High-Throughput Screening for Novel Prostate Cancer Drug Targets –Getting Personal

Prostate cancers form a heterogeneous group of diseases and there is a need for novel biomarkers, and for more efficient and targeted methods of treatment. In this thesis, the potential of microarray data, RNA interference (RNAi) and compound screens were utilized in order to identify novel biomarkers, drug targets and drugs for future personalized prostate cancer therapeutics. First, a bioinformatic mRNA expression analysis covering 9873 human tissue and cell samples, including 349 prostate cancer and 147 normal prostate samples, was used to distinguish in silico prevalidated putative prostate cancer biomarkers and drug targets. Second, RNAi based high-throughput (HT) functional profiling of 295 prostate and prostate cancer tissue specific genes was performed in cultured prostate cancer cells. Third, a HT compound screen approach using a library of 4910 drugs and drug-like molecules was exploited to identify potential drugs inhibiting prostate cancer cell growth. Nine candidate drug targets, with biomarker potential, and one cancer selective compound were validated in vitro and in vivo. In addition to androgen receptor (AR) signaling, endoplasmic reticulum (ER) function, arachidonic acid (AA) pathway, redox homeostasis and mitosis were identified as vital processes in prostate cancer cells. ERG oncogene positive cancer cells exhibited sensitivity to induction of oxidative and ER stress, whereas advanced and castrate-resistant prostate cancer (CRPC) could be potentially targeted through AR signaling and mitosis. In conclusion, this thesis illustrates the power of systems biological data analysis in the discovery of potential vulnerabilities present in prostate cancer cells, as well as novel options for personalized cancer management.

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.

Collagen binding integrins and cancer testis antigens in prostate cancer and melanoma

Camilla Pelo Collagen Binding Integrins and Cancer Testis Antigens in Prostate Cancer and Melanoma Department of Biochemistry, MediCity Research Laboratory, University of Turku, Finland Annales Universitatis Turkuensis, Painosalama Oy, Turku, Finland 2016 ABSTRACT Prostate cancer is the second most common cancer in men worldwide. The incidence of melanoma, in turn, is increasing faster than any other cancer incidences. In Finland, more than 5000 prostate cancer and 1200 new melanoma cases are diagnosed each year. One approach to further understand the cellular processes involved in prostate cancer and melanoma is to gain better knowledge about alterations in gene expression and their potential impact on the progression of the diseases. This thesis is focused on expression studies in two gene families; integrins and cancer testis antigens (CT antigens), in human prostate adenocarcinoma and advanced human melanoma. Integrins are heterodimeric transmembrane receptors which regulate many important cellular processes such as cell proliferation, migration and survival. CT antigens are frequently expressed in different types of cancers, but are only expressed in testis in healthy individuals. CT antigens are also highly immunogenic proteins. Due to the properties mentioned above, integrins and CT antigens can function as target molecules for the development of cancer diagnostics and drugs. One of the main purposes of this thesis was to study the expression of the four collagen binding integrins α1β1, α2β1, α10β1, α11β1 and the cancer testis antigen 16 (CT16) in cancer cell lines and human tissues of prostate cancer and metastatic melanoma. Additional aims included studies on the biological role of CT16 and the abundance of CT16 in sera of advanced melanoma patients. The prognostic and diagnostic significance of CT16 and the collagen binding integrins were also evaluated. Expression studies on collagen binding integrins and the CT antigen CT16 in melanoma and prostate cancer were limited and the biological role of CT16 was unknown. In this thesis, the expression levels of α2β1 and α11β1 were found to be significantly altered in prostate cancer tissues. Integrin α2β1 decreased gradually during disease progression while α11 was elevated in prostate carcinoma compared to healthy tissues. In advanced melanoma, enhanced levels of α2 were associated with a significant shorter overall survival in advanced melanoma. In this thesis, CT16 was identified as a frequently expressed melanoma CT antigen with an anti-apoptotic function. To conclude, this thesis presents α2β1 and CT16, as potential and promising biomarkers for advanced melanoma. This thesis reports also the first functional study of CT16. Keywords: Collagen binding integrins, α1β1, α2β1, α10β1, α11β1, Cancer Testis antigens, CT16, melanoma, prostate cancer, expression

Auria Biopankin hyödynnettävyys tutkittavien rekrytoinnissa kliinisiin lääketutkimuksiin

Kliininen lääketutkimus on ihmiseen kohdistuva interventiotutkimus, jonka tavoitteena on selvittää lääkeaineiden farmakodynaamisia sekä farmakokineettisiä ominaisuuksia ihmiselimistössä. Tutkittavien rekrytointi kliinisiin lääketutkimuksiin voi olla haastavaa ja hidasta, mikä vaikeuttaa tutkimuksen suorittamista. Biopankki on yksikkö, johon on säilöttynä ihmisperäisiä näytteitä ja niihin liitettyjä terveystietoja, joita voidaan käyttää biopankkitutkimusten tekemiseen. Vuonna 2013 astui voimaan Biopankkilaki (688/2012), jonka myötä Suomeen on perustettu yhdeksän biopankkia. Tämän projektin tarkoituksena oli selvittää, miten Auria Biopankin näyte- ja tietorekisteriä voitaisiin hyödyntää tutkittavien rekrytoimiseksi kliinisiin lääketutkimuksiin. Projekti on toteutettu yhteistyössä Auria Biopankin kanssa. Projektia varten valittiin kuusi lääkekehityksen kannalta mielenkiintoista ja ajankohtaista sairautta, jotka ovat: Alzheimerin tauti, idiopaattinen keuhkofibroosi (IPF), multippeliskleroosi eli MS-tauti, eturauhassyöpä, ei-pienisoluinen keuhkosyöpä sekä monisysteemiatrofia (MSA). Jokaiselle sairaudelle suoritettiin näyte- ja tietorekisterihaku Auria Biopankin rekisteristä. Hakua varten luotiin sairauskohtaiset tutkittavien mukaanotto- ja poissulkukriteerit, suoritettujen todellisten kliinisten lääketutkimusten kriteereitä apuna käyttäen. Näyte- ja tietorekisterihaun tarkoituksena oli selvittää, kuinka monta kriteereitä vastaavaa henkilöä biopankin rekisterissä on, ja voidaanko heiltä biopankin välityksellä tiedustella halukkuutta ottaa osaa kliiniseen lääketutkimukseen. Projektin toisena osana luotiin toimintamalleja sekä mallikirjeitä biopankin välityksellä tapahtuvaa yhteydenottoa varten. Varsinaista yhteydenottoa sopiviin henkilöihin ei tämän projektin yhteydessä suoritettu. Suurinosa Auria Biopankin näytteistä on kerätty sairaalahoidon yhteydessä, joten oletettavasti erikoissairaanhoidossa hoidettavat sairaudet ovat kattavammin edustettuina biopankin rekisterissä. Tietorekisterihaun tuloksista tämä ei kuitenkaan ole suoraan havaittavissa. Valituista sairauksista eturauhassyöpää sairastavia kriteerit täyttäviä potilaita löytyi hieman alle 130, kun Alzheimerin taudin, IPF:n, ja MSA:n kohdalla luku jäi alle kymmeneen. MS-tautia sairastavia henkilöitä löytyi alle 20 ja ei-pienisoluista keuhkosyöpää sairastavia noin 10. Biopankkilain mukainen biopankkitoiminta on Suomessa vielä uutta. Aktiivisten biopankkisuostumusten määrä tulee todennäköisesti kasvamaan voimakkaasti seuraavien vuosien aikana, mikä mahdollistaa biopankkien näyte- ja tietorekisterien kasvun ja tiedon nykyistä paremman hyödyntämisen. Mitä enemmän on biopankkisuostumuksen antaneita henkilöitä, sitä enemmän on henkilöitä tavoitettavissa biopankin välityksellä myös kliinisiin lääketutkimuksiin liittyen.