Epiregulin expression and secretion is increased in castration-resistant prostate cancer.

INTRODUCTION In prostate cancer, long-term treatment directed against androgens often leads to the development of metastatic castration-resistant prostate cancer, which is more aggressive and not curatively treatable. Androgen deprivation results in elevated epiregulin expression in LNCaP cells which is a ligand of EGFR. This study aims to reveal the expression and regulation of epiregulin in different prostate cancer stages enabling a more specific molecular characterization of different prostate carcinoma types. METHODS Five different prostate carcinoma cell lines were used to characterize the epiregulin expression on the RNA and protein levels. Epiregulin expression and its correlation with different patient conditions were further analyzed using clinical prostate cancer tissue samples. Additionally, the regulation of epiregulin biosynthesis was examined at transcriptional, post-transcriptional and release level. RESULTS An increased epiregulin secretion is detected in castration-resistant prostate cancer cell lines and prostate cancer tissue samples indicating a correlation of epiregulin expression with tumor recurrence, metastasis and increased grading. Analysis regarding the activity of different transcription factors suggests the involvement of SMAD2/3 in the regulation of epiregulin expression. In addition, miR-19a, -19b, and -20b are involved in post-transcriptional epiregulin regulation. The release of mature epiregulin occurs via proteolytic cleavage by ADAM17, MMP2, and MMP9 which are increased in castration-resistant prostate cancer cells. DISCUSSION The results demonstrate epiregulin regulation by different mechanism and suggest a potential role as a diagnostic tool to detect molecular alterations in prostate cancer progression. Additionally, although EGFR inhibitors false in prostate cancer, epiregulin could be a therapeutic target for patients with castration-resistant prostate cancer

Stromal-epithelial interaction induces GALNT14 in prostate carcinoma cells

IntroductionCell-cell communication is an important process in healthy tissue but also gains enhanced attention regarding pathological tissue. To date, the tumor microenvironment is gradually brought into focus when studying tumorigenesis. In the prostate gland, stromal and epithelial cells greatly interact to maintain homeostasis or tissue integrity. This study focuses on an indirect communication via soluble factors.MethodsTo investigate the cell-cell interaction via soluble factors, the prostate carcinoma cell line LNCaP and the stromal primary cells p21 were co-cultured without direct contact and RNA was isolated at defined time points. Differences in gene expression were finally analyzed by RNA sequencing.ResultsRNA sequencing revealed a time-depending differential expression profile. Selected factors were subsequently characterized at molecular level and analyzed in human prostate tissue of different developmental stages as well as pathology. GALNT14 was one of the highest induced co-culture-specific genes in LNCaP cells. Detection in healthy tissue and BPH revealed an age-dependent decrease in GALNT14 expression. Moreover, in prostate carcinoma, GALNT14 expression heavily varied independent of the Gleason score.ConclusionOverall, this work provides a basis for further studies related to paracrine stromal-epithelial interaction in prostate carcinoma and highlights the importance of GALNT14

Changes in gene and miRNA expression after neuroendocrine trans-differentiation of prostate cancer cell line LNCaP

Das Prostatakarzinom ist bei Männern die am häufigsten diagnostizierte Krebserkrankung und wird in späten Stadien mit einer Androgen-deprivierenden Therapie (ADT) behandelt. Eine dauerhafte ADT führt jedoch zu einer neuroendokrinen trans-Differenzierung (NETD) von Prostatakarzinomzellen, welche in vielen Fällen in einem aggressiveren Tumorrezidiv resultiert. Obwohl die Ursache und auch einige wichtige Signalwege der NETD bereits bekannt sind, gibt es aktuell keine Behandlungs-möglichkeiten. Daher müssen weitere Gene identifiziert werden, die während der NETD eine wichtige Rolle spielen, um neue therapeutische Ansätze zu liefern. Des Weiteren wurde bislang die Rolle von miRNAs in der NETD nicht genauer untersucht. Zunächst wurde ein in vitro Modell der NETD von LNCaP Zellen etabliert, um anschließend ein mRNA und miRNA Expressionsprofil von neuroendokrin (NE)-ähnlichen LNCaP Zellen zu erstellen. Dabei wurde festgestellt, dass nach NETD von LNCaP Zellen sowohl die mRNA als auch die miRNA Expression stark verändert ist. Im Anschluss daran wurden putative Zielgene für deregulierte miRNAs vorhergesagt und die Bindung der miRNA an das Zielgen verifiziert. Dabei konnten AKT3, CCND1 und CREB5 als Zielgene der miR-17 Familie identifiziert werden. Durch die Reduktion der miR-17 Familie nach NETD könnte die induzierte Expression der drei Gene, aufgrund ihrer anti-apoptotischen Wirkung, die Apoptose-Resistenz und Androgen-Unabhängigkeit der NE-ähnlichen LNCaP Zellen erklären. Des Weiteren wurde CPEB1 als Zielgen der reprimierten miR-19a, -301a und-425 identifiziert. Das induzierte Protein CPEB1 leitet neben der Zellseneszenz, in einigen Geweben auch die neuronale Differenzierung ein. Außerdem wurde VEGFC als Zielgen der reprimierten miR-128 identifiziert. VEGFC ist in NE-ähnlichen LNCaP Zellen ebenfalls induziert und stimuliert die Vaskularisierung des Tumors. Zum Schluss wurde der Effekt der miR-17 Familie, sowie miR-19a, -301a und -425 auf LNCaP Zellen mittels verschiedener in vitro Assays untersucht. Dabei konnte festgestellt werden, dass Mitglieder der miR-17 Familie eine tumorsuppressive Wirkung und miR-19a, -301a sowie -425 eine onkogene Wirkung auf LNCaP Zellen haben. Somit konnte in dieser Arbeit gezeigt werden, dass nach NETD neben der mRNA, auch die miRNA Expression verändert ist und diese direkte Auswirkungen auf zelluläre Eigenschaften der LNCaP Zellen besitzen.Prostate cancer (PCa) is the most commonly diagnosed cancer in men worldwide and is treated with an androgen deprivating therapy (ADT) in late stages. However, permanent ADT leads to a neuroendocrine trans-differentiation (NETD) of PCa cells, which results in a recurrence of a more aggressive tumor. Although the cause and some important signaling pathways of the NETD are already known, there are no treatment options available jet. Therefore, additional genes must be identified that play a significant role during NETD of PCa cells to provide new therapeutic approaches. Furthermore, the role of miRNAs in the NETD of PCa cells has not been further investigated. First, an in vitro model of NETD of LNCaP cells was established to subsequently generate an mRNA and miRNA expression profile of neuroendocrine (NE) -like LNCaP cells. After NETD of LNCaP cells both the mRNA and the miRNA expression is stongly altered. Subsequently, putative target genes for deregulated miRNAs were predicted and the binding of miRNA to the target gene was verified. AKT3, CCND1 and CREB5 could be identified as target genes of the miR-17 family. The reduced expression of miR-17 family after NETD of LNCaP cells induced the expression of the three genes, which could explain the apoptosis resistance and androgen independence of NE-like LNCaP cells due to their anti-apoptotic effects. Furthermore, CPEB1 was identified as a target gene of the repressed miR-19a, -301a and -425. In addition to cell senescence, the induced CPEB1 protein also induces neuronal differentiation in several tissues. Furthermore VEGFC was identified as a target gene of the repressed miR-128. VEGFC is also induced in NE-like LNCaP cells and stimulates vascularization of the tumor. Finally, the effect of miR-17 family, as well as miR-19a, -301a, and -425 on LNCaP cells was investigated by various in vitro assays. Members of miR-17 family have a tumor suppressive effect while miR-19a, -301a and -425 have an oncogenic effect on LNCaP cells. In this work it could be shown that NETD of LNCaP cells leads to changes in mRNA and miRNA expression and that these miRNAs have direct effects on cellular properties of LNCaP cells

The deregulation of miR-17/CCND1 axis during neuroendocrine transdifferentiation of LNCaP prostate cancer cells

<div><p>Prostate carcinoma contain foci of neuroendocrine transdifferentiation, resulting in an increase of androgen-independent neuroendocrine-like (NE) tumor cells, whose number significantly correlates with tumor aggressiveness and thus lower survival rate. Neuroendocrine transdifferentiation of prostate cancer cells and a potential role of miRNAs within this process are poorly understood. MicroRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression. The aim of this project was to identify new genes and miRNAs involved in neuroendocrine transdifferentiation. LNCaP prostate cancer cells were differentiated to NE-like cancer cells and microarray analyses were performed. Microarray results have been validated for the eight most deregulated mRNAs and microRNAs via qRT-PCR and analyzed with different algorithms to predict new targets for deregulated microRNAs. The induced CyclinD1 gene could be validated as new target gene for the repressed miR-17 family containing miR-17, miR-20a, miR-20b, miR-106a and miR-106b via reporter gene assays and Western Blot. Functional analysis of miR-17 family shows a high influence on cell proliferation, colony forming ability and apoptosis in LNCaP cells. Our data demonstrate wide changes in mRNA and microRNA expression during neuroendocrine transdifferentiation of LNCaP cells and confirm new mRNA-miRNA interactions with potential roles in NE-transdifferentiation of prostate carcinoma.</p></div

Quantification of CCND1 and miRNA expression in neuroendocrine transdifferentiated LNCaP cells compared to untreated LNCaP cells and predicted miRNA target sites.

<p>The expression of CCND1 and miR-17 family miRNAs (A) that were assumed to be elevated or reduced according to their signals in microarray (black bars) was assessed by qRT-PCR (grey bars). CCND1 was predicted to be elevated while miR-17, miR-20a, miR-20b, miR-106a, miR-106b and miR-93 were predicted to be reduced in NE-transdifferentiated LNCaP as compared to untreated cells (*, p<0.05). (B) A schematic representation of the predicted miRNA interaction site and the mutated seed sequences are shown. (C) The 3’UTR region of CCND1 is depicted.</p

Differentially expressed miRNAs in neuroendocrine differentiated LNCaP cells compared to untreated LNCaP cells.

<p>Differentially expressed miRNAs in neuroendocrine differentiated LNCaP cells compared to untreated LNCaP cells.</p

Classification of deregulated genes in LNCaP after NETD according to GO terms with p-value <0.05.

<p>(A) GO enrichment results for repressed genes (Top 20). (B) GO enrichment results for induced genes (Top 20).</p

Validation of differentially expressed genes in NE-differentiated LNCaP cells compared to untreated cells.

<p>The expression of eight mRNAs (A, B) and miRNAs (C, D) that were assumed to be elevated or reduced according to their signals in microarray (black bars) was assessed by qRT-PCR (grey bars). Results represent the mean from 4 independent NE-transdifferentiations performed in duplicates (*, p<0.05).</p

Neuroendocrine transdifferentiation of LNCaP cells through androgen deprivation.

<p>(A) LNCaP cells were cultivated in media with charcoal-stripped FCS (CS-FCS) or control FCS for 14 days. Transdifferentiated cells show dendrite like cell processes indicated by black arrows. (B) Successful transdifferentiation was validated through time course expression analysis of AR, PSA, NSE, NTS and TUBB3 in transdifferentiated LNCaP cells compared to untreated cells during androgen deprivation for 14 days by RT-PCR. Beta-actin and GAPDH served as endogenous controls.</p