Breast cancer is a heterogeneous disease, composed of distinct entities with differing underlying pathogenic processes. One such entity is the so-called HER2 subtype, which is characterized by amplification and/or overexpression of this member of the human epidermal growth factor receptor (HER) family. HER2 is an orphan receptor with intrinsic tyrosine kinase activity [1
] whose activation results from the dynamic heterodimerization of HER receptors members [2
]. This activates a large repertoire of transforming signaling molecules and pathways that are, to a great extent, shared by HER members.
Excess HER2 signaling leads to numerous oncogenic processes, including cell proliferation and survival [1
]. The major signaling pathways activated by HER2 include the RAS-Raf1-Mek-Erk and the PI3K-Akt pathways. Akt signaling leads to mTOR activation. The mTOR signaling complex 1 (mTORC1) helps maintaining protein synthesis through phosphorylation of at least two direct targets, eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) and ribosomal protein S6 kinases (S6Ks) [3
] that regulate the activity of EIF4F, a heterotrimeric complex required for the cap-dependent ribosome recruitment phase of translation initiation.
Activation of the Ras-MAPK-Erk and PI3K-Akt-mTOR pathways both culminate in activation of transcriptional programs, as well as cyclin dependant kinases, that lead to progression through the cell cycle. Current evidence indicates that, through either of these pathways, HER2 signaling can regulate c-Myc, a multifunctional transcription factor involved in cell cycle progression (see [4
] and references therein). In particular, mTORC1 activity might contribute to cell cycle progression in HER2 overexpressing cells, as c-Myc expression is critically dependent upon EIF4F activity in cells with high Akt activity [5
]. Consistent with this, inhibition of mTORC1 by RAD001 (everolimus) potently inhibits cell cycle progression of HER2 overexpressing breast cancer cells [7
In addition to their deregulated proliferation, HER2 overexpressing cells exhibit altered survival signals. Breast cancer cells overexpressing HER2 are resistant to an array of cytotoxic agents and radiation damage [8
]. In particular, anti-apoptotic signals associated with alterations of the downstream Ras-MAPK-Erk and PI3K-Akt-mTOR pathways contribute to chemo- and radioresistance. If targeting these survival signals is expected to be of therapeutic benefit in combination with cytotoxic approaches, a well-designed inhibition of some of these survival signals could have a more radical effect and directly promote tumor destruction. Indeed, some of the survival signals harbored by HER2 overexpressing cells might directly contribute to cancer progression by allowing cancer cells to survive to constitutive death signals. The existence of such signals is suggested, at least in part, by the fact that the kinase cascade triggered by the hyperactivity of receptors of the HER family can be "addictive" to cancer cells [10
]. Such apparent addiction seems to result from the fact that hyperactivity of HER pathways has tumor promoting (survival) effects, but also tumor suppressive (death promoting) ones [11
]. Death signals downstream of EGFR signaling have been reported, but not fully described in molecular details [10
]. Moreover, it has remained unknown whether similar signals are initiated downstream of HER2. Investigating whether constitutive death and compensatory survival signals exist in HER2 overexpressing cells is of importance, as it may lead to the identification of a critical event in the HER2 network that needs to be altered by current targeted therapies, or that could be directly targeted without altering the rest of the network with great therapeutic benefit.
An investigation of the roles played by the Bcl-2 family of proteins in the survival of HER2 overexpressing cells may prove very useful to address this issue. This family of interacting proteins represents an integrating node towards which converge numerous death and survival signals in mammalian cells, including these induced by oncogenic signals [13
]. Anti-apoptotic Bcl-2 homologues preserve mitochondrial integrity by opposing the activity of multi-domain pro-apoptotic Bcl-2 family members Bax and Bak, which display sequence conservation throughout three Bcl-2 homology (BH) domains (BH1-3), and that of their upstream effectors, the BH3-only proteins (e.g. Bim, Puma, Bad...). This occurs essentially by physical interactions between anti- and pro-apoptotic members which allows the former to negatively control the activation, and the activity, of pro-apoptotic Bax and Bak (themselves essential actors of the apoptotic response of mammalian cells to multiple stimuli). Anti-apoptotic Bcl-2 homologues (e.g. Bcl-2, Bcl-xL, Mcl-1) control the sensitivity to conventional pro-apoptotic therapy of tumor cells. In certain instances, their expression is necessary to maintain the survival of cancer cells [14
], indicating that they may be required to counteract constitutive death signals. There is substantial evidence that the balance between anti- and pro-apoptotic proteins of the Bcl-2 family is biased in favor of survival proteins during breast carcinogenesis. Most breast cancers arise from epithelial cells that express Bcl-2, Bcl-xL and Mcl-1 [16
], and enhanced expression of these proteins is almost systematically found in transformed mammary epithelial cells. Signaling pathways downstream of HER2 have numerous anti-apoptotic effects on Bcl-2 family members [18
In this study, we investigated whether and how the imbalance in favor of survival proteins of the Bcl-2 family, which is induced by the sustained activity of signaling pathways downstream of HER2, contributes to survival maintenance in HER2 overexpressing breast cancer cells. We herein demonstrate that such cells undergo apoptosis upon depletion of Mcl-1, and that this Mcl-1 dependence is due to their constitutive expression of the pro-apoptotic protein Bim. The latter expression is a direct consequence of oncogenic signaling, as it is due to mTORC1 dependent expression of c-Myc, which occupies regions within the Bim promoter.