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Generation of CD44-specific aptamers for targeted approaches in the treatment of pancreatic cancer
Generation of CD44-specific aptamers for targeted approaches in the treatment of pancreatic cancer
Pancreatic ductal adenocarcinoma (PDAC) has become one of the leading causes of cancer related death worldwide due to its aggressive local behavior and early metastatic spread. The heterogenic composition of the tumor itself and its microenvironment decreases the efficacy of common chemotherapeutic drugs. As a result, a combination of high-dose cytostatic agents, the FOLFIRINOX-regimen, has to be employed as a treatment strategy for advanced PDAC. But even this strict therapeutic setup promises a survival benefit of only a couple of months, while posing the risk of severe adverse side effects. Many studies have shown that overexpression of the cell surface protein CD44 is common in pancreatic ductal adenocarcinoma and is associated with advanced local aggressiveness of tumors, formation of metastases and significant reduction in overall survival. CD44 interacts with hyaluronic acid and other components of the extracellular matrix and is involved in signal transduction on known tumor-promoting subcellular pathways. Furthermore, CD44 and especially its isoform CD44v6, which contains an alternatively spliced exon, convey undesirable cancer stemness traits to PDAC tumors. To overcome the limitations and difficulties of chemotherapeutic treatment in PDAC, in this study a targeted drug delivery system is implemented. An exceptionally elegant approach to this case is comprised of aptamers, RNA-molecules that are often referred to as ‘nucleic acid antibodies’. The binding affinity of aptamer oligonucleotides can be prompted towards any desired molecular target through a process of mutagenesis and evolutionary selection (the SELEX-cycle). The molecules are significantly smaller than antibodies, nontoxic, nonimmunogenic and easily modifiable by incorporation of altered nucleotides into their RNA-backbone. In this work, aptamers with selective specificity for CD44 were generated, their binding properties were evaluated, and the molecules were modified by incorporation of 5- Fluorouracil in place of every Uracil residue. The respective drug is part of the FOLFIRINOX-regimen in the clinical treatment of PDAC. No specific aptamer selectivity could be achieved for the exon variant 6 of CD44 in an experimental subset. The 5FU- modified CD44-specific aptamers showed a strong cytostatic effect on pancreatic cancer cells in vitro, which scaled with the degree of cellular CD44 expression and was specific for 5FU administration. Interestingly, the generated aptamers exerted supraadditive inhibitory effects on pancreatic cancer cell growth. The observation could be explained by in silico modeling of the molecular interaction, which revealed severe obstruction of the hyaluronan binding pocket of CD44 by the generated aptamers. More work is needed to obtain a deeper understanding of the mechanistic processes that are caused by treatment with the generated CD44-specific aptamers. However, in general, this novel approach is a step toward overcoming the limitations that are associated with PDAC treatment on multiple molecular levels.
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Peterhansl, Julian
2023
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Peterhansl, Julian (2023): Generation of CD44-specific aptamers for targeted approaches in the treatment of pancreatic cancer. Dissertation, LMU München: Faculty of Medicine
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Abstract

Pancreatic ductal adenocarcinoma (PDAC) has become one of the leading causes of cancer related death worldwide due to its aggressive local behavior and early metastatic spread. The heterogenic composition of the tumor itself and its microenvironment decreases the efficacy of common chemotherapeutic drugs. As a result, a combination of high-dose cytostatic agents, the FOLFIRINOX-regimen, has to be employed as a treatment strategy for advanced PDAC. But even this strict therapeutic setup promises a survival benefit of only a couple of months, while posing the risk of severe adverse side effects. Many studies have shown that overexpression of the cell surface protein CD44 is common in pancreatic ductal adenocarcinoma and is associated with advanced local aggressiveness of tumors, formation of metastases and significant reduction in overall survival. CD44 interacts with hyaluronic acid and other components of the extracellular matrix and is involved in signal transduction on known tumor-promoting subcellular pathways. Furthermore, CD44 and especially its isoform CD44v6, which contains an alternatively spliced exon, convey undesirable cancer stemness traits to PDAC tumors. To overcome the limitations and difficulties of chemotherapeutic treatment in PDAC, in this study a targeted drug delivery system is implemented. An exceptionally elegant approach to this case is comprised of aptamers, RNA-molecules that are often referred to as ‘nucleic acid antibodies’. The binding affinity of aptamer oligonucleotides can be prompted towards any desired molecular target through a process of mutagenesis and evolutionary selection (the SELEX-cycle). The molecules are significantly smaller than antibodies, nontoxic, nonimmunogenic and easily modifiable by incorporation of altered nucleotides into their RNA-backbone. In this work, aptamers with selective specificity for CD44 were generated, their binding properties were evaluated, and the molecules were modified by incorporation of 5- Fluorouracil in place of every Uracil residue. The respective drug is part of the FOLFIRINOX-regimen in the clinical treatment of PDAC. No specific aptamer selectivity could be achieved for the exon variant 6 of CD44 in an experimental subset. The 5FU- modified CD44-specific aptamers showed a strong cytostatic effect on pancreatic cancer cells in vitro, which scaled with the degree of cellular CD44 expression and was specific for 5FU administration. Interestingly, the generated aptamers exerted supraadditive inhibitory effects on pancreatic cancer cell growth. The observation could be explained by in silico modeling of the molecular interaction, which revealed severe obstruction of the hyaluronan binding pocket of CD44 by the generated aptamers. More work is needed to obtain a deeper understanding of the mechanistic processes that are caused by treatment with the generated CD44-specific aptamers. However, in general, this novel approach is a step toward overcoming the limitations that are associated with PDAC treatment on multiple molecular levels.