Clear Cell Renal Cell Carcinoma Cell Lines Research Articles

Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics.

Metabolic characteristics of kidney cancers have mainly been obtained from the most frequent clear cell renal cell carcinoma (CCRCC) studies. Moreover, the bioenergetic perturbances that affect metabolic adaptation possibilities of papillary renal cell carcinoma (PRCC) have not yet been detailed. Therefore, our study aimed to analyze the in situ metabolic features of PRCC vs. CCRCC tissues and compared the metabolic characteristics of PRCC, CCRCC, and normal tubular epithelial cell lines. The protein and mRNA expressions of the molecular elements in mammalian target of rapamycin (mTOR) and additional metabolic pathways were analyzed in human PRCC cases compared to CCRCC. The metabolic protein expression pattern, metabolite content, mTOR, and metabolic inhibitor sensitivity of renal carcinoma cell lines were also studied and compared with tubular epithelial cells, as “normal” control. We observed higher protein expressions of the “alternative bioenergetic pathway” elements, in correlation with the possible higher glutamine and acetate consumption in PRCC cells instead of higher glycolytic and mTOR activity in CCRCCs. Increased expression of certain metabolic pathway markers correlates with the detected differences in metabolite ratios, as well. The lower lactate/pyruvate, lactate/malate, and higher pyruvate/citrate intracellular metabolite ratios in PRCC compared to CCRCC cell lines suggest that ACHN (PRCC) have lower Warburg glycolytic capacity, less pronounced pyruvate to lactate producing activity and shifted OXPHOS phenotype. However, both studied renal carcinoma cell lines showed higher mTOR activity than tubular epithelial cells cultured in vitro, the metabolite ratio, the enzyme expression profiles, and the higher mitochondrial content also suggest increased importance of mitochondrial functions, including mitochondrial OXPHOS in PRCCs. Additionally, PRCC cells showed significant mTOR inhibitor sensitivity and the used metabolic inhibitors increased the effect of rapamycin in combined treatments. Our study revealed in situ metabolic differences in mTOR and metabolic protein expression patterns of human PRCC and CCRCC tissues as well as in cell lines. These underline the importance in the development of specific new treatment strategies, new mTOR inhibitors, and other anti-metabolic drug combinations in PRCC therapy.

A Novel Biomarker, FKBP10, for Poor Prognosis Prediction in Patients with Clear Cell Renal Cell Carcinoma.

Objective To screen genes associated with poor prognosis of clear cell renal cell carcinoma (CcRCC) from the public databases HPA (Human Protein Atlas), UALCAN, and GEPIA (Gene Expression Profiling Interactive Analysis) and to investigate the expression of FKBP10 in CcRCC and the effect on prognosis of the patients and the biological behavior of CcRCC cells. Methods The tumor tissues and adjacent noncancerous tissues of 42 patients with CcRCC diagnosed and treated in our hospital were collected, and the general information of the patients was recorded. FKBP10 expression in the tissues was determined by qRT-PCR and western blot, and its relationship with general information and prognosis of patients was analyzed. Knockdown or overexpression experiments were carried out with the human proximal tubule epithelial cell line HK-2 and CcRCC cell lines Caki-1, 786-O, ACHN, and A498 to verify the relationship between FKBP10 expression and cell proliferation and adhesion ability using MTT assay and fibronectin adhesion assay, respectively. Western blot was utilized to examine the protein expression level of c-Myc, cyclin D1, and Bcl-2 in the cells. Results FKBP10 was highly expressed in CcRCC tissues and cells and was correlated with poor prognosis. In addition, FKBP10 expression was positively correlated with CcRCC tumor size and staging and negatively correlated with tumor differentiation. Moreover, knockdown of FKBP10 significantly inhibited the proliferation of CcRCC cells, notably declined the protein expression of c-Myc, cyclin D1, and Bcl-2, and promoted cell adhesion. Conclusion FKBP10 is highly expressed in CcRCC tissues and cells and is associated with poor prognosis in patients. FKBP10 participated in the occurrence and development of CcRCC by promoting cell proliferation and inhibiting apoptosis and adhesion.

Synthetic lethality of cyclin-dependent kinase inhibitor Dinaciclib with VHL-deficiency allows for selective targeting of clear cell renal cell carcinoma

ABSTRACT Clear cell renal cell carcinoma (CC-RCC) remains one of the most deadly forms of kidney cancer despite recent advancements in targeted therapeutics, including tyrosine kinase and immune checkpoint inhibitors. Unfortunately, these therapies have not been able to show better than a 16% complete response rate. In this study we evaluated a cyclin-dependent kinase inhibitor, Dinaciclib, as a potential new targeted therapeutic for CC-RCC. In vitro, Dinaciclib showed anti-proliferative and pro-apoptotic effects on CC-RCC cell lines in Cell Titer Glo, Crystal Violet, FACS-based cell cycle analysis, and TUNEL assays. Additionally, these responses were accompanied by a reduction in phospho-Rb and pro-survival MCL-1 cell signaling responses, as well as the induction of caspase 3 and PARP cleavage. In vivo, Dinaciclib efficiently inhibited primary tumor growth in an orthotopic, patient-derived xenograft-based CC-RCC mouse model. Importantly, Dinaciclib targeted both CD105+ cancer stem cells (CSCs) and CD105− non-CSCs in vivo. Moreover, normal cell lines, as well as a CC-RCC cell line with re-expressed von-Hippel Lindau (VHL) tumor suppressor gene, were protected from Dinaciclib-induced cytotoxicity when not actively dividing, indicating an effective therapeutic window due to synthetic lethality of Dinaciclib treatment with VHL loss. Thus, Dinaciclib represents a novel potential therapeutic for CC-RCC.

DUSP4 promotes the carcinogenesis of CCRCC via negative regulation of autophagic death.

DUSP4 is considered as an oncogenic gene. However, the effect of DUSP4 on the carcinogenesis of clear cell Renal cell carcinoma (CCRCC) is still unclear. In this study, DUSP4 mRNA levels were significantly increased in CCRCC tissues and cell lines. Furthermore, DUSP4 overexpression promotes the proliferation, migration, and tumorigenicity of CCRCC cells while DUSP4 silencing showed the opposite effects. Importantly, both autophagic activity (LC3 conversion rate and LC3 puncta formation) and total death level promoted by DUSP4 silencing were reversed by treatment with 3-MA in CCRCC cells. Moreover, the proliferation and migration of CCRCC cells inhibited by DUSP4 silencing were also recovered by suppression of autophagy with 3-MA. In conclusion, DUSP4 serves as an oncogenic gene in CCRCC carcinogenesis due to its inhibitory effect on autophagic death, indicating the potential value of DUSP4 in the diagnosis and treatment of CCRCC.

Myoferlin silencing inhibits VEGFR2-mediated proliferation of metastatic clear cell renal cell carcinoma

Recently, ramucirumab, a drug that targets vascular endothelial growth factor receptor (VEGFR), was clinically approved; therefore, we evaluated VEGFR2 expression and its predictive roles in tumor progression in clear cell renal cell carcinoma (CCRCC). Since we do not have many options for treating aggressive renal cell carcinoma patients, the application of anti-VEGFR2 therapy might be useful. Myoferlin (MYOF) is a 230 kDa transmembrane multi-C2-domain protein that contributes to plasma membrane repair, fusion, and endocytosis and is overexpressed in several invasive cancer cell lines, including breast, pancreas, and malignant melanoma. It forms a complex with VEGFR2 to inhibit VEGFR2 degradation. In this study, a total of 152 patients who had undergone nephrectomy for CCRCC were enrolled. Based on tissue microarray (TMA) blocks, the positive intensity and high proportion of MYOF showed a statistically significant correlation with the negative intensity (p < 0.001) and low proportion (p < 0.001) of VEGFR2, respectively. In addition, Fuhrman’s nuclear grade ≥3 showed a significant correlation with VEGFR2 expression. In multivariate analysis, CCRCC patients with positive MYOF and negative VEGFR2 expression demonstrated poor clinical outcomes. We confirmed that positive MYOF expression and negative VEGFR2 expression were positively correlated in this CCRCC population. Knocking down MYOF in Caki-1 cells resulted in the downregulation of VEGFR2 at both mRNA and protein levels. Wound healing assays revealed that the loss of MYOF in Caki-1 cells decreased cell confluence compared to that in control cells. We demonstrated that MYOF influences cellular proliferation of the metastatic CCRCC cell line by regulating VEGFR2 degradation. Combined therapies targeting the MYOF and VEGFR2 pathways might be effective against metastatic CCRCC to increase patient survival.

Targeting the Mevalonate Pathway Suppresses VHL-Deficient CC-RCC through an HIF-Dependent Mechanism.

Clear cell renal cell carcinoma (CC-RCC) is a devastating disease with limited therapeutic options available for advanced stages. The objective of this study was to investigate HMG-CoA reductase inhibitors, also known as statins, as potential therapeutics for CC-RCC. Importantly, treatment with statins was found to be synthetically lethal with the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in 90% of CC-RCC driving the disease. This effect has been confirmed in three different CC-RCC cell lines with three different lipophilic statins. Inhibition of mevalonate synthesis by statins causes a profound cytostatic effect at nanomolar concentrations and becomes cytotoxic at low micromolar concentrations in VHL-deficient CC-RCC. The synthetic lethal effect can be fully rescued by both mevalonate and geranylgeranylpyrophosphate, but not by squalene, indicating that the effect is due to disruption of small GTPase isoprenylation and not the inhibition of cholesterol synthesis. Inhibition of Rho and Rho kinase (ROCK) signaling contributes to the synthetic lethality effect, and overactivation of hypoxia-inducible factor signaling resulting from VHL loss is required. Finally, statin treatment is able to inhibit both tumor initiation and progression of subcutaneous 786-OT1-based CC-RCC tumors in mice. Thus, statins represent potential therapeutics for the treatment of VHL-deficient CC-RCC. Mol Cancer Ther; 17(8); 1781-92. ©2018 AACR.

SMAD proteins directly suppress PAX2 transcription downstream of transforming growth factor-beta 1 (TGF-β1) signalling in renal cell carcinoma.

Canonical TGF-β1 signalling promotes tumor progression by facilitating invasion and metastasis, whereby release of TGF-β1, by (for example) infiltrating immune cells, induces epithelial to mesenchymal transition (EMT). PAX2, a member of the Paired box family of transcriptional regulators, is normally expressed during embryonic development, including in the kidney, where it promotes mesenchymal to epithelial transition (MET). PAX2 expression is silenced in many normal adult tissues. However, in contrast, PAX2 is expressed in several cancer types, including kidney, prostate, breast, and ovarian cancer. While multiple studies have implicated TGF-β superfamily members in modulating expression of Pax genes during embryonic development, few have investigated direct regulation of Pax gene expression by TGF-β1. Here we have investigated direct regulation of PAX2 expression by TGF-β1 in clear cell renal cell carcinoma (CC-RCC) cell lines. Treatment of PAX2-expressing 786-O and A498 CC-RCC cell lines with TGF-β1 resulted in inhibition of endogenous PAX2 mRNA and protein expression, as well as expression from transiently transfected PAX2 promoter constructs; this inhibition was abolished in the presence of expression of the inhibitory SMAD, SMAD7. Using ChIP-PCR we showed TGF-β1 treatment induced SMAD3 protein phosphorylation in 786-O cells, and direct SMAD3 binding to the human PAX2 promoter, which was inhibited by SMAD7 over-expression. Overall, these data suggest that canonical TGF-β signalling suppresses PAX2 transcription in CC-RCC cells due to the direct binding of SMAD proteins to the PAX2 promoter. These studies improve our understanding of tumor progression and epithelial to mesenchyme transition (EMT) in CC-RCC and in other PAX2-expressing cancer types.

MiR-199a-3p acts as a tumor suppressor in clear cell renal cell carcinoma

ObjectivesTo explore the biological function and mechanism of miR-199a-3p in clear cell renal cell carcinoma (CCRCC). MethodsWe investigated the expression of miR-199a-3p in CCRCC through quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Over expression of miR-199a-3p was performed in CCRCC cell lines, and cell growth curve, colony formation capacity, cell invasion, wound healing and cell apoptosis assay were used for investigating the roles of miR-199a-3p in CCRCC. ResultsThe expression of miR-199a-3p in CCRCC tissues was significantly lower than that in para-carcinoma tissues. Functional assay showed that over expression of miR-199a-3p influenced cell growth, colony formation, cell invasion, cell migration and cell apoptosis in CCRCC cell lines. ConclusionsOur work suggested that miR-199a-3p was related to cell growth, colony formation, cell invasion, cell migration and cell apoptosis, which might act as a tumor suppressor in CCRCC.

The role of CRP and ATG9B expression in clear cell renal cell carcinoma.

The purpose of the study is to investigate the correlation between the expression of C-reactive protein (CRP) and autophagy-related 9B (ATG9B) and pathological features of clear cell renal cell carcinoma (CCRCC) patients. We also intended to explore the effects of manipulated expression of CRP and ATG9B on the apoptosis and cell cycle progression of CCRCC cell line. ATG9B expression in CCRCC tissues and adjacent renal tissues was analyzed by immunohistochemistry (IHC). Gene expression was determined at transcription and translational levels using real-time quantitative PCR (RT-qPCR) and Western blot. The association between CRP/ATG9B expression and clinical-pathological parameters including age, gender, pathological grades, TNM stage and distant metastasis of the patients was assessed by correlation analysis. siRNA and overexpression plasmids construction were used to manipulate the expression of CRP in human CCRCC cell line 786-O. Cell apoptosis and cell cycle progression were determined using flow cytometry (FCM) and Hoechst 33258 staining. CRP expression correlates with ATG9B expression. The expression of CRP and ATG9B are significantly correlated with TNM staging, distant metastasis, and survival time of CCRCC patients. A high-level of CRP indicates a poor overall survival (OS). In addition, CRP expression influences cell cycle and apoptosis of CCRCC cells. The study reveals that CRP might be a CCRCC development promoter. In addition, there is a close relationship between CRP and ATG9B in CCRCC carcinogenesis.

Elevated expression of KIF18A enhances cell proliferation and predicts poor survival in human clear cell renal carcinoma

The function of kinesin family member 18A (KIF18A) in human renal cell carcinoma (RCC) is unclear. The purpose of the current study was to determine the expression and prognostic significance of KIF18A in RCC. Specimens from 273 RCC patients undergoing nephrectomies were studied. Expression of KIF18A mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR) or quantitative PCR, and the expression of KIF18A protein was examined by immunohistochemistry and western blotting. The expression of KIF18A in clear-cell RCC cell lines was decreased using small interfering RNA targeting KIF18A, and increased by transfection with KIF18A cDNA. The proliferative ability of RCC cells in vitro and in vivo was detected by WST-1 assay and an animal xenograft model with BALB/c nude mice, respectively. The association between KIF18A expression and overall survival was calculated using Kaplan-Meier analysis. The results showed that KIF18A expression was significantly increased in RCC tissues compared with normal kidney tissues. The level of KIF18A expression was significantly associated with tumor stage, histological grade, metastasis and tumor size. Moreover, KIF18A increased the proliferation of RCC cells in vitro and in vivo. KIF18A expression was upregulated in RCC and enhanced the proliferation of RCC cells. Therefore, it appears that KIF18A plays a key role in the carcinogenesis and progression of RCC, and is a novel candidate prognostic marker for RCC patients. Furthermore, silencing KIF18A expression may serve as a new therapeutic strategy against RCC.

Low expression of phosphatase and tensin homolog in clear‑cell renal cell carcinoma contributes to chemoresistance through activating the Akt/HDM2 signaling pathway.

Clear-cell renal cell carcinoma (CCRCC) is the most frequent primary malignancy in the adult kidney. Most patients with advanced CCRCC have poor prognosis as CCRCC remains resistant to chemotherapy. The present study explored the possible mechanism underlying CCRCC resistance to chemotherapy and found that loss of PTEN in CCRCC may be involved. Knockdown of PTEN in the CCRCC cell line ACHN blocked etoposide-induced apoptosis and etoposide-impaired cell proliferation was also inhibited. It has been demonstrated that most chemotherapy drugs exert their anti-cancer effects via p53-mediated apoptosis, and in accordance, with this, the present study showed that treatment with etoposide significantly increased p53 levels. Silencing of PTEN in ACHN inhibited the Akt/HDM2 signaling cascade and depressed p53 expression, and the interaction between HDM2 and p53 was also enhanced. This was further verified in CCRCC tissue specimens from patients The results of the present study suggested that loss of PTEN, which deactivated Akt/HDM2 signaling followed by degradation of p53, may contribute to the development of etoposide resistance in CCRCC.

Engrailed-2 is down-regulated but also ectopically expressed in clear cell renal cell carcinoma

Engrailed-2 (EN2), a member of the homeobox family of genes, encodes a homeodomain-containing transcription factor that is thought to be a potential oncogene in a number of cancers. Because the role of EN2 in clear cell renal cell carcinoma (CCRCC) has not been determined, we investigated its expression in CCRCC tissues and cell lines. Using immunohistochemical (IHC) staining, we found that EN2 protein was expressed in normal renal cells and tubules, but was frequently down-regulated in tissues from patients with CCRCC and in CCRCC cell lines. In addition, we found that EN2, which functions in the nucleus, was completely localized to the cytoplasm of CCRCC cells as detected by IHC and immunofluorescence staining. Furthermore, expression of EN2 protein was negatively correlated with increasing histological grade of CCRCC tumors (P = 0.003). The exact role of EN2 expression in renal carcinoma carcinogenesis requires further investigation.

Expression of Opa interacting protein 5 (OIP5) is associated with tumor stage and prognosis of clear cell renal cell carcinoma

Opa interacting protein 5 (OIP5), overexpressed in some types of human cancers, has been reported to be associated with the carcinogenesis of human cancer. However, the biological function and clinical significance of OIP5 in human Clear Cell Renal Cell Carcinoma (CCRCC) remains unknown. In the present study, we found the expression of OIP5 was markedly upregulated in surgical CCRCC specimens and CCRCC cell lines. Immunohistochemical analysis revealed that paraffin-embedded archival CCRCC specimens exhibited higher levels of OIP5 expression than normal renal tissues. Further statistical analysis suggested the upregulation of OIP5 was positively correlated with the Fuhrman grade (P=0.02), T classification (P=0.015), N classification (P=0.018) and clinical stage (P=0.035). Also, patients with high OIP5 expression dramatically exhibited shorter survival time (P=0.001). In addition, the OIP5 expression was an independent prognostic marker of overall survival of CCRCC patients in a multivariate analysis (P=0.008). Experimentally, we demonstrated that silencing OIP5 in CCRCC cell lines by specific siRNA clearly inhibited cell growth. In conclusion, our findings suggested that OIP5 could be a valuable marker of CCRCC progression and prognosis, and a promising therapeutic target for CCRCC.

Expression and role of vascular endothelial growth inhibitor in sporadic clear cell renal cell carcinoma

To evaluate the expression of vascular endothelial growth inhibitor (VEGI) in sporadic clear cell renal cell carcinoma (CCRCC) and explore its relationships between VEGI expression, pathologic grade and tumor staging. Western blot and immunohistochemical staining were used to detect the expression of VEGI in CCRCC cell line (786-O cells), CCRCC and paired normal kidney tissues. A total of 50 CCRCC cases were recruited. There were 37 males and 13 females with an average age of 53 ± 12 years. The tumor sizes were < 7 cm (n = 33) and ≥ 7 cm (n = 17). Their pathologic grades were G1 (n = 14), G2 (n = 22) and G3 (n = 14) and pathologic stages pT1 (n = 32), 10 pT2 (n = 10) and pT3 (n = 8). VEGI protein was predominantly located in cytoplasm. Compared with normal kidney tissues(mean optic density (MOD) of VEGI staining: 0.40 ± 0.16), it was lower in CCRCC tissues (MOD: 0.11 ± 0.06, P < 0.01). In addition, the positive rate of VEGI expression, the expression intensity and the MOD of VEGI protein were negatively correlated with the pathologic grade of CCRCC (r = -0.640, P < 0.01; r = -0.831, P < 0.01; r = -0.781, P < 0.01 respectively). The MOD of VEGI expression in ≥ 7 cm tumors (MOD, 0.08 ± 0.04) was significantly lower than that in < 7 cm tumors (MOD: 0.12 ± 0.06, P < 0.05). However, there was no correlations between the VEGI protein level and age, gender and pathologic stage of patients (P > 0.05). VEGI protein is predominantly located in cytoplasm. Compared with CCRCC tissues, VEGI protein level is higher in normal ones. In consideration of negative correlations between VEGI expression, pathologic grade and tumor size, it is implied that VEGI may play a negative regulatory role in the occurrence and development of CCRCC.

Kaposi's sarcoma‐derived cell line SLK is not of endothelial origin, but is a contaminant from a known renal carcinoma cell line

Kaposi's sarcoma (KS) is an endothelial cell-derived tumor. Investigations of the molecular mechanisms of KS pathogenesis and the identification of drugs for treatment of KS depend critically on valid cell-culture models. Two major immortalized cell lines are available for KS research. Recently, the KS cell line KS Y-1 has been shown to be cross-contaminated with the T24 urinary bladder cancer cell line (ATCC HTB-4). Here, we show by short tandem repeat profiling that the second KS cell line, SLK, is indistinguishable from the clear-cell renal-cell carcinoma cell line Caki-1. Immunocytochemical detection of cytokeratin expression confirmed the epithelial-cell origin of SLK cells. Our findings indicate that SLK cells are not of endothelial origin and should not be used in future studies as a model for KS-derived endothelial tumor cells. We suggest that in the future, more attention needs to be paid to the authenticity of cells in lines derived from human tissues.

Notch1 activation promotes renal cell carcinoma growth via PI3K/Akt signaling

Both the Notch1 and PI3K/Akt pathways are aberrantly activated in clear cell renal cell carcinoma (CCRCC) and involved in the tumorigenesis. The aim of this study was to test our hypothesis that elevated Notch1 signaling activity exerts its growth-promoting effects via the PI3K/Akt pathway in CCRCC. To investigate the relationship between the two pathways, we enhanced and suppressed the Notch1 activity respectively in a CCRCC cell line through diverse means, and then evaluated ensuing phosphorylated Akt (pAkt) levels. To further study their collaboration in promoting tumor growth, cell proliferation assay, colony formation assay and cell cycle analysis were conducted under several different conditions. Immunostaining of the tissue microarrays was used to determine whether the phenomena we observed also existed in vivo. The results showed that Notch1 signaling was activated in CCRCC tissue samples and cell lines. Notch1 activation increased CCRCC cell proliferation, enhanced anchorage-independent growth, and accelerated G1/S cell cycle progression. Such effects of the Notch1 signaling were, at least in part, mediated by the PI3K/Akt pathway. Correlations between Notch1, pAkt and Ki-67 protein levels in tissue microarrays reinforced our in vitro findings. Taken together, the current study established a functional link between the Notch1 and PI3K/Akt pathways in CCRCC.

Combination of Temsirolimus and tyrosine kinase inhibitors in renal carcinoma and endothelial cell lines

Multitargeted tyrosine kinase inhibitors (TKIs) (such as Sunitinib and Sorafenib) and mTOR inhibitors (such as Temsirolimus) are effective in treating metastatic clear-cell renal cell carcinoma (CCRCC), by acting on different pathways in both tumour and endothelial cells. A study of their combined effect could be of major interest. We studied endothelial and CCRCC cell lines treated with Sunitinib, Sorafenib, Temsirolimus and 2 drug combinations: Sunitinib-Temsirolimus and Sorafenib-Temsirolimus. We studied inhibition of proliferation with an MTT assay under normoxia and hypoxia, VEGF expression by quantitative RT-PCR and ELISA, and angiogenesis with a Matrigel assay. TKIs and Temsirolimus inhibited proliferation of endothelial and tumour cell lines and inhibited angiogenesis. Anti-proliferative effects were more significant on cell lines with VHL gene inactivation and under hypoxic conditions. VEGF expression was induced by TKIs, but inhibited by Temsirolimus. The Sunitinib/Temsirolimus combination had synergistic or additive effects on the proliferation of tumour and endothelial cell lines. The Sorafenib-Temsirolimus combination had additive effects on the proliferation of most tumour cell lines, but not endothelial cell lines. Both combinations had additive effects on the inhibition of angiogenesis. In our model, Sunitinib, Sorafenib and Temsirolimus had anti-tumour and anti-angiogenic effects. The combinations of Sunitinib or Sorafenib with Temsirolimus had additive or synergistic effects on the inhibition of tumour and endothelial cell proliferation, and on the inhibition of angiogenesis. This work could lead to new trials with lower-dose combinations to prevent side effects and enhance efficacy.

Cyclooxygenase-2 Expression and Its Prognostic Significance in Clear Cell Renal Cell Carcinoma

BackgroundThe prognostic value of cyclooxygenase-2 (COX-2) in human renal cell carcinoma (RCC) remains unclear. The purposes of this study are to elucidate the clinical significance of COX-2 in clear cell RCC (CCRCC) and to assess the treatment effect of COX-2 inhibition on CCRCC cell lines.MethodsUsing tumor samples obtained from 137 patients who had undergone nephrectomy at Seoul National University Hospital, we evaluated COX-2 expression on immunohistochemistry. Moreover, we performed the cell proliferation assay using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and cell invasion assay. Thus, we evaluated the effect of meloxicam, an inhibitor of COX-2, in two human CCRCC cell lines.ResultsCancer-specific survival (p=0.038) and progression-free survival (p=0.031) were shorter in the COX-2 high expression group. A multivariate logistic regression model showed that COX-2 expression was an independent risk factor for pTNM stage and Fuhrman nuclear grade. The MTT assay revealed that COX-2 inhibition led to the suppression of the proliferation of CCRCC cell lines. Moreover, it also reduced their invasion capacity.ConclusionsThis study postulates that COX-2 is a poor prognostic indicator in human CCRCC, suggesting that COX-2 inhibition can be a potential therapy in CCRCC.

NOTCH1 functions as an oncogene by regulating the PTEN/PI3K/AKT pathway in clear cell renal cell carcinoma

ObjectivesAlthough NOTCH1 plays a wide-ranging role in controlling cell fate, differentiation, and development, its pathologic roles in clear cell renal cell carcinoma (CCRCC) are still unclear. In the present study, the expression pattern of NOTCH1 was examined in CCRCC tissues, and the interaction of NOTCH1 with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT pathway was investigated in vitro. Materials and methodsThirty-six paired CCRCC and adjacent non-neoplastic renal samples were analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The alteration of NOTCH1, hairy and enhancer of split 1 (HES1), PTEN, AKT (phosphorylated at Ser473) in CCRCC cell line (786-O), and the human normal kidney tubule epithelial cell line (HKC) were analyzed by Western blotting and qRT-PCR, before and after transfection with siRNA against NOTCH1 or the plasmid containing the ORF clone of NOTCH1. The effects of NOTCH1 signaling pathway on cells proliferation, apoptosis, invasion, and migration were detected by MTS assay, flow cytometry analyses, and transwell chamber assay, respectively. ResultsThe NOTCH1 expression levels were significantly increased in CCRCC tissues compared with the adjacent non-neoplastic renal samples, while it had no significant association with the pathologic parameters. NOTCH1 signaling cascade was constitutively active in human CCRCC cell lines. Blocking NOTCH1 signaling resulted in the attenuation of proliferation, invasion, and migration, as well as PTEN up-regulation with decreased AKT phosphorylation. NOTCH1 overexpression had an opposite effect to NOTCH1 knockdown. ConclusionsOur findings indicated that NOTCH1 receptor expression was up-regulated in CCRCC, and that NOTCH1 could regulate PTEN expression and the activity of the PI3K/AKT pathway via HES1 in 786-O and HKC cell lines. These might provide a basis for the designing new therapeutic strategies for CCRCC.

Abstract 3439: Sarcomatoid renal cell carcinoma as a biological example of epithelial to mesenchymal transition (EMT)

Abstract About 8% of clear cell renal cell carcinoma (CCRCC) cases contain areas of a radically different morphology, demonstrating spindle shaped cells and a histological resemblance to sarcomas. These biphasic neoplasms are called sarcomatoid CCRCC (sCCRCC). Patients diagnosed with sCCRCC face a considerably worse prognosis due to an increased propensity for metastasis. Using a set of biphasic CCRCC cases, we showed that sCCRCC has a higher proliferative index and more prononced vimentin expression as compared to the CCRCC counterpart. We selected a CCRCC/sCCRCC case demonstrating overgrowth as well as metastatic spread for extended immunohistochemical characterization. Using mouse xenografts we evaluated a $$$$CCRCC cell-line and put forward the hypothesis that some CCRCC cell-lines may be derived from the sarcomatoid component rather than from the clear cell part, with obvious implications for the validity of in vitro models of CCRCC. We confirm that sCCRCC is derived from the clear cell component and show that sCCRCC morphologically and immunohistochemically may represent a completed epithelial mesenchymal transition (EMT). Furthermore we suggest that TGF-β1 may be the driving force behind sarcomatoid transdifferentiation i.e. EMT in CCRCC. 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 3439. doi:10.1158/1538-7445.AM2011-3439