published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

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

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Η εγκαθίδρυση και ανάπτυξη καρκίνου του προστάτη διαμεσολαβείται από τη δράση μιας πλειάδας ογκογενετικών αυξητικών παραγόντων. Μέχρι σήμερα έχει αναδειχθεί η εμπλοκή του αυξητικού παράγοντα HARP στην ανάπτυξη καρκινικών όγκων διαφορετικής προελεύσεως. Στην παρούσα εργασία, διερευνήθηκε η πιθανή συμμετοχή της HARP στην ανάπτυξη καρκίνου του προστάτη. Με εφαρμογή μιας αντινοηματικής στρατηγικής πραγματοποιήθηκε καταστολή έκφρασης της HARP στην καρκινική κυτταρική σειρά προστάτη LNCaP και μελετήθηκε τόσο ο ρόλος της HARP στην αύξηση και μεταναστευτική ικανότητα των καρκινικών κυττάρων, όσο και η ενδεχόμενη αγγειογενετική δράση της in vitro και in vivo. Η εξωγενώς χορηγούμενη ανασυνδυασμένη HARP ανθρώπου, ήταν μιτογόνος για τα κύτταρα LNCaP. Επιπρόσθετα η καταστολή έκφρασης της ενδογενούς HARP, ανέδειξε την αναγκαιότητα του συγκεκριμένου αυξητικού παράγοντα για τη μετανάστευση των κυττάρων LNCaP καθώς και για την κυτταρική αύξηση τόσο σε συνθήκες εξαρτώμενες όσο και ανεξάρτητες από την προσκόλληση σε υπόστρωμα. Οι επαγόμενες, από τα κύτταρα LNCaP, λειτουργίες των ενδοθηλιακών κυττάρων in vitro και ο σχηματισμός νέων αγγείων in vivo, αναχαιτίστηκαν όταν ανεστάλη η έκφραση της HARP. Ο αυξητικός παράγοντας ινοβλαστών FGF-2 είναι ένας πλειοτροπικός αυξητικός παράγοντας, ο οποίος διαδραματίζει σημαντικό ρόλο στην εγκαθίδρυση και ανάπτυξη καρκίνου του προστάτη. Τα αποτελέσματα της παρούσας διατριβής, κατέδειξαν ότι ο FGF-2 δύναται να επάγει σε σημαντικό ποσοστό τον πολλαπλασιασμό και τη μετανάστευση των κυττάρων LNCaP. Το μόριο της HARP φαίνεται να μεσολαβεί προκειμένου να εκδηλωθούν οι διεγερτικές δράσεις του FGF-2, δεδομένου ότι τελευταίος δεν επηρέασε αντίστοιχες λειτουργίες των κυττάρων LNCaP στα οποία είχε κατασταλεί η έκφραση της HARP. Επιπλέον, ο FGF-2 διέγειρε την έκφραση και έκκριση της HARP από τα κύτταρα LNCaP και αύξησε τη δραστηριότητα λουσιφεράσης πλασμιδιακού οχήματος αναφοράς, στο οποίο είχε κλωνοποιηθεί η ρυθμιστική περιοχή του γονιδίου της HARP. Ο ειδικός αναστολέας του υποδοχέα FGFR-1, SU-5402, αναχαίτισε την επαγόμενη από τον FGF-2 ενεργοποίηση του γονιδίου της HARP και την επακόλουθη έκκριση της πρωτεΐνης, οδηγώντας με τον τρόπο αυτό σε εξασθένιση του κυτταρικού πολλαπλασιασμού. Επώαση των κυττάρων LNCaP με πυροσταφυλικό νάτριο, το οποίο απομακρύνει με έμμεσο τρόπο το υπεροξείδιο του υδρογόνου, ανέδειξε την εξάρτηση των διεγερτικών δράσεων του FGF-2 από την ενδοκυτταρική παραγωγή υπεροξειδίου του υδρογόνου, ενώ ανάσχεση της δραστικότητας του FGFR-1 ανέστειλε τον επαγόμενο από τον FGF-2 σχηματισμό δραστικών μορφών οξυγόνου. Με χρησιμοποίηση ολιγονουκλεοτιδικών δολωμάτων έναντι του ΑΡ-1 και εφαρμογή κατευθυνόμενης μεταλλαξιγένεσης στη ρυθμιστική περιοχή του γονιδίου της HARP, διαπιστώθηκε η εμπλοκή του ΑΡ-1 στην επαγόμενη από τον FGF-2 έκφραση και έκκριση της HARP. Η επίδραση του FGF-2 στα κύτταρα LNCaP, φαίνεται να οφείλεται στη δέσμευση των Fra-1, JunD και της ενεργού μορφής της c-Jun στη ρυθμιστική περιοχή του γονιδίου της HARP. Συμπερασματικά, καταδεικνύεται ο σημαντικός ρυθμιστικός ρόλος του αυξητικού παράγοντα HARP σε ποικίλες βιολογικές διεργασίες των καρκινικών κυττάρων ανθρώπινου προστάτη. Επιπλέον, στην παρούσα εργασία προτείνεται ο ρόλος και ο μηχανισμός δράσης του αυξητικού παράγοντα FGF-2 στα κύτταρα LNCaP, ενώ ταυτόχρονα αντικατοπτρίζεται η πολυπλοκότητα των μονοπατιών αυξητικών παραγόντων που εμπλέκονται στον καρκίνο του προστάτη. / The development and growth of human prostate cancer is mediated by many tumor cell-derived growth factors. Heparin affin regulatory peptide (HARP) seems to be involved in the progression of several tumors of diverse origin. In the present work, we sought to determine if HARP is implicated in human prostate cancer. An antisense strategy for inhibition of HARP expression in the human prostate cancer cell line LNCaP was used to study the role of HARP on cancer cell growth, migration and angiogenic potential in vitro and in vivo. Exogenous human recombinant HARP was mitogenic for LNCaP cells. By decreasing the expression of endogenous HARP, we found that HARP was essential for LNCaP cell migration, as well as anchorage-dependent and independent growth. Endothelial cell functions in vitro and blood vessel formation in vivo induced by LNCaP cells were also inhibited when HARP expression was diminished. Fibroblast growth factor 2 (FGF-2) is a pleiotropic growth factor that has been implicated in prostate carcinoma formation and progression. In the present study we found that exogenous FGF-2 significantly increased human prostate cancer LNCaP cell proliferation and migration. HARP seems to be an important mediator of FGF-2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF-2 significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full length promoter of HARP gene. The FGFR1-specific inhibitor SU-5402 blocked the FGF-2-increased HARP gene activation and the consequent protein release, leading to impairment of LNCaP cell proliferation. Treatment of LNCaP cells with the hydrogen peroxide scavenger pyruvate, pointed to the dependence of FGF-2-induced HARP expression and LNCaP cell proliferation on hydrogen peroxide generation, and blockade of FGFR1 activity abrogated the FGF-2-induced production of reactive oxygen species. Activator protein-1 (AP-1) seems to be involved in FGF-2-stimulated HARP expression and secretion by LNCaP cells, as revealed using AP-1 decoy oligonucleotides and point mutation analyses in the HARP gene promoter. Binding of AP-1 complexes consisting of Fra-1, JunD and phospho-c-Jun, to the HARP promoter seems to be amenable for FGF-2 effect. These results point to an important regulator role of HARP in diverse biological activities in human prostate cancer cells. Furthermore, the present work establishes the role and the mode of activity of FGF-2 in LNCaP cells and reflects the many-sidedness of growth factor pathways within prostate cancer.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a metalloproteinase which becomes upregulated in prostate cancer and has been implicated in processes of prostate cancer metastasis. Here, we show that MT1-MMP is minimally expressed in nonmalignant primary prostate cells, moderately expressed in DU-145 cells, and highly expressed in invasive PC-3 and PC-3N cells. Using MT1-MMP promoter reporters and mobility shift assays, we show that Sp1 regulates MT1-MMP expression in DU-145, PC-3, and PC-3N cells and in PC3-N cells using chromatin immunoprecipitation analysis and silencing RNA. Investigation of signaling pathways in these cells showed that DU-145 cells express constitutively phosphorylated extracellular stress-regulated kinase (ERK), whereas PC-3 and PC-3N cells express constitutively phosphorylated AKT/PKB and c-Jun NH2 terminal kinase (JNK). We show that MT1-MMP and Sp1 levels are decreased in PC-3 and PC-3N cells when PI-3K and JNK are inhibited, and that MT1-MMP levels are decreased in DU-145 cells when MEK is inhibited. Transient transfection of PC-3 and PC-3N cells with a dominant-negative JNK or p85, and DU-145 cells with a dominant negative ERK, reduced MT1-MMP promoter activity. We also identified the insulin-like growth factor (IGF-1R) as an upstream regulatory component of MT1-MMP in PC-3N and LNCaP cells, which express high and low levels of the enzyme, respectively. Treatment of PC-3N cells with an IGF-1R specific inhibitor decreased MT1-MMP promoter activity, RNA and protein levels. Additionally, treatment of LNCaP cells with a synthetic androgen to increase IGF-1R levels and subsequent treatment with IGF-I increased MT1-MMP promoter activity, RNA and protein levels. Analysis of MT1-MMP and IGF-1R expression in human prostate cancer tissues demonstrated that MT1-MMP expression was high in the apical cytoplasmic regions of PIN and prostate cancer and less intense in the basalateral cytoplasmic membrane regions of benign glands. IGF-1R was expressed in normal glands and highly expressed in prostate cancer. In conclusion, we have identified several novel mechanisms regulating MT1-MMP expression in prostate cancer cell lines as well as differential localization of the enzyme in human prostate cancer tissues. These results provide insight into the complex mechanisms of prostate cancer metastasis and may be useful for developing future diagnostic procedures or therapies.

For cancer cells to initiate cell migration and progress to metastasize, epithelial genes must be silenced and the expression of mesenchymal genes must be upregulated. During prostate carcinogenesis, E-cadherin expression is downregulated through multiple mechanisms, the majority of which combine to silence E-cadherin expression through transcriptional regulation at the level of the E-cadherin promoter. Recently it has been discovered that there is transcriptional upregulation of the mesenchymal cadherin, N-cadherin during prostate cancer metastasis. Although N-cadherin expression can be detected in human prostate cancer and in prostate carcinoma cell lines, the mechanisms controlling the transcriptional regulation of N-cadherin in cancer are uncharacterized. This body of work offers the first evidence for the mechanisms controlling the transcriptional upregulation of N-cadherin expression in prostate carcinoma. We utilized anchorage independent culture to induce downregulation of N-cadherin expression, and then analyzed the necessary events for N-cadherin upregulation when cells attached to Fibronetin (FN). In order to determine the functional regions of the N-cadherin proximal promoter that were involved in the upregulation of N-cadherin expression, we cloned regions of the human N-cadherin 5’ proximal promoter, and regions of the first intron of the N-cadherin gene into a luciferase reporter vector. It was determined that the bHLH transcription factor Twist1 controlled the upregulation of N-cadherin transcription in PC-3 cells, through β1 integrin dependent nuclear localization of Twist1. A cis-element located in the first intron of the N-cadherin gene was shown to be necessary for Twist1 mediated effects on the N-cadherin promoter. We then determined the requirements for cell-type specific expression of the N-cadherin promoter. It was determined that an additional cis-element located in the first intron of the N-cadherin gene was necessary to repress N-cadherin promoter activity in cells lacking N-cadherin. Through deletion analysis of the N-cadherin promoter luciferase construct, a DNA binding site for the transcription factor FoxP1 was discovered. FoxP1 binds to the repressive cis-element in vitro, and mutation of the FoxP1 DNA binding site eliminated cell-type specific activity of the N-cadherin promoter. Therefore, we have documented that the aberrant expression of N-cadherin in prostate carcinoma involves alterations in both positive and negative transcriptional regulators.

Aim: The aims of this essay were first to see if there were any factors that could have an inpact on participating in supportive care groups and activities after a prostatic cancer diagnosis. The second aim was to examine what kind of support the patients would chose.   Methods: Data was collected with a survey handed out to the prostate cancer patients visiting the urologist reception at the hospital in Uppsala, during two weeks in the fall of 2011.   Main Results: Men show very little interest in participating in supportive care groups and activities. When asked to chose which kind of support they could consider, individual sessions and group sessions were the most common choice.   Conclusion: Men diagnosed with prostate cancer chose not to participate in supportive care. Further studies are required to determine what may be the reason to that. / Syfte: Syftet med detta arbete var att se om olika faktorer kunde påverka deltagande i stödverksamhet efter att patienten fått diagnosen prostatacancer, samt vilken form av psykosocialt stöd patienterna föredrar.    Metod: Metoden som använts var en enkätstudie med både kvantitativ ansats, som delades ut under hösten 2011 till prostatacancerpatienter på urologmottagningen, Akademiska sjukhuset.   Huvudresultat: Män anger att de inte är intresserade av att delta i stödverksamhet efter diagnos och eventuell behandling. Vid behov ansåg de att enskilda eller gruppsamtal var mest relevanta som stödverksamheter. Informanterna ansåg att rehabilitering med samtal och yoga hade minst relevans.   Slutsats: Män som drabbats av prostatacancer väljer att inte delta i stödverksamhet. Behov finns därför av att utföra mer studier för att klargöra orsakerna till detta.

As the most frequently diagnosed cancer in North American men, prostate cancer can progress to the androgen independent stage after initial response to androgen ablation therapy. The molecular mechanisms involved in the hormonal progression of prostate cancer are not completely understood. Here, we analyze changes in the transcriptome of prostate cancer cells at different stages of progression to reveal potential mechanisms. Applying Affymetrix GeneChip technology, we identified the transcriptomes in response to stimulation of androgen and PKA pathways in human prostate cancer cells. In addition to PSA, other common target genes were identified. Genes differentially expressed in response to androgen and stimulation of the PKA pathway in vitro were also differentially expressed during hormonal progression in vivo. Upon androgen stimulation, androgen receptor binds to a functional androgen response element within the promoter region of SESN1, a p53 targeted gene, and represses its expression. The expression of SESN1 was induced by castration in LNCaP xenografts, but the expression was eventually suppressed again in the androgen independent stage of prostate cancer. Knockdown of SESN1 promoted the proliferation of prostate cancer cells. Expression patterns of androgen-regulated genes in androgen independent tumours were revealed to be more similar to that from before castration than to the tumors under androgen ablation. The β-catenin, a potent coactivator of the androgen receptor, and Wnt pathway was deregulated in androgen-independent tumours. There was increased nuclear colocalization and interaction of androgen receptor and β-catenin with hormonal progression of prostate cancer. This study provides insight into hormonal effects on prostate cancer and possible pathways involved in the development of androgen independent disease, as well as potential therapeutic targets.

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.