Breast cancer is the second most common type of cancer among women in the US and the second leading cause of cancer death. HER2+ breast cancers represent ~20% of all cancer types, are highly invasive, and can be treated by using targeted therapies against the HER2 receptor. However, these therapies are challenged by the development of drug resistance, often induced by the presence of mutations in the cell-membrane proteins and receptors and/or by alternative signaling pathways that cross-talk with- or transactivate HER2+ triggered signaling. This study was aimed at investigating the cell membrane proteome of SKBR3 cells, representative of HER2+ breast cancers, and the signaling landscape and cellular responses elicited by the cell membrane receptors when the cells are stimulated with either growth factors or therapeutic drugs. It was hypothesized that the identification of a broad range of cell membrane proteins with roles in cancer progression and signaling crosstalk will lead to a more comprehensive understanding of the biological processes that sustain the proliferation of cancer cells, and will guide the selection of more efficient drug targets. The project was conceptualized in three stages: (1) profiling the cell membrane proteins of SKBR3 cells, (2) determining the functional role of the detected cell membrane proteins in the context of cancer hallmarks and exploring their mutational profile, and (3) analyzing the cellular events that occur in response to treatment with a single therapeutic agent or a combination of drugs. Mass spectrometry technologies were used for performing proteomic and phosphoproteomic profiling of SKBR3 cells, detecting changes in the abundance of the detected proteins, and identifying the presence of mutations in the cell membrane proteins. Orthogonal enrichment methods were developed for profiling the low-abundance cell membrane proteins, for generating a rich landscape of cell membrane receptors with various functional roles and relevance to the cancer hallmarks, and for enabling the detection of potentially new drivers of aberrant proliferation. The analysis of serum-starved, stimulated (with growth factors), or inhibited (with kinase inhibitors) cells revealed alternative protein players and crosstalk activities that determine the fate of cells, and that may fuel the development of resistance to treatment with drugs. The proteome profiles that were generated in this project expand the opportunities for targeting cancer-relevant processes beyond proliferation, which is commonly attempted, broadening the landscape to also include apoptosis, invasion, and metastasis. Altogether, the findings that emerged from this work will lay the ground for future studies that aim at developing more complex and effective targeted cancer treatment approaches. / Doctor of Philosophy / Breast cancer is one of the most common cancers among women in the US and the second major contributor to cancer-related deaths. Several therapies that have been developed for the treatment of cancer target the HER2 receptor, which is overexpressed in ~20% of breast cancers and results in a highly invasive cancer phenotype. However, most patients receiving these therapies observe cancer reoccurrence within a year due to the development of resistance to the therapeutic drug. The current challenge stands in identifying novel protein targets, and in developing new therapies that can be used in combination with the existing approaches to eradicate cancer. Research has indicated that proteins located at the cell membrane play crucial roles in cancer progression and invasion due to their involvement in cell response to stimuli and in initiating signaling cascades within the cell. Knowledge about the cell membrane proteins of HER2+ breast cancer cells is limited due to the challenges associated with their isolation. Therefore, this project was aimed at profiling the cell membrane proteins of HER2+ breast cancer cells, and their intra-cellular signaling activity, to provide insights into the behavior of these cells and to support the identification of potentially novel drug targets. The three objectives of the work were to (1) isolate the cell membrane proteins through various approaches using cell culture conditions that would encourage or discourage cancer cell growth, (2) identify the cancer-relevant signaling pathways and processes represented by the detected cell membrane proteins, and (3) investigate the behavior of cancer cells when treated with drugs. To approach these objectives, a powerful analytical technology, called mass spectrometry, was utilized. Mass spectrometry can accurately and simultaneously detect the presence of the proteins in a biological sample. Our study identified cell membrane proteins that are involved in cancer progression through various signaling pathways, and how these proteins interact with each other to drive the behavior of cells. The study also provided insights into how cancer cells respond when they are treated with various drugs, uncovering to the scientific community a variety of proteins with potential therapeutic value. Lastly, this study sheds light on the complex biology of breast cancer and highlights the importance of continued research to develop more effective treatments.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/115309 |
| Date | 02 June 2023 |
| Creators | Karcini, Arba |
| Contributors | Biological Sciences, Lazar, Maria Iuliana, Hsu, Bryan, Hauf, Silke, Chen, Jing |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | Creative Commons Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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