N6 -Methyladenosine (m6 A) readers are dysregulated in renal cell carcinoma.

N6 -Methyladenosine (m6 A) is the most common modification of messenger RNA (mRNA) in mammals. It critically influences RNA metabolism and plays an essential role in virtually all types of bioprocesses including gene expression, tissue development, self-renewal and differentiation of stem cells, stress response and circadian clock control. It plays a crucial role in carcinogenesis and could be used as a prognostic and a diagnostic tool and as a target for new anticancer therapies. m6 A modification is dynamically and reversibly regulated by three types of proteins. Methyltransferases, so-called "writers" add a methyl group to the adenosine, which can be removed by demethylases, also called "erasers." m6 A-specific RNA-binding proteins, from here on referred to as "readers," preferentially bind to the m6 A site and mediate biological functions, such as translation, splicing or decay of RNA. In this study, we examined the expression of the six m6 A readers HNRNPA2B1, HNRNPC, YTHDC1 and YTHDF1-3 in clear cell renal carcinoma (ccRCC). We show that on mRNA level the expression of all six m6 A readers is significantly downregulated compared to normal renal tissue and on protein level five out of six readers are dysregulated. Lower levels of some m6 A readers are correlated with advanced stage and grade as well as associated with a shorter overall, progression-free and cancer-specific survival. In summary, we could show that m6 A readers are dysregulated in ccRCC and might therefore act as a tumor marker, could give further information on the individual prognosis and be a target of innovative cancer therapy.

The N6 -methyladenosine (m6 A) erasers alkylation repair homologue 5 (ALKBH5) and fat mass and obesity-associated protein (FTO) are prognostic biomarkers in patients with clear cell renal carcinoma.

OBJECTIVES: To comprehensively investigate the role of the N6 -methyladenosine (m6 A) erasers ALKBH5 and FTO in clear cell renal cell carcinoma (ccRCC), other RCC subtypes, and oncocytoma with respect to prognostic value and biomarker potential. PATIENTS AND METHODS: The collection of tissue samples was performed within the framework of the Biobank at the Centre for Integrated Oncology Cologne-Bonn. The gene expressions of alkylation repair homologue 5 (ALKBH5) and fat mass and obesity-associated protein (FTO) were determined using quantitative real-time polymerase chain reaction. ALKBH5 and FTO expressions were further investigated in ccRCC, papillary RCC, chromophobe RCC, sarcomatoid RCC, oncocytoma, and benign renal tissue using tissue microarrays. RESULTS: ALKBH5 mRNA, as well as ALKBH5 and FTO protein expressions, was significantly downregulated in ccRCC compared to normal tissue and most of the other studied tumour entities. Decreased mRNA levels of ALKBH5 and FTO correlated with a shortened overall and cancer-specific survival following nephrectomy. CONCLUSIONS: Taken together, our present data indicate that the m6 A-demethylases ALKBH5 and FTO are dysregulated in ccRCC and could be used as prognostic biomarkers.

Mitochondrial PIWI-interacting RNAs are novel biomarkers for clear cell renal cell carcinoma.

PURPOSE: PIWI-interacting RNAs (piRNAs) have been suggested to serve as biomarkers in cancer. In this study, we validated the expression profile of two piRNAs derived from mitochondria, piR-34536 and piR-51810, in tissue and serum of a cohort of clear cell renal cell carcinoma (ccRCC) patients. METHODS: Tissue and serum samples of patients with ccRCC were collected prospectively in our biobank. Total RNA was isolated from 118 ccRCC tissues, 75 normal renal tissues as well as 30 serum samples from patients with ccRCC, and 15 serum samples from patients with non-malignant diseases. The expression of piRNAs was determined using quantitative real-time PCR. RESULTS: Both piR-34536 and piR-51810 were downregulated in ccRCC compared to non-malignant renal tissue. Decreased tissue piRNA levels were significant and independent predictors of shortened progression-free, cancer-specific and overall survival of ccRCC patients. The piRNA levels in serum did not differ in ccRCC patients and control subjects. CONCLUSIONS: The expression of piR-34536 and piR-51810 in ccRCC tissues may be used as prognostic biomarkers in ccRCC.

5'-tRNA Halves are Dysregulated in Clear Cell Renal Cell Carcinoma.

PURPOSE: In various malignancies RNA fragments are dysregulated. Our study was designed to determine the expression of 4, 5'-tRNA halves in the tissue and serum of patients with clear cell renal cell carcinoma. MATERIALS AND METHODS: Tissue and serum samples of patients with clear cell renal cell carcinoma and nonmalignant disease were collected prospectively in our biobank. We isolated total RNA from 95 clear cell renal cell carcinomas and 50 normal renal tissues as well as serum RNA from 27 patients with clear cell renal cell carcinoma and 13 with nonmalignant urological disease. To specifically determine the expression of 5'-tRNA halves we dephosphorylated and ligated an adaptor nucleotide to the 3' end of the tRNA halves. The expression levels of 4, 5'-tRNA halves (5'-tRNA-Arg-CCT, 5'-tRNA-Glu-CTC, 5'-tRNA-Leu-CAG and 5'-tRNA-Lys-TTT) were then measured by TaqMan® based quantitative reverse transcription-polymerase chain reaction. RESULTS: All studied 5'-tRNA halves were down-regulated in clear cell renal cell carcinoma tissues, indicating a potential role as a tumor suppressor. Furthermore, we noted decreased expression of 5'-tRNA halves in patients with adverse clinicopathological parameters. All 5'-tRNA halves were expressed at lower levels in nonorgan confined clear cell renal cell carcinoma. The 5'-tRNA-Lys-TTT halves inversely correlated with ISUP (International Society of Urological Pathology) grade. In patients with clear cell renal cell carcinoma 5'-tRNA-Arg-CCT, 5'-tRNA-Glu-CTC and 5'-tRNA-Lys-TTT halves circulated at lower levels than in control subjects, indicating relevance as noninvasive biomarkers. CONCLUSIONS: In patients with clear cell renal cell carcinoma 5'-tRNA halves have potential as diagnostic and prognostic biomarkers. The 5'-tRNA halves may act in a tumor suppressive manner, which requires further research to confirm.

The knockdown of the mediator complex subunit MED30 suppresses the proliferation and migration of renal cell carcinoma cells.

BACKGROUND: The mediator complex consists of 33 subunits and plays a central role in transcription. Studies have already described the involvement of individual subunits, especially in carcinogenesis. With regard to the subunit MED30, this has, so far, only been confirmed in gastric and breast carcinoma. The role of MED30 in urological tumours is unknown. MATERIALS AND METHODS: First, a database analysis using cBioPortal was performed for the mRNA expression and survival analysis of MED30 in clear cell renal cell carcinoma (ccRCC) and papillary RCC (pRCC). The immunohistochemical analysis (IHC) against MED30 was performed on tissue microarrays (TMA), with benign, ccRCC, pRCC samples, and ccRCC-metastases. Intensity evaluation was performed using the IRS (Immunoreactive Score). The ccRCC cell lines ACHN and A-498 were used for the functional investigation of proliferation, migration, and invasion after the knockdown of MED30 by siRNA. RESULTS: In a database analysis by cBioPortal, it was shown that mRNA overexpression of MED30 in the pRCC was significantly associated with a poorer overall survival and progression-free survival. In the IHC, pRCC showed the highest level of MED30 expression, unfortunately without significant results in the survival analysis. The knockdown of MED30 resulted in a significant decrease in proliferation, migration, and invasion in ccRCC. CONCLUSION: In summary, MED30 seems to be involved in the progression of the RCC.

Identification of the dopamine transporter SLC6A3 as a biomarker for patients with renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is among the most common human malignancies. METHODS: In order to provide better understanding of the molecular biology of ccRCC and to identify potential diagnostic/prognostic biomarker and therapeutic targets, we utilized a microarray to profile mRNA expression of corresponding normal and malignant renal tissues. Real-time PCR, Western Blot and immunohistochemistry were applied to study the expression of candidate biomarkers. ccRCC cell lines were treated with sertraline to inhibit the dopamine transporter SLC6A3. RESULTS: Differential expression of fourteen mRNAs, yet not studied in ccRCC in depth, was confirmed using qPCR (upregulation: SLC6A3, NPTX2, TNFAIP6, NDUFA4L2, ENPP3, FABP6, SPINK13; downregulation: FXYD4, SLC12A1, KNG1, NPHS2, SLC13A3, GCGR, PLG). Up-/downregulation was also confirmed for FXYD4, KNG1, NPTX2 and SLC12A1 by Western Blot on the protein level. In contrast to the mRNA expression, protein expression of the dopamine transporter SLC6A3 was lower in ccRCC compared to normal renal tissue. Immunohistochemistry indicated that this decrease was due to higher concentrations of SLC6A3 in the proximal tubules. Immunohistochemical analyses further demonstrated that high SLC6A3 expression in ccRCC tissue was correlated with a shorter period of recurrence-free survival following surgery. Treatment of ccRCC cells with the SLC6A3 inhibitor sertraline induced dose-dependent cell-death. CONCLUSION: Our study identified several novel biomarkers with diagnostic potential and further investigations on sertraline as therapeutic agent in ccRCC patients are warranted.

Adipocyte Precursor-Derived NRG1 Promotes Resistance to FGFR Inhibition in Urothelial Carcinoma.

Aberrations of the fibroblast growth factor receptor (FGFR) family members are frequently observed in metastatic urothelial cancer (mUC), and blocking the FGF/FGFR signaling axis is used as a targeted therapeutic strategy for treating patients. Erdafitinib is a pan-FGFR inhibitor, which has recently been approved by the FDA for mUC with FGFR2/3 alterations. Although mUC patients show initial response to erdafitinib, acquired resistance rapidly develops. Here, we found that adipocyte precursors promoted resistance to erdafitinib in FGFR-dependent bladder and lung cancer in a paracrine manner. Moreover, neuregulin 1 (NRG1) secreted from adipocyte precursors was a mediator of erdafitinib resistance by activating human epidermal growth factor receptor 3 (ERBB3; also known as HER3) signaling, and knockdown of NRG1 in adipocyte precursors abrogated the conferred paracrine resistance. NRG1 expression was significantly downregulated in terminally differentiated adipocytes compared with their progenitors. Pharmacologic inhibition of the NRG1/HER3 axis using pertuzumab reversed erdafitinib resistance in tumor cells in vitro and prolonged survival of mice bearing bladder cancer xenografts in vivo. Remarkably, data from single-cell RNA sequencing revealed that NRG1 was enriched in platelet-derived growth factor receptor-A (PDGFRA) expressing inflammatory cancer-associated fibroblasts, which is also expressed on adipocyte precursors. Together, this work reveals a paracrine mechanism of anti-FGFR resistance in bladder cancer, and potentially other cancers, that is amenable to inhibition using available targeted therapies. SIGNIFICANCE: Acquired resistance to FGFR inhibition can be rapidly promoted by paracrine activation of the NRG1/HER3 axis mediated by adipocyte precursors and can be overcome by the combination of pertuzumab and erdafitinib treatment. See related commentary by Kolonin and Anastassiou, p. 648.

Epigenetic regulation of microRNA expression in renal cell carcinoma.

The underlying mechanisms of microRNA deregulation in cancer cells include epigenetic modifications, which play a crucial role in carcinogenesis. We demonstrate that numerous microRNAs are induced in renal cell carcinoma cell lines after treatment with inhibitors of the DNA-methyltransferase (5-aza-2'-deoxycytidine) and the histone-deacetylase (suberoylanilide hydroxamic acid). We provide evidence that enrichment of H3 and H3K18 acetylation at the miR-9 promoter is causative for re-expression, while DNA hypermethylation remains unchanged. Our experiments show that the treatment with the epigenetic drugs causes re-expression of silenced microRNAs with putative tumor suppressive function in ccRCC cell lines.

Depletion of the m6A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.

BACKGROUND: N6-methyladenosine (m6A) is one of the most common RNA modifications in eukaryotes and has effects on RNA structure and stability. Recent studies have shown that m6A methylation is involved in human carcinogenesis. In the present study, we investigated the effects of m6A demethylases FTO and ALKBH5 on renal cell carcinoma (RCC) cell lines. METHODS: The epithelial-mesenchymal in vitro knockdowns of FTO and ALKBH5 induced by antisense oligonucleotides (LNA-GapmeR system) were established in RCC cell lines. Their effects on migration and proliferation were investigated subsequently. The influence of FTO and ALKBH5 knockdown on key epithelial-mesenchymal transition (EMT) genes was analyzed. RESULTS: Inactivation of FTO and ALKBH5 resulted in decreased proliferation and motility in all cell lines examined (ACHN, Caki-1, 769-P). Vimentin (VIM) was downregulated after the knockdown of FTO and ALKBH5, indicating an EMT switch. CONCLUSIONS: Knockdown of the m6A erasers FTO and ALKBH5 inhibits the malignant potential in the cell cultures studied by means of an EMT switch.

CDCP1 expression is frequently increased in aggressive urothelial carcinoma and promotes urothelial tumor progression.

The prognosis of patients with advanced urothelial carcinoma (UC) remains poor and improving treatment continues to be a major medical need. CUB domain containing protein 1 (CDCP1) is a known oncogene in various types of solid cancers and its overexpression is associated with impaired prognosis. However, its role in UC remains undetermined. Here we assessed the clinical relevance of CDCP1 in two cohorts of UC at different stages of the disease. Immunohistochemistry showed that CDCP1 is highly expressed in advanced UC, which significantly correlates with shorter overall survival. Importantly, the basal/squamous UC subtype showed significantly enriched CDCP1 at the mRNA and protein levels. The functional role of CDCP1 overexpression was assessed taking advantage of ex vivo organoids derived from the CDCP1pcLSL/+ transgenic mouse model. Furthermore, CDCP1 knockout UC cell lines were generated using CRISPR/Cas9 technology. Interestingly, CDCP1 overexpression significantly induced the activation of MAPK/ERK pathways in ex vivo organoids and increased their proliferation. Similarly, CDCP1 knockout in UC cell lines reduced their proliferation and migration, concomitant with MAPK/ERK pathway activity reduction. Our results highlight the relevance of CDCP1 in advanced UC and demonstrate its oncogenic role, suggesting that targeting CDCP1 could be a rational therapeutic strategy for the treatment of advanced UC.

Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide.

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria.

Systematic expression analysis of m6A RNA methyltransferases in clear cell renal cell carcinoma.

Objectives: To investigate the regulation of the N-6-methyladenosine (m6A) methyltransferases METTL3, METTL14, WTAP, KIAA1429, and METTL4, referred to as "m6A writers," in clear cell renal cell carcinoma (ccRCC), and other RCC subtypes in respect of the potential prognostic value. Patients and methods: Tissue samples were collected within the framework of the Biobank at the Center for Integrated Oncology Bonn. The expression of the methyltransferases was systematically determined in clear cell renal carcinoma (ccRCC) on the RNA (real-time PCR) and protein level (immunohistochemistry). Additionally, protein expression of the m6A writers was further investigated in papillary RCC, chromophobe RCC, sarcomatoid RCC, oncocytoma, and normal renal tissue (immunohistochemistry). Results: The expression of all m6A-methyltransferases was significantly downregulated in ccRCC compared to benign renal tissue. Low m6A-methyltransferase levels were correlated with higher histological grade, advanced pT-stage, pN-stage, and metastatic disease. Reduced m6A-methyltransferase expression was associated with shorter overall survival. Conclusion: In conclusion, m6A-methyltransferases are dysregulated in ccRCC and might act as tumor suppressor genes, which could be of particular importance for future diagnostic and therapeutic options.

CircEHD2, CircNETO2 and CircEGLN3 as Diagnostic and Prognostic Biomarkers for Patients with Renal Cell Carcinoma.

BACKGROUND: Circular RNA (circRNA) plays an important role in the carcinogenesis of various tumors. It is assumed that circRNAs have a high tissue and tumor specificity, thus they are discussed as cancer biomarkers. The knowledge about circRNAs in clear cell renal carcinoma (ccRCC) is limited so far, and thus we studied the expression profile of seven circRNAs (circCOL5A1, circEHD2, circEDEM2, circEGNL3, circNETO2, circSCARB1, circSOD2) in a cohort of ccRCC patients. METHODS: Fresh-frozen normal and cancerous tissues were prospectively collected from patients with ccRCC undergoing partial/radical nephrectomy. Total RNA was isolated from 121 ccRCC and 91 normal renal tissues, and the circRNA expression profile was determined using quantitative real-time PCR. RESULTS: circEHD2, circENGLN3, and circNETO2 were upregulated in ccRCC compared with non-malignant renal tissue. circENGLN3 expression was highly discriminative between normal and cancerous tissue. None of the circRNAs was correlated with clinicopathological parameters. High circEHD2 and low circNETO2 levels were an independent predictor of a shortened progression-free survival, cancer-specific survival, and overall survival in patients with ccRCC undergoing nephrectomy. CONCLUSIONS: The analysis of circRNAs may provide diagnostic and prognostic information. Thus, circRNAs could help to optimize the individual treatment and ultimately improve ccRCC patients' survival.

Comprehensive Analysis of the ATP-binding Cassette Subfamily B Across Renal Cancers Identifies ABCB8 Overexpression in Phenotypically Aggressive Clear Cell Renal Cell Carcinoma.

BACKGROUND: ATP-binding cassette (ABC) transporters play a crucial role in the development of multidrug resistance in diverse cancer entities. OBJECTIVE: Our study was designed to comprehensively analyze the ABC subfamily B (ABCB) in renal cell carcinoma (RCC) using The Cancer Genome Atlas (TCGA) datasets. DESIGN, SETTING, AND PARTICIPANTS: We performed systematic survival analyses of ABCB1-10 using the TCGA datasets for clear cell, papillary, and chromophobe RCC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Results were validated via quantitative polymerase chain reaction in a clear cell RCC (ccRCC) cohort containing 152 samples. Afterward, ABCB8 protein expression was assessed in a tissue microarray RCC cohort (n = 144) by immunohistochemistry with subsequent quantitative image analysis. In vitro, antisense oligonucleotide-induced ABCB8 knockdowns were established in ACHN and CAKI1 following functional analyses. RESULTS AND LIMITATIONS: Various ABCB members have prognostic value among the three most occurring RCC subtypes. Of note, ABCB8 was identified as the most prognostic ABCB gene in the RCC TCGA cohorts. Further, ABCB8 proved to be an independent predictor of shortened cancer-specific survival in three independent cohorts. In vitro, specific ABCB8 knockdown reduced viability and migration capacity in ACHN and CAKI1. CONCLUSIONS: ABCB8 was identified as a promising prognostic biomarker. Functional analyses suggest a tumor-promoting role of ABCB8 in ccRCC. PATIENT SUMMARY: In this study, the transporter gene ABCB8 proved to be a risk predictor of a worse clinical course in clear cell renal cell carcinoma. In the renal cell carcinoma cell culture model, depletion of this gene led to a reduction in the malignant potential, and inhibition of this gene may therefore possess a therapeutic value.

The contrasting roles of Dysferlin during tumor progression in renal cell carcinoma.

BACKGROUND: The vesicle fusion protein Dysferlin (DYSF) is mainly known as a membrane repair protein in muscle cells. Mutations of DYSF lead to muscular dystrophies and cardiomyopathies. In contrast to other members of the Ferlin protein family, few is known about its role in cancer. Our study was designed to investigate the expression and functional properties of DYSF in ccRCC and its association with clinicopathological parameters and survival. MATERIAL AND METHODS: TCGA cohort: mRNA expression data of DYSF were extracted from TCGA for patients with ccRCC (n = 603; ccRCC n = 522, benign n = 81). Study cohort: mRNA expression of DYSF in ccRCC was determined using qPCR (n = 126; ccRCC n = 82, benign n = 44). Immunohistochemical staining against DYSF was performed on tissue microarrays to validate protein expression (n = 172; ccRCC n = 142, benign n = 30). Correlations between mRNA/protein expression and clinicopathological data were statistically tested. Following siRNA-mediated knockdown of DYSF in ccRCC cell line ACHN, cell migration, invasion and proliferation were investigated. RESULTS: Both DYSF mRNA and protein expression are significantly up-regulated in ccRCC tissue. DYSF mRNA expression decreased during tumor progression: lower expression levels were measured in higher stage/grade and metastatic ccRCC with independent prognostic significance for overall and cancer-specific survival. In contrast, protein expression correlated positively with pathological parameters. Overexpression showed tendency toward poor survival. Accordingly, knockdown of DYSF suppressed migration and invasion of ccRCC cells. CONCLUSION: DYSF mRNA and protein expression are opposingly involved in tumor progression of ccRCC. DYSF could be used as a prognostic biomarker to predict survival of patients with ccRCC.

Downstream neighbor of SON (DONSON) is associated with unfavorable survival across diverse cancers with oncogenic properties in clear cell renal cell carcinoma.

A precise stratification of our patients is essential and can support clinicians to determine the right therapy. The aim of this study was to identify clinically relevant genes using The Cancer Genome Atlas (TCGA) datasets. A comprehensive pan-cancer analysis of 30 distinct tumor entities (N = 9022) identified the largely unknown gene Downstream neighbor of SON (DONSON) to be particularly associated with unfavorable overall survival in clear cell renal cell carcinoma (KIRC). This prognostic potential of DONSON was validated in an independent KIRC cohort via quantitative real-time PCR (n = 152). Further, DONSON protein expression was evaluated via immunohistochemical staining followed by quantitative image analysis using the image analysis software QuPath on a renal cancer tissue microarray (n = 270). Interestingly, DONSON overexpression was preferentially associated with poor survival in 9 of the 30 entities, suggesting tumor-independent oncogenic properties of this largely unknown gene. A particularly strong association of DONSON to an aggressive phenotype was evident in KIRC and proved to be a strong independent predictor of unfavorable overall survival in two additional cohorts on the mRNA and protein level. In our KIRC cell culture model, we observed a substantial attenuation of proliferative activity and migration capacity of the KIRC cells Caki1 and 769p. In conclusion, we identified DONSON as a robust biomarker for risk stratification in KIRC in three independent cohorts and provide evidence that DONSON is linked to a malignant phenotype in the KIRC cell culture model.

Knockdown of Myoferlin Suppresses Migration and Invasion in Clear-Cell Renal-Cell Carcinoma.

BACKGROUND/AIM: Myoferlin (MYOF) has emerged as an oncogenic protein in various human cancer types. This study was conducted to investigate comprehensively the expression and functional properties of MYOF in clear-cell renal-cell carcinoma (ccRCC) with respect to its value as diagnostic biomarker and therapeutic target. MATERIALS AND METHODS: mRNA and protein expression of MYOF were assessed by quantitative polymerase chain reaction and immunohistochemistry. siRNA-mediated knockdown of MYOF was performed in the RCC cell line ACHN followed by proliferation, migration and invasion assays. RESULTS: MYOF mRNA and protein expression were significantly up-regulated in ccRCC. Higher mRNA levels were measured in advanced tumors. MYOF protein expression was increased in tumors with higher histological grades, and those with positive lymph node and surgical margin status. MYOF knockdown led to reduction of migration and invasion in ACHN cells, whereas expression of angiogenesis-associated genes tyrosine-protein kinase receptor-2 (TIE2), angiopoietin 2 (ANG2) and caveolin-1 (CAV1) was up-regulated following knockdown. CONCLUSION: MYOF may serve as a diagnostic biomarker of tumor progression and a potential therapeutic target in ccRCC.

Downstream Neighbor of SON (DONSON) Expression Is Enhanced in Phenotypically Aggressive Prostate Cancers.

Downstream neighbor of Son (DONSON) plays a crucial role in cell cycle progression and in maintaining genomic stability, but its role in prostate cancer (PCa) development and progression is still underinvestigated. Methods: DONSON mRNA expression was analyzed with regard to clinical-pathological parameters and progression using The Cancer Genome Atlas (TCGA) and two publicly available Gene Expression Omnibus (GEO) datasets of PCa. Afterwards, DONSON protein expression was assessed via immunohistochemistry on a comprehensive tissue microarray (TMA). Subsequently, the influence of a DONSON-knockdown induced by the transfection of antisense-oligonucleotides on proliferative capacity and metastatic potential was investigated. DONSON was associated with an aggressive phenotype in the PCa TCGA cohort, two GEO PCa cohorts, and our PCa TMA cohort as DONSON expression was particularly strong in locally advanced, metastasized, and dedifferentiated carcinomas. Thus, DONSON expression was notably upregulated in distant and androgen-deprivation resistant metastases. In vitro, specific DONSON-knockdown significantly reduced the migration capacity in the PCa cell lines PC-3 and LNCaP, which further suggests a tumor-promoting role of DONSON in PCa. In conclusion, the results of our comprehensive expression analyses, as well as the functional data obtained after DONSON-depletion, lead us to the conclusion that DONSON is a promising prognostic biomarker with oncogenic properties in PCa.

Blimp-1Deltaexon7: a naturally occurring Blimp-1 deletion mutant with auto-regulatory potential.

Blimp-1 is a master regulator of terminal B cell differentiation and plays a pivotal role in various developmental processes. In addition to full length Blimp-1, a Blimp-1 mRNA lacking exon 7 (Blimp-1Delta7) has been described to occur in murine B cells. The activity and function of the mutant mRNA-encoded protein (Blimp-1Delta7), lacking three crucial zinc fingers necessary for DNA interaction, is completely unknown. Since isoforms of other prdm family proteins affect each other's functions, we wondered whether Blimp-1Delta7 still plays a role in B cells, independent of direct DNA binding. In this study, we found that Blimp-1Delta7 is preferentially expressed in naïve CD19(+) B cells. A fraction of Blimp-1Delta7 migrates to the nucleus, colocalizes with HDAC2 and is found at sites of repressed chromatin, although it does not bind to the Blimp-1 DNA consensus site. Unexpectedly, Blimp-1 and Blimp-1Delta7 homodimerize as well as heterodimerize with each other. Ectopic expression of Blimp-1Delta7 in WEHI 231 cells, a Blimp-1-negative murine lymphoma line, leads to cessation of proliferation and enhancement of apoptosis. Importantly, LPS-induced differentiation is suppressed in the presence of Blimp-1Delta7. This is in agreement with our finding that Blimp-1Delta7 interferes with endogenous Blimp-1 expression. Thus, our data suggest an auto-regulatory mechanism of Blimp-1 activation.

YRNA Expression Profiles are Altered in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Noncoding RNAs play an important role in human carcinogenesis. YRNAs, a novel class of noncoding RNAs, have been identified as biomarkers in breast cancer patients. OBJECTIVE: To test the hypothesis that YRNA expression is dysregulated in clear cell renal cell carcinoma (ccRCC). DESIGN, SETTING, AND PARTICIPANTS: We first measured the expression of all known YRNAs (hY1, hY3, hY4, and hY5) in a screening cohort (30 ccRCC and 15 normal renal tissues). Subsequently, hY3 and hY4 were validated in an independent cohort (88 ccRCC and 59 normal renal tissues). Finally, hY3 and hY4 levels in serum samples from 30 ccRCC and 15 control individuals were measured. YRNAs were detected using quantitative real-time polymerase chain reaction. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Relative expression values were analyzed using the Mann-Whitney-U test and Kaplan Meier estimates. RESULTS AND LIMITATIONS: Expression of hY3 and hY4 was increased in ccRCC samples compared with normal renal tissue, whereas hY1 and hY5 levels were similar. Expression levels of hY4 correlated with ccRCC stage and the presence of lymph node metastases. Neither hY3 nor hY4 were circulating at different levels in ccRCC patients and control individuals. CONCLUSIONS: The expression of hY3 and hY4 is altered in ccRCC and associated with advanced disease. PATIENT SUMMARY: In this report we studied the expression of noncoding YRNA in clear cell renal cell carcinoma tissue. We observed increased hY3 and hY4 expression levels in cancer tissues. However, expression levels were not different in the serum of patients with cancer or benign disease.