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
). 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 (). 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
(). 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 () 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
). 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
). 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 (). Consistent with these findings, Viloria-Petit et al found that VEGF expression was elevated in cetuximab-resistant A431 cells developed in vivo
). 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
). 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 (–). 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
). 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.