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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.
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.
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.
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 EGFR is a member of the ErbB family of receptor tyrosine kinases. Four ErbB members have been identified to date: EGFR (ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). EGFR plays a critical role in development and in neoplastic processes of cell proliferation, 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. 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 (1).
The EGFR is recognized as a central regulator of proliferation and progression in many human cancers. In general terms, tumor EGFR expression correlates inversely with clinical outcome (2). A series of potent EGFR inhibitors (cetuximab, panitumumab, erlotinib and gefitinib) have been developed in recent years, and several thousand cancer patients have now been treated in the context of controlled clinical trials (3–6). Despite great clinical promise, with 10–20% of patients manifesting highly favorable response to EGFR inhibition, the majority of cancer patients demonstrate either intrinsic resistance or acquired resistance to EGFR inhibitor therapies (7–11). The identification of resistance mechanisms to EGFR inhibitors remains critical to the successful advancement of these promising molecular targeting agents. In this communication, we present the development of cetuximab-, gefitinib-, or erlotinib-resistant H&N tumor cells following chronic exposure to these agents. In addition, we explored the molecular mechanisms associated with the resistant phenotype by characterizing specific molecular and cellular distinctions between EGFR inhibitor-resistant and sensitive parental cells. These studies provide valuable insight regarding molecular mechanisms of acquired resistance to EGFR targeting agents and thereby provide a model to explore strategies to overcome acquired resistance to EGFR targeting agents.
The human head and neck (HNSCC) cell lines, SCC-1 (UM-SCC1) was kindly provided by Dr. Thomas E. Carey (University of Michigan) and were cultured routinely in DMEM supplemented with 10% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 1% penicillin and streptomycin. Cell culture media and supplements were obtained from Life Technologies, Inc. (Gaithersburg, MD). Gefitinib (ZD1839, Iressa™) was generously provided by AstraZeneca (Macclesfield, UK). Erlotinib (OSI-774, Tarceva™) was generously provided by Genentech, Inc (San Francisco, CA). Cetuximab (C225, Erbitux™) was generously provided by ImClone Systems Inc. (New York, NY). Primary antibodies against p-MAPK (Thr202/Tyr204), p-AKT(Ser473) and STAT3 (Tyr705) were obtained from Cell Signaling Technology (Beverly, MA). Anti-p-EGFR(Tyr1173) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and anti-α-tubulin antibody was obtained from Oncogene Research Products (Cambridge, MA). Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences Pharmingen (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO).
Over a period of 12 months, tumor cells in culture were continuously exposed to increasing concentrations of cetuximab, gefitinib, or erlotinib. Commencing with the IC50 of EGFR inhibitors for a particular tumor cell line, the exposure dose was progressively doubled every 10–14 days until 7~8 dose doublings had been successfully achieved. In parallel, controlled parental cells were exposed to corresponding vehicle for the drugs, i.e., PBS for cetuximab and DMSO for gefitinib and erlotinib. The established resistant cell lines were then maintained in continuous culture with the maximally achieved dose of EGFR inhibitor that still allowed cellular proliferation.
Exponentially growing cells were seeded in 6 well plates. Following the treatment, cells were then washed with PBS and fixed/stained with 0.5% crystal violet. Plates were air dried overnight and dye was eluted with 0.1 M sodium citrate (pH 4.2) in ethanol (1:1). Elution was transferred to 96 well plates, and the absorbance was read at 540 nm to determine cell viability.
Parental or resistant cells were harvested by trypsin, washed with PBS, then fixed in 95% ethanol and stored at 4°C for up to 7 days prior to DNA analysis. After the removal of ethanol by centrifugation, cells were incubated with phosphate-citric acid buffer (0.2 M Na2HPO4, pH 7.8, 4 mM citric acid) at room temperature for 45 min. After centrifugation, cells were then stained with a solution containing 33 μg/ml PI, 0.13 mg/ml RNase A, 10 mM EDTA and 0.5% Triton X-100 at 4 °C for 24 h. Stained nuclei were analyzed for DNA-PI fluorescence using a Becton Dickinson FACScan flow cytometer. Resulting DNA distributions were analyzed by Modfit (Verity Software House Inc., Topsham, ME) for the proportion of cells in G1, S, and G2/M phases of the cell cycle.
Following treatment, cells were lysed with Tween-20 lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 μg/ml of leupeptin and aprotinin) and sonicated. Equal amounts of protein were analyzed by SDS-PAGE. Thereafter, proteins were transferred to nitrocellulose membranes and analyzed by specific primary antibodies as indicated in the experiment. Proteins were detected via incubation with HRP-conjugated secondary antibodies and ECL chemiluminescence detection system.
Apoptosis was detected by flow cytometry via the examination of altered plasma membrane phospholipid packing by lipophilic dye Annexin V. Using Annexin V as a fluorescein isothiocyanate (FITC) conjugate in combination with propidium iodide (PI) as an exclusion dye for cell viability, this assay not only can detect apoptotic cells but also discriminate between apoptosis and necrosis. Briefly, treated cells were harvested by trypsin, washed twice with PBS and were then re-suspended in binding buffer at a concentration of 1 × 106 cells/ml according to the manufacturer’s instruction. Thereafter, 5 μl of Annexin V-FITC and 5 μl of PI were added into 100 μl of cell suspension and incubated for 30 min at RT in the dark. After adding 400 μl of binding buffer, labeled cells were counted by flow cytometry within 30 min. All early apoptotic cells (Annexin V-positive, PI-negative), necrotic/late apoptotic cells (double positive) as well as living cells (double negative) were detected by FACSCalibur flow cytometer and subsequently analyzed by Cell Quest software (Becton Dickinson, San Diego, CA). Argon laser excitation wavelength was 488 nm, while emission data were acquired at wavelength 530 nm (FL-1 channel) for FITC and 670 nm (FL-3 channel) for PI.
Athymic nude mice (3–4-week-old females) were obtained from Harlan Bioproducts for science (Indianapolis, IN) and maintained in a laminar air-flow cabinet under aseptic conditions. The care and treatment of experimental animals was in accordance with institutional guidelines. Human cancer cells (~1 × 106) were injected subcutaneously (s.c.) into the dorsal flank area of the mice. Following the establishment of tumor, cetuximab was administered via i.p. at a dose of 0.2 mg twice per week for 4 consecutive weeks, and gefitinib or erlotinib were given by oral gavage at a dose of 1 mg, 5 days per week for 4 consecutive weeks. Tumor volume was determined by direct measurement with calipers and calculated by the formula; π/6 × (large diameter) ×(small diameter)2.
Tumor angiogenesis was evaluated by Matrigel plug neovasculation assay as described previously (12). Briefly, athymic mice were injected subcutaneously along the flank area with 0.5 ml Matrigel. After 24 hrs, a suspension of SCC-1 cells (1 × 106 in 5 μl) was soaked with a polyvinyl sponge (2 × 2 × 1.5 mm). The sponge was then introduced into a surgically created micropocket in the center of Matrigel plug formed within the mouse abdominal wall. The wound was then closed with a suture. Ten days later, mice were injected with 0.2 ml of a 50 mg/ml FITC-Dextran (MW, ~2,000 kDa) solution via the tail vein for the purpose of visualizing vessels within the Matrigel plug. After 15 min, mice were sacrificed, and the Matrigel plugs were removed and fixed in 10% formalin solution. To visualize the general layout of the Matrigel plug and the presence of perfused blood vessels, phase-contrast microscopy and fluorescence microscopy were used respectively. The intensity of fluorescence was further quantified by Adobe Photoshop software (Adobe Systems, Mountain View, CA).
Survival following radiation exposure was defined as the ability of the cells to maintain their clonogenic capacity and to form colonies. Briefly, after exposure to radiation, cells were trypsinized, counted, and seeded for colony formation in 35 mm dishes at 50–5000 cells/dish. Following incubation intervals of 14–21 days, colonies were stained with crystal violet and manually counted. Colonies consisting of 50 cells or more were scored, and 4–10 replicate dishes containing 10–150 colonies/dish were counted for each treatment.
The effect of EGFR inhibitors on apoptosis induction was evaluated using Student’s t test
The HNSCC cell line SCC-1 was used to develop resistance to the EGFR inhibitors cetuximab, erlotinib and gefitinib. As described in “Materials and Methods”, treatment started at the IC50 of each drug which caused 50% inhibition of cell proliferation and the exposure dose was progressively doubled every 10–14 days until 7–8 dose doublings had been achieved. The cetuximab resistant lines (Cet-R) were treated up to a maximal dose of 640–1280 nM of cetuximab, whereas the gefitinib- (Gef-R) and erlotinib-resistant (Erl-R) lines reached a maximal dose of 6.4 μM each. After the establishment of EGFR inhibitor resistant lines, we characterized their resistant phenotype by performing cell proliferation assays when challenged with EGFR inhibitors (Fig. 1). We consistently observed higher proliferative potential and a 10-fold increase or greater in the IC50 for all EGFR inhibitor-resistant cell lines as compared with parental cells (ΔIC50). Cell cycle analysis demonstrated that Cet-R, Gef-R and Erl-R cells did not exhibit a G1 arrest or marked reduction in S phase when challenged with cetuximab, gefitinib or erlotinib as compared to the sensitive parental controls (Supplementary Fig. S1). These results indicate that characteristic cell cycle checkpoints in EGFR inhibitor-resistant lines are no longer affected by EGFR blockade. We then confirmed the establishment of stable EGFR inhibitors-resistant cells in a drug-free culture system. Results demonstrated that EGFR inhibitor-resistant SCC-1 cells still exhibited the resistant phenotype even when cells were cultured in drug-free medium for at least 9 months (Supplementary Fig. S2).
Building upon these results, we used a mouse xenograft model to determine if the resistance to EGFR inhibitors developed in vitro would retain the resistance phenotype in vivo. To perform these experiments, we inoculated the EGFR inhibitor sensitive lines (Parental) and the EGFR-inhibitor resistant lines into the dorsal flank of athymic nude mice. After two weeks, systemic administration of either vehicle or the corresponding EGFR inhibitor was delivered to mice. These in vivo results, presented in Fig. 2, indicate that EGFR inhibitor-resistant cells established in culture maintain their resistant phenotype in the in vivo xenograft model system. Taken together, these results indicate that we have developed SCC-1 cell lines resistant to cetuximab, erlotinib and gefitinib. In addition, these cells can grow in the absence of drug for long periods of time and maintain their resistant phenotype as well as maintaining a resistant phenotype in vivo.
To determine if the EGFR signaling cascade was altered in EGFR-inhibitor resistant lines, we investigated the active EGFR, AKT, MAPK & STAT3, Using immunoblotting analysis, we found that treatment with EGFR inhibitors efficiently blocked EGF-stimulated activation of EGFR, AKT, MAPK & STAT3 in a dose-dependent manner in parental cells but not in Cet-R, Gef-R and Erl-R cells (Fig. 3). These results not only confirm the resistant characteristic of resistant cells to EGFR inhibitor but also imply that alternative signaling pathways related to MAPK, AKT or STAT3 may play an important role in the development of resistance to EGFR targeting agents.
To determine if EGFR-inhibitor resistant lines had decreased apoptotic potential, we measured apoptosis using Annexin V/PI staining in parental and resistant SCC-1 cells. As shown in Fig. 4, treatment with cetuximab, gefitinig or erlotinib resulted in the significant induction of apoptosis in a dose dependent manner in parental cells. However, there was no significant change of early apoptotic cell populations in Cet-R, gef-R or Erl-R cells following EGFR inhibitor exposure. These results indicate that EGFR-inhibitor resistant lines may be able to escape the characteristic apoptotic response induced by EGFR inhibitors.
Alteration of EGFR signaling in EGFR inhibitors-resistant cells may cause changes in angiogenic potential. To test this hypothesis, we investigated tumor vascularization by subcutaneously implanting tumor cell-imbedded Matrigel plugs into nude mice as described previously (13). In this assay, parental and EGFR inhibitor-resistant SCC-1 cells were introduced into separate Matrigel plug in the same mouse. Following implantation with tumor cells, blood capillaries were observed growing from the edge of the Matrigel plugs toward the implanted tumor as visualized by fluorescent microscopy. As shown in Fig. 5, plugs with parental SCC-1 cells were visibly clear, with limited identifiable blood vessels migrating into the Matrigel. In contrast, plugs containing Cet-R, Gef-R or Erl-R cells showed extensive vascularization and growth of vessels towards the tumor core that was confirmed by the enhanced intensity of fluorescence (bottom panel). These results suggest that the development of acquired resistance to EGFR inhibitors in vitro can enhance mechanisms involved in angiogenesis.
To determine if EGFR inhibitor-resistant cells have increased resistance to radiation treatment, we evaluated EGFR inhibitor resistant lines using clonogenic survival assays (14). Fig. 6 depicts radiation-survival curves for Cet-R, Gef-R, Erl-R and the corresponding parental SCC-1 cells. The results indicated that EGFR inhibitor-resistant cells had a higher survival rate when treated with 3, 6, or 9 Gy of radiation as compared to parental cells. The reduced cell death in resistant cells was further confirmed by evaluating the apoptosis profile of tumor cells following radiation treatment using Annexin V/PI flow cytometric analysis as described above. As shown in Fig. 7, treatment with radiation resulted in the induction of apoptosis in a dose dependent manner in the parental cells. However, there was no significant change in the apoptotic cell populations when Cet-R, Gef-R or Erl-R cells were treated with radiation. Taken together, these results indicate that tumor cells with acquired resistance to EGFR targeting inhibitors manifest radioresistance possibly by altered abilities to effectively induce apoptosis.
Molecular inhibition of EGFR signaling is under active investigation as a promising cancer treatment strategy. Despite broad enthusiasm regarding the potential value of EGFR target modulation in cancer therapy, acquired resistance to EGFR inhibitors has been widely observed in preclinical model systems and in cancer patients who initially respond well to treatment (11, 15–18). Similar to the development of acquired resistance to other molecular targeted agents, such as Gleevec (19) and Herceptin (20), acquired resistance to EGFR inhibitors may limit therapy options, as EGFR inhibitor-resistant tumors may also become cross-resistant to other drug or treatment modalities with different mechanisms of action (21). Efforts to better understand the underlying mechanisms of acquired resistance to EGFR inhibitors, and potential strategies to overcome resistance, are highly needed.
In the current study we present the development of a HNSCC tumor cell line (SCC-1) resistant to cetuximab, gefitinib and erlotinib to explore mechanisms of resistance to EGFR blockade (Fig. 1). To determine if acquired resistance in this in vitro model was stable, we cultured resistant cells in the presence or absence of cetuximab, gefitinib or erlotinib for nine months. After nine months in drug-free culture, cells maintained their resistance to each individual EGFR inhibitor indicating that the molecular changes that occur in acquired resistance to EGFR blockade are stable (Supplementary Fig. S2). Furthermore, cells with acquired resistance to cetuximab, gefitinib or erlotinib could form xenografts in athymic nude mice and maintain their resistant phenotype when challenged with EGFR inhibitors in vivo (Fig. 2). Results from cell cycle analysis (Supplementary Fig. S1) provide a potential clue to the resistant phenotype which suggests that cells with acquired resistance to EGFR inhibitors have a large population of cells in S-phase of the cell cycle relative to parental controls. Furthermore, cells with acquired resistance to EGFR inhibitors maintain this population in S-phase after challenge with EGFR inhibitors, whereas the parental cells arrest primarily in G1. These findings suggest that cells with acquired resistance to EGFR inhibitors receive proliferative signals independent of signaling from the EGFR, and maintain cells in S-phase even when challenged with EGFR inhibitors.
Although EGFR inhibitors are known to inhibit the PI3K/AKT pathway, the consistent observation of elevated level of p-AKT in our resistant cells (Fig. 3) indicates constitutive activation of AKT as an important mediator of EGFR resistance. Our findings are consistent with the findings of Yamasaki et al. where they observed acquired resistance to erlotinib following constitutively active Akt transfection in A431 cells (15). Janmaat et al. also reported that persistent activity of the PI3K/AKT and/or MAPK pathway associated with gefitinib-resistance of NSCLC cell lines and that simultaneous inhibition of both pathways reduces tumor survival more effectively than inhibition of each pathway alone (22). These data are consistent with other reports showing that loss of PTEN results in PI3K and AKT hyperactivity and resistance to gefitinib in MDA-468 breast cancer cells (23, 24). Reconstitution of PTEN in these cells re-established EGFR-driven AKT signaling and thereby restored gefitinib sensitivity (25). More interestingly, recent studies from our group and the Engelman group demonstrate that sensitive cancers may adapt to activate the PI3K-AKT pathway as they become resistant via activation of alternative receptor tyrosine kinases, such as ErbB3, c-Met and IGFR (26–28). Taken together, these data suggest that AKT-mediated survival-signaling pathways play a key role in resistance to anti-EGFR therapy and that the use of inhibitors that target the AKT pathway in resistant tumors may be beneficial for clinical response.
Beyond targeting AKT, an alternative strategy to overcome acquired resistance to EGFR inhibitors is to combine EGFR agents with angiogenesis targeting agents. This approach is highly appealing in view of observations that the angiogenic process is involved in the development of resistance to anti-EGFR therapy. By using the matrigel plug neovascularization assay, we demonstrate that plugs with acquired resistance to cetuximab, gefitinib or erlotinib exhibit a higher vessel density than parental cells (Fig. 5). Consistent with these findings, Viloria-Petit et al found that VEGF expression was elevated in cetuximab-resistant A431 cells developed in vivo (18). In addition, Ciardiello et al determined that VEGF expression was elevated in gefitinib-resistant cells when screening a panel of colon cancer cell lines (30). Thus, up-regulation of VEGF may contribute to increased angiogenesis and contribute to resistance to EGFR inhibitors. Combining anti-angiogenic agents with EGFR inhibitors may confer the resistance to anti-EGFR therapy. To test this possibility, combined treatment of EGFR targeting agent and VEGFR targeting agent, ZD6474, has been evaluated in gefitinib and cetuximab-resistant tumor cells (31, 32). The results demonstrated that the combined treatment achieved significantly greater tumor growth inhibition in both sensitive and resistant tumor cells. Furthermore, ZD6474 inhibited tumor growth in cells that were refractory to anti-EGFR therapy. These results provide a clinical rationale for further investigation of anti-angiogenenic agents as a potential treatment option for EGFR inhibitor-resistant tumors.
Acquired resistance to EGFR inhibitors presents a clinical problem not only due to the development of resistance to EGFR blocking agents, but also due to potential manifestation of resistance to other drug or treatment modalities with distinct mechanisms of action. Data from the present study demonstrates that following chronic exposure to EGFR targeting agents, tumor cells with acquired resistance to EGFR inhibitors developed resistance to ionizing radiation (Fig. 6–7). Consistent with our findings, previous reports indicate that dose and schedule of drug administration in combination with radiation appeared to be a critical element to obtain radiosensitization of tumor cells (33, 34). Stea et al. suggested that the optimal combination of gefitinib and irradiation occurred only with a short pre-incubation period of 30 min followed by 8 hrs of continuous exposure to gefitinib post-radiation. On the other hand, a prolonged pre-incubation interval of 24 hrs not only reduced radiosensitivity, but actually conferred radioprotection in tumor cells (33). These results suggest that the optimal dose of drug for radiosensitivity modulation and sequencing of modalities should be considered with caution. Furthermore, these results warrant consideration in the design of future clinical strategies for EGFR-radiation combinations. Specifically, it may prove most advantageous to deliver EGFR inhibitors during or after radiation as opposed to before radiation. Recent data from Milas et. al. similarly suggest benefit from continuing EGFR inhibitor therapy following radiation treatment (35).
In summary, the work presented herein describes the establishment of resistant tumor cell lines to cetuximab, gefitinib or erlotinib derived from H&N SCC-1 cells. Our results demonstrate that following long-term exposure to anti-EGFR agents, tumor cells acquire resistance not only to anti-EGFR therapy but also to radiation therapy. Several lines of data point to potential resistance mechanisms involving the activation of alternative survival pathways, such as PI3K/AKT, pro-apoptotic and angiogenic cascades. Proteomic- and genomic-based approaches are current underway to further examine molecular and cellular profiles of resistant cell lines to better understand molecular mechanisms that enable bypass of anti-EGFR effects. Utilizing cetuximab-, gefitinib- and erlotinib-resistant cells, this model system provides a valuable resource to further define molecules involved in EGFR targeting, as well as potential methods to overcome treatment resistance.
The development of acquired resistance to EGFR inhibitors is emerging as a potential treatment barrier for EGFR targeted therapy. In the current study, we establish and characterize EGFR inhibitor-resistant tumor cells against three leading EGFR inhibitors to investigate molecular mechanisms of acquired resistance and consider potential strategies that may help overcome resistance. Several lines of data suggest resistance mechanisms that involve the activation of alternative survival pathways, such as AKT and angiogenic signaling. These data provide a rationale for the investigation of AKT or angiogenesis targeting agents as worthy treatment approaches for tumors that manifest EGFR inhibitor-resistance. In addition, results from the current study provide data regarding the combination of EGFR inhibitors with radiation. In the context of EGFR-inhibitor resistance, the data suggests that it may prove most advantageous to deliver EGFR inhibitors concurrent or immediately following radiation as opposed to prior to radiation.
Grant support: This work was supported in part by NIH R01 CA 113448-01 (PMH)
We gratefully thank Dr. for expert guidance regarding Matrigel plug assay and Kathleen Schell for her assistance in the flow cytometry facility at the University of Wisconsin Comprehensive Cancer Center. We also wish to thank ImClone (Cetuximab), AstraZeneca (Gefitinib) and Genentech/OSI (Erlotinib) for kindly providing anti-EGFR agents for experimental studies.