Cell-cycle arrest and senescence in TP53-wild type renal carcinoma by enhancer RNA-P53-bound enhancer regions 2 (p53BER2) in a p53-dependent pathway

Although renal cell carcinoma remains one of the most malignant cancers, our understanding of progression and recurrence of this disease is limited. The present study explored the precise role of miR-155-5p in renal cancer metastasis. The expression of miR-155-5p in renal carcinoma clinical tissues and cells was determined using quantitative real time-polymerase chain reaction. The role of miR-155-5p on tumor cell growth were examined using CCK-8 and colony formation assays. Transwell assay was utilized to identify the role of miR-155-5p on the invasion and migration of renal cancer cells. Markers of epithelial-mesenchymal transition were determined using western blot. The in vivo effects of miR-155-5p on renal cancer cell growth, apoptosis, and metastasis were explored using xenograft mice. Luciferase reporter assay was performed to identify the potential target of miR-155-5p. Levels of miR-155-5p were significantly elevated in renal cancer tissues and cell lines. Suppression of miR-155-5p decreased the growth, colony formation, migration, and invasiveness of renal cancer cells. In contrast, overexpression of miR-155-5p led to opposite effects on renal cancer cells. Mechanically, the apoptosis-inducing factor was identified as the target of miR-155-5p. Interference of miR-155-5p significantly increased mRNA and protein expression of the apoptosis-inducing factor, whereas overexpression of miR-155-5p remarkably suppressed the apoptosis-inducing factor levels in renal cancer cells. The xenograft model identified that suppression of miR-155-5p restrained tumor growth and promoted apoptosis, whereas overexpression of miR-155-5p decreased apoptosis and accelerated tumor growth. Moreover, the number of lung metastasis nodules were decreased following injection with anti-miR-155-5p transfected cells, whereas the nodules were remarkably increased after overexpression of miR-155-5p. In addition, in vitro and in vivo assays both confirmed that suppression of miR-155-5p increased the expression of E-cadherin and decreased levels of N-cadherin and Snail, whereas overexpression of miR-155-5p accelerated epithelial-mesenchymal transition progression in renal cancer cells. These findings demonstrate that miR-155-5p enhances metastasis and epithelial-mesenchymal transition by targeting the apoptosis-inducing factor, suggesting that miR-155-5p represents a novel therapeutic target for renal cancer.

Cisplatin is widely employed in combating renal cancers. However, a major challenge is the resistance of cisplatin by renal cancer cells. Increased miR-222 expression is known to promote this resistance and so ways of downregulating the expression of miR-222 is widely sought after as a means of combating the resistance. The study was performed to explore the effects of miR-222 on cisplatin resistance of renal cancer cell strains and related mechanisms. The expression of miR-222 and Dickkopf-3 (DKK3) was regulated to explore the effects of miR-222 and DKK3 on cisplatin chemotherapy in renal cancer cisplatin-resistant cell strains, and to figure out the regulatory relationship between miR-222 and DKK3. In renal cancer tissues and cell lines, miR-222 was highly expressed and DKK3 was lowly expressed. Renal cancer cisplatin-resistant cells had markedly higher miR-222 expression and lower DKK3 expression than normal renal cancer cells, suggesting a potential involvement of miR-222 and DKK3 in the cisplatin resistance in renal cancer cells. Down-regulated miR-222 expression led to stronger inhibition of the renal cancer cell growth by cisplatin and markedly higher DKK3 expression. Dual-luciferase reporter assay results showed that miR-222 had a targeted inhibition on DKK3. Co-transfection of miR-222 mimic and shDKK3, together with the up-regulation of miR-222 and DKK3 expressions, resulted in a higher sensitivity of renal cancer cells to cisplatin chemotherapy than the up-regulation of miR-222 expression alone. Down-regulated miR-222 expression can remove the inhibition of DKK3 expression and increase the sensitivity of renal cancer cells to cisplatin chemotherapy.

To investigate whether microRNA-588 was involved in the development and progression of renal cancer, and to explore its possible regulatory mechanisms. Tumor tissues excised from renal carcinoma and adjacent normal tissues were selected for the experiment. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to analyze the expression level of microRNA-588 in tissue specimens. The relationship between the expression of microRNA-588 and the prognosis of patients with renal cell carcinoma was also evaluated. Subsequently, two renal cancer cell lines, including769-P and 786-O, were selected for functional experiments in vitro. Eukaryotic initiation factor 5A2 (pcDNA-EIF5A2) or microRNA-588 mimics was transfected into 769-P cells, respectively. Meanwhile, si-EIF5A2 or microRNA-588 inhibitor was transfected into 786-O cells. After that, the mRNA expression level of EIF5A2 was detected by qRT-PCR. The invasiveness and metastasis abilities of the two cell lines were evaluated via transwell assay. Furthermore, the levels of EIF5A2 and epithelial-mesenchymal transition (EMT)-related proteins were analyzed using Western blot. Luciferase reporter gene assay was used to confirm that microRNA-588 could directly regulate EIF5A2 expression. QRT-PCR and Western blot were performed to explore the mRNA and protein expressions of EIF5A2 in patients with highly or lowly-expressed microRNA-588. The correlation between the two molecules was evaluated using linear analysis. Through the above experiments, it was verified whether microRNA-588 could enhance the invasiveness and metastasis of renal cancer by targeting EIF5A2. MicroRNA-588 expression in tumor tissues of patients with renal carcinoma was significantly decreased with the increase of tumor diameter and stage. A higher level of microRNA-588 indicated significantly longer overall survival of patients. This suggested that microRNA-588 expression was negatively correlated with the prognosis of patients. Overexpression of microRNA-588 remarkably reduced the invasion and metastasis abilities of 769-P cells, as well as the expressions of EMT-related proteins. However, opposite results were observed in 786-O cells after knockdown of microRNA-588. Reporter gene assay confirmed that microRNA-588 could target bind to EIF5A2. In 769-P cells, up-regulated microRNA-588 significantly inhibited the mRNA and protein expressions of EIF5A2. However, down-regulated microRNA-588 in 786-O cells significantly enhanced the expressions of EIF5A2 at both mRNA and protein levels. Linear analysis verified that microRNA-588 was negatively correlated with EIF5A2 at the mRNA level. Additionally, the up-regulation of EIF5A2 in 769-P cells enhanced the malignancy of cancer cells and the expressions of EMT-related proteins. However, in 786-O cells, opposite results were observed after knockdown of EIF5A2. MicroRNA-588 was lowly expressed in renal cancer tissues and cell lines. This might lead to an increase in the protein level of EIF5A2, eventually promoting tumor invasion and metastasis.

359 ONCOGENIC FUNCTIONS OF SECRETED FRIZZLED-RELATED PROTEIN 2 (SFRP2) IN HUMAN RENAL CANCER

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-kappaB site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-kappaB activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies.

Preliminary study on the role of the C5orf46 gene in renal cancer

362 ROLE OF SECRETED FRIZZLED-RELATED PROTEIN 3 (SFRP3) IN HUMAN RENAL CELL CARCINOMA

Wnt/beta-catenin signaling is involved in renal cancer. Among Wnt antagonist families, sFRP3 is generally thought to be an inhibitor of Wnt signaling in several cancers. Here we reported the functional significance of sFRP3 in renal cell carcinoma. We performed immunohistochemistry on kidney tissue microarray and confirmed the expression level of sFRP3 in normal kidney and renal cancer cell lines (primary and metastatic). We transfected sFRP3 to the primary renal cancer cell lines and performed several functional analysis (MTS, colony formation, apoptosis, cell cycle, invasion, angiogenesis, and in vivo study). We also knocked down sFRP3 mRNA in metastatic cell lines using siRNA technique and performed several functional analysis. We found the level of sFRP3 protein was high in normal kidney, low in primary renal cancer tissues and high in metastatic renal cancer tissues. We performed dual sFRP3 mRNA expression experiments to determine the role of sFRP3 using primary and metastatic renal cancer cell lines. Functional analysis showed increased numbers of viable and invaded cells, tube formation and decreased numbers of apoptotic cells in sFRP3 transfected A498 cells. Promotion of tumor growth was also observed in nude mice injected with sFRP3 transfected A498 cells. In contrast, the number of viable cells and invasive cells was decreased in sFRP3 mRNA knocked down metastatic cells (ACHN and Hs891.T). To investigate the mechanism of sFRP3 function, we performed microarray analysis to see which genes were up or down regulated by sFRP3 expression. Among these genes, MMP-3 and ANGPT1 were significantly up-regulated in sFRP3 transfected cells. This is the first report to show that sFRP3 expression promotes cell growth, invasion, and inhibition of apoptosis in renal cancer cells. Furthermore, our results suggest that sFRP3 may play a role in invasion and angiogenesis by enhancing MMP-3 and ANGPT1 expression. The changes in sFRP3 expression levels in normal, primary cancer, and metastatic renal cancer tissues suggest that sFRP3 may play dual roles as a tumor suppressor gene and an oncogene during renal cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2355.

Esophageal cancer-related gene 4 (ECRG4) has been proposed as a putative tumor suppressor gene in several tumors. However, the role and regulation of ECRG4 in the pathogenesis of human renal cancer remain largely unknown. Our current study revealed that expression of ECRG4 is downregulated in renal cell lines and renal cancer tissues. ECRG4 expression was significantly associated with histological grade of tumors (p < 0.001), primary tumor stage (p = 0.017), and distant metastasis (p = 0.017). Low expression of ECRG4 was an independent prognostic indicator for survival of renal cancer patients. Silencing of ECRG4 expression in renal cell lines was associated with its promoter methylation. Moreover, ectopic expression of ECRG4 markedly inhibited cell proliferation and invasion in renal cancer cell lines. These results indicated that ECRG4 is frequently silenced by the methylation of promoter in renal cell cancers. ECRG4 may be a tumor suppressor in renal cancer and serve as a prognostic marker.

Renal cell carcinoma (RCC) is one of the most common malignancies in the urinary system. Due to the lack of early symptoms, diagnosis of RCC usually occurs at late stages or after cancer metastasis leading to poor prognosis. Therefore, it is crucial to study early molecular mechanisms and biomarkers. Previous studies have suggested that microRNAs are involved in RCC initiation and development, making them a good candidate for early diagnosis and therapy. MiR146b-5P plays important roles in the progression of multiple cancers including thyroid cancer, pancreatic cancer, cervical cancer. However, it is not clear whether and how miR146b-5P is involved in RCC. In this study, we aimed to investigate the function of miR146b-5P in RCC. We examined the expression levels of miR146b-5p in renal cancer tissue and cell lines. We also explored the effects of blocking miR146b-5p in renal tumor growth and inflammatory signaling. Finally, we determined if miR146b-5p regulates tumorigenesis through TRAF6. We found that miR146b-5p levels were significantly increased in renal cancer tissue and renal cancer cells. Blocking miR146b-5p suppressed renal tumor growth and enhanced inflammatory response through increased TRAF6 expression. These effects were eliminated in TRAF6 knockout mice. Our results suggest that enhanced miR146b-5p expression may be a biomarker for RCC and modulating miR146b-5p and TRAF6 levels represent a potential therapeutic strategy for RCC.

Krüppel-like factor 4 (KLF4) is a transcription factor that can have divergent functions in different malignancies. The expression and role of KLF4 in renal cell cancer remain unclear. The purpose of this study is to determine epigenetic alterations and possible roles of KLF4 in renal cell carcinoma. The KLF4 expression in primary renal cell cancer tissues and case-matched normal renal tissues was determined by protein and messenger RNA analyses. The epigenetic alterations were detected by methylation-specific PCR and Sequenom MassARRAY. Kaplan-Meier curves and the log-rank test were used for the survival analysis. The effects of KLF4 on cell growth and epithelial-to-mesenchymal transition (EMT) were determined in renal cancer cell lines after viral-based and RNA activation-mediated overexpression of KLF4. In vivo antitumor activity of KLF4 was evaluated by using stably KLF4-transfected renal cancer cells. KLF4 expression was dramatically decreased in various pathological types of renal cancer and associated with poor survival after nephrectomy. Hypermethylation of KLF4 promoter mainly contributed to its expression suppression. In vitro assays indicated that KLF4 overexpression inhibited renal cancer cell growth and survival. KLF4 overexpression also suppressed renal cancer cell migration and invasion by altering the EMT-related factors. In vivo assay showed that ectopic expression of KLF4 also inhibited tumorigenicity and metastasis of renal cancer. Our results suggest that KLF4 is a putative tumor suppressor gene epigenetically silenced in renal cell cancers by promoter CpG methylation and that it has prognostic value for renal cell progression.

BackgroundCell autophagy has been proposed to be involved in drug resistance therapy. However, how the long non-coding RNA (lncRNA) reduces risks of drug resistance in renal cancer (RC) cells needs a thorough inquiry. This study was assigned to probe the effect and mechanism of HOTAIR on sunitinib resistance of RC.MethodsClinical RC tissues and para-carcinoma tissues were obtained to detect the expressions of miR-17-5p, HOTAIR and Beclin1. Sunitinib-resistant cells (786-O-R and ACHN-R) were constructed using parental RC cells (786-O and ACHN). The resistance of 786-O-R and ACHN-R cells to sunitinib was examined. Western blot and qRT-PCR were assayed to obtain the expressions of miR-17-5p, HOTAIR and Beclin1. The effects of HOTAIR knockdown or miR-17-5p overexpression/knockdown on cell autophagy and sunitinib resistance were measured by MDC staining, immunofluorescence and Western blot. The sensitivity of RC cells to sunitinib and change in cell clone formation after sunitinib treatment were assessed by CCK-8 assay and colony formation assay, respectively. The relationships among HOTAIR, miR-17-5p and Beclin1 were verified by dual-luciferase reporter gene and RIP assay. The role of HOTAIR knockdown in sunitinib resistance was verified in nude mice.ResultsHOTAIR expression in sunitinib-resistant cells is higher than that in parental cells. Knockdown of HOTAIR in sunitinib-resistant cells lead to refrained sunitinib resistance and cell autophagy both in vivo and in vitro. Activation of autophagy could raise resistance to sunitinib in RC cells, while inhibition of autophagy could improve the sensitivity of sunitinib-resistant cells to sunitinib. HOTAIR could compete with miR-17-5p to regulate Beclin1 expression. Knockdown of miR-17-5p in parental cells increases cell resistant to sunitinib, and overexpression of miR-17-5p in sunitinib-resistant cells increases cell sensitive to sunitinib.ConclusionHOTAIR negatively targets miR-17-5p to activate Beclin1-mediated cell autophagy, thereby enhancing sunitinib resistance in RC cells.

Introduction: Tumor genotyping using fluid samples such as blood can potentially allow tracking of dynamic changes in mutational profiles over time and allow better access than biopsies. We present a method to detect somatic mutations from a blood draw, where circulating tumor cell (CTC) enrichment above 10% of total cell numbers allows the use of standard gene panels typically used to analyze tissue-based biopsies. Methods: Clinical samples were obtained from 9 prostate cancer (PC) patients and 6 renal cell cancer (RCC) patients, followed by CTC enrichment using the IsoFluxTM System. Cells were lysed and DNA amplified by whole genome amplification (WGA) using the NGS Kit (Fluxion Biosciences) and quantified via qPCR. CTCs defined as CK+, CD45- nucleated cells (DAPI+) for cell enumeration. Analytical samples were prepare by spiking tumor derived cell lines into whole blood and parallel analysis. Next-generation sequencing was performed using 3 targeted cancer panels on the Ion torrentTM PGM platform: the Ion ampliseqTM cancer hot spot panel (50 genes; 6 PC samples), Oncomine (143 genes; 3 PC samples), and a 29-gene panel of actionable mutations in RCC (6 samples). Data was analyzed using a customized variant calling/filtering pipeline. Variant filtering and functional interpretation was performed using VarSeqTM. All data was analyzed in a blinded manner. Results: Our method was able to isolate CTCs from all patient samples. WGA DNA concentrations were at a range of 25-164 ng/μL (median, 69) in PC and 29-180 ng/μL (median, 69) in RCC. CTC purity after the first enrichment step was in 2.9-33.7% (median, 10.5%) of PC samples and 1.9-33% (median, 14.5%) of RCC; final CTC purity is estimated at 5-40%. We found 1 variant/sample using hotpot, 12/sample using Oncomine, and 3/sample using the RCC panel. Conclusions: Our assay consistently detected somatic variants from blood draw using standard gene panels in both PC and RCC. Obtaining repeat tumor biopsies from patients during treatment and/or at time of progression is both challenging and impractical from a clinical perspective. Our assay provides molecular characterization using standard blood draws and will be prospectively validated in clinical trials. Citation Format: Robert J. Amato, Reynolds Brobey, Mehdi Dehghani, Kevin Rosenblatt, Glauco Souza, Hubert Tseng, Jeff Jensen, Cristian Ionescu-Zanetti. Somatic mutation detection via sequencing using circulating tumor cell samples from patients with renal cell and prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B09.

Introduction and Objective: The Src family of protein kinases (SFKs) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals in various malignancies. The pleotropic functions of SFKs in cancer make them promising targets for intervention. We investigated the role of miR-205 in inhibition of Src-mediated oncogenic pathways in renal cancer. Methods: The methods employed in this study include quantitative-real time PCR; western blot; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viabiltity, clonability, migration and invasiveness of prostate cancer cells. Luciferase reporter and mutational assays; generation of miR-205 stable cells and in-vivo study in nude mice was also performed. Results: The expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a non-malignant cell line, and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3’UTR sequences complementary to either Src, Lyn or Yes, which was abolished by mutations in these 3’UTR regions. Over-expression of miR-205 in A498 cells reduced Src, Lyn and Yes expression both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phosphoSrc-regulated ERK1/2 pathway. Cell motility factor- FAK and STAT3 activation was also inhibited by miR-205. Transient as well as stable over-expression of miR-205 in A498 cells resulted in induction of G0/G1 cell cycle arrest and apoptosis as indicated by decreased levels of cyclin D1 and cMyc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study demonstrating that miRNA-205 inhibits proto-oncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer. Conclusion: This study demonstrates that miRNA-205 inhibits proto-oncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2011-2936

Objective To observe serum protein change between Uygur with renal cell cancer (RCC) and Han with RCC and to construct different ethnic(including Han and Uygur) diagnostic model of RCC in XinJiang and screen serum differentially expressed protein between Han with renal cancer and Uygur with renal cancer in Xinjiang;Methods To use weak cation exchange and hydrophobic surface protein chip (CM10) and Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) to detect the serum protein.To detected serum specimens from patients (36 Han and 24 Uygur)with RCC and 60 healthy controls so as to construct different ethnic RCC diagnostic model and screen out disparate serum proteins between Han with RCC and Uygur with RCC.Results Obvious disparity existed between healthy controls and RCC patients in Uygur.Diagnostic model was composed and established by six proteins (5935,5945,5911,13 766,13 965,6887).The specificity and sensitivity were 91.67％ (22/24) and 100.00％ (24/24) ; Obvious disparity existed between healthy controls and RCC patients in Han.Total were four proteins peaks (5937,5345,5947,5912),and diagnostic model composed and established.The specificity and sensitivity were 94.44％ (34/36)and 91.67％ (33/36) ;disparity existed between Uygur RCC patients and Han RCC patients in terms of serum protein finger prints.Three proteins peaks M/Z 6887,13 766,13 965 existed diagnostic model of Uygur patients group while lacked in Han patients group,meanwhile,M/Z 5345 existed diagnostic model of Han patients group while lacked in Uygur patients group.Conclusion It has difference for protein finger prints between Han with RCC and Uygur with RCC in Xinjing.M/Z 6887,13 766,13 965,5345 may be differential markers between Uygur with RCC and Han with RCC and M/Z 6887,13 766,13 965 may be Keratin,transthyretin and interferon-inducedtransmembrane protein-1. Key words: Renal cell carcinoma; Han; Uygur; Proteomics