Thesis

Structure/Function Studies of PEAK1 Subcellular Localization and Secretome-Inducing Capacity in Stem Cells

Breast cancer is one of the most common cancers among women and is responsible for ~14% of cancer mortality in women. Critically the tumor microenvironment and specifically the stromal portion is increasingly being recognized as a critical factor in cancer development, progression, and metastasis (which is the leading cause of cancer mortality). Multiple reports have recently highlighted the importance of a mesenchymal stem cells (MSCs) as being an important catalyst for cancer progression. It has been shown that PEAK1 (Pseudopodium Enriched Atypical Kinase 1) is a crucial factor in breast cancer and works to act as a switch to shift that works in concert with TGFβ and fibronectin to promote tumor progression. Interestingly, it has been shown that PEAK1 is highly expressed within the tumor stroma and specifically within MSCs and fibroblasts. Using an in vitro model, we sought to identify what domains of PEAK1 are capable of localizing to the actin cytoskeleton within these two cell systems. With the use of multiple N and C terminus truncations we demonstrate that PEAK1 is capable of localizing to the actin cytoskeleton when the ATR is intact. Another aim was to identify if MSCs overexpressing PEAK1 levels might lead to a secretome profile promoting in cancer proliferation, and if a knockdown of PEAK1 may lead to a loss of the proliferative capabilities of the MSC ix secretome, While overexpressing PEAK1 in a transient fashion and permanent knockdowns did not show an corresponding increase or decrease in the rates of proliferation in Breast Cancer Cells (BCCs) further work such as a constant overexpression of PEAK1 is needed to validate what effect (if any) PEAK1 has on the secretome of the stromal cell population) To identify whether high PEAK1 levels within the tumor stroma promote cancer development we utilized in vitro methods of assessing cell proliferation. Specifically, we utilized conditioned media (CM) from MSCs that had either been transfected with constructs to overexpress PEAK1 or been transduced with a vector to knockdown PEAK1. By utilizing this CM we found that the secretome profile of MSCs with high or low PEAK1 do not significantly alter HER2+ BCC proliferation.

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