Clear Renal Cell Carcinoma Samples Research Articles

Prognostic Significance of Elevated UCHL1, SNRNP200, and PAK4 Expression in High-Grade Clear Cell Renal Cell Carcinoma: Insights from LC-MS/MS Analysis and Immunohistochemical Validation.

Recent advancements in proteomics have enhanced our understanding of clear cell renal cell carcinoma (CCRCC). Utilizing a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by immunohistochemical validation, we investigated the expression levels of UCHL1, PAK4, and SNRNP200 in high-grade CCRCC samples. Our analysis also integrated Reactome pathway enrichment to elucidate the roles of these proteins in cancer-related pathways. Our results revealed significant upregulation of UCHL1 and SNRNP200 and downregulation of PAK4 in high-grade CCRCC tissues compared to non-cancerous tissues. UCHL1, a member of the ubiquitin carboxy-terminal hydrolase family, showed variable expression across different tissues and was notably involved in the Akt signaling pathway, which plays a critical role in cellular survival in various cancers. SNRNP200, a key component of the RNA splicing machinery, was found to be essential for proper cell cycle progression and possibly linked to autosomal dominant retinitis pigmentosa. PAK4's role was noted as critical in RCC cell proliferation and invasion and its expression correlated significantly with poor progression-free survival in CCRCC. Additionally, the expression patterns of these proteins suggested potential as prognostic markers for aggressive disease phenotypes. This study confirms the upregulation of UCHL1, SNRNP200, and PAK4 as significant factors in the progression of high-grade CCRCC, linking their enhanced expression to poor clinical outcomes. These findings propose these proteins as potential prognostic markers and therapeutic targets in CCRCC, offering novel insights into the molecular landscape of this malignancy and highlighting the importance of targeted therapeutic interventions.

The vasculogenic mimicry related signature predicts the prognosis and immunotherapy response in renal clear cell carcinoma

BackgroundClear cell carcinoma of the kidney is a common urological malignancy characterized by poor patient prognosis and treatment outcomes. Modulation of vasculogenic mimicry in tumor cells alters the tumor microenvironment and the influx of tumor-infiltrating lymphocytes, and the combination of its inducers and immune checkpoint inhibitors plays a synergistic role in enhancing antitumor effects.MethodsWe downloaded the data from renal clear cell carcinoma samples and vasculogenic mimicry-related genes to establish a new vasculogenic mimicry-related index (VMRI) using a machine learning approach. Based on VMRI, patients with renal clear cell carcinoma were divided into high VMRI and low VMRI groups, and patients’ prognosis, clinical features, tumor immune microenvironment, chemotherapeutic response, and immunotherapeutic response were systematically analyzed. Finally, the function of CDH5 was explored in renal clear cell carcinoma cells.ResultsVMRI can be used for prognostic and immunotherapy efficacy prediction in a variety of cancers, which consists of four vasculogenic mimicry-related genes (CDH5, MMP9, MAPK1, and MMP13), is a reliable predictor of survival and grade in patients with clear cell carcinoma of the kidney and has been validated in multiple external datasets. We found that the high VMRI group presented higher levels of immune cell infiltration, which was validated by pathological sections. We performed molecular docking prediction of vasculogenic mimicry core target proteins and identified natural small molecule drugs with the highest affinity for the target protein. Knockdown of CDH5 inhibited the proliferation and migration of renal clear cell carcinoma.ConclusionsThe VMRI identified in this study allows for accurate prognosis assessment of patients with renal clear cell carcinoma and identification of patient populations that will benefit from immunotherapy, providing valuable insights for future precision treatment of patients with renal clear cell carcinoma.

Development and validation of a tumor tissue based multivariate biomarker for predicting angiogenesis inhibitor clinical benefit in renal cell carcinoma (RCC).

467 Background: Angiogenesis inhibitors, including vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs), are standard of care therapy (alone and in combination) for several advanced cancers, including RCC. There is an unmet need for a biomarker to identify patients with RCC most likely to benefit from VEGFR TKIs to guide individualized treatment decision making. Hence, we sought to develop and validate a biomarker for predicting single-agent systemic VEGFR TKI benefit in patients with RCC. Methods: Candidate biomarkers were selected by co-expression patterns in VEGFR TKI sensitive/resistant tumor types from pan-solid tumor TCGA expression profiling data and the literature. The angiogenesis inhibitor treatment response score (Angio TRS) was developed as a multivariate expression-based algorithm from RNA-based quantitative transcriptional profiling (qTP) performed in parallel with clinical FFPE tumor based comprehensive genomic profiling, with the Angio TRS High/Low threshold set at the median of clear cell RCC samples. The locked Angio TRS (and High/Low status) was then validated in a cohort of adult RCC patients treated with a systemic line of single agent VEGFR TKI therapy within a prospective observational trial; group outcomes (by time to next therapy [TTNT]) were compared by univariate analysis, Cox proportional hazards modeling (adjusting for age, biologic sex, histology, nuclear grade [incorporating sarcomatoid features], therapy line, and TKI type), and unadjusted Kaplan Meier analysis. Laboratory information for IMDC risk group status was not available. Results: Across 3,721 solid tumor tissue samples, median Angio TRS was highest in known VEGFR TKI sensitive tumors (RCC, thyroid carcinoma, sarcoma). Angio TRS was then validated in a separate cohort of 86 patients with RCC treated with single agent systemic VEGFR TKI (median follow-up 29.2 months; 76% male, 77% clear cell, 14% with sarcomatoid features, 84% 1st line, and 71% treated with sunitinib, pazopanib or axitinib); 52% of patients were Angio TRS High. By univariate analysis, Angio TRS status (p=0.008) and nuclear grade 4 (with or without sarcomatoid features; p=0.06 & 0.07) were most significantly associated with VEGFR TKI TTNT; by multivariate analysis, only Angio TRS status was significantly associated with TTNT (High vs. Low median TTNT 15.8 vs. 5.6 months, adjusted hazard ratio 0.46, p=0.012). Conclusions: Angio TRS is a multivariate expression-based algorithm performed on FFPE tumor tissue, validated to be prognostic of clinical outcome for patients with RCC treated with single agent VEGFR TKI when controlling for clinical factors. Angio TRS may support individualized treatment decision making in patients with advanced RCC. Additional independent validation in a cohort of Kaiser Permanente patients will be presented. Clinical trial information: NCT03061305 .

Antitumor activity of RUNX3: Upregulation of E-cadherin and downregulation of the epithelial-mesenchymal transition in clear-cell renal cell carcinoma.

RUNX3 is a transcription factor and tumor suppressor that is silenced or inactivated in diverse tumors. The effect of RUNX3 on the epithelial-mesenchymal transition in clear-cell renal cell carcinoma (CCRCC) remains unclear. We determined the expression of RUNX3 and E-cadherin in tumor tissues and adjacent normal tissues of 30 CCRCC patients; established cultured CCRCC cells with the overexpression of RUNX3; and examined the in vivo tumorigenic function of RUNX3 in a nude mouse xenograft model of CCRCC. RUNX3 and E-cadherin were downregulated in human CCRCC samples. Cell lines with RUNX3 overexpression had reduced cell proliferation, invasion, and migration, a prolonged cell cycle, increased apoptosis, and increased expression of E-cadherin. In the nude mouse xenograft model of CCRCC, tumors with the overexpression of RUNX3 had smaller volumes and weights and had increased expression of E-cadherin. In conclusion, RUNX3 overexpression increased the level of E-cadherin and inhibited the proliferation, invasion, and migration of CCRCC in vitro and in vivo. RUNX3 has potential use as a biomarker for prognostic monitoring of CCRCC and as a therapeutic target for the treatment of this cancer.

Whole exome sequencing reveals molecular mechanisms of clear cell renal cell carcinoma with sarcomatoid differentiation

Objective: To investigate the molecular mechanisms of clear cell renal cell carcinoma (CCRCC) with sarcomatoid differentiation (CCRCCS) and to explore new therapeutic targets for CCRCCS. Methods: Whole exome sequencing was performed on the carcinomatous and sarcomatoid components of five CCRCCS cases collected from January 2017 to October 2018. A highly frequent non-synonymous mutation of cadherin 23 (CDH23) was revealed by whole exome sequencing and further studied in additional samples. The sequencing of CDH23 in 40 specimens with CCRCCS and 50 specimens with CCRCC collected from January 2008 to October 2018 were conducted using Sanger sequencing. Immunohistochemistry was carried out to detect the protein expression of CDH23 in the additional 90 cases. Results: Carcinomatous and sarcomatoid components of CCRCCS shared most of the somatic single-nucleotide variants (SSNVs) as revealed through whole exome sequencing, while the sarcomatoid component had higher overall SSNVs than carcinomatous component. A highly frequent non-synonymous mutation of CDH23 (p.Arg1804Gln) was observed both in carcinomatous and sarcomatoid components of CCRCCS that resulted in the alteration in the highly conserved calcium-binding site mediating the functions of cadherins. In the additional 90 specimens, CDH23 mutation was much frequently detected in CCRCCS than that in CCRCC samples and even the high grade CCRCC. CDH23 protein was not or weakly expressed in most CCRCCS specimens with CDH23 mutation. There was an correlation between CDH23 gene mutation and negative expression of its protein (r=0.598, P<0.01). Conclusions: The present study reveals, for the first time, that the mutation of CDH23 (p.Arg1804Gln) is a genetic risk factor for CCRCCS. It is associated with the decreased expression of CDH23 protein, resulting in the absence of cadherin function of CDH23, indicating that CDH23 mutation may be involved in the sarcomatoid transformation in CCRCCS. Thus, CDH23 might be a potential therapeutic target for CCRCCS.

A novel highly frequent single‑nucleotide polymorphism site of cadherin 23 in clear cell renal cell carcinoma with sarcomatoid differentiation based on whole exome sequencing.

Clear cell renal cell carcinoma (CCRCC) with sarcomatoid differentiation (CCRCCS) displays invasive behavior, poor prognosis, and poor therapeutic response. The present study was aimed to gain new insights into the molecular mechanisms of sarcomatoid transformation, and identify new prognostic and therapeutic targets for CCRCCS. Whole exome sequencing was performed on matched carcinomatous and sarcomatoid elements from five specimens with CCRCCS. A non‑synonymous single‑nucleotide polymorphism (SNP) of cadherin23 (CDH23) was further studied through Sanger sequencing in expanded 40specimens with CCRCCS and 50specimens with CCRCC. Carcinomatous and sarcomatoid elements shared most somatic single‑nucleotide variants (SSNVs) as revealed through whole exome sequencing. Sarcomatoid element had higher overall SSNVs than carcinomatous element. A highly frequent mutation of CDH23 (rs3802711) was observed in CCRCCS that resulted in an alteration in the highly conserved calcium‑binding site in the three‑dimensional (3D) structure mediating the functions of cadherins. In the expanded 90 specimens, CDH23 SNP (rs3802711) was a highly frequent mutation in CCRCCS than that in all CCRCC samples and even high grade CCRCC. Cox multivariate analysis indicated that CDH23 (rs3802711) genotype was an independent prognostic factor affecting the overall survival of the cohort. CDH23 gene and protein were negatively or weakly expressed in most CCRCCS specimens with CDH23 mutation. The present study revealed, for the first time, that the CDH23 (rs3802711) was a highly genetic risk factor for CCRCCS. It was associated with the decreased expression of CDH23 protein, resulting in the absence of cadherin function of CDH23, indicating that the CDH23 mutation may be involved in the sarcomatoid transformation in CCRCCS. Collectively, a novel and specific SNP of CDH23 was identified in CCRCCS and a new candidate cadherin involved in EMT was revealed. Furthermore, a new prognostic evaluation factor and potential therapeutic target for CCRCCS was identified.

Expression of BCAN gene in clear renal cell carcinoma and its clinical significance

Objective To analyze the expression of BCAN gene in clear renal cell carcinoma(ccRCC)and its effect on the prognosis of patients. Methods The expression of BCAN in ccRCC in Oncomine database was explored. The clinical data and information about target gene expression of patients with ccRCC were obtained from the TCGA database for statistical analysis. Gene enrichment analysis was carried out using GSE73731 data set of clear renal cell carcinoma samples in GEO database. Protein related to BCAN was analyzed by String database. Results Among the patients with ccRCC, the patients with low BCAN expression were significantly lower than the high expression group in terms of pathological stage and T stage(P 0.05). The overall survival of patients in the low expression group of BCAN was better than that in the high expression group(P=0.033). The ccRCC samples in the high expression group of BCAN gene were mainly concentrated in KRAS signaling pathway. Conclusions BCAN can promote the invasive ability of tumor cells through various ways, which is expected to be one of the important biomarkers for the poor prognosis of ccRCC. Key words: Carcinoma, Renal Cell; Brevican

Expression of pituitary transforming gene 1 in renal clear cell carcinoma and its association with prognosis

Objective To inyestigate the expression of pituitary transforming gene 1 (PTTG1) in renal clear cell carcinoma (RCCC) tissues and explore its association with the prognosis. Methods PTTG1 mRNA and protein levels were assessed in 41 paired RCCC tissues and adjacent normal tissues by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and immunohistochemistry respectively. Further immunohistochemical analysis was implemented in 89 samples of RCCC to evaluate the associations between PTTG1 levels and the clinical characteristics in RCCC. Results FQ-PCR and immunohistochemical analysis demonstrated that the PTTG1 mRNA and protein levels were significantly higher in RCCC compared to normal tissues (P=0.007). In addition, the PTTG1 protein level in 89 RCCC samples was found to be significantly correlated with Fuhrman grade (P=0.026) and TNM stage (P=0.031), whereas it was not associated with gender (P=0.123), age (P=0.338) and recurrence (P=0.285). The five-year survival rate was 35.3% and 72.7% in patients with high and low PTTG1 levels respectively. The survival time of RCCC patients with high PTTG1 level was significantly lower than those with low PTTG1 1evel (P=0.029). Conclusion The overexpression of PTTG1 indicates a poor prognosis in RCCC patients and, therefore, PTTG1 may serve as a novel prognostic marker for RCCC. Key words: Pituitary transforming gene 1; Renal clear cell carcinoma; Survival rate; Prognosis

Study on the mechanism behind lncRNA MEG3 affecting clear cell renal cell carcinoma by regulating miR-7/RASL11B signaling.

The goal of this research was to study the relationships between maternally expressed gene 3 (MEG3), microRNA-7 (miR-7), and RASL11B, and explore their influence on the progression of clear cell renal cell carcinoma (CCRCC). Microarray analysis was conducted using the data provided by The Cancer Genome Atlas. The expression levels of MEG3 and miR-7 in CCRCC and adjacent tissue samples were ascertained by quantitative real-time polymerase chain reaction (qRT-PCR). The cell proliferation activity was unmasked by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell apoptosis and cell cycle were investigated by flow cytometry. A dual luciferase reporter assay was used to verify target relationships. Wound healing assay and transwell assay were used to detect cell migration and invasion ability. Decreased MEG3 expression was observed in CCRCC tissues and cells. Overexpression of MEG3 accelerated apoptosis; inhibited cell proliferation, migration and invasion; and induced G0/G1 phase cell cycle arrest in CCRCC. MiR-7, directly binding to MEG3, was overexpressed in the CCRCC tissues and could inhibitthe apoptosis and promote the migration and invasion of CCRCC cells. RASL11B, lowly expressed in CCRCC, was a target of miR-7. After the overexpression of RASL11B, G0/G1 phase cell cycle arrest was induced; cell apoptosis was promoted; and the proliferation, invasion, and migration of CCRCC cells were inhibited. MEG3 could up-regulate RASL11B to inhibit the cell proliferation, invasion, and migration; induce G0/G1 cell cycle arrest; and promotecell apoptosis by suppressing miR-7 in CCRCC.

CD73 expression in primary and metastatic renal cell carcinoma (RCC).

643 Background: Agents targeting the PD-1 pathway have improved outcomes in RCC. CD73 may be an additional mechanism which tumors can exploit for immune evasion. It is regulated by hypoxia inducible factor (HIF) and converts AMP to adenosine. The resulting increase in extracellular adenosine can inhibit T-cell effector function. We evaluated CD73 expression in primary and metastatic tumor samples in patients with RCC. Methods: A commercial TMA (US Biomax) consisting of 31 primary clear cell RCC samples (2 with sarcomatoid features) with 8 matched and 1 unmatched metastases was used to assess CD73 expression. A genitourinary pathologist (OH) confirmed pathology and grade. Immunohistochemistry (IHC) was performed using a monoclonal anti-CD73 antibody (Cell Signaling; D7F9A). A combined score (CS: % of cells positive x intensity) was employed to quantify CD73 expression. Expression levels between matched primary and metastatic tissue was compared using Wilcoxon signed-rank test. Correlation of CD73 expression (CS &gt; 0) in primary tumor with baseline clinical and pathologic characteristics was assessed using Kruskal-Wallis and Fisher's exact tests. Overall survival (OS) was estimated using the Kaplan-Meier method. Comparison between CD73 expression groups used log rank test. Results: CD73 expression was seen in 19% (n = 6) of primary and 66.7% (n = 6) of metastatic samples. Among matched primary and metastatic samples (n = 8 each), median CS was 25 (Q1-Q3:0-105) in metastatic samples while none of the corresponding primary samples demonstrated CD73 expression (median CS = 0, p = 0.062). CD73 expression in primary tumor samples did not correlate with baseline clinical or pathologic features. Five-year OS was 50% in patients who expressed CD73 in primary tumors compared to 84% in those who did not (p = 0.26). Conclusions: We observed a numerical increase in CD73 expression in metastatic tissue compared to primary RCC samples and worse 5 year OS. The small number of samples has limited statistical significance. Further studies are needed to examine the impact of CD73 expression on outcomes in RCC patients treated with currently approved checkpoint inhibitors and the investigational CD73 antagonists that are in early phases of development.

Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma.

Inhibitor of growth family member 4 (ING4) is a candidate tumor suppressor that serves important roles in tumor growth and angiogenesis. In the present study ING4 expression was assessed in clear cell renal carcinoma (CCRC) tissues and its association with the progression of CCRC was determined. The expression of ING4 in 125 patients with CCRC was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunohistochemical methods. A total of 40 adjacent normal renal tissues were used as control samples. The results identified that ING4 expression was positive in 100% of normal renal tissues, but in only 82.3% CCRC samples. RT-qPCR and western blotting results demonstrated that expression levels of ING4 mRNA and protein were significantly decreased in CCRC compared with in normal tissues (P<0.0001). ING4 expression was not associated with sex, age or tumor volume (P>0.05), but was associated with the nuclear grade of renal cancer (P<0.0001), the clinical stage of CCRC (P<0.0001) and lymphatic metastasis (P<0.0001). The results of the present study indicated that dysregulation of ING4 expression may be involved in the initiation and progression of CCRC.

Notch Pathway Is Activated via Genetic and Epigenetic Alterations and Is a Therapeutic Target in Clear Cell Renal Cancer

Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. Transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls revealed overexpression of NOTCH ligands and receptors in tumor tissues. Examination of the TCGA RNA-seq data set also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival. Parallel DNA methylation and copy number analysis demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligand JAGGED1 was overexpressed and associated with loss of CpG methylation of H3K4me1-associated enhancer regions. JAGGED2 was also overexpressed and associated with gene amplification in distinct CCRCC samples. Transgenic expression of intracellular NOTCH1 in mice with tubule-specific deletion of VHL led to dysplastic hyperproliferation of tubular epithelial cells, confirming the procarcinogenic role of NOTCH in vivo Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression, and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain. Treatment with the γ-secretase inhibitor LY3039478 led to inhibition of CCRCC cells in vitro and in vivo In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target.

Alteration of ASIC1 expression in clear cell renal cell carcinoma.

BackgroundAcidic extracellular pH is a major feature of tumor tissue. Acid-sensing ion channels (ASICs) represent an H+-gated subgroup of the degenerin/epithelial Na+ channel family and are activated by acidic microenvironment. Little is known about the expression and clinical significance of ASICs in solid tumors. The purpose of this study was to examine the expression of ASIC1 in human clear cell renal cell carcinoma (CCRCC) and to determine if the expression of ASIC1 is associated with clinicopathological features.MethodsThe expression of ASIC1 in CCRCC tissues at the mRNA and protein levels was determined by real-time quantitative polymerase chain reaction and Western blot analysis, respectively. A tissue microarray was used to assess the expression of ASIC1 protein in tumor tissue and matched adjacent normal tissues from 75 patients with CCRCC.ResultsASIC1 expression was detected in normal renal and CCRCC samples. The expressions of ASIC1 protein and mRNA were significantly decreased in the CCRCC tissues compared with matched normal renal tissues (P<0.05). The staining density measurement showed that the expression of ASIC1 was significantly decreased in stage I (P=0.037), stage II (P=0.026), and stage III (P=0.026), grades I–II CCRCC (P=0.004), and CCRCC from male patients (P=0.00002). However, no significant difference was observed for ASIC1 expression between CCRCC and normal tissue in patients with stage IV CCRCC (P=0.236), patients with grades III–IV CCRCC (P=0.314), and female patients (P=0.095). Spearman correlations demonstrated that ASIC1 expression did not correlate to tumor stage (correlation coefficient [CC =0.168], P=0.149) and the age of patients (CC −0.147, P=0.688) but showed a positive correlation to higher tumor grades (CC =0.270, P=0.018).ConclusionASIC1 is downregulated in CCRCC. ASIC1 expression may be potentially used as a novel biomarker and even a CCRCC therapeutic target. Further efforts will be made to clarify the mechanism of ASIC1 in occurrence, progression, and metastasis of CCRCC.

High expression of APRIL correlates with poor prognosis in clear cell renal cell carcinoma

IntroductionThe members of the Tumor Necrosis Factor Superfamily (TNFSF), including A PRoliferation Inducing Ligand (APRIL), have been studied in RCC and other tumors. In this study, we investigated the expression of APRIL in resected clear cell renal cell carcinoma (CCRCC) samples by immunohistochemistry (IHC) and analyzed its association with the clinicopathologic characteristics and prognosis of the patients. MethodsWe examined 206 CCRCC samples from patients who underwent radical or partial nephrectomy at Seoul National University Hospital between 1999 and 2002. Tissue microarray (TMA) blocks were made, and immunohistochemical staining for APRIL expression was performed. ResultsWe classified the IHC results as high expression or low expression. Of the 206 cases, 89 cases (43.2%) were classified as showing high expression, and 117 cases (56.8%) showed low expression. The high expression of APRIL was significantly correlated with higher Fuhrman nuclear grade and higher pathologic stage (p=0.000 and 0.004), and we observed that the high expression of APRIL was significantly correlated with the overall survival of the patients (p=0.045) and cancer-specific survival (p=0.020), but was not correlated with disease-free survival (p=0.106). In multivariate analysis adjusted for Fuhrman nuclear grade and pathologic stage, the high expression of APRIL was not an independent prognostic factor for CCRCC, as determined by overall survival (p=0.830) and cancer-specific survival (p=0.792). ConclusionsWe found that the high expression of APRIL in CCRCCs was correlated with high Fuhrman nuclear grade, high pathologic stage, and poor overall and cancer-specific survival of the patients. However, it did not correlate with disease-free survival and was not an independent prognostic factor.

Preclinical study of bladder cancer diagnosis using tumor homing peptide fluorescent molecular probe

Objective To evaluate the features of optical molecular imaging of bladder tumor cells labeled by tumor homing peptide fluorescent molecular probe, and to explore the theoretical foundation of optical molecular imaging for bladder cancer diagnosis. Methods After prepared the FITC-CSNRDARRC fluorescent molecular probe, laser scanning confocal microscope, immuno fluorescence and multispectral fluorescence in vivo optical molecular imaging system have been used to evaluate the binding sites, the affecting factors of binding rates, the specificity and the targets. BIU-87 bladder tumor cell line, BIU-87 bladder tumor cell line, 68 cases of paraffin bladder tumor tissue samples, 16 cases of paraffin glandular bladder inflammatory samples, 43 cases of paraffin renal clear cell carcinoma samples, 68 cases of paraffin gastric adenocarcinoma samples, 29 cases of urine exfoliated cells suspected bladder cancer and BIU-87 bladder cancer nude xenograft have been used in this study. Results The binding site of FITC-CSNRDARRC fluorescent molecular probe were at the nucleus of labeled bladder tumor cells. The binding rates were correlated linearly with the dose of probe and the grade of pathology. The in vitro and in vivo studies demonstrate that the probe has a binding specificity with bladder tumor. When the FITC-CSNRDARRC fluorescent molecular probe labeled tumor cells, bright green spots were observed under laser scanning confocal microscope. The bright green spots were more apparent after stained by DAPI again. The tissue samples and tumor cells in the urine can be successful labeled and identified by fluorescence microscope. Optical molecular imaging of in vivo xenograft tumor tissues showed fluorescent spots under EMCCD. Conclusions The labeled loci of single cell by FITC-CSNRDARRC probe have been identified. The spatial resolution of optical molecular image is related to sensitivity of CCD, and the optical molecular imaging cannot be imaged by the conventional endoscope camera. Key words: Urinary bladder neoplasms; Tumor homing peptide; Fluorescent molecular probe; Optical molecular imaging; Molecule diagnosis

Cytoplasmic expression of pontin in renal cell carcinoma correlates with tumor invasion, metastasis and patients' survival.

Renal cell carcinoma (RCC) is the most lethal of all genitourinary malignancies. Distant metastasis represents the major cause of death in patients with RCC. Recent studies have implicated the AAA+ ATPase pontin in many cellular activities that are highly relevant to carcinogenesis. In this study, we demonstrate for the first time that pontin was up-regulated in RCC, and plays a previously unknown pro-invasive role in the metastatic progression of RCC through epithelial-to-mesenchymal transition (EMT) pathway. 28 pairs of freshly frozen clear cell RCC samples and the matched normal renal tissues analyzed by quantitative RT-PCR and western blotting demonstrated that pontin was up-regulated in clear cell RCC tissues than in normal renal tissues. In addition, immunohistochemistry was used to evaluate subcellular pontin expression in 95 RCC patients, and found that overexpression of pontin in cytoplasm positively correlated with the metastatic features, predicting unfavorable outcomes of RCC patients. Furthermore, in vitro experiments show pontin was predominantly expressed in cytoplasm of RCC cell lines, and a significant suppression of cell migration and invasion in pontin siRNA treated RCC cell lines was observed. Mechanistic studies show that pontin depletion up-regulated the E-cadherin protein and down-regulated vimentin protein, and decreased nuclear β-catenin expression, suggesting the involvement of EMT in pontin induced metastatic progression. Together, our data suggest pontin as a potential prognostic biomarker in RCC, and provide new promising therapeutic targets for clinical intervention of kidney cancers.

Transglutaminase 2 expression and its prognostic significance in clear cell renal cell carcinoma.

Background:A few recent studies have demonstrated a possible role of transglutaminase 2 (TG2) in tumorigenesis or progression of renal cell carcinoma (RCC). The aim of this study was to examine TG2 expression and its clinicopathologic significance in a large number of human clear cell RCCs (CCRCCs).Methods:We analyzed 638 CCRCC patients who underwent partial or radical nephrectomy between 1995 and 2005. The expression of TG2 was determined by immunohistochemistry and categorized into four groups, according to staining intensity: negative (0), mild (1+), moderate (2+), and strong (3+).Results:TG2 staining intensity was negative in 8.5% of CCRCC (n=54), 1+ in 32.6% (n=208), 2+ in 50.5% (n=322), and 3+ in 8.5% (n=54). Strong TG2 expression was correlated with high Fuhrman nuclear grade (p=.011), high T category (p=.049), metastasis (p=.043) and male sex (p<.001) but not with N category.The survival analysis showed a significant association between strong TG2 expression and worse overall and cancer-specific survival (p=.027 and p=.010, respectively). On multivariate analysis, strong TG2 expression was a marginally significant prognostic indicator for Fuhrman nuclear grade and TNM staging (p=.054).Conclusions:Our study is the first to demonstrate the clinicopathologic significance of TG2 expression in a large number of human CCRCC samples. Strong TG2 expression was associated with high nuclear grade and poor prognosis.

Quantitative Proteomics Analysis of Differentially Expressed Proteins Involved in Renal Clear Cell Carcinoma by Shotgun Approach Coupled with Stable Isotope Dimethyl Labeling

Renal clear cell carcinoma (RCC) when detected incidentally by ultrasonic imaging generally shows an advanced disease stage of being diagnosed metastatic as a result of lacking specific biomarkers at an early stage. To date, RCC is still ranked as the most common renal cancer and considered as one of the most treatment-resistant metastatic malignancies. In this study, a quantitative proteomics approach by means of gel-free shotgun proteomics methodology and stable isotope dimethyl labeling coupled with nano-liquid chromatography/ tandem mass (nanoLCMS/ MS) have been employed to identify 18 up-regulated and 48 down-regulated proteins in RCC samples. It is worth noting that binding and structural proteins in renal clear cells accounted for 43% and 33% of up-regulated proteins respectively, while catalytic enzymes occupied as high as 73% of down-regulated proteins. Collectively, instead of one universal tumorigenesis enzyme being identified, the pathogenesis of RCC may involve a variety of protein factors including ANXA2, LGALS1, VIM and TPM1 related to metastasis, angiogenesis, tumor invasion as well as tumor growth, in addition to CRYAB, GSTA1, CALB1 and HSPD1, which are linked to apoptosis function. The upshot of this study highlighted by a series of down-regulated proteins suggests that the clear-cut decrease of ATP generation components related to mitochondrial dysfunction and altered energy metabolism may be involved in RCC carcinogenesis.

Профиль метилирования группы генов микроРНК при светлоклеточном почечноклеточном раке; связь с прогрессией рака

MicroRNA regulates gene expression, is involved in many cellular processes, and plays an important role in the development of cancer. The regulation of the expression of miRNA genes can be achieved by methylating their CpG islands, which is shown in different types of tumors. The methylation of miRNA genes in clear cell renal cell carcinoma (CCRCC) has mainly been studied for the miR-9 and miR-34 families. The methylation of six miRNA genes (miR-124a-2, -124a-3, -9-1, -9-3, -34b/c, -129-2) was investigated with the use of representative set of CCRCC samples (46 cases). Methylation of three genes miR-124a-2, -124a-3, and -129-2 was studied in kidney tumors for the first time. Methylation analysis was performed using methyl specific PCR. It is shown that the frequency of methylation of six genes (miR-124a-2, -124a-3, -9-1, -9-3, -34b/c and -129-2) was significantly higher in tumor samples than in samples of histologically normal tissue (P < 3 x 10(-5) by Fisher's exact test). These results suggest the properties of tumor suppressors for the six miRNA genes indicated in CCRCC. We also found correlations between the methylation frequency of some miRNA genes and signs of the progression of CCRCC (tumor size, clinical stage, loss of differentiation, and metastasis).

Down-regulation of human leukocyte antigen class I (HLA-I) is associated with poor prognosis in patients with clear cell renal cell carcinoma

Human leukocyte antigen class I (HLA-I) molecules are transmembrane glycoproteins that have been reported to be down-regulated in multiple types of human malignancies, including clear cell renal cell carcinoma (CCRCC). However, only one study has investigated its prognostic value in CCRCC. In the present study, HLA-I protein expression was analyzed in 120 archived, paraffin-embedded CCRCC samples and 10 adjacent normal tissues using immunohistochemistry. The correlation between HLA-I expression and clinicopathological factors was evaluated by the χ2 test. Patients’ overall survival was analyzed by the Kaplan–Meier method. HLA-I down-regulation was observed in 38.3% (46/120) of renal tumor samples, but only in 10% (1/10) of adjacent normal tissues. Statistical analysis showed a significant correlation of HLA-I expression with TNM stage, lymph node metastasis, and Fuhrman grade. Patients with tumors displaying down-regulation of HLA-I showed significantly shorter overall survival (P=0.021, log-rank test). More importantly, multivariate analysis indicated that down-regulation of HLA-I was an independent prognostic factor for CCRCC patients (P=0.033). Overall, our data suggest that HLA-I down-regulation is associated with tumor progression and a poor prognosis in CCRCC patients, and emphasize the importance of HLA-I in natural and therapeutic immune surveillance of patients with CCRCC.