Expression and prognostic significance of the inhibitor of apoptosis protein (IAP) family and its antagonists in chronic lymphocytic leukaemia

Abstract

Impaired apoptosis is still considered to be an important event in the development and progression of chronic lymphocytic leukaemia (CLL). However, mechanisms of this defect have not been fully elucidated. In this study, expression of inhibitor of apoptosis proteins, IAPs (cIAP1, cIAP2, XIAP and survivin), and their antagonists (Smac/DIABLO and HtrA2/Omi) was comprehensively analysed in 100 untreated CLL patients, using flow cytometry and Western blot techniques.

Expression of anti-apoptotic cIAP1 and cIAP2 in leukaemic cells was significantly higher than in non-tumour lymphocytes (p = 0.000001 and p = 0.014, respectively), whereas the IAP-antagonist, Smac/DIABLO, was decreased in CLL (p = 0.010). Higher expression of all analysed IAPs (cIAP1, p = 0.002; cIAP2, p = 0.026; XIAP, p = 0.002; survivin, p = 0.00006) and lower levels of Smac/DIABLO (p = 0.006) were found in patients with progressive disease, compared to those with stable CLL. High baseline expression of cIAP1 and survivin correlated with worse response to treatment. Co-expression of these proteins was associated with shorter overall survival of CLL patients (p = 0.005).

In conclusion, CLL cells show the apoptosis-resistant profile of IAPs/IAP-antagonist expression. Upregulation of IAPs is associated with a progressive course of the disease. Co-expression of cIAP1 and survivin seems to be an unfavourable prognostic factor in CLL patients. Further studies with longer follow up are warranted to confirm and expand these findings.

Introduction

Chronic lymphocytic leukaemia (CLL) is a heterogenous disease with a highly variable clinical course.¹ Although CLL is the most common leukaemia in western countries, its biology is still poorly understood. CLL is characterised by the relentless accumulation of monoclonal mature CD5⁺ B cells in lymphoid organs, bone marrow, and peripheral blood. Virtually all circulating CLL lymphocytes are long-lived cells arrested in the G₀/early G₁ phase of the cell cycle.² The progressive expansion of lymphocytes has led to the notion that defective apoptosis rather than increased cell proliferation is the primary cause of CLL. The microenvironment likely plays a prominent role because the malignant cells progressively accumulate in vivo, whereas they rapidly undergo apoptosis when cultured in vitro.³

Apoptosis, defined as programmed cell death, is executed through the activity of caspases, cysteine proteases which are regulated by a number of pro- and anti-apoptotic proteins. Bcl-2 family proteins are major regulators of mitochondria-dependent apoptosis.⁴ Several of them were found to be overexpressed in CLL⁵ which is associated with resistance to apoptosis, poor response to chemotherapy (alkylating agents or purine analogues), and advanced stages of the disease.6, 7 Furthermore, increased Bcl-2/Bax rates have been correlated with refractory disease, its progression, and shorter survival.⁸ Other important apoptosis-regulating proteins include the p53 protein family.⁹ Mutations or deletions of the p53 gene are known to be altered in a number of haematological malignancies, including progressive CLL.¹⁰

An essential checkpoint in apoptosis regulation is the control of caspase activation by the more recently discovered family of inhibitor of apoptosis proteins (IAPs). IAPs are the group of proteins which block both the mitochondrial-dependent and independent pathways of apoptosis. They also participate in the regulation of the cell cycle and intracellular signal transduction.11, 12 The family consists of eight proteins containing baculoviral IAP repeat (BIR) domains which allow them to bind to caspases, inhibiting these enzymes. They are: cIAP1 and cIAP2 (cellular inhibitors of apoptosis 1 and 2), XIAP (X-chromosome binding IAP), ILP-2 (IAP–like protein 2), livin, NAIP (neuronal apoptosis inhibitory protein), survivin and BRUCE (Apollon).13, 14, 15, 16, 17 The majority of IAP activities are dependent on the BIR domain. Among the mammalian IAPs, c-IAP1, cIAP2, and XIAP have three BIRs in the N-terminal portion of the molecule and a RING finger at the C-terminus, NAIP contains three BIRs without RING, survivin and BRUCE each have just one BIR.11, 12, 13 The BIR2 domain is capable of binding and inhibiting caspase-3 and -7, whereas BIR3 is an inhibitory segment for caspase-9. BIR1 does not display the inhibitory ability of caspase activation. BIR domains are also associated with the binding of cIAP1 and cIAP2 to TNF receptor associated factors (TRAF1 and TRAF2), the XIAP interaction with TAB1 protein and, as a consequence, with the activation of the nuclear factor κB (NF-κB) pathway. Additionally, the RING domain allows some proteins (XIAP, c-IAP1, c-IAP2 and livin) to act as E3 ubiquitin ligases. The E3 ubiquitin ligase activity of the IAPs is capable of promoting ubiquitination and proteasomal degradation of caspases, TRAF2 and several other partners. Furthermore, both cIAP1 and cIAP2 additionally contain the caspase recruitment domain (CARD) located between the BIR and RING domains. The functional significance of this domain for the anti-apoptotic function of IAPs is largely untested, but amino-terminal fragments of human c-IAP1 and c-IAP2 that retain only the BIR domains are sufficient to block apoptosis, implying that the CARD domain is not absolutely required. Other domains of potential interest in the IAP family include a functionally intact ubiquitin-conjugating (UBC) domain in BRUCE. This protein could conceivably provide a functional connection between apoptosis proteins and the ubiquitin proteasome pathway for protein degradation. The NAIP protein contains a P-loop consensus sequence similar to some ATP/GTP-binding proteins.12, 13, 14, 15, 16, 17 The structure of cIAP1, cIAP2 and XIAP allows them to inhibit caspase-3, -7 and -9 and ubiquitinate caspase-3, -7 and the IAP-antagonist, Smac/DIABLO. Additionally, they indirectly inhibit apoptosis through NF-κB activation. Most probably, survivin directly inhibits caspase-9 through the BIR domain and, indirectly, caspase-3 through p21Cip/WAF1. The other potential mechanism of survivin anti-apoptotic activity is the inhibition of apoptosis-inducing factor (AIF) released from the mitochondria (reviewed in 14). More recent reports brought a new insight into the activity of IAPs, including action through the external pathway of caspase activation.

The function of IAPs is negatively regulated by several proteins. Currently, three proteins are known to bind to different IAPs and inhibit their activity: Smac/DIABLO (second mitochondrial derived activator of caspase/direct IAP binding protein with low pI), HtrA2/Omi (high temperature requirement A) and XAF1 (XIAP-associated factor 1).18, 19, 20 HtrA2/Omi and Smac/DIABLO are localised within the mitochondria and are released into the cytosol upon apoptotic stimuli. Cytosolic HtrA2/Omi results in displacement of XIAP from caspases and loss of their suppressive effect on caspase activity. Moreover, HtrA2/Omi was shown to be able to degrade XIAP protein. It is suggested that HtrA2/Omi may also be responsible for the proteolytic degradation of XIAP, cIAP1 and cIAP2. Smac/DIABLO is also an important molecule that regulates the function of IAPs. It can bind to the BIR domain of IAPs (XIAP, cIAP1, cIAP2, survivin, livin, and BRUCE), thereby interfering with either caspase-3/-7 or caspase-9 inhibition. XAF1 resides in the nucleus and can affect a relocation of the XIAP protein from the cytoplasm to the nucleus. It has been reported that XAF1 is mainly expressed in normal tissues but is missing or present at low levels in most cancer cell lines, which implies a tumour-suppressing function.18, 19, 20

There is growing evidence that IAP proteins can be involved in the pathogenesis of human malignancies.21, 22, 23 Overexpression of IAPs was found in a wide variety of cancer cell lines and primary tumour biopsy samples, including primary acute leukaemias and B-cell lymphomas.24, 25, 26, 27, 28

Thus far, there are only a few clinical observations concerning IAP expression in CLL.29, 30, 31, 32 To date, there are no reports regarding the potential prognostic significance of these proteins and their antagonists in the disease. Therefore, the primary aim of this study was to perform a complex analysis of the IAP family members and their antagonists’ content in leukaemic cells in vivo, in untreated CLL patients in comparison to healthy controls. Additionally, we aimed to assess possible differences in expression of these proteins between patients with stable and progressive CLL. Finally, we also tried to establish a potential prognostic significance of the IAP expression.