Expression of the microtubule-associated protein 2 (MAP2) as a potential independent prognostic marker in prostate cancer.

PURPOSE: Investigation of Microtubuli-associated Protein 2 (MAP2) expression and its clinical relevance in prostate cancer. MATERIAL AND METHODS: MAP2 expression was immunohistochemically analysed on radical prostatectomy specimens using whole block sections (n = 107) and tissue microarrays (TMA; n = 310). The staining intensity was evaluated for carcinoma, benign tissue and prostatic intraepithelial neoplasia. Expression data were correlated with clinicopathological parameters and biochemical recurrence-free survival. Additionally, MAP2 protein expression was quantitatively analysed in the serum of histologically confirmed prostate carcinoma patients and the control group using a commercial enzyme-linked immunosorbent assay. RESULTS: MAP2 staining was significantly stronger in neoplastic tissue than in non-neoplastic prostatic glands, both in whole block sections (p < 0.01) and in TMA sections (p < 0.05). TMA data revealed significantly stronger MAP2 staining in high-grade tumors. Survival analysis showed a significant correlation between strong MAP2 staining in carcinoma and shortened biochemical recurrence-free survival after prostatectomy (p < 0.001). Multivariate Cox regression analysis confirmed MAP2 as an independent predictor for an unfavourable course. Mean MAP2 serum levels for non-PCA vs. PCA patients differed significantly (non-PCA = 164.7 pg/ml vs. PCA = 242.5 pg/ml, p < 0.001). CONCLUSION: The present data support MAP2 as a novel biomarker in PCA specimens. MAP2 is correlated with tumor grade and MAP2 high-expressing PCA is associated with an increased risk of biochemical recurrence after radical prostatectomy. Future studies are necessary to evaluate MAP2 as a valuable immunohistochemical biomarker in preoperative PCA diagnostic procedures, in particular with regard to treatment modalities.

Clinical implications of AGR2 in primary prostate cancer: Results from a large-scale study.

Human anterior gradient-2 (AGR2) has been implicated in carcinogenesis of various solid tumours, but the expression data in prostate cancer are contradictory regarding its prognostic value. The objective of this study is to evaluate the expression of AGR2 in a large prostate cancer cohort and to correlate it with clinicopathological data. AGR2 protein expression was analysed immunohistochemically in 1023 well-characterized prostate cancer samples with a validated antibody. AGR2 expression levels in carcinomas were compared with matched tissue samples of adjacent normal glands. AGR2 expression levels were dichotomized and tested for statistical significance. Increased AGR2 expression was found in 93.5% of prostate cancer cases. AGR2 levels were significantly higher in prostate cancer compared with normal prostate tissue. A gradual loss of AGR2 expression was associated with increasing tumour grade (ISUP), and AGR2 expression is inversely related to patient survival, however, multivariable significance is not achieved. AGR2 is clearly upregulated in the majority of prostate cancer cases, yet a true diagnostic value appears unlikely. In spite of the negative correlation of AGR2 expression with increasing tumour grade, no independent prognostic significance was found in this large-scale study.

Androgen Receptor Splice Variants Contribute to the Upregulation of DNA Repair in Prostate Cancer.

BACKGROUND: Canonical androgen receptor (AR) signaling regulates a network of DNA repair genes in prostate cancer (PCA). Experimental and clinical evidence indicates that androgen deprivation not only suppresses DNA repair activity but is often synthetically lethal in combination with PARP inhibition. The present study aimed to elucidate the impact of AR splice variants (AR-Vs), occurring in advanced or late-stage PCA, on DNA repair machinery. METHODS: Two hundred and seventy-three tissue samples were analyzed, including primary hormone-naïve PCA, primary metastases, hormone-sensitive PCA on androgen deprivation therapy (ADT) and castration refractory PCA (CRPC group). The transcript levels of the target genes were profiled using the nCounter platform. Experimental support for the findings was gained in AR/AR-V7-expressing LNCaP cells subjected to ionizing radiation. RESULTS: AR-Vs were present in half of hormone-sensitive PCAs on androgen deprivation therapy (ADT) and two-thirds of CRPC samples. The presence of AR-Vs is highly correlated with increased activity in the AR pathway and DNA repair gene expression. In AR-V-expressing CRPC, the DNA repair score increased by 2.5-fold as compared to AR-V-negative samples. Enhanced DNA repair and the deregulation of DNA repair genes by AR-V7 supported the clinical data in a cell line model. CONCLUSIONS: The expression of AR splice variants such as AR-V7 in PCA patients following ADT might be a reason for reduced or absent therapy effects in patients on additional PARP inhibition due to the modulation of DNA repair gene expression. Consequently, AR-Vs should be further studied as predictive biomarkers for therapy response in this setting.

Prostate-specific membrane antigen expression in hepatocellular carcinoma: potential use for prognosis and diagnostic imaging.

AIM: The prostate-specific membrane antigen (PSMA) is currently being established as a potent diagnostic marker in many tumor types. So far, its evidence in hepatocellular carcinoma (HCC) is sparse. The aim of our study was a comprehensive evaluation of PSMA expression and its prognostic role in patients with hepatocellular carcinoma as well as feasibility test of PSMA as an agent for diagnostic imaging. METHODS: The cohort for immunohistochemistry consisted of 153 patients with HCC. For validation purposes the HCC cohort (n = 359) of The Cancer Genome Atlas was analyzed on transcript level as well. RESULTS: On immunohistochemistry, non-tumorous liver tissue showed PSMA expression on canalicular membranes in all cases. In tumor tissue two patterns of expression, with a canalicular (41.1% of tumors) and a neovascular (89.9% of tumors) staining were seen. Completely negative for both two patterns were only 4.1% of tumors; conversely, 79.2% of the tumors showed high levels of PSMA protein expression at any location. At mRNA level higher FOLH1 (PSMA) expression rates were statistically significant and independently associated with longer overall survival times.Additionally, a case report of successful diagnostic 68Ga-PSMA-11 PET/CT in a patient with HCC progression on multiple therapy lines is provided. CONCLUSIONS: Majority of hepatocellular carcinomas show high levels of PSMA expression on tumor vessels and on canalicular membrane of the tumor cells. Putative diagnostic, prognostic and therapeutic value of PSMA in HCC warrants further clinically oriented investigations.

Where is the limit of prostate cancer biomarker research? Systematic investigation of potential prognostic and diagnostic biomarkers.

BACKGROUND: The identification of appropriate biomarkers is essential to support important clinical decisions in patients with prostate cancer. The aim of our study was a systematic bioinformatical analysis of the mRNA expression of all genes available for the prostate adenocarcinoma cohort of The Cancer Genome Atlas (TCGA), regarding their potential prognostic and diagnostic role. METHODS: The study cohort comprises 499 patients (TCGA prostate cancer cohort). mRNA expression data were available for approx. 20,000 genes. The bioinformatical statistical pipeline addressed gene expression differences in tumor vs. benign prostate tissue (including gene set enrichment analysis, GSEA) in samples from tumors with different aggressivenesses (Gleason score), as well as prognostic values in multistep survival analyses. RESULTS: Among all genes analyzed, 1754 were significantly downregulated and 1553 genes were significantly upregulated in tumor tissue. In GSEA, 16 of 30 top enriched biological processes were alterations of epigenetic regulation at different levels. Significant correlation with Gleason Score was evident for 8724 genes (range of Pearson r-values 0.09-0.43; all p < 0.05). In univariate Cox regression analyses, mRNA expression of 3571 genes showed statistically significant association with biochemical recurrence-free survival with a range of hazard ratios 0.3-3.8 (p-value 7.4e- 07 to 0.05). Among these, 571 genes were independently associated with biochemical recurrence in multivariate analysis. Access to the full database including results is provided as supplement. CONCLUSIONS: In our systematic analysis we found a big number of genes of potential diagnostic and prognostic value, many of which have not been studied in prostate cancer to date. Due to the comprehensive nature of this analysis and free access to the results, this study represents a reference database for prostate cancer researchers which can be used as a powerful tool for validation purposes and planning of new studies.

Apelin and apelin receptor expression in renal cell carcinoma.

BACKGROUND: The APLNR (apelin receptor) has been shown to be an essential gene for cancer immunotherapy, with deficiency in APLNR leading to immunotherapy failure. The aim of this study is to investigate the expression of APLN (apelin) and APLNR in patients with renal cell carcinoma (RCC), and its association with clinicopathological parameters and survival. METHODS: Three well-characterised patient cohorts with RCC were used: Study cohort 1 (clear-cell RCC; APLN/APLNR mRNA expression; n = 166); TCGA validation cohort (clear-cell RCC; APLN/APLNR mRNA expression; n = 481); Study cohort 2 (all RCC subtypes; APLNR protein expression/immunohistochemistry; n = 300). Associations between mRNA/protein expression and clinicopathological variables/patients' survival were tested statistically. RESULTS: While APLN showed only very weak association with tumour histological grade (TCGA cohort), APLNR/mRNA protein expression correlate significantly with ccRCC aggressiveness. APLNR is expressed in tumour vasculature and tumour cells at different levels, and these expression levels associate with tumour aggressiveness in opposing directions. APLNR expression was negatively correlated with PD-L1 expression by tumour cells in a subset of patients with ccRCC. APLNR expression in either compartment is an independent prognostic factor for survival of patients with ccRCC. CONCLUSION: The APLNR/APLN-system appears to play an important role in ccRCC, warranting further clinical investigation.

A tyrosine O-prenyltransferase catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL.

A putative prenyltransferase gene sirD has been identified in the gene cluster encoding the biosynthesis of the phytotoxin sirodesmin PL in Leptosphaeria maculans. The gene product was found to comprise 449 aa, with a molecular mass of 51 kDa. In this study, the coding region of sirD was amplified by PCR from cDNA, cloned into pQE70, and overexpressed in Escherichia coli. The overproduced protein was purified to apparent homogeneity, and characterized biochemically. The dimeric recombinant SirD was found to catalyse the O-prenylation of L-Tyr in the presence of dimethylallyl diphosphate; this was demonstrated unequivocally by isolation and structural elucidation of the enzymic product. Therefore, SirD catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL. K(m) values for L-Tyr and dimethylallyl diphosphate were determined as 0.13 and 0.17 mM, respectively. Interestingly, SirD was found to share significant sequence similarity with indole prenyltransferases, which catalyse prenyl transfer reactions onto different positions of indole rings. In contrast to indole prenyltransferases, which accept indole derivatives, but not Tyr or structures derived thereof, as substrates, SirD also prenylated L-Trp, resulting in the formation of 7-dimethylallyltryptophan. A K(m) value of 0.23 mM was determined for L-Trp. Turnover numbers of 1.0 and 0.06 S(-1) were calculated for L-Tyr and L-Trp, respectively.

Tryptophan aminopeptidase activity of several indole prenyltransferases from Aspergillus fumigatus.

Recently, five indole prenyltransferases from Aspergillus fumigatus have been proven biochemically to be responsible for prenylations of diverse substrates. In this study, we show peptidase activities of 7-DMATS, FgaPT1, CdpNPT, and FtmPT1, with preference for linear peptides containing a tryptophanyl moiety at the N terminus. Testing of 31 peptides revealed that these enzymes shared similar substrate specificity and accepted H-L-Trp-L-Ala-OH and H-L-Trp-Gly-OH as best substrates for aminopeptidase activity. By using H-L-Trp-Gly-OH as substrate, Km values at 350, 380, 300, and 420 microM and enzymatic rate constants kcat/Km at 0.51, 0.24, 0.53, and 0.14 mM(-1)s(-1) were determined for 7-DMATS, FgaPT1, CdpNPT, and FtmPT1, respectively. In contrast to prenyltransferase activities, the aminopeptidase activities were strongly or completely inhibited by EDTA. Mn2+ increased the aminopeptidase activities of FtmPT1 and CdpNPT up to 4- and 6-fold, respectively. To the best of our knowledge, this is the first report on the catalytic promiscuity of prenyltransferases.

Potential of a 7-dimethylallyltryptophan synthase as a tool for production of prenylated indole derivatives.

Recently, a gene for a 7-dimethylallyltryptophan synthase (7-DMATS) was identified in Aspergillus fumigatus and its enzymatic function was proven biochemically. In this study, the behaviour of 7-DMATS towards aromatic substrates was investigated and compared with that of the 4-dimethylallyltryptophan synthase FgaPT2 from the same fungus. In total, 24 simple indole derivatives were tested as potential substrates for 7-DMATS. With an exception of 7-methyltryptophan, all of the substances were accepted by 7-DMATS and converted to their prenylated derivatives, indicating a more flexible substrate specificity of 7-DMATS in comparison to that of FgaPT2. The relative activities of 7-DMATS towards these substrates were from 4% to 89% of that of L-tryptophan, much higher than that of FgaPT2. Structural elucidation of the isolated enzymatic products by nuclear magnetic resonance and mass spectrometry analysis proved unequivocally the prenylation at position C7 of the indole ring. Overnight incubation with eight substances showed that the conversion ratios were in the range of 55.9% to 99.7%. This study provided an additional example that prenylated indole derivatives can be effectively produced by using the overproduced and purified 7-DMATS.

A 7-dimethylallyltryptophan synthase from Aspergillus fumigatus: overproduction, purification and biochemical characterization.

A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 aa, with a molecular mass of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60, and overexpressed in Escherichia coli. The soluble His(6)-fusion protein was purified to apparent homogeneity, and characterized biochemically. The enzyme was found to catalyse the prenylation of Trp at the C-7 position of the indole moiety, in the presence of dimethylallyl diphosphate (DMAPP); therefore, it functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. K(m) values were 67 microM for DMAPP, and 137 microM for l-Trp. Geranyl diphosphate was not accepted as prenyl donor, while Trp-containing dipeptides were found to be aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although Ca(2+) enhanced the reaction velocity slightly. The enzyme is the second dimethylallyltryptophan synthase identified in A. fumigatus. Interestingly, it shares a sequence identity of only 31 % at the amino acid level with another known dimethylallyltryptophan synthase, FgaPT2, from the same fungus; FgaPT2 prenylates l-Trp at the C-4 position of the indole ring. Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.