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1. Abstract
Breast cancer is the second most common cancer worldwide, and the one of the leading causes of cancer death in women. Due to recent advances in therapeutic approaches and early detection programs the breast cancer survival rate has seen a dramatic improvement. Consequently, it is now breast cancer recurrence, not the primary tumor, which is the leading cause of breast cancer related death. These relapses are due to malignant cells that survive treatment. Therefore, further characterization of the treatment-resistant cells is needed in order to improve the therapeutic approaches.
The overexpression of oncogenes drives tumorigenesis. In human breast cancer the epidermal growth factor receptor type-2 (Her2 also called Neu) is overexpressed in 20% of breast cancer, whereas the oncogenic transcription factor c-Myc is over-expressed in 16% of breast cancers. We model breast cancer through the use of an inducible mouse model in which the oncogenes NEU and c-MYC are under the control of a doxycycline inducible promoter. By administering a diet supplemented with doxycycline we are able to induce tumorigenesis, follow primary tumor development, and additionally inactivate the oncogene expression through withdrawal of doxycycline from the mouse diet. In essence, this would mimic a perfect therapeutic setting in which we are able to completely eradicate expression of the oncogenes, yet, these mice eventually succumb to tumor recurrence in the majority of cases. Therefore the aim of my thesis is to eliminate or disrupt the treatment-resistant cells so that they are unable to give rise to the tumor relapses even after the oncogene withdrawal.
We have taken an in vitro approach to screen candidates, which would interfere with the re- formation of the mammary gland following oncogene inactivation. To do so, we cultured primary mouse mammary epithelial cells, taken from the transgenic mice, in an in vitro 3D organotypic culture system. The structures, which grow in vitro closely, resemble the polarized, lumen filled acini which mammary glands are comprised of.
In these cultures, we tested different inhibitors for molecules which we confirmed were activated within the tumors derived from the c-MYC/NEU mouse model. Specifically, we tested inhibitors for Focal Adhesion Kinase, Integrin Linked Kinase, for signaling pathways, which regulate survival and membrane polarity as well an inhibitory antibody for E-cadherin bridges, which play an important role in establishing cell to cell contacts and cellular epithelial polarity.
Initial results of the in vitro assay show a decrease in the number of cells surviving following treatment with inhibitors. In order to determine the characteristics of the cells surviving treatment and their tumorigenic potential, we injected them into the cleared mammary fat pad of immunodeficient mice.
In the meantime using inducing a second round of induction in vitro we have seen that the survivors show reduced ability to re-induce. We also performed immunofluorescence staining on the treated cells to investigate the effect of the inhibitors on the re-polarized acinar structures.
The first indications from these experiments hint that introduction of inhibitors at the time of oncogene inactivation severely disrupts reconstitution of the polarized acinar mammary gland structure. A clear distribution, ie (basal/luminal) of the structural molecular components, ZO-1, E-cad was missing, suggesting an inhibition in the re-establishment of polarity in the surviving structures.
Further studies are necessary to conclude that these inhibitors are able to target the treatment- resistant cells, but our first promising results encourage this line of research.
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2. Aim of the thesis
The aim of my research work is to optimize primary tumor treatment to reduce the probability of tumor recurrence. We plan to affect the tumor cells that potentially survive withdrawal of the oncogenes by a synthetic lethal approach of targeted therapy and additional treatments at the same time point.
We intend to address the importance of basement membrane interactions for the survival of refractory cells. Although we do not yet know the mechanism by which cells survive and re- polarize after doxycycline withdrawal (for model see 2.Abstract), it is very likely that the placement of transformed cells at the rim of solid spheres –allowing interaction with a basement membrane— is a determinant of cell survival upon de-induction of oncogenes. Obvious targets of interference are integrin molecules, focal adhesion kinase (FAK) and downstream signaling pathways. We utilize blocking antibodies against E-Cadherin and small molecule inhibitors against FAK as well as ILK in a first approach. These treatments will be applied to solid spheres at the same time when they are weaned from doxycycline to induce apoptosis of the outer cell layer. Doxycycline naïve acini will serve as toxicity control.
Following these treatments, we will analyze remaining structures for their phenotype, overall number of surviving cells in comparison to controls and will test the remaining cells for their ability to give rise to relapses in the cleared fat pad of immune-compromised mice.
