Volker Jung

The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

Analysis of serum microRNAs (miR-26a-2*, miR-191, miR-337-3p and miR-378) as potential biomarkers in renal cell carcinoma

INTRODUCTION Emerging evidence suggest that microRNAs could serve as non-invasive biomarker for cancer patients. Our study was designed to analyze circulating serum microRNAs in patients with renal cell carcinoma (RCC). MATERIALS AND METHODS Serum RNA was isolated from patients with clear cell RCC (ccRCC) and non-malignant disease; an artificial microRNA (cel-miR-39) was spiked-in prior the isolation procedure to control isolation efficiency. The levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 in serum were determined using quantitative real-time PCR; the microRNA levels were normalized to cel-miR-39. RESULTS First, miR-26a-2*, miR-191, miR-337-3p and miR-378 were quantified in serum of each 25 patients with ccRCC and non-malignant disease. The level of miR-378 was significantly increased in ccRCC patients, and thus chosen for validation. The analysis of miR-378 in the validation cohort with 117 RCC patients and 123 control subjects did not confirm a different level of miR-378. Also, miR-378 was not correlated to pT-stage, lymph node/distant metastasis, vascular invasion and Fuhrman grade. CONCLUSIONS The analysis of circulating serum levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 is unlikely to provide helpful diagnostic/prognostic information in RCC patients.

Factor interaction analysis for chromosome 8 and DNA methylation alterations highlights innate immune response suppression and cytoskeletal changes in prostate cancer

BackgroundAlterations of chromosome 8 and hypomethylation of LINE-1 retrotransposons are common alterations in advanced prostate carcinoma. In a former study including many metastatic cases, they strongly correlated with each other. To elucidate a possible interaction between the two alterations, we investigated their relationship in less advanced prostate cancers.ResultsIn 50 primary tumor tissues, no correlation was observed between chromosome 8 alterations determined by comparative genomic hybridization and LINE-1 hypomethylation measured by Southern blot hybridization. The discrepancy towards the former study, which had been dominated by advanced stage cases, suggests that both alterations converge and interact during prostate cancer progression. Therefore, interaction analysis was performed on microarray-based expression profiles of cancers harboring both alterations, only one, or none. Application of a novel bioinformatic method identified Gene Ontology (GO) groups related to innate immunity, cytoskeletal organization and cell adhesion as common targets of both alterations. Many genes targeted by their interaction were involved in type I and II interferon signaling and several were functionally related to hereditary prostate cancer genes. In addition, the interaction appeared to influence a switch in the expression pattern of EPB41L genes encoding 4.1 cytoskeleton proteins. Real-time RT-PCR revealed GADD45A, MX1, EPB41L3/DAL1, and FBLN1 as generally downregulated in prostate cancer, whereas HOXB13 and EPB41L4B were upregulated. TLR3 was downregulated in a subset of the cases and associated with recurrence. Downregulation of EPB41L3, but not of GADD45A, was associated with promoter hypermethylation, which was detected in 79% of carcinoma samples.ConclusionAlterations of chromosome 8 and DNA hypomethylation in prostate cancer probably do not cause each other, but converge during progression. The present analysis implicates their interaction in innate immune response suppression and cytoskeletal changes during prostate cancer progression. The study thus highlights novel mechanisms in prostate cancer progression and identifies novel candidate genes for diagnostic and therapeutic purposes. In particular, TLR3 expression might be useful for prostate cancer prognosis and EPB41L3 hypermethylation for its detection.

Frequent mitotic errors in tumor cells of genetically micro-heterogeneous glioblastomas

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity as to both histomorphology and genetic changes, displaying a wide variety of numerical chromosome aberrations the most common of which are monosomy 10 and trisomy 7. Moreover, GBM in vitro are known to have variable karyotypes within a given tumor cell culture leading to rapid karyotype evolution through a high incidence of secondary numerical chromosome aberrations. The aim of our study was to investigate to what extent this mitotic instability of glioblastoma cells is also present in vivo. We assessed the spatial distribution patterns of numerical chromosome aberrations in vivo in a series of 24 GBM using two-color in situ hybridization for chromosomes 7/10, 8/17, and 12/18 on consecutive 6-µm paraffin-embedded tissue slides. The chromosome aberration patterns were compared with the histomorphology of the investigated tumor assessed from a consecutive HE-stained section, and with the in vitro karyotype of cell cultures established from the tumors. All investigated chromosomes showed mitotic instability, i.e., numerical aberrations within significant amounts of tumor cells in a scattered distribution through the tumor tissue. As to chromosomes 10 and 17, only monosomy occurred, as to chromosome 7 only trisomy/polysomy, apparently as a result of selection in favor of the respective aberration. Conversely, chromosomes 8, 12, and 18 displayed scattered patterns of monosomy as well as trisomy within a given tumor reflecting a high mitotic error rate without selective effects. The karyotypes of the tumor cell cultures showed less variability of numerical aberrations apparently due to clonal adaptation to in vitro conditions. We conclude that glioblastoma cells in vivo are characterized by an extensive tendency to mitotic errors. The resulting clonal diversity of chromosomally aberrant cells may be an important biological constituent of the well-known ability of glioblastomas to preserve viable tumor cell clones under adaptive stress in vivo, in clinical terms to rapidly recur after antitumoral therapy including radio- or chemotherapy.

Genomic and Expression Analysis of the 3q25-q26 Amplification Unit Reveals TLOC1/SEC62 as a Probable Target Gene in Prostate Cancer

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined. (Mol Cancer Res 2006;4(3):169–76)

Detection of novel amplicons in prostate cancer by comprehensive genomic profiling of prostate cancer cell lines using oligonucleotide-based arrayCGH.

Background The purpose of this study was to prove the feasibility of a longmer oligonucleotide microarray platform to profile gene copy number alterations in prostate cancer cell lines and to quickly indicate novel candidate genes, which may play a role in carcinogenesis. Methods/Results and Findings Genome-wide screening for regions of genetic gains and losses on nine prostate cancer cell lines (PC3, DU145, LNCaP, CWR22, and derived sublines) was carried out using comparative genomic hybridization on a 35,000 feature oligonucleotide microarray (arrayCGH). Compared to conventional chromosomal CGH, more deletions and small regions of gains, particularly in pericentromeric regions and regions next to the telomeres, were detected. As validation of the high-resolution of arrayCGH we further analyzed a small amplicon of 1.7 MB at 9p13.3, which was found in CWR22 and CWR22-Rv1. Increased copy number was confirmed by fluorescence in situ hybridization using the BAC clone RP11-165H19 from the amplified region comprising the two genes interleukin 11 receptor alpha (IL11-RA) and dynactin 3 (DCTN3). Using quantitative real time PCR (qPCR) we could demonstrate that IL11-RA is the gene with the highest copy number gain in the cell lines compared to DCTN3 suggesting IL11-RA to be the amplification target. Screening of 20 primary prostate carcinomas by qPCR revealed an IL11-RA copy number gain in 75% of the tumors analyzed. Gain of DCTN3 was only found in two cases together with a gain of IL11-RA. Conclusions/Significance ArrayCGH using longmer oligonucleotide microarrays is feasible for high-resolution analysis of chomosomal imbalances. Characterization of a small gained region at 9p13.3 in prostate cancer cell lines and primary prostate cancer samples by fluorescence in situ hybridization and quantitative PCR has revealed interleukin 11 receptor alpha gene as a candidate target of amplification with an amplification frequency of 75% in prostate carcinomas. Frequent amplification of IL11-RA in prostate cancer is a potential mechanism of IL11-RA overexpression in this tumor type.

Silencing of the SEC62 gene inhibits migratory and invasive potential of various tumor cells

Sec62 is part of the protein translocation apparatus in the membrane of the endoplasmic reticulum (ER). In yeast, Sec62 participates in the post‐translational translocation of proteins into the ER, but its function in mammals remains elusive. Previously we described the amplification and over‐expression of the SEC62 gene in prostate cancer cell lines and the protein has been described as a potential target gene in prostate cancer. In the current study we show that in the tumor tissue of prostate cancer patients Sec62 protein levels are elevated compared with tumor‐free tissue derived from the same patients or from prostates of control group patients and that the higher Sec62 protein content correlates with an increasing de‐differentiation of the cells. Therefore, up‐regulation of Sec62 protein content indeed is a phenomenon associated with prostate cancer progression. Analysis of a multi‐tissue tumor array showed that in addition to prostate cancer, overproduction of Sec62 is observed in various other tumors, most significantly in tumors of the lung and the thyroid. To examine the tumor‐related functions of Sec62, we silenced the SEC62 gene in the prostate cancer cell‐line PC3 as well as in a set of other tumor cell‐lines with two different siRNAs. In general, after silencing of SEC62 the cell migration and the invasive potential of the cells was blocked or at least dramatically reduced while cell viability was hardly affected. Thus, the SEC62 gene may indeed be considered as a target gene in the therapy of various tumors.

Sec62 bridges the gap from 3q amplification to molecular cell biology in non-small cell lung cancer.

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

In situ hybridization analysis of genes coding collagen IV α1 chain, laminin β1 chain, and s‐laminin in prostate tissue and prostate cancer: Increased basement membrane gene expression in high‐grade and metastatic lesions

Recent immunohistochemical data have shown that invasive prostate cancer cells are separated from the host tissue by basement membranes (BM), and express associated adhesive molecules that bind to these de novo synthesized extracellular matrices.

Collecting Duct Carcinomas Represent a Unique Tumor Entity Based on Genetic Alterations

Collecting duct carcinoma (CDC) is a rare renal neoplasm that is associated with poor prognosis due to its highly aggressive course and limited response to immuno- or chemotherapy. Histologically, CDC is defined as a subtype of renal cell carcinomas, but in some cases, it is difficult to differentiate from urothelial carcinomas (UC). Therefore the aim of this study was to determine genetic alterations of CDC in comparison to that of urothelial carcinomas of the upper urinary tract (UUT-UC) to clarify the histological origin of this rare tumor entity. Twenty-nine CDC samples were obtained from seven different German centers and compared with twenty-six urothelial carcinomas of the upper urinary tract. Comparative genomic hybridization (CGH) was used to investigate the genetic composition of patients’ tumors and allowed the detection of losses and gains of DNA copy numbers throughout the entire genome. The clinical data were correlated with CGH results. CGH analysis of CDC revealed DNA aberrations in many chromosomes. DNA losses were more frequently observed than gains, while high-level amplifications were not detected. The mean frequency of CDC chromosomal aberrations (4.9/case) was slightly lower than that in UUT-UC (5.4/case). Recurrent CDC DNA losses occurred at 8p (n=9/29), 16p (9/29), 1p (n=7/29) and 9p (n=7/29), and gains occurred in 13q (n=9/29). In contrast to CDC, the most frequently detected UUT-UC DNA aberration was a loss at 9q (n=13/26). DNA losses at 9q, 13q and 8q as well as gains at 8p showed significant variations in UUT-UC compared to CDC. There was no correlation between the patients’ clinical course and the presence or absence of these recurrent genetic alterations. CDCs are characterized by a different genetic pattern compared to UUT-UC. Regarding the published data on renal cell carcinoma, we conclude that CDC appears to be a unique entity among kidney carcinomas.