Dissertation
Utilization of forward genetic approaches to elucidate mechanisms of cancer initiation and drug resistance
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2020
DOI: 10.17077/etd.005285
Abstract
Cancer is responsible for almost 10 million deaths worldwide per year and there are 17 million new cancer diagnoses made during the same time period. Fortunately, we have made great strides in advancing cancer therapy from broad-spectrum, toxic chemotherapies that kill all fast dividing cell to highly targeted therapies that specifically inhibit oncogenic mechanisms, leading to reduction of side effects and increased survival. And most recently, we are utilizing the patients’ own immune system to kill cancer cells. Unfortunately, some patients with certain cancer types have not benefited as much from these targeted therapies—such as liver cancer—or have developed resistance to the targeted and immunotherapies—as is the case for Raf inhibitor therapy for melanoma. Thus, there is a crucial need for the identification of factors contributing to cancer development and subsequent drug resistance in these malignancies.
I have approached both of these needs by utilizing the Sleeping Beauty (SB) transposon system to perform forward genetic mutagenesis screens in both mouse models and human cell culture models of cancer. The SB system uses an oncogenic transposon to create a random population of mutagenized cells, recapitulating the stepwise accumulation of mutation in which cancer arises. Evolutionary pressures either in a mouse model of cancer initiation or in cells under a drug selection allow selective clonal expansion of cells harboring advantageous mutations. Identification of common mutational events in the tumor or resistant cell population leads to a list of genes implicated in driving tumorigenesis or drug resistance.
For one project, I used a mouse model of hepatocellular carcinoma (HCC) that mimics the specific microenvironment that enriches for HCC development to identify novel genes involved in tumorigenesis. One gene, Gli2, is a transcription factor in the Hedgehog signaling pathway and when overexpressed drives HCC development in mice. Additionally, GLI2 is highly expressed in both damaged human liver and human HCCs, potentially demonstrating a role for the protein in proper liver regeneration and repair but also in HCC development. Another gene identified as a candidate driver of HCC, Fign, is a microtubule severing ATPase that is also overexpressed in a subset of human HCC. When overexpressed in hepatocytes, Fign promotes migration and invasion towards growth factors, demonstrating an oncogenic and metastatic-promoting role. These phenotypes however are dependent on the ability of Fign to sever microtubules, suggesting that Fign could potentially be a beneficial therapeutic target in a subset of HCC.
For my second project, I aimed to address clinically relevant drug resistance issues by identifying mechanisms human cancer cells use to develop resistance to cancer therapies. To do this, I created a novel genetic screening method to perform transposon mutagenesis screens in human cells using the SB system. Utilizing this new method, I performed a screen to identify mechanisms human BRAFV600-mutant melanomas employ to drive resistance to targeted BRAF and MEK inhibitors. Resistance drivers identified include both known and unexplored resistance mechanisms including a DBL guanine exchange factor (GEF)-driven resistance mechanism. However, there is significant heterogeneity in the resistance mechanisms across cell lines, demonstrating the need for robust analysis of these mechanisms across a panel of cell lines or patient tumors to best recapitulate the mechanisms present in human populations.
I have also focused on discovering therapeutic ways to target these identified mechanisms of resistance. The GEF-driven mechanism activates the RAC/PAK pathway and potentially other signaling pathways to reestablish ERK signaling and proliferation in the presence of BRAF and MEK inhibitors. Fortunately, I have identified that the combination of a Raf dimer blocker and a Src inhibitor can abrogate most resistance mechanisms identified in our screens, demonstrating a viable drug regimen for treating a subset of BRAF inhibitor resistant patients.
Details
- Title: Subtitle
- Utilization of forward genetic approaches to elucidate mechanisms of cancer initiation and drug resistance
- Creators
- Charlotte Rose Feddersen
- Contributors
- Adam J Dupuy (Advisor)John F Engelhardt (Committee Member)Michael D Henry (Committee Member)Dawn E Quelle (Committee Member)Tina L Tootle (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Anatomy and Cell Biology
- Date degree season
- Spring 2020
- DOI
- 10.17077/etd.005285
- Publisher
- University of Iowa
- Number of pages
- xiii, 165 pages
- Copyright
- Copyright 2019 Charlotte Rose Feddersen
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 142-159).
- Public Abstract (ETD)
Cancer is a disease driven by mutations in genes that allow normal cells to grow too fast and outcompete the neighboring cells, forming a tumor that can destroy the normal tissue that is meant to be there. These mutations can also allow cancer cells to travel to other areas of the body and destroy tissue there. Even when we find a drug that kills cancer cells, mutations can cause the cancer cells to develop resistance to these drugs, similar to how bacteria can develop resistance to antibiotics.
In order to treat cancer more effectively, we need to identify what mutations allow cancer to develop, migrate, and acquire drug resistance. When we identify these mutations, we can design new therapies to specifically target these mutations. For some cancers, identifying these mutations is difficult due to a variety of technical and biological reasons, so we use other models to more easily identify these mutations. My research uses two models to identify mutations that cause liver cancer and mutations that cause drug resistance in melanoma.
My results show that some human liver cancers may be driven by increased levels of a protein called FIGN that makes liver cells more able to move towards nutrients and invade into areas where they are not meant to be. Future research will explore if we can target this protein with a drug to prevent it from promoting movement and invasion.
My results also show that melanoma uses many different ways to acquire resistance to a common drug called vemurafenib that is used clinically to treat advanced melanoma. However, I show that if a strong Raf inhibitor and a Src inhibitor (two gene families that are known to drive cancer) are combined, I can prevent many of the resistance mechanisms melanoma uses. Further research is needed before we can try this combination therapy in clinic; however, it is a promising therapy since both inhibitors are already being used in the clinic as single agents.
- Academic Unit
- Anatomy and Cell Biology
- Record Identifier
- 9983966299002771
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