Breast cancer is the most common malignancy and the second-leading cause of cancer-related deaths in women in the United States. Despite extensive studies regarding breast cancer, much is still unknown about the biological mechanism by which a normal cell turns cancerous. One critical factor involved in this process is Bcl-2, which is highly expressed in 40–80% of breast cancer patients and is also common in other tumors.1
The conventional view of Bcl-2 as a proto-oncogene focused on its ability to prevent apoptosis that contributes to the elimination of cancerous cells.2
Apoptosis is tightly regulated by the interplay of the Bcl-2 family proteins characterized by the presence of up to four conserved ‘Bcl-2 homology' (BH) domains.2
Members of the Bcl-2 family are classified as either anti-apoptotic (e.g. Bcl-2) or pro-apoptotic (e.g. Bax and Bak, Bid).3
The anti-apoptotic Bcl-2 proteins block apoptosis by sequestering pro-apoptotic ones from inducing apoptosis. Of particular relevance, the molecular surfaces of Bcl-2's possess a BH3-binding groove, an extended hydrophobic cleft formed by the juxtaposition of the BH1–3 domains, which can accommodate the α
-helical BH3 domain of the proapoptotic Bcl-2 molecules.4, 5
Thus, the interactions between anti- and pro-apoptotic Bcl-2 proteins regulate the balance of cells' life and death and determine the propensity of cells to succumb to apoptosis.
Although intense efforts have focused on the anti-apoptotic role of Bcl-2, recent evidence; however, uncovered a critical role of Bcl-2 to concomitantly prevent autophagy.6, 7
In contrast to the ‘self-destruct' apoptosis, autophagy (‘self-eating') involves the lysosome-dependent bulk degradation of cytoplasmic components, through a centrally important double-membrane-bound vesicle, the autophagosome. Formed within the cell, autophagosomes serve to surround, sequester, and finally, seal off extraneous cellular components from the rest of the inside of the cell. Autophagosomes subsequently fuse with lysosomes, having their sequestered contents degraded and the resulting macromolecules recycled.8, 9
Although initially recognized as a response to nutrient deprivation, autophagy is now implicated as being essential to a variety of cellular processes including stress adaptation, development, immunity, and protection against neurodegeneration and cancer.8, 9
Bcl-2 suppresses autophagy by directly targeting Beclin1, a component of the class III PI3K complex involved in the autophagosome formation.6, 10
An allelic loss of beclin1
impairs autophagy and renders mammary cells tumor-prone, suggesting that defects in Beclin1-mediated autophagy are essential for malignant transformation.11, 12
Although the precise mechanism governing Beclin1-mediated tumor suppression is still elusive, recent studies have demonstrated that autophagy defects in tumors cause accumulation of unwanted protein aggregates such as the p62 and ER chaperons, oxidative stress, and genome damage, all of which concomitantly fuel tumor growth.13, 14
It is within this context we postulate that the inhibition of the tumor suppressor Beclin1 by Bcl-2 may contribute to the oncogenic potential of Bcl-2. In support of this view, it has been shown that when Beclin1 function is left unchecked by Bcl-2, excessive levels of autophagy induce cell death in breast cancer cells.6
Moreover, knocking down bcl-2
results in autophagic rather than apoptotic cell death in MCF7 cells, suggesting that an alternative and/or additional mechanism involving autophagy may have a role in Bcl-2-mediated oncogenesis.15
Most notably, our recent study on a viral Bcl-2 (vBcl-2) encoded by γ
-herpesvirus 68 (γ
-HV68) clearly indicated that the inhibition of autophagy by the vBcl-2–Beclin1 interaction directly contributes to persistent infection, a prerequisite for the malignant transformation of infected cells.16
All these observations raise the strong possibility that autophagy is tumor-suppressive and that Bcl-2-mediated blockade of autophagy may contribute to Bcl-2 oncogenicity.
Structural analysis revealed that the interaction of Beclin1 with Bcl-2 is reminiscent of that of pro-apoptotic Bcl-2 proteins, in that Beclin1 has a putative α
-helical BH3 domain that allows it to dock into the hydrophobic groove of Bcl-2.7, 17
As the BH3-binding groove of Bcl-2 is engaged by both pro-apoptotic and pro-autophagic molecules, mutations in the groove that block Beclin1 binding also eliminate binding to pro-apoptotic Bcl-2's and vice versa
thus far, further complicating efforts to genetically dissect the in vivo
role of Bcl-2 in cancer. As such, the role of Bcl-2 antagonism of autophagy in tumor development remains ill defined and the underlying mechanism is unclear.
In this study, we first used loss-of-function mutagenesis to identify functional domains that can differentiate between Bcl-2's anti-autophagic binding and its anti-apoptotic interaction. Further, using an in vitro MCF7 breast cancer cell-culture model and an in vivo mouse xenograft model, we observed that a Bcl-2 mutant that no longer inhibits apoptosis but retains its anti-autophagy function promotes the tumorigenic properties of MCF7 cells to a level similar to wild-type (WT) Bcl-2. This effect was specifically due to the inhibition of autophagy and was dependent on having an intact Beclin1 binding. Our findings thus demonstrate an oncogenic role of Bcl-2-mediated autophagy inhibition in breast cancer. Unlike what was previously thought, that anti-apoptosis features prominently in the functions of Bcl-2 in vivo, our study suggests that the anti-autophagic property of Bcl-2 may be, at least in some contexts, eminently exploited in human cancer, thereby making it an attractive target for cancer therapy.