| Abstract |
Breast cancer is one of the most common types of cancer. On a global scale, it is the second most responsible cause for female death. Indicatively, in United Kingdom breast cancer represents 20% of all cancer types and is the main cause of death of women at the ages between 35-55 years old. In order to characterize different stages of this disease types the expression levels of Estrogen Receptors (ERs), Progesteron Receptors (PRs) as well as Epidermal Growth Factor Receptor (EGFR) are evaluated utilizing histopathological assessment.
Indeed, Insulin-like Growth Factor-I (IGF-I), Epidermal Growth Factor (EGF) and the hormone Estradiol (E2) have an important role in the development and biological behavior of breast cancer cells. Usually, the fist therapeutic step for breast cancer is hormone therapy. However, in hormone resistant tumor, cancer cell survival is promoted by developing alternative growth factor signaling pathways. Such a candidate signaling pathway is the IGF-I/EGF crosstalk. Moreover, particularly important for breast cancer is the interaction between the IGF-I/EGF axis and E2 signaling pathway which is a focus of study at a cellular level. The principal aims of the present PhD thesis include the investigation of IGF-I/EGF and E2 signaling pathways, breast cancer cell adhesion capability assessment and also, the identification of putative intracellular signaling mediators. In addition, the role of tumor suppression gene p53 was studied on cell adhesion and as a mediator of IGF-I receptor (IGF-IR) signaling. Finally, caffeine, a widely consumed substance, known to affect basic downstream signaling molecules of IGF-I/EGF and E2 pathways was investigated.
IGF-I/EGF and E2 regulate several estrogen- responsive breast cancer biological functions, including cellular proliferation and motility. Cell growth is influenced by the cellular microenvironment, the extracellular matrix (ECM). ECM interactions are fundamental for carcinogenicity. Thus, ECM modifications and molecular interactions between the ECM molecules and cancer cells modulate cellular functions. To be more accurate, ECM components are involved in cellular functions of tumor cells, affecting metastasis and consequently, cancer development. Fibronectin, an abundant ECM component in breast cancer tissues, is a high molecular weight glycoprotein and has been characterized as a crucial biomarker of aggressiveness. In the present study, IGF-I/EGF and E2 were demonstrated to increase adhesion of MCF-7 cell line onto fibronectin. In addition, IGF-I/EGF and E2 mediated adhesion required IGF-I Rparticipation. The above factors were shown to activate intracellular cancer-related signaling molecules which affect functions like proliferation, adhesion and motility. Such signaling mediators include Extracellular signal-regulated kinases (Erk1/2) that belong to Mitogen- Activated Protein Kinase (MAPK) family and Phosphatidylinositol 3-kinase (PI-3 kinase). Intracellularly, Erk1/2 inhibition prevents IGF-I/EGF/E2- dependent MCF-7 adhesion, indicating the importance of Erk1/2 as an intracellular signaling mediator of IGF-I/EGF/E2 mediated adhesion. Moreover, IGF-IR expression profile can be altered by Erk1/2 (full designation). Specifically, Erk1/2 pathway inhibition prevents both the expression and the activation of IGF-IR. Furthermore, IGF-IR mRNA and protein expression are sytongly enhanced by EGF and E2 action.
In addition, data using confocal microscopy showed that incubation with IGF-I and EGF induced microfilament reorganization of the MCF-7 actin cytoskeleton. This reorganization seemed to be modified by the inhibition of Erk1/2, which is the main cell mediator of IGF-I and EGF. Focal Adhesion Kinase (FAK), which is activated through phosphorylation during adhesion onto a respective substrate, was designated to play a crucial role in this adhesion study. It is important to mention that incubation with IGF-I induced the co-localization of IGF-IR and FAK, which was visible close to the cell membranes of MCF-7 cells. The later indicated that IGF-IR may be considered as a meeting point for the IGF-/EGF- and E2- dependent cell adhesion onto fibronectin.
Additionally, the possible role of the tumor suppressor gene p53 on the adhesion ability of MCF-7 breast cells was examined. Results of the present study showed that IGF-IR plays a key role in these cells’ adhesion. However, the activities of tumor suppressor genes can affect IGF-IR signaling and its role in breast cancer cells. The transcription of IGF-IR gene is negatively regulated by protein products of tumor suppressor genes, including the breast cancer 1 gene (BRCA1) and the p53 gene. Furthermore, the p53 gene loss of function has been shown to modify the expression of IGF-IR, and this mechanism did not include a direct binding on DNA. According to the present study, p53 did not modulate the expression of IGF-IR. Moreover, p53 did not affect the adhesion capacity of these cells onto fibronectin.
This PhD thesis also investigated the role of caffeine in breast cancer cell functions. Coffee is a beverage daily consumed on a world scale. Data that correlate coffee consumption and danger of breast cancer occurrence are controversial. The role of coffee concerning the risk of breast cancer occurrence differs due to the heterogeneity of the disease and ER expression based breast cancer categorization. Caffeine, whose chemical identity is 1,3,7-trimethylxanthine has in this study been used in its original in order to investigate breast cancer cell functions.
Previous studies have shown that treatment of breast cancer cell lines with caffeine affects cell proliferation. Caffeine was shown to exhibit anti-cancer properties, by decreasing growth of MCF-7 (ERα+) and MDA-MB231 (ERα-) cells. Moreover, it has been argued that caffeine acts as an anti-estrogen and modulates cell cycle pathways including ER/cyclin D1 and IGF-IR/pAkt. Thus, in the third part of the present thesis, cell lines with different ER status have been used. More specifically, the cell lines used were stably transfected MCF-7 cells that lack ERα expression, denominated MCF-7/SP10 (ERα-) cells and transfected with vector control MCF-7/csh cells (negative control). The results of the present study indicate that caffeine in a manner independent of ER status did not affect cell proliferation, but appeared to modulate cell adhesion though molecules that are related to IGF-I/EGF and E2 signaling pathways.
To summarize, the results of the present thesis suggest that IGF-IR plays a crucial role in IGF-I/EGF and E2 signaling pathways which modulate cell adhesion, function that is closely correlated to cell motility and cancer development. Furthermore, the thesis presents data associated to the role of the tumor suppressor p53 gene on breast cancer cell behavior. Data on the effect of caffeine on breast cancer cell functions and on the modulation of molecules that participate in the IGF-I/EGF and E2 signaling pathways are also shown. The presented data, may contribute to the development of targeted therapeutic methods for the treatment of breast cancer.
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