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Access Type
WSU Access
Date of Award
January 2024
Degree Type
Thesis
Degree Name
M.S.
Department
Cancer Biology
First Advisor
Larry H. Matherly
Abstract
Activation of stimulator of interferon genes (STING) activates the transcription factor pIRF3, promoting transcription of genes encoding type I interferons including IFN-β and other cytokines that stimulate broader immune responses. Targeting the STING pathway is especially compelling given its importance in promoting cancer progression and metastasis. Cyclic dinucleotides (CDNs) function as STING agonists. Inspired by pre-clinical studies, two phase I clinical trials with CDNs including ADU-S100 were conducted but concluded early due to lack of efficacy. CDNs have been implicated as traversing the plasma membrane by the reduced folate carrier (SLC19A1; RFC). While previous exploratory studies have shown that depletion of the proton-coupled folate transporter (SLC46A1; PCFT) in THP-1 cells had nominal effects on CDN-mediated STING activation, overexpression of PCFT did increase STING signaling. Limited clinical responses may reflect the impact of physiologic variables governing RFC and PCFT not previously considered. Understanding mechanisms of CDN uptake by RFC and PCFT under physiologic conditions may elucidate the role of folate transporters on CDN transport and STING signaling. To study the impact of RFC levels on CDN transport and STING signaling, we used an engineered R1-11/Tet-on-RFC HeLa model. RFC dose-dependence of ADU-S100 uptake and level of STING activation was confirmed by monitoring IFN-β mRNA transcripts (RT-qPCR) and phosphorylation of IRF3 by Western blotting. Induction of IFN-β was observed in THP-1 cells in response to ADU-S100 treatment at pH 7.2, the RFC pH optimum. ADU-S100 showed a low affinity for binding to RFC as measured by direct competition for transport with [3H]methotrexate. ADU-S100 treatment showed no pIRF3 or IFN-β production in an engineered R1-11/Tet-on-PCFT HeLa model, suggesting no transport via PCFT. Lastly, the engineered R1-11/Tet-on-RFC/PCFT HeLa model reveals an increase in STING activity as measured by Western blotting and RT-qPCR. In conclusion, RFC mediates ADU-S100 uptake and STING activation under physiologic conditions. Whereas PCFT does not directly mediate ADU-S100 uptake, an indirect effect is possible. Additional CDNs with better substrate specificity for RFC should be explored to improve the efficacy of CDN-based cancer therapeutics.
Recommended Citation
Mcquithey, Holly R., "The Impact Of Folate Transporters On The Import Of Cyclic Dinucleotides And Sting Signaling" (2024). Wayne State University Theses. 947.
https://digitalcommons.wayne.edu/oa_theses/947