Renal Cell Carcinoma Samples Research Articles

Abstract 1155: Inhibitory effect of CYP1B1 on antitumor activities induced by dysregulated CDC20 and DAPK1 in renal cell carcinoma

Abstract Introduction and Objective: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC. Methods: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses. Results: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p<0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples. Conclusions: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC. Citation Format: Yozo Mitsui, Inik Chang, Shinichiro Fukuhar, Hiroshi Hirata, Ryan Kenji Wong, Soichiro Yamamura, Varahram Shahryari, Guoren Deng, Saini Sharanjot, Shahana Majid, Hiroaki Shiina, Rajvir Dahiya, Yuichiro Tanaka. Inhibitory effect of CYP1B1 on antitumor activities induced by dysregulated CDC20 and DAPK1 in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1155.

MicroRNA-195 targets VEGFR2 and has a tumor suppressive role in ACHN cells via PI3K/Akt and Raf/MEK/ERK signaling pathways.

Increasing evidence indicates that dysregulation of miR-195 may contribute to the occurrence and development of multiple types of human malignancies. However, the function and the mechanism of miR-195 in clear cell renal cell carcinoma (ccRCC) are still not fully understood. In the present study, we used qRT-PCR to detect the expression of miR-195 in ccRCC tissues and normal kidney tissues. MTT assay was performed to detect the cell viability of miR-195. Migration and invasion were evaluated by Transwell migration and Matrigel invasion assays, respectively. Additionally, apoptosis levels were evaluated using TUNEL assays, and signaling pathway changes were determined by western blot analysis. We observed that miR-195 was downregulated in clear cell renal cell carcinoma samples compared with normal renal samples. We identified that overexpression of miR-195 inhibited ACHN cell viability, migration, invasion, and it also induced cell apoptosis by targeting VEGFR2 via PI3K/Akt and Raf/MEK/ERK signaling pathways. These findings indicate that miR-195 has a tumor suppressive role in ACHN cells and miR-195 may be a promising candidate target for prevention and treatment of renal cell carcinoma.

ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma.

BackgroundActivating transcription factor 2 (ATF2) is a basic helix-loop-helix transcription factor, which has been shown to participate in the pathobiology of numerous cancers. However, the role of ATF2 in renal cell carcinoma (RCC) remains unclear.MethodsATF2 knockdown and overexpression studies were performed in RCC cells to evaluate changes in cell viability, cell cycle, apoptosis, migration and invasion. Xenograft models were used to examine the tumorigenic and metastatic capability of RCC cells upon ATF2 suppression. The expression of ATF2 in human RCC samples was determined using immunohistochemistry on a tissue microarray.ResultsATF2 knockdown in RCC cells reduced their proliferative and metastatic potentials, whereas ATF2 overexpression enhanced these properties. Mechanistic studies revealed that the transcription of CyclinB1, CyclinD1, Snail and Vimentin was directly regulated by ATF2 in RCC cells. Moreover, ATF2 was shown to be highly expressed in RCC tissues, especially in tumors with metastases. High expression of ATF2 correlated with aggressive clinico-pathological characteristics and predicted poor prognosis of RCC patients.ConclusionsATF2 exerts an oncogenic role in RCC and could serve as an important prognostic biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0383-2) contains supplementary material, which is available to authorized users.

Clinical potential of TCF21 methylation in the diagnosis of renal cell carcinoma.

The aim of the present study was to investigate the clinical potential of transcription factor (TCF) 21 methylation in the diagnosis of renal cell carcinoma (RCC). TCF21 methylation levels were quantified in renal tissues (55 cases of RCC tissue and 22 cases of normal tissue) and urine samples (33 cases of urine samples with RCC and 15 cases of normal urine samples) using pyrosequencing. Spearman's rank correlation coefficient was used to investigate the correlation between TCF21 methylation levels and clinical parameters (gender, age, smoking history, Fuhrman grade and clinical stage). The receiver operating characteristic (ROC) curve was utilized to evaluate the accuracy of predictive diagnosis of RCC. TCF21 methylation levels were significantly increased in RCC samples compared with normal renal tissues and urine samples. The Spearman's correlation analysis revealed that the TCF21 methylation level was positively associated with age (P=0.002), smoking (P=0.017) and Fuhrman grade (P=0.045) in RCC tissues and was positively associated with tumor size (P<0.001), Fuhrman grade (P=0.017) and clinical stage (P=0.017) in urine samples. ROC curves revealed that the cut-off value, sensitivity and specificity were 23.61, 89.00 and 61.90%, respectively in tissue samples, and 26.84, 79 and 100%, respectively in urine samples. Furthermore, there were significant differences in the area under the curve between the tissue and urine samples (P=0.004). The results of the present study indicate that TCF21 may be used as a biomarker for diagnosing RCC, and TCF21 methylation levels in urine samples may be a useful means of diagnosing RCC.

MicroRNA-126 inhibits tumor cell invasion and metastasis by downregulating ROCK1 in renal cell carcinoma.

MicroRNAs (miRNAs) are involved in cancer development and progression. Renal cell carcinoma (RCC) frequently undergoes metastasis and has a high mortality rate. The current study measured miRNA-126 (miR-126) expression levels in 128 pairs of clear cell RCC and adjacent normal kidney tissue samples by reverse transcription-quantitative polymerase chain reaction, and analyzed the association between miR-126 and various clinicopathological parameters. In addition, cell proliferation, wound healing and cell invasion assays were conducted using RCC cells overexpressing miR-126. Potential miR-126 target genes and the signaling pathways that may be regulated by miR-126 were then examined. miR-126 expression was significantly reduced in patients with metastatic RCC compared with patients without metastasis. Consistently, overexpression of miR-126 in RCC cells significantly inhibited cell proliferation, migration and invasion in vitro compared with negative control miRNA. A luciferase reporter assay demonstrated that miR-126 targets Rho associated coiled-coil containing protein kinase 1 (ROCK1) by directly binding the 3′-untranslated region. Furthermore, western blotting identified miR-126 as an important regulator of the AKT and extracellular signal-regulated 1/2 signaling pathways. The results of the present study indicate that miR-126 inhibits RCC cell proliferation, migration and invasion by downregulating ROCK1. These findings suggest that miR-126 may be valuable as a potential target for therapeutic intervention in RCC.

AGS16F Is a Novel Antibody Drug Conjugate Directed against ENPP3 for the Treatment of Renal Cell Carcinoma.

New cancer-specific antigens are required for the design of novel antibody-drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody-mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial.

MP92-18 GOLPH3 IS OVEREXPRESSED AND CONTRIBUTES TO APOPTOTIC-RESISTANCE AND METASTASIS IN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: The enhancer of zeste homolog 2 (EZH2) gene has been recognized as a proto-oncogene and linked to human malignancies. Here, we determined EZH2 expression in primary renal cell carcinoma (RCC) specimens and explored its potential role in prosurvival and metastasis of RCC. METHODS: EZH2 protein expression in RCC specimens was determined by immunohistochemistry. siRNA targeting EZH2 were transfected into RCC CaKi-2, 786-O, and 769-P cells. Cell cycle and apoptosis were detected by flow cytometry following EZH2 knockdown. Transwell assay were used to determine invasive potential in response to EZH2 inhibition. Western blot was performed to detect the expression of cell cycle and apoptosis associated proteins. RESULTS: EZH2 was higher expressed in RCC sample than in paired adjacent nontumorous tissue. 76.5% of RCC sample showed dense staining of EZH2, whereas the neighboring unaffected cells stained at the normal level. After RCC cells were transfected with siRNA targeting to EZH2, all three cells were arrested in G1/S phase (P 0.05, compared to control siRNA transfected cells). In addition, significant increased apoptosis were observed in the three cell lines following EZH2 silencing (P 0.05, compared to control cells). Furthermore, inhibition of EZH2 resulted in decreased invasive capability of RCC cells in vitro. Mechanically, EZH2 knockdown led to upregulation of p21, cleaved caspase-9, -3, and PARP. Downregulation of PCNA and MMP-2 was also observed following EZH2 silencing. CONCLUSIONS: EZH2 is overexpressed in RCC. Moreover, it plays a critical role in both proliferation and apoptotic-resistance in RCC cell lines. In addition, EZH2 can enhance invasive potential of RCC cell in vitro. These data suggest that inhibition of EZH2 could provide an advantage in improving therapeutic effect of RCC.

MP85-02 OVEREXPRESSION OF VCAN IS ASSOCIATED WITH METASTASIS AND UNFAVORABLE PROGNOSIS IN PATIENTS WITH RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2016MP85-02 OVEREXPRESSION OF VCAN IS ASSOCIATED WITH METASTASIS AND UNFAVORABLE PROGNOSIS IN PATIENTS WITH RENAL CELL CARCINOMA Yozo Mitsui, Hiroaki Shiina, Shinichiro Fukuhara, Miho Hiraki, Naoko Arichi, Hiroaki Yasumoto, Rajvir Dahiya, and Yuichiro Tanaka Yozo MitsuiYozo Mitsui More articles by this author , Hiroaki ShiinaHiroaki Shiina More articles by this author , Shinichiro FukuharaShinichiro Fukuhara More articles by this author , Miho HirakiMiho Hiraki More articles by this author , Naoko ArichiNaoko Arichi More articles by this author , Hiroaki YasumotoHiroaki Yasumoto More articles by this author , Rajvir DahiyaRajvir Dahiya More articles by this author , and Yuichiro TanakaYuichiro Tanaka More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2268AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The supportive stroma around a solid tumor consists of specific extracellular matrix components, and plays important roles for activating the microenvironment of primary and secondary tumor sites. Versican (VCAN), a large aggregating chondroitin sulfate proteoglycan, is secreted into the extracellular environment. VCAN promotes tumor progression and enhances metastasis in several cancers, and we hypothesized that overexpression of VCAN is associated with the pathogenesis of renal cell carcinoma (RCC). METHODS The expression of VCAN was analyzed using 3 renal tumor cell lines (Caki-2, ACHN, 786-O), and 84 matched clear cell RCC and normal renal tissues. We also examined whether down-regulation of VCAN had an effect on the viability, or migratory and invasion properties of RCC cells. RESULTS The mRNA expression of VCAN in the RCC cell lines was significantly higher than in the nonmalignant HK-2 cell line and also significantly higher in RCC samples as compared to normal kidney samples (p<0.001). The higher expression of VCAN was significantly correlated with systematic metastasis (p<0.001). When the expression level of the VCAN mRNA transcript was divided into 2 groups based on median values, higher expression was significantly associated with 5-year cause-specific survival after radical nephrectomy (p<0.05). After transfection of VCAN siRNA, cell viability, migration, and invasion ability were significantly inhibited in the RCC cell lines. We further analyzed genes affected by VCAN knockdown based on gene microarray and observed that TNF-alpha was up-regulated and MMP7 and CXCR4 were down-regulated. CONCLUSIONS VCAN expression is up-regulated in RCC and associated with poor prognosis. VCAN depletion reduces tumorigenesis in RCC cell lines by inducing TNF-alpha-mediated apoptosis. In addition, VCAN can promote RCC development and metastasis by inducing MMP7 and CXCR4 expression. Thus, VCAN may be an attractive target for novel diagnostic and therapeutic strategies for RCC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1098 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yozo Mitsui More articles by this author Hiroaki Shiina More articles by this author Shinichiro Fukuhara More articles by this author Miho Hiraki More articles by this author Naoko Arichi More articles by this author Hiroaki Yasumoto More articles by this author Rajvir Dahiya More articles by this author Yuichiro Tanaka More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma - Tissue Microarray of Human Samples.

Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of “inhibitor of caspase-activated DNase” (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

Decreased apoptosis repressor with caspase recruitment domain confers resistance to sunitinib in renal cell carcinoma through alternate angiogenesis pathways

Apoptosis repressor with caspase recruitment domain (ARC), an endogenous inhibitor of apoptosis, is upregulated in a number of human cancers, thereby conferring drug resistance and giving a rationale for the inhibition of ARC to overcome drug resistance. Our hypothesis was that ARC would be similarly upregulated and targetable for therapy in renal cell carcinoma (RCC). Expression of ARC was assessed in 85 human RCC samples and paired non-neoplastic kidney by qPCR and immunohistochemistry, as well as in four RCC cell lines by qPCR, Western immunoblot and confocal microscopy. Contrary to expectations, ARC was significantly decreased in the majority of clear cell RCC and in three (ACHN, Caki-1 and 786-0) of the four RCC cell lines compared with the HK-2 non-cancerous human proximal tubular epithelial cell line. Inhibition of ARC with shRNA in the RCC cell line (SN12K1) that had shown increased ARC expression conferred resistance to Sunitinib, and upregulated interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). We therefore propose that decreased ARC, particularly in clear cell RCC, confers resistance to targeted therapy through restoration of tyrosine kinase-independent alternate angiogenesis pathways. Although the results are contrary to expectations from other cancer studies, they were confirmed here with multiple analytical methods. We believe the highly heterogeneous nature of cancers like RCC predicate that expression patterns of molecules must be interpreted in relation to respective matched non-neoplastic regions. In the current study, this procedure indicated that ARC is decreased in RCC.

Kidney cancer. Heavy metals as a risk factor

The association between exposure to heavy metals and cancer has been extensively studied, although the mechanisms involved are far from being completely understood. Concerning renal cell carcinoma, several heavy metals have been implicated as risk factors, namely cadmium (Cd) and lead (Pb). Herewith, we investigated the presence and distribution of heavy metals in samples of renal cell carcinoma, as well as adjacent renal tissue (control samples), in patients submitted to radical or partial nephrectomy for renal cell carcinoma. Samples from renal tumour and adjacent renal tissue were processed and observed by Scanning Electron Microscopy coupled with X-Ray Microanalysis (SEM-XRM), in order to detect and quantify heavy metals in situ, using the JEOL JSM-6301F microscope. Our results revealed a significant difference in the composition of heavy metals between the renal adjacent tissue (control) and the tumour tissue. No heavy metal particles were detected in the adjacent (control) tissue, by this technique, but the presence of heavy metals such as chromium (Cr), iron (Fe) and copper (Cu) were detected among other particles which could be seen in the tumour tissue samples. Our results might suggest a possible role of heavy metals in the oncogenic pathway of renal cell carcinoma. However, there is controversy regarding this topic: 1 - Is this anomalous sequestration of heavy metals just an epiphenomenon or is this a hint of a causal mechanism? 2 - Can heavy metals be used as biomarkers with potential diagnostic or prognostic interest? For example, could they be used to improve the diagnostic value of renal biopsy in clinical practice?

Genomic profiling of the genes on chromosome 3p in sporadic clear cell renal cell carcinoma

Somatic mutations of the BRCA1 associated protein-1 (BAP1) gene, which maps to 3p21, have been found in several tumors including malignant mesothelioma, uveal melanoma, and renal cell carcinoma (RCC). The role of BAP1 inactivation in tumor development remains unclear. It has been reported that Vhl knock-out mice did not develop RCC, but Vhl knock-out mice with single allele loss of Bap1 in nephron progenitor cells developed RCC, indicating that Bap1 inactivation may be essential in murine renal tumorigenesis. To clarify the role of BAP1 in human RCC development, we performed mutation analyses, including copy number detection of BAP1 and assessment of allelic imbalance using microsatellite polymorphisms on 3p, in 45 RCC samples derived from 45 patients without VHL or BAP1 germline mutation. Additionally, we analyzed the sequences of the VHL, PBRM1, and SETD2 genes, and examined promoter methylation of VHL. Using immunostaining, we also checked for expression of BAP1 protein, which is normally located in the nuclei. None of the RCCs had biallelic deletion of BAP1, but five (11.1%) showed a biallelic mutation (four with a sequence-level mutation with monoallelic loss and one with a biallelic sequence-level mutation); these cells were negative for nuclear BAP1 staining. These patients had worse recurrence-free survival than the patients without a biallelic mutation (p=0.046). However, there were no significant differences in worse outcome by multivariate analysis combined with age, T stage, histological subtype, infiltration and vascular invasion. In 35 RCCs (77.8%), monoallelic loss of BAP1 was accompanied by VHL biallelic mutation or VHL promoter hypermethylation. In five RCCs (11.1%), we detected 3p loss-of-heterozygosity, but the copy number of BAP1 was normal. Surprisingly, nuclear staining of BAP1 was negative in 10 out of 31 tumors (32.3%) with hemizygous normal BAP1, suggesting that haploinsufficiency may relate to RCC development.

Identification of long-non coding RNA UCA1 as an oncogene in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, which is associated with poor prognosis and high recurrence. Long non‑coding RNAs (lncRNAs) have been reported to be dysregulated in cancer and to be important in the regulation of carcinogenesis, thus suggesting that this class of molecules may be used as biomarkers in cancer. The lncRNA urothelial carcinoma associated 1 (UCA1) has been observed to be upregulated and to function as an oncogene in certain types of cancer; however, the role of UCA1 in RCC remains to be elucidated. The present study aimed to determine the expression and function of UCA1 in RCC. Quantitative polymerase chain reaction (qPCR) was used to determine the expression levels of UCA1 in 46 paired RCC and adjacent normal tissue samples. Furthermore, qPCR was used to determine the expression levels of UCA1 in four RCC cell lines compared with the human embryonic kidney 293T cell line. The impact of UCA1 on cell migration, proliferation and apoptosis was investigated by wound scratch assay, MTT and flow cytometry, respectively. The results of the present study demonstrated that UCA1 expression levels were significantly increased in RCC tissues and cells, as compared with the controls. Ectopic expression and gene silencing of UCA1 in RCC cell lines exerted opposite effects on cellular proliferation, migration and apoptosis, and the results suggested that UCA1 may function as an oncogene in RCC. These results indicated that UCA1 may be considered as a promising biomarker for diagnosis, and a therapeutic target in RCC. Further research is required to elucidate the role and target genes of UCA1 in RCC.

T-cell receptor (TCR) repertoire in metastatic renal cell carcinoma (RCC) patients treated with first-line vascular endothelial growth factor receptor blockade.

501 Background: Reports have demonstrated an inverse relationship between suppression of immune surveillance mechanisms and activation of the vascular endothelial growth factor receptor (VEGFR) pathway suggesting that T cell repertoires may impact response to VEGFR blockade. We evaluated the association between clinical outcomes and T cell repertoire in metastatic RCC patients receiving a front-line anti-VEGFR, pazopanib. Methods: Pre-treatment RCC tumors were analyzed from VEG105192, a phase III study of mRCC patients randomized (2:1) to pazopanib (paz) 800 mg daily vs placebo (pbo) for TCR gamma (TCRG) and TCR beta (TCRB) CDR3 regions. Using the Adaptive Biotechnologies immunoSEQ Assay, we assessed TCR clonality, a measure of total repertoire represented by expanded clones, and entropy, a measure of evenness and diversity. PD-L1 was evaluated by immunohistochemistry (IHC) H-Scores. The goal of the study was to determine if the repertoire of T cell clones was associated with progression-free survival as a clinical endpoint. Results: In the cohort with available tissue, the median PFS was 10.8 and 5.5 months (mos) for paz and pbo, respectively. TCRB (n = 114) and TCRG (n = 43 pbo, 109 paz) clonality ranged from 0-0.31 and 0-0.98, and entropy from 1-12.1 and 0-10.37, respectively. TCRB and TCRG entropy were highly correlated (Spearman’s R = 0.92, n = 114). Samples from the pbo-treated group with higher TCRG entropy, defined as the top 25th percentile, were associated with an improved median PFS (12.8 months) when compared to the lower 75th percentile (3.1 months, P = 0.023); similar trends were seen for TCRB entropy. Neither entropy nor clonality was associated with maximal reduction in tumor volume in the paz-treated group. PD-L1 H-scores were not associated with entropy or clonality (P &gt; 0.05). Conclusions: Our data suggests that RCC samples with higher entropy are associated with a favorable prognosis. Identification of tumors with restricted TCRB/G chain usage and less diverse repertoire, as represented by lower entropy and higher clonality, may impact responses to VEGFR blockade and requires further study. Clinical trial information: NCT00334282.

CYP1B1 promotes tumorigenesis via altered expression of CDC20 and DAPK1 genes in renal cell carcinoma.

BackgroundCytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC.MethodsExpression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses.ResultsFirst, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p < 0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples.ConclusionsCYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.

MiR-362-3p targets nemo-like kinase and functions as a tumor suppressor in renal cancer cells.

MicroRNAs (miRNAs) have been demonstrated to exhibit abnormal expression patterns in various types of human cancer. The aim of the present study was to identify a novel tumor suppressor microRNA (miR) and investigate its physiological function and mechanism in renal cell carcinoma (RCC). The expression levels of miRNA (miR)‑362‑3p expres were measured in 47 pairs of RCC and adjacent normal tissue samples, using reverse transcription-quantitative polymerase chain reaction analysis. In addition, miR‑362‑3p was transfected into renal cancer cells to investigate its role in the regulation of cell proliferation, migration, invasion, apoptosis and cell cycle. Identification of the target gene of miR‑362‑3p was performed using luciferase reporter assays and western blot analyses. The results demonstrated that the expression levels of miR‑362‑3p were downregulated in the RCC tissue samples, compared with the adjacent normal tissue samples. The upregulation of miR‑362‑3p using a synthesized mimic suppressed the proliferation, migration and invasion of the renal cancer cells, and induced cell apoptosis and G1 phase arrest. Further experiments demonstrated that the overexpression of miR‑362‑3p resulted in decrease expression levels of nemo-like kinase. These results suggested that miR-362-3p functions as a tumor suppressor in RCC, and may serve as a potential molecular target in the treatment of RCC.

Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma.

Aberrant expression of cancer genes and non-canonical RNA species is a hallmark of cancer. However, the mechanisms driving such atypical gene expression programs are incompletely understood. Here, our transcriptional profiling of a cohort of 50 primary clear cell renal cell carcinoma (ccRCC) samples from The Cancer Genome Atlas (TCGA) reveals that transcription read-through beyond the termination site is a source of transcriptome diversity in cancer cells. Amongst the genes most frequently mutated in ccRCC, we identified SETD2 inactivation as a potent enhancer of transcription read-through. We further show that invasion of neighbouring genes and generation of RNA chimeras are functional outcomes of transcription read-through. We identified the BCL2 oncogene as one of such invaded genes and detected a novel chimera, the CTSC-RAB38, in 20% of ccRCC samples. Collectively, our data highlight a novel link between transcription read-through and aberrant expression of oncogenes and chimeric transcripts that is prevalent in cancer.

MicroRNA-22 functions as a tumor suppressor by targeting SIRT1 in renal cell carcinoma.

Accumulating evidence demonstrates that microRNA-22 (miR-22) was deregulated in many types of cancers and was involved in various cellular processes related to carcinogenesis. However, the exact roles and mechanisms of miR-22 remain unknown in human renal cell carcinoma (RCC). Here, the relationship between miR-22 expression pattern and clinicopathological features of patients with EOC were determined by real-time quantitative RT-PCR (qRT-PCR). Furthermore, the role of miR-22 and possible molecular mechanisms in EOC were investigated by several in vitro approaches and in a nude mouse model. Results from qRT-PCR showed that miR-22 was significantly downregulated in RCC samples compared with corresponding non-cancerous tissues, which was significantly associated with tumor stage and lymph node metastasis. Functional study demonstrated that enforced overexpression of miR-22 in renal cancer cells inhibited proliferation, migration and invasion, and induced cell apoptosis in vitro, and suppressed tumor growth in vivo. In addition, SIRT1 was identified as a direct target of miR-22 by a luciferase reporter assay. Overexpression of miR-22 activated p53 and its downstream target p21 and PUMA, and the apoptosis markers cleaved CASP3 and PARP, and inhibited epithelial-mesenchymal transition (EMT). These findings showed that miR-22 functioned as tumor suppressor in RCC and blocked RCC growth and metastasis by directly targeting SIRT1 in RCC, indicating a potential novel therapeutic role in RCC treatment.

MiR-96 suppresses renal cell carcinoma invasion via downregulation of Ezrin expression

BackgroundThe present study examined the role of microRNA (miR)-96 in renal cell carcinoma (RCC) invasion.MethodsThe expression of miR-96 was detected by quantitative reverse transcription-polymerase chain reaction in human RCC cell lines with high (Caki-1) and low (786-O) metastatic potential. Invasive ability and Ezrin expression were assessed in Caki-1 and 786-O cells transfected with a miR-96 mimic or inhibitor using wound healing assays, Transwell assays and western blotting. Expression of miR-96 and Ezrin was also examined in primary RCC samples from 17 patients with metastatic disease and 46 patients who maintained remission during a follow-up period of 37 months.ResultsmiR-96 expression was significantly lower in Caki-1compared to786-O cells. The invasive ability of Caki-1 and 786-O cells increased following transfection of cells with miR-96 inhibitor, whereas it decreased following transfection with miR-96 mimic. Ezrin levels were negatively correlated with miR-96 in RCC, and inhibition of Ezrin expression suppressed the miR-96-induced change in invasive ability. The negative correlation between miR-96 and metastasis/Ezrin expression was also observed in human RCC specimens.ConclusionsThese results suggest that miR-96 suppresses RCC invasion by modulating Ezrin expression.

Abstract 1686: CD70 as a target in non-Hodgkin lymphoma (NHL) and renal cell carcinoma (RCC)

Abstract Background CD70 is a member of the TNF superfamily expressed primarily on activated lymphocytes. CD70 interacts with CD27 to regulate B and T cell functions. Among normal, nonlymphoid tissues, CD70 is only expressed on stromal cells of the thymic medulla and mature dendritic cells. CD70 expression in RCC and NHL highlights its potential as a target for cancer therapy. Methods An indirect immunohistochemistry (IHC) assay was developed using an anti-CD70 mouse monoclonal antibody (Ryan 2010), and validated in a CLIA-compliant laboratory. Control cell lines had receptor numbers and IHC staining intensities of ∼190,000 (3+), ∼21000 (3+), ∼4000 (1+) and 0 (0). Archived tumor tissue from consenting patients with relapsed/refractory (RR) NHL or metastatic RCC were stained and read by a board-certified pathologist at Quest Diagnostics, reporting the percentage of cells staining for CD70, intensity, and subcellular localization of the stain. Results The rates and homogeneity of combined membrane and non-membrane CD70 staining intensity ≥1+ and 3+ for each tumor type are shown in the table below. The majority of NHL (88%) and RCC (80%) samples exhibited membranous CD70 staining ≥1+; &amp;gt;75% exhibited 3+ staining. Among the NHL and RCC subtypes sampled, high levels of 3+ intensity were observed in diffuse large B cell lymphoma (DLBCL; N = 44, 84%), follicular lymphoma (FL; N = 26, 77%), mantle cell lymphoma (MCL; N = 6, 67%), clear cell RCC (ccRCC; N = 138, 93%), and papillary RCC (pRCC; N = 15, 67%). No chromophobe RCC samples showed membranous staining (N = 5). CD70 expression in NHL and RCC tumor samples% cells in sample expressing CD70Samples positive for membrane and non-membrane CD70 expression, n (%)NHLRCCDLBCL, N = 44Other NHL, N = 41Clear Cell, N = 138Other RCC, N = 32&amp;gt;/ = 1+ intensity3+ intensity&amp;gt;/ = 1+ intensity3+ intensity&amp;gt;/ = 1+ intensity3+ intensity&amp;gt;/ = 1+ intensity3+ intensity02 (5)7 (16)6 (15)12 (29)4 (3)9 (7)11 (34)14 (44)1-209 (20)10 (23)11 (27)17 (41)12 (9)26 (19)7 (22)7 (22)21-404 (9)6 (14)8 (20)3 (7)14 (10)19 (14)2 (6)3 (9)41-604 (9)3 (7)3 (7)4 (10)10 (7)18 (13)2 (6)5 (16)61-807 (16)7 (16)5 (12)1 (2)12 (9)26 (19)4 (13)2 (6)81-10018 (41)11 (25)8 (20)4 (10)86 (62)40 (29)6 (19)1 (3) Conclusions CD70 is expressed in the majority of DLBCL, FL, MCL, ccRCC, and pRCC samples tested. Based on the unmet medical needs of these diseases, limited expression of CD70 on normal tissues, predominant membrane expression of CD70, and observed preclinical antitumor activity (Jeffrey 2013), a CD70-targeted antibody-drug conjugate, SGN-CD70A, has been advanced into clinical development in CD70-positive RR NHL and metastatic RCC (NCT# NCT02216890). Citation Format: Kenneth W. Wood, Elaina M. Gartner, Mechthild Jonas, Maureen Ryan, Dana A. Kennedy. CD70 as a target in non-Hodgkin lymphoma (NHL) and renal cell carcinoma (RCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1686. doi:10.1158/1538-7445.AM2015-1686