Pathology - Theses
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ItemInvestigating the predictors of sensitivity to the Chk1 inhibitor PF-00477736 in melanoma( 2013)Chk1 and Chk2 are two of the most important and highly studied checkpoint kinases that help protect cells from endogenous or exogenous insults which could damage DNA. When activated they arrest cell cycle at specific checkpoints in order for cells to repair damage before resuming the cell cycle and mitosis. Recently, several inhibitors of Checkpoint kinases have been developed which were designed to sensitise TP53 mutant cancers with defective G1 checkpoints to DNA damaging chemotherapy. However, a recent study has demonstrated that TP53 mutation alone does not predict sensitivity to Chk1 inhibitors. Moreover, inhibition of Chk1 by PF-00477736 (PF-736) as a single agent was also recently shown to be effective in mediating p53-dependent apoptosis in lymphoma cells by increasing the level of MYC oncogene-induced DNA damage. Other reports have also shown that mutant BRAF and RAS (NRAS) which are the predominant oncogenes found in melanoma could induce DNA damage in epithelial cells and thyroid cancers, respectively. This led to the hypothesis that melanomas which are predominantly TP53 wild-type and BRAF or NRAS mutant would be responsive to Chk1 inhibitors as single agents. Screening for drug sensitivity across a panel of 30 melanoma cell lines showed a wide range of IC50 values following 3 days treatment with PF-736. BRAF and TP53 mutation status were not found to affect sensitivity to PF-736. Due to unavailability of NRAS mutation data at the time of analysis, it is still unconfirmed whether BRAF and NRAS as the source of oncogene DNA damage in melanoma influence the sensitivity of melanoma to PF-736 treatment. Several cell lines categorised as sensitive was revealed to have high Total Growth Inhibition (TGI) values. This suggests that there may be genetic variations in the cells that have high TGI particularly in cell death activation pathways that affect the sensitivity of these cells to Chk1 inhibition. Previously, it has been reported that up-regulation of p53 negative-regulators such as Mdm4 and Bcl-2 family anti-apoptotic proteins were responsible for the defective p53-mediated apoptosis responses in p53 wild-type melanomas. Overexpression of BCL2 or BCLxl was able to prevent apoptosis in C002-M1, a cell line that underwent apoptosis following PF-736 treatment. However, MCL1 overexpression did not result in similar effect. Assessment of Mdm4 basal protein levels did not reveal any significant correlation with PF-736 IC50. Further assessment utilising MDM4 knockdown by shRNA in the A375 cell line expressing high Mdm4 protein expression did not result in sensitisation to PF-736. These data suggested that the increased levels of Bcl-2 or Bcl-XL proteins were able to decrease the level of apoptosis but did not significantly alter sensitivity to PF-736 in C002-M1 cell line. Additionally knockdown of MDM4 levels did not alter PF-736 IC50 in a cell line with high Mdm4 protein expression. Hence it is possible that other cellular characteristics such as the level of inherent DNA damage recently reported to be important in sensitivity of melanoma to Checkpoint kinase inhibition, could provide more insight in revealing factors that determine the sensitivity of PF-736 in melanoma. γH2AX is a marker of double strand break which can be utilised as a measure of DNA damage. In an attempt to assess γH2AX as a biomarker of sensitivity, the levels of inherent γH2AX and fold change following PF-736 treatment was determined. The result showed that high level of inherent γH2AX showed a significant correlation with low PF-736 IC50, suggesting that high levels of endogenous DNA damage is associated with sensitivity to PF-736. Furthermore, the average fold increase in levels of γH2AX after PF-736 treatment across the entire cell line panel also significantly correlated with PF-736 IC50. Additionally, we discovered that increase in pChk2 T68 levels following PF-736 treatment also correlated with response to PF-736 in the 30 melanoma cell line panel. Increase in pChk1 S345 following Chk1 inhibition, previously reported as a potential pharmacodynamic biomarker of Chk1 inhibition was confirmed across the panel following the same PF-736 treatment demonstrating the biomarkers identified were associated with Chk1 inhibition. A significant component of this project was to identify predictors of sensitivity using a pharmacogenomic whole genome-wide approach and a candidate approach. For the candidate approach, gene expression of selected genes from pathways related to the DNA damage response pathway for the 30 melanoma cell line panel were analysed with IC50 from SRB assay utilising linear regression analysis to search for significant correlation between sensitivity to PF-736 and gene expression. Gene expression of NBN, CHK2, RAD21 and RAD54B, which are components of the DSB repair particularly HR repair, significantly associated with sensitivity to PF-736. An unbiased Pearson correlation analysis between whole-genome gene expression microarray data and IC50 data from SRB assay of the 30 melanoma cell line panel also showed good correlation between DSB repair genes and sensitivity to PF-736. From this analysis, RAD21 and RAD54B were showed to have a good correlation with sensitivity to PF-736. An SVM prediction model built using the gene expression data pre-processed using the Pearson correlation analysis to include only genes that are differentially expressed across the panel and their sensitivity to PF-736 based on IC50, to discover a gene set that can classify melanoma cell lines between sensitive and resistant to PF-736. The result showed that the SVM model was able to predict at 100% accuracy with a cut-off of 2048 nM. Some of the genes included in the gene set for the SVM prediction model were genes that are important for DSB repair particularly HR such as RAD21, RAD54B and NSMCE2 confirming the importance of this specific DNA repair pathway in association with sensitivity to PF-736 particularly in melanoma. In conclusion, this study has revealed the importance of the DSB Response pathway in predicting the sensitivity to the Chk1 specific inhibitor PF-736 in melanoma. As there are also several other cancers which exhibit oncogene-induced DNA damage and aberrant DSB repair pathways, this study may help in predicting the sensitivity of these types of cancers in with single agent Chk1 therapy. Furthermore, this project has verified that melanoma cell lines are sensitive to Chk1 inhibition therapy as a single agent and that the level of inherent DNA damage may provide insight into the sensitivity of melanoma to Chk1 inhibition. Moreover, candidate and genome-wide pharmacogenomic analyses have revealed the importance of DSB repair pathway and Bcl-2 family anti-apoptotic genes in predicting sensitivity to Chk1 inhibition. Although functional study on C002-M1 cell line did not show evidence that the level of Bcl-2 family anti-apoptotic proteins can affect sensitivity, the western blot data, candidate gene and genome wide predictor analysis across the 30 melanoma cell line panel showed that there is a significant correlation between Bcl-2 family anti-apoptotic proteins particularly Bcl-XL to sensitivity to PF-736, which warrants further investigation. As a future direction, the current predictor model will need to be tested with a validation set of samples, to test its robustness in predicting sensitivity to single agent Chk1 inhibition therapy. To progress further the assessment of the usefulness of Chk1 inhibition therapy in melanoma, an investigation on Chk1 inhibition in combination with DNA damaging chemotherapy which was shown to be promising in the preliminary study, will need to be carried out.
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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.
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ItemMolecular characterisation of IFN-γ induced Stat1-independent and Erk1/2- and AP-1-dependent signalling pathway and genes that respond to IFN-γ( 2013)Interferon (IFN)-γ regulates a diverse range of biological activities that includes anti-pathogenic, anti-cancer and immunoregulatory effects. IFN-γ mediates these activities by regulating changes in gene expression via intracellular pathways. Although the canonical IFN-γ-induced Jak/Stat1 signalling pathway is the primary mechanism regulating gene expression, IFN-γ activates multiple intracellular cascades. There is evidence suggesting that Stat1-independent pathways are important for IFN-γ activity since a third of IFN-γ regulated genes (IRGs) were regulated in the absence of Stat1 (Gil, Bohn et al. 2001) and Stat1-/- mice were more resistant to viral challenge than mice deficient in type I and II IFN receptors (Gil, Bohn et al. 2001; Shresta, Sharar et al. 2005). Our laboratory identified the downstream components of a novel Stat1-independent signalling pathway that activated Erk1/2 and AP-1 and regulated transcription of IRGs (Gough, Sabapathy et al. 2007). However, the upstream components of this pathway have not been characterised. Since Stat1 was not required for IFN-γ-induced activation of Erk1/2 and AP-1 I hypothesised that this pathway is entirely independent of canonical IFN-γ Jak/Stat1 signalling. In support of this, studies have shown that IFN-γ-induced engaged multiple membrane-proximal kinases such as the PI-3K/Akt and adapter proteins such as MyD88 independent of Stat1 and/or Jak proteins. Therefore I hypothesised that Jak1 and Jak2 were not required for activation of the IFN-γ induced Stat1-independent Erk1/2 and AP-1 pathway or to regulate transcription of IRGs. Therefore, alternate kinases or membrane associated proteins would be recruited to the IFN-γ receptor (Ifngr) to activate Erk1/2 and AP-1 and regulate gene expression. The most appropriate method to investigate IFN-γ-induced Jak1 and Jak2-independent signalling is the use of a doubly deficient Jak1/Jak2 model. However, since Jak1-deficient and Jak2-deficient mice are not viable, Jak1-/-/Jak2-/- mice are not viable either and there is no published literature on a doubly deficient Jak1/Jak2 model. The studies in this thesis developed and fully characterised the first reported doubly deficient Jak1/Jak2 model. In a novel finding the signalling analyses performed using doubly deficient Jak1/Jak2 cells demonstrated that IFN-γ-induced activation of Erk1/2 and likely AP-1 was independent of Jak1 and Jak2. Therefore building on previous findings by our laboratory the data in this thesis established that IFN-γ-induced a Jak1/Jak2/Stat1-independent and Erk1/2/AP-1-dependent pathway. Since Jak1 and 2 were not involved IFN-γ must engage another mechanism to active downstream Erk1/2. The studies in this thesis excluded a role for c-Src; therefore alternate signalling molecules must be recruited to the Ifngr. Gene expression analyses showed that transcription of the Immediate Early Genes c-Jun and Nfkbia appeared to be independent of Jak2 but not Jak1. Further analyses performed using a larger gene subset of genome wide studies are required to identify a subset of IRGs potentially regulated by Jak1/Jak2/Stat1-independent and Erk1/2/AP-1-dependent pathway. The studies in this thesis suggest that the Erk1/2 and AP-1 pathway may represent a primary yet transient response to IFN-γ that regulates gene expression independent of canonical Jak/Stat1 signal transduction. Determination of the cellular and biological effects regulated by the non-canonical Erk1/2/AP-1 pathway may have important implications for our understanding of the anti-viral, anti-cancer and immunomodulatory effects of IFN-γ.