Role Of X-linked Inhibitor Of Apoptosis Research Articles

XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis

BackgroundBreast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women. Despite improvement in diagnosis, the survival rate of this disease has still not improved. X-linked Inhibitor of Apoptosis (XIAP) has been shown to be over-expressed in various cancers leading to poor overall survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored.MethodsWe examined the expression of XIAP in more than 1000 Middle Eastern breast cancer cases by immunohistochemistry. Apoptosis was measured by flow cytometry. Protein expression was determined by western blotting. Finally, in vivo studies were performed on nude mice following xenografting and treatment with inhibitors.ResultsXIAP was found to be over-expressed in 29.5% of cases and directly associated with clinical parameters such as tumor size, extra nodal extension, triple negative breast cancer and poorly differentiated breast cancer subtype. In addition, XIAP over-expression was also significantly associated with PI3-kinase pathway protein; p-AKT, proliferative marker; Ki-67 and anti-apoptotic marker; PARP. XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; embelin and found that embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. Finally, breast cancer cells treated with combination of embelin and PI3-kinase inhibitor; LY294002 synergistically induced apoptosis and caused tumor growth regression in vivo.ConclusionThese data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells.

Abstract 17: Xiap over-expression is a poor prognostic marker in breast cancer and can be targeted to induce efficient apoptosis

Abstract Breast cancer is the most common cancer in females and is ranked only second to lung cancer in cancer-related deaths all over the world in women. Despite improvement in diagnosis and management of breast cancer, the survival rate of this disease for stage 4 disease is still only 22%. X-linked Inhibitor of Apoptosis (XIAP) is a member of the inhibitor of apoptosis family of proteins that has been shown to be over-expressed in a variety of cancers including breast cancer leading to an overall poor survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored. We therefore examined the expression of XIAP in a cohort of 964 breast cancer cases by immunohistochemistry and found that XIAP was over-expressed in 29.5% (284/964) of the cases and was found to be directly associated with clinical parameters such as tumor size (p = 0.0044), extra nodal extension (p = 0.0041) and poorly differentiated breast cancer (p<0.0001). In addition, XIAP over-expression was also significantly associated with activated AKT (p<0.0001), Ki-67 (p<0.0001) and PARP (p<0.0001). Finally, XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; Embelin on a panel of breast cancer cell lines and found that Embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. In order to identify the exact pathway involved in XIAP inhibition, we treated breast cancer cells with different doses of Embelin and found that there was down-regulation of XIAP leading to activation of caspases-9, -3 and PARP. We also found that Embelin treatment caused cleavage of caspase-8 and truncation of Bid leading to activation of the mitochondrial apoptotic pathway and efficient apoptosis. Finally, as our clinical data demonstrated a significant association between XIAP over-expression and activation of AKT in breast cancer samples, we synergistically induced apoptosis in breast cancer cells using sub-optimal doses of Embelin and PI3-kinase inhibitor; LY294002. These data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and XIAP can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells. Citation Format: Azhar R. Hussain, Maqbool Ahmed, Rong Bu, Shaham Beg, Alanood M. Alrashed, Roxanne Melosantos, Dahish S. Ajarim, Shahab Uddin, Khawla S. AlKuraya. Xiap over-expression is a poor prognostic marker in breast cancer and can be targeted to induce efficient apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 17. doi:10.1158/1538-7445.AM2015-17

X-Linked Inhibitor of Apoptosis Regulates Lung Fibroblast Resistance to Fas-Mediated Apoptosis

The accumulation of apoptosis-resistant fibroblasts within fibroblastic foci is a characteristic feature of idiopathic pulmonary fibrosis (IPF), but the mechanisms underlying apoptosis resistance remain unclear. A role for the inhibitor of apoptosis (IAP) protein family member X-linked inhibitor of apoptosis (XIAP) has been suggested by prior studies showing that (1) XIAP is localized to fibroblastic foci in IPF tissue and (2) prostaglandin E₂ suppresses XIAP expression while increasing fibroblast susceptibility to apoptosis. Based on these observations, we hypothesized that XIAP would be regulated by the profibrotic mediators transforming growth factor (TGF)β-1 and endothelin (ET)-1 and that increased XIAP would contribute to apoptosis resistance in IPF fibroblasts. To address these hypotheses, we examined XIAP expression in normal and IPF fibroblasts at baseline and in normal fibroblasts after treatment with TGF-β1 or ET-1. The role of XIAP in the regulation of fibroblast susceptibility to Fas-mediated apoptosis was examined using functional XIAP antagonists and siRNA silencing. In concordance with prior reports, fibroblasts from IPF lung tissue had increased resistance to apoptosis compared with normal lung fibroblasts. Compared with normal fibroblasts, IPF fibroblasts had significantly but heterogeneously increased basal XIAP expression. Additionally, TGF-β1 and ET-1 induced XIAP protein expression in normal fibroblasts. Inhibition or silencing of XIAP enhanced the sensitivity of lung fibroblasts to Fas-mediated apoptosis without causing apoptosis in the absence of Fas activation. Collectively, these findings support a mechanistic role for XIAP in the apoptosis-resistant phenotype of IPF fibroblasts.

Hypoxia‐induced downregulation of XIAP in trophoblasts mediates apoptosis via interaction with IMUP‐2: Implications for placental development during pre‐eclampsia

The regulation of trophoblast apoptosis is essential for normal placentation, and increased placental trophoblast cell apoptosis is the cause of pathologies such as intrauterine growth retardation (IUGR) and pre-eclampsia. X-linked inhibitor of apoptosis (XIAP) is expressed in trophoblasts, but little is known about the role of XIAP in placental development. In the present study, the function of XIAP in the placenta and in HTR-8/SVneo trophoblasts under hypoxic conditions was examined. In addition, the correlation between XIAP and immortalization-upregulated protein-2 (IMUP-2) was demonstrated in HTR-8/SVneo trophoblasts under hypoxia, based on a previous study showing that increased IMUP-2 induces trophoblast apoptosis and pre-eclampsia. XIAP was downregulated in pre-eclamptic placentas (P < 0.05). In HTR-8/SVneo trophoblasts, XIAP expression was decreased and the expression of apoptosis-related genes was increased in response to hypoxia. Ectopic expression of hypoxia inducible factor (HIF)-1α in HRT-8 SV/neo cells induced the nuclear translocation of XIAP and alterations of XIAP protein stability. Furthermore, hypoxia induced nuclear translocated XIAP co-localized with upregulated IMUP-2 in trophoblast nuclei, and the interaction between XIAP and IMUP-2 induced apoptosis in HRT-8 SV/neo cells. The present results suggest that hypoxia-induced down-regulation of XIAP mediates apoptosis in trophoblasts through interaction with increased IMUP-2, and that this mechanism underlies the pathogenesis of pre-eclampsia.

Immunohistochemical detection of antiapoptotic protein X-linked inhibitor of apoptosis in mammary carcinoma

An immunohistochemical survey of X-linked inhibitor of apoptosis (XIAP) expression in mammary carcinoma was performed. XIAP, the most potent of the inhibitor of apoptosis family of caspase inhibitors, has been linked to tumor aggressiveness and therapeutic resistance in several malignancies and is considered an attractive target for cancer drug discovery. Routinely processed sections from 94 ductal carcinomas, 9 lobular carcinomas, and 10 ductal carcinomas with lobular components or features were subjected to citrate-based antigen retrieval, immunostained with anti-XIAP (BD Biosciences, Franklin Lakes, NJ), Envision+ reagents (Dako, Carpinteria, CA), and diaminobenzidine. Positive staining was found in 22.7% of grade 1, 44% of grade 2, and 89.5% of grade 3 ductal carcinomas. Strong staining occurred in no cases of grade 1, 13% of grade 2, and 55.2% of grade 3 ductal carcinomas. XIAP staining increased overall with grade of ductal carcinoma in situ as well. The staining intensity of invasive carcinoma correlated with that of the corresponding ductal carcinoma in situ in 70% of cases. Ductal carcinomas overall showed more staining than lobular carcinomas. XIAP is most strongly and commonly detected in grade 3 ductal carcinoma. The degree of XIAP expression appears frequently to be determined in the preinvasive intraductal phase of tumorigenesis. These findings suggest a possible role of XIAP in the more aggressive clinical behavior of grade 3, compared with lower-grade ductal carcinomas.

XIAP mRNA levels but not XAF1 or XIAP/XAF1 mRNA levels predict pathological response in bladder cancer patients treated with neoadjuvant chemotherapy

20030 Background: The relation between apoptosis-related molecules and chemosensitivity has been extensively studied. In recent years, attention has shifted to a new family of inhibitor of apoptosis proteins (IAPs). XIAP (X- linked inhibitor of apoptosis) is the most versatile and potent member of the IAP family. To date, the overexpression of XIAP has been detected in various cancers. XAF1 (X-linked inhibitor of apoptosis associated factor 1) is a new protein identified for its ability to interact with XIAP. Neither XIAP nor XAF1 or XIAP/XAF1 mRNA expression have been studied in bladder cancer patients. Methods: The expression of XIAP and XAF1 mRNA was analyzed by a real time quantitative fluorogenic PCR method in a group of 17 patients with locally advanced bladder cancer treated with a combination of neoadjuvant Gemcitabine and Cisplatin. The prognostic significance of XIAP and XAF1 mRNA expression and the correlation with several clinicopathological variables was evaluated. Results: XIAP and XAF1 mRNA expression was detected in all 17 (100%) case samples. The levels of XIAP mRNA expression showed a moderate variation among samples. In contrast, XAF1 and XIAP/XAF1 mRNA levels showed significant variation among samples. Bivariate correlation analyses revealed a significant positive Spearman direct correlation coefficient between the XIAP expression and the pathological response. No significant correlation was found for XAF1 expression as well as for the XIAP/XAF1 ratio and clinical and pathological response. Conclusions: This is first study to address the role of XIAP, its negative regulator XAF1, and the XIAP/XAF1 ratio in bladder cancer patients. The positive correlation between the XIAP mRNA expression and the pathological response is in line with a previous study from our group in which a correlation was found between XIAP expression and survival. All these observations point to a complex role of XIAP in tumor biology. XAF1 mRNA expression in bladder carcinomas did not achieve significance as an independent predictive and prognostic factor in a bivariate analysis. Further studies are necessary in order to better assess a possible clinical value for XIAP and XAF1 as predictive and prognostic markers in cancer patients. No significant financial relationships to disclose.

Synergy is achieved by complementation with Apo2L/TRAIL and actinomycin D in Apo2L/TRAIL-mediated apoptosis of prostate cancer cells: role of XIAP in resistance.

Tumors have an inherent immunogenicity that can be exploited by immunotherapy. However, often tumors develop mechanisms that render them resistant to most immunologic cytotoxic effector mechanisms. This study examines the underlying mechanism of resistance to Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-mediated apoptosis. We studied prostate tumor cell lines for their sensitivity to Apo2L/TRAIL-mediated apoptosis in the presence and absence of the sensitizing agent actinomycin D (Act D). Apoptosis was determined by flow cytometry and signaling for apoptosis by Western blot. Treatment with subtoxic concentrations of Act D significantly sensitizes the tumor cells (CL-1, DU-145, and PC-3 prostate tumor cells) to Apo2L/TRAIL-mediated apoptosis. The cytotoxicity of Act D-sensitized prostate tumor cells was a result of synergistic activation of caspases (caspase-3, -9, and -8), detectable after 6 hr of treatment. Treatment with Apo2L/TRAIL alone, although it was insufficient to induce apoptosis, resulted in the loss of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytoplasm in the absence of significant caspases activation. These findings suggested that a major apoptosis resistance factor blocking the Apo2L/TRAIL apoptotic signaling events is present downstream of the mitochondrial activation. The expression of receptors and anti-apoptotic proteins were examined in Act D-sensitized CL-1 cells. The earliest and the most pronounced change induced by Act D was down-regulation of X-linked inhibitor of apoptosis (XIAP) and up-regulation of Bcl-xL/-xS proteins. The role of XIAP in resistance was demonstrated by overexpression of Smac/DIABLO, which inhibited inhibitors of apoptosis (IAPs) and sensitized the cells to Apo2L/TRAIL. Apo2L/TRAIL receptors (DR4, DR5, DcR1, and DcR2), c-FLIP, Bcl-2, and other IAP members (c-IAP1 and c-IAP2) were marginally affected at later times in the cells sensitized by Act D. This study suggests that the combination of Act D-induced down-regulation of XIAP (Signal I) and Apo2L/TRAIL-induced release of cytochrome c (Signal II) leads to the reversal of resistance to Apo2L/TRAIL-mediated apoptosis in the tumor cells. The sensitization of tumor cells to Apo2L/TRAIL by Act D is of potential clinical application in the immunotherapy of drug/Apo2L/TRAIL refractory tumors.