Expression of CIDE proteins in clear cell renal cell carcinoma and their prognostic significance
Clear cell renal cell carcinoma (ccRCC) is the major and aggressive subtype of renal cell carcinoma. It is known to derive its histologic appearance from accumulation of abundant lipids and glycogens. The cell death-inducing DFF45-like effector (CIDE) family has been characterized as the lipid droplet proteins involved in the metabolism of lipid storage droplets. The purpose of this study was to evaluate the expression of CIDE proteins in ccRCC cells and to investigate their prognostic significance. We examined consecutive patients with sporadic ccRCC, who underwent nephrectomy, to measure their mRNA and protein expression of CIDE proteins. We found that Cidec and ADRP expression were significantly up-regulated in ccRCC, compared with normal kidney tissues. Cideb was down-regulated. We also found that Cideb was expressed more in low-grade ccRCC than in high-grade tumors. To further clarify the relationship between Cideb expression and patient prognosis, we evaluated 57 ccRCC patients followed up for 120 months. Reduced ccRCC Cideb expression was associated with a higher Fuhrman nuclear grade. Patients with high Cideb expression had better overall survival rate than those with low expression (p < 0.05). Cideb expression was an independent predictor of survival (p = 0.001). Although the biologic function of Cideb in ccRCC remains unknown, the expression level of Cideb might be a novel predictor of prognosis in ccRCC.Electronic supplementary materialThe online version of this article (doi:10.1007/s11010-013-1605-y) contains supplementary material, which is available to authorized users.
Introduction<!><!>Oil Red O staining<!>Electron microscopy<!><!>Immunohistochemistry<!>Western blot<!>Statistical analyses<!><!>Discussion<!>
<p>Renal cell carcinoma (RCC) is the most common malignant tumor in the adult kidney, accounting for about 3 % of human malignancies [1]. On the basis of current genetic knowledge and histologic findings, RCC can be classified into at least four major subtypes: clear cell (ccRCC), papillary, chromophobe, and collecting duct carcinoma [2, 3]. Among them, ccRCC represents the most common subtype (83 %) [4]. Unfortunately, ccRCCs show an extremely variable clinical course, which cannot be predicted. Additional prognostic markers are needed for a more accurate determination of the prognosis and the improvement of therapeutic strategies.</p><p>Clear cell RCC is characteristically of a bright golden color, and the clear appearance of tumor cells is due to cellular storage of lipid and glycogen [5–7]. Some epidemiologic studies have shown that obesity is a risk factor for RCC [8, 9]. In general, previous studies suggested that obese patients (with a BMI >30 kg/m2) were associated with a high proportion of ccRCC [10–12]. It is apparent that ccRCC cells possess some abnormalities in the metabolism of lipids and glycogen.</p><p>The cell death-inducing DFF45-like effector (CIDE) family has been characterized as the crucial lipid droplet (LD) proteins involved in the formation and stabilization of lipid storage droplets [13, 14]. The CIDE family is composed of three members: Cidea, Cideb, and Cidec (CIDE-3 or Fat-specific protein 27) [14]. Previous studies have suggested that Cidea is predominantly expressed in brown adipose tissue [15], and mammary glands [16], while Cidec is expressed at high levels in white adipose tissue [17]. Cideb is strongly expressed in the liver and kidney, in both mice and humans [18, 19].</p><p>The presence of abundant LDs suggests that LD proteins are associated with the development of ccRCC. It has been reported adipose differentiation-related protein (ADRP), a LD protein, is highly up-regulated both at the transcriptional and protein levels in ccRCC [20]. The role of CIDE family proteins has not been evaluated. In this study, we measured the CIDE family protein expression in ccRCC using real-time quantitative PCR and western blot. CIDE family expression levels were correlated with the malignancy of ccRCC. We examined 57 consecutive patients with ccRCC to evaluate Cideb expression level in primary tumors and its prognostic significance.</p><!><p>Clinical and histopathological characteristics</p><!><p>The frozen sections of fresh renal tissues and ccRCC samples were stained with Oil Red O. In brief, cryopreserved tissues were cut into 10-μm sections, and fixed in 10 % formalin for 5 min. Sections were washed in 60 % isopropanol for 2 min, then incubated in Oil Red O (Sigma, USA) working solution for 15 min. The stained tissues were washed using 60 % isopropanol, and then water, to remove residual staining. Slides were counterstained in hematoxylin for 2 min. The slides were mounted with aqueous mounting media and glycerin jelly, and examined under light microscopy.</p><!><p>The tissues for electron microscopy were fixed in 2.5 % cold glutaraldehyde overnight, at 4 °C. Tissues were then rinsed for 1 h in cold phosphate buffer solution (PBS, 0.1 M, pH 7.4) and fixed in 1 % osmium tetroxide for 1 h. Tissues underwent gradient acetone dehydration, were Epon 812 resin embedded, and ultra-thin sections (70 nm) were cut onto slides. Sections were stained with uranyl acetate and lead citrate for JEM-1011 transmission electron microscope observation.</p><!><p>Primer sequences</p><!><p>We had 57 pathologically confirmed ccRCC patients with long-term follow-up, and obtained the patient's formalin-fixed paraffin-embedded specimens. All samples were dewaxed in xylene three times for 5–10 min, rehydrated in descending alcohol gradients for 5 min, and blocked for endogenous peroxidase (3 %H2O2 in 80 %methanol) for 20 min. Antigen retrieval was performed using two treatments in 10 mM sodium citrate in a microwave for 15 min. After blocking non-specific antigen with normal goat serum for 30 min, the slides were incubated with mouse anti-Cideb monoclonal antibody (dilution at 1:200) overnight at 4 °C. Slides were incubated with 100–200 μl of labeled secondary antibody for 30 min at room temperature. Visualization was performed using diaminobenzidine (DAB). The slides were counterstained in hematoxylin for 2 min, dehydrated in ethanol, and mounted. The Cideb expression was evaluated in ccRCC. Low expression was considered as absence or <20 % expression in RCC, and high expression was considered as ≥20 % expression in RCC.</p><!><p>From 10 frozen tumor tissues and 10 normal renal tissues, total samples were separated in a 10 % SDS-PAGE gel and transferred onto a Immobilon-Polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, MA, USA). After blocking with 5 % skimmed milk, the membrane was incubated with mouse anti-Cideb monoclonal antibody (dilution at 1:2,000), rabbit anti-Cidea antibody (dilution at 1:1,000), rabbit anti-Cidec (dilution at 1:4,000), or mouse anti-GAPDH (dilution at 1:1,000). After washing, membranes were incubated for 1 h with a 1:5,000 dilution of peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin (Santa Cruz Biotechnology, Santa Cruz, CA) and expression characterized by chemiluminescence.</p><!><p>Statistical analysis was performed using the SPSS 13.0 software. Statistical analyses were performed with independent samples for t test and cox proportional hazards regression model. p value <0.05 was considered as statistically significant.</p><!><p>Increased lipid droplets in ccRCC. Images of renal sections stained with Oil Red O from normal renal and ccRCC (a, b). The red
color spots in Oil Red O staining represent the lipid droplets in the ccRCC(B). scale
bars 50 μm. c transmission electron micrograph of renal sections from ccRCC(original magnification ×5000). L,lipid droplets. (Color figure online)</p><p>a RT-PCR results of CIDE family in ccRCC and adjacent noncancerous tissues. b Expression of CIDE protein in ccRCC and adjacent noncancerous tissues. β-tubulin was used as an internal control</p><p>a Relative mRNA expression by quantitative real-time PCR analysis.*p = 0.018. The data are shown as mean ± SE. b Western blot analysis of low grade and high grade ccRCC</p><p>Correlation between Cideb immunostain and Fuhrman nuclear grade in clear cell RCC. a liver(positive control) b normal renal c grade 1 ccRCC d grade 2 ccRCC e grade 3 ccRCC f grade 4 ccRCC (original magnification ×400)</p><p>Univariate and multivariate analyses of cancer-specific survival in 57 patients with ccRCC</p><p>RR risk ratio, 95 % CI 95 % confidence interval</p><p>The association of survival with different levels of Cideb expression is illustrated in 57 ccRCC patients (p < 0.01). Patients with high expression of Cideb had longer survival than those with low expression of Cideb</p><!><p>It is well known that the clear appearance of tumor cells results from cellular storage of lipid and glycogen [7]. Clear-cell RCC with lower nuclear grade show a typical "clear-cell" appearance. However, as nuclear grade increases, the "clear-cell" character diminishes, and the number of LD decreases. Some LD proteins (such as ADRP, adipose differentiation-related protein or adipophilin) have been shown to have a role in clear-cell renal carcinoma differentiation [20]. The microvessel density in ccRCC tends to decrease as the tumor grade increases [22, 23].</p><p>The CIDE family regulates lipid metabolism and plays an important role in the development of metabolic disorders including obesity, insulin resistance, and hepatic steatosis [15, 17, 24–26]. We identified CIDE family members which were associated with LD storage in ccRCC. Compared with normal kidney tissues, there was significant up-regulation of Cidec and down-regulation of Cideb in ccRCC, but little change in Cidea. Cidea was a BAT-specific marker, while Cidec was most highly expressed in WAT [14]; thus, the different ccRCC expression levels suggest that the lipid storage in ccRCC is more related to WAT than BAT. In addition, we observed that the mRNA levels of Cidec in high-grade ccRCC was slightly higher than that in low-grade ccRCC, but the protein levels of Cidec were similar. Several possibilities might be involved in the regulation of Cidec, such as protein degradation, transcriptional regulation, etc.. Normally, Cideb is expressed at a high level in liver and kidney tissues [18]. Although CIDE proteins have been shown to regulate the biosynthesis and storage of LDs in adipocytes and hepatocytes [14, 18, 25], the function of CIDE proteins in ccRCC has not been determined.</p><p>It is well known that ccRCC contains abundant lipids in the cytoplasm, including triglycerides, cholesterol esters, and phospholipids. These lipids impart the typical gross "yellow" appearance [1, 21, 27]. We confirmed the abundant lipid accumulation in ccRCC using Oil Red O and electron microscopy. We found that Cidec protein expression significantly increased, while Cidea expression did not change in ccRCC, compared with normal renal tissue. The different Cidea and Cidec expression levels in ccRCC suggest that lipid storage in ccRCC is more related to WAT than BAT. The down-regulation of Cideb in ccRCC suggests it may prevent the formation of ccRCC cells. We speculated that Cideb could promote lipid secretion in renal cells, similar to its functions in liver cells [18]. These data strongly suggest that the decreasing level of Cideb protein is implicated in lipid uptake and storage in clear-cell RCC. Moreover, Cideb expression levels were likely to reflect microscopic morphologic appearances and the degree of malignancy of clear-cell RCC.</p><p>Using IHC, we found that the expression of Cideb in both grade 3 and grade 4 were lower than that in grade 1 and grade 2, while the protein level of Cideb in grade 1 were similar to grade 2. The differences of Cideb in the ccRCC were similar to ADRP [28]. The higher Fuhrman nuclear grade was associated with poor prognosis [29]. Multivariate analysis confirmed that reduction in the expression of Cideb was an independent prognostic factor related to shorter progression-free survival (p = 0.001). Thus, low expression of Cideb might be applied as a novel prognostic marker. Collectively, these findings suggest that ccRCC has abnormal lipid metabolism. We observed that the expression of Cideb was largely decreased at both mRNA and protein levels in ccRCC. Our data indicate that the loss of Cideb expression is an important event in lipogenesis and progression of ccRCC.</p><p>The mechanism of abnormal lipid metabolism in ccRCC has not been fully elucidated. It is noteworthy that lipogenesis is part of the malignant process in RCC [30]. In this study, we have shown that low Cideb expression is correlated with higher nuclear grade of the ccRCC and poor clinical outcome. A further, larger study should be performed to extend and validate the precise mechanism by which Cideb regulates lipid metabolism and tumor progression in ccRCC.</p><!><p>Supplementary material 1 (DOCX 20 kb)</p><p>Ming Yu, Hui Wang and Jun Zhao Contributed equally to this study.</p>