Regulation of Apoptosis by Viral Oncogenes

Primary epithelial cells become transformed as a result of the combined action of deregulation of cell cycle control and inhibition of programmed cell death (apoptosis). Expression of the human adenovirus (Ad2/5) E1A oncogene releases normal restrictions on cell cycle progression through interactions with the retinoblastoma tumor suppressor protein and its relatives, and with the p300 and CBP transcriptional co-activators. The cellular response to this deregulation of cell growth control by E1A is the stabilization of the p53 tumor suppressor protein and the induction of p53-dependent apoptosis of transformed cells, and p53-independent apoptosis in virus infected cells. The activation of this apoptotic program prevents transformation and can diminish virus replication. The adenovirus E1B oncogene encodes an apoptosis inhibitor, E1B 19K, that is required to inhibit apoptosis, which thereby sustains transformation and productive infection. E1B 19K is a viral homologue of cellular BCL-2, and its expression blocks apoptosis induced by E1A and death receptor signaling during infection, and by p53 during transformation. E1B 19K functions as a general apoptosis inhibitor by binding to and inhibiting pro-apoptotic BCL-2 family members BAX and BAK that propagate cell death signaling through mitochondria. The long-term focus of the White laboratory has been to determine the mechanism by which E1A and E1B 19K regulate apoptosis, and the role of apoptosis in infection and oncogenic transformation. As deregulation of apoptosis is a common feature of many disease states, knowledge gained by this pursuit should provide new opportunities for the development of novel therapies, particularly for cancer treatment.

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E1A Sensitizes Cells to TNF-alpha by Down-Regulating c-FLIP-Short

Tumor necrosis factor-a (TNF-alpha) activates both apoptosis and NF-kB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested. c-FLIP is a TNF-alpha induced gene that inhibits caspase-8 activation during TNF-alpha signaling. Adenovirus infection and E1A expression sensitizes cells to TNF-alpha by allowing apoptosis in the absence of inhibitors of gene expression suggesting that it may be disabling a survival signaling pathway. E1A promotes TNF-alpha-mediated activation of caspase-8 suggesting that sensitivity is occurring at the level of the death-inducing signaling complex (DISC). Furthermore, E1A expression down-regulates c-FLIP-Short expression and prevents its induction by TNF-alpha. Viral FLIP expression rescued E1A-mediated sensitization to TNF-alpha by restoring the resistance of caspase-8 to activation thereby, preventing cell death. E1A inhibits TNF-alpha-dependent induction of c-FLIP-Short mRNA and stimulates ubiquitination and proteasome-dependent degradation of c-FLIP-Short protein (Fig. 1). Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-kB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis refractory tumor cells.

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Mechanism of Inhibition of TNF-alpha-Mediated Apoptosis by E1B 19K

The adenovirus E1B 19K gene product is a potent inhibitor of death receptor-mediated apoptosis, including that mediated by TNF-alpha during viral infection. TNF-alpha is a component of the immune response by the infected host to virus infection that acts to eliminate infected cells through apoptosis induction. TNF-alpha receptor 1 (TNFR-1) activation by TNF-alpha recruits adaptor molecules that facilitate caspase-8 activation. Caspase-8 cleaves the BCL-2 family member BID to truncated BID (tBID), which translocates to mitochondria, interacts with and induces conformational changes in BAX and BAK, which facilitates oligomerization of BAX and BAK into a high molecular mass protein complex. These BAX-BAK oligomers promote cytochrome c and Smac/DIABLO release from mitochondria, which stimulate caspase-9 and caspase-3 activation and apoptotic cell death of infected cells (Fig. 2). Oligomerized BAX and BAK may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K inhibits neither caspase-8 activation nor caspase-8-dependent BID cleavage. E1B 19K is constitutively bound to BAK and TNF-alpha signaling induces an interaction between E1B 19K and BAX. E1B 19K binding to BAK and BAX, blocks BAX-BAK oligomerization, cytochrome c and Smac/DIABLO release from mitochondria, and caspase-9 activation. Thus, E1B 19K blocks TNF-alpha-mediated death signaling by inhibiting BAK and a specific form of BAX in mitochondria. E1B 19K thereby inhibits the release of pro-apoptotic mitochondrial proteins and prevents caspase activation and apoptosis, which sustains virus infection. (Fig. 3). Since either BAX or BAK is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both BAX and BAK may be required to inhibit their oligomerization and the release proteins from mitochondria which promote caspase activation and cell death. Inhibition of BAX and BAK oligomerization by E1B 19K bears striking similarity to the means by which bacterial immunity proteins block pore formation by bacterial toxins, which have structural homology to BAX and BAK.

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BAX and BAK Function to Limit Adenovirus Replication Through Apoptosis Induction

Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by E1B 19K. Unlike apoptosis mediated by death receptors, infection with pro-apoptotic E1B 19K mutant viruses does not induce cleavage of BID, but nonetheless induces changes in BAX and BAK conformation and promotes their oligomerization. These events are associated with release of cytochrome c and Smac/DIABLO from mitochondria, caspase activation and apoptosis (Fig. 4). In wild-type adenovirus infected cells, E1B 19K binds to BAK, precluding BAX-BAK interaction and BAX conformational changes (Fig. 5). Infection with E1B 19K mutant viruses induces apoptosis in wild-type and BAX or BAK deficient baby mouse kidney epithelial cells (BMKs), but not in those deficient for both. In the absence of BAX and BAK, apoptosis during virus infection is blocked, allowing more efficient replication of the virus. Thus, BAX and BAK-mediated apoptosis severely limits adenoviral replication, demonstrating that BAX and BAK function as an antiviral response at the cellular level. By encoding a BAX and BAK inhibitor (E1B 19K), adenovirus escapes this cell-activated apoptotic response to ensure efficient virus replication.

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BAX and BAK Mediate p53-Independent Suppression of Tumorigenesis

In oncogenic transformation of primary epithelial cells, E1A expression causes accumulation of p53 that implements cell death by apoptosis as a means to inhibit transformation. E1A expression in p53 deficient cells relieves this p53-dependent apoptosis, producing transformed cells with high frequency. While transformed and immortal, these cells retain their p53-independent apoptotic response and are not tumorigenic. BAX and BAK are essential regulators of pro-apoptotic signaling and the disruption of apoptosis is linked to the development of cancer. To investigate the role of BAX and BAK in tumorigenesis, primary BMKs from wild-type, BAX or BAK, or BAK and BAK deficient mice were transformed by expression of adenovirus E1A and inactivation of p53. In wild-type BMKs the expression of E1A and inactivation of p53 is sufficient for transformation but not tumorigenesis. In contrast, transformed BAX and BAK deficient BMKs have a profound defect in apoptosis and form highly invasive carcinomas. Transformed BMKs deficient for either BAX or BAK are also tumorigenic but only when heterozygous for the remaining bax or bak allele, the expression of which is lost in most resulting tumors. Thus, BAX and BAK function to suppress tumorigenesis, and their deficiency is selected for in vivo. Identification of the p53-independent, BAX- and BAK-dependent apoptotic signaling pathway responsible for suppression of tumorigenesis that is inactivated in BAX and BAK deficient cells, should provide insight into novel means for restoring apoptotic function to tumor cells that have lost it. These findings also explain why the DNA tumor virus adenovirus encodes three oncogenes: E1A to inactivate RB and drive proliferation; E1B 55K to disable p53 and inactivate cell cycle checkpoint control; and E1B 19K to disable p53-independent apoptosis triggered from the tumor or infected cell microenvironment (Fig. 6).

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DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells

Expression of adenovirus E1A deregulates cell proliferation to facilitate viral DNA replication, prompting the initiation of apoptosis signaled primarily through proapoptotic BAK in productively infected cells. We discovered that in uninfected cells, BAK is complexed with the anti-apoptotic BCL-2 family member Myeloid Cell Leukemia 1 (MCL-1). E1A expression during infection resulted in the specific down-regulation of MCL-1 through destabilization of the protein and loss of the mRNA. Upon loss of the MCL-1-BAK complex, BAK complexed with either BAX in proapoptotic E1B mutant adenovirus-infected cells, or with the adenovirus BCL-2 homolog E1B 19K in cells infected with the wild-type virus in which apoptosis is inhibited. Loss of MCL-1 was required to initiate the apoptotic pathway in infected cells as restoration of MCL-1 expression rescued infected cells from E1A-induced apoptosis. Analogous to E1A expression, DNA damage down-regulates MCL-1, and adenovirus infection resulted in the accumulation of phosphorylated H2AX and ataxia-telangiectasia mutant protein (ATM), hallmarks of DNA double-strand breaks. Thus, MCL-1 may function by maintaining BAK in an inactive state, and the loss of MCL-1 upon activation of the DNA damage response, perhaps through replication stress induced in virus infected cells, may be required to initiate the apoptotic response. This activation of a DNA damage response by an oncogene that results in down-regulation of anti-apoptotic MCL-1 may convey sensitivity to chemotherapeutic drugs.

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Hypoxia and Defective Apoptosis Drive Genetic Instability and Tumorigenesis

See feature article by Louisa Flintof on this work in the October 2004 issue of Nature Reviews Cancer.

Genomic instability is a hallmark of cancer development and progression, and characterizing the stresses that create and the mechanisms by which cells respond to genomic perturbations is essential. We discovered that antiapoptotic BCL-2 family proteins promote tumor formation of transformed baby mouse kidney (BMK) epithelial cells by antagonizing BAX- and BAK-dependent apoptosis. Cell death in vivo correlated with hypoxia and induction of PUMA (p53 up-regulated modulator of apoptosis). Strikingly, carcinomas formed by transformed BMK cells in which apoptosis was blocked by aberrant BCL-2 family protein function displayed prevalent, highly polyploid, tumor giant cells. Examination of the transformed BMK cells in vivo revealed aberrant metaphases and ploidy changes in tumors as early as 9 days after implantation, which progressed in magnitude during the tumorigenic process. An in vitro ischemia system mimicked the tumor microenvironment, and gain of BCL-2 or loss of BAX and BAK was sufficient to confer resistance to apoptosis and to allow for accumulation of polyploid cells in vitro. These data suggest that in vivo, even in cells in which p53 function is compromised, apoptosis is an essential response to hypoxia and ischemia in the tumor microenvironment and that abrogation of this response allows the survival of cells with abnormal genomes and promotes tumorigenesis.

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Caspase dependent Processing Activates the Pro-Apoptotic Activity of Deleted in Breast Cancer-1 During Tumor Necrosis Factor alpha Mediated Death Signaling

Deleted in Breast Cancer 1 (DBC 1) was initially cloned from a homozygously deleted region in breast and other cancers on human chromosome 8p21, although no function is known for the protein product it encodes. We identified the generation of amino terminally truncated versions of DBC 1 during Tumor Necrosis Factor (TNF) a mediated apoptosis. Full-length 150 kDa DBC 1 underwent caspase dependent processing during TNF a mediated death signaling, to produce p120 DBC 1 and p66 DBC 1 carboxy terminal fragments. Endogenous DBC 1 localized to the nucleus in healthy cells, but localized to the cytoplasm during TNF a mediated apoptosis, consistent with the loss of the amino terminus containing the nuclear localization signal. Overexpression of an amino terminal truncated DBC 1, resembling p120 DBC 1, caused mitochondrial clustering, mitochondrial matrix condensation, and sensitized cells to TNF a mediated apoptosis. The carboxy terminal coiled-coil domain of DBC 1 was responsible for the cytoplas mic and mitochondrial localization, and for the death promoting activity of DBC 1. Thus, caspase dependent proc essing of DBC 1 may act as a feed-forward mechanism to promote apoptosis and possibly also tumor suppression. D BC 1, like its homolog Cell cycle and Apoptosis Regulatory Protein 1 (CARP 1), may function in the regulation of apoptosis.

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Key Roles of BIM-driven Apoptosis in Epithelial Tumors and Rational Chemotherapy

Defective apoptosis not only promotes tumorigenesis, but also can confound chemotherapeutic response. We discovered that the proapoptotic BH3-only protein BIM is a tumor suppressor in epithelial solid tumors and is also a determinant in paclitaxel sensitivity in vivo. Furthermore, the H-ras/mitogen-activated protein kinase (MAPK) pathway conferred resistance to paclitaxel that is dependent on functional inactivation of BIM. Whereas paclitaxel induces BIM accumulation and BIM-dependent apoptosis in vitro and in tumors in vivo, the H-ras/MAPK pathway suppresses this BIM induction by phosphorylating BIM and targeting BIM for degradation in proteasomes. The proteasome inhibitor Velcade (P-341, Bortezomib) restores BIM induction, abrogates H-ras-dependent protection against paclitaxel, and promotes BIM-dependent tumor regression, suggesting the potential benefits of combinatorial chemotherapy of Velcade and paclitaxel. Tumorigenesis results in the acquisition of mutations that promote tumor growth and chemoresistance, and relating tumor genotype to prognostic indications and to effective treatment regimens is essential for successful therapeutic outcome. Determining the mechanism of apoptosis induction by the chemotherapeutic drug paclitaxel revealed that BIM suppresses tumorigenesis and is required for paclitaxel responsiveness. The targeting of BIM for degradation in proteasomes by the H-ras/MAPK pathway is the molecular basis for paclitaxel resistance in tumors with activating mutations in RAS, and paclitaxel responsiveness is restored by joint administration of the proteasome inhibitor Velcade. Thus rational combinatorial chemotherapy using proteasome inhibitors to enhance chemosensitivity to paclitaxel in tumors where the H-ras/MAPK pathway is activated may be therapeutically beneficial.

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Autophagy Promotes Tumor Cell Survival and Restricts Necrosis, Inflammation, and Tumorigenesis

Defective apoptosis renders immortalized epithelial cells highly tumorigenic, but how this is impacted by other common tumor mutations is not known. In apoptosis-deficient cells, inhibition of autophagy by AKT activation or by allelic disruption of beclin1, confers sensitivity to metabolic stress by inhibiting an autophagy-dependent survival pathway. While autophagy acts to buffer metabolic stress, the combined impairment of apoptosis and autophagy promotes necrotic cell death in vitro and in vivo. Thus inhibiting autophagy under conditions of nutrient limitation can restore cell death to apoptosis-refractory tumors, but this necrosis is associated with inflammation and accelerated tumor growth. Thus, autophagy may function in tumor suppression by mitigating metabolic stress, and in concert with apoptosis, by preventing death by necrosis.