ccRCC Metastasis Research Articles

Metastasis-associated mutations in clear cell renal cell carcinoma.

600 Background: Over 30% of patients with clear cell renal cell carcinoma (ccRCC) exhibit metastasis at the time of diagnosis and exhibit poor outcome. About 20-50% of patients with localized disease eventually develop metastasis after nephrectomy. The goal of the current study was to identify target gene mutations associated with ccRCC metastasis. Methods: In this IRB approved study, genomic DNA from 128 ccRCC resected specimens (128 unique patients) were profiled using a custom targeted next-generation sequencing (NGS) panel comprising 70 frequently mutated genes and prognostic SNPs in renal cancer. The specimen cohort consisted of 78 primary (29 stage I-III, 30 stage IV, 19 unknown) and 50 metastatic (10 lung, 9 bone, 25 other sites, 6 unknown) lesions. Following hybrid capture, sequencing was performed (MiSeq, Illumina) and variants identified using CLCbio (Qiagen). Chi-square test was used to test for significance. Results: The median specimen had 4 non-synonymous mutations (123/128 samples had at least one mutation). 66 genes showed a non-synonymous mutation in at least one specimen. There was no significant difference between total mutation count between primary and metastatic specimens (Mann-Whitney test). TSC1 mutation was significantly enriched in metastatic (8/50) compared with primary lesions (3/78). No TSC1 mutation was found in the 29 localized (stage I-III) primary lesions. Interestingly, TSC1 maps to 9q34 and our prior copy number studies indicated that loss of 9q is also enriched in metastatic lesions. Of the 11 specimens bearing TSC1 mutation in this cohort, 5 also had loss of 9q. In 44 specimens with known sites of resection, SETD2 and TSC1 mutations were not found in metastases to the lung but found in 30-40% of lesions at other sites, KDM5C and TSC1 mutations were not found in metastases to the bone while 25-30% of lesions at other sites exhibited these mutations. Due to the limited sample size, significance could not be determined for these associations. Conclusions: Preliminary results of this study implicated TSC1 in the metastasis of ccRCC. Since TSC1 mutation is known to confer sensitivity to mTOR inhibitors, our initial finding of absence of TSC1 mutation in lung and bone sites of metastasis may have therapeutic implications.

Impact of histologic subtype and sarcomatoid transformation on metastasis in renal cell carcinoma: a single institute experience in 149 patients.

To compare the metastatic pattern and outcome of clear cell RCC (ccRCC) and papillary RCC (pRCC), and to assess the impact of sarcomatoid transformation on the disease spread and prognosis. This IRB-approved, HIPAA-compliant retrospective study included 149 consecutive patients (108 men; mean age 58 years; range 25-86) with metastatic RCC (ccRCC = 116, pRCC = 33), identified from imaging database. All available imaging studies and electronic records of these patients were reviewed to document pathological features, distribution and timing of metastasis, and survival. The metastatic pattern and survival was first compared between the ccRCC and pRCC groups, and then between those with and without sarcomatoid transformation; all 27 cases of sarcomatoid transformation occurred in the ccRCC group. Metastases were noted at presentation in 62 (42%) and after median 13 months in the remaining 87 (58%) patients. Lymph nodes (134/149), lung (125/149), and bone (60/149) were the most common metastatic sites, which did not differ between the RCC subtypes. Pancreatic (p = 0.0014) and renal (p = 0.046) metastases were more common in ccRCC, lymphangitic spread (p = 0.0003) and peritoneal metastasis (p = 0.039) more common in pRCC. In ccRCC, sarcomatoid transformation was associated with high-grade tumors (p < 0.0001), more frequently demonstrated lymphangitic (p = 0.016), pleural (p = 0.0018), and peritoneal metastases (p = 0.0002), and had shorter metastasis-free survival and overall survival (log-rank test, p < 0.0001). In the absence of sarcomatoid transformation, ccRCC had longer metastasis-free interval (median 22 months compared to 6 months) (p = 0.0238) and overall survival (median survival 48 months vs. 25 months) (p = 0.0193) compared to pRCC. Histologic subtype, as well as the presence of sarcomatoid transformation, affects the metastatic pattern and metastasis-free survival of RCC. In the absence of sarcomatoid transformation, ccRCC has a better outcome than pRCC.

Fast clearance of lipid droplets through MAP1S-activated autophagy suppresses clear cell renal cell carcinomas and promotes patient survival.

Clear cell renal cell carcinoma (ccRCC) is composed of cells whose cytoplasm filled with lipid droplets, subcellular organelles coated with adipocyte differentiation-related protein (ADFP) for the storage of triacylglycerol converted from excess free fatty acids. Mammalian cells primarily use the autophagy-lysosome system to degrade misfolded/aggregated proteins and dysfunctional organelles such as lipid droplets. MAP1S (originally named C19ORF5) is an autophagy activator and promotes the biogenesis and degradation of autophagosomes. Previously, we reported that MAP1S suppresses hepatocellular carcinogenesis in a mouse model and promoted the survival of patients with prostate adenocarcinomas by increasing the degradation of aggregated proteins and dysfunctional mitochondria. Here we show that a suppression of MAP1S in renal cells causes an impairment of autophagic clearance of lipid droplets. In contrast, an overexpression of MAP1S causes an activation of autophagy flux and a reduction of lipid droplets so less DNA double strand breakage is induced. The levels of MAP1S in normal renal cells are dramatically higher than those in the ccRCC tissues and cell lines derived from renal cell carcinomas. High levels of MAP1S are associated with a reduced malignancy and metastasis of ccRCC and predict a better survival of ccRCC patients. Therefore, autophagy defects in the degradation of lipid droplets triggered by the MAP1S deficiency may enhance the initiation and development of ccRCC and reduce the survival of ccRCC patients.

MicroRNA-21 stimulates epithelial-to-mesenchymal transition and tumorigenesis in clear cell renal cells.

Clear cell renal cell carcinoma (ccRCC) metastasis may result from epithelial-to-mesenchymal transition and mesenchymal stem cells that contribute to the development of the primary tumor. In this study, it was demonstrated that microRNA-21 (miR-21) acts as an oncogenic driver of ccRCC. ccRCC spheres were isolated and it was shown that they exhibited cancer stem cell-like properties, including the formation of self-renewing spheres. Spheres showed increased expression of stem cell-related transcription factors and epithelial-mesenchymal transition (EMT) markers. In addition, secondary sphere formation capacity was assessed after miR-21 transfection. miR-21 accelerated the formation of ccRCC spheres, which shared molecular characteristics with the spontaneous ccRCC spheres. It was demonstrated that miR-21 overexpression facilitates ccRCC sphere formation. Thus, a single miRNA may have an impact on the formation of highly tumorigenic cancer spheres in kidney cancer.

Renal cell carcinoma with metachronous metastasis to the contralateral adrenal gland and urinary bladder: A case report

The adrenal gland and urinary bladder are rare localizations of metastases of renal cell carcinoma (RCC). In the present study, a case of metastasis to the contralateral adrenal gland and urinary bladder of clear cell-type RCC (ccRCC) in the left kidney is reported in a male who had undergone left radical nephrectomy at 55 years of age. Computed tomography (CT) revealed a mass in the right adrenal gland 5 years following surgery, and type-B ultrasound demonstrated a 1.0-cm solid space-occupying lesion in the right wall of the bladder. Consequently, laparoscopic right adrenalectomy and transurethral resection of the bladder tumor were performed. Furthermore, based on histopathological examination and immunohistochemical staining, the patient was pathologically diagnosed with contralateral adrenal gland and urinary bladder metastasis of ccRCC. CT performed at the 1-year follow-up detected multiple solid space-occupying lesions in the right kidney. Therefore, the patient was treated with sunitinib targeted therapy. To date, the patient is generally in good condition, without evident drug side effects or complaints of discomfort.

PinX1 serves as a potential prognostic indicator for clear cell renal cell carcinoma and inhibits its invasion and metastasis by suppressing MMP-2 via NF-κB-dependent transcription.

PIN2/TRF1-interacting telomerase inhibitor 1 (PinX1) is a novel cloned gene which has been identified as a major haploinsufficient tumor suppressor essential for maintaining telomerase activity, the length of telomerase and chromosome stability. This study explored the clinical significance and biological function of PinX1 in human clear cell renal cell carcinoma (ccRCC). The clinical relevance of PinX1 in ccRCC was evaluated using tissue microarray and immunohistochemical staining in two independent human ccRCC cohorts. Our data demonstrated that PinX1 expression was dramatically decreased in ccRCC tissues compared with normal renal tissues and paired adjacent non-tumor tissues. Low PinX1 expression was significantly correlated with depth of invasion, lymph node metastasis and advanced TNM stage in patients, as well as with worse overall and disease-specific survival. Cox regression analysis revealed that PinX1 expression was an independent prognostic factor for ccRCC patients. Moreover, PinX1 inhibited the migration and invasion of ccRCC by suppressing MMP-2 expression and activity via NF-κB-dependent transcription in vitro. In vivo studies confirmed that PinX1 negatively regulated ccRCC metastasis and the expression of MMP-2 and NF-κB-p65. These findings indicate that PinX1 suppresses ccRCC metastasis and may serve as a ccRCC candidate clinical prognostic marker and a potential therapeutic target.

Association of genome-wide copy number alterations with metastasis of clear cell renal cell carcinoma.

428 Background: About one-third of patients with clear cell renal cell carcinoma (ccRCC) exhibit metastasis at the time of diagnosis and show poor prognosis. The genetic and epigenetic alterations associated with metastasis in ccRCC have variably been studied, and their role in the metastatic process is unclear. The goals of the current study were to identify genomic copy number alterations (CNAs) associated with ccRCC metastasis and examine their clinical utility. Methods: In this IRB-approved study, genome-wide copy number profiling was performed on DNA from 144 ccRCC (81 primary and 63 metastatic lesions). Differential CNAs between primary and metastatic lesions and between different metastatic sites were identified using Fisher’s exact test. Associations between CNAs and overall survival (OS) were tested using the log rank statistic and Kaplan-Meier method. Genomic profiling data of 437 and 240 primary ccRCC (TCGA and PMID: 23797736, respecitively) were used for verification. Results: Between primary and metastatic lesions, 25 CNAs were significantly different (p&lt;0.05). Of the 11 more frequent in metastatic lesions, nine retained significance when comparing stage IV and stage I TCGA ccRCC. For 368 TCGA locally-invasive tumors (stages I, II, and III), three CNAs (loss of 9p24.3-p13.3, 9p12-q11, and 9q21.12-q21.33) were associated with inferior survival (p=0.002). In the second dataset of 214 locally-invasive lesions, loss of 18q11.2-q23 correlated with shorter OS (p=0.025). Across metastatic lesions, nine CNAs were found to be significantly enriched in lung lesions and three in bone. In a subset of 127 ccRCC with known metastatic status at 5 years after diagnosis, two of these CNAs (gain of 7q36.1-36.3 in lung and loss of 22q13.2 in bone) were significantly enriched in the corresponding primary specimens. Conclusions: This study identified CNAs associated with ccRCC metastasis and common sites of metastasis that have the potential to serve as biomarkers to assist in better risk stratification of patients with this disease. Integrated analyses of genes mapping to the loci of genomic imbalance would further our understanding of the biology of metastasis in renal cancer.

Hypoxia-induced overexpression of stanniocalcin-1 is associated with the metastasis of early stage clear cell renal cell carcinoma.

BackgroundAlthough metastasis of clear cell renal cell carcinoma (ccRCC) is predominantly observed in late stage tumors, early stage metastasis of ccRCC can also be found with indefinite molecular mechanism, leading to inappropriate clinical decisions and poor prognosis. Stanniocalcin-1 (STC1) is a glycoprotein hormone involved in calcium/phosphate homeostasis, which regulates various cellular processes in normal development and tumorigenesis. This study aimed to investigate the role and mechanism of regulation of STC1 in the metastasis of early stage ccRCC.MethodsSTC1 mRNA and protein expression was determined in ccRCC surgical specimens, RCC cell lines, and human kidney tubule epithelial cell line HKC by real-time polymerase chain reaction (RT-PCR) and western blotting. Immunohistochemistry staining (IHC) and immunofluorescence were also used to examine the expression and localization of STC1 in ccRCC tissues and cancer cells. Knockdown and overexpression studies were conducted in vitro in RCC cell lines using small interfering RNAs (siRNA) and lentiviral-mediated gene delivery to evaluate the role of STC1 in cell proliferation, anchorage-dependent and independent growth, cell cycle control, and migration and invasion.ResultsSTC1 mRNA and protein expression were significantly up-regulated in tumors when compared with non-tumor tissues, with the greatest increase in expression observed in metastatic tissues. Clinicopathological analysis revealed that STC1 mRNA expression was associated with Fuhrman tumor grade (P = 0.008) and overall Tumor Node Metastasis (TNM) staging (P = 0.018). STC1 expression was elevated in T1 stage metastatic tumors when compared with localized tumors, and was positively correlated with average tumor diameter. Silencing of STC1 expression by Caki-1 and A498 resulted in the inhibition of cell proliferation, migration, and invasion, meanwhile down-regulation of STC1 impaired epithelial–mesenchymal transition (EMT) of ccRCC cell lines. Overexpression of STC1 in Caki-2 enhanced cell growth and proliferation but not migration and invasion. Further investigation identified hypoxia and HIF-1α as candidate regulators of STC1 expression.ConclusionsOur findings demonstrate a role for STC1 in metastasis of early stage ccRCC and suggest that STC1 may be a biomarker of potential value both for the prognosis of this disease and for guiding clinical decisions regarding surgical strategies and adjuvant treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0421-4) contains supplementary material, which is available to authorized users.

Piwi-Interacting RNAs (piRNAs) Are Dysregulated in Renal Cell Carcinoma and Associated with Tumor Metastasis and Cancer-Specific Survival.

Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the up-regulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.

KCNJ1 inhibits tumor proliferation and metastasis and is a prognostic factor in clear cell renal cell carcinoma.

Potassium inwardly rectifying channel, subfamily J, member 1 (KCNJ1), as an ATP-dependent potassium channel, plays an essential role in potassium balance. KCNJ1 variation is associated with multiple diseases, such as antenatal Bartter syndrome and diabetes. However, the role of KCNJ1 in clear cell renal cell carcinoma (ccRCC) is still unknown. Here, we studied the expression and function of KCNJ1 in ccRCC. The expression of KCNJ1 was evaluated in ccRCC tissues and cell lines by quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry analysis. The relationship between KCNJ1 expression and clinicopathological characteristics was analyzed. p3xFLAG-CMV-14 vector containing KCNJ1 was constructed and used for transfecting ccRCC cell lines 786-O and Caki-2. The effects of KCNJ1 on cell proliferation, invasion, and apoptosis were detected in ccRCC cell lines using cell proliferation assay, transwell assay, and flow cytometry, respectively. We found that KCNJ1 was low-expressed in ccRCC tissues samples and cell lines, and its expression level was significantly associated with tumor pathology grade (P = 0.002) and clinical stage (P = 0.023). Furthermore, the KCNJ1 expression was a prognostic factor of ccRCC patient's survival (P = 0.033). The re-expression of KCNJ1 in 786-O and Caki-2 significantly inhibited cancer cell growth and invasion and promoted cancer cell apoptosis. Moreover, knockdown of KCNJ1 in HK-2 cells promoted cell proliferation. Collectively, these data highlight that KCNJ1, low-expressed in ccRCC and associated with poor prognosis, plays an important role in ccRCC cell growth and metastasis.

Downregulated miR-646 in clear cell renal carcinoma correlated with tumour metastasis by targeting the nin one binding protein (NOB1).

Background:Nin one binding protein (NOB1) was identified as a potential oncogene in human glioma and miR-646 plays an important role in human growth and development. However, the underlying molecular mechanisms of NOB1 in tumorigenicity and its correlation with miR-646 in renal cell carcinoma (RCC) have not been investigated.Methods:We performed bioinformatic analysis to explore miRNA targeting NOB1. The expression of NOB1 and miR-646 from 100 cases of clear cell RCC (ccRCC) and 30 cases of adjacent non-tumour tissues were detected by quantitative real-time PCR. The expression of miR-646 was correlated with NOB1 expression, tumour features and patient metastasis-free survival. The effect of overexpression of mir-646 on renal cancer cell proliferation was detected by colony formation in soft agar. Using a xenograft tumour model, we observed the in vivo tumorigenesis effect of miR-646 and NOB1.Results:miR-646 negatively regulated NOB1 and inhibited the proliferation and migration of renal cancer cells. There was a significant upregulation of NOB1 in ccRCC and it was further increased in metastatic cases, while miR-646 was downregulated in tumour tissues and further decreased in metastatic ccRCC. Additionally, expression of miR-646 was inversely correlated with the expression of NOB1. The downregulation of miR-646 also indicated a higher probability of developing metastasis. Most importantly, miR-646 expression was an independent predictor of ccRCC metastasis by the univariate analysis and binary logistic regression model (both P<0.05). Colony formation in soft agar and xenograft tumour model suggested that miR-646 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, suppression of NOB1 increased the phosphorylation of several proteins in MAPK pathway.Conclusions:Downregulated miR-646 in ccRCC was associated with tumour metastasis through MAPK pathway by targeting NOB1. miR-646 and NOB1 may play an important role in the development of ccRCC.

Overexpression of SATB1 is associated with biologic behavior in human renal cell carcinoma.

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of SATB1 in RCC remains unclear. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of SATB1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of SATB1 was significantly associated with depth of invasion (P<0.001), lymph node status (P = 0.001) and TNM stage (P = 0.009). SATB1 knockdown inhibited the proliferation, migration and invasion of 786-O cells, whereas SATB1 overexpression promoted the growth and aggressive phenotype of ACHN cells in vitro. Furthermore, SATB1 expression was positively correlated with ZEB2 expression (P = 0.013), and inversely linked to levels of SATB2 and E-cadherin (P = 0.005 and P<0.001, respectively) in ccRCC tissues. Our data provide a basis for the concept that overexpression of SATB1 may play a critical role in the acquisition of an aggressive phenotype for RCC cells through EMT, providing new insights into the significance of SATB1 in invasion and metastasis of ccRCC, which may contribute to fully elucidating the exact mechanism of development and progression of RCC.

MiR-141 Is a Key Regulator of Renal Cell Carcinoma Proliferation and Metastasis by Controlling EphA2 Expression

Although microRNAs (miRNA) have been revealed as crucial modulators of tumorigenesis, our understanding of their roles in renal cell carcinoma (RCC) is limited. Here we sought to identify human miRNAs that act as key regulators of renal carcinogenesis. We performed microarray-based miRNA profiling of clear cell RCC (ccRCC) and adjacent normal tissues and then explored the roles of miR-141 both in vitro and in vivo, which was the most significantly downregulated in ccRCC tissues. A total of 74 miRNAs were dysregulated in ccRCC compared with normal tissues. miR-141 was remarkably downregulated in 92.6% (63/68) ccRCC tissues and would serve as a promising biomarker for discriminating ccRCC from normal tissues with an area under the receiver operating characteristics curve of 0.93. Overexpression of miR-141 robustly impaired ccRCC cell migratory and invasive properties and suppressed cell proliferation by arresting cells at G0-G1 phase in vitro and in human RCC orthotopic xenografts. Significantly, the antitumor activities of miR-141 were mediated by its reversal regulation of erythropoietin-producing hepatocellular (Eph) A2 (EphA2), which then relayed a signaling transduction cascade to attenuate the functions of focal adhesion kinase (FAK), AKT, and MMP2/9. In addition, a specific and inverse correlation between miR-141 and EphA2 expression was obtained in human ccRCC samples. Finally, miR-141 could be secreted from the ccRCC donor cells, and be taken up and function moderately in the ccRCC recipient cells. miR-141 serves as a potential biomarker for discriminating ccRCC from normal tissues and a crucial suppressor of ccRCC cell proliferation and metastasis by modulating the EphA2/p-FAK/p-AKT/MMPs signaling cascade.

Renal Cell Carcinoma Cancer Stem Cells as Therapeutic Targets

Although early stage disease detection and treatment options for clear cell renal cell cancer (ccRCC) improved in recent years, prognosis of patients with late stage ccRCC remains poor, mostly due to development of tyrosine kinase inhibitors and mammalian target of rapamycin inhibitors resistance followed by disease progression. Cancer stem cells (ccRCC-CSC) model has focused a significant attention in recent years as a potential explanation for the tumor heterogeneity, drug resistance, disease recurrence and metastasis of ccRCC and other cancers. Cancer stem cells have been proposed to be responsible for tumor initiation, repopulation and growth that cause patients to succumb to renal cancer. Precise identification of ccRCC-CSC populations and definition of hierarchy of cells within ccRCC tumor including tumor initiating cells and tumor progenitor cells will facilitate accurate characterization of drug targets and ultimately contribute to more personalized and effective care. This mini-review discusses the potential strategies to inhibit the signaling pathways underlying stemness in an effort to treat renal cancer. Mechanism that could be exploited as a therapeutic target against drug resistant ccRCC-CSCs is summarized.

Downregulation of FOXO3a Promotes Tumor Metastasis and Is Associated with Metastasis-Free Survival of Patients with Clear Cell Renal Cell Carcinoma

To explore the mechanisms underlying clear-cell renal cell carcinoma (ccRCC) metastasis using transcriptional profiling and bioinformatics analysis of ccRCC samples, and to elucidate the role of FOXO3a in ccRCC metastasis. Gene expression profiling was performed using four primary metastatic and five primary nonmetastatic ccRCC samples. The mRNA and protein levels of FOXO3a in ccRCC samples were investigated by real-time reverse transcription PCR and immunohistochemistry, respectively. The association between metastasis-free survival of patients with ccRCC and FOXO3a mRNA levels was analyzed. Biologic functions of FOXO3a in renal cancer cell lines were investigated. The influence of FOXO3a on tumor metastasis was also studied in vivo orthotopic xenograft tumor model. Finally, the mechanism by which FOXO3a attenuation could increase invasion and migration of tumor cells was explored. Bioinformatics analysis of the profiling data identified FOXO3a as a key factor in ccRCC metastasis. FOXO3a expression was decreased in primary metastatic ccRCC samples. Patients with low FOXO3a mRNA levels had poor metastasis-free survival (P = 0.003). Knocking down FOXO3a induced tumor cell invasion and migration in the nonmetastatic ccRCC cells. Induced FOXO3a overexpression in SN12-PM6 cells could inhibit tumor metastasis in vivo. Downregulation of FOXO3a increased SNAIL1 expression, thereby activating the epithelial-mesenchymal transition (EMT) of RCC cell lines. The loss of FOXO3a induced EMT of tumor cells by upregulating SNAIL1, which promoted tumor cells metastasis in vitro and in vivo. Thus, FOXO3a could be considered as an independent prognostic factor in ccRCC metastasis and could be a marker of occult metastases.

MP29-16 FABP7 AS A POTENTIAL TARGET AND MARKER IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2014MP29-16 FABP7 AS A POTENTIAL TARGET AND MARKER IN CLEAR CELL RENAL CELL CARCINOMA Kazuhiro Nagao, Nachi Shinohara, Frank Smit, Sander Jannink, Mirjam de Weijert, Keita Kobayashi, Yoshihisa Kawai, Hiroaki Matsumoto, Takahiko Hara, Hideyasu Matsuyama, Yuji Owada, Peter Mulders, and Egbert Oosterwijk Kazuhiro NagaoKazuhiro Nagao More articles by this author , Nachi ShinoharaNachi Shinohara More articles by this author , Frank SmitFrank Smit More articles by this author , Sander JanninkSander Jannink More articles by this author , Mirjam de WeijertMirjam de Weijert More articles by this author , Keita KobayashiKeita Kobayashi More articles by this author , Yoshihisa KawaiYoshihisa Kawai More articles by this author , Hiroaki MatsumotoHiroaki Matsumoto More articles by this author , Takahiko HaraTakahiko Hara More articles by this author , Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author , Yuji OwadaYuji Owada More articles by this author , Peter MuldersPeter Mulders More articles by this author , and Egbert OosterwijkEgbert Oosterwijk More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.758AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To identify potential targets in clear cell renal cell carcinoma (ccRCC), we performed a transcriptome analysis of ccRCC and normal kidney specimens. The transcriptome analysis revealed numerous highly overexpressed genes related to hypoxia, pH regulation and metabolisms in ccRCC compared to normal kidney specimens. FABP7 (Fatty Acid Binding Protein 7) is supposed to be involved in fatty acid synthesis in cancer cells and was highly overexpressed in ccRCC specimens compared to normal kidney specimens. We confirmed the significance of FABP7 as a potential target and marker in ccRCC. METHODS We performed a gene expression analysis against the total RNA extracted from 60 ccRCC and 20 normal kidney specimens on the human exon 1.0 ST array (Affimetrix). The expression profiles were analysed by unsupervised hierarchical average linkage clustering algorithm. Among the overexpressed genes, FABP7 had the highest mean difference between ccRCC and normal kidney in the profile. The expression levels were further studied by immunohistochemical analysis in 19 advanced ccRCC cases treated by molecular targeted drugs. The mRNA expression levels were evaluated in 8 ccRCC cell lines and the effect of gene knockdown was studied in a highly expressed cell line (A498). RESULTS Among the overexpressed genes, FABP7 had the highest mean expression level in mRNA, while the expression levels varied depending on the ccRCC cases. Our immunohistochemical analysis for the ccRCC specimens demonstrated significant correlation between FABP7 expression and distant metastasis (p<0.05). Based on proliferation assay (MTT assay), functional suppression of FABP7 resulted in significant growth inhibition of A498 cells (p<0.05). CONCLUSIONS Functional suppression for FABP7 downregulated the cell viability in a ccRCC cell line (A498). FABP7 expression level was significantly correlated with distant metastasis. FABP7 could be a potential therapeutic target and diagnostic marker to detect metastasis of ccRCC. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e311 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kazuhiro Nagao More articles by this author Nachi Shinohara More articles by this author Frank Smit More articles by this author Sander Jannink More articles by this author Mirjam de Weijert More articles by this author Keita Kobayashi More articles by this author Yoshihisa Kawai More articles by this author Hiroaki Matsumoto More articles by this author Takahiko Hara More articles by this author Hideyasu Matsuyama More articles by this author Yuji Owada More articles by this author Peter Mulders More articles by this author Egbert Oosterwijk More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

High expression of the secreted protein dickkopf homolog 4: roles in invasion and metastasis of renal cell carcinoma and its association with Von Hippel-Lindau gene.

The aim of this study was to investigate the effects of the dickkopf homolog 4 (DKK4)/Wnt/β-catenin signaling pathway on tumorigenesis and metastasis in clear cell renal cell carcinoma (ccRCC), as well as to elucidate the underlying mechanisms. We examined the expression of DKK4 in 30 cases of ccRCC and matched adjacent normal tissues, and investigated its correlation with clinicopathological characteristics. Stable DKK4-transfected cells were established, and DKK4 functional analyses were performed, including a T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay, and experiments on cell viability, apoptosis, invasive capability and tumor growth in vivo. Finally, western blot analysis was performed to detect Von Hippel-Lindau (VHL) expression in 50 clinical specimens. The expression levels of the DKK4, β-catenin and β-catenin downstream target genes, cyclin D1 and c-myc, were determined in the these specimens, as well as in RCC4(-), T3-14(+) cell lines by qRT-PCR and western blot analysis. The same tests were also performed in human embryonic kidney (HEK)293 cells which were transfected with the pCDH-DKK4 plasmid. After 6 weeks the tumor weight significantly increased in the mice transfected with the tumor cells. DKK4 mRNA and protein expression levels were significantly upregulated (p<0.001). DKK4 was distinctly overexpressed (68.0%) in all patient tissues. VHL(-) samples accounted for 60.0% of all samples, while DKK4 expression was significantly upregulated in 50% of these samples, indicating a correlation with VHL(-) expression (r=0.403, p<0.05). We also observed reduced expression levels of cyclin D1, c-myc and β-catenin (to a greater extent) in the VHL(-), RCC4(-) and T3-14(+) cells, as well as in the stably transfected HEK293 cells. DKK4 may be an oncogene, and its upregulated expression may be involved in the pathogenesis of ccRCC as a downstream gene of VHL. By activating other pathways apart from the Wnt/β-catenin pathway, DKK4 may play an important role in ccRCC tumorigenesis and metastasis.

Dicer is down-regulated in clear cell renal cell carcinoma and in vitro Dicer knockdown enhances malignant phenotype transformation

ObjectivesAlthough emerging evidence has shown that the deregulation of micro-ribonucleic acid (RNA) biogenesis machinery is involved in various human malignancies, this role has not been investigated in clear cell renal cell carcinoma (ccRCC). This study aims to determine whether Dicer, a key enzyme responsible for biogenesis of microRNA, is deregulated in ccRCC. The biological roles of Dicer in vitro are also determined. Materials and methodsThe expression of Dicer at messenger RNA and protein levels was detected by real-time quantitative polymerase chain reaction and western blot, respectively, in human kidney tubule epithelial cell line, nonmetastatic 786-O ccRCC cell line, and metastatic ACHN ccRCC cell line, as well as in 42 cases of ccRCC surgical specimens including 14 cases with distant metastasis and their corresponding adjacent normal renal tissues. Dicer expression levels in specimens were also measured by immunohistochemical staining. Knockdown of Dicer expression in 786-O and ACHN ccRCC cell lines was achieved by transfecting short interfering RNA against Dicer. The effects of Dicer on cell proliferation, migration, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, flow cytometric analyses, and Boyden chamber Transwell assay, respectively. ResultsCompared with human kidney tubule epithelial cell line, Dicer expression levels were significantly down-regulated in 786-O and ACHN ccRCC cell lines, with the metastatic ACHN ccRCC cell line having even lower levels. Meanwhile, Dicer expression levels were significantly down-regulated in ccRCC surgical specimens compared with adjacent normal renal tissues, with the metastatic ones further reduced, and Dicer messenger RNA levels were significantly correlated with overall tumor-node-metastasis stage of ccRCC. In vitro, the knockdown of Dicer significantly promoted cell proliferation, migration, and invasion. ConclusionsReduced expression of Dicer may play a role in the tumorigenesis of ccRCC and further decline may be associated with distant metastasis of ccRCC.

Tubulin cofactor A functions as a novel positive regulator of ccRCC progression, invasion and metastasis

Microtubules (Mts), which consist of α/β-tubulin heterodimers, are involved in cancer development and metastasis. Tubulin cofactor A (TBCA) plays crucial roles in modulating tubulin folding and α/β-tubulin heterodimer polymerization. Here, we identified the aberrant expression of TBCA in clear cell renal cell carcinoma (ccRCC) specimens as well as cell lines and revealed the function of TBCA as a novel positive regulator in ccRCC progression, invasion and metastasis. qRT-PCR, Western blot and immunohistochemistry assays confirmed that TBCA was significantly highly expressed in ccRCC specimens and cell lines compared to their corresponding normal kidney tissues and HKC. Accordingly, the influence of TBCA on cell proliferation, apoptosis and invasion/migration was detected through overexpression and knockdown of endogenous TBCA protein level in ccRCC cells via plasmids. Silencing of TBCA expression inhibited the proliferation of 786-O cells and Caki-1 cells and promoted the apoptosis of 786-O cells. Down-regulation of TBCA expression also reduced the invasion and migration ability of 786-O cells. Interestingly, overexpression of TBCA did not induce biocharacteristics that directly contrasted to those of TBCA knockdown. Importantly, exploration of the mechanism showed that TBCA could function via modulating cytoskeleton integration and influencing cell cycle progress. Furthermore, down-regulation of TBCA expression in 786-O and Caki-1 cells affected cytoskeleton integration and cell size, induced S/G2 cell cycle arrest and led to cyclineA/E and CDK2 aberrant expression. By investigating novel roles of TBCA in regulation of ccRCC cell progression, invasion and metastasis, our study identified that TBCA may be a potential molecular target for ccRCC therapy.

302 DOWN-REGULATION OF NDUFB6 CAUSED BY THE GENE COPY NUMBER LOSS AT 9P24.1-P13.3 IS IMPLICATED IN AUGMENTED CELL GROWTH OF METASTATIC RENAL CELL CARCINOMAS

You have accessJournal of UrologyKidney Cancer: Basic Research (II)1 Apr 2013302 DOWN-REGULATION OF NDUFB6 CAUSED BY THE GENE COPY NUMBER LOSS AT 9P24.1-P13.3 IS IMPLICATED IN AUGMENTED CELL GROWTH OF METASTATIC RENAL CELL CARCINOMAS Takahiro Narimatsu, Keiko Matsuura, Toru Inoue, Chisato Nakada, Yasuhiro Sumino, Takeo Nomura, Fuminori Sato, Masatsugu Moriyama, and Hiromitsu Mimata Takahiro NarimatsuTakahiro Narimatsu Oita, Japan More articles by this author , Keiko MatsuuraKeiko Matsuura Oita, Japan More articles by this author , Toru InoueToru Inoue Oita, Japan More articles by this author , Chisato NakadaChisato Nakada Oita, Japan More articles by this author , Yasuhiro SuminoYasuhiro Sumino Oita, Japan More articles by this author , Takeo NomuraTakeo Nomura Oita, Japan More articles by this author , Fuminori SatoFuminori Sato Oita, Japan More articles by this author , Masatsugu MoriyamaMasatsugu Moriyama Oita, Japan More articles by this author , and Hiromitsu MimataHiromitsu Mimata Oita, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1686AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Although it is well known that deregulation of VHL-HIF1 pathway is responsible for the carcinogenesis of clear cell renal cell carcinoma (ccRCC), molecular mechanisms responsible for the development of metastasis are still poorly investigated. The first aim of this study is to identify copy number alteration (CNA) that is specifically detected in metastasis but not in primary lesion. The second aim is to identify a gene whose deregulation is responsible for the development of metastasis. METHODS For the array comparative genomic hybridization (CGH) analysis, genomic DNAs were extracted from the formalin-fixed, paraffin-embedded tissues. Twenty cases of primary ccRCCs and their corresponding metastases were analyzed using 4x44K oligonucleotide CGH array (Agilent Inc.). For the gene expression analysis, total RNA was extracted from frozen samples of 30 primary ccRCCs. Expression levels of the 58 genes were analyzed by qRT-PCR. To investigate the biologic function of candidate genes, RCC cell lines, 786-O and 769-P were used. Cell proliferation, invasiveness and apoptosis were analyzed before and after the infection of lenti-virus encoding candidate genes to the RCC cell lines. RESULTS By comparing CNAs of primary ccRCCs with their corresponding metastases, we found that gene copy number loss at 9p24.1-p13.3 was more frequently found in metastases than in primary lesions. Out of 58 genes located at 9p24.1-p13.3, we identified 2 genes, NDUFB6 and LRRC19, both of which were down-regulated due to copy number loss. Next, using lenti-virus encoding each gene, we introduced the two genes into 786-O and 769-P cells in which both the 2 genes were down-regulated. We found that, when NDUFB6 was overexpressed in both cell lines, cell proliferation was significantly suppressed (figure shown below), although apoptotic activity and invasion activity were unchanged. CONCLUSIONS Down-regulation of NDUFB6 caused by gene copy number loss at 9p24.1-p13.3 may bring about growth advantage to metastatic ccRCC cells. We propose that NDUFB6 is a new candidate for a tumor suppressor and that its down-regulation may play an important role in the development of metastasis of ccRCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e123 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takahiro Narimatsu Oita, Japan More articles by this author Keiko Matsuura Oita, Japan More articles by this author Toru Inoue Oita, Japan More articles by this author Chisato Nakada Oita, Japan More articles by this author Yasuhiro Sumino Oita, Japan More articles by this author Takeo Nomura Oita, Japan More articles by this author Fuminori Sato Oita, Japan More articles by this author Masatsugu Moriyama Oita, Japan More articles by this author Hiromitsu Mimata Oita, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...