Dissertation
Elucidating mechanisms that enhance metastatic phenotypes through pharmacological ascorbate and fluid shear stress resistance
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2025
DOI: 10.25820/etd.008072
Abstract
The motivation for initiating Chapter 2 was due to the clinical outcomes in the Phase I and II PDAC pharmacological ascorbate (P-AscH) trials. Patients treated with intravenous injections of P-AscH in combination with standard-of-care therapy demonstrated a significant increase in overall survival and progression free survival. Despite these encouraging results, approximately 30% of patients did not respond to this treatment regimen. To elucidate mechanisms of resistance to P-AscH, I developed two pancreatic cancer cell lines. Despite originating from the same cancer type, the two P-AscH resistant (AR) cell lines developed unique mechanisms to tolerate treatments to P-AscH.
To take an unbiased approach in identifying pathways which may confer P-AscH resistance, I conducted transcriptomic analysis on the AR cell lines. While the two AR cell lines displayed differences in redox regulation, both lines exhibited a substantial increase in epithelial- to-mesenchymal transition (EMT) gene signature. We further assessed the in vivo relevance and found that AR cells demonstrated significantly enhanced tumor colonization. Our findings demonstrate distinct mechanisms for AcsR cells to tolerate high doses of P-AscH. Although catalase has historically been demonstrated as a key factor in controlling redox flux, catalase may not be necessary for all cell types. In summary, our data indicate a potential route for pancreatic cancer to tolerate high levels of P-AscH- and may explain how some patients do not respond to this treatment regimen.
In Chapter 3, I explored the effects of fluid shear stress (FSS) exposure on cancer cells. During metastasis circulating tumor cells (CTCs) are exposed to hemodynamic forces, such as fluid shear stress (FSS). Recently we showed that viable CTCs resist mechanical destruction when exposed to FSS by a mechano-adaptive mechanism that depends on RhoA-actomyos activity. Since the RhoA-actomyosin axis is implicated in other cellular behaviors that might contribute to metastasis, we explored the hypothesis that exposure to FSS alters the biology of viable CTCs to promote metastatic colonization.
Our findings demonstrate that brief pulses of FSS exposure can enhance invasive behavior, anchorage-independent proliferative capacity, metastatic colonization, and rapidly alter the metabolome. The phenotypic changes driven by exposure to FSS depend, in part, on RhoA activation but may also reflect other mechanisms by which cancer cells respond to FSS. In summary, our data indicate that FSS exposure in the circulation not only represents a physical barrier that CTCs must overcome to survive, but it also provides instructive cues that may enhance the metastatic behavior of some CTCs.
Chapter 4 represents a convergence of findings and exploration of novel pathways relevant to chapters 2 and 3. I conducted two independent Sleeping Beauty (SB) mutagenesis screens to confer drivers to 1) pharmacological ascorbate resistance and 2) fluid shear stress resistance. The SB screen using P-AscH as the selection agent revealed ASAH1 as a potential candidate for P-AscH resistance. ASAH1 encodes for acid ceramidase (AC) which has been shown to alleviate redox stress and inhibit apoptotic pathways during cancer progression. This transcript has also been implicated in regulating the cytoskeleton and enhance cellular stiffening, which may have additional implications towards FSS resistance phenotypes.
In the screen conducted with FSS as the selection agent, CRYBG1 was identified as a resistance candidate. Indeed, CRYBG1 did robustly enhance FSS resistance in both PANC-1 and MIA cell lines. Notably, previous research suggests that CRYBG1 can be a tumor suppressor in prostate cancer and melanoma preventing the metastatic phenotype from emerging. Despite these reports, my results suggest that CRYBG1 not only can enhance metastatic phenotypes such as anchorage-independent growth, but also can confer cross-resistance to high doses of P-AscH treatments. The mechanisms by which CRYBG1 is active during FSS and P-AscH exposure need to be further developed, however these findings highlight novel mechanisms which are not currently being studied.
In the Appendix, I discuss specific mechanisms by which treatments of P-AscH can regulate the cell cycle, which differs between normal and tumorigenic cell lines. This work is some of the first to explore alterations to biological pathways outside redox mechanisms. These insights provide supplemental insights that complement the work in Chapter 3.
Collectively, this thesis provides new insights into mechanisms that may drive metastatic tumor progression. The pathways upregulated in response to P-AscH and FSS resistance highlight promising directions for future research. These findings emphasize the potential roles of redox stress and biomechanical forces in promoting metastatic disease. Understanding these mechanisms may ultimately aid in the development of targeted therapies aimed at mitigating different forms of cancer cell resistance and limiting metastatic progression.
Details
- Title: Subtitle
- Elucidating mechanisms that enhance metastatic phenotypes through pharmacological ascorbate and fluid shear stress resistance
- Creators
- Amanda Nicole Pope
- Contributors
- Michael Henry (Advisor)Joseph Cullen (Advisor)Dawn Quelle (Committee Member)Adam Dupuy (Committee Member)Prabhat Goswami (Committee Member)Joseph Caster (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Cancer Biology)
- Date degree season
- Summer 2025
- DOI
- 10.25820/etd.008072
- Publisher
- University of Iowa
- Number of pages
- xviii, 164 pages
- Copyright
- Copyright 2025 Amanda Nicole Pope
- Language
- English
- Date submitted
- 07/15/2025
- Description illustrations
- illustrations, graphs, tables
- Description bibliographic
- Includes bibliographical references (pages 154-164).
- Public Abstract (ETD)
The spread of cancer to distant regions of the body, known as metastasis, accounts for approximately 90% of cancer related deaths. Metastasis is a highly orchestrated process in which cancer cells must adapt and overcome various challenges to colonize foreign tissue environments. In addition to surviving chemotherapy and radiation, cancer cells must thrive under these harsh conditions to support continued outgrowth. The liquid tumor microenvironment a cancer cell is exposed to during its time in the circulation also imposes hemodynamic forces, including fluid shear stress, which must also be endured. Resistance to both chemical and mechanical stressors have been implicated in promoting metastasis.
In this work, I explore distinct mechanisms which contribute to metastatic phenotypes in two contexts: (1) resistance to pharmacological ascorbate (P-AscH) and (2) adaptation to fluid shear stress (FSS). I generated two separate P-AscH resistant pancreatic cancer cell lines, which exhibited unique regulatory pathways to combat oxidative damage. Despite differences in specific mechanisms, both lines demonstrated enhanced metastatic behavior, likely mediated through epithelial-to-mesenchymal transition (EMT). Additionally, recent work from our lab has shown that cancer cells have innate resistance to FSS, compared to primary epithelial cell lines. Further investigation revealed that exposure to FSS induces genomic and metabolic alterations that are associated with increased metastatic potential.
Collectively, these results reveal that resistance to P-AscH and FSS can promote metastasis by (1) altering redox homeostasis which in turn activates pro-metastatic pathways and (2) FSS can promote metastasis by altering biological pathways within the cell.
- Academic Unit
- Biomedical Science Program
- Record Identifier
- 9984948341102771
Metrics
9 Record Views