Pathology - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemThe role of infection in the aetiology of prostate cancer( 2015)An infectious aetiology for prostate cancer has been conjectured for decades but the evidence gained from questionnaire-based and sero-epidemiological studies is weak and inconsistent and a causal association with any infectious agent remains to be established. The work described in this thesis questions whether the inconsistency in evidence could be related to tumour heterogeneity (high-grade or low-grade) or the nature of infections. It was decided to focus on high-grade disease, as this the most aggressive form, and on evidence of persistent infection, as transient infection was considered unlikely to play a causal role. Quantitative molecular methods were used first to seek evidence of single-organism infection comparing high-grade with lower-grade tumours. The potential of targeted 16S rRNA gene sequencing and total RNA sequencing was then evaluated regarding its utility to characterise microbial communities within high-grade tumours. Archival tissue blocks were retrieved for 52 high-grade and 76 low-grade prostate cancers. DNA samples were extracted and screened by RealTime-PCR using validated and standardised assays for the following candidate organisms C. trachomatis, U. urealyticum, U. parvum, M. genitalium, BKV, HSV-1 and 2, P. acnes types IA, IB and II, T. vaginalis and XMRV DNA. Samples were screened for 16 HPV genotypes by PCR and flow cytometry. Prevalence of M. genitalium, U. urealyticum, and HSV was low and did not differ by tumour grade. The prevalence of P. acnes type IA, IB, II was higher than for other agents, however no evidence of an association was detected. Neither BKV, XMRV, T. vaginalis, U. parvum, C. trachomatis nor HPV DNA were detected. Given the sensitivity and specificity of the methods used for the candidate organisms, the absence or low levels of detectable microbial DNA indicate a low probability that candidates contributed to cancer risk. A massively parallel sequencing (MPS) approach was used to characterise any resident microbial communities and the relative abundance of bacterial constituents within tissue. DNA and RNA were extracted from 20 snap-frozen tissue samples from high-grade prostate tumours (10 high-grade prostate cancer cores and matched unaffected prostate tissue). Prior to sequencing, broad-range PCR was applied to DNA across three hypervariable regions (V2-V4) of the 16S rRNA gene to enrich for bacterial species. As 16rRNA gene sequencing is only able to detect bacterial/archael microorganisms, MPS was also applied to cDNA from total RNA that was extracted from the same tissue samples to detect other microorganisms (viral, bacterial and protozoal origin) that may be associated with prostate cancer. Sample cDNA was sequenced and the data were queried for 16S rRNA sequences and the presence of expressed viral genes. Partial 16S rRNA sequencing identified Enterobacteriaceae species common to all samples and P. acnes in 95%. Total RNA sequencing detected endogenous retroviruses that provided proof of concept. As this part of the study was exploratory, associations between the organisms identified and prostate cancer risk could not be ascertained. Further studies, specifically designed to detect associations between the disease phenotype and aetiological agents, are required.
-
ItemDelineating the tumor suppressive role of Scribble in prostate cancer progression and metastasis( 2013)Cell and tissue polarity are distinguished by the asymmetrical distribution of cytoplasmic and membrane components that allows the formation of structurally and functionally distinct domains within cells, and the organisation of multilayered tissues. This asymmetry is required for many cellular processes including migration, interaction with the microenvironment, diversification of cell shapes and development. To establish cellular polarity several polarity regulators are required, such as Scribble, whose loss results in deregulated cellular functions. Scribble is localised at the baso-lateral membrane, which is crucial for its normal functionality. Mislocalisation, which appears as diffuse expression within the cytoplasm, also results in disrupted cellular polarity and therefore impacts polarity regulated cellular functions. Scribble is widely accepted as an evolutionarily conserved tumor suppressor that is often deregulated in many human epithelial cancers, and is generally considered to contribute to tumor progression. Loss of cell and tissue polarity is a hallmark of epithelial cancers suggesting a crucial role for polarity regulators in suppressing tumor formation and progression(1). Interestingly, Scribble is also found to be overexpressed in many epithelial cancers, including prostate cancer, while mislocalization in prostate cancer rather than overexpression correlates with poor patient outcome in the clinic. Scribble has been shown to be a weak initiator of prostate neoplasia in mice owing to elevated Ras/MAPK signaling. Furthermore, Scribble depletion and K-Ras-oncogenic activation have been shown to cooperate in vivo, resulting in an increased incidence of invasive prostate carcinoma compared to single mutants, indicating that Scribble loss can contribute to tumor progression in the presence of an additional oncogenic mutation. Nonetheless, the molecular mechanisms underpinning tumour progression in the context of Scribble loss are not well understood. To directly assess the role of Scribble in prostate carcinogensis and metastasis, in vitro functional assessment of PC3 prostate cancer cells expressing constructs to knockdown (shSCRIB7), overexpress (hSCRIB) or mislocalize (SCRIB P305L) Scribble together with in vivo experimental models of metastasis have been performed. This work shows that Scribble is upregulated in PC-3 cells and that Scribble plays a role in coordinating directed cell migration and invasion, but does not mediate PC-3 cell cycle progression or proliferation in vitro. In addition, by employing the well characterized prostate cancer transgenic mouse model (PBCre+;Ptenfl/fl), in combination with Scribble depletion, it appears that loss of Scribble and Pten cooperate to facilitate invasion, associated with a reduction in the cell:cell adhesion molecule E-cadherin. Collectively, these findings establish that Scribble functions to coordinate prostate cancer cell migration and invasion and that deregulation of Scribble (either by over-expression, depletion or mislocalisation) causes aberrant migration and invasion. Further investigations into understanding how Scribble and the polarity network can regulate migration and invasion during prostate cancer on a molecular level may provide valuable insights into the mechanisms behind prostate cancer progression and prove to hold prognostic value in the clinic, as well as identify novel routes of therapeutic intervention.