Quantitative Analysis of Novel Prostate Cancer Markers in Tissue

Prostate cancer is a heterogeneous disease affecting an increasing number of men all over the world, but particularly in the countries with the Western lifestyle. The best biomarker assay currently available for the diagnosis of the disease, the measurement of prostate specific antigen (PSA) levels from blood, lacks specificity, and even when combined with invasive tests such as digital rectal exam and prostate tissue biopsies, these methods can both miss cancers, and lead to overdiagnosis and subsequent overtreatment of cancers. Moreover, they cannot provide an accurate prognosis for the disease. Due to the high prevalence of indolent prostate cancers, the majority of men affected by prostate cancer would be able to live without any medical intervention. Their latent prostate tumors would not cause any clinical symptoms during their lifetime, but few are willing to take the risk, as currently there are no methods or biomarkers to reliably differentiate the indolent cancers from the aggressive, lethal cases that really are in need of immediate medical treatment. This doctoral work concentrated on validating 12 novel candidate genes for use as biomarkers for prostate cancer by measuring their mRNA expression levels in prostate tissue and peripheral blood of men with cancer as well as unaffected individuals. The panel of genes included the most prominent markers in the current literature: PCA3 and the fusion gene TMPRSS2-ERG, in addition to BMP-6, FGF-8b, MSMB, PSCA, SPINK1, and TRPM8; and the kallikrein-related peptidase genes 2, 3, 4, and 15. Truly quantitative reverse-transcription PCR assays were developed for each of the genes for the purpose, time-resolved fluorometry was applied in the real-time detection of the amplification products, and the gene expression data were normalized by using artificial internal RNA standards. Cancer-related, statistically significant differences in gene transcript levels were found for TMPRSS2-ERG, PCA3, and in a more modest scale, for KLK15, PSCA, and SPINK1. PCA3 RNA was found in the blood of men with metastatic prostate cancer, but not in localized cases of cancer, suggesting limitations for using this method for early cancer detection in blood. TMPRSS2-ERG mRNA transcripts were found more frequently in cancerous than in benign prostate tissues, but they were present also in 51% of the histologically benign prostate tissues of men with prostate cancer, while being absent in specimens from men without any signs of prostate cancer. PCA3 was shown to be 5.8 times overexpressed in cancerous tissue, but similarly to the fusion gene mRNA, its levels were upregulated also in the histologically benign regions of the tissue if the corresponding prostate was harboring carcinoma. These results indicate a possibility to utilize these molecular assays to assist in prostate cancer risk evaluation especially in men with initially histologically negative biopsies.Eturauhassyöpä on heterogeeninen tauti, joka koskettaa kasvavaa määrää miehiä ympäri maailman, mutta erityisesti länsimaista elämäntyyliä edustavissa maissa. Eturauhassyöpädiagnostiikan tarpeisiin paras tällä hetkellä tarjolla oleva biomerkkiainemääritys, prostataspesifisen antigeenin (PSA) pitoisuuden määritys verestä, ei ole tarpeeksi spesifinen käyttötarkoitukseensa. Vaikka kyseiseen testiin yhdistetään peräsuolen kautta tehtävä eturauhasen tunnustelu ja eturauhasesta otettujen koepalojen tutkiminen kudostutkimustasolla, voi osa syövistä jäädä diagnosoimatta. Toisaalta niitä voidaan diagnosoida tarpeettomasti, mikä voi johtaa myös ylihoitamiseen. Mainitut diagnoosimenetelmät eivät myöskään pysty ennustamaan taudinkehitystä. Koska suuri osa eturauhassyövistä on hitaasti kasvavia ja kliinisesti merkityksettömiä, suurin osa miehistä, joilla syöpä todetaan, pystyisi elämään latentin kasvaimen kanssa ilman lääketieteellistä hoitoakin koko elinaikansa, ilman että kasvain ehtii aiheuttaa kliinisiä oireita missään vaiheessa. Harvat potilaat kuitenkaan valitsevat tätä vaihtoehtoa, koska käytössä ei ole menetelmiä tai biomerkkiaineita, joilla voitaisiin luotettavasti todeta, mitkä syövät kuuluvat hitaasti etenevään alaluokkaan, ja mitkä taas ovat aggressiivisia ja välitöntä lääketieteellistä hoitoa vaativia syöpiä. Tässä väitöskirjatyössä tutkittiin kahdentoista uuden biomerkkiaineeksi ehdotetun geenin soveltuvuutta eturauhassyöpädiagnostiikkaan. Niiden lähetti-RNA-tasoja mitattiin sekä eturauhassyöpää sairastavien että terveiden henkilöiden eturauhaskudoksessa ja veressä. Tutkittuihin geeneihin kuuluivat alan kirjallisuudessa tällä hetkellä näkyvimmin esiintyvät merkkiaineet PCA3-geeni ja fuusiogeeni TMPRSS2-ERG, sekä BMP-6-, FGF-8b-, KLK2-, KLK3-, KLK4-, KLK15-, MSMB-, PSCA-, SPINK1- ja TRPM8-geenit. Kunkin geenin ilmenemistason mittaamiseen kehitettiin absoluuttisesti kvantitatiivinen käänteiskopiointi-PCR-määritys, jossa monistustuotteet havaittiin reaaliaikaisesti aikaerotteisen fluorometrian avulla, ja tulokset normalisoitiin keinotekoisen, sisäisen standardi-RNA:n avulla. Viiden tutkitun geenin – TMPRSS2-ERG:n, PCA3:n, KLK15:n, PSCA:n ja SPINK1:n –ilmenemistasoissa havaittiin tilastollisesti merkitseviä, syöpään viittaavia muutoksia. PCA3-RNA:ta löydettiin verestä vain jo-levinneiden syöpien tapauksissa, mikä rajoittanee menetelmän käyttöä syövän varhaiseen havaitsemiseen verestä. TMPRSS2- ERG-lähetti-RNA:ta löydettiin useammin eturauhaskudoksesta, jos kyseessä oli syöpäkudos, mutta myös 51 prosentista niitä histologisesti hyvänlaatuisia kudoksia, jotka olivat peräisin syöpää sisältävistä eturauhasista. Tätä merkkiainetta ei kuitenkaan havaittu miehillä, joilla ei ollut todettu merkkejä eturauhassyövästä. PCA3-geenin osoitettiin yli-ilmenevän 5.8-kertaisesti syöpäkudoksessa, mutta sen ilmeneminen oli lisääntynyt syöpäisissä eturauhasissa myös histologisesti hyvänlaatuisilla kudosalueilla. Tulokset antavat aiheen olettaa, että molekyylitason määrityksiä voitaisiin mahdollisesti käyttää eturauhassyöpäriskin arvioinnissa erityisesti, jos potilaan ensimmäisestä eturauhaskoepalasta ei kudostasolla havaita syöpää.Siirretty Doriast

Cancer-associated Changes in the Expression of TMPRSS2-ERG, PCA3, and SPINK1 in Histologically Benign Tissue From Cancerous vs Noncancerous Prostatectomy Specimens.

To investigate whether messenger ribonucleic acid (mRNA) expression of TMPRSS2-ERG fusion gene, a suggested prostate cancer (PCa) biomarker, was specific to cancerous lesions alone and to study the expression of SPINK1 and PCA3 mRNAs in the same cohort to also explore the proposed mutual exclusivity of TMPRSS2-ERG and SPINK1 expression

Global expression of AMACR transcripts predicts risk for prostate cancer - a systematic comparison of AMACR protein and mRNA expression in cancerous and noncancerous prostate.

The high false negative rates for initial prostate biopsies refer a large number of the men for repeat biopsies each year. Therefore, biomarkers associated with high risk of the presence of malignancy in histologically benign biopsies could provide a tool to discriminate the patients who need repeat biopsy or intensive follow-up from those who do not. Here we examined the diagnostic applicability of alpha-methylacyl CoA racemase (AMACR) and androgen receptor (AR) mRNA expression and AMACR protein levels in benign and cancerous prostatic tissue

Prostate cancer risk SNP rs10993994 is a trans-eQTL for SNHG11 mediated through MSMB

How genome-wide association studies-identified single-nucleotide polymorphisms (SNPs) affect remote genes remains unknown. Expression quantitative trait locus (eQTL) association meta-analysis on 496 prostate tumor and 602 normal prostate samples with 117 SNPs revealed novel cis-eQTLs and trans-eQTLs. Mediation testing and colocalization analysis demonstrate that MSMB is a cis-acting mediator for SNHG11 (P < 0.01). Removing rs10993994 in LNCaP cell lines by CRISPR/Cas9 editing shows that the C-allele corresponds with an over 100-fold increase in MSMB expression and 5-fold increase in SNHG11 compared with the T-allele. Colocalization analysis confirmed that the same set of SNPs associated with MSMB expression is associated with SNHG11 expression (posterior probability of shared variants is 66.6% in tumor and 91.4% in benign). These analyses further demonstrate variants driving MSMB expression differ in tumor and normal, suggesting regulatory network rewiring during tumorigenesis

Validation of novel biomarkers for prostate cancer progression by the combination of bioinformatics, clinical and functional studies

The identification and validation of biomarkers for clinical applications remains an important issue for improving diagnostics and therapy in many diseases, including prostate cancer. Gene expression profiles are routinely applied to identify diagnostic and predictive biomarkers or novel targets for cancer. However, only few predictive markers identified in silico have also been validated for clinical, functional or mechanistic relevance in disease progression. In this study, we have used a broad, bioinformatics-based approach to identify such biomarkers across a spectrum of progression stages, including normal and tumor-adjacent, premalignant, primary and late stage lesions. Bioinformatics data mining combined with clinical validation of biomarkers by sensitive, quantitative reverse-transcription PCR (qRT-PCR), followed by functional evaluation of candidate genes in disease-relevant processes, such as cancer cell proliferation, motility and invasion. From 300 initial candidates, eight genes were selected for validation by several layers of data mining and filtering. For clinical validation, differential mRNA expression of selected genes was measured by qRT-PCR in 197 clinical prostate tissue samples including normal prostate, compared against histologically benign and cancerous tissues. Based on the qRT-PCR results, significantly different mRNA expression was confirmed in normal prostate versus malignant PCa samples (for all eight genes), but also in cancer-adjacent tissues, even in the absence of detectable cancer cells, thus pointing to the possibility of pronounced field effects in prostate lesions. For the validation of the functional properties of these genes, and to demonstrate their putative relevance for disease-relevant processes, siRNA knock-down studies were performed in both 2D and 3D organotypic cell culture models. Silencing of three genes (DLX1, PLA2G7 and RHOU) in the prostate cancer cell lines PC3 and VCaP by siRNA resulted in marked growth arrest and cytotoxicity, particularly in 3D organotypic cell culture conditions. In addition, silencing of PLA2G7, RHOU, ACSM1, LAMB1 and CACNA1D also resulted in reduced tumor cell invasion in PC3 organoid cultures. For PLA2G7 and RHOU, the effects of siRNA silencing on proliferation and cell-motility could also be confirmed in 2D monolayer cultures. In conclusion, DLX1 and RHOU showed the strongest potential as useful clinical biomarkers for PCa diagnosis, further validated by their functional roles in PCa progression. These candidates may be useful for more reliable identification of relapses or therapy failures prior to the recurrence local or distant metastases

Correction: Validation of Novel Biomarkers for Prostate Cancer Progression by the Combination of Bioinformatics, Clinical and Functional Studies.

[This corrects the article DOI: 10.1371/journal.pone.0155901.]

Analysis of AR-FL and AR-V1 in Whole Blood of Patients with Castration Resistant Prostate Cancer as a Tool for Predicting Response to Abiraterone Acetate

PURPOSE: Reliable molecular diagnostic tools are still unavailable for making informed treatment decisions and monitoring the response in patients with castration resistant prostate cancer. We evaluated the significance of whole blood circulating androgen receptor transcripts of full length (AR-FL) and splice variants (AR-V1, AR-V3 and AR-V7) as biomarkers of abiraterone acetate treatment resistance in patients with castration resistant prostate cancer. MATERIALS AND METHODS: After retrospective analysis in 112 prostate specimens AR-FL, AR-V1, AR-V3 and AR-V7 were evaluated in 185 serial blood samples, prospectively collected from 102 patients with castration resistant prostate cancer before and during abiraterone acetate therapy via reverse transcription quantitative polymerase chain reaction. RESULTS: AR-FL was present in all samples while AR-V1, AR-V3, AR-V7 and at least 1 of them was detected in 17%, 55%, 65% and 81% of castration resistant prostate cancer blood samples, respectively. The highest amount of AR-V1 was found in blood of patients whose response time was short and medium in comparison to extended. Patients with a higher level of AR-FL and/or AR-V1 had the shortest progression-free survival and overall survival (p <0.0001). CONCLUSIONS: Blood circulating AR-FL or AR-V1 can serve as blood based biomarkers for identification of the primary resistance to abiraterone acetate and the tool to monitor de novo resistance development during abiraterone acetate treatment