XAF1 Expression Research Articles

XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is well known as an antagonist of XIAP-mediated caspase inhibition. Although XAF1 serves as a tumor-suppressor gene, the role of XAF1 in cellular senescence remains unclear. We found that XAF1 expression was increased by genotoxic agents, such as doxorubicin and ionizing radiation in pulmonary microvascular endothelial cells, consequently leading to premature senescence. Conversely, downregulation of XAF1 in premature senescent cells partially overcame endothelial cell senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by XAF1 induction. XAF1 expression was transcriptionally regulated by Bromodomain 7 (BRD7). XAF1 induction with interferon-gamma (IFN-γ) treatment was abrogated by BRD7 knockdown, which resulted in blocking interferon-induced senescence. In lung cancer cells, XAF1 tumor suppressor activity was decreased by BRD7 knockdown, and inhibition of tumor growth by IFN-γ did not appear in BRD7-depleted xenograft tumors. These data suggest that XAF1 is involved in BRD7-associated senescence and plays an important role in the regulation of endothelial senescence through a p53-dependent pathway. Furthermore, regulation of the BRD7/XAF1 system might contribute to tissue or organismal aging and protection against cellular transformation.

XAF1 inhibits cell proliferation and induces apoptosis in human lung adenocarcinoma cell line A549 in vitro

背景与目的XAF1是重要的肿瘤细胞生长抑制因子，其低表达与多种肿瘤细胞有关。研究肿瘤抑制基因XAF1对人肺腺癌细胞株A549的作用及机制。方法利用重组腺病毒Ad5/F35-XAF1和对照腺病毒Ad5/F35-NULL瞬时转染A549细胞，用逆转录聚合酶链式反应（reverse transcriptase polymerase chain reaction, RT-PCR）和Western blot方法检测A549细胞株中XAF1 mRNA和蛋白质的表达；MTT检测细胞增殖率、流式细胞仪检测细胞凋亡率，并用Western blot法检测凋亡相关蛋白的表达。结果腺病毒介导的XAF1瞬时转染肺腺癌A549细胞后，XAF1 mRNA及蛋白表达水平明显提高，并能明显抑制该细胞增殖和促进细胞凋亡，蛋白质印记法显示凋亡相关蛋白PARP、Caspase-3、Caspase-8的裂解条带。结论恢复XAF1基因在人肺腺癌A549细胞中表达后，能明显抑制该肿瘤细胞增殖并促进其凋亡，其机制可能与XAF1激活肺癌细胞相关凋亡途径有关。

Abstract 597: Negative regulation of TNFα-induced NFκB activation by XAF1 via inhibition of the TRADD/TRAF2/RIP1 signal complex

Abstract Introduction: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) was originally implicated as a tumor suppressor due to its inhibitory effect on anti-apoptotic XIAP. It contains an N-terminus tumor necrosis factor (TNF) receptor associated factor (TRAF) Zn finger domain that is highly conserved in TRAF family members. XAF1 expression can be induced by TNFα; however, its role in the signal cascade initiated by TNFα/TNF receptor (TNFR) 1 has not yet been clearly elucidated. Here we report that XAF1 can negatively regulate TNFα-induced nuclear factor kappa B (NFκB) activation via inhibition of signaling molecule TRAF2 and signal complex formation downstream of TNFα/TNFR1. Methods: To investigate the effects of XAF1 on TNFα signal transduction machinery, we assessed TNF-induced expression of the NFκB response gene IL-8 by real-time PCR, and phosphorylation of inhibitor of NFκB (IκB) by immunoblotting with an anti-phospho-IκB antibody, in HEK293 cells with an inducible XAF1 expression vector. Interaction between TRAF2 or RIP1 and XAF1 was investigated by immunoprecipitation. NFκB transcriptional activation potential, induced by ectopic TRAF2 or RIP1 with or without co-expressed XAF1, was quantified by luciferase assays. Inhibition of TRAF2 ubiquitination by XAF1 was also assessed by immunoblot. Transient transfection of plasmids expressing TRADD, TRAF2, RIP1 and/or XAF1 followed by immunoprecipition was performed to further investigate the role of XAF1 on the signal complex formation. Results: Luciferase assays and real-time PCR studies showed that XAF1 inhibited TNFα-induced NFκB activation and IL-8 expression. Both TRAF2 and RIP1 interacted with XAF1 according to our immunoprecipitation results but only TRAF2 activity was inhibited by XAF1. Poly-ubiquitination of TRAF2 was attenuated when XAF1 was present. In addition, immunoprecipitation and immunoblot data showed that XAF1 interfered with RIP1/TRADD association and regulated TRADD and RIP1 turnover. Discussion: Our data demonstrated an inhibitory effect of XAF1 on TNFα-induced NFκB activation and gene expression. A possible mechanism may be via inhibition of TRAF2, an E3 ubiquitin ligase responsible for signal transduction leading to NFκB activation. XAF1 inhibited both TRAF2-mediated NFκB activation and TRAF2 ubiquitination, which is essential for its signal delivery function. XAF1 also interacted with RIP1 yet did not block RIP1-induced NFκB promoter activity. However, the binding of RIP1 to TRADD, an adaptor protein of TNFR1-mediated signal pathway, was disrupted by XAF1. Furthermore, degradation of TRADD and RIP1 was accelerated when XAF1 was present. These data suggested that XAF1 may act as a TNF-induced negative regulator of NFκB activation by inhibiting TRAF2 ubiquitination and by regulating the integrity of the signal complex of TRADD/TRAF2/RIP1. Citation Format: Boren Lin, Da Xu, Douglas W. Leaman. Negative regulation of TNFα-induced NFκB activation by XAF1 via inhibition of the TRADD/TRAF2/RIP1 signal complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2014-597

Tumor suppressor XAF1 induces apoptosis, inhibits angiogenesis and inhibits tumor growth in hepatocellular carcinoma

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1), a XIAP-binding protein, is a tumor suppressor gene. XAF1 was silent or expressed lowly in most human malignant tumors. However, the role of XAF1 in hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the effect of XAF1 on tumor growth and angiogenesis in hepatocellular cancer cells. Our results showed that XAF1 expression was lower in HCC cell lines SMMC-7721, Hep G2 and BEL-7404 and liver cancer tissues than that in paired non-cancer liver tissues. Adenovirus-mediated XAF1 expression (Ad5/F35-XAF1) significantly inhibited cell proliferation and induced apoptosis in HCC cells in dose- and time- dependent manners. Infection of Ad5/F35-XAF1 induced cleavage of caspase -3, -8, -9 and PARP in HCC cells. Furthermore, Ad5/F35-XAF1 treatment significantly suppressed tumor growth in a xenograft model of liver cancer cells. Western Blot and immunohistochemistry staining showed that Ad5/F35-XAF1 treatment suppressed expression of vascular endothelial growth factor (VEGF), which is associated with tumor angiogenesis, in cancer cells and xenograft tumor tissues. Moreover, Ad5/F35-XAF1 treatment prolonged the survival of tumor-bearing mice. Our results demonstrate that XAF1 inhibits tumor growth by inducing apoptosis and inhibiting tumor angiogenesis. XAF1 may be a promising target for liver cancer treatment.

Melphalan-Induced Apoptosis of EBV-Transformed B Cells through Upregulation of TAp73 and XAF1 and Nuclear Import of XPA

Melphalan (Mel) is widely used to treat patients with hematologic cancer, including multiple myeloma, but its mechanism of action in EBV-transformed B cells is poorly described. In this study, we demonstrate a novel mechanism by which transcriptionally active p73 (TAp73) induces translocation of X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) and xeroderma pigmentosum group A (XPA) during apoptosis caused by Mel treatment. We observed that Mel induced significant generation of reactive oxygen species (ROS) and subsequent apoptosis, as well as an early phosphorylation of p38 MAPK that preceded expression of the mitochondria membrane potential disruption-related molecules and the cleavage of caspases. In particular, Mel led to upregulation of TAp73, XAF1, and Puma and induced XPA nuclear import and translocation of Bax into mitochondria. Mel-induced apoptosis was inhibited by pretreatment with the ROS scavenger 4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC) and the p38 MAPK inhibitor SB203580. We supposed that ROS generation might be the first event in Mel-induced apoptosis, because APDC blocked the increase in ROS, p38 MAPK, and TAp73, but SB203580 did not block ROS generation. Moreover, Mel elicited activation of ATR, and APDC inhibited phosphorylation of ATR but not SB203580. APDC and SB203580 completely blocked XPA and Bax translocation. We conclude that Mel promotes TAp73-mediated XAF1 and Puma expression via ROS generation and ATR/p38 MAPK pathway activation, thereby triggering apoptosis. Our results provide evidence of a novel alternate regulatory mechanism of TAp73 and reveal that Mel may be a therapeutic drug for curing EBV-related malignancies.

ROS and ERK1/2-mediated caspase-9 activation increases XAF1 expression in dexamethasone-induced apoptosis of EBV-transformed B cells

Dexamethasone (Dex) inhibits the growth of diverse types of cancer cells and is utilized clinically for the therapy of hematological malignancies. In this study, we investigated the molecular mechanisms of Dex action in the apoptosis of Epstein-Barr virus (EBV)-transformed B cells. We showed that Dex inhibited the proliferation of EBV-transformed B cells and induced apoptosis by activating caspase-9, -3 and -8. While activation of caspase-9 was triggered as early as 2 h after Dex treatment, cleavage of caspase-8 was deferred and was found 8 h after the exposure. Dex-dependent activation of caspase-8 was blocked by the specific caspase-9 inhibitor, z-LEHD-fmk. Moreover, Dex significantly increased the expression of X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) and induced the translocation of XAF1 into the cytosol. Cytosolic XAF1 with Puma induced the translocation of Bax into mitochondria. Dex led to up-regulation of reactive oxygen species (ROS) generation and the phosphorylation of ERK1/2 after the exposure. We speculated that ROS generation might be the first event of Dex-induced apoptosis because ROS inhibitor NAC abrogated ROS production and ERK1/2 activation, but PD98059 did not block ROS production. NAC and PD98059 also suppressed the translocation of XAF1, Puma and Bax into mitochondria. These results demonstrated that Dex-mediated activation of caspase-9 via ROS generation and ERK1/2 pathway activation resulted in the activation of caspase-8 and the increment of XAF1, thereby induced apoptosis of EBV-transformed B cells. These findings suggest that Dex constitutes a probable therapy for EBV-associated hematological malignancies.

XAF1 is frequently methylated in human esophageal cancer

To explore epigenetic changes in the gene encoding X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) during esophageal carcinogenesis. Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction (MSP) in four esophageal cancer cell lines (KYSE30, KYSE70, BIC1 and partially methylated in TE3 cell lines), nine cases of normal mucosa, 72 cases of primary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza-deoxycytidine (5-aza-dc), a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immunohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clinicopathological data was analyzed by logistic regression. MSP results were as follows: loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation (completely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells); all nine cases of normal esophageal mucosa were unmethylated; and 54/72 (75.00%) samples from patients with esophageal cancer were methylated, and 25/72 (34.70%) matched adjacent tissues were methylated (75.00% vs 34.70%, χ(2) = 23.5840, P = 0.000). mRNA level of XAF1 measured with semi-quantitative reverse transcription polymerase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue (χ(2)= 9.143, P = 0.002). XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal cancer, 24/32 samples were methylated, and 8/32 esophageal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation (Fisher's exact test, P = 0.004). Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metastasis with respect to methylation of XAF1 for the 72 tissue samples from patients with esophageal cancer. XAF1 is frequently methylated in esophageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.

Abstract 195: Cell death pathway activation during monocytic / macrophagic differentiation of hematopoietic tumor cell lines

Abstract Apoptosis is a phenomenon of cellular degeneration highly conserved evolutionarily, being observed not only during development, but also associated with a number of diseases. Apoptosis is regulated by a complex set of proteins and accomplished by biochemical mechanisms that act to initiate and complete the destruction of the cell. Recently, typical biochemical and morphological changes associated to cell death were correlated with other vital cellular events, such as proliferation and differentiation. Despite the general tendency to treat apoptosis and cell differentiation as the result of different signaling pathways, several studies have shown that there is a high degree of morphological similarity between these events. The molecules involved in the regulation of apoptosis-like processes of differentiation have not been fully described. Since the main endogenous inhibitor of caspase-3 is the protein XIAP, which in turn is regulated by XAF1, we analyzed the expression of these proteins in the context of differentiation of different cell lines. The objective of this study was to evaluate the activation pathways of cell death in the process of monocytic/macrophage differentiation. We have observed that the expression of XAF1 protein decreases during monocytic/macrophage differentiation of HL60 cell line induced by PMA, but levels of its mRNA do not change. Interestingly, we have found that HL60 cell lines overexpressing either Bcl-XL or Bcl-2 cell lines have decreased expression of XAF1 due to methylation of its promoter. Also, besides rendering cells resistant to apoptosis as expected, HL60 cells overexpressing either Bcl-XL or Bcl-2 were more prone to differentiate by PMA treatment than parental cells. The correlation between high levels of mRNA expression of Bcl-2 with low expression of XAF1 mRNA was also observed in the analysis of samples from patients with follicular lymphoma retrieved from Oncomine database (www.oncomine.com). These results demonstrate the differential activation of pathways of cell death in monocytic / macrophagic differentiation, suggesting that a descrease in XAF1 expression is a necessary event during this process. Furthemore, our results reveal a transcriptional regulatory loop exerted by Bcl-2 family of proteins that had not yet been described. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 195. doi:10.1158/1538-7445.AM2011-195

XAF1 expression and regulatory effects of somatostatin on XAF1 in prostate cancer cells

BackgroundSomatostatin prevents cell proliferation by inducing apoptosis. Downregulation of the XAF1 transcript may occur during the development of prostate cancer. It is interesting to evaluate the potential regulatory effects of somatostatin on XAF1 expression during the development of prostate cancer cells.MethodsXAF1 mRNA and protein expression in human prostate epithelial cells RWPE-1, androgen dependent prostate cancer LNCaP, and androgen independent DU145 and PC3 cells were evaluated using RT-PCR and Western blot. The regulation of XAF1 mRNA and protein expression by somatostatin and its analogue Octreotide was evaluated.ResultsSubstantial levels of XAF1 mRNA and proteins were detected in RWPE-1 cells, whereas prostate cancer cells LNCaP, DU145 and PC3 exhibited lower XAF1 expression. Somatostatin and Octreotide up-regulated XAF1 mRNA and protein expression in all prostate cancer cell lines.ConclusionsXAF1 down-regulation may contribute to the prostate cancer development. The enhanced XAF1 expression by somatostatin indicates a promising strategy for prostate cancer therapy.

Abstract 4515: Tumor suppressor XAF1 inhibits hepatocellular carcinoma growth and angiogenesis

Abstract Background and Aims: X-linked inhibitor of apoptosis (XIAP)-associated factor 1(XAF1) is a novel identified XIAP antagonist and a potential tumor suppressor. Our previous study has shown that stable overexpression of XAF1 could induce apoptosis and inhibit tumorigenesis in hepatocellular carcinoma (HCC). However, the therapeutic effect and mechanism of XAF1 on HCC remains unknown. Materials and Methods: Recombinant adenovirus Ad5/F35-XAF1 and control virus Ad5/F35-Null were generated. Cellular proliferation was detected by methyl thiazolyl tetrazolium (MTT). Cell apoptosis was determined by Flow cytometry using Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). The expression of genes was detected by RT-PCR, Western Blot and immunohistochemistry (IHC). Tumor growth in vivo was determined in a nude mice xenograft model. Microvessel density (MVD) was measured by CD31 IHC staining. Results: The expression of XAF1 was extremely low or undectable in four HCC cell lines (SMMC7721, Hep3B, HepG2, Bel7404) and 30 human HCC tissues compared with the paired non-cancer liver tissues, while the high expression of XIAP was detected in HCC tissues than in normal live tissues. Infection of Ad5/F35-XAF1 virus increased the expression of XAF1 in all 4 HCC cell lines, significantly inhibited cell proliferation and induced cell apoptosis in a dose- and -time dependent manner, resulting in the cleavage of caspase-3, −8, −9 and PARP, release of cytochrome c, upregulation of pro-apoptotic protein Bax and BNIP3L, and downregulation of XIAP. Furthermore, intra-tumoral injection of Ad5/F35-XAF1 virus significantly induced apoptosis in vivo and inhibited HCC xenograft growth. Notably, Ad5/F35-XAF1 virus treatment significantly upregulated the expression of angiopoietin-2 protein and decreased MVD in tumor xenograft tissues. Furthermore, adeno-XAF1 treatment significantly prolonged the survival time of animals bearing tumor xenografts. No obvious pathological changes in main organs were observed in XAF1 virus-treated mice. Conclusion: Our results show that restoration of XAF1 expression obviously induces cell apoptosis and inhibits the HCC both in vitro and in vivo. Expression of XAF1 could significantly suppressed tumor angiogenesis probably through upregulating angiopoietin-2 expression to remold the newly-born vessels. Our results suggest that XAF1 may have a potential candidate for HCC gene therapy. The project was supported by National Nature Science foundation of China No. 30500221 (Tu SP) and NIH grant R01CA120915 (Yang CS). *Corresponding author: Shui-Ping Tu, E-mail: Tushuiping@yahoo.com. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4515.

Identification of a functional p53 responsive element within the promoter of XAF1 gene in gastrointestinal cancer cells

It has been reported that XAF1 expression in gastric cancer is negatively correlated with p53. Our purpose was to clarify the regulatory mechanism of p53 on XAF1 expression. The effects of overexpressed wild-type and mutant p53 on XAF1 expression were evaluated. Binding capacity of core XAF1 promoter sequence to the recombinant p53 protein was examined. Site-directed mutation of putative p53 binding sequence and p53 knockdown by siRNA were performed. The protein expression and promoter activities of XAF1 in cells with null p53 were higher than that with wild-type and mutant p53. Ectopic overexpression of wild-type p53 suppressed XAF1 expression. A half-site (-95 to -86 nt) and a quarter-site (-4 to +1 nt) of p53 responsive element were found within XAF1 promoter. Both sequences bound to recombinant p53 effectively and specifically. Site-mutation of p53 responsive sequences abrogated the binding capacity. However, only the mutation of half-site increased XAF1 promoter activities. Suppression of p53 not only decreased the binding capacity of p53 responsive halfsite but also increased XAF1 transcription. In conclusion, we demonstrated that p53 could suppress the transcription of XAF1 through interaction with a high affinity responsive element (-95 to -86 nt) within XAF1 promoter, indicating a novel exclusive mechanism between these two tumor suppressors.

XAF1 as a prognostic biomarker and therapeutic target in pancreatic cancer

XAF1 (X chromosome-linked inhibitor of apoptosis [XIAP]-associated factor 1) is a novel XIAP modulator that negatively regulates the anti-apoptotic effects of XIAP and sensitizes cells to other cell death triggers. It has been reported to be downregulated in a variety of human cancer cell lines. However, the role of XAF1 in pancreatic carcinogenesis remains unclear. In the present study, we investigated the prognostic values of XAF1 expression and its regulation in cancer cell growth and apoptosis both in vitro and in vivo. From the immunohistochemistry staining of tissue microarray, 40 of 89 (44.9%) pancreatic specimens showed low levels of XAF1 expression. Statistical analysis suggested the downregulation of XAF1 was significantly correlated with tumor staging (P = 0.047) and those patients with low XAF1 levels had shorter survival times (P = 0.0162). Multivariate analysis indicated that XAF1 expression was an independent prognostic indicator of the survival of patients with pancreatic cancer (P = 0.007). Furthermore, we found that restoration of XAF1 expression mediated by Ad5/F35 virus suppressed cell proliferation and induced cell cycle arrest and apoptosis, accompanied by the activation of caspases 3, 8, and 9 and poly(ADP-ribose) polymerase as well as increased level of cytochrome c and Bid cleavage. Notably, XAF1 restoration robustly decreased survivin expression rather than XIAP. In addition, in vivo s.c. xenografts from Ad5/F35-XAF1 treatment, which showed less cellular proliferation and enhanced apoptosis, were significantly smaller than those from control groups. Our findings document that XAF1 is a valuable prognostic marker in pancreatic cancer and could be a potential candidate for cancer gene therapy.

Combination of DNA methylation inhibitor 5‐azacytidine and arsenic trioxide has synergistic activity in myeloma

XAF1 is a newly identified candidate tumour-suppressor gene that can antagonise XIAP and sensitise cells to cell death triggers. This study was undertaken to study the effect of 5-azacytidine (AZA) on XAF1 expression in myeloma cells and efficacy of 5-AZA and arsenic trioxide (ATO) combination treatment in myeloma in vivo and in vitro. XAF1 expression was analysed by semi-quantitative PCR and western blotting. Methylation specific PCR was used to detect methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with various concentrations of 5-AZA and ATO. Expression of XAF1 mRNA variants were confirmed by gel electrophoresis and sequencing. Untreated RPMI 8226 cell expresses two variants of XAF1 mRNA. Untreated XG-7 cell has no expression of XAF1. Hypermethylation of XAF1 promoter CpG islands was detected in both cell lines. Both cell lines express full-length XAF1 transcript after treated with 2.5 mumol/L 5-AZA for 72 h. Our studies demonstrated that 5-AZA exhibits anti-myeloma synergy with ATO. In addition, ATO alone, 5-AZA alone, or combination of 5-AZA and ATO was effective in slowing myeloma growth and prolonging survival of myeloma-loaded nude mice. The findings suggested that 5-AZA and ATO may be an effective combination in the therapy of myeloma patients.

Combination of DNA Methylation Inhibitor 5-Azacytidine and Arsenic Trioxide Has Synergistic Activity in Multiple Myeloma

This study was undertaken to study the effect of 5-azacytidine on XAF1 expression in myeloma cells and efficacy of 5-azacytidine and arsenic trioxide combination treatment in myeloma in vivo and in vitro. XAF1 expression was analyzed by semi-quantitative PCR and Western blotting. Methylation specific PCR (MSP) was used to detect methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with various concentrations of arsenic trioxide and 5-azacytidine. Expression of XAF1 mRNA variants were confirmed by gel electrophoresis and sequencing. Untreated RPMI 8226 cell expresses two variants of XAF1 mRNA. Untreated XG-7 cell has no expression of XAF1. Hypermethylation of XAF1 promoter CpG islands was detected in both cell lines. Both cell lines express full-length XAF1 mRNA transcript after treated with 2.5 μmol/L 5-azacytidine for 72 h. Our studies demonstrated that 5-azacytidine exhibits antimyeloma synergy with arsenic trioxide. 5-azacytidine treatment reversed XAF1 promoter region hypermethylation status, leading to expression of full-length XAF1 transcript. In addition, combination of 5-azacytidine and arsenic trioxide was effective in slowing myeloma growth and prolonging survival of myeloma-loaded nude mice. The findings suggested that 5-azacytidine and arsenic trioxide may be an effective combination in the therapy of myeloma patients.

Expression der pro-apoptotischen Apoptoseinhibitor-(IAP)Antagonisten XAF1, Smac/DIABLO und HtrA2 in Keimzelltumoren des Hodens

Down-regulation of the IAP antagonistis XAF1, Smac/DIABLO and HtrA2, has been related to the onset and progression of various malignancies. We examined the mRNA-expression of these pro-apoptotic parameters in testicular germ cell tumors (TGCT) and normal testicular tissue and correlated their expression levels to clinicopathological tumour features. Real-time RT-PCR was used to quantify the mRNA-expression of XAF1, Smac/DIABLO and HtrA2 in normal testicular tissue (n = 18), carcinoma in situ (n = 4), seminomas (n = 64), and non-seminomatous germ cell tumors (n = 35). Compared to normal testicular tissue, the expression levels of XAF1 were increased in TGCT (p < 0.001), whereas those of Smac/DIABLO and HtrA2 were decreased (p < 0.001 and p < 0.001). Smac/DIABLO expression levels showed a significant trend towards a gradual decrease from normal testicular tissue to CIS and seminomas and finally to NSGCT (p < 0.001). Moreover, XAF1 and HtrA2 expression levels gradually increased with progression of clinical tumour stage in seminoma patients (p = 0.001 and p = 0.018), their expression levels being strongly intercorrelated (Spearman rho correlation coefficient: 0.674; p < 0.001). These data suggest that a down-regulation of Smac/DIABLO and HtrA2 is implicated in the development and progression of TGCT, whereas overexpression of XAF1 in TGCT might contribute to their extraordinary sensitivity to chemotherapy. Regarding the additional correlation of XAF1 and HtrA2 expression with clinical tumour stage in seminoma patients, it appears reasonable to further evaluate these three IAP antagonists as molecular parameters for the prediction of treatment response and prognosis of TGCT patients.

XAF1 mRNA expression improves progression-free and overall survival for patients with advanced bladder cancer treated with neoadjuvant chemotherapy

The aim of this study was to investigate whether mRNA expression of the apoptosis-associated genes, XAF1 and XIAP, in bladder cancer patients correlates with response to neoadjuvant treatment. Gene expression was analyzed by a real-time quantitative PCR method in paired samples from 14 bladder cancer patients treated with a combination of neoadjuvant gemcitabine and cisplatin. The prognostic significance of XAF1 and XIAP mRNA expression as well as the correlation with several clinical and pathological findings were evaluated. The clinical response in the XAF1-high subset (n = 5) was remarkably higher compared with the XAF1-low subset (n = 9) (100% vs. 44.4%; P = 0.038). These results translated into a notably improvement of progression-free survival (PFS) in the XAF1-high subset (log-rank P = 0.012). In addition, patients in the XAF1-high subset had a 3.9-fold decreased chance of dying from the disease (hazard ratio for death (HR), 0.257; (CI 95%), 0.043-1.536, P = 0.036). When we evaluated the expression of XIAP, although an inverse correlation was found between expression and pathological response, there were no statistically significant associations with the clinical response, the length of PFS, and OS. This is one of the few studies to address the role of XAF1 in a clinical setting. The data presented here identify XAF1 as a novel predictive and prognostic factor in bladder cancer patients. Furthermore, our observations are in line with previous studies, which point towards XAF1 as a tumor-suppressor gene. Nonetheless, additional studies, both mechanistic and translational, are warranted and may help not only in corroborating the role of XAF1 as a prognostic marker, but also as a potential target for anticancer therapy.

Interferon-γ Induces X-linked Inhibitor of Apoptosis-associated Factor-1 and Noxa Expression and Potentiates Human Vascular Smooth Muscle Cell Apoptosis by STAT3 Activation

Interferon (IFN)-gamma actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-gamma signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-gamma on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-gamma consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs. STAT3 activation resulted in nuclear translocation of this transcription factor. By selectively inhibiting STAT3 and not STAT1 signaling, we identified a number of candidate IFN-gamma-inducible, STAT3-dependent gene products by microarray analysis. Results for selected genes, including the pro-apoptotic molecules X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa, were verified by real time quantitative reverse transcription-PCR and immunoblot analyses. IFN-gamma-induced STAT3 and STAT1 signaling in VSMCs demonstrated reciprocal inhibition. STAT3 activation by IFN-gamma sensitized VSMCs to apoptosis triggered by both death receptor- and mitochondrial-mediated pathways. Knock down of XAF1 and Noxa expression inhibited the priming of VSMCs to apoptotic stimuli by IFN-gamma. Finally, we confirmed the in vivo relevance of our observations using a chimeric animal model of immunodeficient mice bearing human coronary artery grafts in which the expression of XAF1 and Noxa as well as the pro-apoptotic effects induced by IFN-gamma were dependent on STAT3. The data suggest STAT1-independent signaling by IFN-gamma via STAT3 that promotes the death of human VSMCs.

Frequent Alteration of XAF1 in Human Colorectal Cancers: Implication for Tumor Cell Resistance to Apoptotic Stresses

X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) is a candidate tumor suppressor located at the chromosome 17p13 region, but the molecular basis underlying its inactivation in human tumors and growth-inhibiting function has not been well defined. We explored the candidacy of XAF1 as a suppressor in colorectal tumorigenesis. XAF1 expression was characterized by polymerase chain reaction-based cloning, isoform-specific polymerase chain reaction, ribonuclease protection, and immunoblot assays. Allelic loss of the gene was evaluated by loss of heterozygosity (LOH) assay, and promoter CG dinucleotide (CpG) site methylation was determined using bisulfite sequencing. The effect of XAF1 on tumor growth was examined using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, colony formation, and viability assays. Expression of 5 XAF1 variants including 2 novel transcripts was down-regulated concomitantly in 11 of 20 (55%) cell lines and 26 of 65 (40%) primary tumors. XAF1 reduction was tumor-specific and showed a correlation with advanced stage and high grade of tumor. LOH of the gene was found in 12 of 33 (36%) tumors. Promoter CpG site methylation was observed frequently in both cell lines and tumor tissues including many LOH tumors, suggesting that biallelic inactivation of XAF1 might be common in colorectal cancers. XAF1 expression suppressed tumor cell growth and enhanced cellular response to various apoptotic stimuli, such as 5-fluorouracil, etoposide, H(2)O(2), gamma-irradiation, ultraviolet, and tumor necrosis factor-alpha, whereas knockdown of its expression protected cells from the stresses. Genetic and epigenetic alteration of XAF1 is a common event in colorectal tumorigenesis and contributes to the malignant tumor progression by providing survival advantages for tumor cells under various stress conditions.

XAF1 Mediates Tumor Necrosis Factor-α-induced Apoptosis and X-linked Inhibitor of Apoptosis Cleavage by Acting through the Mitochondrial Pathway

Tumor necrosis factor-alpha (TNF-alpha) and Fas ligand induce apoptosis by interacting with their corresponding membrane-bound death receptors and activating caspases. Since both systems share several components of the intracellular apoptotic cascade and are expressed by first trimester trophoblasts, it is unknown how these cells remain resistant to Fas ligand while sensitive to TNF-alpha. XAF1 (X-linked inhibitor of apoptosis (XIAP)-associated factor 1) is a proapoptotic protein that antagonizes the caspase-inhibitory activity of XIAP. Here, we demonstrated that XAF1 functions as an alternative pathway for TNF-alpha-induced apoptosis by translocating to the mitochondria and promoting XIAP inactivation. In addition, we showed that the overexpression of XAF1 sensitized first trimester trophoblast cells to Fas-mediated apoptosis. Furthermore, we also determined that the differential expression of XAF1 in first and third trimester trophoblast cells was due to changes in XAF1 gene methylation. Our results establish a novel regulatory pathway controlling trophoblast cell survival and provide a molecular mechanism to explain trophoblast sensitivity to TNF-alpha and the increased number of apoptotic trophoblast cells observed near term. Aberrant XAF1 expression and/or localization may have consequences for normal pregnancy outcome.

Silencing of the XAF1 gene by promoter hypermethylation in cancer cells and reactivation to TRAIL-sensitization by IFN-β

BackgroundXIAP-associated factor 1 (XAF1) is a putative tumor suppressor that exerts its proapoptotic effects through both caspase-dependent and – independent means. Loss of XAF1 expression through promoter methylation has been implicated in the process of tumorigenesis in a variety of cancers. In this report, we investigated the role of basal xaf1 promoter methylation in xaf1 expression and assessed the responsiveness of cancer cell lines to XAF1 induction by IFN-β.MethodsWe used the conventional bisulfite DNA modification and sequencing method to determine the methylation status in the CpG sites of xaf1 promoter in glioblastoma (SF539, SF295), neuroblastoma (SK-N-AS) and cervical carcinoma (HeLa) cells. We analysed the status and incidence of basal xaf1 promoter methylation in xaf1 expression in non-treated cells as well as under a short or long exposure to IFN-β. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-β-mediated sensitization to TRAIL-induced cell death.ResultsWe found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-β across the four cancer cell lines studied. At the basal level, aberrant promoter methylation was linked to xaf1 gene silencing. After a short exposure, the IFN-β-mediated reactivation of xaf1 gene expression was related to the degree of basal promoter methylation. However, in spite of continued promoter hypermethylation, we find that IFN-β induced a transient xaf1 expression, that in turn, was followed by promoter demethylation upon a prolonged exposure. Importantly, we demonstrated for the first time that IFN-β-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-β-mediated TRAIL sensitivity.ConclusionTogether, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-β treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer.