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(1) Background: C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) are promising theranostic targets. However, it is unclear whether CXCR4 and FAP positivity mark distinct microenvironments, especially in solid tumors. (2) Methods: Using Random Forest (RF) analysis, we searched for entity-independent mRNA and microRNA signatures related to CXCR4 and FAP overexpression in our pan-cancer cohort from The Cancer Genome Atlas (TCGA) database — representing n = 9242 specimens from 29 tumor entities. CXCR4- and FAP-positive samples were assessed via StringDB cluster analysis, EnrichR, Metascape, and Gene Set Enrichment Analysis (GSEA). Findings were validated via correlation analyses in n = 1541 tumor samples. TIMER2.0 analyzed the association of CXCR4 / FAP expression and infiltration levels of immune-related cells. (3) Results: We identified entity-independent CXCR4 and FAP gene signatures representative for the majority of solid cancers. While CXCR4 positivity marked an immune-related microenvironment, FAP overexpression highlighted an angiogenesis-associated niche. TIMER2.0 analysis confirmed characteristic infiltration levels of CD8+ cells for CXCR4-positive tumors and endothelial cells for FAP-positive tumors. (4) Conclusions: CXCR4- and FAP-directed PET imaging could provide a non-invasive decision aid for entity-agnostic treatment of microenvironment in solid malignancies. Moreover, this machine learning workflow can easily be transferred towards other theranostic targets.
Gene expression in eukaryotic cells is regulated by the combinatorial action of numerous gene-regulatory factors, among which microRNAs (miRNAs) play a fundamental role at the post-transcriptional level. miRNAs are single-stranded, small non-coding RNA molecules that emerge in a cascade-like fashion via the generation of primary and precursor miRNAs. Mature miRNAs become functional when incorporated into the RNA induced silencing complex (RISC). miRNAs guide RISCs to target mRNAs in a sequence-specific fashion. To this end, base-pairs are usually formed between the miRNA seed region, spanning nucleotide positions 2 to 8 (from the 5' end) and the 3'UTR of the target mRNA. Once miRNA-mRNA interaction is established, RISC represses translation and occasionally induces direct or indirect target mRNA degradation. Interestingly, miRNAs are expressed not only in every multicellular organism but are also encoded by several viruses, predominately by herpesviruses. By controlling both, cellular as well as viral mRNA transcripts, virus-encoded miRNAs confer many beneficial effects on viral growth and persistence. Murine cytomegalovirus (MCMV) is a ß-herpesvirus and so far, 29 mature MCMV-encoded miRNAs have been identified during lytic infection. Computational analysis of previously conducted photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking immunoprecipitation (PAR-iCLIP) experiments identified a read cluster within the 3' untranslated region (3'UTR) of the immediate early 3 (IE3) transcript in MCMV. Based on miRNA target predictions, two highly abundant MCMV miRNAs, namely miR-m01-2-3p and miR-M23-2-3p were found to potentially bind to two closely positioned target sites within the IE3 PAR-iCLIP peak. To confirm this hypothesis, we performed luciferase assays and showed that activity values of a luciferase fused with the 3'UTR of IE3 were downregulated in the presence of miR-m01- 2 and miR-M23-2. In a second step, we investigated the effect of pre-expression of miR-m01-2 and miR-M23-2 on the induction of virus replication. After optimizing the transfection procedure by comparing different reagents and conditions, plaque formation was monitored. We could demonstrate that the replication cycle of the wild-type but not of our MCMV mutant that harbored point mutations in both miRNA binding sites within the IE3-3'UTR, was significantly delayed in the presence of miR-m01-2 and miR-M23-2. This confirmed that miR-m01-2 and miR-M23-2 functionally target the major transcription factor IE3 which acts as an indispensable regulator of viral gene expression during MCMV lytic infection. Repression of the major immediate early genes by viral miRNAs is a conserved feature of cytomegaloviruses. The functional role of this type of regulation can now be studied in the MCMV mouse model.
MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing's syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing's disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes.
Das Mammakarzinom ist weltweit die häufigste krebsbedingte Todesursache bei Frauen. Fortschritte in der Therapie ermöglichen zwar eine Verlängerung der Lebens- dauer, jedoch kommt es dadurch vermehrt zur Bildung von Metastasen im zentralen Nervensystem (ZNS). Die Diagnostik und Behandlung von ZNS-Metastasen sind be- grenzt und die Lebensqualität sowie Lebensdauer der Betroffenen nimmt bei zerebraler Metastasierung rapide ab. Ziel aktueller Forschungsprojekte ist daher, Biomarker zu identifizieren, die Hinweise auf eine Brustkrebserkrankung oder Metastasierung liefern. So soll eine kostengünstige, risikoarme und minimalinvasive Methode etabliert werden, die zuverlässige Daten über die Prognose und dementsprechende Therapien erbringt. Diese Arbeit hatte daher die Absicht, mithilfe von qPCR Expressionsprofile von miRNAs aus Serumproben von Brustkrebspatientinnen zu erstellen und deren Funktion als prog- nostische Biomarker für eine Metastasierung ins ZNS zu erweisen. Anhand von Metas- tasierung und Rezeptorstatus wurden die Proben in Untergruppen eingeteilt und statis- tisch mit einer gesunden Kontrollgruppe verglichen.
Insgesamt zeigte sich bei 26 miRNAs eine signifikante Dysregulation der Expression bei mindestens einer der Untergruppen. Insbesondere bei ZNS-Metastasen war das Expres- sionsmuster bei miRNA-122-5p, miRNA-296-5p, miRNA-490-3p und miRNA-576-3p sig- nifikant erhöht, während die Expression von miRNA-130a-3p, miRNA-148b-3p und miRNA-326 signifikant reduziert war. Basierend auf den Übereinstimmungen unserer Er- gebnisse mit den Daten bisheriger Forschungsprojekten wiesen vier miRNAs eine po- tenzielle Funktion als Biomarker für Metastasen auf: miRNA-122-5p, miRNA-490-3p und miRNA-130a-3p, miRNA-326. Bei ZNS-Metastasen zeigten besonders miRNA-122-5p und miRNA-490-3p statistisch relevante Veränderungen.
Um den Einfluss von miRNAs auf den gesamten Körper darzustellen, wurde mithilfe ver- schiedener Datenbanken nach entsprechenden Zielgenen und Signalwegen für die 26 identifizierten miRNAs recherchiert. Neben dem Einfluss auf Stoffwechselwege und Er- krankungen, zeigte sich bei acht Targets ein Zusammenhang mit der Entstehung von Krebs.
Ergänzend zur Identifikation von miRNA-Expressionsprofilen wurden Zellkulturversuche mit zerebralen Endothel- (cerebEND) und Brustkrebszellen (4T1) durchgeführt. Verwendet wurden zwei cerebEND- und eine 4T1-Zellreihe von Mäusen, von denen eine ce- rebEND-Kultur zuvor in der Arbeitsgruppe Burek mit einem miRNA-210-Vektor trans- fiziert wurde.
Studien belegen den Einfluss von miRNA-210 auf den mitochondrialen Stoffwechsel, Angiogenese, Reaktionen auf DNA-Schäden, Apoptose und Zellüberleben sowie auf die Proteine BRCA1, PARP1 und E-Cadherin und schreiben ihr damit eine Funktion in der Krebsentstehung und Metastasierung zu.
Zur Bestimmung der Proliferation und Aktivität der transfizierten cerebEND-210-Zellen im Verhältnis zur unbehandelten Kontrolle, wurden BrdU-Proliferationsassays und MTT- Assays mit verschiedenen Zellzahlen durchgeführt. Bei der Untersuchung der Prolifera- tion zeigte sich in beiden Versuchen eine erhöhte Aktivität der cerebEND-210-Zellen, da miRNA-210 vermutlich auch hier das Zellüberleben gesichert hat. Zudem wurde die An- heftung der Brustkrebszellen an den zerebralen Endothelzellen im Adhäsionsversuchs überprüft. Hierbei wurde eine Abnahme der Adhäsion der cerebEND-210-Zellen beo- bachtet. Vermutet wird eine Veränderung des Phänotyps der Rezeptorbindungen der cerebEND-210-Zellen.
Die Ergebnisse der Zellkulturversuche dienen als Grundlage für weitere Experimente.
Non-coding RNAs (ncRNAs) are a type of genetic material that do not encode proteins but regulate the gene expression at an epigenetic level, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). The role played by ncRNAs in many physiological and pathological processes has gained attention during the last few decades, as they might be useful in the diagnosis, treatment and management of several human disorders, including endocrine and oncological diseases. Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine cancer, still characterized by high mortality and morbidity due to both endocrine and oncological complications. Despite the rarity of this disease, recently, the role of ncRNA has been quite extensively evaluated in ACC. In order to better explore the role of the ncRNA in human ACC, this review summarizes the current knowledge on ncRNA dysregulation in ACC and its potential role in the diagnosis, treatment, and management of this tumor.
Patients affected by gastroenteropancreatic–neuroendocrine tumors (GEP–NETs) have an increased risk of developing osteopenia and osteoporosis, as several factors impact on bone metabolism in these patients. In fact, besides the direct effect of bone metastasis, bone health can be affected by hormone hypersecretion (including serotonin, cortisol, and parathyroid hormone-related protein), specific microRNAs, nutritional status (which in turn could be affected by medical and surgical treatments), and vitamin D deficiency. In patients with multiple endocrine neoplasia type 1 (MEN1), a hereditary syndrome associated with NET occurrence, bone damage may carry other consequences. Osteoporosis may negatively impact on the quality of life of these patients and can increment the cost of medical care since these patients usually live with their disease for a long time. However, recommendations suggesting screening to assess bone health in GEP–NET patients are missing. The aim of this review is to critically analyze evidence on the mechanisms that could have a potential impact on bone health in patients affected by GEP–NET, focusing on vitamin D and its role in GEP–NET, as well as on factors associated with MEN1 that could have an impact on bone homeostasis.
Merkel cell carcinoma (MCC) is a deadly skin cancer, and about 80% of its cases have been shown to harbor integrated Merkel polyomavirus in the tumor cell genome. Viral oncoproteins expressed in the tumor cells are considered as the oncogenic factors of these virus-positive Merkel cell carcinoma (VP-MCC). In contrast, the molecular pathogenesis of virus-negative MCC (VN-MCC) is less well understood. Using gene expression analysis of MCC cell lines, we found histone methyltransferase PRDM8 to be elevated in VN-MCC. This finding was confirmed by immunohistochemical analysis of MCC tumors, revealing that increased PRDM8 expression in VN-MCC is also associated with increased H3K9 methylation. CRISPR-mediated silencing of PRDM8 in MCC cells further supported the histone methylating role of this protein in VN-MCC. We also identified miR-20a-5p as a negative regulator of PRDM8. Taken together, our findings provide insights into the role of PRDM8 as a histone methyltransferase in VN-MCC tumorigenesis.
Wilms tumor (WT) or nephroblastoma is the most common kidney tumor in childhood. Several genetic alterations have been identified in WT over the past years. However, a clear-cut underlying genetic defect has remained elusive. Growing evidence suggests that miRNA processing genes play a major role in the formation of pediatric tumors, including WT.
We and others have identified the microprocessor genes DROSHA and DGCR8 as key players in Wilms tumorigenesis. Exome sequence analysis of a cohort of blastemal-type WTs revealed the recurrent hotspot mutations DROSHA E1147K and DGCR8 E518K mapping to regions important for catalyic activity and RNA-binding. These alterations were expected to affect processing of miRNA precursors, ultimately leading to altered miRNA expression. Indeed, mutated tumor samples were characterized by distinct miRNA patterns. Notably, these mutations have been observed almost exclusively in WT, suggesting that they play a specific role in WT formation.
The aim of the present work was to first examine the mutation frequency of DROSHA E1147K and DGCR8 E518K in a larger cohort of WTs, and to further characterize these microprocessor gene mutations as potential oncogenic drivers for WT formation.
Screening of additional 700 WT samples by allele-specific PCR revealed a high frequency of DROSHA E1147K and DGCR8 E518K mutations, with the highest incidence found in tumors of high-risk histology. DROSHA E1147K was heterozygously expressed in all cases, which strongly implies a dominant negative effect. In contrast, DGCR8 E518K exclusively exhibited homozygous expression, suggestive for the mutation to act recessive.
To functionally assess the mutations of the microprocessor complex in vitro, I generated stable HEK293T cell lines with inducible overexpression of DROSHA E1147K, and stable mouse embryonic stem cell (mESC) lines with inducible overexpression of DGCR8 E518K. To mimic the homozygous expression observed in WT, DGCR8 mESC lines were generated on a DGCR8 knockout background. Inducible overexpression of wild-type or mutant DROSHA in HEK293T cells showed that DROSHA E1147K leads to a global downregulation of miRNA expression. It has previously been shown that the knockout of DGCR8 in mESCs also results in a significant downregulation of canonical miRNAs. Inducible overexpression of wild type DGCR8 rescued this processing defect. DGCR8 E518K on the other hand, only led to a partial rescue. Differentially expressed miRNAs comprised members of the ESC cell cycle (ESCC) and let-7 miRNA families whose antagonism is known to play a pivotal role in the regulation of stem cell properties. Along with altered miRNA expression, DGCR8-E518K mESCs exhibited alterations in target gene expression potentially affecting various biological processes.
We could observe decreased proliferation rates, most likely due to reduced cell viability. DGCR8-E518K seemed to be able to overcome the block of G1-S transition and to rescue the cell cycle defect in DGCR8-KO mESCs, albeit not to the full extent like DGCR8-wild-type. Moreover, DGCR8-E518K appeared to be unable to completely block epithelial-to-mesenchymal transition (EMT). Embryoid bodies (EBs) with the E518K mutation, however, were still able to silence the self-renewal program rescuing the differentiation defect in DGCR8-KO mESCs.
Taken together, I could show that DROSHA E1147K and DGCR8 E518K are frequent events in WT with the highest incidence in high-risk tumor entities. Either mutation led to altered miRNA expression in vitro confirming our previous findings in tumor samples. While the DROSHA E1147K mutation resulted in a global downregulation of canonical miRNAs, DGCR8 E518K was able to retain significant activity of the microprocessor complex, suggesting that partial reduction of activity or altered specificity may be critical in Wilms tumorigenesis.
Despite the significant differences found in the miRNA and mRNA profiles of DGCR8 E518K and DGCR8-wild-type mESCs, functional analysis showed that DGCR8 E518K could mostly restore important cellular functions in the knockout and only slightly differed from the wild-type situation. Further studies in a rather physiological environment, such as in a WT blastemal model system, may additionally help to better assess the subtle differences between DGCR8 E518K and DGCR8 wild-type observed in our mESC lines. Together with our findings, these model systems may thus contribute to better understand the role of these microprocessor mutations in the formation of WT.
Context
Cushing’s syndrome (CS) is a rare disease of endogenous hypercortisolism associated with high morbidity and mortality. Diagnosis and classification of CS is still challenging.
Objective
Circulating microRNAs (miRNAs) are minimally invasive diagnostic markers. Our aim was to characterize the circulating miRNA profiles of CS patients and to identify distinct profiles between the two major CS subtypes.
Methods
We included three groups of patients from the German Cushing’s registry: ACTH-independent CS (Cortisol-Producing-Adenoma; CPA), ACTH-dependent pituitary CS (Cushing’s Disease; CD), and patients in whom CS had been ruled out (controls). Profiling of miRNAs was performed by next-generation-sequencing (NGS) in serum samples of 15 CS patients (each before and after curative surgery) and 10 controls. Significant miRNAs were first validated by qPCR in the discovery cohort and then in an independent validation cohort of 20 CS patients and 11 controls.
Results
NGS identified 411 circulating miRNAs. Differential expression of 14 miRNAs were found in the pre- and postoperative groups. qPCR in the discovery cohort validated 5 of the significant miRNAs from the preoperative group analyses. Only, miR-182-5p was found to be significantly upregulated in the CD group of the validation cohort. Comparing all CS samples as a group with the controls did not reveal any significant differences in expression.
Outcome
In conclusion, our study identified miR-182-5p as a possible biomarker for CD, which has to be validated in a prospective cohort. Furthermore, our results suggest that presence or absence of ACTH might be at least as relevant for miRNA expression as hypercortisolism itself.
microRNA-Genexpressionsprofile in Blut-, Haut- und Nervenproben von Patienten mit Polyneuropathien
(2020)
Die Polyneuropathie (PNP) ist die häufigste Störung des peripheren Nervensystems bei Erwachsenen. Die Suche nach der Ursache bleibt in vielen Fällen erfolglos, ist aber unverzichtbar, da die Therapiewahl von der Ätiologie der Erkrankung abhängt. Geeignete Biomarker könnten die Differentialdiagnose unter Umständen erleichtern. microRNAs (miRNAs) sind in dieser Hinsicht vielversprechend, da in vielen Studien bei Nervende- und regenerationsprozessen sowie in neuropathischen Schmerzmodellen eine Dysregulation beschrieben wurde.
In dieser Studie wurde die Expression zweier miRNAs, miR-103a und miR-let-7d, sowie eines Zielmoleküls der miR-103a, des Kalziumkanals Cav1,2, in einer großen Kohorte von PNP-Patienten unterschiedlicher Ätiologie in Blut, Haut- und Nervenbiopsien untersucht. Insgesamt wurden 116 Patienten und 22 Kontroll-probanden in die Studie eingeschlossen. Nach der Isolation von RNA aus weißen Blutzellen (WBC), Haut- und Nervenbiopsien folgte die Expressionsbestimmung mittels qRT-PCR.
Während sich jeweils Unterschiede zwischen PNP-Patienten und Kontrollen und zwischen Patienten mit entzündlicher und solchen mit nicht-entzündlicher PNP zeigten, wurden keine Unterschiede in der Expression zwischen den ätiologischen Subgruppen oder zwischen Patienten mit schmerzhafter und schmerzloser PNP festgestellt. In den Nervenbiopsien der Patientenkohorte ergab sich eine inverse Korrelation der miR-103a und ihrem Zielgen Cacna1c, die darauf hinweisen könnte, dass Cacna1c von der miR-103a negativ reguliert wird.
Da in unserer Patientenkohorte keine Unterschiede zwischen den PNP-Subgruppen auftraten, scheint der Einsatz der miR-103a und miR-let-7d als diagnostische Biomarker zur ätiologischen Einordnung einer PNP nicht gerechtfertigt. Dennoch deuten unsere Ergebnisse auf eine mögliche Rolle der untersuchten miRNAs bei Entstehung und Verlauf von PNP hin. Für ein tieferes pathophysiologisches Verständnis der miRNAs vor allem bei entzündlichen Neuropathien, könnte die Untersuchung von weiteren miRNAs und Zielgenen Aufschluss geben.