The expression of cholinesterases in human renal tumours varies according to their histological types

The change in the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in neoplastic colon and lung prompted us to study the possible effect of cancer on the expression of cholinesterases (ChEs) in kidney. Samples of papillary renal cell carcinoma (pRCC), conventional RCC (cRCC), chromophobe RCC (chRCC) and renal oncocytoma (RON), beside adjacent non-cancerous tissues, were analyzed. In pRCC both AChE and BuChE activities were statistically increased; in cRCC and chRCC only AChE activity increased and in RON neither AChE nor BuChE activities were affected. Abundant amphiphilic AChE dimers (G(2)(A)) and fewer monomers (G(1)(A)) were identified in healthy kidney as well as in all tumour classes. Incubation with PIPLC revealed glycosylphosphatidylinositol in AChE forms. BuChE is distributed between principal G(4)(H), fewer G(1)(H), and much fewer G(4)(A) and G(1)(A) species. RT-PCR showed similar amounts of AChE-H, AChE-T and BuChE mRNAs in healthy kidney. Their levels increased in pRCC but not in the other tumour types. The data support the idea that, as in lung tumours, in renal carcinomas expression of ChE mRNAs, biosynthesis of molecular components and level of enzyme activity change according to the specific kind of cell from which tumours arise.     

Heterogeneity of Hippo signalling activity in different histopathologic subtypes of renal cell carcinoma

This study aimed to reveal the prognostic role of the Hippo pathway in different histopathological subtypes of renal cell carcinoma (RCC). The TCGA-KIRC (n = 537), TCGA-KIRP (n = 291) and TCGA-KICH (n = 113), which contain data about clear cell (ccRCC), papillary (pRCC) and chromophobe RCC (chRCC), respectively, were investigated. Gene Set Variation Analysis was used to compare the activity of many pathways within a single sample. Oncogenic pathway-related expression differed between cases of ccRCC involving low and high Hippo pathway activity. There were two subsets of ccRCC, in which the cancer exhibited lower and higher Hippo signalling activity, respectively, compared with normal tissue. In the ccRCC cohort, lower Hippo pathway activity was associated with a higher clinical stage (p < 0.001). The Hippo pathway (HR = 0.29; 95% CI = 0.17–0.50, p < 0.001), apoptosis (HR = 6.02; 95% CI = 1.47–24.61; p = 0.013) and the p53 pathway (HR = 0.09; 95% CI = 0.02–0.36; p < 0.001) were identified as independent prognostic factors for ccRCC. The 5-year overall survival of the ccRCC patients with low and high Hippo pathway activity were 51.9% (95% CI = 45.0–59.9) and 73.6% (95% CI = 67.8–79.9), respectively. In conclusion, the Hippo pathway plays an important role in the progression of ccRCC. Low Hippo pathway activity is associated with poor outcomes in ccRCC, indicating the tumour suppressor function of this pathway.     

Overexpression of Wip1 Is Associated with Biologic Behavior in Human Clear Cell Renal Cell Carcinoma

Wild-type p53-induced phosphatase (Wip1 or PPM1D) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of Wip1 in RCC remains unclear. The present study investigated its abnormal expression and dysfunctions in clear cell renal cell carcinoma (ccRCC) in vitro. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of Wip1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of Wip1 was significantly associated with depth of invasion (P<0.001), Distant metastasis (P = 0.001), lymph node status (P<0.001) and Fuhrman grade (P<0.001). Wip1 knockdown inhibited the proliferation, migration and invasion of 786-O and RLC-310 cells, whereas Wip1 overexpression promoted the growth and aggressive phenotype of 786-O and RLC-310 cells in vitro. The uni- and multivariate analyses indicated that expression of Wip1 was an independent predictor for survival of ccRCC patients (P = 0.003, P = 0.027 respectively). Wip1- negative patients had a higher tumor-free/overall survival rate than patients with high Wip1 expression (P = 0.001, P = 0.002 respectively). Overexpression of Wip1 is useful in the prediction of survival in ccRCC patients.     

UBE3C Promotes Growth and Metastasis of Renal Cell Carcinoma via Activating Wnt/β-Catenin Pathway

Renal cell carcinoma (RCC) is the most common primary malignancy of the kidney and one of the most lethal genitourinary malignancies. Clear-cell renal cell carcinoma (ccRCC) has an extremely poor prognosis because of a high potential for tumor growth, vascular invasion, metastasis and recurrence. Unfortunately, the mechanism of RCC growth and metastasis is not well understood. In this report, we for the first time demonstrated ubiquitin protein ligase E3C (UBE3C) as a driving factor for RCC growth and metastasis. UBE3C expression was increased in ccRCC tissues compared with adjacent normal tissues. ccRCC patients with high UBE3C protein expression in tumors were associated with significantly worse postoperative survival. Knockdown of UBE3C expression in ACHN cells inhibited cell proliferation, migrations and invasiveness in vitro while overexpression of UBE3C in 786-O cells exerted the opposite effects. UBE3C up-regulated β-catenin protein levels and promoted β-catenin nuclear accumulation, leading to the activation of the Wnt/β-catenin signal pathway in RCC cells. Collectively, these observations suggest that UBE3C plays an important role in RCC development and progression, and UBE3C may be a novel target for prevention and treatment of ccRCC.     

Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma

Background

The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients.

Principal Findings

Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 γ-carboxylated, suggesting these molecules to be active in vivo.

Significance

These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.     

Quantitative Phosphoproteomics Analysis Uncovers PAK2- and CDK1-Mediated Malignant Signaling Pathways in Clear Cell Renal Cell Carcinoma

Clear cell Renal Cell Carcinoma (ccRCC) is among the 10 most common cancers in both men and women and causes more than 140,000 deaths worldwide every year. In order to elucidate the underlying molecular mechanisms orchestrated by phosphorylation modifications, we performed a comprehensive quantitative phosphoproteomics characterization of ccRCC tumor and normal adjacent tissues. Here, we identified 16,253 phosphopeptides, of which more than 9000 were singly quantified. Our in-depth analysis revealed 600 phosphopeptides to be significantly differentially regulated between tumor and normal tissues. Moreover, our data revealed that significantly up-regulated phosphoproteins are associated with protein synthesis and cytoskeletal re-organization which suggests proliferative and migratory behavior of renal tumors. This is supported by a mesenchymal profile of ccRCC phosphorylation events. Our rigorous characterization of the renal phosphoproteome also suggests that both epidermal growth factor receptor and vascular endothelial growth factor receptor are important mediators of phospho signaling in RCC pathogenesis. Furthermore, we determined the kinases p21-activated kinase 2, cyclin-dependent kinase 1 and c-Jun N-terminal kinase 1 to be master kinases that are responsible for phosphorylation of many substrates associated with cell proliferation, inflammation and migration. Moreover, high expression of p21-activated kinase 2 is associated with worse survival outcome of ccRCC patients. These master kinases are targetable by inhibitory drugs such as fostamatinib, minocycline, tamoxifen and bosutinib which can serve as novel therapeutic agents for ccRCC treatment.     

Connexin43 is associated with the progression of clear cell renal carcinoma and is regulated by tangeretin to sygergize with tyrosine kinase inhibitors

BackgroundThe roles of Connexin43 (Cx43) in clear cell renal cell carcinoma (ccRCC) microenviroment remains to be poorly defined.MethodsThe expression profile, prognosis and immune analysis of Cx43 in various cancers, particularly in ccRCC were performed using TCGA database, and various biological function assays were applied to explore the physiological role of Cx43 and tangeretin in ccRCC. Western blot were applied to examine the protein expression and Kunming mice were used to evaluate preliminary safety or anti-tumor activity of tangeretin and sunitinib.ResultsCompared with the normal group, higher expression levels of Cx43 in ccRCC, and distinct associations between Cx43 expression and ccRCC prognosis or immune infiltration, were found. Notably, the expression of Cx43 was found to be highly correlated with that of receptor tyrosine kinases (RTKs), particularly with VEGFR1, VEGFR2 and VEGFR3. The expression of Cx43 and EGFR was also found to be higher in ccRCC than that in the para-cancerous specimens. Knocking down Cx43 expression decreased RCC cell viability, cell migration, p-EGFR, MMP-9 and survivin expression. Using 14 Chinese medicine monomers, tangeretin was screened and found to inhibit tumor cell viability and Cx43 expression. Tangeretin also enhanced the sensitivity of RCC cells to tyrosine kinase inhibitors (TKIs) sunitinib and sorafenib. However, the same concentration of tangeretin exerted a less prominent effect on normal renal cell viability.ConclusionsCx43 is strongly associated with RTK expression and ccRCC progression, while tangeretin can inhibit RCC cell malignancy by inhibiting Cx43 expression and enhance the sensitivity of RCC cells to TKIs.     

Expression of Ror2 Mediates Invasive Phenotypes in Renal Cell Carcinoma

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.     

MiR-506 Is Down-Regulated in Clear Cell Renal Cell Carcinoma and Inhibits Cell Growth and Metastasis via Targeting FLOT1

Background

Some microRNAs (miRNAs) are abnormally expressed in cancer and contribute to tumorigenesis. In the present study, we investigated the role of miR-506 in clear cell renal cell carcinoma (ccRCC).

Methods

miR-506 expression was detected in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 and ccRCC specimens by quantitative real-time-PCR. We assessed the association of miR-506 expression with pathology and prognosis in ccRCC patients. We over-expressed and knocked-down miR-506 expression in two renal cancer cell lines, 786-O and ACHN, and assessed the impact on cell proliferation, migration and invasion. A luciferase reporter assay was conducted to confirm the target gene of miR-506 in renal cancer cell lines.

Results

miR-506 was significantly down-regulated in renal cancer cell lines and ccRCC specimens. Low miR-506 expression in ccRCC specimens was associated with an advanced clinical stage and poor prognosis. miR-506 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Over-expression of miR-506 in renal cancer cells decreased cell growth and metastasis, In contrast, down-regulation of miR-506 expression promoted renal cancer cell growth and metastasis. FLOT1, a potential target gene of miR-506, was inversely correlated with miR-506 expression in ccRCC tissues. Consistent with the effect of miR-506, knockdown of FLOT1 by siRNA inhibited cell malignant behaviors. Rescue of FLOT1 expression partially restored the effects of miR-506.

Conclusions

miR-506 exerts its anti-cancer function by directly targeting FLOT1 in renal cancer, indicating a potential novel therapeutic role in renal cancer treatment.     

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved. The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking. To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels. In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mU·mg(-1), P = 0.031) and conventional RCC (2.64 ± 1.49 mU·mg(-1), P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mU·mg(-1)). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mU·mg(-1), P = 0.031). Glycosylphosphatidylinositol-linked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney. Acetylcholinesterase mRNAs with exons E1c and E1e, 3'-alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney. The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000-fold lower in human kidney than in colon. Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10-fold in pRCC. The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol-anchored acetylcholinesterase explains the splicing to acetylcholinesterase-H mRNA. The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed.     

葡萄糖6-磷酸脱氢酶调控细胞周期蛋白E1和细胞周期蛋白依赖性激酶2促进细胞增殖及其在肾透明细胞癌中的预后价值

肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)是一种转移率高、预后差的细胞代谢性疾病,对其有效诊疗及预后分子标志物的研究十分重要。葡萄糖6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase, G6PD)在ccRCC中高表达,并提示患者不良预后,其促进ccRCC细胞增殖的分子机制有待进一步揭示。本研究发现,降低G6PD可抑制细胞周期G₁/S期转化并显著抑制ccRCC细胞增殖。G6PD可在细胞水平调控G₁/S期转化及增殖相关因子Cyclin D1,CDK4,CDK6,Cyclin E1和CDK2基因表达。TCGA数据库分析结果表明,ccRCC 中Cyclin D1,Cyclin E1 和 CDK2的mRNA 水平显著升高,而CDK4表达无明显差异,CDK6表达却显著降低。相关性分析结果显示,G6PD与Cyclin D1呈显著负相关(P<0.0001),G6PD与CDK4,CDK6之间无显著相关性(P>0.05),G6PD与Cyclin E1(P<0.0001)以及CDK2(P<0.05)显著正相关。进一步免疫组化检测结果表明,Cyclin E1和 CDK2在ccRCC肿瘤组织中表达显著升高。生存预后分析结果显示,Cyclin D1高表达提示ccRCC患者整体预后更为良好,CDK4和CDK6表达水平在ccRCC患者总生存率预测中无意义;而Cyclin E1和CDK2高表达均可提示ccRCC患者预后不良。进一步细胞水平检测发现,Cyclin E1、CDK2表达降低可显著逆转G6PD促进ccRCC细胞增殖的能力。综上,与增殖相关因子Cyclin D1,CDK4和CDK6相比,G6PD有可能通过促进Cyclin E1和CDK2表达升高而发挥促进 ccRCC肿瘤细胞增殖的作用,并且这3者的异常高表达有望成为ccRCC患者不良预后的独立生存预测因素。     

Genetic Variants in RKIP Are Associated with Clear Cell Renal Cell Carcinoma Risk in a Chinese Population

Background

Raf-1 kinase inhibitor protein (RKIP) plays a critical role in tumor development by regulating cell functions such as invasion, apoptosis and differentiation. Down-regulation of RKIP expression has been implicated in the development and progression of renal cell carcinoma (RCC). Herein, we hypothesized that genetic polymorphisms in RKIP might be associated with susceptibility and progression of RCC.

Methods

A total of 5 tagging single-nucleotide polymorphisms (tSNPs) in RKIP were selected and genotyped by SNapShot method in a case-control study of 859 RCC patients and 1004 controls. The logistic regression was used to evaluate the genetic association with occurrence and progression of RCC. The functionality of the important SNP was preliminary examined by qRT-PCR.

Result

We found that the rs17512051 in the promoter region of RKIP was significantly associated with decreased clear cell RCC (ccRCC) risk (TA/AA vs. TT: P = 0.039, OR = 0.78, 95%CI = 0.62–0.99). Another SNP (rs1051470) in the 3′UTR region of RKIP was marginally associated with increased ccRCC risk (TT vs. CC+CT: OR = 1.45, 95%CI = 1.01–2.09). In the stratified analysis, the protective effect of rs17512051 was more predominant in the subgroups of male, non-smokers, non-drinkers as well as subjects without history of diabetes. Furthermore, we observed higher RKIP mRNA levels in the presence of the rs17512051A allele in normal renal tissues.

Conclusion

Our results suggest that the potentially functional RKIP rs17512051 polymorphism may affect ccRCC susceptibility through altering the endogenous RKIP expression level. Risk effects and the functional impact of this polymorphism need further validation.     

Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin are well-known therapeutic targets for renal cell carcinoma (RCC). Sunitinib is an agent that targets VEGF receptors and is considered to be a standard treatment for metastatic or unresectable clear cell RCC (ccRCC). However, ccRCC eventually develops resistance to sunitinib in most cases, and the mechanisms underlying this resistance are not fully elucidated. In the present study, we established unique primary xenograft models, KURC1 (Kyoto University Renal Cancer 1) and KURC2, from freshly isolated ccRCC specimens. The KURC1 xenograft initially responded to sunitinib treatment, however finally acquired resistance. KURC2 retained sensitivity to sunitinib for over 6 months. Comparing gene expression profiles between the two xenograft models with different sensitivity to sunitinib, we identified interleukin 13 receptor alpha 2 (IL13RA2) as a candidate molecule associated with the acquired sunitinib-resistance in ccRCC. And patients with high IL13RA2 expression in immunohistochemistry in primary ccRCC tumor tends to have sunitinib-resistant metastatic site. Next, we showed that sunitinib-sensitive 786-O cells acquired resistance in vivo when IL13RA2 was overexpressed. Conversely, shRNA-mediated knockdown of IL13RA2 successfully overcame the sunitinib-resistance in Caki-1 cells. Histopathological analyses revealed that IL13RA2 repressed sunitinib-induced apoptosis without increasing tumor vasculature in vivo. To our knowledge, this is a novel mechanism of developing resistance to sunitinib in a certain population of ccRCC, and these results indicate that IL13RA2 could be one of potential target to overcome sunitinib resistance.