IAP-targeted therapies for cancer.

DNA damage, chromosomal abnormalities, oncogene activation, viral infection, substrate detachment and hypoxia can all trigger apoptosis in normal cells. However, cancer cells acquire mutations that allow them to survive these threats that are part and parcel of the transformation process or that may affect the growth and dissemination of the tumor. Eventually, cancer cells accumulate further mutations that make them resistant to apoptosis mediated by standard cytotoxic chemotherapy or radiotherapy. The inhibitor of apoptosis (IAP) family members, defined by the presence of a baculovirus IAP repeat (BIR) protein domain, are key regulators of cytokinesis, apoptosis and signal transduction. Specific IAPs regulate either cell division, caspase activity or survival pathways mediated through binding to their BIR domains, and/or through their ubiquitin-ligase RING domain activity. These protein-protein interactions and post-translational modifications are the subject of intense investigations that shed light on how these proteins contribute to oncogenesis and resistance to therapy. In the past several years, we have seen multiple approaches of IAP antagonism enter the clinic, and the rewards of such strategies are about to reap benefit. Significantly, small molecule pan-IAP antagonists that mimic an endogenous inhibitor of the IAPs, called Smac, have demonstrated an unexpected ability to sensitize cancer cells to tumor necrosis factor-alpha and to promote autocrine or paracrine production of this cytokine by the tumor cell and possibly, other cells too. This review will focus on these and other developmental therapeutics that target the IAPs in cancer.

Both cIAP1 and cIAP2 regulate TNFalpha-mediated NF-kappaB activation.

The cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2) proteins have been implicated in the activation of NF-kappaB by TNFalpha; however, genetic deletion of either cIAP1 or 2 did not support a physiologically relevant role, perhaps because of functional redundancy. To address this, we used combined genetic and siRNA knockdown approaches and report that cIAP1 and 2 are indeed critical, yet redundant, regulators of NF-kappaB activation upon TNFalpha treatment. Whereas NF-kappaB was properly activated by TNFalpha in cultured and primary cells deficient in either cIAP1 or 2, removal of both cIAPs severely blunted its activation. After treatment with TNFalpha, cIAP1 and 2 were rapidly recruited to the TNF receptor 1, along with the adapter protein TNF receptor associated factor 2. Importantly, either cIAP1 or 2 was required for proper TNF receptor 1 signalosome function. In their combined absence, polyubiquitination of receptor interacting protein 1, an upstream event necessary for NF-kappaB signaling, was attenuated. As a result, phosphorylation of the inhibitor of kappaB kinase beta was diminished, and signal transduction was severely blunted. Consequently, cells missing both cIAP1 and 2 were sensitized to TNFalpha-mediated apoptosis. Collectively, these data demonstrate that either cIAP1 or 2 is required for proper Rip1 polyubiquitination and NF-kappaB activation upon TNFalpha treatment.

Detachment-induced upregulation of XIAP and cIAP2 delays anoikis of intestinal epithelial cells.

Detachment of normal epithelial cells from the extracellular matrix triggers apoptosis, a phenomenon called anoikis. Conversely, carcinoma cells tend to be relatively more anoikis-resistant than their normal counterparts, and this increased resistance represents a critical feature of the malignant phenotype. Mechanisms that control susceptibility and resistance to anoikis are not fully understood. It is now known that detachment of non-malignant epithelial cells triggers both pro- and antiapoptotic signals, and it is the balance between these signals and the duration of detachment that determine further fate of the cells. Detachment-induced antiapoptotic events delay anoikis and if cells reattach relatively soon after detachment they survive. Direct regulators of apoptosis responsible for this delay of anoikis are unknown. We found that detachment of non-malignant intestinal epithelial cells triggers upregulation of inhibitors of apoptosis protein (IAP) family, such as X-chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis-2 (cIAP2). We demonstrated that this upregulation requires detachment-dependent activation of the transcription factor nuclear factor-kappaB. We further observed that various IAP antagonists accelerate anoikis, indicating that upregulation of the IAPs delays detachment-triggered apoptosis. We conclude that the IAPs are important regulators of the balance between detachment-triggered life and death signals. Perhaps, not by coincidence, these proteins are often upregulated in carcinomas, tumors composed of cells that tend to be anoikis-resistant.

Evidence for apoptosis in the fetal Down syndrome brain.

In Down syndrome, enhanced apoptosis (programmed cell death) may play a role in the pathogenesis of characteristic early mental retardation and precocious neurodegeneration of Alzheimer type. Various apoptosis-associated proteins (Bax, Bcl-2, Fas, p53, Hsp70, neuronal apoptosis inhibitory protein-like immunoreactivity) were investigated in four different cortical regions and the cerebellum of one fetal Down syndrome (35 weeks' gestation) postmortem brain sample compared with a control brain sample. The most impressive finding was an at least fivefold elevation of Bax protein together with decreased Bcl-2 values in all Down syndrome cerebral regions investigated. In addition, antiapoptotic, presumably caspase-inhibitory, principles like heat shock protein 70 and neuronal apoptosis inhibitory protein were also reduced. Whereas Fas protein, an important member of receptor-mediated apoptosis, was inconsistently altered, a rather surprising finding was reduced proapoptotic, regulatory protein p53 in four of five regions. The findings are in good agreement with the proposed role of the Bcl-2 protein family in regulating developmental (naturally occurring) apoptotic neuronal death and further suggest that developmental apoptosis may be inappropriately commandeered by so far undefined pathologic processes in Down syndrome.

Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence from CD34(+) hematopoietic stem cells: implications for ex vivo tumor purging and autologous stem cell transplantation.

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma patients.

Neuronal apoptosis inhibitory protein (NAIP)-like immunoreactivity in brains of adult patients with Down syndrome.

In Down syndrome (DS), enhanced apoptosis (programmed cell death) may play a role in the pathogenesis of characteristic early mental retardation and precocious neurodegeneration of Alzheimer-type. The human IAP (inhibitor of apoptosis proteins) genes (NAIP, c-IAP-2/HIAP-1, c-IAP-1/Hiap-2, XIAP, survivin) are an evolutionary conserved family of proteins which prevent cell death across species, implying that they act at a central, highly conserved point in the cell death cascade. Evidence for downregulation of NAIP-mRNA in fetal DS (23rd week of gestation), as found by subtractive hybridization technique challenged studies at the protein level in adult DS brain specimen. NAIP-like immunoreactivity was determined in four different regions of cerebral cortex and cerebellum in 9 adult DS patients with Alzheimer-like neuropathologic lesions, 9 Alzheimer disease (AD) patients as compared to 9 controls. For the first time, NAIP-IR could be demonstrated in different cortical regions of the human brain. Compared to control subjects, western blotting demonstrated significantly decreased levels in parietal and occipital cortex in DS and in frontal and occipital cortex in AD. While the mode of NAIP action is unknown, inhibition of certain caspases has already been demonstrated for other IAP-family members (c-IAP1, c-IAP2 and XIAP). Although decreased NAIP-IR of certain brain regions in DS and AD awaits further confirmation, the results suggest that alterations of apoptosis regulatory (inhibitory) proteins may be another feature of neurodegeneration in DS and AD.

Penetration and intracellular routing of nucleus-directed peptide-based shuttles (loligomers) in eukaryotic cells.

Loligomers are multitasking, peptide-based shuttles that are able to penetrate cells and self-localize into distinct cellular compartments. In particular, loligomer 4 incorporates internalization and nuclear import sequences as well as reporter groups. The intracellular routing of loligomer 4 was analyzed by microscopy and flow cytometry, to define and demonstrate localization events. Electron micrographs of CHO cells exposed to a biotinylated derivative of loligomer 4 as well as confocal images of CHO cells treated with rhodamine-labeled loligomer 4 indicate their presence in the cytosol, endocytic vesicles, and the nucleus of CHO cells. Loligomer 4 accumulates irreversibly inside cells. Uptake of loligomer 4 by six mammalian cell lines (Daudi, EL4, CHO, COS-7, VERO, and HeLa) was proven by flow cytometry, establishing the generality of the principle. Cells presented as monolayers typically were less able to endocytose the construct than cells grown in suspension. Cellular accumulation of loligomer 4 varied between cell lines with COS-7 and VERO cells showing the highest level of uptake. Plasmids harboring reporter genes could be transported efficiently inside CHO cells, suggesting that loligomer 4 either alone or noncovalently associated with large macromolecules can effectively reach the nucleus of cells. In summary, loligomer 4 constructs provide a simple synthetic platform for the design of guided intracellular agents.

The inhibitors of apoptosis (IAPs) and their emerging role in cancer.

The inhibitor of apoptosis protein family has been characterized over the past 5 years, initially in baculovirus and more recently in metazoans. The IAPs are a widely expressed gene family of apoptotic inhibitors from both phylogenic and physiologic points of view. The diversity of triggers against which the IAPs suppress apoptosis is greater than that observed for any other family of apoptotic inhibitors including the bcl-2 family. The central mechanisms of IAP apoptotic suppression appear to be through direct caspase and pro-caspase inhibition (primarily caspase 3 and 7) and modulation of and by the transcription factor NF-kappaB. Although evidence for a direct oncogenic role for the IAPs has yet to be delineated, a number of lines of evidence point towards this class of protein playing a role in oncogenesis. The strongest evidence for IAP involvement in cancer is seen in the IAP called survivin. Although not observed in adult differentiated tissue, survivin is present in most transformed cell lines and cancers tested to date. Survivin has been shown to inhibit caspase directly and apoptosis in general, moreover survivin protein levels correlate inversely with 5 year survival rates in colorectal cancer. Recent data has also implicated survivin in cell cycle control. The second line of evidence for IAP involvement in cancer comes from their emerging role as mediators and regulators of the anti-apoptotic activity of v-Rel and NF-kappaB transcription factor families. The IAPs have been shown to be induced by NF-kappaB or v-Rel in multiple cell lines and conversely, HIAP1 and HIAP2 have been shown to activate NF-kappaB possibly forming a positive feed-back loop. Overall a picture consistent with an IAP role in tumour progression rather than tumour initiation is emerging making the IAPs an attractive therapeutic target.

Shiga-like toxin purges human lymphoma from bone marrow of severe combined immunodeficient mice.

Shiga-like toxin-1 (SLT-1) is a bacterial toxin that kills cells by inhibiting protein synthesis. SLT-1 is composed of one cytotoxic A-subunit and five B-subunits that bind to CD77, a cell-surface glycolipid. In the human hematopoietic system, CD77 expression is restricted to a subset of activated B cells and derived cancers. Here we report that SLT-1 treatment of murine bone marrow ex vivo effectively cures severe combined immunodeficient mice of a human B-cell lymphoma xenograft while sparing normal hematopoietic precursor cells. Flow cytometry results using fluorescein isothiocyanate-labeled SLT-1 B-subunit show the high prevalence of expression of SLT-1 receptors (CD77) in human non-Hodgkin's lymphomas, especially follicular lymphomas. These results suggest the use of SLT-1 for the purging of human bone marrow before autologous bone marrow transplant in the case of CD77+ B-cell lymphomas as just one of many possible uses.

Identification of binding proteins for nuclear localization signals of the glucocorticoid and thyroid hormone receptors.

Nuclear entry of proteins the size of the glucocorticoid and thyroid hormone receptors appears to be mediated by an interaction of nuclear localization signals (NLSs) within the proteins and specific NLS-binding proteins. NLSs have been identified in the hinge region of both receptors. We have identified the cellular binding proteins of the glucocorticoid receptor NLS and the thyroid hormone receptor NLS after cross-linking of radiolabeled signal peptides to subcellular fractions. Two S49 lymphoma cytosolic polypeptides of 60 and 76 kilodaltons (kDa) were specifically bound to either the glucocorticoid or thyroid hormone receptor NLS. The two binding sites demonstrated saturable binding. A competitive binding assay showed that the binding sites were specific for NLSs and that a mutated NLS was a poor competitor for the binding of labeled glucocorticoid receptor NLS. However, competition studies with peptides unrelated to NLSs, yet resembling NLSs in that they had a net positive charge, revealed that the 60-kDa entity demonstrated greater specificity for binding to NLSs than did the 76-kDa polypeptide. Glucocorticoid receptor NLS and thyroid hormone receptor NLS-binding proteins of 60 and 76 kDa were also identified in nuclear fractions. Although the unoccupied glucocorticoid receptor resides in the cytoplasm, while the unoccupied thyroid hormone receptor is always found in the nucleus, the hinge NLS interactions do not specify these different localizations of the unoccupied receptors. Rather, the data support roles for the hinge NLS in general steps of nuclear import and the 60-kDa cross-linked product as a chaperone of both receptors into the nucleus. Its cellular localization also suggests a role for the 76-kDa cross-linked product as a chaperone, but its relatively less stringent binding specificity may indicate that this polypeptide has a different physiological function.

Nuclear and nuclear envelope binding proteins of the glucocorticoid receptor nuclear localization peptide identified by crosslinking.

The molecular mechanisms underlying the nuclear entry of steroid receptors and possible regulation of steroid hormone action during receptor passage across the nuclear envelope have not been elucidated. A nuclear localization signal has been identified in the hinge region of the glucocorticoid receptor. A synthetic peptide corresponding to this sequence was radio-iodinated and incubated with high salt- and detergent-extracted rat liver nuclei or nuclear envelope in the presence of crosslinker. After SDS-PAGE, two nuclear polypeptides of 60 and 76 kDa which had been specifically crosslinked were identified by autoradiography. A 60 kDa polypeptide was also crosslinked in the nuclear envelope fraction. ATP and elevated temperatures enhanced the crosslinking of both nuclear peptides. Finally, we showed that the pattern of crosslinking of the simian virus 40 large tumour antigen nuclear localization signal was identical to that of the glucocorticoid receptor signal to the nuclear polypeptides. The crosslinked peptides are good candidates for nuclear importers of the glucocorticoid receptor. In addition, the data suggest that these binding sites may be part of a general mechanism for nuclear entry of proteins.

Microsomal dexamethasone binding sites identified by affinity labelling.

Binding studies with [3H]dexamethasone identified a class of binding sites on male rat liver microsomes. The binding sites were glucocorticoid-dependent and specific for glucocorticoids and progestins. Scatchard binding parameters, competition studies with triamcinolone acetonide, a synthetic glucocorticoid which competes well for the glucocorticoid receptor, and immunoblotting with an antiglucocorticoid receptor antibody indicated that these sites are distinct from the cytosolic glucocorticoid receptor. Affinity labelling experiments with [3H]dexamethasone 21-mesylate revealed two specifically labelled peptides, one at approx. 66 kDa and a doublet at 45 kDa. The 66 kDa peptide had been previously identified in serum and may be present as a result of serum contamination of the microsomal preparation. The 45 kDa doublet, on the other hand, had been shown to be absent from rat serum. The characteristics of the 45 kDa peptide(s) were identical to those of the dexamethasone binding site identified in the binding studies. [3H]Dexamethasone binding characteristics and affinity labelling of microsomal subfractions, separated by isopycnic centrifugation, showed that the binding sites are located in the endoplasmic reticulum. The identification and role of the 45 kDa peptide doublet remain to be determined.