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Dissertation
Targeting resistance mechanisms with selenium compounds in the treatment of clear cell renal cell carcinoma
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2023
DOI: 10.25820/etd.006849
Abstract
ccRCC poses a significant global health challenge as its incidence continues to rise, resulting in a substantial annual mortality rate. ccRCC tumors are highly immunogenic and show resistance to conventional chemotherapy and radiotherapy. The treatment landscape for ccRCC has been revolutionized by the discovery of antiangiogenic TKIs and ICIs. Nonetheless, even in initially responding patients, resistance to these therapies eventually emerges. Resistance (intrinsic and acquired), lack of durable response, serious adverse events, and cost represent major clinical challenges and underline the need to identify additional ‘druggable’ targets. Identifying and targeting resistance biomarkers in ccRCC is a hot area of research, with the goal of enhancing the effectiveness of existing treatments and mitigating resistance, ultimately improving patient outcomes and quality of life. Utilizing cost-effective and safe compounds like selenium to target resistance biomarkers is of utmost importance in optimizing ccRCC therapeutics.
In ccRCC, TGF-β1 plays a pivotal role in driving the transition from epithelial non-sarcomatoid to dedifferentiated sarcomatoid ccRCC, associated with increased aggressiveness and metastatic potential. Additionally, PD-L1 and VEGF play roles in the pathogenesis of ccRCC, and they are overexpressed in ccRCC cell lines with and without sarcomatoid differentiation. These biomarkers are associated with immune evasion, drug resistance, and increased tumor angiogenesis. Studies were carried out to evaluate the in vitro effect of nontoxic concentrations of MSA and the in vivo effect of the therapeutic doses of SLM on the expression levels of TGF-β1, PD-L1, and VEGF in ccRCC cell lines, and patient tumor biopsies.
Various ccRCC cell lines were evaluated for their expression of TGF-β1, PD-L1, and VEGF. The 786-O, RCJ41T1, RCJ41T2, and RCJ41M cells were picked up for further investigation. An examination of the interaction between MSA and TGF-β1 was conducted through a simple mixing experiment and a CETSA, revealing a direct and specific interaction between MSA and TGF-β1. Subsequently, the effect of MSA on TGF-β1 expression was explored with varying doses and exposure times, demonstrating a dose- and time-dependent downregulation in the expression of TGF-β1, PD-L1, and VEGF. In light of these findings, the relationship between TGF-β1 and PD-L1/VEGF expression was investigated by creating a TGF-β1 KO cell line. Initially, the created TGF-β1 KO cell line was characterized in terms of growth rate and TGF-β1, PD-L1, VEGF, and HIF-2α expression. Growth curve analysis indicated that the parental cells exhibited notably faster growth compared to the TGF-β1 KO cells, which is in line with expectations as TGF-β1 is considered to be a growth factor. Regarding the expression of the biomarkers, it was shown a decrease in TGF-β1 expression, with no change in the expression of PD-L1 and VEGF after 3 days of selection. However, after 7 days of selection, a complete KO of TGF-β1 led to a downregulation of PD-L1 VEGF and HIF-2α expression, confirming the role of TGF-β1 as an upstream regulator of PD-L1, VEGF and HIF-2α. The effect of MSA treatment on TGF-β1, PD-L1, and VEGF expression in the KO cells was also examined, revealing an unexpected upregulation of PD-L1 and VEGF. One possible explanation of this phenomenon is the activation of alternative pathways, such as PI3K/Akt/mTOR, resulting in an increase in HIFs expression, subsequently leading to higher PD-L1 and VEGF levels.
Moreover, the effect of MSA, axitinib, and the combination on the ccRCC cell lines viability was tested showing an additive antiproliferative effect of the combination. Finally, SLM was used to test the effect of its addition on axitinib in the treatment of ccRCC nude mice xenograft model. The in vivo results showed no difference between the axitinib-treated group and the combination group. Further investigation revealed that the in vivo results may have been subject to artifacts due to inappropriate preparation and mixing of the drugs prior to administration. A repeat of the in vivo experiment with separate administration and fresh preparation of the medication will be conducted in the future.
Additionally, the importance of oncogenic and tumor suppressor microRNA as resistance biomarkers in ccRCC has been investigated. The expression of various microRNAs in ccRCC and human biopsies was identified. Similarly, the expression of the same microRNAs has been also identified and compared in untreated patient kidney samples and patient kidney samples after being treated with SLM and currently available therapeutics for ccRCC including ipilimumab, nivolumab, axitinib, pembrolizumab, and cabozantinib. The results revealed higher expression levels of miRNA-210, -155, and -34a in the untreated compared to the treated tissue, however, no definitive conclusion could be drawn from this experiment because the treated and the untreated tissues were not obtained from the same patients.
In conclusion, the addition of selenium compounds to standard-of-care treatment of ccRCC such as axitinib showed an additive antiproliferative effect in vitro and has the potential to exhibit synergistic effects in animal experiments, through downregulation of resistance biomarkers including TGF-β1, PD-L1, and VEGF.
Details
- Title: Subtitle
- Targeting resistance mechanisms with selenium compounds in the treatment of clear cell renal cell carcinoma
- Creators
- Aseel Owayed Awad Rataan
- Contributors
- Aliasger K. Salem (Advisor)Nicole K. Brogden (Committee Member)Reza Nejadnik (Committee Member)Sean M. Geary (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Pharmacy
- Date degree season
- Autumn 2023
- DOI
- 10.25820/etd.006849
- Publisher
- University of Iowa
- Number of pages
- xvii, 85 pages
- Copyright
- Copyright 2023 Aseel Owayed Awad Rataan
- Language
- English
- Date submitted
- 11/30/2023
- Description illustrations
- illustrations, tables, graphs
- Description bibliographic
- Includes bibliographical references (pages 74-85).
- Public Abstract (ETD)
- ccRCC is a type of kidney cancer with a significant global health concern due to its increasing prevalence and high mortality rate. These tumors are resistant to standard treatments like chemotherapy and radiation. Fortunately, there have been improvements in treatment with the use of specific drugs known as antiangiogenic TKIs and ICIs. However, even with these treatments, resistance eventually develops, and this resistance is a major challenge in medical care. Scientists are working on finding new targets for treatment, and one of these targets is a protein called TGF-β1, which plays a role in making this specific type of cancer more aggressive. Other proteins like PD-L1 and VEGF also contribute to the cancer's growth and resistance. We studied the effect of selenium compounds on these proteins, both in cells (in the lab setting) and in ccRCC mice models. We found that selenium when combined with the standard treatment called axitinib, can slow down the growth of ccRCC cells in the lab setting. However, when we tested this combination in mice, it didn't show the expected results, possibly because of issues with how the drugs were mixed and given. Hence, we are planning to repeat this experiment with a better method in the future. Additionally, we looked at small pieces of the genetic material called microRNAs in ccRCC and in patient samples before and after treatment with various drugs. We found some differences in the levels of certain microRNAs in treated and untreated samples, but more research is needed to draw firm conclusions. In summary, adding selenium to the standard treatment for ccRCC appears to slow down the cancer's growth in the lab, and it may have even better effects in animal experiments if administered correctly. We also explored the role of certain genetic material in the disease and how it changes with treatment.
- Academic Unit
- Pharmacy; Craniofacial Anomalies Research Center
- Record Identifier
- 9984546541802771
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