Breast cancer is the most prevalent form of cancer in women and accounts for 519,000 annual deaths (WHO Statistics). It has long been established that oestrogen (E2) stimulates tumour growth of oestrogen receptor (ER) positive breast cancer and is involved in the pathogenesis of the disease. Consequently, therapeutic approaches targeting the ER were developed. The use of endocrine therapy is an integral component in treating breast cancer however resistance to such drugs is a major limitation. Unfortunately, even initially responding tumours eventually develop resistance - acquired resistance. The aim of this study was to determine which intracellular pathways may be important in conferring acquired endocrine resistance. In order to do so, a three-stage MCF-7 cell model emulating the clinical development of acquired endocrine was used. MCF-7/LCC1 (LCC1) and MCF-7/LCC9 (LCC9) cells lines were derived from the oestrogen dependent and antioestrogen sensitive MCF-7 cell line. LCC1 cells remain responsive to endocrine therapies but their growth is not dependent on oestrogenic stimulus. LCC9 cells, on the other hand are fully resistant to endocrine therapies and completely oestrogen independent. A number of different cell membrane receptors and intracellular pathways have been implicated in endocrine resistance including HER receptor family, PI3K/Akt & MEK/ERK pathways. These pathways are of particular interest since they are able to activate ER in the absence of oestrogenic stimulus. It is likely that several pathways may be important in conferring resistance to endocrine therapies therefore the experiments in this study focussed on the transcriptional regulation of HER receptors, the activation of the Akt pathway and its implication to basic cellular processes. Following E2 treatment (48h), HER2/3/4 mRNA and protein levels were reduced in MCF- 7 and LCC1 but not in the endocrine-resistant LCC9 cell line as measured by QRT-PCR and Western blotting. The anti-estrogen fulvestrant (ICI 182,780) reversed the E2 modulation. A previous study has shown that ER and the HER2 promoter compete for limiting amounts of SRC-1 in oestrogen-responsive ZR-75-1 cells, causing HER2 repression after E2 stimulation (Newman et al.,Oncogene, 19, 490-7, 2000). ER RNAi abolished E2 repression of HER2 in MCF-7 and LCC1 cells. Furthermore, LCC9 cells have reduced SRC-1 recruitment to ER (assessed by ChIP) allowing SRC-1 to bind to the HER2 promoter. SRC-1 RNAi reduced HER2 transcription in MCF7 cells in a manner similar to E2 whilst it did not restore E2 repression in LCC9 suggesting that the latter cells have alternative mechanisms regulating HER2 transcription. RNAis against the other two p160 co-activators TIF2 and AIB1 did not restore E2 mediated HER2 repression in LCC9 cells. The importance of redundancy between p160 co-activators was also determined by performing double knockouts. SRC-1/TIF2 and TIF2/AIB1 double siRNAs had little effect on HER2 mRNA levels however SRC-1/AIB1 siRNA restored oestrogen mediated downregulation of HER2 transcription in LCC9 cells. This data indicates that SRC-1 and AIB1 co-activators play a role in the transcriptional regulation of HER receptor particularly in MCF-7 and LCC1 cells. The regulation of this transcriptional mechanism is altered in resistant LCC9 cells but, as evidenced by the double knockouts, p160 coactivators are still able to affect HER expression in these cells. This mechanism was further studied in primary breast cancer tumour material. The importance of the Akt pathway in this cell line model was also investigated as phospho-Akt levels are elevated in LCC1 and LCC9 cells. This in turn was shown to activate mTOR and ER (Ser167 residue phosphorylation) thereby contributing to increased growth and ligand independent activation of the oestrogen receptor respectively. Activation of PI3K and PTEN is unchanged in LCC1 and LCC9 cells suggesting that these proteins are not responsible for elevated Akt phosphorylation. In contrast, these cells do express higher levels of phospho-IGFR due to the high availability of receptor ligands (IGFI & IGFII). This is likely to be, at least partially, responsible for the elevated Akt activation. Moreover, the role of Akt isoforms was also determined as they are known to have different functions. The levels of Akt 2 phosphorylation are higher in endocrine resistant cell lines in comparison to parental MCF-7 cells. Interestingly, the Akt 3 phosphorylation is present in all cell lines whilst Akt 1 phosphorylation is minimal. Nevertheless, Akt RNAi studies reveal that Akt 1 and 2 siRNA dramatically reduce growth in MCF-7, LCC1 and LCC9 cells. These results suggest that Akt 2 phosphorylation may play a part in conferring endocrine resistance but the other isoforms are also important for normal cellular growth. The cell cycle profiles of LCC1 and LCC9 are very similar to MCF-7. Similarly, migration levels are unchanged in endocrine resistant cell lines. However, in the presence of antioestrogenic drugs, apoptosis in LCC1 and LCC9 cells in reduced in comparison to the parental MCF-7 cell line. Furthermore, LCC1 and LCC9 cells have higher invasion rates. The deregulation of HER receptor expression and elevated Akt activation may together confer survival advantage in LCC1 and LCC9 cells whilst also increasing their invading potential.

The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells. / Doctor of Philosophy (Ph.D.)

A interação entre as vias de sinalização dos estrógenos e fatores de crescimento está relacionada a maior agressividade dos tumores de mama. Assim, o objetivo deste trabalho foi verificar a interação entre as vias do E2 e do EGF no controle da expressão dos genes HNRPK, PAWR e PHLDA1 nas células MCF-7 (ER+) e MDA-MB-231 (ER-). Nas células MCF-7, o EGF e o E2 diminuíram a expressão do PAWR e aumentaram a expressão de PHLDA1. A inibição do ER pelo ICI resultou no aumento da expressão de PAWR sendo que a adição do E2 ou do EGF diminuiu sua expressão sem retomar os níveis observados nos tratamentos com E2 ou EGF isoladamente. Para o gene PHLDA1, o efeito do E2 e do EGF não variou após o tratamento com ICI. Nas células MDA-MB-231, a ação do EGF foi mais efetiva sobre a expressão de PAWR. A via ERK1/2 é importante na ativação do ER pelo EGF. O efeito do EGF sobre o PHLDA1 ocorre através da ativação das vias ERK1/2 e p38 MAPK. Estes resultados mostram a interação entre as vias do E2 e do EGF no controle da expressão do PAWR, mas não do PHLDA1. / The crosstalk between estrogens and growth factors pathways has been associated with breast cancer aggressiveness. Based on this, the present study aimed to determine the possible crosstalk between E2 and EGF pathways on the HNRPK, PAWR and PHLDA1 expression regulation in MCF-7 (ER+) and MDA-MB-231 (ER-) cells. In MCF-7 cells, treatments with E2 and EGF decreased PAWR expression and increased PHLDA1 expression. The ER inhibition by the ICI treatment resulted in increased PAWR expression. The E2 or EGF addition down-regulated its expression without a return to the levels observed after the E2 or EGF treatments alone. To the PHLDA1 gene, the effect of E2 and EGF treatments did not change after the ICI treatment. In MDA-MB-231 cells, the EGF effect was more significant on the PAWR gene expression control. The ERK1/2 pathway is important to the ER activation by EGF. The EGF effect over the PHLDA1 is dependent on the ERK1/2 and p38 MAPK activation. These findings suggest a crosstalk between E2 and EGF pathways on the PAWR expression control but not to PHLDA1.

A interação entre as vias de sinalização dos estrógenos e fatores de crescimento está relacionada a maior agressividade dos tumores de mama. Assim, o objetivo deste trabalho foi verificar a interação entre as vias do E2 e do EGF no controle da expressão dos genes HNRPK, PAWR e PHLDA1 nas células MCF-7 (ER+) e MDA-MB-231 (ER-). Nas células MCF-7, o EGF e o E2 diminuíram a expressão do PAWR e aumentaram a expressão de PHLDA1. A inibição do ER pelo ICI resultou no aumento da expressão de PAWR sendo que a adição do E2 ou do EGF diminuiu sua expressão sem retomar os níveis observados nos tratamentos com E2 ou EGF isoladamente. Para o gene PHLDA1, o efeito do E2 e do EGF não variou após o tratamento com ICI. Nas células MDA-MB-231, a ação do EGF foi mais efetiva sobre a expressão de PAWR. A via ERK1/2 é importante na ativação do ER pelo EGF. O efeito do EGF sobre o PHLDA1 ocorre através da ativação das vias ERK1/2 e p38 MAPK. Estes resultados mostram a interação entre as vias do E2 e do EGF no controle da expressão do PAWR, mas não do PHLDA1. / The crosstalk between estrogens and growth factors pathways has been associated with breast cancer aggressiveness. Based on this, the present study aimed to determine the possible crosstalk between E2 and EGF pathways on the HNRPK, PAWR and PHLDA1 expression regulation in MCF-7 (ER+) and MDA-MB-231 (ER-) cells. In MCF-7 cells, treatments with E2 and EGF decreased PAWR expression and increased PHLDA1 expression. The ER inhibition by the ICI treatment resulted in increased PAWR expression. The E2 or EGF addition down-regulated its expression without a return to the levels observed after the E2 or EGF treatments alone. To the PHLDA1 gene, the effect of E2 and EGF treatments did not change after the ICI treatment. In MDA-MB-231 cells, the EGF effect was more significant on the PAWR gene expression control. The ERK1/2 pathway is important to the ER activation by EGF. The EGF effect over the PHLDA1 is dependent on the ERK1/2 and p38 MAPK activation. These findings suggest a crosstalk between E2 and EGF pathways on the PAWR expression control but not to PHLDA1.