Renal Cell Carcinoma Specimens Research Articles

MiR‑539 suppresses proliferation and induces apoptosis in renal cell carcinoma by targeting high mobility group A2

Renal cell carcinoma (RCC) is one of the most common urinary malignancies with a high rate of morbidity. MicroRNAs (miRNAs) have been shown to be critical post-transcriptional regulators in tumorigenesis. The present study aimed to investigate the effect of miRNA (miR)-539 on the proliferation and apoptosis of RCC. The expression of miR-539 and high mobility group AT-hook 2(HMGA2) were examined in clinical RCC specimens. The 786-O RCC cell line was also used and was transfected with miR-539 mimics or inhibitors. The correlation between miR-539 and HMGA2 was confirmed using a luciferase reporter assay. Cell viability and apoptosis were detected using MTT and flow cytometry assays. The protein levels of HMGA2, AKT, phosphorylated (p)-AKT, mammalian target of rapamycin (mTOR) and p-mTOR were analyzed using western blot analysis. The results revealed that miR-539 was negatively correlated with the expression of HMGA2 in clinical RCC specimens. Further experiments identified HMGA2 as a direct target of miR-539. The overexpression of miR-539 downregulated the expression of HMGA2, reduced cell proliferation and promoted cell apoptosis, whereas the knockdown of miR-539 led to the opposite results. miR-539 also suppressed the phosphorylation of AKT and mTOR, without altering the levels of total AKT and mTOR. Taken together, the results of the present study indicated that miR-539 negatively regulated the expression of HMGA2 in clinical specimens and in vitro. miR539 inhibited cell proliferation and induced apoptosis in RCC cells. This regulatory effect of miR-539 may be associated with the AKT signaling pathway. Therefore, miR-539 may be used as a biomarker for predicting the progression of RCC.

T-cell Responses in the Microenvironment of Primary Renal Cell Carcinoma-Implications for Adoptive Cell Therapy.

In vitro expansion of large numbers of highly potent tumor-reactive T cells appears a prerequisite for effective adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TIL) as shown in metastatic melanoma (MM). We therefore sought to determine whether renal cell carcinomas (RCC) are infiltrated with tumor-reactive T cells that could be efficiently employed for adoptive transfer immunotherapy. TILs and autologous tumor cell lines (TCL) were successfully generated from 22 (92%) and 17 (77%) of 24 consecutive primary RCC specimens and compared with those generated from metastatic melanoma. Immune recognition of autologous TCLs or fresh tumor digests was observed in CD8+ TILs from 82% of patients (18/22). Cytotoxicity assays confirmed the tumoricidal capacity of RCC-TILs. The overall expansion capacity of RCC-TILs was similar to MM-TILs. However, the magnitude, polyfunctionality, and ability to expand in classical expansion protocols of CD8+ T-cell responses was lower compared with MM-TILs. The RCC-TILs that did react to the tumor were functional, and antigen presentation and processing of RCC tumors was similar to MM-TILs. Direct recognition of tumors with cytokine-induced overexpression of human leukocyte antigen class II was observed from CD4+ T cells (6/12; 50%). Thus, TILs from primary RCC specimens could be isolated, expanded, and could recognize tumors. However, immune responses of expanded CD8+ RCC-TILs were typically weaker than MM-TILs and displayed a mono-/oligofunctional pattern. The ability to select, enrich, and expand tumor-reactive polyfunctional T cells may be critical in developing effective ACT with TILs for RCC. In summary, TILs isolated from primary RCC specimens could recognize tumors. However, their immune responses were weaker than MM-TILs and displayed a mono-/oligofunctional pattern. The ability to select and expand polyfunctional T cells may improve cell therapy for RCC. Cancer Immunol Res; 6(2); 222-35. ©2018 AACR.

Cancer-Specific Survival Stratification Derived from Tumor Expression of Tissue Inhibitor of Metalloproteinase-2 in Non-Metastatic Renal Cell Carcinoma.

Degradation of the extracellular matrix is a prerequisite for the processes of cancer cell invasion and metastasis. The purpose of our study was to assess the association of matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9) and their inhibitors (TIMP-1 and TIMP-2) with renal cell carcinoma (RCC) progression and cancer-specific survival (CSS), using immunohistochemical analysis of 60 formalin-fixed, paraffin-embedded sections of tumor tissue and normal tissue near the tumor from surgical T1-3bN0M0 RCC specimens. Significant overexpression of MMP-2 in tumor and normal tissue was correlated with advanced stages, tumor size, sarcomatous differentiation and clinical symptoms. Overall survival was 31.7% (55.2% M0, 9.7% M1) and CSS 56.7% (100% M0, 16.1% M1) with a follow-up of 76 (5-230) months. Fuhrman grade [HR 2.87 (95% CI: 1.28-6.45); p=0.01], tumor size [HR 1.13 (95% CI: 1.03-1.26); p=0.009] and low TIMP-2 expression [HR 0.35 (95% CI: 0.16-0.78); p=0.01] were independent predictive factors of CSS and stratified the patients into three groups with different rates of 10-year CSS; [100%, 73.9% and 20.5% for the good, intermediate and poor prognosis group respectively (p=0.000006)] . This study offers strong evidence that TIMP-2 expression in tumor tissue may play a crucial role in progression and poor prognosis in human localized and locally advanced RCC.

Overexpression of CAPN2 promotes cell metastasis and proliferation via AKT/mTOR signaling in renal cell carcinoma.

The calpain 2 (CAPN2) is upregulated in various malignant carcinomas. Previous studies have reported that CAPN2 functioned as an oncogenic factor in human cancers. However, its clinical role and potential effects on cell metastasis and proliferation in renal cell carcinoma (RCC) remain unknown. In this study, we evaluated the mRNA and protein levels of CAPN2 in human RCC specimens, matched normal specimens, and RCC cell lines using quantitative Real-time PCR (RT-PCR) and western blot. Immunohistochemistry of 74 RCC tissues in a tissue microarrays (TMAs) and normal kidney tissues were performed. Kaplan-Meier survival curve analyses were conducted to measure the correlation between CAPN2 and tumor prognosis. Cell migration, invasion and proliferation were detected by transwell assays and Cell Counting Kit-8 (CCK-8) assays. CAPN2 exhibited a significant overexpression in human RCC tissues and cell lines compared with adjacent non-tumor tissues and normal human proximal tubule epithelial cell line HK-2. Strong staining of CAPN2 was associated with higher clinical stage and histological grade. In addition, sh-CAPN2 could significantly inhibit migration, invasion and proliferation of 769-P and CAKI-1 cells. Conversely, increased cell biological behaviors were observed in CAPN2-OV CAKI-2 cells. Moreover, the subsequent mechanism investigation suggested that CAPN2 promoted tumor progression by activating AKT/mTOR signaling, enhancing epithelial mesenchymal transition (EMT) and MMP9 levels. The present study indicates that CAPN2 may act as a prominent indicator for RCC progression and a novel therapeutic target for RCC patients.

Regulation of spindle and kinetochore-associated protein 1 by antitumor miR-10a-5p in renal cell carcinoma.

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA‐10a‐5p (miR‐10a‐5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR‐10a‐5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR‐10a‐5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR‐10a‐5p in RCC cell lines (786‐O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore‐associated protein 1 (SKA1) was identified as an antitumor miR‐10a‐5p target by genome‐based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI‐treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR‐10a‐5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR‐10a‐5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.

DNA-methylation-mediated repression of miR-766-3p promotes cell proliferation via targeting SF2 expression in renal cell carcinoma.

Aberrant expression of microRNA (miRNA) emerges as an important role in a wide range of human malignances, and further identification as well as validation of the change of these endogenous non-protein-coding transcripts is warranted. Here, we identify a novel epigenetic regulation of miR-766-3p and investigate its biological function as well as clinical significance in renal cell carcinoma (RCC). Bisulfate analysis elucidates that the promoter of miR-766-3p is highly methylated in RCC tissues compared to non-tumorous tissues. Notably, the downregulation of miR-766-3p is obviously associated with clinical stage and worse prognosis in RCC patients. Upregulated miR-766-3p attenuates cell-cycle progression via targeting SF2 expression and additional SF2/P-AKT/P-ERK signaling pathway. Moreover, high level of SF2, as a novel oncoprotein in RCC, was significantly associated with poor survival in a large cohort of RCC specimens. Taken together, our study presents a road map for the prediction and validation of miR-766-3p/SF2 axis and thus imparts a therapeutic way for further RCC progression.

MiRNA-200a induce cell apoptosis in renal cell carcinoma by directly targeting SIRT1.

Accumulating evidence indicates that microRNAs are implicated in tumor initiation and progression through negatively regulating oncogenes or tumor suppressor genes. In the present study, we report that the expression of miR-200a was significantly lower in renal cell carcinoma (RCC) specimens and RCC cell lines. Restoration of miR-200a suppressed cell growth, arrested cell cycle progression, and promoted cell apoptosis in RCC cell lines. We next used qRT-PCR array technology to identify Sirtuin 1 (SIRT1) as one of the downregulated proteins during miR-200a overexpression in 786-O cells. Following a further assay by luciferase reporter system, SIRT1 was validated as a direct target of miR-200a. Moreover, siRNA-mediated knockdown of SIRT1 could partially phenocopy the effects of miR-200a overexpression. In contrast, overexpression of truncated SIRT1 (without an endogenous 3'-UTR) could rescue the effect of miR-200a overexpression on 786-O cells, which suggested that SIRT1 3'-UTR is targeted by miR-200a specifically. These observations provide further evidence for a critical tumor-suppressive role of the miR-200a in RCC in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 upregulation in RCC.

Establishment of patient-derived renal cell carcinoma (RCC) models based on orthotopic xenografts (PDX) and cancer stem cell (CSC) isolation to provide prognostic and predictive information.

e16055 Background: The introduction of VEGF/VEGFR- and mTOR, and immune checkpoint-targeted agents has radically changed metastatic RCC treatment; however, predictive biomarkers are still lacking and the potential for curative impact of such treatments in earlier disease stages is hotly debated. To identify potential diagnostic, prognostic, and predictive biomarkers, we developed patient-derived preclinical RCC models based on PDX obtained from freshly dissociated cancer tissues and combining isolation of cell populations endowed with self renewal and multi-lineage differentiation abilities (CSC). Methods: 182 surgical RCC specimens (clear cell, n = 110; papillary type I/II, n = 23, chromophobe/oncocytoma, n = 26, other, n = 23; stage I-III, n = 124, stage IV, n = 28, n/a n = 30) were collected; tumor spheroids were obtained and characterized in 57 cases, using a stem-cell isolating medium supplemented with EGF/b-FGF; 30 cancer samples were orthotopically injected in immunocompromised mice and were able to engraft in 67% of cases (G3-G4 cases more frequently than G1-G2 cases, p = 0.04). Results: PDX tumors recapitulated histological appearance, sarcomatoid features when they were present, and tumor heterogeneity of their human counterpart; interestingly, PDX engraftment was significantly correlated with shorter DFS in the corresponding patient population (p = 0.12). We conducted proteomic analysis by Reverse Phase Protein Array (RPPA) in 21 patient-derived CSC models in vitro: a panel of established (HIF, VEGFR, mTOR) and novel (EGFR(Y1148)), AKT, PI3K) oncogenic signals were deregulated in our isolated cell populations; molecular endpoints were correlated with grading and with angiogenesis and mTOR pathways (p < 0.05). Data analysis is ongoing to ascertain whether specific RPPA-signatures can potentially complement clinical prognostic information and suggest candidate biomarkers and potential targets for therapy. Conclusions: In the era of personalized therapy, the combined use of PDX and RPPA from tumor specimens may be very useful for drug testing and patient stratification in RCC.

MP52-03 CLASSIFICATION OF KIDNEY TUMORS WITH 1064 NM DISPERSIVE RAMAN SPECTROSCOPY

You have accessJournal of UrologySurgical Technology & Simulation: Instrumentation & Technology I1 Apr 2017MP52-03 CLASSIFICATION OF KIDNEY TUMORS WITH 1064 NM DISPERSIVE RAMAN SPECTROSCOPY Miki Haifler, Isaac Pence, Benjamin Ristau, Andres Correa, shreyas Joshi, Richard Greenberg, David Chen, Marc Smaldone, Alexander Kutikov, Rosalia Viterbo, Robert Uzzo, Amnon Zisman, Anita Mahadevan-Jansen, and Chetan Patil Miki HaiflerMiki Haifler More articles by this author , Isaac PenceIsaac Pence More articles by this author , Benjamin RistauBenjamin Ristau More articles by this author , Andres CorreaAndres Correa More articles by this author , shreyas Joshishreyas Joshi More articles by this author , Richard GreenbergRichard Greenberg More articles by this author , David ChenDavid Chen More articles by this author , Marc SmaldoneMarc Smaldone More articles by this author , Alexander KutikovAlexander Kutikov More articles by this author , Rosalia ViterboRosalia Viterbo More articles by this author , Robert UzzoRobert Uzzo More articles by this author , Amnon ZismanAmnon Zisman More articles by this author , Anita Mahadevan-JansenAnita Mahadevan-Jansen More articles by this author , and Chetan PatilChetan Patil More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1633AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The number of small, incidentally detected renal masses increase steadily. About 6,000 benign cases are misclassified radiographically as malignant and removed surgically yearly. Raman spectroscopy (RS) has been widely demonstrated for tissue characterization, however current implementations with either 785 or 830 nm near-infrared excitation have been ineffectual in tissues with intense auto-fluorescence such as the kidney. Recently, a RS system using 1064 nm light source was described which may have greater sensitivity for malignant and benign tissue discrimination due to decreased bulk tissue auto-fluorescence. Our aim was to evaluate the ability of 1064nm RS to distinguish normal and malignant renal tissue. METHODS Ex vivo specimens of Renal Cell Carcinoma and healthy human kidney were obtained from the Cooperative Human Tissue Network at Vanderbilt university. Measurements were made using of a benchtop dispersive 1064 nm Raman system. Multiple spectra were acquired from at least 5 physical locations across each specimen. A total of 93 measurements were used for the final analysis. The resulting spectra were put into a machine learning algorithm, sparse multinomial logistic regression (SMLR), to predict class membership of healthy and malignant tissues, and cross-validated using a leave-one-specimen out approach. Posterior probabilities of group classifications were extracted. Spectral bands that robustly differentiated between malignant and benign tissue were identified by the SMLR algorithm. A quantitative metric based on SMLR outputs called feature importance, defined as the product of the mean weight and frequency of usage of each feature, guided the association of spectral features with biological indicators of healthy and diseased Kidney tissue. RESULTS The SMLR algorithm identified 152 significant Raman spectral bands. most important features are depicted in figure 1. Correct classification by the SMLR algorithm was obtained in 93.33% of the trials with sensitivity, specificity, negative and positive predictive value of 93.2%, 88.6, 92.9% and 89.2% respectively. CONCLUSIONS RS can accurately differentiate normal and malignant renal tissue. This suggests implications for utilizing RS for optical biopsy and surgical guidance in nephron sparing surgery. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e704 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Miki Haifler More articles by this author Isaac Pence More articles by this author Benjamin Ristau More articles by this author Andres Correa More articles by this author shreyas Joshi More articles by this author Richard Greenberg More articles by this author David Chen More articles by this author Marc Smaldone More articles by this author Alexander Kutikov More articles by this author Rosalia Viterbo More articles by this author Robert Uzzo More articles by this author Amnon Zisman More articles by this author Anita Mahadevan-Jansen More articles by this author Chetan Patil More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Downregulation of microRNA-15a suppresses the proliferation and invasion of renal cell carcinoma via direct targeting of eIF4E.

The downregulation of microRNA-15a has been reported in several human tumors. However, its expression and functional importance in renal cell carcinoma (RCC) remain unknown. The aim of the present study was to investigate its expression, biological functions and underlying mechanisms in RCC tumorigenesis. The expression levels of miR-15a were examined by qRT-PCR in 40 RCC specimens and adjacent-paired normal tissues. Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and Transwell assays were used to explore the potential influence of miR-15a transfection on RCC cell proliferation, the cell cycle, cell apoptosis, and cell invasion. Luciferase reporter assays were performed to confirm the potential target of miR-15a, in combination with qRT-PCR, western blotting and immunohistochemical assays. We found that miR-15a was significantly downregulated in most RCC specimens compared with adjacent normal tissues (P<0.01). Overexpression of miR-15a inhibited cellular growth, suppressed invasion and arrested cells at the G1/G0 phase, and induced cell apoptosis in RCC cells. Luciferase assays revealed that miR-15a directly targeted the binding site of the 3′-untranslated region (3′-UTR) of eIF4E, and inhibited its expression at both mRNA and protein levels. eIF4E expression was negatively associated with miR-15a expression in RCC tissues. eIF4E overexpression treatment partially abrogated the inhibitory effect of miR-15a on cell proliferation and invasion, as well as inactivated P13K/AKT/mTOR signaling in RCC cells. In conclusion, the present study indicated that miR-15a downregulation was associated with cell proliferation and invasion by directly targeting eIF4E during RCC progression. Thus, it may serve as a potential tumor suppressor and therapeutic target for the treatment of RCC.

OSR1 is a novel epigenetic silenced tumor suppressor regulating invasion and proliferation in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most malignant tumors in human. Here, we found that odd-skipped related transcription factor 1 (OSR1) was downregulated in 769-P and 786-O cells due to promoter CpG methylation. OSR1 expression could be restored by pharmacological demethylation treatment in silenced cell lines. Knockdown of OSR1 in two normal expressed cell lines- A498 and ACHN promoted cell invasion and cellular proliferation. RNA-Sequencing analysis showed that expression profile of genes involved in multiple cancer-related pathways was changed when OSR1 was downregulated. By quantitative real-time PCR, we confirmed that depletion of OSR1 repressed the expression of several tumor suppresor genes involved in p53 pathway, such as p53, p21, p27, p57 and RB in A498 and ACHN. Moreover, knockdown of OSR1 suppressed the transcriptional activity of p53. Of note, OSR1 depletion also led to increased expression of a few oncogenic genes. We further evaluated the clinical significance of OSR1 in primary human RCC specimens by immunohistochemical staining and found that OSR1 expression was downregulated in primary RCC and negatively correlated with histological grade. Thus, our data indicate that OSR1 is a novel tumor suppressor gene in RCC. Downregulation of OSR1 might represent a potentially prognostic marker and therapeutic target for RCC.

Expression of Pax2 and E-cadherin in renal cell carcinoma: correlation with clinicopathologic parameters

Objective To investigate the expression of paired box 2 (Pax2) and E-cadherin in human renal cell carcinoma (RCC) and their correlation with clinicopathologic parameters. Methods The RCC tissue microarrays containing 85 renal cell carcinoma specimens and 35 normal kidney tissue specimens were used to detect Pax2 and E-cadherin expressions by immunohistochemistry. Results The positive expression rate of Pax2 was 77.6% (66/85) in RCC specimens, which was significantly higher than that in 35 normal kidney tissue specimens (P 0.05). The expression of E-cadherin in RCC was significantly related to the size of tumor, histological classification, pathological grade and clinical stage (P<0.05). The positive expression rate of E-cadherin in survival group was significantly higher than that in death group (P<0.05). Conclusions The abnormal expressions of Pax2 and E-cadherin may play a crucial stage in development of human RCC. Detection of the expressions of Pax2 and E-cadherin can be used in the evaluation of malignant degree and prognosis of RCC. Key words: PAX2 transcription factor/ME; Cadherins/ME; Kidney neoplasms/ME

Clinicopathological significance of ganglioside DSGb5 expression in renal cell carcinoma.

Disialosyl globopentaosylceramide (DSGb5) is a ganglioside originally isolated from tissue extracts of renal cell carcinoma (RCC) with metastasis. Previous in vitro experiments have suggested that DSGb5 promotes metastasis by enhancing the migration of RCC cells and downregulating NK cell cytotoxicity against RCC cells. In this study, we investigated the clinicopathological significance of DSGb5 expression in RCC and outcomes of RCC patients. A total of 156 RCC patients who underwent surgical treatments at our hospital from January 2007 through December 2012 were analyzed in this study. The expression of DSGb5 in RCC specimens was examined by immunohistochemical staining with monoclonal antibody 5F3. The immunostaining intensity of RCC tissues was assessed in comparison with that in benign renal tubules as an internal positive control. The relationship between DSGb5 expression and clinicopathological characteristics was investigated and recurrence free survival following surgery was evaluated. Microvascular invasion was observed in 68% (n=19/28) and in 45% (n=58/128) of the DSGb5 high expression group and low expression group, respectively (p=0.031). Of 156 patients with a median follow up of 51months, 18 patients (12%) developed metastasis following surgery. Patients in the DSGb5 high expression group showed significantly lower recurrence-free survival as compared with those in the DSGb5 low expression group (log-rank P=0.047). In the present study, DSGb5 expression was associated with microvascular invasion in RCC tissues, and patients with DSGb5 high expression showed significantly lower recurrence-free survival rates. These findings suggest that DSGb5 expressed in RCC is correlated with metastasis and is a potential predictor for identifying patients who experience metastasis after surgery.

The Piezo Actuator-Driven Pulsed Water Jet System for Minimizing Renal Damage after Off-Clamp Laparoscopic Partial Nephrectomy.

In the setting of partial nephrectomy (PN) for renal cell carcinoma, postoperative renal dysfunction might be caused by surgical procedure. The aim of this study was to clarify the technical safety and renal damage after off-clamp laparoscopic PN (LPN) with a piezo actuator-driven pulsed water jet (ADPJ) system. Eight swine underwent off-clamp LPN with this surgical device, while off-clamp open PN was also performed with radio knife or soft coagulation. The length of the removed kidney was 40 mm, and the renal parenchyma was dissected until the renal calyx became clearly visible. The degree of renal degeneration from the resection surface was compared by Hematoxylin-Eosin staining and immunostaining for 1-methyladenosine, a sensitive marker for the ischemic tissue damage. The mRNA levels of neutrophil gelatinase-associated lipocalin (Ngal), a biomarker for acute kidney injury, were measured by quantitative real-time PCR. Off-clamp LPN with ADPJ system was successfully performed while preserving fine blood vessels and the renal calix with little bleeding. In contrast to other devices, the resection surface obtained with the ADPJ system showed only marginal degree of ischemic changes. Indeed, the expression level of Ngal mRNA was lower in the resection surface obtained with the ADPJ system than that with soft coagulation (p = 0.02). Furthermore, using the excised specimens of renal cell carcinoma, we measured the breaking strength at each site of the human kidney, suggesting the applicability of this ADPJ to clinical trials. In conclusion, off-clamp LPN with the ADPJ system could be safely performed with attenuated renal damage.

The Correlation of Tissue-Based Biomarkers in Primary and Metastatic Renal Cell Carcinoma Lesions: A Tissue Microarray Study

Purpose The study was aimed to determine the correlations of tissue-based biomarker expressions between primary and metastatic specimens of renal cell carcinoma and with several well-known prognostic clinicopathological parameters. Materials and Methods The immunohistochemistry (IHC) was used to determine the expression levels of 9 tissue-based markers calculated in H-score expressed by percentage of expression multiplied by the intensity score (0, 1, 2, and 3 points). Using 17 patients' 38 specimens paired with primary renal lesion and its metastatic lesions collected between 2004 and 2015, Tissue microarray with IHC was performed with BAP1, PBRM1, pS6, PTEN, TGase2, PD-L1, CA9, PSMA, and Ki-67 on formalin-fixed paraffin-embedded sections. Pearson correlation and accuracy test were performed to analyze the correlation between primary and metastatic tissues. Results The 17 patients' mean age was 56.9 years old, mean tumor size was 7.9 cm, and the male to female ratio was 13:4 (76.5%:23.5%), respectively. Three patients had 2, 3, and 3 metastatic tissues, and the rest of 14 patients had only one metastatic tissue. The H-score (PSMA and Ki67) and intensity score (pS6 and PSMA) showed that some differential significant markers were identified which had statistical correlations of expression levels between primary and metastatic lesions among 9 markers. However, no real correlation of PSMA, Ki67, and pS6 markers were found their expressions of between primary and metastatic tissues because of their skewed expressions. Conclusions Tissue markers failed to correlate their expression levels in primary lesions with those of metastatic lesions. Key Words: Renal cell carcinoma; Metastasis; Tissue microarray; Biomarker; Immunohistochemistry.

MiR-144-3p serves as a tumor suppressor for renal cell carcinoma and inhibits its invasion and metastasis by targeting MAP3K8

MicroRNAs (miRNAs) are important regulators involved in various cancers, including renal cell carcinoma (RCC). The role of the miRNAs involved in RCC progress and metastasis is largely unknown. Here, miRNA microarray analysis was performed to screen the significant miRNAs involved in RCC progress, and miR-144-3p was chosen for further study. We found that the expression of miR-144-3p was significantly lower in RCC specimens and cell lines. In addition, low expression level of miR-144-3p is correlated with tumor progression and poor survival in RCC patients. Based on in vitro assays, we found that miR-144-3p significantly inhibit cancer cell proliferation and progression. Furthermore, function studies revealed that miR-144-3p was significantly correlated with the metastasis potential by affecting the epithelial-mesenchymal transition (EMT). Moreover, Mitogen-activated protein kinase 8 (MAP3K8) is direct target of miR-144-3p, while the expression levels of MAP3K8 were inversely correlated with the expression levels of miR-144-3p in RCC tissues. Overall, our findings demonstrate that miR-144-3p targeted the MAP3K8 pathway to reduce tumor cells proliferation and metastasis in RCC, suggesting that this axis may provide a novel therapeutic target for RCC therapy.

FK506 Binding Protein 10 Is Overexpressed and Promotes Renal Cell Carcinoma

Introduction: FK506 binding proteins (FKBPs) function as oncogenes or tumor suppressors by interacting with steroid hormone receptors, kinases, or other cellular factors in addition to intracellular ligands FK506 and rapamycin. In this study, we aimed at evaluating the expression and function of FKBPs in renal cell carcinoma (RCC). Materials and Methods: Thirty-four RCC specimens were analyzed by whole transcriptome sequencing. Small interfere RNA was employed to knockdown FKBP10 in 786-O and A-704 cells, and cell proliferation, cell cycle progression, invasion and migration were evaluated. Results: FKBP10 was different from other members of FKBPs with most significant upregulation in almost all RCC specimens, compared to normal mucosa. FKBP10 expression was high in additional 38 RCC specimens and RCC cell lines compared to paired non-tumor tissues and normal renal tubule cells. FKBP10 knockdown led to cell cycle arrest at G0/G1 phase, and reduced cell proliferation, invasion, and migration in 786-O and A-704 cells. In addition, heat shock protein 90 was significantly downregulated after FKBP10 knockdown. Conclusions: FKBP10 is overexpressed and promotes RCC and is a new promising biomarker and therapy target for RCC.

Poor Prognosis Associated with High Levels of Thymidine Phosphorylase and Thrombocytosis in Patients with Renal Cell Carcinoma

Purpose: The study aimed to investigate the expression of thymidine phosphorylase (TP) in renal cell carcinoma (RCC) and its correlation with clinicopathological features and thrombocytosis, and also to determine their prognostic significance. Patients and Methods: TP expression in 127 RCC specimens was evaluated with immunohistochemistry assays. Platelet (PLT) counts of patients before surgery were recorded. Correlations among TP expression, PLT and the clinicopathological features of the patients and their prognostic values were studied statistically. Results: Sixty-eight patients of high TP expression (54%) and 59 of low TP expression (46%) were detected. There were 25 patients with thrombocytosis (20%). High TP expression was significantly associated with tumor stage (p = 0.004), histological grade (p = 0.001) and thrombocytosis (p = 0.012). The 5-year overall survival rate was 88.1% in patients with low TP expression, whereas it was 61.8% in patients with high TP expression (p < 0.001). The 5-year survival rates for patients with and without thrombocytosis were 16.0% and 88.2%, respectively (p < 0.001). The multivariate analysis showed that high TP expression and thrombocytosis would play a role as independent prognostic factors in RCC patients. Conclusion: High TP expression and thrombocytosis can be regarded as independent prognostic factors of poor survival in patients with RCC.

MiR-144 inhibits cell proliferation of renal cell carcinoma by targeting MTOR.

MicroRNAs (miRNAs) modulate the expression of tumorigenesis-related genes and play important roles in the development of various types of cancers. It has been reported that miR-144 is dysregulated and involved in multiple malignant tumors, but its role in renal cell carcinoma (RCC) remains elusive. In this study, we demonstrated miR-144 was significantly downregulated in human RCC. The decreased miR-144 correlated with tumor size and TNM stage. Moreover, overexpression of miR-144 in vitro suppressed RCC cell proliferation and G2 transition, which were reversed by inhibition of miR-144. Bioinformatic analysis predicted that mTOR was a potential target of miR-144, which was further confirmed by dual luciferase reporter assay. Additionally, the examination of clinical RCC specimens revealed that miR-144 was inversely related to mTOR. Furthermore, knocking down mTOR with siRNA had the same biological effects as those of miR-144 overexpression in RCC cells, including cell proliferation inhibition and S/G2 cell cycle arrest. In conclusion, our results indicate that miR-144 affects RCC progression by inhibiting mTOR expression, and targeting miR-144 may act as a novel strategy for RCC treatment.

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.