The role of Organic Cation Transporters in the pharmacokinetics of clinically relevant DNA damaging agents : in vivo and in silico studies

In cellular systems, nutrient homeostasis - defined as the ability of a system to maintain in equilibrium nutrient concentrations within fluctuating gradients relative to the internal and external environment - is essential to thrive in harsh conditions. Between the intracellular and extracellular space of the cell membrane, a plethora of molecular and cellular signalling cues interact to coordinate the maintenance of cellular homeostasis. One of the main mechanisms that controls this interaction is orchestrated by membrane proteins. These membrane proteins exist in several classes, structures and functions and are specialized in controlling the traffic of all sorts of nutrients and molecules across the membrane.         The aim of this study was to investigate the structure-function relationship of Organic Cation Transporters (OCTs), one of the most important family of membrane proteins. OCTs are involved in the physiological uptake of various nutrients and recent evidences show that these transporters may be far more important in regulating the entry of xenobiotics and therapeutic drugs into cells. The soil nematode Caenorhabditis elegans has provided powerful insights into the roles of OCTs. This nematode uses the membrane-bound transporter OCT-1 as an uptake transporter, which has been classified as an organic cation transporter based on the transport of the prototypical substrate, the ammonium cation tetraethylammonium (TEA) (Wu et al. 1999). Nearly a decade and a half later, it was shown that OCT-1 transports the antioxidant ergothioneine (Cheah et al., 2013). DNA sequence analysis revealed the existence of additional and related transporters such as OCT-2 in C. elegans; however, the roles of OCT-2 have not been described so far. With this in mind, we set out to investigate whether OCT-1 and OCT-2 might coordinate and share the responsibilities in the uptake of cationic compounds in C. elegans.  In this thesis, we unveil for the first time the role of OCT-2 in C. elegans.  We showed that, unexpectadly, OCT-1 has little role in the uptake of cationic compounds; nonetheless it exerts regulatory functions on oct-2 expression. In the absence of oct-1, oct-2 expression is significantly upregulated and, thus, it is OCT-2 that serves as the major uptake transporter for clinically relevant chemotherapeutic drugs such as doxorubicin, cisplatin and methotrexate used for several cancer treatments. Our research therefore refutes the initial hypothesis that OCT-1 is/acts as a direct uptake transporter in C. elegans.    In this work, we established an in vivo screening method to uncover the uptake efficiency of cationic drugs by OCT-1 and OCT-2 by monitoring DNA damage-induced germ cell apoptosis. We observed that deletion of oct-1 gene and/or RNAi-driven downregulation of oct-1 causes upregulation of oct-2, therefore stimulating the uptake of chemotherapeutic drugs and potential toxic metabolites which cause deleterious effects on the animal. Moreover, worms with defective DNA repair pathways were particularly sensitive to the chemotherapeutic agents when oct-2 was upregulated. Importantly, depletion of oct-2 completely impeded the uptake of these drugs thus preventing their genotoxic effects, and hence leading to drug resistance phenotypes. Additionally, we performed an in silico modeling-based comparative screening of OCT-1 and OCT-2, and showed that this approach selectively discriminated amongst ligands that bind robustly to OCT-2 and not OCT-1. We validated this approach in vivo, and demonstrated that OCT-2-dependent transport of DNA-damaging compounds successfully sensitized worms with defective DNA repair pathways, and this effect was reversed once oct-2 was downregulated.  Collectively, these experimental methods serve as a proof of concept in studying key characteristics of relevant cationic transporters. Applying in silico methods for the preselection of target molecules and subsequent validation with our OCTs-based in vivo model will undoubtedly increase the success of chemotherapeutics and screening of newly synthesized molecules with a cost-efficient model system. Our work has pivotal implications, as it indicates that (i) hyperactive uptake transporters are likely to import abnormally high concentrations of genotoxic compounds and metabolites over many years causing genomic instability and eventually cancers and (ii) these uptake transporters may hold the key to the mechanisms of drug resistance observed in many types of cancers.  In short, these results underscore the importance of uptake transporters in regulating the entry of chemotherapeutic drugs into cells and raises the possibility that drug-resistance and drug-sensitive responses observed in cancer patients could be governed at the level of drug uptake. This study is therefore pivotal as it exploits uptake transporters as a novel approach to expand on several drug screening programs using C. elegans.  Thus, our work has immediate applications to a broad range of disciplines. Finally, in a related theme, we present the discovery of a new DNA repair mechanism whereby lesions created by the nucleoside 5-hydroxymethyuracil (5-hmU) are removed by the base excision repair pathway. In this work, we examined the in vivo roles of four base-excision DNA repair enzymes in C. elegans - the two DNA glycosylases UNG-1 and NTH-1 and the two AP endonucleases APN-1 and EXO-3 - in processing the oxidatively modified product of thymine, 5-hmU. C. elegans UNG-1 has been previously characterized in vitro to remove uracil, while NTH-1 was shown to remove thymine glycol, 5-formyl cytosine and 5-hmU. Likewise, the two AP endonucleases have been characterized and both can incise abasic sites and remove 3′-blocking lesions at DNA single strand breaks. However, APN-1 is distinct from EXO-3, as it possesses two additional activities, a 3′- to 5′-exonulease activity and a nucleotide incision repair activity that acts directly on certain oxidatively modified bases. Herein, we used C. elegans mutants and observe that ung-1 mutants exhibited a decrease in brood size and lifespan, and an elevated level of germ cell apoptosis when challenged with 5-hmU. Similar phenotypes were seen with apn-1 mutant, which were exacerbated by RNAi downregulation of apn-1 in the ung-1 mutant. The nth-1 or exo-3 mutants displayed wild type phenotypes towards 5-hmU. We propose a model suggesting that UNG-1 is involved in removing 5-hmU incorporated into the genome and the resulting abasic site is cleaved by APN-1 or EXO-3. In the absence of UNG-1, the 5-hmU is removed by NTH-1, which creates a genotoxic 3′-blocking lesion that requires the action of the 3′-diesterase or 3′- to 5′-exonuclease activity of APN-1. Our data provide the first evidence that C. elegans UNG-1 possesses the ability to remove 5-hmU in vivo, which may have been replaced with SMUG1-like activity in mammalian cells.

Keyphrases: Drug uptake and resistance, Organic Cation Transporters, C. elegans, Apoptosis, DNA damage and repair pathways, In silico modeling, Gene expression, DNA repair pathway inhibitor, 5-hydroxymethyluracil, DNA glycosylases, AP endonucleases