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.Uusien eturauhassyövän hoitokohteiden identifiointi tehoseulontamenetelmiä hyväksi käyttäen – kohti täsmähoitoa Eturauhassyöpä on monimuotoinen ja epäyhtenäinen joukko sairauksia, joiden hoitamiseksi tarvitaan uusia tehokkaampia merkkiaineita, sekä kohdennettuja hoitovaihtoehtoja. Tässä väitöstutkimuksessa yhdistettiin tieto geenien ilmentymisestä geenin hiljentämisen mahdollistavaan RNA-interferenssi (RNAi) -tekniikkaan sekä lääketehoseulontoihin uusien merkkiaineiden, lääkehoidon kohteiden sekä lääkeaineiden löytämiseksi, ja kohdennettujen eturauhassyöpähoitojen mahdollistamiseksi. Aluksi hyödynsimme tietoja geenien ilmentymisestä 9873:ssa ihmiskudos- ja solunäytteessä erityisesti eturauhas- (n = 147) ja eturauhassyöpäkudoksessa (n = 349) ilmentyvien geenien havaitsemiseen. Seuraavaksi 295:n eturauhassyöpäkudokselle ominaisen geenin vaikutusta viljeltyjen eturauhassyöpäsolujen kasvuun tutkittiin RNAi–tehoseulontatekniikkaa hyödyntäen. Samanaikaisesti 4910:n eri lääkeaineen tehoa eturauhassyöpäsolujen kasvun estossa tutkittiin lääketehoseulontoja hyväksi käyttäen. Yhdeksän uuden lupaavan lääkehoidon kohteen sekä yhden syöpäsolujen kasvua estävän lääkeaineen toiminta varmennettiin jatkotutkimuksissa. Tulokset osoittivat, että androgeenireseptorin (AR) signaloinnin lisäksi solulimakalvoston toiminta, arakidonihappoaineenvaihdunta, hapetus-pelkistys –tasapainotila ja tuman jakautuminen, mitoosi, ovat tärkeitä eturauhassyöpäsolujen kasvulle. Uudet lääkehoidon kohdegeenit ilmentyivät eri eturauhassyövissä ja osoittivat, että ERG syöpägeeniä ilmentävät syöpäsolut olivat herkkiä oksidatiiviselle stressille ja solulimakalvoston toiminnan häiriölle, kun taas mitoosin estoa voitaisiin mahdollisesti hyödyntää pitkälle edenneiden ja hormonihoidoille vastustuskykyisten eturauhassyöpien hoidossa. Yhteenvetona voidaan todeta, että tämän väitöstutkimuksen tulokset havainnollistavat systeemibiologisen tutkimuksen mahdollisuudet uusien syöpähoitojen kehityksessä.Siirretty Doriast

Molecular insights into the zinc homeostasis of breast and prostate cancer cells

Zinc ion (Zn2+) is essential to life as a structural or catalytic component of proteins. The cell has developed an elaborate molecular network over the extensive evolutionary timeline to maintain zinc homeostasis. Any disruption of such a network will lead to zinc dyshomeostasis, resulting in health problems such as cancers. Zinc dyshomeostasis is an intriguing phenomenon in breast and prostate cancers, with breast cancer cells exhibiting higher intracellular Zn2+ levels compared to their corresponding normal epithelial cells, in contrast to the low Zn2+ levels in prostate cancer cells compared to the normal prostate counterpart. Such contrasting zinc profiles of breast and prostate cancer cells provide an avenue for this PhD project to investigate the Zn2+ homeostasis of breast and prostate cancer cells by a systematic approach of gene profiling via quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence confocal microscopy and proteomic analysis with a panel of cell lines including two breast cancer cell lines (MCF-7, MDA-MB-231), two prostate cancer cell lines (PC3 and DU145), along with normal breast epithelial and prostate epithelial cell lines (MCF10A, RWPE-1). A systematic approach of gene profiling, immunofluorescence confocal microscopy and proteomic analysis was carried out, using a panel of cell lines which include two breast cancer cell lines (MCF-7, MDA-MB-231), two prostate cancer cell lines (PC3 and DU145), along with normal breast epithelial and prostate epithelial cell lines (MCF10A, RWPE-1). The systematic experimental approach of this project returned the meaningful findings, which enhances our knowledge and understanding of the zinc homeostasis in breast and prostate cancer cells. The molecular clues uncovered here should not only contribute to the elucidation of molecular network of zinc homeostasis but should also serve as potential molecular targets for anti-cancer drug development. As zinc dyshomeostasis is increasingly linked to the development and progression of cancers such as breast and prostate cancers, and zinc is emerging as an important signalling messenger in normal and cancerous cells, the findings of this project provide avenues for further studies on breast and prostate cancers

Novel urinary and serological markers of prostate cancer using proteomics techniques: an important tool for early cancer diagnosis and treatment monitoring

In Africa, Prostate cancer (PCa) is the most frequently diagnosed solid organ tumour in males and use of prostate specific antigen (PSA) is presently fraught with diagnostic inaccuracies. Not least, in a multi-ethnic society like South Africa, proteome differences between African, Caucasian and Mixed-Ancestry PCa patients are largely unknown. Hence, discovery and validation of affordable, non-invasive and reliable diagnostic biomarkers of PCa would expand the frontiers of PCa management. We have employed two high-throughput proteomics technologies to identify novel urine- and blood-based biomarkers for early diagnosis and treatment monitoring of prostate cancer in a South African cohort as well as elucidate proteome differences in patients from our heterogeneous cohort. We compared the urinary proteomes of PCa, Benign Prostatic Hyperplasia (BPH), disease controls comprising patients with other uropathies (DC) and normal healthy controls (NC) both by pooling and individual discovery shotgun proteomic assessment on a nano-Liquid chromatography (nLC) coupled Hybrid Quadrupole-Orbitrap Mass Spectrometer platform. In-silico verification of identified biomarkers was performed using the Human Protein Atlas (HPA) as well as SRMAtlas; and verified potential biomarkers were experimentally prevalidated using a targeted parallel reaction monitoring (PRM) proteomics approach. Further, we employed the CT100+ antigen microarray platform to assess the differential humoral antibody response of PCa, DC and BPH patients in our cohort to a panel of 123 tumour-associated cancer antigens. Candidate antigen biomarkers were analyzed for ethnic group variation in our cohort and potential cancer diagnostic and immunotherapeutic inferences were drawn. Using these approaches, we identified 5595 and 9991 non-redundant peptides from the pooled and individual experiments respectively. While nine proteins demonstrated ethnic trend, 37 and 73 proteins were differentially expressed by pooled and individual analysis respectively. All 32 verified biomarkers were prevalidated with parallel reaction monitoring. Good PRM signals for 12 top ranking biomarker was observed, including PSA and prostatic acid phosphatase. We also identified 41 potential diagnostic and immunotherapeutic antigen biomarkers. Proteogenomic functional pathway analyses of differentially expressed antigens showed similar enrichments of biologic processes. We identified herein novel urinary and blood-based potential diagnostic biomarkers and immunotherapeutic targets of PCa in a South African PCa Cohort using multiple proteomics approaches

Zinc Signaling in Physiology and Pathogenesis

The essential trace element zinc plays indispensable roles in multiple cellular processes. It regulates a great number of protein functions, including transcription factors, enzymes, adapters, and growth factors as a structural and/or catalytic factor. Recent studies have highlighted another function of zinc as an intra- and intercellular signaling mediator, which became recognized as the “zinc signal”. Indeed, zinc regulates cellular signaling pathways, which enable conversion of extracellular stimuli to intracellular signals, and controls various intracellular and extracellular events, and thus zinc mediates communication between cells. The zinc signal is essential for physiology, and its dysregulation causes a variety of diseases, such as diabetes, cancer, osteoarthritis, dermatitis, and dementia. This Special Issue focuses on crucial roles of zinc signaling in biological processes in molecular and physiological basis, addressing the future directions and questions underlying this unique phenomenon. Because there is growing interest and attention in physiopathological contribution of zinc signal, we believe this Special Issue will provide very timely information on it and thus should appeal to a wide range of readers

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds

Drug Discovery

Natural products are a constant source of potentially active compounds for the treatment of various disorders. The Middle East and tropical regions are believed to have the richest supplies of natural products in the world. Plant derived secondary metabolites have been used by humans to treat acute infections, health disorders and chronic illness for tens of thousands of years. Only during the last 100 years have natural products been largely replaced by synthetic drugs. Estimates of 200 000 natural products in plant species have been revised upward as mass spectrometry techniques have developed. For developing countries the identification and use of endogenous medicinal plants as cures against cancers has become attractive. Books on drug discovery will play vital role in the new era of disease treatment using natural products

Molecular and cellular expression of novel genes associated with breast cancer

An improved and individualised breast cancer care can be pursued with the use of tumour markers that are associated with risk assessment, diagnosis, prognosis, prediction of treatment outcome, monitoring of the disease and development of novel therapies. As such, this study proposed to characterise and evaluate the potential for the management of breast cancer of four novel unpublished breast-associated genes, the breast-associated UniGene clusters (BUC6, BUC9, BUC10 and BUC11), which were identified in our laboratory by database mining. The first aspect of this research project was to undertake extensive in silico analysis and in vitro expression analysis for all genes using sequencing and RT-PCR based assays on a variety of tissues of normal and cancerous origin in order to further select the most promising candidates for potential use in cancer care. In light of all the data collected, the work was subsequently focused on the BUC11 gene. SinceBUC11 mRNA was found to be expressed at very low levels in normal tissues except in some non-essential tissues where it is expressed at a higher level, BUC11 was not considered a cancer testis antigen. BUC11 mRNA was also found at high levels in all testicular cancer tissues tested as well as in a quarter of prostate cancer tissues tested and at lower levels in a small proportion of melanomas, and thus BUC11 was not considered to be a tumour-specific gene

Computational Methods for the Analysis of Genomic Data and Biological Processes

In recent decades, new technologies have made remarkable progress in helping to understand biological systems. Rapid advances in genomic profiling techniques such as microarrays or high-performance sequencing have brought new opportunities and challenges in the fields of computational biology and bioinformatics. Such genetic sequencing techniques allow large amounts of data to be produced, whose analysis and cross-integration could provide a complete view of organisms. As a result, it is necessary to develop new techniques and algorithms that carry out an analysis of these data with reliability and efficiency. This Special Issue collected the latest advances in the field of computational methods for the analysis of gene expression data, and, in particular, the modeling of biological processes. Here we present eleven works selected to be published in this Special Issue due to their interest, quality, and originality