Although many cancers initially respond to EGFR kinase inhibitors, they invariably become resistant to this therapy. To date, most studies evaluating acquired resistance mechanisms to EGFR TKIs have focused on NSCLCs with EGFR
mutations. In those highly sensitive cancers, laboratory models and patient specimens have identified secondary mutations in EGFR
(T790M) and amplification of the MET
oncogene as mechanisms of acquired resistance (14
). However, it is widely appreciated that some cancers with amplified wild-type EGFR
also derive significant clinical benefit from EGFR TKIs, but there has been a paucity of investigations into potential resistance mechanisms in this population (11
). In this study, we developed a drug-resistant model of the amplified EGFR
wild-type A431 cells. Unlike the parental cells, the A431 GR cells maintain PI3K signaling in the presence of gefitinib. By immunoprecipitating PI3K, we determined that PI3K signaling is maintained in the resistant cells by an activated IGFIR pathway. In these cells, this pathway appears to be activated at least in part by loss of IGFBP-3. Importantly, treating the A431 GR cells with a combination of an EGFR and an IGFIR inhibitor was sufficient to reverse the resistant phenotype. Additionally, we found that resistance to another EGFR wild-type cell line, HN11, also maintained Akt phosphorylation in the presence of gefitinib and was effectively treated with a combination of EGFR and IGFIR inhibitors.
Many studies have suggested that downregulation of PI3K signaling is required for RTK inhibitors to work effectively (24
). For example, EGFR
-amplified cells with PTEN loss have intrinsic resistance to EGFR inhibitors, even though inhibiting EGFR led to downregulation of Erk signaling (26
). Furthermore, ectopic expression of an oncogenic PI3K mutant in a highly sensitive EGFR
mutant cell line (HCC827) is sufficient to confer resistance to gefitinib (24
). Interestingly, in cell-line models of acquired resistance to EGFR inhibitors using EGFR
mutant cell lines HCC827 and H3255, both the Erk and PI3K pathways are maintained in the resistant cells (via a T790M mutation and MET amplification, respectively) (21
). However, unlike those models, the A431 GR and HN11 GR in the study herein continued to downregulate Erk in response to gefitinib but maintained PI3K/Akt signaling. Thus, these models further support the notion that maintenance of PI3K signaling is essential for a cancer cell to become resistant to EGFR TKIs. Indeed, in these models, the IGFIR pathway was sufficient to maintain p-Akt in the presence of gefitinib. Surprisingly, in the parental A431 cells but not the more highly sensitive EGFR
mutant HCC827 cells, addition of exogenous IGF-I ligand was able to maintain Akt, Erk, and mTOR activity in the presence of gefitinib. The inability of exogenous IGF-I to activate these downstream pathways in the mutant EGFR
HCC827 cells despite evidence of ligand-induced IGFIR phosphorylation is intriguing and requires additional investigation.
The parental A431 cells can be made resistant to EGFR TKIs by treatment with exogenous IGF-I. Thus, it was not surprising that the sensitivity of these cells was affected by the presence or absence of fetal bovine serum, as this is a rich source of IGFs. In fact, as shown in Figure A, in the absence of serum, gefitinib modestly inhibited Akt in the resistant cells. Importantly, the in vivo experiments demonstrated that under “physiological” conditions, the emergence of resistance was thwarted by initial treatment of the A431 xenografts with a combination of EGFR and IGFIR inhibitors (Figure B). Such a treatment paradigm has potential clinical ramifications. Perhaps, if patients are treated with therapies designed to combat potential mechanisms of resistance, as is done for infectious diseases such as tuberculosis and HIV, longer-term remissions would be achieved with combinatorial therapy delivered immediately after diagnosis. These results also imply that the testing of drugs that specifically block mechanisms of resistance to targeted therapies, such as IGFIR antagonists in this study, may not provide significant clinical activity when used as single agents but will be effective when used in combination.
It is noteworthy that IGFBP-3 expression is markedly downregulated in the A431 gefitnib-resistant cells. This result fits well the published correlation between EGFR and IGFBP-3 overexpression in esophageal cancers. According to this report, IGFBP-3 expression is at least partially under the control of EGFR activity, although this mechanism is not completely understood. Further, EGF stimulation in A431 cells upregulates IGFBP-3, whereas treatment with AG1478, an EGFR TKI, results in its downregulation (38
). These observations are consistent with our findings that A431 GR cells, which are developed under chronic EGFR inhibition, have suppressed IGFBP-3 expression. This may account for the cells’ adaptation to utilizing the IGFIR pathway to activate PI3K/AKT signaling when grown under conditions of EGFR inhibition. In our study, reexposure of the A431 GR cells to IGFBP-3 resensitized both the PI3K pathway and cell survival to the effects of gefitinib. IGFBP-3 has long been established as a potent negative regulator of IGFIR activation and is believed to block ligand from binding to receptor, although it may also have IGF-independent antiproliferative activities (39
). IGFBP-3 has been shown to inhibit the proliferation and invasiveness of cancer cells (40
). Finally, treatment with recombinant human IGFBP-3 increases the sensitivity of BT474/HerR (Herceptin resistant) cells to trastuzumab in vitro and has shown potent single-agent activity in mice bearing trastuzumab-resistant xenografts (43
The results from a recent phase I study evaluating an anti-IGFIR antibody in patients with advanced human malignancies also highlights the primary role of IGFBP-3 in regulating IGFIR signaling (44
). In this study, there was 1 impressive complete response that occurred in a patient with Ewing sarcoma. This is particularly intriguing because the EWS/FLI-1 translocation characteristic of Ewing sarcoma leads to loss of IGFBP-3 expression via direct transcriptional repression by the resulting fusion protein (45
). Thus, it is tempting to speculate that these molecular events lead to activation of and “addiction” to IGFIR and thereby explain the complete response observed in this clinical trial. It will be interesting to learn whether loss of IGFBP expression serves as a marker of addiction to IGFIR signaling in other cell lines and tumor types as well.
Unlike EGFR T790M mutation and MET amplification, activation of IGFIR pathway via loss of IGFBP expression is not a genetic event that will easily be identified in patient specimens. Although one could envision using immunohistochemistry or quantitative RT-PCR to evaluate for these effects, it is likely that such results will be less clear. Since antibodies targeting IGFIR and small-molecule IGFIR inhibitors are entering the clinic, we will likely learn of the effectiveness of combining EGFR and IGFIR inhibitors soon. If clinically tolerated, this combination may be an optimal approach to delaying the onset of resistance as we observed in A431 xenografts. In fact, some combinations of irreversible EGFR TKIs to inhibit T790M, MET inhibitors, and IGFIR antagonists may ultimately find their way to front-line therapy for patients that have EGFR-driven cancers. This approach may eliminate a significant proportion of the tumors that develop acquired resistance and potentially prolong the benefit of anti-EGFR therapy.