Cetuximab has shown great promise in clinical oncology, but is limited by intrinsic as well as acquired resistance. These resistance issues present a clinical obstacle for the optimal advancement of this promising molecular targeting agent. In this study we investigated mechanisms of acquired resistance to cetuximab using previously established cetuximab-resistant tumor clones following long-term exposure to cetuximab (Wheeler et al., 2008
). Sub-cellular analysis of EGFR localization in cetuximab-resistant clones indicated a marked increase in nuclear expression of the EGFR (). Emerging evidence over the last decade has indicated that nEGFR has been detected in highly proliferative tissues and linked with poor clinical outcome in breast, oropharyngeal SCC, and ovarian cancer (Lo and Hung, 2006
; Psyrri et al., 2005
; Xia et al., 2008
). Furthermore, nEGFR functions as a transcription factor for cyclin D1, iNOS, B-myb and Aurora Kinase A (Cao et al., 1995
; Hanada et al., 2006
; Hung et al., 2008
; Lin et al., 2001
; Lo et al., 2005a
; Lo et al., 2006b
; Lo and Hung, 2006
; Marti et al., 1991
) as well as phosphorylating and stabilizing PCNA (Wang et al., 2006
). These data suggest that nEGFR provides a “second compartment” of proliferation mediated by the EGFR, the first compartment being the classical membrane-bound EGFR signaling through the RAS/RAF/MEK/MAPK pathway. Analysis of cetuximab-resistant clones indicated a marked upregulation of cyclin D1, B-myb and PCNA demonstrating that nEGFR was functional in cetuximab-resistant clones. Together, these observations suggest a potential model of resistance to cetuximab therapy ().
Potential mechanism for resistance to cetuximab
It has been reported that EGF stimulation can induce the accumulation of EGFR in the nucleus (Hsu and Hung, 2007
). This finding led us to investigate the levels of ligand expression in cetuximab-resistant cells (). In addition to EGF, we found several EGFR ligands to be increased. Furthermore, we found that all ligands that were upregulated had the potential to induce translocation of the EGFR to the nucleus with varying degrees (). These data suggest that deregulation of EGFR ligands have the potential to drive EGFR to the nucleus. Furthermore, blockade of TACE, the enzyme necessary for release of EGFR ligands from the membrane (Borrell-Pagès et al., 2003
; Sunnarborg, 2002
; Sunnarborg et al., 2002
), dramatically decreased nEGFR in all cetuximab-resistant clones, further strengthening a role for ligands in this process (). If EGFR ligands can drive nEGFR, then ligand alone should induce moderate levels of resistance to cetuximab. In we tested this hypothesis by adding individual ligands or ligand plus cetuximab and measured the resistance. Each ligand resulted in a varying degree of resistance but appeared that HB-EGF and β-cellulin, which induced the more accumulation of EGFR in the nucleus (), correlated with the most robust resistance (). Taken together these findings indicate that EGFR ligands may be a critical determinant of inducing resistance to cetuximab and may be a viable screening tool for therapeutic response to cetuximab. Similar findings have been reported for additional HER family ligands in the resistance to HER family inhibitor therapy (Rajput et al., 2007
; Ritter et al., 2007
). Collectively our results and these reports suggest that surveillance of HER family ligands levels may provide an indicator of therapeutic response to cetuximab and other HER family inhibitors.
Recent experiments have shown that EGF can induce nuclear translocation of the EGFR. In , we expanded on this finding and showed that EGF, HB-EGF, AR, and β-cellulin drive EGFR to the nucleus. Furthermore, we showed that dasatinib could block this translocation. These findings suggest that EGFR movement to the nucleus is dependent, in part, on SFK, and this may be an early signal in translocation. Dasatinib treatment of cetuximab-resistant clones led to a dramatic decrease in the nEGFR (). Taken together these findings suggest that overexpression of EGFR ligands initiate movement of the EGFR to the nucleus, mediated by SFK. Blockade of SFK may represent a unique therapeutic target to block nEGFR in patients.
Our results also imply that blockade of SFK led to increased EGFR on the plasma membrane and not the nucleus (). This is most likely due to the fact that SFK's are an initial signal for movement of the EGFR from the membrane to the nucleus. We postulated that increased membrane EGFR and loss of the “second compartment” of EGFR signaling in the nucleus after SFK blockade may restore sensitivity to cetuximab. This was seen in where the combinatorial therapy of dasatinib and cetuximab had greater impact than either agent alone. The overall findings from this series of experimentations indicate that targeting both the EGFR and SFK may provide a valuable clinical strategy to explore. Investigations into which Src family member may regulate EGFR translocation to the nucleus remain to be elucidated.
It is difficult to determine if nEGFR itself can drive resistance since the presence of ligand appears to be necessary. We took steps to distinguish these two processes by overexpressing EGFR tagged to a nuclear localization sequence (EGFR-NLS) in the cetuximab-sensitive HP parental lines in vitro and in vivo ( and ). Three different EGFR-NLS/Myc-expressing clones, was localized to the nucleus, was tyrosine phosphorylated and resulted in the upregulation of Cyclin D1 and B-myb (). Each EGFR-NLS/Myc-expressing clone had increases in resistance to cetuximab in vitro (). In addition, when these tumor cells were utilized in mouse xenograft models and challenged repeatedly with cetuximab, all three clones showed no significant difference in growth relative to the IgG control, whereas the HP and the vector only controls had statistically significant tumor growth control with cetuximab (). Collectively these data support the hypothesis that nEGFR, in part, can play a role in resistance to cetuximab therapy.
Nuclear EGFR has been strongly correlated with poor overall survival in several tumor types (Lo et al., 2005b
; Psyrri et al., 2008
; Xia et al., 2008
). In summary, this is the first report indicating that nEGFR may play a functional role in the response to molecular therapeutics agents. The findings reported herein have clinical implications for the design of optimal therapeutic strategies. In this model, we demonstrate that HER family ligands are upregulated in the cells with acquired-resistance to cetuximab. This in turn enhances the translocation of the EGFR to the nucleus. This process appears to be dependent on SFK. Once in the nucleus, EGFR performs several functions that mediate proliferation of tumor cells. In patients where this mechanism may be prominent, SFK inhibitor, dasatinib represents an approach inhibit nuclear translocation, leading to removal of nEGFR and its functions with re-sensitization to cetuximab therapy. Furthermore, these findings suggest that nEGFR may prove a viable molecular target. This may be undertaken by targeting nEGFR-expressing tumors with EGFR TKI (erlotinib and gefitinib) as they can diffuse across the membrane and abrogate nEGFR kinase function such as phosphorylation of PCNA. Strategies along these lines have already been suggested (Wheeler et al., 2008
). In addition, targeting with dasatinib and cetuximab either concomitantly or sequentially may be a viable clinical approach for individuals with nuclear expression of EGFR.