Eric A. Armstrong

Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members

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.

Dual-Agent Molecular Targeting of the Epidermal Growth Factor Receptor (EGFR) Combining Anti-EGFR Antibody with Tyrosine Kinase Inhibitor

Molecular inhibition of epidermal growth factor receptor (EGFR/HER1) signaling is under active investigation as a promising cancer treatment strategy. We examined the potency of EGFR inhibition achieved by combining anti-EGFR monoclonal antibody and tyrosine kinase inhibitor, which target extracellular and intracellular domains of the receptor, respectively. We specifically studied the combination of cetuximab (Erbitux, C225; ImClone Systems, New York, NY) with either gefitinib (Iressa, ZD1839; AstraZeneca, Macclesfield, UK) or erlotinib (Tarceva, OSI-774; Genentech, South San Francisco, CA) across a variety of human cancer cells. The combination of cetuximab plus gefitinib or erlotinib enhanced growth inhibition over that observed with either agent alone. As measured by immunostaining, inhibition of EGFR phosphorylation with the combination of cetuximab plus gefitinib or erlotinib was augmented over that obtained with single-agent therapy in head and neck (H&N) cancer cell lines. Phosphorylation inhibition of downstream effector molecules [mitogen-activated protein kinase (MAPK) and AKT] also was enhanced in tumor cells treated with the combination of cetuximab plus gefitinib or erlotinib. Flow cytometry and immunoblot analysis demonstrated that treatment of H&N tumor cells with cetuximab in combination with either gefitinib or erlotinib amplified the induction of apoptosis. Following establishment of cetuximab-resistant cell lines, we observed that gefitinib or erlotinib retained the capacity to inhibit growth of lung and H&N tumor cells that were highly resistant to cetuximab. Treatment with gefitinib or erlotinib, but not cetuximab, also could further inhibit the activation of downstream effectors of EGFR signaling in cetuximab-resistant cells, including MAPK and AKT. These data suggest that tyrosine kinase inhibitors may further modulate intracellular signaling that is not fully blocked by extracellular anti-EGFR antibody treatment. Finally, animal studies confirmed that single EGFR inhibitor treatment resulted in partial and transient tumor regression in human lung cancer xenografts. In contrast, more profound tumor regression and regrowth delay were observed in mice treated with the combination of cetuximab and gefitinib or erlotinib. Immunohistochemical staining, which demonstrated significant reduction of the proliferative marker proliferating cell nuclear antigen in mice treated with dual EGFR inhibitors, further supported this in vivo observation. Together, these data suggest that combined treatment with distinct EGFR inhibitory agents can augment the potency of EGFR signaling inhibition. This approach suggests potential new strategies to maximize effective target inhibition, which may improve the therapeutic ratio for anti-EGFR-targeted therapies in developing clinical trials.

Mechanisms of Enhanced Radiation Response following Epidermal Growth Factor Receptor Signaling Inhibition by Erlotinib (Tarceva)

Erlotinib (Tarceva) is an orally available HER1 (epidermal growth factor receptor, EGFR) tyrosine kinase inhibitor advancing through clinical trials for the treatment of a range of human malignancies. In this study, we examine the capacity of erlotinib to modulate radiation response and investigate specific mechanisms underlying these interactions in human tumor cell lines and xenografts. The impact of erlotinib on cell cycle kinetics was analyzed using flow cytometry, and the impact on apoptosis was evaluated via fluorescein-labeled pan-caspase inhibition and poly(ADP-ribose) polymerase cleavage. Radiation-induced EGFR autophosphorylation and Rad51 expression were examined by Western blot analysis. Radiation survival was analyzed using a clonogenic assay and assessment of in vivo tumor growth was done using a mouse xenograft model system. Microarray studies were carried out using 20 K human cDNA microarray and select genes were validated using quantitative reverse transcription-PCR (RT-PCR). Independently, erlotinib and radiation induce accumulation of tumor cells in G(1) and G(2)-M phase, respectively, with a reduction of cells in S phase. When combined with radiation, erlotinib promotes a further reduction in S-phase fraction. Erlotinib enhances the induction of apoptosis, inhibits EGFR autophosphorylation and Rad51 expression following radiation exposure, and promotes an increase in radiosensitivity. Tumor xenograft studies confirm that systemic administration of erlotinib results in profound tumor growth inhibition when combined with radiation. cDNA microarray analysis assessing genes differentially regulated by erlotinib following radiation exposure identifies a diverse set of genes deriving from several functional classes. Validation is confirmed for several specific genes that may influence radiosensitization by erlotinib including Egr-1, CXCL1, and IL-1beta. These results identify the capacity of erlotinib to enhance radiation response at several levels, including cell cycle arrest, apoptosis induction, accelerated cellular repopulation, and DNA damage repair. Preliminary microarray data suggests additional mechanisms underlying the complex interaction between EGFR signaling and radiation response. These data suggest that the erlotinib/radiation combination represents a strategy worthy of further examination in clinical trials.

Epidermal Growth Factor Receptor cooperates with Src Family Kinases in 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.

Insulin-like Growth Factor-I Receptor Signaling Blockade Combined with Radiation

Signaling through the insulin-like growth factor-I receptor (IGF-IR) is implicated in cellular proliferation, apoptosis, carcinogenesis, metastasis, and resistance to cytotoxic cancer therapies. Targeted disruption of IGF-IR signaling combined with cytotoxic therapy may therefore yield improved anticancer efficacy over conventional treatments alone. In this study, a fully human anti-IGF-IR monoclonal antibody A12 (ImClone Systems, Inc., New York, NY) is examined as an adjunct to radiation therapy. IGF-IR expression is shown for a diverse cohort of cell lines, whereas targeted IGF-IR blockade by A12 inhibits IGF-IR phosphorylation and activation of the downstream effectors Akt and mitogen-activated protein kinase. Anchorage-dependent proliferation and xenograft growth is inhibited by A12 in a dose-dependent manner, particularly for non-small cell lung cancer lines. Clonogenic radiation survival of H226 and H460 cells grown under anchorage-dependent conditions is impaired by A12, demonstrating a radiation dose-enhancing effect for IGF-IR blockade. Postradiation anchorage-independent colony formation is inhibited by A12 in A549 and H460 cells. In the H460 xenograft model, combining A12 and radiation significantly enhances antitumor efficacy compared with either modality alone. These effects may be mediated by promotion of radiation-induced, double-stranded DNA damage and apoptosis as observed in cell culture. In summary, these results validate IGF-IR signal transduction blockade as a promising strategy to improve radiation therapy efficacy in human tumors, forming a basis for future clinical trials.

Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab

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 (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (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 KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines 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 mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor 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 (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

A Phosphatidylinositol 3-Kinase-dependent Pathway That Differentially Regulates c-Raf and A-Raf

Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.

Establishment and Characterization of a Model of Acquired Resistance to Epidermal Growth Factor Receptor Targeting Agents in Human Cancer Cells

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 Food and Drug Administration approval in oncology. These strategies of EGFR inhibition have shown major tumor regressions in approximately 10% to 20% of advanced cancer patients. Many tumors, however, 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 greatly needed. Experimental Design: To develop cell lines with acquired resistance to EGFR inhibitors we utilized the human head and neck squamous cell carcinoma 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, mitogen-activated protein kinase, AKT, and signal transducer and activator of transcription 3, 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 showed cross-resistance to ionizing radiation. Conclusions: We have developed EGFR inhibitor–resistant human head and neck squamous cell carcinoma cell lines. This model provides a valuable preclinical tool to investigate molecular mechanisms of acquired resistance to EGFR blockade.

p53 Modulates Acquired Resistance to EGFR Inhibitors and Radiation

There is presently great interest in mechanisms of acquired resistance to epidermal growth factor receptor (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.

Development and Characterization of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma Tumorgrafts

Purpose: To develop a clinically relevant model system to study head and neck squamous cell carcinoma (HNSCC), we have established and characterized a direct-from-patient tumorgraft model of human papillomavirus (HPV)–positive and HPV-negative cancers. Experimental Design: Patients with newly diagnosed or recurrent HNSCC were consented for donation of tumor specimens. Surgically obtained tissue was implanted subcutaneously into immunodeficient mice. During subsequent passages, both formalin-fixed/paraffin-embedded as well as flash-frozen tissues were harvested. Tumors were analyzed for a variety of relevant tumor markers. Tumor growth rates and response to radiation, cisplatin, or cetuximab were assessed and early passage cell strains were developed for rapid testing of drug sensitivity. Results: Tumorgrafts have been established in 22 of 26 patients to date. Significant diversity in tumorgraft tumor differentiation was observed with good agreement in degree of differentiation between patient tumor and tumorgraft (Kappa 0.72). Six tumorgrafts were HPV-positive on the basis of p16 staining. A strong inverse correlation between tumorgraft p16 and p53 or Rb was identified (Spearman correlations P = 0.085 and P = 0.002, respectively). Significant growth inhibition of representative tumorgrafts was shown with cisplatin, cetuximab, or radiation treatment delivered over a two-week period. Early passage cell strains showed high consistency in response to cancer therapy between tumorgraft and cell strain. Conclusions: We have established a robust human tumorgraft model system for investigating HPV-positive and HPV-negative HNSCC. These tumorgrafts show strong correlation with the original tumor specimens and provide a powerful resource for investigating mechanisms of therapeutic response as well as preclinical testing. Clin Cancer Res; 19(4); 855–64. ©2012 AACR.