Shimada K, Matsuyoshi S, Nakamura M, Ishida E, Konishi N.. Phosphorylation status of Fas-associated death domain-containing protein (FADD) is associated with prostate cancer progression. J Pathol 206: 423-432
Department of Pathology, Nara Medical University, School of Medicine, Nara, 634-8521, Japan. The Journal of Pathology
(Impact Factor: 7.43).
08/2005; 206(4):423-32. DOI: 10.1002/path.1791
It has recently been demonstrated that phosphorylation of FADD at serine 194 plays an important role in the induction of apoptosis by anti-cancer drugs in human prostate cancer cells. The present study has assessed whether this phosphorylation status is valuable as a marker for human prostate cancer progression, and has investigated its biological role in cell growth. Immunohistochemical studies revealed much higher phosphorylation of FADD at serine 194 in normal epithelial cells than in cancer cells, although FADD was found to be highly expressed to the same extent in both cases. The positivity for phosphorylated FADD was significantly lower for patients with a Gleason score greater than or equal to 7, a positive surgical margin, extracapsular or seminal vesicle invasion. In addition, a relationship was also apparent in cancer cells refractory to neoadjuvant hormonal therapy. Interestingly, in Gleason score 3 + 4 tumours, the positivity for FADD phosphorylation was statistically increased by neoadjuvant hormonal therapy, resulting in a reduced percentage of cases with a positive surgical margin and extracapsular invasion. In vitro data showed different functions of phosphorylated and non-phosphorylated FADD: in normal epithelial cells, overexpression of a phosphorylation-mimicking mutant FADD (S194E) caused G2/M cell-cycle arrest, while a non-phosphorylation-mimicking mutant (S194A) had no effect, whereas S194A overexpression resulted in cell cycle progression and enhanced colony-forming activity in cancer cells, but S194E FADD was without influence. These results clearly demonstrate that transition from phosphorylated FADD to the non-phosphorylated form might be associated with carcinogenesis and that induction of FADD phosphorylation could therefore be a target for chemohormonal therapy of human prostate cancer. Moreover, assessment of FADD phosphorylation may be useful as a new biomarker to predict cancer progression.
Available from: Tomomi Fujii
- "Tumor stage and grade were noted at the time of diagnosis before the collection of specimens. We followed the same tissue fixation and processing procedure as described in a previous report [15-17]. After deparaffinization, the sections were heated for 5 min in 10 mM of sodium citrate buffer (pH 6.0) in a pressure cooker. "
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ABSTRACT: Reactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression. In the present research, we examined the pathobiological role of NADPH oxidase (NOX)4-mediated generation of reactive oxygen species (ROS) in urothelial carcinoma (UC) of the urinary bladder, and demonstrated the utility of ROS labeling in urine cytology.
NOX4 gene was silenced in vivo and in vitro by NOX4 siRNA transfection with or without atlocollagen. Cell cycle and measurement of ROS were analyzed by flowcytometry. Orthotopic implantation animal model was used in vivo experiment. NOX4 expression in urothelial carcinoma cells was observed by immunohistochemical analysis using surgical specimens of human bladder cancer. Urine cytology was performed after treatment with ROS detection reagents in addition to Papanicolaou staining.
NOX4 was overexpressed in several UC cell lines and the NOX inhibitor, diphenylene iodonium reduced intracellular ROS and induced p16-dependent cell cycle arrest at the G1 phase. Moreover, silencing of NOX4 by siRNA significantly reduced cancer cell growth in vivo as assessed in an orthotopic mouse model. Immunohistochemistry demonstrated high expression of NOX4 in low grade/non-invasive and high grade/invasive UC including precancerous lesions such as dysplasia but not in normal urothelium. Then, we assessed the usefulness of cytological analysis of ROS producing cells in urine (ROS-C). Urine samples obtained from UC cases and normal controls were treated with fluorescent reagents labeling the hydrogen peroxide/superoxide anion and cytological atypia of ROS positive cells were analyzed. As a result, the sensitivity for detection of low grade, non-invasive UC was greatly increased (35% in conventional cytology (C-C) vs. 75% in ROS-C), and the specificity was 95%. Through ROS-C, we observed robust improvement in the accuracy of follow-up urine cytology for cases with previously diagnosed UC, especially in those with low grade/non-invasive cancer recurrence (0% in C-C vs. 64% in ROS-C).
This is the first report demonstrating that ROS generation through NOX4 contributes to an early step of urothelial carcinogenesis and cancer cell survival. In addition, cytology using ROS labeling could be a useful diagnostic tool in human bladder cancer.
Available from: Satoshi Anai
- "Before the collection of specimens, as appropriate, and tumor stage and grade were noted at the time of diagnosis. We followed the same tissue fixation and processing procedure as described in a previous report[21,22]. After deparaffinization, sections were heated for 5 minutes in 10 mM of sodium citrate buffer (pH 6.0) in a pressure cooker. "
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ABSTRACT: Survival rate for patients presenting muscle invasive bladder cancer is very low, and useful therapeutic target has not been identified yet. In the present study, new COX2 downstream signals involved in urothelial carcinoma cell survival were investigated in vitro and in vivo.
COX2 gene was silenced by siRNA transfection. Orthotopic implantation animal model and transurethral instillation of siRNA with atelocollagen was constructed to examine the effects of COX2 knockdown in vivo. Cell cycle was examined by flowcytoketry. Surgical specimens derived from patients with urinary bladder cancer (all were initially diagnosed cases) were used for immunohistochemical analysis of the indicated protein expression in urothelial carcinoma cells.
Treatment with the COX2 inhibitor or knockdown of COX2 reduced expression of casein kinase (CK) 2 α, a phophorylated Akt and urokinase type plasminogen activator (uPA), resulting in p27 induction, cell cycle arrest at G1 phase and cell growth suppression in human urothelial carcinoma cell lines expressing COX2. Silencing of CK2α exhibited the similar effects. Even in UMUC3 cells lacking the COX2 gene, COX2 inhibition also inhibited cell growth through down-regulation of the CK2α-Akt/uPA axis. The mouse orthotropic bladder cancer model demonstrated that the COX2 inhibitor, meloxicam significantly reduced CK2α, phosphorylated Akt and uPA expression, whereas induced p27 by which growth and invasiveness of bladder cancer cells were strongly inhibited. Immunohistochemically, high expression of COX2, CK2α and phosphorylated form of Akt was found in high-grade, invasive carcinomas as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes.
COX2-dependent and independent activation of CK2α-Akt/uPA signal is mainly involved in urothelial carcinoma cell survival, moreover, not only COX2 but also CK2α could be direct targets of COX2 inhibitors.
Available from: PubMed Central
- "The phosphorylated form of FADD (p-FADD) has recently been reported to regulate apoptotic activity . Although the role of p-FADD in ESCC outcome is unclear, higher levels of p-FADD protein correlated with reduced survival in patients with lung adenocarcinomas  and prostate cancer . "
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ABSTRACT: Cancer of the esophagus is a deadly malignancy, and development of biomarkers that predict survival is an urgent need. The apoptotic pathways have been hypothesized as important in progression of esophageal squamous cell carcinoma (ESCC). We investigated a panel of proteins that regulate apoptosis as candidate of biomarkers of prognosis in ESCC.
Tissue microarray (TMA) including 313 surgically-resected cases of ESCC specimens was built for immunohistochemical interrogation. We evaluated seven genes in the FasL-Fas apoptotic pathway - FasL, Fas, FAS-associated death domain protein (FADD), phosphorylated-FADD, and caspase 8 and 10, and the antiapoptotic protein bcl-2. We studied pathway integrity and relations to risk and clinical factors, and determined the prognostic significance of each marker.
Five markers showed strong inter-marker correlations (r > or = 0.28, p < 0.001), including FasL, Fas, FADD, and caspases 8 and 10. FasL and FADD also showed modest correlations with one or more cancer risk factors, but none of the markers was significantly associated with either tumor stage or lymph node metastasis, the only two clinical factors that predicted survival in these ESCC cases. Multivariate-adjusted proportional hazard regression models showed no association between protein expression and risk of death for any of the seven markers examined.
Individual biomarkers in the apoptosis pathway do not appear to predict survival of patients with ESCC.
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