37 research outputs found
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 ZielmolekĂĽle, KLK14, KLK15 und
RECK zeigten ihr Potenzial als unabhängige Prognosemarker für das
Prostatakarzinom, die KLKs dabei mit positiver und RECK mit negativer
Assoziation fĂĽr 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 fĂĽr 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. WeiterfĂĽhrende Fragestellungen 73
5\. ZUSAMMENFASSUNG 75
6\. SUMMARY 76
7\. LITERATURVERZEICHNIS 77
8\. EIGENE VERĂ–FFENTLICHUNGEN 88
9\. ABKĂśRZUNGEN 89
10\. Lebenslauf 92
11\. Danksagung 93In ĂĽber 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 fĂĽr die Anoikisresistenz der Tumorzellen sowie
eine Beteiligung von Caspase-8 an der p16-vermittelten Anoikis belegt werden.
Capan-1 Pankreaskarzinom-Zellen exprimierten ĂĽberwiegend 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-naïve 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–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