Medicine (St Vincent's)

Breast cancer is the most frequently diagnosed malignancy in females and accounts for the highest cancer related mortality worldwide. According to the available statistics, more than 90% of breast cancer related deaths occur not because of the primary tumour but as a result of the secondary metastases. Therefore, understanding the precise mechanisms involved in the progression of secondary metastases from the primary tumour is of utmost importance as a prognostic indicator for estimating metastatic risk, deciding the treatment plan, and monitoring of treatment response. The process of Epithelial to Mesenchymal Transition (EMT) has been widely accepted as a major mechanism that is involved in the metastatic cascade. The aim of the current thesis was to explore the effects of epithelial versus mesenchymal manipulation of breast cancer cells on the sequential events of metastatic progression. Since it is widely accepted that one of the cardinal features during EMT is loss or down-regulation E-cadherin, we modulated this molecule in our experimental model, the MDA-MB-468 (MDA-468) breast cancer cell line, to generate cells with a stable forced epithelial phenotype and epithelial cells with stable mesenchymal traits. The E-cadherin overexpressing cells were generated by transfecting MDA-468 cells with a plasmid vector encoding full length E-cadherin (468-CDH1), while dominant negative E-cadherin expressing cells were produced by transfecting the cells with a plasmid vector encoding a chimera of the E-cadherin cytoplasmic tail connected to the interleukin 2 receptor extracellular domain (468-dnCDH1). In addition, E-cadherin knocked down cells were generated using short hairpin technology (468-shCDH1-B and 468-shCDH1-D). The EMT-inducing effects of epidermal growth factor (EGF) and hypoxia were characterised in terms of EMT marker expression, morphology, proliferation, and migration. These assessments were also performed on the E-cadherin manipulated cells and also to examine the effects of these manipulations on EGF response. The effective introduction of each manipulation was confirmed, however, very little effect on EMT marker expression was seen. Morphology was affected by the shCDH1-mediated knock down, but not other treatments. Cell migration was inhibited by CDH1 transfection and stimulated in the most complete shCDH1 knock down, as was invasive colony outgrowth. Opposite trends on proliferation were noted, with tendencies for higher proliferation with more functional E-cadherin, and reduced proliferation in shCDH1 cells. Importantly, the published effects of EGF on apoptosis of MDA-468 cells were not seen, and instead an intense but reversible EMT was seen in the cells that became detached. Despite the lack of constitutive effects on EMT marker status, sufficient behavioural responses were seen to warrant in vivo analysis. When these E-cadherin manipulated cells were inoculated into the mammary fat pad of SCID mice, the cells in which E-cadherin was optimally knocked down (468-shCDH1-B) formed significantly slower growing, smaller tumours compared to the vector control while the growth rate was also diminished, albeit less dramatically, in the moderately E-cadherin knocked down (468-shCDH1-D) cells. The 468-dnCDH1 cells did not develop tumours efficiently and those that formed displayed a significantly slower growth than the vector control. This contradicts the accepted knowledge that loss of E-cadherin is indicative of aggressive tumour behaviour. However, there is a possibility that these tumours are associated with late diagnosis due to their smaller size, escaping chemotherapeutics due to their slower proliferation rate, yet being capable of forming secondary metastases, thus indeed being more aggressive. In contrast, the 468-CDH1 tumours grew marginally faster than the vector control. Overall, the results are suggestive of E-cadherin facilitating the establishment of MDA-468 primary tumours thus indicating the necessity of re-evaluation of the role of E-cadherin other than its function as a tumour suppressor. All tumour groups, except 468-shCDH1-B, displayed typical histological features of MDA-468 tumours, including widespread central necrosis surrounded by a thin rim of viable tumour tissue while the degree of necrosis was significantly lower in 468-shCDH1-B group. Vimentin positive cells, depicting EMT was seen at the invasion front and the tumour-necrosis border, whereas in 468-shCDH1-B tumours almost all cells were vimentin positive, in contrast to the lack of constitutive effects in vitro. Thus, E-cadherin knockdown promoted an intense EMT in vivo, while hypoxia and stromal influences also induced EMT in the MDA-468 cells. The local lymphovascular invasion (LVI) was a common feature associated with all tumours, and the majority of invaded tumour cells were E-cadherin positive, including the tumour emboli seen in 468shCDH1. A gradual transition from a vimentin positive mesenchymal phenotype, to concurrent vimentin and E-cadherin positive metastable status, to an E-cadherin-expressing epithelial phenotype was observed in some LVI, highlighting the occurrence of EMT reversal through mesenchymal to epithelial transition (MET). These observations provide evidence of an extremely early MET in metastatic progression. Observation of E-cadherin positive tumour cells in both local and distant lymph nodes also further emphasise the mechanism MET during tumour progression. Lung metastases were common across all tumour groups and although no significant difference was observed among the groups despite their different E-cadherin status, an obvious trend was seen towards primary tumours with E-cadherin expression having an advantage for forming secondary deposits in the lungs. The overall findings of the current thesis suggest that EMT is a transient process that rather swiftly reverts through MET during the establishment of secondary tumours even at the primary site. These studies highlight the requirement for a re-appraisal of the precise role of E-cadherin in tumour progression.

Epithelial to mesenchymal transition, EMT, is a developmental process that may be adopted by tumour cells to facilitate metastasis. EMT changes resulting in a more mesenchymal phenotype have been associated with a “migrating stem cell” pattern in breast and other carcinoma cells. PMC42 human breast cancer cells can be induced by epidermal growth factor (EGF) to a more mesenchymal state and adopt cancer stem cell characteristics. This study investigated the cancer stem cell properties in EGF-induced EMT in the PMC42 system, which consists of the predominantly mesenchymal PMC42-ET cell line and its epithelial-like subline, PMC42-LA. Quantitative reverse transcriptase polymerase chain reaction, qRT-PCR, was used to measure relative gene expression levels following EGF-induced EMT ofPMC42 cells in two- and three-dimensional cell cultures. Stem cell properties were evaluated through immunocytochemistry and two-channel fluorescence-activated cell sorting (FACS). CD44+/CD24- expression, a marker of breast cancer stem cells (BCSC), was measured after EGF-induced EMT. Ten ng/mL EGF treatment increased the number of cells expressing CD44+/CD24- in PMC42-ET but not in PMC42-LA cells as shown by FACS analysis. A mammosphere formation assay, which measures the ability of cells to form complex structures, was used to assess the functionality of the stem like properties exhibited by the different cell populations. This evidence of stemness was found in the BCSC-like population (CD44+/CD24-) of PMC42-ET cells. Expression of embryonic stem cell markers such as ABCG2 and ITGA6, EMT-BCSC related genes generated from EMT and BCSC bioinformatic comparison, and CD24-correlated genes (SerpinE1, EMP1, EMP3, AXL and VIM) were examined in the PMC42 system. EGF-induced EMT increased those gene expressions in the PMC42 system. PLP2 and VIL2, components of the Invasiveness Gene Signature derived from BCSC (Liu et al., 2007), showed no change in expression during EGF-induced EMT of PMC42. In conclusion, transcriptional changes demonstrating an increased stem-like or BCSC-like characteristic was observed in the PMC42 system following an EGF induced EMT. These results suggest the existence of a subpopulation of PMC42 cells which inherently resemble BCSC and provide new insights into the potential role of these subpopulations in EMT-associated metastasis.

Medicine (St Vincent's) - Theses

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