Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809).     

Alterations of the WNT7A Gene in Clear Cell Renal Cell Carcinomas

WNT7A (wingless-type MMTV integration site family, member 7A) is a known tumor suppressor gene of non-small cell lung carcinomas (NSCLC) and is frequently inactivated due to CpG-island hypermethylation in human cancers. The members of WNT family are involved in cell signaling and play crucial roles in cancer development. In the present work hypermethylation of the WNT7A gene was detected in 66% (29/44) of analyzed clear cell renal cell carcinomas (RCCs) using methyl-specific PCR (MSP). Moreover, bisulfite sequencing confirmed intensive hypermethylation of the 5′-CpG island of the WNT7A gene. Methylation analysis revealed positive correlations between tumor stage, Fuhrman nuclear grade and WNT7A hypermethylation. Additionally, restoration of WNT7A gene expression in the A498 cell line by 5-aza-2′-deoxycytidine treatment confirmed a direct contribution of hypermethylation in silencing of the WNT7A gene. High frequency of loss of heterozygosity (LOH) was demonstrated on chromosome 3p25 in regions surrounding the WNT7A gene. The frequent down-regulation of WNT7A gene expression was detected in 88% (15/17) of clear cell RCCs. We have also shown that the WNT7A gene possesses tumor suppression function by colony-formation and cell proliferation assays in RCC cell lines. In summary, the WNT7A gene is inactivated by genetic/epigenetic alterations in clear cell RCC and demonstrates tumor suppressor properties.     

Identification of Potential Serum Proteomic Biomarkers for Clear Cell Renal Cell Carcinoma

Objective

To investigate discriminating protein patterns and serum biomarkers between clear cell renal cell carcinoma (ccRCC) patients and healthy controls, as well as between paired pre- and post-operative ccRCC patients.

Methods

We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to identify patients with ccRCC. A total of 162 serum samples were analyzed in this study, among which there were 58 serum samples from ccRCC patients, 40 from additional paired pre- and post-operative ccRCC patients (n = 20), and 64 from healthy volunteers as healthy controls. ClinProTools software identified several distinct markers between ccRCC patients and healthy controls, as well as between pre- and post-operative patients.

Results

Patients with ccRCC could be identified with a mean sensitivity of 88.38% and a mean specificity of 91.67%. Of 67 m/z peaks that differed among the ccRCC, healthy controls, pre- and post-operative ccRCC patients, 24 were significantly different (P<0.05). Three candidate peaks, which were upregulated in ccRCC group and showed a tendency to return to healthy control values after surgery, were identified as peptide regions of RNA-binding protein 6 (RBP6), tubulin beta chain (TUBB), and zinc finger protein 3 (ZFP3) with the m/z values of 1466.98, 1618.22, and 5905.23, respectively.

Conclusion

MB-MALDI-TOF-MS method could generate serum peptidome profiles of ccRCC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

Identification of Metastamirs as Metastasis-associated MicroRNAs in Clear Cell Renal Cell Carcinomas

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2''-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.     

肾透明细胞癌相关微小RNA的研究进展

微小核糖核酸(miRNAs)是一类长约22个核苷酸的非编码单链小核糖核酸分子,miRNA通过与靶mRNA 3'端非翻译序列完全或部分互补结合,导致靶mRNA降解或转录后翻译抑制,从而调控靶基因的表达.最新研究显示人类血清/血浆中miRNA表达稳定,并在肿瘤患者血清中发现多种miRNA,其中的一些已经被证实与肾癌发生及发展相关,以往miRNA与肾癌的研究方.向多集中于肾癌组织,尽管发现很多有差异的miRNA,但不同研究者之间的结果常难以相互验证,而最近研究证实血清miRNA具有组织相关性和器官特异性,并对某些肿瘤具有高敏感性和特异性,因此其有望成为新的肿瘤标志物.肾癌是国内泌尿系统的第二常见恶性肿瘤,而且其近年来发病率和死亡率有逐年增高的趋势.由于肾透明细胞癌是肾癌的主要亚型,因此本文就血清miRNA在肾透明细胞癌的表达及其作用的研究进展作一综述.     

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.     

The Profile of Heparanase Expression Distinguishes Differentiated Thyroid Carcinoma from Benign Neoplasms

IntroductionThe search for a specific marker that could help to distinguish between differentiated thyroid carcinoma and benign lesions remains elusive in clinical practice. Heparanase (HPSE) is an endo-beta-glucoronidase implicated in the process of tumor invasion, and the heparanase-2 (HPSE2) modulates HPSE activity. The aim of this study was to evaluate the role of heparanases in the development and differential diagnosis of follicular pattern thyroid lesions.MethodsHPSE and HPSE2 expression by qRT-PCR, immunohistochemistry evaluation, western blot analysis and HPSE enzymatic activity were evaluated.ResultsThe expression of heparanases by qRT-PCR showed an increase of HPSE2 in thyroid carcinoma (P = 0.001). HPSE activity was found to be higher in the malignant neoplasms than in the benign tumors (P<0.0001). On Western blot analysis, HPSE2 isoforms were detected only in malignant tumors. The immunohistochemical assay allowed us to establish a distinct pattern for malignant and benign tumors. Carcinomas showed a typical combination of positive labeling for neoplastic cells and negative immunostaining in colloid, when compared to benign tumors (P<0.0001). The proposed diagnostic test presents sensitivity and negative predictive value of around 100%, showing itself to be an accurate test for distinguishing between malignant and benign lesions.ConclusionsThis study shows, for the first time, a distinct profile of HPSE expression in thyroid carcinoma suggesting its role in carcinogenesis.     

LncRNAs Expression Signatures of Renal Clear Cell Carcinoma Revealed by Microarray

Background

Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. They are aberrantly expressed in many types of cancers. In this study, we described lncRNAs profiles in 6 pairs of human renal clear cell carcinoma (RCCC) and the corresponding adjacent nontumorous tissues (NT) by microarray.

Methodology/Principal Findings

With abundant and varied probes accounting 33,045 LncRNAs in our microarray, the number of lncRNAs that expressed at a certain level could be detected is 17157. From the data we found there were thousands of lncRNAs that differentially expressed (≥2 fold-change) in RCCC tissues compared with NT and 916 lncRNAs differentially expressed in five or more of six RCCC samples. Compared with NT, many lncRNAs were significantly up-regulated or down-regulated in RCCC. Our data showed that down-regulated lncRNAs were more common than up-regulated ones. ENST00000456816, {"type":"entrez-nucleotide","attrs":{"text":"X91348","term_id":"1418768","term_text":"X91348"}}X91348, {"type":"entrez-nucleotide","attrs":{"text":"BC029135","term_id":"23271405","term_text":"BC029135"}}BC029135, {"type":"entrez-nucleotide","attrs":{"text":"NR_024418","term_id":"212286160","term_text":"NR_024418"}}NR_024418 were evaluated by qPCR in sixty-three pairs of RCCC and NT samples. The four lncRNAs were aberrantly expressed in RCCC compared with matched histologically normal renal tissues.

Conclusions/Significance

Our study is the first one to determine genome-wide lncRNAs expression patterns in RCCC by microarray. The results displayed that clusters of lncRNAs were aberrantly expressed in RCCC compared with NT samples, which revealed that lncRNAs differentially expressed in tumor tissues and normal tissues may exert a partial or key role in tumor development. Taken together, this study may provide potential targets for future treatment of RCCC and novel insights into cancer biology.     

Blood Levels of Carbonic Anhydrase 9 Correlate with Clear Cell Renal Cell Carcinoma Activity

Introduction  Biomarkers for early detection of renal cell carcinoma (RCC) may help diagnose minimal residual disease in patients at risk for RCC, can guide anti-angiogenic therapy, or may help identify candidates for adjuvant treatment. In this study, we investigated whether blood levels of carbonic anhydrase 9 (CA9) correlate with RCC tumor burden and therefore disease activity. Methods  CA9 is a von Hippel–Lindau–hypoxia inducible factor target upregulated in clear cell RCC. We used an anti-CA9 antibody (M75)-based enzyme-linked immunosorbent assay test to measure CA9 levels in blood obtained before and after nephrectomy for clinically localized disease in patients with: (1) clear cell RCC, (2) papillary and chromophobe RCC or oncocytoma, or (3) benign kidney lesions, and we compared these samples to blood drawn from normal control individuals. Results  We observed a significant (p < 0.006) decrease in the blood levels of CA9, after nephrectomy for localized disease, in the majority of patients with clear cell RCC (57%). In contrast, patients with nonclear cell RCC, benign disease, or those having undergone debulking nephrectomy for metastatic disease did not have a decrease in CA9 blood levels after nephrectomy. Preliminary longitudinal follow up measurements of CA9 levels in a small group of patients indicated that rising CA9 levels may correlate with disease progression. Conclusions  Plasma CA9 levels correlate with disease activity in a subset of clear cell RCC patients and should be considered in future multiplex RCC biomarker development algorithms.     

Assessment of Intratumoral Micromorphology for Patients with Clear Cell Renal Cell Carcinoma Using Susceptibility-Weighted Imaging

Background

Multiple treatment options exist for the management of renal cell carcinomas. Preoperative evaluation of clear cell renal cell carcinoma (CRCC) grades is important for deciding upon the appropriate method of therapy. We hypothesize that susceptibility weighted imaging (SWI) is sensitive enough to detect intratumoral microvessles and microbleeding in renal cell carcinoma, which can be used to grade CRCC.

Material and Methods

Retrospective reviews of 37 patients with pathologically proven CRCCs were evaluated. All patients underwent SWI examinations. The characteristics of intratumoral susceptibility signal intensity (ITSS) includes the likelihood of the presence of ITSS, morphology of ITSS, dominant structure of ITSS and ratio of ITSS area to tumor area, which were all assessed on SWI. The results were compared using the nonparametric Mann-Whitney test.

Results

ITSS was seen in all patients except 4 patients with low-grade CRCCs. There was no significant difference between low and high-grade CRCCs when looking at the likelihood of the presence of ITSS. There was a significant difference in the mean score of dominant structures between low and high-grade CRCCs. Specifically, more dominant vascular structures and less hemorrhage were seen in low-grade tumors (2.15±1.05) compared to high-grade tumors (1.27±0.47) (P<0.005). The ratio of ITSS area to tumor area was also significantly higher for the high-grade group (1.55±0.52) than that for the low-grade group (0.88±0.43) on SWI (P<0.005).

Conclusion

SWI is useful for grading CRCCs.     

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.     

Tumor-Specific Hypermethylation of Epigenetic Biomarkers,Including SFRP1, Predicts for Poorer Survival in Patients from the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) Project

The recent publication of the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) project has provided an immense wealth and breadth of data providing an invaluable tool for confirmation and expansion upon previous observations in a large data set containing multiple data types including DNA methylation, somatic mutation, and clinical information. In clear cell renal cell carcinoma (CCRCC) many genes have been demonstrated to be epigenetically inactivated by promoter hypermethylated and in a small number of cases to be associated with clinical outcome. This study created two cohorts based on the Illumina BeadChip array used to confirm the frequency of tumor-specific hypermethylation of these published hypermethylated genes, assess the impact of somatic mutation or chromosomal loss and provide the most comprehensive assessment to date of the association of this hypermethylation with patient survival. Hypermethylation of the Fibrillin 2 (FBN2) gene was the most consistent epigenetic biomarker for CCRCC across both cohorts in 40.2% or 52.5% of tumors respectively. Hypermethylation of the secreted frizzled-related protein 1 (SFRP1) gene and the basonuclin 1 (BNC1) gene were both statistically associated with poorer survival in both cohorts (SFRP1 - p = <0.0001 or 0.0010 and BNC1 - p = <0.0001 or 0.0380) and represented better independent markers of survival than tumor stage, grade or dimension in one cohort and tumor stage or dimension in the other cohort. Loss of the SFRP1 protein can potentially activate the WNT pathway and this analysis highlighted hypermethylation of several other WNT pathway regulating genes and demonstrated a poorer survival outcome for patients with somatic mutation of these genes. The success of demethylating drugs in hematological malignances and the current trials in solid tumors suggest that the identification of clinically relevant hypermethylated genes combined with therapeutic advances may improve the effectiveness and usefulness of such drugs in clear cell renal cell carcinoma.     

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.