized the human head and neck squamous cell carcinoma (HNSCC) tumor cell line SCC-1. Cells were treated with increasing concentrations of cetuximab, gefitinib or erlotinib and characterized for the molecular changes in the EGFR-inhibitor resistant lines relative to the EGFR-inhibitor sensitive lines.

Results

EGFR inhibitor-resistant lines were able to maintain their resistant phenotype in both drug-free medium and in athymic nude mouse xenografts. In addition, EGFR inhibitor-resistant lines showed a markedly increased proliferation rate. EGFR inhibitor-resistant lines had elevated levels of phosphorylated EGFR, MAPK, AKT and STAT3 which were associated with reduced apoptotic capacity. Subsequent in vivo experiments indicated enhanced angiogenic potential in EGFR inhibitor-resistant lines. Finally, EGFR inhibitor-resistant lines demonstrated cross resistance to ionizing radiation.

Conclusions

We have developed EGFR inhibitor-resistant HNSCC cell lines. This model provides a valuable preclinical tool to investigate molecular mechanisms of acquired resistance to EGFR blockade.

The epidermal growth factor receptor (EGFR) is a central regulator of proliferation and progression in human cancers. Five EGFR inhibitors, two monoclonal antibodies and three TKIs, have recently gained FDA approval in oncology (cetuximab, panitumumab, erlotinib, gefitinib and lapatinib). These strategies of EGFR inhibition demonstrate major tumor regressions in approximately 10–20% of advanced cancer patients. However, many tumors eventually manifest acquired resistance to treatment. In this study we established and characterized a model to study molecular mechanisms of acquired resistance to the EGFR monoclonal antibody cetuximab. Using high-throughput screening we examined the activity of 42 receptor tyrosine kinases in resistant tumor cells following chronic exposure to cetuximab. Cells developing acquired resistance to cetuximab exhibited increased steady-state EGFR expression secondary to alterations in trafficking and degradation. In addition, cetuximab-resistant cells manifested strong activation of HER2, HER3 and cMET. EGFR upregulation promoted increased dimerization with HER2 and HER3 leading to their transactivation. Blockade of EGFR and HER2 led to loss of HER3 and PI(3)K/Akt activity. These data suggest that acquired-resistance to cetuximab is accompanied by dysregulation of EGFR internalization/degradation and subsequent EGFR-dependent activation of HER3. Taken together these findings suggest a rationale for the clinical evaluation of combinatorial anti-HER targeting approaches in tumors manifesting acquired resistance to cetuximab.

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.