(Author)
Regulation of Xiap Protein in Breast Cancer
Breast Cancer
Breast cancer is the type of cancer that develops in the tissues of the breast. Two most commonly reported types of breast cancer are “ductal carcinoma” and “lobular carcinoma”. Ductal carcinoma is initiated in the lining of the milk ducts, which are thin tubes involved in carrying of milk from lobules of the breast to the nipple. Ductal carcinoma can be in the form of Invasive ductal carcinoma (IDC) as well as Ductal carcinoma in situ (DCIS). IDC is the most commonly found type of breast cancer with nearly 80% of all cases of breast cancers. DCIS is among the most common type of non-invasive breast cancer. On the other hand, lobular carcinoma is the form of breast cancer that develops in the lobules or milk glands of the breast. Inflammatory breast cancer (IBC) is another type of uncommon yet extremely hostile form of breast cancer. IBC constitutes about 1-5% of all breast cancer cases in the U.S. Breast cancer can spread from ducts or lobules of the breast to other surrounding areas of the body. It can develop in both men and women; however chances of breast cancer development in men are rare. Obesity, consumption of alcohol, and decreased physical activity are some of the factors behind breast cancer development. Early recognition of breast cancer can increase the five-year survival rate by about 90%.
In the U.S., nearly 232,670 new cases of breast cancer were found in females and 2,360 new cases have been reported in males in the year 2014. Nearly 40,000 deaths have been found in females, and 430 deaths have been reported in males in the U.S. in 2014 (National Cancer Institute, cancer.gov).
X-Linked Inhibitor Of Apoptosis Protein (XIAP)
XIAP gene is involved in making proteins that are found in many cells of the body including immune cells, and is located on the X chromosome. This protein is the member of the inhibitor of apoptosis (IAP) family of proteins. XIAP protein is also known as baculoviral IAP repeat-containing protein 4 (BIRC) and inhibitor of apoptosis protein 3 (IAP3). It is also referred to as human IAP-like Protein (hILP) as it is slightly different from the human IAPs. This protein is usually localized in the cytoplasm of cells, but few studies are also showing its localization in the nucleus (Aird 7).
Initially, IAPs were found in baculoviruses, but XIAP is among those homologous proteins which were discovered in mammals. This protein is found to have three baculoviral IAP repeat (BIR) domains followed by one UBA domain and a RING finger. BIR domains are found to be cysteine- and histidine-rich domains. UBA domain is helpful in binding of XIAP protein to ubiquitin. RING domain is composed of E3 ubiquitin ligase that is important in ubiquitination of other proteins and autoubiquitination, i.e. self-ubiquitination. XIAP has transcript size of about 9.0kb having 1.8kb of open reading window.
XIAP protein helps in protecting cells from the intrinsic and extrinsic pathways of apoptosis, which is the self-destructing phenomenon found in cells. It works by stopping the action of enzymes known as caspases having an important role in apoptosis. It has to be noted that disturbance in the process of apoptosis is commonly found in almost all types of cancers. XIAP is found to have significant ability to act on caspases that is why it is also referred to as the most potent caspase inhibitor known to scientists. Viral infection is also involved in the process of apoptosis, and XIAP protein also helps in this case. XIAP protein also has other important functions in the body as for example it has an important role in the development of cancer chemoresistance. It is also a potent pro-survival signaling molecule.
XIAP is found to be involved in malignant effects of various cancer cells particularly cell invasion, angiogenesis, proliferation, and chemoresistance. Researchers have found that increased XIAP expression has a strong role with malignancy but the molecular mechanisms behind the XIAP accumulation in various malignant cells of cancer need further studies. The ability of XIAP to stop caspases is considered to have an important role in chemoresistance in breast cancer. This ability is also responsible for the adverse prognostic factor.
Particularly, the XIAP protein acts on caspase enzymes 3, 7, and 9, and inhibits them. The BIR2 domain of XIAP helps in the inhibition of caspase 3 and 7 whereas BIR3 domain binds to and helps in the inhibition of caspase 9. After inhibiting the caspase-3 and caspase-7 activity, the BIR2 domain of XIAP attaches to the active-site substrate groove, inhibiting the access of the normal protein substrate that can result in apoptosis. The RING domain uses E3 ubiquitin ligase activity and helps IAPs to catalyze the process of ubiquitination of self, which is the process of addition of ubiquitin protein to the substrate protein. BIR domain is found to be sufficient in performing the protein’s function as mutations affecting the RING Finger are not found to have a significant affect on the process of apoptosis.
It has been reported that the stabilization of XIAP elevates its half-life (Aird 16). XIAP is found to be stabilized through different mechanisms including phosphorylation and complex formation.
Regulation Of XIAP In The Human Body
XIAP protein is found to be commonly expressed in most normal cells of the body. It is involved in binding and inhibiting caspases, and ubiquitinating other proteins that is why it is thought to be an important anti-apoptotic and pro-survival protein. In the body, XIAP can be regulated transcriptionally, translationally, and posttranslationally.
Regulation of XIAP at Transcriptional level. Researchers have found that a number of transcription factors are involved in the regulation of XIAP at transcriptional level. These factors can positively or negatively regulate XIAP proteins as, for example, nuclear factor kappa B (NFκB), STAT5, and Sp1 positively regulate XIAP, and p53 negatively regulates XIAP (Aird 10).
Working of XIAP with NFκB. XIAP can be regulated by NFκB (Aird 1). In fact, it is one of the most widely studied regulatory processes of XIAP. NFκB is a protein complex that is involved in the transcription of DNA. It is found in almost all types of animal cells and have an important role in different stimuli such as stress, ultraviolet radiation, free radicals, and antigens of bacteria and viruses. NFκB is also found to be highly upregulated in IBC. Activation of NFκB results in elevation of XIAP transcription that are found to be involved in the resistance of cells to various therapeutic agents such as paclitaxel, doxorubicin, and cisplatin.
Regulation of XIAP at Translational level. XIAP can also be regulated at the translational level. Researchers have found that XIAP has an extraordinarily long 5’ UTR (1.7 kb) having an internal ribosomal entry site (IRES). IRES is involved in the beginning of translation in the middle of a messenger RNA (mRNA) sequence as a part of the process of protein synthesis. IRES elements are usually considered as viral elements but some of the cellular RNAs, which are important for the survival of cells, have also been found to have two distinct 5’ UTRs (one long and one short). Researchers have found that XIAP can be translated during the times of cellular stress such as those caused by irradiation, serum starvation, and therapeutic strategies. This translation is helpful in overcoming the problem of apoptosis usually produced by stress causing agents. In this case, XIAP IRES is found to be actively translated in the times of cellular stress (Aird 13).
Researchers have found only a few RNA binding proteins to be involved in positive or negative regulation of XIAP IRES (Aird 13). La autoantigen – an RNA binding protein that is found to be attached to XIAP IRES – is cleaved under the process of apoptosis. This protein is found in abundance in the cytoplasm, where it attaches to XIAP IRES and helps in the XIAP translation to stop cell death. Researchers have found that decreased interaction of La autoantigen and XIAP IRES results in decreased XIAP IRES activity.
Heterogeneous nuclear ribonucleoprotein C1 and C2 (hnRNPC1/2), RNA binding proteins, are also found to be accumulated in the cytoplasm in pre-apoptotic cells. Here, they attach to the XIAP IRES to overcome the problem of death signals. Researchers have found that hnRNPC1/2 expression is increased in ischemic brains resulting in elevated XIAP expression. hnRNPC1/2 can be induced during cellular stress that can result in XIAP IRES-mediated translation and therapeutic resistance of cancer cells.
Murine double minute 2 homolog (MDM2) also known as E3 ubiquitin-protein ligase Mdm2, a protein that is commonly overexpressed in cancers and can destabilize p53, has also been found to positively regulate the IRES initiated translation of XIAP. Usually, MDM2 is dephosphorylated by the cyclin G1-PP2A complex – a complex made by a combination of cyclin G1 and protein phosphatase 2A – during periods of cellular stress. This dephosphorylated form stays in the cytoplasm and interacts with the XIAP IRES resulting in resistance to therapeutic agents.
La autoantigen, MDM2, and hnRNPC1/2 are found to act as positive regulators of XIAP IRES; however, Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an RNA binding protein, is found to negatively regulate XIAP IRES mediated translation. This protein also localizes in the cytoplasm during the times of cellular stress and attaches to the core RNP binding site; however, it does not compete with La autoantigen for binding, showing that there is different mechanism behind its regulation of XIAP IRES (Aird 15).
Regulation of XIAP at Post-Translational level.XIAP is also regulated post-translationally. Researchers have found that most of the mechanisms behind the post-translational regulation result in the reduction of autoubiquitination and subsequent degradation of the XIAP protein via the proteasome (Aird 16). Phosphorylation is found to be one of the most important mechanisms behind XIAP autoubiquitination. XIAP can be phosphorylated at serine 87 by Akt1 and Akt2, and/or Raf-1. Process of phosphorylation is found to decrease the autoubiquitination of XIAP in the presence of various apoptotic stimulators resulting in reduced drug-induced apoptosis.
Among the factors that can increase the stability of XIAP are Protein kinase B (PKB, also known as Akt), Protein kinase C (PKC), and notch proteins. The mechanism behind the regulation of XIAP stability through PKC needs further investigation. In one research, it has been reported that phorbol-12-myristate-13-acetate (PMA), which is a potent activator of PKC, stops ubiquitination caused by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) treatment; however, further investigations are required on the direct attachment of PKC to XIAP and its phosphorylation to stop its ubiquitination. However, it is clear that PKC-mediated stabilization of XIAP leads to drug resistance.
Notch-1 is found to increase the stability of the XIAP protein. Researchers have reported that half-life of the XIAP protein increases by about four times as a result of overexpression of Notch-1. Direct interaction has been found between the transcriptional activation domain (TAD domain) of Notch-1 and the RING domain of XIAP that reduces the binding accessibility of E2 ligases resulting in stabilization of the XIAP protein. Notch expression can also modulate the expression of survivin, which is the member of the IAP family, in an aggressive subset of breast cancer cells. Survivin is also thought to form complex with XIAP that is also involved in chemoresistance of these cells. Survivin degradation is also found to be related to XIAP destabilization that can also result in increased apoptosis.
Working of XIAP with Akt. Akt is found to be highly active in many types of cancers. Stabilization of XIAP through Akt could be a potent anti-apoptotic mechanism. Researchers have also reported that many signaling pathways can modify the stability of XIAP as, for example, it has been reported that overexpression of cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins (cFLIPs) can increase XIAP stability through Akt pathway resulting in the resistance of cells to TRAIL-induced apoptosis. This interaction of Akt and XIAP is also responsible for influencing cancer cells to resist apoptotic-inducing effects of therapeutic agents (Aird 16).
Researchers have reported that XIAP can also influence the stimulation of Akt. Overexpression of XIAP can also result in elevated phosphorylated Akt. Moreover, reduced XIAP expression can result in decreased phospho-Akt (p-Akt) resulting in Akt cleavage in some cancer cells such as ovarian cancer cells. XIAP siRNA can result in a significant reduction of p-AKT expression in SUM190 cell lines of IBC cells, but the mechanism behind the XIAP regulation of AKT activation needs further studies (Aird 19).
Constitutive expression of XIAP is related to attaching and stopping the terminal effects in apoptotic processes, and regulating the survival of cells through different pathways such as Phosphoinositide 3-kinase (PI3-K)/Akt pathway that is involved in the stimulation of Akt by phosphorylation at ser473 in the C-terminal activation domain. On the other hand, phosphorylation of XIAP as a result of Akt is found to save XIAP from the process of ubiquitination and destruction in cancer cells. Recently, researchers have reported that a complex regulatory system can work between XIAP and the Akt signaling pathway through feedback mechanism. The knocking down of XIAP is related to the stimulation of caspase-9 through Akt signal transduction in breast cancer cells (Rajput, plosone.org ).
Role of XIAP in Breast Cancer
XIAP protein is usually upregulated in cancer cells such as breast cancer. This upregulation is also found to be responsible for the development of resistance of cells to various therapeutic agents. Researchers have found that increased transcription of XIAP is among the most important processes behind the increased therapeutic resistance of cancer cells (Aird 10).
Role Of XIAP In Therapeutic Resistance Of IBC Cells. In IBC, ErbB2 overexpression and activation is commonly found. ERbB2 gene is involved in the production of growth factor receptors, and growth factors are involved in stimulating the cell growth and cell division. However, problem of undergoing the process of apoptosis can result in therapeutic resistance, and this problem is commonly found in patients of IBC undergoing therapy with ErbB-targeting agents.
Researchers have found that XIAP expression is inversely related to the sensitivity of cells to therapeutic agents. Several mechanisms are involved in this process that are related to different therapeutic agents such as TRAIL: cisplatin, doxorubicin, and paclitaxel, and two ErbB-targeting agents, a lapatinib-analog (GW583340) and trastuzumab.
Marked and specific overexpression of XIAP in cells can be involved in resistance to trastuzumab and GW583340. This overexpression is found to be caused by IRES-mediated XIAP translation. Stable type of XIAP overexpression utilizing a lentiviral system has the ability to reverse the sensitivity to therapeutic agents in the IBC cells. Furthermore, XIAP down-regulation in cells, which are resistant to therapeutic agents can cause reduced viability and elevated apoptosis, showing that XIAP is actively involved in survival of cells which are resistant to some therapeutic agents such as trastuzumab and GW583340.
Resistant cells are also found to have an increased antioxidant expression and capability. Researchers have found that inhibition of XIAP function can overcome this increase in antioxidant potential, showing that XIAP is also involved in oxidative stress-induced apoptosis (Aird v) in cancers.
Conclusion and Future Directions
XIAP is an important protein in the inhibition of caspases. It helps in reduction of apoptosis. Although XIAP has been proposed to have a role in the cell cycle regulatory functions due to its nuclear localization but still it needs further studies (Aird 7).
Various mechanisms behind its working and its interaction with other proteins require further studies. Those studies could help in the development of optimum therapeutic strategies against breast cancer. Researchers are also of the opinion that the development of XIAP inhibitors can help in the development of agents that can overcome the problem of resistance of cells against therapy (Aird v).
XIAP can also be used as a biomarker of response to various therapeutic agents in breast cancers. Studies have shown its role in sensitivity or resistance of breast cancer cells to different therapeutic agents having different mechanisms of action. This shows that checking the levels of XIAP after treatment can help in confirming the efficacy of treatment. If the levels of XIAP in patients are not affected, they should be moved to different therapeutic strategy.
Regulation of XIAP as a result of STAT5, Sp1, and p53 need further investigations. More than a decade has been passed that scientists are aware of XIAP IRES but still the activity of XIAP IRES needs further research. Moreover, the mechanism behind the XIAP IRES upregulation during the times of cellular stress and regulation of the XIAP IRES in the context of dying cells require further investigations (Aird 13). To understand the mechanism behind the regulation of XIAP IRES with the help of RNA binding proteins further research is also required.
List Of Abbreviations
Akt - Protein kinase B
BIR - Baculoviral IAP repeat
BIRC - Baculoviral IAP repeat-containing protein 4
cFLIPs - cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins
DCIS - Ductal carcinoma in situ
FLICE - Cellular Fas-associated death domain-like interleukin-1-beta converting enzyme
hILP - Human IAP-like Protein
hnRNPC1/2 - Heterogeneous nuclear ribonucleoprotein C1 and C2
IAP - Inhibitor of apoptosis family of proteins
IAP3 - Inhibitor of apoptosis protein 3
IBC - Inflammatory breast cancer
IDC - Invasive ductal carcinoma
IRES - Internal ribosomal entry site
MDM2 – Mouse/Murine double minute 2 homolog
p-Akt - phospho-Akt
PI3-K - Phosphoinositide 3-kinase
PKB - Protein kinase B
PKC - Protein kinase C
PMA - Phorbol-12-myristate-13-acetate
PP2A - Protein phosphatase 2A
TAD - Transcriptional activation domain
Works Cited
Aird, Katherine Marie. Role of X-linked inhibitor of apoptosis protein in therapeutic resistance of inflammatory breast cancer cells. Diss. DUKE UNIVERSITY, 2010.
National Cancer Institute. “Breast Cancer”. National Institutes of Health. National Cancer Institute, 2014. Web. 02 Nov. 2014.
Rajput, Shashi, et al. "Targeted apoptotic effects of thymoquinone and tamoxifen on XIAP mediated Akt regulation in breast cancer." PloS one 8.4 (2013): e61342.
