MiR-744-5p inhibits the proliferation, invasion, and migration of clear-cell renal cell carcinoma cells by targeting CCND1 / 南方医科大学学报

OBJECTIVE@#To explore the role of miR-744-5p/CCND1 axis in clear-cell renal cell carcinoma (ccRCC).@*METHODS@#We examined the expression levels of miR-744-5p in 65 pairs of ccRCC and adjacent tissue specimens and in 5 ccRCC cell lines and human renal tubular epithelial (HK2) cells using qRT-PCR. The ccRCC cell lines 786-O and OSRC2 were transfected with miR-744-5p mimic, CCND1 mimic, or their negative control mimics, and the changes in cell proliferation, migration, and invasion were evaluated with CCK-8, wound healing, and Transwell assays. The downstream target molecules of miR-744-5p were predicted by bioinformatics analysis, and the expression level of CCND1 in ccRCC cells was verified by qRT-PCR and Western blotting. The relationship between miR-744-5p and CCND1 was further validated by dual luciferase reporter assay, and the role of the miR-744-5p/CCND1 axis in ccRCC was explored by rescue experiments.@*RESULTS@#MiR-744-5p was significantly downregulated in ccRCC tissues and cell lines (all P < 0.05), and its overexpression inhibited the proliferation, migration, and invasion of ccRCC cells (all P < 0.05). Bioinformatics analysis and dual luciferase reporter assay showed that CCND1 was a downstream target of miR-744-5p. The results of rescue experiments showed that upregulation of CCND1 could partially reverse the inhibitory effect of miR-744-5p overexpression on ccRCC cell proliferation, migration, and invasion (all P < 0.05).@*CONCLUSION@#MiR-744-5p inhibits the malignant phenotype of ccRCC cells by targeting CCND1, and the miR-744-5p/CCND1 axis may be a novel target for diagnosis and treatment of ccRCC.

Untargeted contrast-enhanced ultrasound versus contrast-enhanced computed tomography: a differential diagnostic performance (DDP) study for kidney lesions

OBJECTIVES: Histopathology is the 'gold standard' for diagnosing renal cell carcinoma but is limited by sample size. Contrast-enhanced ultrasound can differentiate malignant and benign lesions, but the Chinese guidelines on the management of renal cell carcinoma do not include this method. The purpose of this study was to compare the diagnostic parameters of contrast-enhanced ultrasound against those of contrast-enhanced computed tomography for detecting kidney lesions, with histopathology considered the reference standard. METHODS: Patients with suspected kidney lesions from prior grayscale ultrasonography and computed tomography were included in the analysis (n=191). The contrast-enhanced ultrasound, contrast-enhanced computed tomography, and histopathology data were collected and analyzed. A solid, enhanced mass was considered a malignant lesion, and an unenhanced mass or cyst was considered a benign lesion. The Bosniak criteria were used to characterize the lesions. RESULTS: Contrast-enhanced ultrasound and contrast-enhanced computed tomography both detected that 151 patients had malignant tumors and 40 patients had benign tumors. No significant differences in the tumors and their subtypes were reported between contrast-enhanced ultrasound and histopathology (p=0.804). Chromophobe renal cell carcinoma was detected through contrast-enhanced computed tomography (n=1), but no such finding was reported by contrast-enhanced ultrasound. A total of 35 cases of papillary renal cell carcinoma were reported through contrast-enhanced ultrasound while 32 were reported through histopathology. CONCLUSIONS: Contrast-enhanced ultrasound might be safe and as accurate as histopathology in diagnosing kidney lesions, especially renal cell carcinoma. Additionally, this study provides additional information over histopathology and has an excellent safety profile. Level of evidence: III.

Prognostic significance of the dynamic changes of systemic inflammatory response in metastatic renal cell carcinoma

ABSTRACT Purpose: To elucidate the prognostic value of systemic inflammatory response in patients with metastatic renal cell carcinoma (mRCC) who are treated with sunitinib, we evaluated the prognostic role of C-reactive protein (CRP) kinetics. This study also compared prognostic models containing CRP kinetics and neutrophil-to-lymphocyte ratio (NLR) kinetics. Materials and Methods: A consecutive cohort of 94 patients with mRCC who were treated with sunitinib was retrospectively included from Fudan University Shanghai Cancer Center. According to dynamic changes in CRP and the NLR, patients were divided into three groups for analysis of CRP and NLR kinetics. The associations between survival and potential prognostic factors were assessed. The incremental value of prognostication was evaluated. Results: A significant difference (P<0.001) in overall survival (OS) was observed among the three groups of CRP kinetics. The median OS of the non-elevated group was nearly 1.3-fold longer than that of the normalized group (33.0 vs. 26.3 months), and two times longer than that of the non-normalized group (33.0 vs. 14.0 months). Multivariate analysis showed that CRP and NLR kinetics were independent prognostic indicators. The model containing CRP kinetics had a better predictive accuracy than that with NLR kinetics, which was supported by the C-index (0.731 vs. 0.684) and the likelihood ratio χ2 test (79.9% vs. 44.9%). Conclusion: Our study suggests that dynamic changes in CRP can better predict survival in patients with mRCC who are treated with sunitinib. Routine assessment of CRP before and after targeted therapy would help identify patients at risk of a poor outcome.

Immunohistochemical expressionof sodium-dependent glucose transporter - 2 (SGLT-2) in clear cell renal carcinoma: possible prognostic implications

ABSTRACT Purpose: Glucose is a major energy resource for tumor cell survival and growth, and its influx into cells is mainly carried out by facilitative glucose transporters (GLUTs). Sodium - dependent glucose transporters (SGLTs) have been highlighted as playing important roles in diabetic treatment. However, their potential roles in cancer remain unclear. We examined expression patterns of SGLTs in tumor tissues together with conventional pathological variables to determine prognostic significance in patients with renal cell carcinoma (RCC). Materials and Methods: Nephrectomy specimens were obtained from 68 patients. GLUT - 1, - 2 and SGLT - 1, - 2 expression in tumor and adjacent normal tissues were analyzed by immunohistochemical staining, and intensity was quantified using an image analyzer. Results: The four glucose transporters evaluated were broadly distributed in tumor tissues as well as throughout the normal parenchyma. There was no significant correlation between transporter expression and conventional pathological variables. However, increased SGLT - 2 expression was significantly associated with shorter overall survival (p < 0.01), regardless of metastatic status. Conclusions: We propose possible prognostic significance of SGLT - 2 expression in human RCC. Given that glucose is a major energy resource for tumor cells and that glucose transport is largely mediated by SGLT, SGLT - 2 may serve as a possible therapeutic target in RCC.

Enhancing immune effects of a DNA vaccine against kidney cancer using CD40L as an adjuvant

The use of specific combinations of antigens and adjuvant represents a promising approach for increasing the immunogenicity of DNA vaccines. In the present study, we evaluated the immunity and antitumor effects of DNA vaccines with G250 as the target antigen in a mouse model of renal cell carcinoma. We constructed two recombinant plasmids, pVAX1-G250 and pVAX1-CD40L. The recombinant plasmids were injected into mice by intramuscular injection and electrical pulse stimulation. ELISA and ELISPOT experiments were performed to evaluate the corresponding humoral and cellular immune responses following immunization. To further investigate the antitumor potential of the DNA vaccines, we established a tumor-bearing mouse model expressing G250 target antigen. Our results showed that immunization with the combination of the two plasmids exerted the strongest anti-tumor effects. Therefore, our findings demonstrated the effectiveness of CD40L as an adjuvant for DNA vaccines and highlighted the promising use of these vaccines for the treatment of tumors.

Analysis of various potential prognostic markers and survival data in clear cell renal cell carcinoma

ABSTRACT Purpose Clear cell renal cell cancers frequently harbor Von Hippel-Lindau gene mutations, leading to stabilization of the hypoxia-inducible factors (HIFs) and their target genes. In this study, we investigated the relationship between vascular endothelial growth factor (VEGF), HIF-1α, HIF-2α, p53 positivity, microvessel density, and Ki-67 rates with prognostic histopathologic factors (Fuhrman nuclear grade, stage, and sarcomatoid differentiation) and survival in clear cell renal cell carcinomas. Material and Methods Seventy-two nephrectomy specimens diagnosed as clear cell renal cell carcinoma between 2000 and 2012 were reevaluated. Immunohistochemically VEGF, HIF-1α, HIF-2α, p53, CD34 (for microvessel density evaluation), and Ki-67 antibodies were applied to the tumor areas. The relationships of these antibodies with prognostic factors and survival rates were evaluated with statistical analyses. Results Mean survival time was 105.6 months in patients with ccRCC. Patients with high expression of VEGF, HIF-1α and HIF-2α positivity, a high Ki-67 proliferation index, and a high microvessel density evaluation score had a shorter survival time (p<0.05). Conclusions Our findings supported that with the use of these immunohistochemical markers, prognosis of renal cell carcinoma may be predicted at the first step of patient management. New treatment modalities targeted to HIF-1α and HIF-2α might be planned as well as VEGF-targeted therapies in the management of clear cell renal cell carcinomas.

Increased Tissue Factor Expression is an Independent Predictor of Mortality in Clear Cell Carcinoma of the Kidney

Purpose Increased expression of tissue factor (TF), a primary initiator of the extrinsic coagulation pathway, has been associated with a worse prognosis in a variety of solid tumors. We report for the first time the correlation of the immunohistochemical expression of tissue factor with clinical and pathological outcomes in clear cell carcinomas of the kidney. Materials and Methods immunohistochemical expression of tissue factor was evaluated in 58 paraffin-embedded samples of clear cell carcinomas of the kidney treated at the same university hospital, that was correlated with clinical and pathological variables and with overall survival. Results high intensity tissue factor expression (TF area > 10µm2) was observed in 22.4% of the tumors (13 patients), and was an important predictor of overall mortality, both in univariate and multivariate analysis (p < 0.05). Median overall survival for both groups was 66 months; 78.2 months for patients in the group of lower TF expression and 27.5 months for patients in the group of higher TF expression (log rank p < 0.001). The hazard ratio for mortality was 9.7 (CI 3.7-25.6) for tumors with increased TF expression. Conclusions Increased immunohistochemical expression of TF was an important independent predictor of mortality in a contemporary cohort of patients with clear cell carcinoma of the kidney. Further studies are necessary to define the role of TF in clinical practice. .

Expression of VEGF-A, HIF-1 A, CD34 and Ki67 in clear cell renal cell carcinomas and their relationship with conventional prognostic markers / Expression of VEGF-A, HIF-1 A, CD34 and Ki67 in clear cell renal cell carcinomas and their relationship with conventional prognostic markers.

Clear cell renal carcinoma is the most frequent type of renal carcinoma. Recently, attention has been focused in the expression of angiogenic factors by these tumors, which would justify in part their capacity to grow, invade and disseminate, stating a worse evolution of those patients with an unfavorable angiogenic profile. 83 samples of nephrectomy with a diagnosis of clear cell renal cell carcinoma were studied. Clinical and pathological data were collected. Tumors were studied to assess immunohistochemical expression of the following markers: VEGF-A, HIF-1α, CD34 and Ki67. Results indicated a direct linear relationship between expressions of these four markers. Besides, the expression of HIF-1α was directly related to Furhman grade, invasion of the renal vein and tumor stage. Likewise, tumor proliferation index, assessed with Ki67, was directly related to the presence of necrosis, capsular invasion and advanced tumor stage. Regarding the expression of CD34, vascular density was inversely related to tumor necrosis and overall survival. These findings are controversial compared with the available literature. Then, a research scenery would be open, where the importance of generating prospective and more standardized studies are highlighted to determine the role of these angiogenic factors in tumor evolution and prognostic evaluation of these tumors.

Insulin Receptor Expression in Clear Cell Renal Cell Carcinoma and Its Relation to Prognosis

PURPOSE: Both insulin and insulin-like growth factor (IGF)-1 signaling are key regulators of energy metabolism, cellular growth, proliferation, and survival. The IGF-1 receptor (IGF-1R) is overexpressed in most types of human cancers including renal cell carcinoma (RCC) with poor prognosis. Insulin receptor (IR) shares downstream effectors with IGF-1R; however, the expression and function of IR in the tumorigenesis of renal cancer remains elusive. Therefore, we examined the expression of IR and its prognostic significance in clear cell RCC (CCRCC). MATERIALS AND METHODS: Immunohistochemical staining for IR was performed on 126 formalin-fixed paraffin-embedded CCRCC tissue samples. Eight of these cases were utilized for western blot analysis. The results were compared with various clinico-pathologic parameters of CCRCC and patient survival. RESULTS: IR was expressed in the nuclei of CCRCC tumor cells in 109 cases (87.9%). Higher IR expression was significantly correlated with the presence of cystic change, lower Fuhrman nuclear grade, lower pathologic T stage, and lower TNM stage, although it wasn't significantly related to diabetes status and patient survival. Western blot analyses supported the results of the immunohistochemistry studies. CONCLUSION: IR expression in CCRCC may be associated with favorable prognostic factors.

Insulin Receptor Expression in Clear Cell Renal Cell Carcinoma and Its Relation to Prognosis

PURPOSE: Both insulin and insulin-like growth factor (IGF)-1 signaling are key regulators of energy metabolism, cellular growth, proliferation, and survival. The IGF-1 receptor (IGF-1R) is overexpressed in most types of human cancers including renal cell carcinoma (RCC) with poor prognosis. Insulin receptor (IR) shares downstream effectors with IGF-1R; however, the expression and function of IR in the tumorigenesis of renal cancer remains elusive. Therefore, we examined the expression of IR and its prognostic significance in clear cell RCC (CCRCC). MATERIALS AND METHODS: Immunohistochemical staining for IR was performed on 126 formalin-fixed paraffin-embedded CCRCC tissue samples. Eight of these cases were utilized for western blot analysis. The results were compared with various clinico-pathologic parameters of CCRCC and patient survival. RESULTS: IR was expressed in the nuclei of CCRCC tumor cells in 109 cases (87.9%). Higher IR expression was significantly correlated with the presence of cystic change, lower Fuhrman nuclear grade, lower pathologic T stage, and lower TNM stage, although it wasn't significantly related to diabetes status and patient survival. Western blot analyses supported the results of the immunohistochemistry studies. CONCLUSION: IR expression in CCRCC may be associated with favorable prognostic factors.

Serum protein profiling to identify biomarkers for small renal cell carcinoma.

Diagnostic biomarkers for early detection of renal cell carcinoma (RCC) are in great need. In the present study, we compared the serum protein profiles of patients with small RCC to those of healthy individuals to identify the differentially expressed proteins with potential to serve as biomarkers. Serum samples were collected from 10 patients with small RCC and 10 healthy individuals. The serum protein expression profiles were analyzed by two-dimensional (2-D) gel electrophoresis. Twenty-seven proteins with differences in expression levels between RCC patients and healthy volunteers were identified. Of these, 19 were expressed at different levels and eight were expressed in serum from the RCC group, but not from the control group. Six differentially expressed proteins identified by using mass spectrometry included coagulation factor XIII B, complement C3 and its precursor, misato homolog 1 (isoform CRA_b), hemopexin, and alpha-1-B-glycoprotein. Some of these serum proteins are known regulators of tumor progression in human malignancies. In conclusion, we successfully applied 2-D gel electrophoresis and identified six serum proteins differentially expressed between patients with small RCC and healthy volunteers. These proteins may provide novel biomarkers for early detection and diagnosis of human RCC.

MET Expression in Sporadic Renal Cell Carcinomas

Although germline mutations of met proto-oncogene on human chromosome 7q31-34 have been known as useful molecular markers of hereditary papillary renal cell carcinoma (RCC), the expression of MET, a product of met proto-oncogene, has not been fully studied in sporadic RCC, along with its clinical significance. We investigated the expression of MET by immunohistochemistry in 182 cases of renal neoplasm encompassing 145 RCC, 25 urothelial carcinomas of renal pelvis, and 12 oncocytomas. MET was diffusely and strongly expressed in 90% of papillary RCC, all collecting duct carcinomas, and 92% of urothelial carcinomas of renal pelvis. On the contrary, clear cell RCC, chromophobe RCC, and oncocytomas were negative or focally positive for MET expression. In clear cell RCC, MET expression was positively correlated with high nuclear grade, presence of infiltrative growth, tumoral necrosis, papillary architecture, sarcomatoid component, tumoral involvement of the renal pelvis or ureter, involvement of the calyx, and lymphatic invasion. In conclusion, diffuse and strong expression of MET in papillary RCC and collecting duct carcinoma might be helpful in discriminating from the other subtypes of RCC with tubular or papillary growth. In case of MET expression observed in clear cell RCC, it might correlate with those clinicopathological parameters implying aggressive behavior.

Cyclooxygenase-2 and p53 Expression as Prognostic Indicators in Conventional Renal Cell Carcinoma

The aim of this study was to investigate the relationship of cyclooxygenase (COX) -2 and p53 expression with prognosis in patients with conventional renal cell carcinoma (RCC). Formalin-fixed, paraffin-embedded tissue sections of conventional RCC from 92 patients, who had undergone radical nephrectomy, were examined for COX-2 and p53 expression by immunohistochemistry and compared with clinicopathological variables. The COX-2 expression significantly correlated only with tumor size (p=0.049), whereas the p53 expression profoundly correlated with the TNM stage (p=0.024), M stage (p=0.001), and metastasis (synchronous or metachronous; p= 0.004). The COX-2 overexpression did not significantly associate with p53 positivity (p=0.821). The survival rate of patients correlated with the p53 expression (p < 0.0001) but not with the COX-2 expression (p=0.7506). Multivariate analyses indicated that tumor size, M stage, and p53 expression were independent prognostic factors for cancer-specific survival. The COX-2 expression was not an independent factor. These results show that the increased expression of p53 was associated with metastasis and a worse prognosis in conventional RCC, which suggests that p53 might have played an important role in the progression of conventional RCC. The increased expression of COX-2 was associated only with tumor size, but may not be an important prognostic factor in conventional RCC. No association was observed between COX-2 overexpression and p53 positivity in conventional RCC.

Identification of Proteins Differentially Expressed in the Conventional Renal Cell Carcinoma by Proteomic Analysis

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).

Sarcomatoid renal cell carcinoma with osteosarcomatous differentiation--a case report.

A rare case of sarcomatoid renal cell carcinoma (RCC) with predominantly osteosarcomatous differentiation occurring in a 36-year-old male is reported. Immunohistochemistry excluded the possibility of primary osteosarcoma of the kidney.

Mechanism of Immune Response During Immunotherapy

Tumor immunology embraces an extensive array of biological phenomena that include interactions between neoplastic cells and the innate and adaptive immune response. Among immune cells, T cells have taken the center stage because they can be easily demonstrated to specifically recognize autologous cancer cells. However, their role is limited and other components of the immune response are likely necessary for the completion of cancer rejection. Metastatic melanoma and renal cell carcinoma (RCC) are malignancies strongly predisposed to regress in response to the systemic administration of high-dose interleukin (IL)-2. Several clinical Studies in extensive cohorts of patients have shown that this treatment can induce complete or partial clinical regressions of metastatic disease in 15 to 20% of patients who receive this treatment.1-6 Although IL-2 has direct pluri-potent effects on cells with immune and inflammatory function, it remains unexplained which cell subset is implicated in mediating tumor regression. In a quest to characterize the mechanism of action of IL-2 during the course of immunotherapy, we have investigated the early changes in transcriptional profiles of circulating mononuclear cells and microenvironment of melanoma metastases following high dose IL-2 administration (720,000 IU/kg) by serial sampling of blood cells and tumors in the form of fine needle aspirate (FNA).7 Furthermore, studies are currently ongoing to characterize the proteomic profiling of RCC patients undergoing the same treatment using protein arrays (manuscript in preparation). The predominant activation of genes related to inflammation and activation of mononuclear phagocytes lead us to further characterize this cell subset in the context of stimulation with a panel of soluble factors potentially present in the circulation and tumor microenvironment.

Suppression of NF-kappaB activation in normal T cells by supernatant fluid from human renal cell carcinomas

T lymphocytes from patients with renal cell carcinoma (RCC) show reduced immune function and impaired activation of the transcription factor, NF-kappaB. We determined the mechanism of NF-kappaB suppression in T cells of RCC patient and determined whether supernatant fluid from RCC explants (RCC-S) induced the same phenotype of NF-kappaB suppression in normal T cells that is observed in patient T cells. The pattern of kappaB-binding activity in T cells of RCC patient was altered as compared to that seen in T cells obtained from normal volunteers. In some patients, no activation of RelA/NFkappaB1-binding activity was detectable, while in others kappaB-binding activity was modestly induced but the duration was reduced. IkappaBalpha was degraded normally following stimulation in both normal controls and T cells from RCC patients. RCC-S did not alter the cytoplasmic levels of RelA and NF-kappaB1 but did suppress their nuclear localization and inhibited the activation of RelA/NF-kappaB1 binding complexes. These results show that RCC-S can induce in normal T cells the same phenotype of impaired NF-kappaB activation that is detected in T cells of RCC patient. It also appears that NF-kappaB suppression by RCC-S may contribute to the immunosuppression of host immunity.