Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Prostate cancer (CaP) has the highest incidence of any malignancy affecting South African males. The aetiology of prostate carcinoma indicate that ethnicity is one of the most important risk factors. The causes of these ethnic differences are unknown but presumably involve both environmental and genetic factors. Carcinoma of the prostate is androgen dependent, and it has been suggested that variations in androgen metabolism and synthesis may affect an individuals' risk. Therefore, genes involved in these pathways are candidates for determining CaP susceptibility. In this study two candidate genes in the androgen biosynthetic and metabolic pathway were analysed, viz., the androgen receptor gene (AR), involved in androgen transport and transcriptional activation, and the cytochrome p450c17a gene (CYP17), important for testosterone biosynthesis. Comprehensive mutation detection assays were designed (appropriate for analysis of archival paraffin-embedded material) for almost the entire coding region (excluding polymorphic repeat sequences), and including all splice site junctions of the AR gene, as well as the entire coding region of CYP17. The aim of this study was thus to determine the type and frequencies of genetic variants of these androgen metabolism genes within the diverse South African population, and to determine if the observed ethnic variation in the incidence and progression of CaP can be explained by ethnic-based genetic differences. For high sensitivity mutation detection, the most powerful of the pre-screening methods was used, namely denaturing gradient gel electrophoresis (DGGE). 20 CaP and 25 control benign prostatic hyperplasia (BPH) tissue samples were screened in order to identify possible mutations. Blood samples from the same patients were analysed in order to determine whether mutations are germline and therefore present in all cells of the body. Additional blood samples from the Western Province Blood Transfusion Service (WPBTS) (Refer to section 2.1.2, Table) were also analysed in order to determine the frequency of identified polymorphisms within the general population. Certain polymorphisms were further analysed in paraffin-embedded wax material (exclusively from Blacks) to determine the distribution of these polymorphisms in the Black population. Direct sequencing of mutant-containing DNA fragments was performed to determine the exact location and nature of mutation. Using the AR- DGGE assay 4 novel mutations were identified as well as a previously reported codon 211 (E211) polymorphism. With the CYP17- DGGE assay, 3 novel single nucleotide polymorphisms (SNPs) were detected. Three base variants occured, in codons 36 (L36), 46 (H46) and 65 (S65), as well as intronic substitutions in intron 4 (IVS+58G4C) and intron 6 (IVS-25C7A). Frequencies of SNPs were measured in the CaP and BPH samples. In conclusion, the identified polymorphisms could be used as markers in determining CaP susceptibility and may thus facilitate the identification of individuals with a high- or low-risk of developing carcinoma of the prostate. / AFRIKAANSE OPSOMMING: Prostaatkanker vertoon die hoogste voorkoms van enige kwaardaardigheid wat Suid-Afrikaanse mans aantas. Die etiologie van prostaatkarsinoom dui aan dat etnisiteit een van die mees belangrike risikofaktore is. Oorsake van hierdie etniese verskille is onbekend, maar vermoedelik is omgewing en genetiese faktore albei betrokke. Karsinoom van die prostaat is androgeenafhanklik en daar is voorgestel dat variasies in androgeenmetabolisme en androgeensintese 'n persoon se risiko mag affekteer. Gevolglik, is gene betrokke in hierdie paaie kandidate vir die bepaling van prostaatkanker vatbaarheid. In hierdie studie het ons twee kandidaat gene in die androgeen biosintetiese en metaboliese pad geanaliseer, naamlik, die androgeen reseptor geen (AR), betrokke in androgeen vervoer en aktivering van transkripsie, en die sitokroom p450c17a geen (CYP17), belangrik vir testosteroon biosintese. Ons het omvattende mutasie-bespeurings-essai-sisteme ontwikkel (ook uitvoerbaar op argivale paraffien-bewaarde materiaal), wat amper vir die hele koderende streek van die AR geen gebruik kan word (uitsluitend herhalende polimorfiese reekse) en wat alle splytpunt-aansluitings van die AR geen insluit, asook vir die hele koderende streek van CYP17. Die doel van hierdie studie was dus om die tipe en frekwensies van genetiese variante van androgeen metabolisme gene in ons diverse Suid-Afrikaanse bevolking te bepaal, en om vas te stel of die waarneembare etniese wisseling in die insidensie en vordering van prostaatkanker verstaan kan word deur etnies gebaseerde genetiese verskille. Die mees sensitiewe tegniek wat tans beskikbaar is vir vooraf-sifting vir onbekende mutasies is gekies, naamlik denaturerende gradiënt gel elektroforese (DGGE). Om moontlike mutasies op te spoor, het ons 20 prostaatkanker en 25 benijne prostaathiperplasie (BPH) monsters geanaliseer. Analise was gedoen op bloedmonsters van dieselfde pasiënte om vas te stel of kiemlyn mutasies (in alle liggaamselle) teenwoordig is. Bykomstige bloedmonsters (van die Westelike Provinsie Bloedoortappingsdiens) is ook geanaliseer om die frekwensie van bespeurde polimorfismes in die algemene bevolking te bepaal. Argivale paraffien-bewaarde materiaal (eksklusief van Swartes) is ook geanaliseer om die verspreiding van sekere polimorfismes in die Swart bevolking te bepaal. Direkte DNA volgorde bepaling van mutante DNA fragmente is uitgevoer om die ligging en tipe van mutasies te bepaal. Met die toepassing van ons AR-DGGE mutasiesisteem het ons 4 nuwe mutasies ontdek asook 'n kodon 211 (E211) polimorfisme wat voorheen gevind is. Vyf enkel nukleotied polimorfismes is met die CYP17-DGGE mutasiesisteem opgespoor. Die polimorfismes sluit in: drie basis veranderinge wat voorkom in kodons 36 (L36), 46 (H46) en 65 (S65), asook introniese substitutisies in intron 4 (IVS+58G4C) en intron 6 (IVS-25C7 A). Frekwensies van die polimorfismes was bereken in die prostaatkanker en BPH monsters. Die resultate aangebied in hierdie tesis dui aan dat die gevonde polimorfismes as merkers gebruik kan word om prostaatkanker vatbaarheid te bepaal en daardeur individue te identifiseer met 'n hoë of lae risiko vir prostaatkarsinoom ontwikkeling.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Background and aims of the study. Prostate cancer (PCa) is one of the most common hormone-dependent cancers in men in Western and also Asian countries. The standard treatment options for localized PCa include surgery and androgen-deprivation therapy (ADT). However, most patients upon ADT therapy invariably relapse and progress to a more aggressive and metastatic stage termed as castration-resistant PCa (CRPC). Accumulating studies indicate that androgen receptor (AR) transcriptional activity is dysregulated during the advanced progression of CRPC. One important mechanism responsible for the growth of CRPC includes increased intra-tumoral androgen synthesis in PCa. Recently, a novel androgen-responsive fusion gene TMPRSS2:ERG formed by fusion between the transmembrane protein TMPRSS2 and transcription factor ERG, has been identified in approximately 50% PCa samples, which results in the aberrant expression of ERG function as oncogenic factor in PCa. Currently, TMPRSS2:ERG is regarded as a significant potential diagnostic and prognostic biomarker for PCa. Estrogen-related receptor alpha-ERRα, the first identified ligand-independent orphan nuclear receptor, is characterized to be up-regulated in advanced cancers, suggesting that ERRα might play important regulatory roles in the malignant progression of PCa. Previous studies showed that ERRα can functionally cross-talk with AR signaling via co-targeting to AR targets and regulate the expression of some steroidogenic enzymes in breast cancer. Based on this background, it is hypothesized that ERRα could functionally regulate the TMPRSS2:ERG fusion gene and play a regulatory role in the development and progression of CRPC through activation of the intracellular androgen synthesis pathway. / Results. 1) The results obtained in this study showed that suppression of ERRα by its specific inverse agonist XCT790 or shRNA-knockdown could induce down-regulation of TMPRSS2:ERG and also its target genes in AR-positive VCaP PCa cells. 2) Ectopic expression of ERRα and/or its coactivator PGC-1α could increase the expression of TMPRSS2:ERG in AR-negative NCI-H660 PCa cells. 3) Two ERRα-DNA binding elements were identified by ChIP assay and sequence analysis in the promoter of TMPRSS2:ERG and both of these two elements could be transactivated by ERRα and PGC-1α. 4) Ectopic expression of TMPRSS2:ERG under the regulation of ERRα enhanced the prostatic cell invasion capacity as shown in the TMPRSS2:ERG infectants of BPH-1 and PC-3 prostatic cells. 5) ERG expressed by the TMPRSS2:ERG fusion could directly transactivate the ERRα gene in prostatic cells. 6) A positive correlation on the expressions between TMPRSS2:ERG and ERRα was demonstrated in a xenograft model of CRPC (VCaP-CRPC). 7) The expression of TMPRSS2:ERG and ERRα showed significant up-regulation and the transactivation activity of ERRα was also enhanced in castration-resistant VCaP-CRPC cells. 8) Ectopic expression of ERRα could promote resistant growth capacity to androgen-deprivation condition in LNCaP PCa cells, whereas shRNA-mediated silence of ERRα could weaken this resistant capacity. Furthermore, ectopic expression of ERRα in LNCaP-ERRα infectants could promote their in vivo growth resistance to castration in SCID mice. 9) Expression of several androgenic enzyme genes, including CYP11A1, CYP17A1 and ARK1C3, were detected to be up-regulated in castration-resistant VCaP-CRPC cells. Moreover, ectopic expression of ERRα could induce the increased expression of these enzyme genes in LNCaP-ERRα infectants, whereas knockdown of ERRα by shRNA could decrease their expression. 10) ERRα could directly transactivate the gene promoters of CYP11A1, CYP17A1 and ARK1C3 which contain ERRE elements prediction by sequence analysis. These results suggested that ERRα could play a role in de novo or intra prostatic androgen synthesis in the PCa cells. / Conclusions. The results obtained in this study suggested that ERRα and TMPRSS2:ERG could form a positive reciprocal loop in PCa cells, and ERRα could also promote the resistant growth capacity of PCa cells resistant to the androgen-deprivation condition in vitro and also castration-resistant growth in vivo via a mechanism of up-regulation of androgenic enzyme genes. The results also suggested that ERRα might play a significant regulatory role in the development and progression of PCa, particularly the advanced CRPC, and also ERRα could be a potential therapeutic target for the treatment of PCa, particularly the advanced PCa-CRPC. / 研究背景與研究目的：前列腺癌作為激素依賴的一種癌症，經常出現在西方和亞洲國家的男性人群中。對於局限性前列腺癌多採用外科手術和去勢的治療。但是大多數病人經過去勢治療后會再次復發並且形成更加惡心幾轉移的前列腺癌，稱之為去勢難治性前列腺癌（CRPC）。越來越多的研究表明在去勢難治性前列腺癌發病過程中，雄激素受體轉錄活性異性增強。其中一個重要機理解釋為前列腺癌細胞自身合成的雄激素增多。進來，在大約50%的前列腺癌病人中新檢測到一個受雄激素受（AR）體調控的融合基因TMPRSS2:ERG，它是由稱為TMPRSS2的一個跨膜蛋白和一個稱為ERG的轉錄因子融合而成，它的出現導致了在前列腺癌中異常的稱為致癌因子的ERG蛋白的高表達。目前，TMPRSS2:ERG已經被作為一個重要的潛在的診斷和預測的標誌物應用在前列腺癌中。作為第一個鑒定的配體不依賴的孤兒受體-ERRα，被證明在晚期的癌症中有很高的表達，預示著ERRα可能在惡性的癌症中起到一個非常重要的調控作用。之前的研究表明通過共同調控AR的下游基因，ERRα同AR信號通路之間有功能性的交叉調控；除此之外，在乳腺癌中，ERRα還可以調控一些類固醇類化合物的合成相關的一些酶的合成。依據上述，我們推定ERRα可能功能性地調控TMPRSS2:ERG融合基因的表達並且通過調控細胞內的雄激素的合成進而在去勢難治性前列腺癌的發生和發展中起到一個非常重要的作用。 / 結果：本論文研究結果總結如下：1）在有AR表達的前列腺癌細胞-VCaP細胞中，通過ERRα特異性的抑制劑XCT790處理或者shRNA介入的干擾ERRα的mRNA的方法來抑制ERRα，下調了TMPRSS2:ERG和它的一些下游調控基因的表達。2）在沒有AR表達的前列腺癌細胞-NCI-H660細胞中，上調ERRα或者它的特異性的共激活因子PGC-1α表達可以提升TMPRSS2:ERG的表達。3）通過ChIP實驗，在TMPRSS2:ERG的啟動子上面，兩個ERRα的DNA結合位點被鑒定出來。並且這兩個位點可以被ERRα和PGC-1α轉錄激活。4）在兩個前列腺細胞BPH-1和PC-3細胞中，在ERRα的調控下高表達TMPRSS2:ERG融合基因可以增強細胞的侵襲能力。5）融合基因TMPRSS2:ERG導致的ERG蛋白的表達可以直接轉錄激活ERRα的表達。6）我們通過VCaP細胞的異種移植建立VCaP-CRPC的體內模型來模擬CRPC過程，在整個過程中，我們發現TMPRSS2:ERG和ERRα有一致性的表達相關性。除此之外，我們根據上述動物模型通建立了VCaP-CRPC細胞系，並且發現在VCaP-CRPC細胞細胞中，TMPRSS2:ERG和ERRα都有被上調並且ERRα的轉錄活性同樣也提升。7）在LNCaP細胞中高表達ERRα可以提升細胞在去除雄激素的環境中生長的能力。但是當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減弱這種生長的能力。用LNCaP-ERRα穩轉ERRα的細胞異種移植建立SCID老鼠體內腫瘤模型，我們發現和LNCaP-pBABE對照組相比，LNCaP-ERRα細胞生長的更快更大。並且在對老鼠進行睪丸切除術后，LNCaP-ERRα組細胞更快適應這種環境并繼續生長，相比之下，LNCaP-pBABE對照組則持續萎縮減小。8）在上述的VCaP-CRPC細胞中，我們發現一些和雄激素合成相關的關鍵的酶包括CYP11A1，CYP17A1和ARK1C3的表達量有顯著地提升。而且在LNCaP-ERRα細胞中同樣檢測到這些酶的表達量的提升。然而當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減降低上述酶的表達。9）我們在CYP11A1，CYP17A1和ARK1C3基因的啟動子區域發現有ERRα結合位點，並且發現這些位點可以被ERRα轉錄激活。 / 結論：本論文的研究結果提示在前列腺癌細胞中，ERRα和TMPRSS2:ERG可以形成一個相互正向調控的循環。除此之外，上調ERRα可以促進細胞在去除雄激素的環境中生長的能力，並且在動物體內可以提升細胞在睪丸去除的環境中的適應和生長能力。這種體內和體外的能力的提升是通過一種潛在的上調前列腺癌細胞的雄激素合成相關的關鍵的酶的表達，進而提升雄激素的含量而得以實現的。上述的結果預示著ERRα可能在前列腺癌發生機發展的過程中起到非常重要的調控作用，尤其在晚期的CRPC中。同時，ERRα也可能作為一個潛在的重要的前列腺癌尤其是晚期的CRPC的治療靶點，尤其是一些潛在ERRα的特異性抑制劑，比如XCT790，可能作為將來用以作為治療前列腺癌的特異性靶點藥物。 / Xu, Zhenyu. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 126-143). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Xu, Zhenyu. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Jia, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.