Shyhmin Huang

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 inhibition in cancer therapy: biology, rationale and preliminary clinical results.

The epidermal growth factor receptor (EGFR), a growth factor receptor involved in the regulation of cellular differentiation and proliferation, is highly expressed by many tumor cells. In light of a relationship between overexpression of EGFR and clinically aggressive malignant disease, EGFR has emerged as a promising target for cancer therapy. In recent years, several molecular strategies have been explored to modulate either the EGFR itself, or the downstream signal beyond the cell surface receptor. One of the most promising current strategies involves the use of anti-EGFR monoclonal antibodies (mAbs), either alone or in combination with conventional cytotoxic modalities such as chemotherapy or radiotherapy. This review focuses primarily on recent progress in the development of anti-EGFR mAbs, and examines their potential in the treatment of cancer.

Head and neck cancer as a clinical model for molecular targeting of therapy: combining EGFR blockade with radiation.

PURPOSE/OBJECTIVE The primary purpose of this presentation is to develop the concept that molecular blockade of specific growth factor receptors and signal transduction pathways in combination with radiation will prove a valuable cancer therapeutic strategy. More specifically, the rationale for molecular blockade of the epidermal growth factor receptor (EGFR) system in combination with ionizing radiation for epithelial tumors, such as squamous cell carcinomas (SCCs) of the head and neck (H&N), is described. METHODS AND MATERIALS Preclinical experimentation with in vitro and in vivo model systems regarding the capacity of EGFR blockade, using the monoclonal antibody C225, to modulate SCC tumor growth behavior and response to radiation is presented. The rationale for new clinical trials that are currently exploring this concept are presented. RESULTS Blockade of the EGFR system in SCC cell lines with C225 induces G1 cell cycle arrest with an associated decrease in the S-phase fraction. Inhibition of tumor cell proliferation is readily measured following C225 exposure and the corresponding alterations in expression of key regulators of the G1-S cell cycle phase transition are identified. Exposure of SCCs to C225 in culture enhances radiosensitivity following single-dose radiation exposure. Profound augmentation of the in vivo radiation response of SCC tumor xenografts in athymic mice is similarly demonstrated following systemic administration of C225. Preliminary studies are presented regarding potential underlying mechanisms of action for this enhanced tumor response to the combination of C225 and radiation including: (a) proliferative growth inhibition, (b) enhancement of radiation-induced apoptosis, (c) inhibition of damage repair, and (d) downregulation of tumor angiogenic response. Preliminary observations from the Phase III multicenter clinical trial examining C225 plus radiation therapy for advanced H&N cancer patients are provided. CONCLUSION Molecular inhibition of the EGFR signal transduction system in combination with radiation represents a promising investigational area in cancer therapeutics. Epithelial tumors that are rich in their expression of EGFR (e.g., SCC of the H&N) hold special promise for receptor blockade approaches. More broadly, the ultimate therapeutic effect of selected molecular agents which block specific growth factor receptors and signaling pathways may be enhanced when delivered in combination with radiation.

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.

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.

Enhancing the antitumor activity of ErbB blockade with histone deacetylase (HDAC) inhibition

Molecular inhibition of the ErbB signaling pathway represents a promising cancer treatment strategy. Preclinical studies suggest that enhancement of antitumor activity can be achieved by maximizing ErbB signaling inhibition. Using cDNA microarrays, we identified histone deacetylase (HDAC) inhibitors as having strong potential to enhance the effects of anti‐ErbB agents. Studies using a 20,000 element (20K) cDNA microarray demonstrate decreased transcript expression of ErbB1 (epidermal growth factor receptor) and ErbB2 in DU145 (prostate) and ErbB2 in SKBr3 (breast) cancer cell lines. Additional changes in the DU145 gene expression profile with potential interaction to ErbB signaling include down‐regulation of caveolin‐1 and hypoxia inducible factor 1‐α (HIF1‐α), and up‐regulation of gelsolin, p19(INK4D) and Nur77. Findings were validated using real time RT‐PCR and Western blot analysis. Enhanced proliferative inhibition, apoptosis induction and signaling inhibition were demonstrated when combining HDAC inhibition with ErbB blockade. These results suggest that used cooperatively, anti‐ErbB agents and HDAC inhibitors may offer a promising strategy of dual targeted therapy. Additionally, microarray data suggest that the beneficial interaction of these agents may not derive solely from modulation of ErbB expression, but may result from effects on other oncogenic processes including angiogenesis, invasion and cell cycle kinetics.