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

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

cAMP and the Fibrous Sheath Protein CABYR (Ca2+-Binding Tyrosine-Phosphorylation-Regulated Protein) Is Required for 4D Sperm Movement

A new life starts with successful fertilization whereby one sperm from a pool of millions fertilizes the oocyte. Sperm motility is one key factor for this selection process, which depends on a coordinated flagellar movement. The flagellar beat cycle is regulated by Ca2+ entry via CatSper, cAMP, Mg2+, ADP and ATP. This study characterizes the effects of these parameters for 4D sperm motility, especially for flagellar movement and the conserved clockwise (CW) path chirality of murine sperm. Therefore, we use detergent-extracted mouse sperm and digital holographic microscopy (DHM) to show that a balanced ratio of ATP to Mg2+ in addition with 18 &micro;M cAMP and 1 mM ADP is necessary for controlled flagellar movement, induction of rolling along the long axis and CW path chirality. Rolling along the sperm&rsquo;s long axis, a proposed mechanism for sperm selection, is absent in sea urchin sperm, lacking flagellar fibrous sheath (FS) and outer-dense fibers (ODFs). In sperm lacking CABYR, a Ca2+-binding tyrosine-phosphorylation regulated protein located in the FS, the swim path chirality is preserved. We conclude that specific concentrations of ATP, ADP, cAMP and Mg2+ as well as a functional CABYR play an important role for sperm motility especially for path chirality

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

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

Response of CCND1 3’UTR and protein expression towards miR-17 family.

<p>(A) The CCND1 3’UTR was cloned behind the luciferase reporter gene of the pMIR vector and the potential binding site for the indicated miRNAs in the 3’UTR was additionally mutated by site directed mutagenesis (CCND1 mut). The reporter gene construct was expressed with the miRNA expression construct or with the empty pSG5 vector as control in the indicated combinations. Results represent the mean of at least four independent experiments performed in duplicates. The dashed line represents the luciferase activity of the empty luciferase reporter plasmid with the empty pSG5 vector which was set to 100% (***, p<0.001). (B) LNCaP cells were transfected either with control vector or miRNA expression vectors. 48 hours post-transfection the protein expression of CCND1 was determined by Western blot using ß-actin as loading control. The densitometrical quantification of Western Blots represents the relative downregulation of CCND1 expression as determined in three independent experiments in relation to the corresponding ß-actin band as loading control.</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

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

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

Deregulated genes in the KEGG pathway “cell cycle” in LNCaP cells after NETD.

<p>The pathway enrichment result for “cell cycle” (hsa04110) shows repressed genes marked with red asterisks and induced genes marked with blue asterisks.</p