Acquired resistance to cetuximab can occur by several distinct mechanisms including (1) mutations in the EGFR that alter response to cetuximab, (2) loss of target (i.e., EGFR), (3) loss of downstream signaling molecules, (4) increase in HER family member ligands, (5) and activation of other RTKs with overlapping signal transduction cascades with EGFR (i.e., cMET, IGF1R). One approach to identifying molecular changes leading to acquired resistance to cetuximab is to develop resistant cells lines by chronically challenging them with cetuximab and evaluating the resulting clones with congenic sensitive parental lines. Previously we reported the development of a cetuximab acquired resistance model using the NSCLC NCI-H226.20
Lung lines with acquired resistance to cetuximab exhibited increased steady-state EGFR expression and activity secondary to alterations in trafficking and degradation.20
This increased steady-state activity resulted in constitutive activation of HER3 and signals to PI(3)K/Akt leading to escape from cetuximab therapy. In the current study, we found that cells with acquired resistance to cetuximab had a higher basal level of active SFKs. This increased SFK activity led to prolonged EGFR activity and was critical for EGFR activation of HER3 and subsequent activation of PI(3)K/Akt and other survival signals. Furthermore, combinatorial treatment of cetuximab-resistant cells with dasatinib could re-sensitize resistant cells to cetuximab growth inhibition. Collectively, these data suggest that SFKs and EGFR cooperate in acquired resistance to cetuximab. Combinatorial therapy targeting these two kinases may have increased clinical efficacy than targeting either molecule alone.
The EGFR plays a crucial role in human malignancies and contributes to tumor formation and metastasis. However, it is well established that Src and its family members can potentiate the transformation ability as well as the mitogenic and tumorigenic activity of the EGFR.6,22,24,25
Full kinase activity of SFKs is a prerequisite for biological cooperation with EGFR as well as for phosphorylation of Y845 on the EGFR. SFK phosphorylation of Y845 on the EGFR is required for DNA synthesis stimulated by EGF as well as endothelin, lysophosphatidic acid, growth hormones and cytokines. Experiments using an EGFR Y845F mutant in mouse fibroblasts resulted in blockade of DNA synthesis.6,15,26
Taken together these findings suggest that Src and its family members are critical for full activation of the EGFR. In addition to direct phosphorylation and cooperation with the EGFR, previous work has shown that SFKs mediate EGFR ligand cleavage leading to proliferation and invasion of HNSCC cancer cells.27
Recent work by Koppikar et al. indicated that combined inhibition of the SFKs and the EGFR abrogated both growth and invasion of HNSCC.18
Collectively these reports provide a persuasive body of evidence indicating a strong relationship between SFKs and the EGFR in cancer.
We found cells with acquired resistance to cetuximab exhibit increased steady-state activity of the EGFR ( and reviewed in ref. 20
). This is in contrast to a recent report suggesting increased ubiquitination (and thus decreased EGFR levels) as a mechanism of acquired resistance to cetuximab therapy in DiFi colorectal cetuximab-resistant cells.28
However, similar to their findings we show that cetuximab-resistant cells have increased basal level of active SFKs resulting in hyper-phosphorylation of the Y845 and Y1173 of the EGFR (). Taken together these results indicate that SFK activation in both NSCLC and CRC cancer lines with acquired resistance to cetuximab may have a common mechanism of resistance.
SFKs become active upon binding to several RTKs including the EGFR, PDGFR, FGFR, CSF-1R, as well as integrins, and cell-cell adhesion molecules.22
Data in indicates that blockade of EGFR through TKIs or siRNA results in the loss of active, but not total, SFK in cells with acquired resistance to cetuximab. Collectively, these data suggest that the EGFR, rather than an alternative RTK, is responsible for the binding and activation of SFKs. These data strengthen the conclusions of this communication that co-targeting SFKs and the EGFR may have increased effectiveness than targeting either molecule alone.
One consequence of increased SFK activity is enhanced EGFR phosphorylation (), which could be abrogated by treatment with dasatinib in cetuximab-resistant clones, but not parental cells. Not only did this treatment lead to decreased phosphorylation of the EGFR, but also resulted in decreased survival of cetuximab-resistant clones (). Our data indicates that dasatinib influenced the HER3-PI(3)K/Akt survival pathway in cells with acquired resistance to cetuximab ( and ).
Currently there are nine known family members of the Src family. When measuring active levels of SFKs in cells with acquired resistance to cetuximab, we observed a different banding pattern when compared to parental controls ( and ). The parental cell lines, when immunoblotted with the SFKY416 antibody, showed a single active band. However, three independent cetuximab-resistant clones had multiple banding patterns. This may suggest that cetuximab-resistant cells have increased activity of several different members of the Src family. This may account for the increased SFK activity in resistant cells. However, dasatinib treatment resulted in loss of the active band(s) in the HP, HC1, HC4 and HC8 clones () but did not alter proliferative potential of cetuximab sensitive parental lines (). This suggests that cells with acquired resistance to cetuximab may have acquired multiple active forms of SFKs but also acquired an oncogene addiction to these signaling molecules.
It has been reported that increased SFK activity in NSCLC can promote the survival of EGFR-dependent cell lines by enhancing phosphorylation of the EGFR and HER3.16
We previously reported that escape from cetuximab therapy is centered around activation and recruitment of HER3.20
This escape mechanism has been reported by other investigators.29,30
Blockade of SFK activity by dasatinib, at high doses, in resistant cells lead to decreased phosphorylation of EGFR at Y845 (data not shown) and led to decreased HER3 and subsequent PI(3)K/Akt activity (). These findings support the conclusions by Zhang et al. which reported experiments blocking SFKs in EGFR-dependent NSCLC that resulted in decreased phosphorylation of HER3.16
Furthermore, they concluded that SFKs are active in NSCLC and cooperate with the EGFR to promote survival. Here we demonstrate that SFKs and the EGFR cooperate in acquired resistance to cetuximab. Experiments aimed at modulating SFKs and EGFR levels in cetuximab-sensitive lines are ongoing. Taken together these results indicate that SFKs play a strong role in mediating signals from the EGFR to HER3 and subsequently the PI3(K)/Akt pathway and suggest that increased SFK activity may lead to escape from cetuximab therapy.
In we targeted both the EGFR and SFKs using dasatinib and cetuximab to determine if combinatorial therapy directed at both SFKs and the EGFR would result in increased anti-proliferative activity than either agent alone. Our data suggested that the combination had a profound impact on proliferative potential and that the combination could abrogate active HER3 and PI(3)K/Akt pathways. These data, in conjunction with Lu et al.28
strengthen the hypothesis that SFKs and the EGFR cooperate in both NSCLC and CRC and may be a common molecular mechanism to escape cetuximab challenge.
In conclusion, NSCLC with acquired resistance to cetuximab show a remarkable increase in the activity of Src family kinases. This enhanced activity of SFKs leads to increased activity of the EGFR leading to activation of HER3 and the PI(3)K/Akt and ultimately escape from cetuximab therapy. Treatment of cetuximab-resistant cells with dasatinib results in re-sensitization to cetuximab therapy. Collectively, our data suggest a strong rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab.