Inhibitor of Apoptosis (IAP) proteins as therapeutic targets for radiosensitization of human cancers

Radiotherapy initiates a variety of signaling events in cancer cells that eventually lead to cell death in case the DNA damage cannot be repaired. However, the signal transduction pathways that mediate cell death in response to radiation-inflicted DNA damage are frequently disturbed in human cancers, contributing to radioresistance. For example, aberrant activation of antiapoptotic programs such as high expression of Inhibitor of Apoptosis (IAP) proteins has been shown to interfere with the efficacy of radiotherapy. Since IAP proteins have been linked to radioresistance in several malignancies, therapeutic targeting of IAP proteins may open new perspectives to overcome radioresistance. Therefore, molecular targeting of IAP proteins may provide novel opportunities to reactivate cell death pathways that mediate radiation-induced cytotoxicity. A number of strategies have been developed in recent years to antagonize IAP proteins for the treatment of cancers. Some of these approaches have already been translated into a clinical application. While IAP protein-targeting agents are currently being evaluated in early clinical trials alone or in combination with conventional chemotherapy, they have not yet been tested in combination with radiation therapy. Therefore, it is a timely subject to discuss the opportunities of antagonizing IAP proteins for radiosensitization. Preclinical studies demonstrating the potential of this concept in relevant in vitro and in vivo models underscore that this combination approach warrants further clinical investigation. Thus, combination protocols using IAP antagonists together with radiotherapy may pave the avenue to more effective radiation-based treatment options for cancer patients.     

The inhibitors of apoptosis: there is more to life than Bcl2

The inhibitor of apoptosis (IAP) genes constitute a highly conserved family found in organisms as diverse as insects and mammals. These genes encode proteins that directly bind and inhibit caspases, and thus play a critical role in deciding cell fate. The IAPs are in turn regulated by endogenous proteins (second mitochondrial activator of caspases and Omi) that are released from the mitochondria during apoptosis. Overexpression of the IAPs, particularly the X-chromosome-linked IAP, has been shown to be protective in a variety of experimental animal models of human neurodegenerative diseases. Furthermore, overexpression of one or more of the IAPs in cancer cell lines and primary tumor samples appears to be a frequent event. IAP gene amplification and translocation events provide genetic evidence that further strengthens the case for classifying the IAPs as oncogenes. Therapeutic strategies that interfere with IAP expression or function are under investigation as an adjuvant to conventional chemotherapy- and radiation-based cancer therapy. This paper reviews the structure and function of the IAP family members and their inhibitors, and surveys the available evidence for IAP dysregulation in cancer.     

Expression of the IAP protein family is dysregulated in pancreatic cancer cells and is important for resistance to chemotherapy

Pancreatic cancer is one of the most aggressive human tumors with a 5-year survival rate of only 3% and a striking resistance to chemotherapy and radiotherapy. The search for new therapeutic approaches includes strategies exploiting the deregulation of apoptotic pathways commonly found in cancer cells. The IAP proteins are inhibitors of apoptosis that have altered activity in numerous cancer types and are implicated in resistance to chemotherapy, and therefore are potentially interesting as therapeutic targets. We investigated alterations in the expression of IAPs and their inhibitors in pancreatic adenocarcinoma by using real-time PCR, in situ hybridization and immunohistochemistry. We found differential expression of various IAPs in this malignancy, and particularly we observed overexpression of cIAP-2, survivin, livin and XIAP. We also looked for correlations between the expression of IAPs and resistance to paclitaxel, doxorubicin, CDDP and 5-fluorouracil, and found that resistance to these drugs correlates most significantly with expression of cIAP-2. Using RNAi to downregulate these proteins we further confirmed that the levels of cIAP-2 and XIAP influence the response to the anti-cancer drugs, although only marginally for 5-FU. We conclude that anti-tumor strategies based on the inhibition of particular IAPs can be useful in targeting pancreatic adenocarcinoma.     

Novel therapeutic targets in lung cancer: Inhibitor of apoptosis proteins from laboratory to clinic

Lung cancer is the leading cause of cancer death worldwide. Despite the introduction of new agents and schedules, chemotherapy still obtains unsatisfactory overall response rates, rare complete remissions and responses of relatively short duration. The inhibitor of apoptosis proteins (IAPS) are a family of caspase inhibitors that selectively bind and inhibit caspases-3, -7, and -9. As caspase activation is central to apoptosis, novel therapeutic drugs that target IAPs enabling apoptosis to occur have potential as a treatment of malignancy. Several agents that target core components of the apoptotic signalling pathway are currently at an early stage of development. This review reports the progress being made in characterising the IAP family, with a focus on the available data relevant to the treatment of lung cancer.     

Inhibitor of apoptosis proteins: new therapeutic targets in hematological cancer?

Apoptosis is an essential process for the selection and survival of lymphocytes. Resistance to apoptosis can promote malignant transformation of hematopoietic cells. Proteins that regulate apoptosis may therefore be critically involved in the development of hematological cancer. A delicate balance between pro- and antiapoptotic mechanisms determines whether a cell death signal can activate the execution of the apoptotic cell death program. The family of inhibitor of apoptosis (IAP) proteins is a recently identified, novel category of apoptosis-regulatory proteins. IAPs can inhibit the activation of caspases that are the executioners of apoptosis, activated by both the extrinsic and intrinsic pathway. IAPs may thereby set the threshold for apoptosis-activation and play a key role in the regulation of apoptotic cell death. IAPs themselves are also subject to strict regulation through feedback mechanisms. This paper focuses on the role of IAP family proteins in the regulation of apoptosis and discusses implications for their involvement in cancer and possible use for cancer therapy, especially in leukemias and lymphomas.     

Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer

Introduction

Inhibitor of apoptosis (IAPs) proteins are a family of proteins that can block apoptosis in normal cells and have been suggested to cause resistance to apoptosis in cancer. Overexpression of oncogenic receptor tyrosine kinases is common in breast cancer; in particular 20% of all cases show elevated Her2. Despite clinical success with the use of targeted therapies, such as Trastuzumab, only up to 35% of Her2-positive patients initially respond. We reasoned that IAP-mediated apoptosis resistance might contribute to this insensitivity to receptor tyrosine kinase therapy, in particular ErbB antagonists. Here we examine the levels of IAPs in breast cancer and evaluate whether targeting IAPs can enhance apoptosis in response to growth factor receptor antagonists and TRAIL.

Methods

IAP levels were examined in a breast cancer cell line panel and in patient samples. IAPs were inhibited using siRNA or cell permeable mimetics of endogenous inhibitors. Cells were then exposed to TRAIL, Trastuzumab, Lapatinib, or Gefitinib for 48 hours. Examining nuclear morphology and staining for cleaved caspase 3 was used to score apoptosis. Proliferation was examined by Ki67 staining.

Results

Four members of the IAP family, Survivin, XIAP, cIAP1 and cIAP2, were all expressed to varying extents in breast cancer cell lines or tumours. MDAMB468, BT474 and BT20 cells all expressed XIAP to varying extents. Depleting the cells of XIAP overcame the intrinsic resistance of BT20 and MDAMB468 cells to TRAIL. Moreover, siRNA-based depletion of XIAP or use of a Smac mimetic to target multiple IAPs increased apoptosis in response to the ErbB antagonists, Trastuzumab, Lapatinib or Gefitinib in Her2-overexpressing BT474 cells, or Gefitinib in EGFR-overexpressing MDAMB468 cells.

Conclusions

The novel findings of this study are that multiple IAPs are concomitantly expressed in breast cancers, and that, in combination with clinically relevant Her2 treatments, IAP antagonists promote apoptosis and reduce the cell turnover index of breast cancers. We also show that combination therapy of IAP antagonists with some pro-apoptotic agents (for example, TRAIL) enhances apoptosis of breast cancer cells. In some cases (for example, MDAMB468 cells), the enhanced apoptosis is profound.     

The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer

Apoptosis is a crucial biological process that prevents uncontrolled cell proliferation and eliminates harmful cells. Resistance to apoptotic stimuli is a hallmark feature of various cancers. One of the mechanisms through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of inhibitor of apoptosis proteins (IAPs). IAPs are a group of structurally related proteins that were initially identified in baculoviruses. Mammalian IAPs block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms. This family of proteins has become increasingly prominent in the field of cancer biology. To date, overexpression of several IAPs has been detected in various cancers. This paper reviews the recent advances in the research of IAPs. The differential expression and the biological significance of each IAP in various cancer types will be discussed. Finally, we review the most recent advances in the research efforts aimed at using IAPs as potential targets for cancer therapy.     

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.     

Intracisternal A particle genes: Distribution in the mouse genome,active subtypes,and potential roles as species‐specific mediators of susceptibility to cancer

Rodents, mice and rats in particular, are the species of choice for evaluating chemical carcinogenesis. However, different species and strains often respond very differently, undermining the logic of extrapolation of animal results to humans and complicating risk assessment. Intracisternal A particles (IAPs), endogenous retroviral sequences, are an important class of transposable elements that induce genomic mutations and cell transformation by disrupting gene expression. Several lines of evidence support a role of IAPs as mouse‐specific genetic factors in responses to toxicity and expression of disease phenotypes. Since multiple subtypes and copies of IAPs are present in the mouse genome, their activity and locations relative to functional genes are of critical importance. This study identified the major “active” subtypes of IAPs (subtype 1/1a) that are responsible for newly transposed IAP insertions described in the literature, and confirmed that (1) polymorphisms for IAP insertions exist among different mouse strains and (2) promoter activity of the LTRs can be modulated by chemicals. This study further identified all the genes in the C57BL/6 mouse genome with IAP subtype 1 and 1a sequences inserted in their proximity, and the major biofunctional categories and cellular signaling networks of those genes. Since many “IAP‐associated genes” play important roles in the regulation of cell proliferation, cell cycle, and cell death, the associated IAPs, upon activation, can affect cellular responses to xenobiotics and disease processes, especially carcinogenesis. This systemic analysis provides a solid foundation for further investigations of the role of IAPs as species‐ and strain‐specific disease susceptibility factors. © 2009 Wiley‐Liss, Inc.     

Targeting inhibitor of apoptosis proteins in combination with dacarbazine or TRAIL in melanoma cells

Melanoma is a highly aggressive malignant tumor with an exceptional ability to develop resistance and no curative therapy is available for patients with distant metastatic disease. The inhibitor of apoptosis protein (IAP) family has been related to therapy resistance in cancer. We examined the importance of the IAPs in the resistance to the commonly used chemotherapeutic agent dacarbazine (DTIC) and the apoptosis inducer TRAIL (TNF-related apoptosis inducing ligand) in malignant melanoma. The data presented show that the expression of IAPs is universal, concomitant and generally high in melanoma cell lines and in patient samples. Depleting IAP expression by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient in all four cell lines examined (FEMX-1, LOX, SKMEL-28 and WM115). The combined treatment of XIAP siRNA and DTIC showed a weak improvement in two of four cell lines, while all four cell lines showed enhanced sensitivity towards TRAIL (AdhCMV-TRAIL) after XIAP depletion. In addition, cIAP-1, cIAP-2 and survivin down-regulation sensitized to TRAIL treatment in several of the cell lines. Cells exposed to TRAIL and XIAP siRNA showed increased DNA-fragmentation and cleavage of Bid, procaspase-8, -9, -7 and -3 and PARP, and change in the balance between pro- and anti-apoptotic proteins, indicating an enhanced level of apoptosis. Furthermore, the combined treatment reduced the ability of melanoma cells to engraft and form tumors in mice, actualizing the combination for future therapy of malignant melanoma.     

Smac mimetic reverses resistance to TRAIL and chemotherapy in human urothelial cancer cells

Purpose

Inhibitors of apoptosis proteins (IAP s) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy.

Results

Single-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis.

Experimental design

A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting.

Conclusion

Our results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.Key words: bladder cancer, smac mimetic, IAP antagonist, chemotherapy, TRAIL     

Exploiting inhibitor of apoptosis proteins as therapeutic targets in hematological malignancies

Resistance to apoptosis is one of the hallmarks of human cancers and contributes to the insensitivity of many cancers to commonly used treatment approaches. Inhibitor of apoptosis (IAP) proteins, a family of anti-apoptotic proteins, have an important role in evasion of apoptosis, as they can both block apoptosis-signaling pathways and promote survival. High expression of IAP proteins is observed in multiple cancers, including hematological malignancies, and has been associated with unfavorable prognosis and poor patients' outcome. Therefore, IAP proteins are currently considered as promising molecular targets for therapy. Indeed, drug-discovery approaches over the last decade aiming at neutralizing IAP proteins have resulted in the generation of small-molecule inhibitors or antisense oligonucleotides that demonstrated in vitro and in vivo antitumor activities in preclinical studies. As some of these strategies have already entered the stage of clinical evaluation, for example, in leukemia, an update on this promising molecular-targeted strategy to interfere with apoptotic pathways is of broad interest.     

Inhibitor of apoptosis proteins as targets for anticancer therapy

Cell death by apoptosis plays a critical role in regulating the subtle balance between cell death and proliferation to maintain tissue homeostasis. Accordingly, tipping the balance in either direction may cause human disease. Too little cell death may promote tumor formation and progression. In addition, killing of cancer cells by current therapies is largely due to induction of apoptosis in tumor cells. Since a hallmark of human cancers is their resistance to apoptosis, there is a demand to develop novel strategies that restore the apoptotic machinery in order to overcome cancer resistance. Inhibitor of apoptosis proteins (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting caspases. Elevated levels of IAPs are found in many human cancers and have been associated with poor prognosis. Recent insights into the role of IAPs have provided the basis for various exciting developments that aim to modulate the expression or function of IAPs in human cancers. Targeting IAPs (e.g., by antisense approaches or small-molecule inhibitors) presents a promising novel approach to either directly trigger apoptosis or to potentiate the efficacy of cytotoxic therapies in cancer cells. Thus, inhibition of IAPs such as X chromosome-linked IAP may prove to be a successful strategy to overcome apoptosis resistance of human cancers that deserves further exploitation.     

Inhibitor of apoptosis proteins as targets for anticancer therapy

Cell death by apoptosis plays a critical role in regulating the subtle balance between cell death and proliferation to maintain tissue homeostasis. Accordingly, tipping the balance in either direction may cause human disease. Too little cell death may promote tumor formation and progression. In addition, killing of cancer cells by current therapies is largely due to induction of apoptosis in tumor cells. Since a hallmark of human cancers is their resistance to apoptosis, there is a demand to develop novel strategies that restore the apoptotic machinery in order to overcome cancer resistance. Inhibitor of apoptosis proteins (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting caspases. Elevated levels of IAPs are found in many human cancers and have been associated with poor prognosis. Recent insights into the role of IAPs have provided the basis for various exciting developments that aim to modulate the expression or function of IAPs in human cancers. Targeting IAPs (e.g., by antisense approaches or small-molecule inhibitors) presents a promising novel approach to either directly trigger apoptosis or to potentiate the efficacy of cytotoxic therapies in cancer cells. Thus, inhibition of IAPs such as X chromosome-linked IAP may prove to be a successful strategy to overcome apoptosis resistance of human cancers that deserves further exploitation.     

Cladribine: from the bench to the bedside – focus on hairy cell leukemia

Cladribine is an adenosine deaminase-resistant deoxyadenosine analog with a unique mechanism of action. It is directly toxic towards both resting and proliferating human lymphocytes and monocytes. The development of this compound into clinical practice best exemplifies rational drug development and the success of translational research. Cladribine is well established as first-line treatment for hairy cell leukemia, inducing an overall response rate of 75–100%, with the majority being complete responses following only a single 7-day treatment course. The 9-year overall survival for hairy cell leukemia is currently 97%. Cladribine has also proven useful in the treatment of a variety of other hematologic malignancies, and its current place in the oncologic therapeutic armamentarium, as well as in potential areas of development, are outlined.     

Cladribine: from the bench to the bedside--focus on hairy cell leukemia

Cladribine is an adenosine deaminase-resistant deoxyadenosine analog with a unique mechanism of action. It is directly toxic towards both resting and proliferating human lymphocytes and monocytes. The development of this compound into clinical practice best exemplifies rational drug development and the success of translational research. Cladribine is well established as first-line treatment for hairy cell leukemia, inducing an overall response rate of 75-100%, with the majority being complete responses following only a single 7-day treatment course. The 9-year overall survival for hairy cell leukemia is currently 97%. Cladribine has also proven useful in the treatment of a variety of other hematologic malignancies, and its current place in the oncologic therapeutic armamentarium, as well as in potential areas of development, are outlined.     

The inhibitor of apoptosis proteins as therapeutic targets in cancer.

Apoptosis is a cell suicide process with a major role in development and homeostasis in vertebrates and invertebrates. Inhibition of apoptosis enhances the survival of cancer cells and facilitates their escape from immune surveillance and cytotoxic therapies. Among the principal molecules contributing to this phenomenon are the inhibitor of apoptosis (IAP) proteins, a family of antiapoptotic regulators that block cell death in response to diverse stimuli through interactions with inducers and effectors of apoptosis. IAP proteins are expressed in the majority of human malignancies at elevated levels and play an active role in promoting tumor maintenance through the inhibition of cellular death and participation in signaling pathways associated with malignancies. Here, we discuss the role of IAP proteins in cancer and options for targeting IAP proteins for therapeutic intervention.     

The epidermal growth factor receptor as a target for gastrointestinal cancer therapy

Opinion statement The epidermal growth factor receptor (EGFR) is a member of the family of transmem-brane protein kinase receptors known as the erbB or HER receptor family. When activated, EGFR phosphorylates and activates other intracellular proteins that affect cell signaling pathways, cellular proliferation, control of apoptosis and angiogenesis. EGFR signaling is best thought of as a network of activating and inactivating proteins with EGFR as the entry point into the network. EGFR overexpression occurs in most GI malignancies and while data are not entirely consistent, EGFR overexpression often confers a poor prognosis in those GI malignancies that have been studied. It often correlates with poorly differentiated histology, more advanced stage and other known poor prognostic markers. The EGFR is a tempting target because of its presence and overexpression on so many tumor types. However, downstream of the EGFR are several proteins that may be activated without EGFR thus allowing blockade to be overcome. Therefore, while blocking the activity of the EGFR protein appears to be a promising anticancer strategy, a simplistic strategy of blocking only EGFR is likely to only impact a minority of patients. It is time for the laboratory and clinical researchers to work closely together to develop this treatment strategy, moving back and forth from clini-cal to laboratory to best understand how to block this network effectively enough to produce a broader antitumor effect. While multiple methods of targeting the EGFR pathway are under development, including the inhibition of downstream proteins, only two modalities have entered clinical trials in GI malignancies: small molecule inhibitors of the intracellular kinase domain of EGFR and antibodies designed to block the extracellular ligand-binding domain of EGFR. EGFR inhibitors are still experimental in every GI malignancy with the notable exception of cetuximab that is approved for second or third-line therapy of metastatic colorectal cancer, used either alone or in combination with irinotecan (Camptosar, Kalamazoo, Mich). Data on clinical applications of these agents in GI malignancies will be the focus of this paper.