Defense Date


Document Type


Degree Name

Master of Science



First Advisor



Genomic instability and acquisition of invasiveness through the basement membrane extracellular matrix (ECM) are two major processes for epithelial cell malignancy in breast cancer. DNA double-strand break repair (DSBR) is one of the processes that get misregulated during breast cancer progression. In addition, radiation induced breaks such as those induced during radiation therapy to treat breast cancer patients are repaired by DSBR, rendering this pathway relevant for therapy as well. DSBR can occur either by homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is accepted as the more error-free pathway. HR is regulated by the cell cycle status such that an increase is observed in G2/M, whereas NHEJ is observed throughout the cell cycle. Previous data show that ECM signaling regulates HR, as well as the kinetics of ionizing radiation (IR) induced complex formation at break sites, or foci kinetics. Both human breast epithelial cell lines and primary mouse mammary epithelial cells were used to show that the ECM receptor β1-integrin is necessary and sufficient in down regulating HR, as well as IR induced foci formation kinetics for the DSBR proteins RAD51, MRE11, and γ-H2AX in single mammary epithelial cells. RAD51 is required for most HR, whereas MRE11 and γ-H2AX function in HR as well as DNA damage signaling. Interestingly, ECM signaling up-regulates HR in cells that have “correct” in vivo-like cell-cell junctions. Based on the observation that single cells and junctioned cells respond to ECM in exact opposite manner, I hypothesized that ECM signaling may interact with cell-cell junction signaling pathways in regulating DNA repair. To test this hypothesis, I asked whether the main breast epithelial adherens junction cadherin, E-cadherin, is involved. I blocked E-cadherin function using a monoclonal antibody MB2. The function blocking was demonstrated by the loss of cell-cell junction interactions and observation of increased cell scattering using phase microscopy. I then asked whether blocking E-cadherin altered the expression and localization of proteins related to DNA repair. Indirect immuno-fluorescence showed that in the E-cadherin blocked non-tumorigenic breast epithelial cell line HMT-3522 S1 there is an up-regulation of nuclear γ-H2AX and RAD51, as well as an increase in the proliferation marker Ki67. In non-proliferative MB2 blocked cells there is an upregulation of γ-H2AX and reduced Ki67. Furthermore, in these proliferative and non-proliferative blocked cells we were able to see lower levels of β-catenin near the cell membrane and an increase in its levels inside the cell especially in the nucleus. The latter has been confirmed also by western blot technique comparing the nuclear and cytoplasmic fraction expression. In addition, western blots showed that total RAD51 level was down-regulated by E-cadherin blocking and γ-H2AX levels were found to be higher in proliferative and non-proliferative MB2 treated cells. MB2 treated cells have a higher frequency of HR in the absence of ECM and in the presence of ECM, MB2 blocking abolishes the ECM effect on HR. Furthermore, in the absence of ECM, RAD51 siRNA treated cells down-regulated HR but the absence of RAD51 did not down regulate HR in the presence of ECM. I was not able to see any difference in the phosphorylated forms of β-catenin such as Tyr-142, Ser-45 and Tyr-86 that has the ability to enter into the nucleus. Therefore, E-cadherin was found to block nuclear β-catenin, RAD51 and γ-H2AX in a proliferation-independent manner. E-cadherin also was necessary for ECM to up-regulate HR. The up-regulation of HR by ECM was only slightly dependent on RAD51 suggesting a novel E-cadherin-dependent and RAD51-independent HR component in breast epithelial cells in contact with ECM as they are in vivo in the normal breast tissue. These experiments will help us to understand the role of E-cadherin and β-catenin in DNA double-stand break repair directly, as well as in combination with ECM signaling. Both alterations in integrin mediated signaling and cell-cell junction integrity contribute to breast cancer progression by rendering breast epithelial cells more invasive. My project will shed light on whether these invasive processes also alter DNA repair and contribute to genome stability. Understanding of the interrelationships among integrin signaling, cell-cell junctions, and genome stability will contribute to understanding normal breast cell processes and open up investigations on how these may go awry in cancer progression.


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