The HER4/4ICD estrogen receptor coactivator and BH3-only protein is an effector of tamoxifen-induced apoptosis

Greater than 40% of breast cancer patients treated with tamoxifen exhibit de novo or acquired tumor resistance. Recent clinical evidence indicates that loss of expression of HER4 is an independent marker for tamoxifen resistance. In direct corroboration with clinical observations, suppression of HER4 expression in the tamoxifen-sensitive MCF-7 and T47D breast tumor cell lines resulted in resistance to tamoxifen-induced apoptosis. Furthermore, HER4 expression was lost in three independent MCF-7 models of acquired tamoxifen resistance. The HER4 intracellular domain (4ICD) is an independently signaling nuclear protein that functions as a potent ERalpha coactivator. In addition, mitochondrial 4ICD functions as a proapoptotic BH3-only protein. Tamoxifen disrupts an estrogen-driven interaction between ERalpha and 4ICD while promoting mitochondrial accumulation of the 4ICD BH3-only protein. BCL-2 inhibition of tamoxifen-induced apoptosis and tamoxifen activation of BAK, independent of BAX, further supports a role for 4ICD during tamoxifen-induced apoptosis. Finally, reintroduction of HER4, but not HER4 with a mutated BH3 domain, restores tamoxifen sensitivity to tamoxifen-resistant TamR cells in a xenograft model. Clinically, breast cancer patients with tumor expression of nuclear 4ICD responded to tamoxifen therapy with no clinical failures reported after 14 years of follow-up, whereas 20% of patients lacking nuclear 4ICD expression succumbed to their disease within 10 years of diagnosis. Our identification of the HER4/4ICD BH3-only protein as a critical mediator of tamoxifen action provides a clinically important role for 4ICD in human cancer and reveals a potential tumor marker to predict patient response to tamoxifen therapy.     

Distinct BH3 domains either sensitize or activate mitochondrial apoptosis,serving as prototype cancer therapeutics

The "BH3-only" proteins of the BCL-2 family require "multidomain" proapoptotic members BAX and BAK to release cytochrome c from mitochondria and kill cells. We find short peptides representing the alpha-helical BH3 domains of BID or BIM are capable of inducing oligomerization of BAK and BAX to release cytochrome c. Another subset characterized by the BH3 peptides from BAD and BIK cannot directly activate BAX, BAK but instead binds antiapoptotic BCL-2, resulting in the displacement of BID-like BH3 domains that initiate mitochondrial dysfunction. Transduced BAD-like and BID-like BH3 peptides also displayed synergy in killing leukemic cells. These data support a two-class model for BH3 domains: BID-like domains that "activate" BAX, BAK and BAD-like domains that "sensitize" by occupying the pocket of antiapoptotic members.     

Functional identification of the apoptosis effector BH3 domain in cellular protein BNIP1

BCL-2 family proteins play a central role in apoptosis regulation in mammals and in C. elegans. Mammalian cellular and viral anti-apoptosis proteins such as BCL-2 and E1B-19K interact with several cellular proteins. Some of these interacting proteins promote apoptosis and belong to the BCL-2 family. Certain BCL-2 family proapoptotic proteins such as BAX and BAK share extensive sequence homology with BCL-2. In contrast, certain pro-apoptotic proteins such as BIK and BID share a single death effector domain, BH3, with other BCL-2 family proteins. By mutational analysis, we show that one of the cellular proteins, BNIP1 (previously Nip-1), that interacts with BCL-2 family anti-apoptosis proteins is a 'BH3 alone' pro-apoptotic protein. Transient transfection of BNIP1 induces a moderate level of apoptosis. Deletions of the N-terminal 32 amino acid region and the C-terminal trans-membrane domain did not significantly affect pro-apoptotic activity. In contrast, deletions encompassing a region containing a motif similar to the BH3-domain abrogated the apoptotic activity. Substitution of BNIP1 BH3 domain for the corresponding sequence in BAX efficiently restored the apoptotic activity of BAX, establishing the functional identity of the BH3 domain of BNIP1. The N-terminal deletions of BNIP1 (that retain the BH3 domain) enhanced the level of interaction with BCL-XL. Mutants containing the BH3 deletions were still able to heterodimerize with BCL-XL while mutants lacking both the N-terminal region and the BH3 domain were unable to heterodimerize, suggesting that BNIP1 may bind to BCL-XL via two different binding motifs.     

Critical requirement of BAX for manifestation of apoptosis induced by multiple stimuli in human epithelial cancer cells

Studies with mouse embryo fibroblasts deficient for the BCL-2 family multidomain proapoptotic proteins BAX and BAK have revealed that both of these proteins are essential for apoptosis induced by multiple stimuli, suggesting that these proapoptotic proteins are functionally overlapping in these cells [M. C. Wei et al., Science (Wash. DC), 292: 727-730, 2001; W. X. Zong et al., Genes Dev., 15: 1481-1486, 2001]. We have determined the effect of several different apoptotic stimuli in a Bax-deficient human epithelial cancer cell line (HCT116BaxKO). We show that this cell line expresses functional BAK protein and is defective in manifestation of apoptosis induced by the BH3-only proteins BIK and BID as well as extrinsic stimuli that engage the death receptors, tumor necrosis factor receptor, tumor necrosis factor-related apoptosis-inducing ligand receptor, and Fas. In addition, this cell line is deficient for apoptosis induced by cytotoxic agents such as UV, staurosporine, and thapsigargin that induce either mitochondrial or endoplasmic reticulum stress. Our results suggest that BAX plays a critical role in the manifestation of apoptosis paradigms induced by multiple stimuli in human epithelial cancer cells. Our results also suggest that the integrity of BAX may have important consequences in the progression of epithelial tumors and in determining the outcome of chemotherapeutic regimens of such tumors.     

New dimension in therapeutic targeting of BCL-2 family proteins

Proteins of the BCL-2 family control the mitochondrial pathway of apoptosis. Targeting these proteins proves to be an attractive strategy for anticancer therapy. The biological context is based on the fact that BH3-only members of the family are specific antagonists of prosurvival members. This prompted the identification of “BH3 mimetic” compounds. These small peptides or organic molecules indeed mimic the BH3 domain of BH3-only proteins: by selectively binding and antagonizing prosurvival proteins, they can induce apoptosis in malignant cells. Some small-molecule inhibitors of prosurvival proteins have already entered clinical trials in cancer patients and two of them have shown significant therapeutic effects. The latest developments in the field of targeting BCL-2 family proteins highlight several new antagonists of prosurvival proteins as well as direct activators of proapoptotic proteins. These compounds open up novel prospects for the development of BH3 mimetic anticancer drugs.     

The irreversible ERBB1/2/4 inhibitor neratinib interacts with the BCL-2 inhibitor venetoclax to kill mammary cancer cells

The irreversible ERBB1/2/4 inhibitor, neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of neratinib to promote greater levels of cell killing.     

BH3-only proteins: integrated control point of apoptosis

Bcl-2 family proteins play central roles in the regulation of most, if not all, apoptotic pathways, and hence this family plays a critical role in oncogenesis. The Bcl-2 homology 3 (BH3)-only members of this family are "proapoptotic," promoting apoptosis by sensing cellular stresses: that is, they are activated or induced in response to stress stimuli. These BH3-only proteins then interfere with the function of "prosurvival" Bcl-2 family members, thereby promoting the progression of apoptosis. It has long been recognized that the induction and activation of each of the BH3-only proteins are subject to the type of stress stimulus. Although it was originally assumed that all the BH3-only proteins exert similar effects on the downstream apoptotic machinery, recent studies have uncovered their distinct functional properties, indicating the operation of more intricate, versatile control mechanisms of apoptosis. In this review, we will summarize recent findings on the diversity in the activation and function of BH3-only proteins. In particular, we focus on the overlapping and individual roles of 2 BH3-only proteins, Puma and Noxa, in the context of the apoptotic response induced by the tumor suppressor p53.     

Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells,thereby enhancing the response to anti-ERBB1/ERBB2 therapy

The present studies focused on defining the mechanisms by which anoikis-resistant (AR) mammary carcinoma cells can be reverted to a therapy-sensitive phenotype. AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA. In AR cells expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. AR cells were resistant to cell killing by multiple anti-tumor cell therapies, including ERBB1/2 inhibitor + MCL-1 inhibitor treatment, and had a reduced autophagic flux response to these therapies, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells. Pre-treatment of anoikis-resistant cells with histone deacetylase inhibitors (HDACIs) for 24 h increased the levels of toxic BH3 domain proteins, reduced MCL-1 levels, and restored/re-sensitized the cell death response of AR tumor cells to multiple toxic therapies. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored the chemo-sensitivity of the tumors and prolonged animal survival. These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.     

Regulation of apoptosis by endoplasmic reticulum pathways

Apoptotic programmed cell death pathways are activated by a diverse array of cell extrinsic and intrinsic signals, most of which are ultimately coupled to the activation of effector caspases. In many instances, this involves an obligate propagation through mitochondria, causing egress of critical proapoptotic regulators to the cytosol. Central to the regulation of the mitochondrial checkpoint is a complex three-way interplay between members of the BCL-2 family, which are comprised of an antiapoptotic subgroup including BCL-2 itself, and the proapoptotic BAX,BAK and BH3-domain-only subgroups. Constituents of all three of these BCL-2 classes, however, also converge on the endoplasmic reticulum (ER), an organelle whose critical contributions to apoptosis is only now becoming apparent. In addition to propagating death-inducing stress signals itself, the ER also contributes in a fundamental way to Fas-mediated apoptosis and to p53-dependent pathways resulting from DNA damage and oncogene expression. Mobilization of ER calcium stores can initiate the activation of cytoplasmic death pathways as well as sensitize mitochondria to direct proapoptotic stimuli. Additionally, the existence of BCL-2-regulated initiator procaspase activation complexes at the ER membrane has also been described. Here, we review the potential underlying mechanisms involved in these events and discuss pathways for ER-mitochondrial crosstalk pertinent to a number of cell death stimuli.     

BH3 peptidomimetics potently activate apoptosis and demonstrate single agent efficacy in neuroblastoma

The major impediment to cure for many malignancies is the development of therapy resistance with resultant tumor progression. Genetic alterations leading to subversion of inherent apoptosis pathways are common themes in therapy resistance. Bcl-2 family proteins play a critical role in regulating mitochondrial apoptosis that governs chemotherapeutic effects, and defective engagement of these pathways contributes to treatment failure. We have studied the efficacy of BH3 peptidomimetics consisting of the minimal death, or BH3, domains of the proapoptotic BH3-only proteins Bid and Bad to induce apoptosis using neuroblastoma (NB) as a model system. We demonstrate that BH3 peptides, modified with an arginine homopolymer for membrane transduction (called r8-BidBH3 and r8-BadBH3, respectively), potently induce apoptosis in NB cells, including those with MYCN amplification. Cell death is caspase 9 dependent, consistent with a requirement for the intrinsic mitochondrial pathway. Substitutions at highly conserved residues within the r8-BidBH3 peptide abolish apoptotic efficacy supporting activity through specific BH domain interactions. Concomitant exposure to r8-BadBH3 and r8-BidBH3 at sublethal monotherapy doses revealed potent synergy consistent with a competitive displacement model, whereby BH3 peptides displace sequestered BH3 proteins to induce cell death. Further, BH3 peptides demonstrate antitumor efficacy in a xenograft model of NB in the absence of additional genotoxic or trophic stressors. These data provide proof of principle that targeted re-engagement of apoptosis pathways may be of therapeutic utility, and BH3-like compounds are attractive lead agents to re-establish therapy-induced apoptosis in refractory malignancies.     

A novel Bcl-x splice product, Bcl-xAK, triggers apoptosis in human melanoma cells without BH3 domain

Pro- and antiapoptotic proteins of the large Bcl-2 family are critical regulators of apoptosis via the mitochondrial pathway. Whereas antiapoptotic proteins of the family share all four Bcl-2 homology domains (BH1-BH4), proapoptotic members may lack some of these domains, but all so far described proapoptotic Bcl-2 proteins enclose BH3. The bcl-x gene gives rise to several alternative splice products resulting in proteins with distinct functions as the antiapoptotic Bcl-xL and proapoptotic Bcl-xS. Here, we describe a novel Bcl-x splice product of 138 amino acids termed Bcl-xAK (Atypical Killer), which encloses the Bcl-2 homology domains BH2 and BH4 as well as the transmembrane domain, but lacks BH1 and BH3. Weak endogenous expression of Bcl-xAK was seen in melanoma and other tumor cells. Interestingly, its overexpression by applying a tetracycline-inducible expression system resulted in significant induction of apoptosis in melanoma cells, which occurred in synergism with drug-induced apoptosis. After exogenous overexpression, Bcl-xAK was localized both in mitochondrial and in cytosolic cell fractions. By these findings, a completely new class of Bcl-2-related proteins is introduced, which promotes apoptosis independently from the BH3 domain and implies additional, new mechanisms for apoptosis regulation in melanoma cells.     

RASSF1A and the BH3-only mimetic ABT-737 promote apoptosis in pediatric medulloblastoma cell lines

The RASSF1A tumor suppressor is potentially the most important candidate gene identified in medulloblastoma to date, being epigenetically silenced in >79% of primary tumors. However, its functional role has not been previously addressed in this tumor type. Here, we demonstrate that expression of RASSF1A promotes the induction of cell death after activation of both the extrinsic and intrinsic apoptotic pathways in medulloblastoma cells. Treatment of UW228-3 cells stably expressing RASSF1A with an anti-CD95 antibody to induce extrinsic apoptosis and etoposide or cisplatin to activate intrinsic apoptosis augmented tumor cell killing in a caspase-dependent manner. This led to increased activation of the pro-apoptotic BCL-2 family member BAX. On the basis of this knowledge, we demonstrate how the loss of RASSF1A function in medulloblastoma cells might be overcome using the novel BH3-only mimetic ABT-737 in combination with chemotherapeutic agents to target the BCL-2 anti-apoptotic members. We show that ABT-737 increased susceptibility to apoptosis induced by DNA damage regardless of RASSF1A expression status through increased activation of BAX. Our findings identify the RASSF1A tumor suppressor as a promoter of apoptotic signaling pathways. Investigation of its mechanism of action has revealed that these pathways can still be promoted in its absence and how these potentially represent novel therapeutic targets for medulloblastoma.     

Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members

We show that the antiapoptotic proteins BCL-2, BCL-XL, MCL-1, BFL-1, and BCL-w each bear a unique pattern of interaction with a panel of peptides derived from BH3 domains of BH3-only proteins. Cellular dependence on an antiapoptotic protein for survival can be decoded based on the pattern of mitochondrial sensitivity to this peptide panel, a strategy that we call BH3 profiling. Dependence on antiapoptotic proteins correlates with sequestration of activator BH3-only proteins like BID or BIM by antiapoptotic proteins. Sensitivity to the cell-permeable BCL-2 antagonist ABT-737 is also related to priming of BCL-2 by activator BH3-only molecules. Our data allow us to distinguish a cellular state we call "primed for death," which can be determined by BH3 profiling and which correlates with dependence on antiapoptotic family members for survival.     

Resistance to UV‐induced apoptosis by β‐HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase

UV exposure is the main etiological agent in the development of non‐melanoma skin cancer (NMSC), but mounting evidence suggests a co‐factorial role for β‐genus HPV types early in tumor initiation or progression. UV damage initiates an apoptotic response, driven at the mitochondrial level by BCL‐2 family proteins, that eliminates damaged cells that may accumulate deleterious mutations and acquire tumorigenic properties. BAK is a pro‐apoptotic BCL‐2 protein that functions ultimately to form pores that permeabilize the mitochondrial outer membrane, thereby committing a cell to death, a process involving changes in BAK phosphorylation and conformation. The E6 protein of β‐type HPV5 signals BAK for proteasomal degradation, a function that confers protection from UV‐induced apoptosis. We find that HPV5 E6 does not constitutively target BAK for proteolysis, but targets the latter stages of BAK activation, following changes in phosphorylation and conformation. A mutational analysis identified the lysine residue on BAK required for proteolysis, and a functional siRNA screen identified the HECT domain E3 ubiquitin ligase HERC1 as being required for E6‐mediated BAK degradation. We show that HERC1 interacts with BAK in E6‐expressing cells that have been damaged by UV, and provide evidence that the interaction of HERC1 with BAK requires access to a hydrophobic surface on BAK that binds BH3 domains of BCL‐2 proteins. We also show that HERC1 contains a putative BH3 domain that can bind to BAK. These findings reveal a specific and unique mechanism used by the HPV5 E6 protein to target BAK.     

Targeting BCL-2 family proteins to overcome drug resistance in non-small cell lung cancer

Cytotoxic chemotherapies are standard of care for patients suffering from advanced non‐small cell lung cancer (NSCLC). However, objective responses are only achieved in 20% of cases and long‐term survival is rarely observed. Clinically applied anticancer drugs exert at least some of their activities by inducing apoptosis. A critical step in apoptotic signal transduction is the permeabilization of the mitochondrial outer membrane (MOM), which is regulated by the BCL‐2 family of proteins. Hence, therapeutic targeting of BCL‐2 proteins is a promising approach to increase the drug‐sensitivity of cancers. To this end we have assessed the impact of conditional expression of the proapoptotic multidomain (BH1‐2‐3) protein BAK, which directly permeabilizes the MOM, and the BH3‐mimetic ABT‐737, which acts indirectly by derepressing BH1‐2‐3 proteins, on apoptosis and drug sensitivity of NSCLC cells. Conditionally expressed BAK sensitized resistant NSCLC cells to drug‐induced apoptosis. In contrast, ABT‐737 was ineffective in those NSCLC cells expressing high levels of the anti‐apoptotic MCL‐1 protein. Tissue microarray analysis of tumor samples from 84 chemotherapy‐naïve NSCLC patients revealed MCL‐1 expression in 56% of cases, thus supporting the relevance of this resistance factor in a clinical setting. Enforced expression of the BH3‐only protein NOXA, which targets MCL‐1, overcame resistance to ABT‐737. Moreover, combining conditionally expressed BAK with ABT‐737 enhanced apoptosis in NSCLC cells independently of their MCL‐1 status. In conclusion, the heterogeneity of apoptosis defects observed in drug‐resistant NSCLC demands individually tailored molecular therapies. Targeting the MOM permeabilizer BAK appears to have a broader apoptogenic activity than the BH3‐only mimetic ABT‐737. © 2007 Wiley‐Liss, Inc.     

Deciphering the molecular events necessary for synergistic tumor cell apoptosis mediated by the histone deacetylase inhibitor vorinostat and the BH3 mimetic ABT-737

The concept of personalized anticancer therapy is based on the use of targeted therapeutics through in-depth knowledge of the molecular mechanisms of action of these agents when used alone and in combination. We have identified the apoptotic proteins and pathways necessary for synergistic tumor cell apoptosis and in vivo antitumor responses seen when the HDAC inhibitor vorinostat is combined with the BH3-mimetic ABT-737 in lymphomas overexpressing Bcl-2. Vorinostat "primes" tumors overexpressing Bcl-2 for rapid ABT-737-mediated apoptosis by inducing expression of the BH3-only gene bmf. Moreover, these synergistic effects of vorinostat/ABT-737 were blunted in cells with an inactive p53 pathway or in cells lacking expression of the p53 target gene, noxa. These studies show the important and complex functional interaction between specific proapoptotic BH3-only proteins and the BH3-mimetic compound ABT-737 and provide the most comprehensive functional link between tumor genotype and the apoptotic and therapeutic effects of HDACi combined with ABT-737.     

Proapoptotic BH3-only BCL-2 family protein BIM connects death signaling from epidermal growth factor receptor inhibition to the mitochondrion

A subset of lung cancers expresses mutant forms of epidermal growth factor receptor (EGFR) that are constitutively activated. Cancers bearing activated EGFR can be effectively targeted with EGFR inhibitors such as erlotinib. However, the death-signaling pathways engaged after EGFR inhibition are poorly understood. Here, we show that death after inhibition of EGFR uses the mitochondrial, or intrinsic, pathway of cell death controlled by the BCL-2 family of proteins. BCL-2 inhibits cell death induced by erlotinib, but BCL-2-protected cells are thus rendered BCL-2-dependent and sensitive to the BCL-2 antagonist ABT-737. BH3 profiling reveals that mitochondrial BCL-2 is primed by death signals after EGFR inhibition in these cells. As this result implies, key death-signaling proteins of the BCL-2 family, including BIM, were found to be up-regulated after erlotinib treatment and intercepted by overexpressed BCL-2. BIM is induced by lung cancer cell lines that are sensitive to erlotinib but not by those resistant. Reduction of BIM by siRNA induces resistance to erlotinib. We show that EGFR activity is inhibited by erlotinib in H1650, a lung cancer cell line that bears a sensitizing EGFR mutation, but that H1650 is not killed. We identify the block in apoptosis in this cell line, and show that a novel form of erlotinib resistance is present, a block in BIM up-regulation downstream of EGFR inhibition. This finding has clear implications for overcoming resistance to erlotinib. Resistance to EGFR inhibition can be modulated by alterations in the intrinsic apoptotic pathway controlled by the BCL-2 family of proteins.     

The apoptotic response in HCT116<Superscript><Emphasis Type="Italic">BAX-/-</Emphasis></Superscript>cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

Background  

Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood.     

Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins

The adenovirus E4orf6 is a viral oncoprotein known to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventually leading to the inhibition of apoptotic activity.     

Inhibition of MCL-1 in breast cancer cells promotes cell death in vitro and in vivo

The present studies have examined approaches to suppress MCL-1 function in breast cancer cells, as a means to promote tumor cell death. Treatment of breast cancer cells with CDK inhibitors (flavopiridol; roscovitine) enhanced the lethality of the ERBB1 inhibitor lapatinib in a synergistic fashion. CDK inhibitors interacted with lapatinib to reduce MCL-1 expression and overexpression of MCL-1 or knock down of BAX and BAK suppressed drug combination lethality. Lapatinib-mediated inhibition of ERK1/2 and to a lesser extent AKT facilitated CDK inhibitor-induced suppression of MCL-1 levels. Treatment of cells with the BH3 domain/MCL-1 inhibitor obatoclax enhanced the lethality of lapatinib in a synergistic fashion. Knock out of MCL-1 and BCL-X_L enhanced lapatinib toxicity to a similar extent as obatoclax and suppressed the ability of obatoclax to promote lapatinib lethality. Pre-treatment of cells with lapatinib or with obatoclax enhanced basal levels of BAX and BAK activity and further enhanced drug combination toxicity. In vivo tumor growth data in xenograft and syngeneic model systems confirmed our in vitro findings. Treatment of cells with CDK inhibitors enhanced the lethality of obatoclax in a synergistic fashion. Overexpression of MCL-1 or knock down of BAX and BAK suppressed the toxic interaction between CDK inhibitors and obatoclax. Obatoclax and lapatinib treatment or obatoclax and CDK inhibitor treatment or lapatinib and CDK inhibitor treatment radiosensitized breast cancer cells. Lapatinib and obatoclax interacted to suppress mammary tumor growth in vivo. Collectively our data demonstrate that manipulation of MCL-1 protein expression by CDK inhibition or inhibition of MCL-1 sequestering function by Obatoclax renders breast cancer cells more susceptible to BAX/BAK-dependent mitochondrial dysfunction and tumor cell death.Key words: MCL-1, Lapatinib, Obatoclax, Flavopiridol, Roscovitine, CDK inhibitor, RTK inhibitor, BCL-2 inhibitor, BAK