Examining Instigators and Suppressors of Metastasis and Mitosis in Pancreatic Cancer

Cancer remains a leading health issue globally, whose burden of disease is predicted to rise. While recent advances have improved diagnostic and prognostic outcomes for many patients, there remains a desperate need for new therapeutic strategies to overcome the multifaceted obstacles of treating the aggressive tumours, such as pancreatic cancer. In order to develop innovative therapeutic strategies, new molecular targets and novel agents or combinations of drugs are required to combat aggressive aspects of cancer, such as metastasis and the development of resistance. Aberrant nuclear factor-κB (NF-κB) activity has been reported in a number of cancer-types including pancreatic cancer. This signalling pathway has been intimately linked with aggressive tumours facilitating tumourigenesis, the epithelial mesenchymal transition (EMT), metastasis, and treatment resistance. Additionally, zinc finger E-box-binding homeobox 1 (ZEB1) is an oncogenic transcription factor that is down-stream of NF-κB activity and facilitates the EMT, metastasis, rapid proliferation, and treatment resistance. Thus, in the current thesis, we examined the potential of targeting these latter aspects of aggressive cancer via investigating NF-κB and ZEB1. The following thesis consists of 6 chapters including: (1) Chapter 1, which comprises an Introduction that is a comprehensive literature review; (2) Chapter 2, Materials and Methods; (3) Results Chapters 3-5, which details and discusses findings; and finally, (4) Chapter 6, that is a concluding analysis of results and also includes future prospective directions. Chapter 3 | Initial studies pertained to targeting the EMT via examining the role of metastasis suppressor, N-myc down-stream regulated gene-1 (NDRG1). NDRG1 plays multifaceted functions in inhibiting oncogenic signalling and suppressing the EMT, which is a precursor state to metastasis. Chapter 3 sought to expand on the role of NDRG1 as a promoter of an epithelial phenotype. Notably, NDRG1 inhibited the oncogenic effects of transforming growth factor-β (TGF-β) in PANC-1 pancreatic cancer cells by promoting expression and co-localisation of E-cadherin and β-catenin at the cell membrane. This was similarly observed in both HT-29 colon and CFPAC-1 pancreatic cancer cells. The increase in E-cadherin expression in PANC-1 cells in response to NDRG1 was mediated by the reduction of three transcriptional repressors of E-cadherin, namely SNAIL, SLUG, and ZEB1. To dissect the mechanisms of how NDRG1 inhibits nuclear SNAIL, SLUG and ZEB1, we assessed involvement of the NF-κB pathway, as its aberrant activation contributes to the EMT. Interestingly, NDRG1 comprehensively inhibited oncogenic NF-κB signalling at multiple sites in this pathway, suppressing NEMO, Iĸĸα, and IĸBα expression, as well as reducing the activating phosphorylation of Iĸĸα/β and IĸBα. NDRG1 also reduced the levels, nuclear co-localisation and DNA-binding activity of NF-κB p65. Further, Iĸĸα, which integrates NF-ĸB and TGF-β signalling to up-regulate ZEB1, SNAIL and SLUG, was identified as an NDRG1 target. Considering these latter findings, therapies targeting NDRG1 could be a new strategy to inhibit metastasis, and as such, we examined novel anti-cancer agents, namely di-2-pyridylketone thiosemicarbazones, which up-regulate NDRG1. These agents down-regulated SNAIL, SLUG and ZEB1 in-vitro and in-vivo using a PANC-1 tumour xenograft model, demonstrating their marked potential. Excitingly, the findings of Chapter 3 highlight the potential suitability of these agents as candidates to target NF-ĸB signalling in cancer. Chapter 4 | Considering the importance of high proliferation in cancer, studies next examined ZEB1 and its effects on the cell cycle and particularly mitosis. Microtubules are polymers of the alternating heterodimeric proteins, α/β-tubulin, that are required for mitotic spindle formation and the separation of chromosomes during mitosis. Microtubule organisation is regulated by microtubule-associated proteins (MAPs). For the first time, we describe a novel role for the epithelial gene repressor, ZEB1, which switches from a chromatin-associated protein during interphase to a MAP that associates with microtubule α- and β-tubulin during mitosis. Additionally, ZEB1 also associates with γ-tubulin, particularly with the microtubule organising center (MTOC). Using confocal microscopy, ZEB1 localisation was predominantly nuclear during interphase, with α/β-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during mitosis, ZEB1 and α/β/γ-tubulin co-localisation was significantly increased during prophase, metaphase, anaphase and telophase, with the association generally peaking during metaphase in a variety of tumour cell-types. Additionally, ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The interaction between ZEB1 and α-tubulin during mitosis was confirmed using the proximity ligation assay (PLA). In contrast to ZEB1, its paralog zinc finger E-box-binding homeobox 2 (ZEB2), was mainly perinuclear and cytoplasmic during interphase, showing some co-localisation with α-tubulin during mitosis. Considering the association between ZEB1 with α/β/γ-tubulin during mitosis, studies investigated ZEB1’s role in the cell cycle. Silencing ZEB1 resulted in a G2-M arrest, which could be mediated by the up-regulation of p21Waf1/Cip1 and p27 via de-repression of its known role as an epithelial repressor. However, it cannot be excluded that the G2/M arrest observed after ZEB1 silencing, is not due to its roles as a MAP. Collectively, ZEB1 appeared to play a role as a MAP during mitosis, and hence, could be functionally involved in this process. Chapter 5 | To expand upon Chapter 4, studies sought to further elucidate the context of ZEB1 in mitosis. Mitosis is a highly orchestrated event that requires specific protein interactions to co-ordinate appropriate chromosomal segregation, cytokinesis and passage through mitosis. A failure to do so can lead to tumourigenesis. ZEB1 has a well-established role as a contributor to an aggressive cancer phenotype, with links to high proliferation rates. Interestingly, ZEB1 has been reported to affect key aspects of proliferation. Namely, ZEB1 transcriptionally represses the expression of cell cycle control proteins, such as the cyclin-dependent kinase inhibitors, p21Waf1/Cip1, p27Kip1 and p15INK4b. In fact, silencing of these latter inhibitors resulted in a G2-M arrest of the cell cycle. In the previous chapter, studies demonstrated a novel switch in the intracellular localisation of ZEB1 between being a well-known chromatin-binding repressor in interphase, to a MAP associating with the microtubular architecture during mitosis. MAP proteins exist in complexes that are able to effect microtubule dynamics. Yes-associated protein 1 (YAP1) has an established role as a MAP that effects microtubule structure, formation and is critical for cytokinesis. Herein, we report the association of ZEB1/YAP1 at the mitotic spindle and at the cleavage furrow using confocal microscopy and the PLA assay. Additionally, the retinoblastoma protein (Rb) is an important tumour suppressor molecule for cell cycle regulation and has been previously shown to negatively regulate ZEB1 expression. Further, de-phosphorylation of Rb also associates with ZEB1, inhibiting its transcriptional activity during interphase. We examined the potential of ZEB1/Rb interaction in the context of mitosis and demonstrate co-localisation at the mitotic spindle as indicated by the Pearson’s coefficient r values and the PLA assay. Our findings also demonstrate ZEB1/Rb in the cleavage furrow, which may indicate a role for the ZEB1/Rb complex in cytokinesis. Thus, this study reports for the first time, newly discovered interactions between ZEB1/YAP1 and ZEB1/Rb during mitosis, which has implications for a role of these complexes in regulating microtubule dynamics. In summary, the findings of this thesis contribute to our understanding of how to potentially target NF-κB signalling and the ZEB1 molecule, which both contribute to the aggressive nature of many cancers. Further, this thesis also expands knowledge regarding the functional roles of ZEB1 which not only acts as a gene repressor, but also has a novel role as a MAP. These findings may aid in the development of novel treatment strategies that could improve cancer outcomes via targeting ZEB1 and more broadly NF-κB signalling using novel thiosemicarbazones