Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients

BACKGROUND: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients. METHODS: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. RESULTS: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic. CONCLUSIONS: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort

Cooperative Effect of miR-141-3p and miR-145-5p in the Regulation of Targets in Clear Cell Renal Cell Carcinoma

Background Due to the poor prognosis for advanced renal cell carcinoma (RCC), there is an urgent need for new therapeutic targets and for prognostic markers to identify high risk tumors. MicroRNAs (miRNAs) are frequently dysregulated in tumors, play a crucial role during carcinogenesis and therefore might be promising new biomarkers. In previous studies, we identified miR-141-3p and miR-145-5p to be downregulated in clear cell RCC (ccRCC). Our objective was to investigate the functional association of these miRNAs, focusing on the cooperative regulation of new specific targets and their role in ccRCC progression. Methods The effect of miR-141-3p and miR-145-5p on cell migration was examined by overexpression in 786-O cells. New targets of both miRNAs were identified by miRWalk, validated in 786-O and ACHN cells and additionally characterized in ccRCC tissue on mRNA and protein level. Results In functional analysis, a tumor suppressive effect of miR-141-3p and miR-145-5p by decreasing migration and invasion of RCC cells could be shown. Furthermore, co-overexpression of the miRNAs seemed to result in an increased inhibition of cell migration. Both miRNAs were recognized as post-transcriptional regulators of the targets EAPP, HS6ST2, LOX, TGFB2 and VRK2. Additionally, they showed a cooperative effect again as demonstrated by a significantly increased inhibition of HS6ST2 and LOX expression after simultaneous overexpression of both miRNAs. In ccRCC tissue, LOX mRNA expression was strongly increased compared to normal tissue, allowing also to distinguish between non-metastatic and already metastasized primary tumors. Finally, in subsequent tissue microarray analysis LOX protein expression showed a prognostic relevance for the overall survival of ccRCC patients. Conclusion These results illustrate a jointly strengthening effect of the dysregulated miR-141-3p and miR-145-5p in various tumor associated processes. Focusing on the cooperative effect of miRNAs provides new opportunities for the development of therapeutic strategies and offers novel prognostic and diagnostic capabilities

Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT- based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro

Renal cell neoplasias: reversion-inducing cysteine-rich protein with Kazal motifs discriminates tumor subtypes, while extracellular matrix metalloproteinase inducer indicates prognosis

BACKGROUND: Matrix metalloproteinases can promote invasion and metastasis, which are very frequent in renal cell carcinoma even at the time of diagnosis. Knowing the reversion-inducing cysteine-rich protein with Kazal motifs (RECK) as an inhibitor of matrix metalloproteinases and the extracellular matrix metalloproteinase inducer (EMMPRIN) protein as inducer, we aimed to determine their expression, localization and possible antagonistic action in the pathogenesis and progression of renal cell tumors in a retrospective study. METHODS: Tumor and adjacent normal tissues of 395 nephrectomized patients were immunostained for RECK and EMMPRIN on a tissue microarray. RESULTS: RECK strongly decreased in renal cell carcinoma compared to normal counterparts (Wilcoxon signed rank test, P < 0.001), and it discriminated tumor entities showing the highest expression in oncocytomas. EMMPRIN, however, could be significantly correlated to pT stage and Fuhrman grading (Spearman’s correlation coefficient r(s) = 0.289 and r(s) = 0.382, respectively). Higher expression of EMMPRIN was associated with decreased overall survival in Kaplan-Meier analysis (P < 0.001), and the EMMPRIN level could independently predict survival for cases without metastasis and involvement of lymph nodes. Decreased RECK expression was confirmed by Western blotting in tissue of eight normal/tumor matches of patients after radical nephrectomy, whereas the EMMPRIN pattern appeared to be heterogeneous. CONCLUSIONS: We propose RECK down regulation in renal cell carcinoma to be an early event that facilitates tumor formation and progression. EMMPRIN, however, as a prognostic tumor marker, increases only when aggressiveness is proceeding and could add an additional step to invasive properties of renal cell carcinoma

Prognostic Role of Survivin and Macrophage Infiltration Quantified on Protein and mRNA Level in Molecular Subtypes Determined by RT-qPCR of KRT5, KRT20, and ERBB2 in Muscle-Invasive Bladder Cancer Treated by Adjuvant Chemotherapy

Objectives: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/BIRC5 and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS). Materials and Methods: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. BIRC5, KRT5, KRT20, ERBB2, and CD68 mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence. Results: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy (p = 0.0138 and p = 0.001, respectively). These results were validated by the quantitation of BIRC5 mRNA by PCR (p = 0.0004 and p = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with BIRC5 mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/BIRC5 levels based on molecular subtype being assessed by KRT20 expression improved the predictive value, with tumors having low survivin/BIRC5 and KRT20 mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, p = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis (p = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or ERBB2-positive tumors was associated with worse DSS. Conclusions: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor–biological axes of high prognostic relevance, which warrant further investigation and validation.</p

The kallikrein-related peptidases 14 and 15 as well as the endogenous metalloproteinase inhibitor RECK as biomarkers for urological tumors and functional relevance of the RECK protein in prostate carcinoma cells

Der Schwerpunkt unserer Forschung in der Klinik für Urologie der Charité – Universitätsmedizin Berlin liegt auf dem Gebiet der Biomarker für Tumoren der Prostata, der Harnblase und der Niere. Aufgrund langjähriger Erfahrungen der Forschungsabteilung im Bereich der Kallikrein-ähnlichen Peptidasen (KLKs, speziell beim Prostatakarzinom) und der Matrix-Metalloproteinasen (MMPs) und ihren Inhibitoren waren die neuesten Vertreter der KLK-Familie und der endogenen MMP-Inhibitoren Gegenstand der Untersuchungen in der hier vorliegenden Habilitationsschrift. Alle drei Zielmoleküle, KLK14, KLK15 und RECK zeigten ihr Potenzial als unabhängige Prognosemarker für das Prostatakarzinom, die KLKs dabei mit positiver und RECK mit negativer Assoziation für ein biochemisches Rezidiv. Beim Harnblasenkarzinom war die verminderte RECK-Expression mit der steigenden Invasivität der Tumoren assoziiert. In Nierenzelltumoren zeigte sich ein anderes Bild, hier konnte RECK auf diagnostischer Ebene sehr gut zwischen Karzinom (mit geringer RECK- Expression) und umliegendem Normalgewebe unterscheiden und die Tumorentitäten differenzieren. Die Biomarker-Arbeiten zu RECK wiesen stets auf den tumorsuppressiven Charakter des Markers hin, der dann in der funktionellen Studie an Prostatakarzinomzellen erstmals für urologische Tumorzellen nachgewiesen wurde. RECK-Überexpression konnte die Invasivität der Zellen drastisch verringern. Die vorgestellten Studien liefern einen wesentlichen Beitrag zur urologischen Tumorforschung, in der geeignete Biomarker sowohl im diagnostischen, vor allem aber im prognostischen Bereich dringend benötigt werden, um betroffene Patienten realistischer und individueller behandeln zu können.The focus of our research in the Department of Urology at the Charité – Universitätsmedizin Berlin is on biomarkers for tumors of the prostate, of the bladder, and of the kidney. Based on long-time experience of our research unit in the fields of kallikrein-related peptidases (KLKs, especially for prostate carcinoma) and matrix metalloproteinases (MMPs) and their inhibitors, recent members of the KLK family and of endogenous MMP inhibitors were analyzed in this work. All three targets, KLK14, KLK15, and reversion-inducing cysteine- rich protein with Kazal motifs (RECK) were ranged as independent prognostic markers for prostate carcinoma, with KLKs positively and RECK negatively associated to biochemical recurrence. For urothelial carcinoma decreased RECK expression was associated with increased invasiveness of the tumors. In renal carcinoma, however, RECK displayed another feature. Here, it was very well able to differentiate between carcinoma (low RECK expression) and adjacent normal tissue as well as to distinguish tumor entities. The studies on RECK as a biomarker always emphasized its tumorsuppressive character which was – for the first time for urological tumor cells - proven in the functional work on prostate carcinoma cells. RECK overexpression dramatically decreased invasiveness of the cells. The studies presented substantially contribute to urological tumor research which urgently needs suitable biomarkers in the diagnostic field, but especially also for prognosis to treat patients in a more reasonable and individualistic way

Molecular Mechanisms of p16 INK4a Mediated Anoikis Induction in Human Pancreatic Carcinoma Cells

0\. TITELBLATT UND INHALTSVERZEICHNIS 1\. EINLEITUNG 1 1.1. Pankreaskarzinome 1 1.2. Der Zellzyklusinhibitor p16INK4a 2 1.3. Anoikis 4 1.4. Die Ras-Familie 6 1.5. Fragestellung 10 2\. MATERIAL UND METHODEN 11 2.1. Material 11 2.2. Methoden 18 3\. ERGEBNISSE 33 3.1. P16-Reexpression restituiert die Anoikissensibilität in Capan-1 Zellen 33 3.2. Inhibition Anoikis-relevanter Signalwege in Capan-1 Zellen 35 3.3. K-Ras Regulation durch p16 in Capan-1 Zellen 39 3.4. K-Ras Inhibition in Capan-1 Zellen 43 3.5. K-ras Restitution in Capan-1/p16 Zellen 46 3.6. Mechanismus der K-Ras Regulation durch p16 in Capan-1 Zellen 58 3.7. Onkogenes K-Ras wird auch in anderen Zellsystemen durch p16 reguliert 62 4\. DISKUSSION 65 4.1. Anoikis-relevante Signalwege 65 4.2. Negative Regulation von K-Ras durch den Tumorsuppressor p16INK4a in Capan-1 Zellen 66 4.3. Onkogenes K-Ras vermittelt Anoikisresistenz in Capan-1 Zellen 67 4.4. Onkogenes K-Ras bestimmt den Transformationscharakter von Capan-1 Zellen 68 4.5. Der p16-bedingte Verlust der Tumorigenität von Capan-1 Zellen bleibt trotz onkogener K-Ras Aktivität bestehen 69 4.6. P16 reguliert K-Ras in Capan-1 Zellen posttranslational 70 4.7. Die Bedeutung der Negativ-Regulation von onkogenem K-Ras durch p16INK4a 72 4.8. Weiterführende Fragestellungen 73 5\. ZUSAMMENFASSUNG 75 6\. SUMMARY 76 7\. LITERATURVERZEICHNIS 77 8\. EIGENE VERÖFFENTLICHUNGEN 88 9\. ABKÜRZUNGEN 89 10\. Lebenslauf 92 11\. Danksagung 93In über 90% humaner Pankreaskarzinome sind sowohl die funktionelle Inaktivierung des Zellzyklus-Inhibitors p16INK4a (p16) als auch eine konstitutive Aktivierung von Kirsten-Ras (K-Ras) nachweisbar. Unsere Voruntersuchungen zeigten eine Restitution der Sensitivität für Anoikis durch p16-Reexpression in humanen Pankreaskarzinom-Zellen, so dass die zu Grunde liegenden molekularen Mechanismen dieser möglicherweise therapeutisch relevanten Reversion in der vorliegenden Arbeit zu untersuchen waren. Am Modell der humanen Pankreaskarzinom-Zelllinie Capan-1 konnte eine essentielle Funktion von onkogenem K-Ras für die Anoikisresistenz der Tumorzellen sowie eine Beteiligung von Caspase-8 an der p16-vermittelten Anoikis belegt werden. Capan-1 Pankreaskarzinom-Zellen exprimierten überwiegend die K-Ras Isoform, auf die auch die detektierbare Ras-Aktivität zurückgeführt werden konnte. Kultivierung der Zellen in Suspension führte zu einem ausgeprägten Anstieg der K-Ras Aktivität. Interessanterweise wiesen Capan-1 Klone mit stabiler p16-Reexpression nicht nur einen vollständigen Verlust der K-Ras Aktivität sowohl unter adhärenten als auch unter Suspensionsbedingungen, sondern auch eine ausgeprägte Reduktion des zellulären K-Ras Gehaltes im Vergleich zu Kontrollzellen auf. Die Verringerung der Ras-Expression von Capan-1 Zellen mit K-rasV12 Antisense Oligonukleotiden führte zu einer erhöhten Anoikisrate, während umgekehrt die Restitution von onkogenem K-Ras in Capan-1/p16 Zellen deren Anoikisfraktion wieder stark reduzierte. Dabei wurde die durch p16-Reexpression verringerte Fähigkeit von Capan-1 Zellen, Kolonien in Soft- Agar zu bilden, durch die stabile Expression von onkogenem K-Ras wiederhergestellt. Der Verlust der Tumorigenität von Capan-1/p16 Zellen in Nacktmäusen blieb jedoch trotz K-Ras Substitution bestehen. Die p16-bedingte K-Ras Regulation zeigte sich statt auf transkriptioneller Ebene in verringerter Proteinstabilität der Ras-Isoform, wahrscheinlich durch eine direkte Assoziation mit p16 und infolgedessen beschleunigten Abbau. Eine Regulation von K-Ras durch p16 in einer weiteren Pankreaskarzinom- und einer Kolonkarzinom-Zelllinie lässt dahinter ein generelles Prinzip vermuten. Die bisherigen Beobachtungen belegen erstmalig eine funktionell relevante Regulation des Onkoproteins K-Ras durch den Tumorsuppressor p16 und charakterisieren die K-Ras Inhibition als notwendiges Ereignis im Kontext der p16 vermittelten Anoikis. Diese neue, funktionelle Interaktion der beiden häufigsten genetischen Alterationen im humanen Pankreaskarzinom bestimmt wahrscheinlich durch das zentrale tumorbiologische Phänomen der Anoikisresistenz den typischen aggressiven Krankheitsverlauf.Over 90% of pancreatic carcinomas exhibit functional inactivation of the cell cycle inhibitor p16INK4a (p16) as well as constitutive activation of Kirsten- Ras (K-Ras). Our previous studies showed a restitution of sensitivity to anoikis by reexpression of p16 in human pancreatic cancer cells so that the basic molecular mechanisms of this possibly therapeutic relevant reversion were to be elucidated in the present thesis work. Exemplary in the human pancreatic carcinoma cell line Capan-1 an essential function of oncogenic K-Ras for anoikis resistance and a participation of Caspase-8 in p16 mediated anoikis was given evidence. Capan-1 pancreatic carcinoma cells mainly express the K-Ras isoform to which all detected Ras activity could be attributed. Cultivating cells in suspension led to a marked increase in K-Ras activity. Interestingly, Capan-1 clones with stable reexpression of p16 exhibited not only a complete loss of K-Ras activity under adhesive as well as suspension conditions, but also a pronounced decrease in the cellular K-Ras content as compared with control cells. Reduction of Ras expression in Capan-1 cells with K-rasV12 antisense oligonucleotides led to an increased rate of anoikis, whilst vice-versa restitution of oncogenic K-ras in Capan-1/p16 cells highly reduced their anoikis fraction. Besides, the ability of Capan-1 cells to form colonies in soft agar, reduced by p16 reexpression, was restored by stable expression of oncogenic K-ras. The Capan-1/p16 acquired loss of tumorigenicity in nude mice, however, remained in spite of K-Ras substitution. In place of the transcriptional level, p16 mediated regulation of K-Ras expression was disclosed in lowered protein stability of the Ras isoform, probably by direct association with p16 and consequently accelerated degradation. The regulation of K-Ras by p16 in a second pancreatic carcinoma cell line and a colon carcinoma cell line points to an underlying general principle. These observations document a functional relevant regulation of the oncoprotein K-Ras by the tumorsuppressor p16 for the first time and characterize the inhibition of K-Ras as an essential event in the context of p16 mediated anoikis. This new, functional interaction of the most prominent genetic alterations in human pancreatic carcinoma probably determines the typical aggressive progress of the disease by the central tumorbiological phenomenon of anoikis resistance

Diagnostic and Prognostic Potential of MicroRNA Maturation Regulators Drosha, AGO1 and AGO2 in Urothelial Carcinomas of the Bladder

Bladder cancer still requires improvements in diagnosis and prognosis, because many of the cases will recur and/or metastasize with bad outcomes. Despite ongoing research on bladder biomarkers, the clinicopathological impact and diagnostic function of miRNA maturation regulators Drosha and Argonaute proteins AGO1 and AGO2 in urothelial bladder carcinoma remain unclear. Therefore, we conducted immunohistochemical investigations of a tissue microarray composed of 112 urothelial bladder carcinomas from therapy-na&iuml;ve patients who underwent radical cystectomy or transurethral resection and compared the staining signal with adjacent normal bladder tissue. The correlations of protein expression of Drosha, AGO1 and AGO2 with sex, age, tumor stage, histological grading and overall survival were evaluated in order to identify their diagnostic and prognostic potential in urothelial cancer. Our results show an upregulation of AGO1, AGO2 and Drosha in non-muscle-invasive bladder carcinomas, while there was increased protein expression of only AGO2 in muscle-invasive bladder carcinomas. Moreover, we were able to differentiate between non-muscle-invasive and muscle-invasive bladder carcinoma according to AGO1 and Drosha expression. Finally, despite Drosha being a discriminating factor that can predict the probability of overall survival in the Kaplan&ndash;Meier analysis, AGO1 turned out to be independent of all clinicopathological parameters according to Cox regression. In conclusion, we assumed that the miRNA processing factors have clinical relevance as potential diagnostic and prognostic tools for bladder cancer