The use of Trastuzumab as an agent to specifically target breast cancers with amplification of the HER2 oncogene was one of the first and most successful applications of targeted therapy for metastatic carcinomas. The broad use of Trastuzumab has resulted in an increasing prevalence of patients whose tumors have developed resistance to the therapy over time and the identification of a significant number who are resistant at the outset. However, the mechanisms underlying resistance to Trastuzumab remain obscure, in part because there is still considerable debate as to the mechanisms that underlie its antitumor activity. There are essentially two schools of thought as to its mechanism of action, one based on inhibition of HER2 functional signaling, the other focused on induction of antibody dependent cytotoxicity (Clynes et al., 2000
; Yakes et al., 2002
Although there is a significant amount of data supporting the latter, most of the hypotheses and experimental data on resistance have been directed at mechanisms that prevent or bypass inhibition of signaling by Trastuzumab. Despite many putative mechanisms described in experimental models, the actual mechanisms of resistance have not been defined in patients, in large part because of the lack of biopsy studies. The experimental models that have been developed have largely focused on mechanisms that cause activation of PI3K/AKT signaling to be less dependent or independent of HER2. These include decreased PTEN function, activation of other receptor tyrosine kinases (e.g. EGFR), or mutational activation of PI3K. Another potential mechanism of resistance, discussed in this paper, is the expression of forms of HER2 that are functionally active, but lack the Trastuzumab epitope. Such tumors would be predicted to remain HER2 dependent for activation of PI3K/AKT signaling but would be resistant to inhibition of the pathway by Trastuzumab. The recent finding that the HER2 kinase inhibitor Lapatinib has antitumor activity in a significant proportion of Trastuzumab-resistant, HER2 overexpressing breast cancer patients suggests that many of these tumors indeed retain a dependence on HER2.
p95-HER2 retains tyrosine kinase activity and has been shown to activate ERK and AKT signaling, but lacks the Trastuzumab binding site (Anido et al., 2006
; Molina et al., 2002
; Xia et al., 2004
). Its expression in human tumors has been correlated with Trastuzumab resistance (Scaltriti et al., 2007
). If this relationship is causal, such tumors would be expected to respond to modalities that inhibit p95-HER2 function or reduce its expression. HER2 is an HSP90 dependent protein that is ubiquitinated and degraded in the proteosome in response to HSP90 inhibitors. We demonstrate that p95-HER2 retains its dependence on HSP90. It is present in the cell in an HSP90 complex and is rapidly and potently degraded in cells exposed to the HSP90 inhibitor SNX-2112. This occurs at comparable concentrations of inhibitor required for degradation of full length HER2. This is compatible with work showing that HSP90 binds to a region in the catalytic domain of HER2 (Tikhomirov and Carpenter, 2003
; Xu et al., 2001
). HSP90 inhibitors rapidly degrade HER2 and inhibit PI3K/AKT and ERK signaling in tumor models with HER2 overexpression, whether or not they express high levels of endogenous or transfected p95-HER2 ((Basso et al., 2002
; Munster et al., 2002
), –). Moreover, we find that HSP90 inhibition potently induces degradation of HER2 and p95-HER2 in vivo
and causes prolonged inhibition of AKT signaling in murine tumor models, at doses that are not toxic to the host.
These data suggest that HSP90 inhibition will be useful in Trastuzumab-resistant tumors that retain HER2 dependence, especially those in which resistance is due to p95-HER2 expression. We have previously demonstrated in tissue culture models that cells transfected with p95-HER2 do not respond to Trastuzumab therapy but are sensitized to the antiproliferative effects of the HER2 kinase inhibitor Lapatinib (Scaltriti et al., 2007
). In this report we demonstrate that the F2#1282 Trastuzumab-resistant xenograft model expresses high levels of p95-HER2. In this model, Trastuzumab treatment appears to further increase p95-HER2, perhaps contributing to resistance. In contrast, Trastuzumab has been shown to decrease p95-HER2 expression in the sensitive BT474 model and this has been adduced as a putative mechanism of Trastuzumab activity (Molina et al., 2001
). Whether upregulation of p95-HER2 expression is necessary for resistance in F2#1282 is not certain, however, it is clear that p95-HER2 expression and mitogenic signaling are not downregulated by Trastuzumab treatment in this model.
In contrast, the growth of F2#1282 tumors is quite sensitive to HSP90 inhibition. A single dose of HSP90 inhibitor is sufficient to induce rapid degradation of both p95-HER2 and full length HER2 and cause prolonged inhibition of AKT and ERK signaling, PARP cleavage, and complete cessation of tumor growth. Similarly, the HER1/2 kinase inhibitor Lapatinib also causes downregulation of HER2 signaling and significantly slows tumor growth. Taken together, these data establish that this Trastuzumab-resistant tumor model remains dependent upon HER2. In further support, we find that a genetically engineered model of p95-HER2 mediated tumorigenesis, the MEF-p95-HER2 model, is also resistant to Trastuzumab, completely dependent upon p95-HER2 expression for survival and highly sensitive to HSP90 inhibition. These data are consistent with the findings of clinical trials of alternative HER2 targeted therapies for patients with HER2 amplified breast cancer that have become resistant to Trastuzumab. Recent trials show that the HER kinase inhibitors, Lapatinib and HKI-272, and the HSP90 inhibitor, 17-AAG, have significant activity in HER2-overexpressing breast tumors that have progressed on Trastuzumab treatment (Geyer et al., 2006
; Modi et al., 2007
). The activity of both of these classes of agents is most likely due to their more potent or different mechanism of inhibition of HER2. This follows the pattern of resistance to other targeted therapies such as BCR-ABL inhibitors in CML or mutant EGFR inhibitors in NSCLC in which resistant tumors often retain their dependence on the targeted oncoprotein. Whether the tumors that are resistant to salvage therapy with a HER kinase or HSP90 inhibitor are still HER2 dependent, but refractory to these inhibitors, or whether they have progressed to a HER2-independent state is unknown.
The current data suggests that either an HSP90 inhibitor or an effective HER kinase inhibitor can inhibit tumors in which resistance is mediated by p95-HER2 and perhaps other HER2-dependent mechanisms. Both modalities effectively inhibit AKT activation, although in F2#1282 the effects of the HSP90 inhibitor on the pathway are much more prolonged. It is not at all clear which modality is superior and, since they inhibit HER2 by different mechanisms, coadministration could conceivably inhibit HER2 function more effectively than either drug alone and with enhanced clinical benefit.
Despite the resistance of F2#1282 tumors to Trastuzumab therapy alone, we have noted that it significantly enhances the antitumor activity of the HSP90 inhibitor. The combination is associated with significant tumor regression compared to the HSP90 inhibitor alone. The mechanisms through which Trastuzumab contributes to antitumor activity are unknown but could include more effective inhibition of the function of full-length HER2 or other effects of Trastuzumab on angiogenesis or tumor immunity. Recently, patients whose tumors had progressed during multiple Trastuzumab-based therapies were treated in a Phase I/II clinical trial, of the HSP90 inhibitor, 17-AAG, in the setting of continued Trastuzumab administration. The results of this trial were quite promising with a 26% objective response rate and 63% evidence of biologic response rate (Modi et al., 2008
). There is no way to know whether the Trastuzumab had any effect on these results, but these and other data suggest that Trastuzumab/HSP90 inhibitor combinations are rational in patients who have not previously been treated as well as those with acquired Trastuzumab resistance.