In the present study, we have shown that, in HER2-positive breast cancer models, the inhibition of the PI3K/AKT/mTOR pathway results in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurs as a result of activation of HER family receptors as evidenced by increased expression of HER3, induction of HER receptors dimerization and phosphorylation and binding of adaptor molecules to HER2 and HER3. Enhanced HER3 protein was observed independently of HER2 overexpression and is due to transcriptional regulation via FoxO transcription factors (; Garrett et al., 2009
; Chandarlapaty et al., 2011
), which are activated upon AKT-mediated nuclear relocalization (Brunet et al., 1999
). Allosteric inhibition of mTORC1 lead to a milder increase in HER2 and HER3 phosphorylation compared with the other PI3K-pathway inhibitors, which was uncoupled to an increase in total HER3 protein and FoxO3a nuclear translocation (data not shown). This may indicate that P-ERK activation following mTORC1 inhibition occurs mainly via the PI3K-RAS signaling (Carracedo et al., 2008
Further evidence that enhanced HER2 signaling is responsible for the observed ERK activation is provided by the observation that HER2 inhibitors prevented ERK activation. On the contrary, small molecule kinase inhibitors of EGFR, IGF-1R or SRC failed to reverse ERK activation secondary to BEZ235 treatment. Taken together, our findings suggest that PI3K inhibition in HER2-overexpressing breast cancer results in hyperactivation of ERK that could potentially result in decreased efficacy of PI3K inhibitors. Anti-HER2 and MEK inhibitors did not only abolish ERK phosphorylation but also increased the anti-proliferative and pro-apoptotic effects of PI3K inhibitors. As a result of our observations, we would propose that a preferred therapeutic strategy in HER2-overexpressing breast cancer would be the administration of PI3K inhibitors in combination with either anti-HER2 agents or MEK inhibitors, instead of PI3K inhibitors given alone.
Our findings provide additional evidence that in cancer cells with constitutive PI3K activation, negative regulatory feedback loops silence compensatory pathways (that is RAS/RAF/ERK) and maintain the dependency on PI3K/AKT/mTOR signaling, a defining feature of oncogene addiction. However, upon pathway (or oncogene) blockade, these inhibitory loops are released and compensatory pathways are activated. We are also learning that these compensatory feedback loops are present at multiple levels of the pathway. Subsequently, different pathways may awake from a ‘dormant state' depending on the level at which the therapeutic intervention occurs. For example, we initially reported that mTORC1 inhibition results in activation of PI3K that resulted in ERK activation (Carracedo et al., 2008
). Here, we show that with direct PI3K blockade another mechanism comes to play, such as ERK activation via RTKs. While activation of RTKs also occurs with mTORC1 allosteric inhibitors, it is less prominent than with PI3K inhibitors. The type of RTK being activated may also vary in a tumor-dependent context since we have previously shown activation of IGF-1R signaling with mTORC1 inhibitors in non-HER2-overexpressing breast cancer cells (O'Reilly et al., 2006
; Tabernero et al., 2008
). In contrast, in HER2-overexpressing cancer cells, HER receptors signaling appears to be responsible for ERK activation. Our findings have implications for future therapeutic strategies, where the selection of drug combinations will depend on both the type of PI3K/AKT/mTOR inhibitor as well as the cancer type. For example, based on the increase of IGF-1R signaling observed with the mTORC1 inhibitors, we are now conducting a clinical trial combining an mTORC1 inhibitor with a monoclonal antibody that inhibits IGF-1R signaling in non-HER2-overexpressing breast cancer. This combination would be less appealing in HER2-positive breast cancers treated with a PI3K inhibitor.
Our study also highlights the role of the RAS/RAF/MEK/ERK pathway as an escape mechanism to PI3K blockade. The ERK cascade is at the heart of signaling networks that govern proliferation, differentiation and cell survival (Kolch, 2000
). RAS is activated by RTKs via the adaptor molecule GRB2 and subsequently signals through RAF, RALGDS or PI3K itself. This pathway is frequently activated in human tumors, often through gain-of-function mutations of the RAS and RAF family members (Davies et al., 2002
). Tumors with mutated BRAF, such as melanoma, are exquisitely sensitive to selective V600-BRAF and MEK inhibitors (Solit et al., 2006
; Flaherty et al., 2009
). Mutations in this pathway may lead to therapeutic resistance to PI3K inhibitors that is reverted by MEK inhibitors. For example, mouse models of mutated KRAS lung cancer are resistant to BEZ235, but combining BEZ235 with an MEK inhibitor results in synergistic tumor shrinkage (Engelman et al., 2008
). In breast cancer, BRAF and RAS mutations are rare, which has supported the use of PI3K inhibitors as single agents. However, BRAF or RAS mutations may not be required for resistance to PI3K inhibitors. For example, resistance to PI3K inhibitors has been described in a subset of basal-like breast cancer cells that have a RAS-driven signature (Hoeflich et al., 2009
). In the present study of HER2-positive breast cancer cells that lack RAS/RAF mutations or a RAS-driven signature, HER2 activation appears to be a novel mechanism of acquired ‘ERK dependence'. As in the case of RAS mutant tumors and tumors with a RAS-driven signature, the addition of an MEK inhibitor should be beneficial in this setting. Our studies confirm this since the addition of an MEK inhibitor (or prevention of pathway activation with anti-HER2 agents) to PI3K inhibitors results in greater inhibition of proliferation, augmented apoptosis and the significant tumor shrinkage observed in vivo
In conclusion, our data support the use of simultaneous inhibition of PI3K/mTOR and ERK pathways in HER2-positive breast cancer instead of the administration of PI3K/mTOR inhibitors used as monotherapy. The inhibition of the ERK pathway can be achieved by either MEK inhibitors or anti-HER2 agents. Clinical trials testing the feasibility of combining PI3K inhibitors with MEK inhibitors are currently being initiated. This combination could be potentially challenging since these two classes of agents may have overlapping toxicities such as skin rash (Shapiro et al., 2009
; Banerji et al., 2010
). On the other hand, the combination of mTOR allosteric inhibitors and trastuzumab has already been shown to be safe (André et al., 2008
) and we have recently started the enrollment of patients with HER2-overexpressing breast cancer in a clinical trial combining the PI3K/mTOR inhibitor BEZ235 with the anti-HER2 monoclonal antibody trastuzumab.