Jaegal Shim et al. (2007) International Worm Meeting "Genetic study of uridine phosphorylase in the function and toxicity of the anti-cancer drug, 5-Fluorouracil using C. elegans."

Seongseop Kim, Jaegal Shim

[International Worm Meeting, 2007]

5-Fluorouracil (5-FU) is a widely used anti-cancer drug for treatment of stomach, pancreatic, breast, colorectal, head and neck cancers. 5-FU has been used more than 40 years but it is still a mainstay drug of colorectal cancer. The anti-cancer drugs of pyrimidine antagonists including 5-FU are inserted into DNA and RNA or inhibit DNA synthesis. Therefore, DNA damage is occurred, and cancer cells become arrested or enter the apoptotic pathway. One of the obstacles of chemotherapy is the anti-cancer drug resistance. To study the resistant mechanism of cancer cells, we used C. elegans as a model system. We treated 5-FU to C. elegans and observed germ-line cell death and larval growth inhibition. Then we searched and studied C. elegans homologues of two enzymes in the 5-FU metabolic pathway. They are dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS). DPD converts 5-FU to inactive molecule, and TS is a major target of 5-FU. Overexpression of DPD and TS gives C. elegans resistance against 5-FU. From this result, we did genetic screen to find out genes related to the 5-FU resistant mechanism. We selected 15 mutants from 72,000 F1 screening. Six mutants are revealed to have mutations in ZK783.2 ORF, and ZK783.2 is uridine phosphorylase homologue. Uridine phosphorylase is one of enzymes in the pyrimidine salvage pathway. Uridine phosphorylase is expressed in most cells from early embryo to late adult. We also tested homologous genes of this pathway whether they are related to 5-FU resistance or not. Using RNAi, we know that some genes are related to 5-FU resistance but others are not. Now we are doing in vitro and in vivo enzyme activity of wild and mutant type uridine phosphorylases, and rescue experiment with human uridine phosphorylase. In addition, we are also doing mapping and cloning of another 5-FU resistant mutant.

Seongseop Kim et al. (2006) East Asia C. elegans Meeting "Genetic study for the anti-cancer drug, 5-Fluorouracil resistant mechanism using C. elegans"

Jaegal Shim, Seongseop Kim

[East Asia C. elegans Meeting, 2006]

5-Fluorouracil (5-FU), which is pyrimidine antagonist, is a widely used anti-cancer drug for treatment of stomach, pancreatic, breast, colorectal, head and neck cancers. 5-FU has been used more than 40 years but it is still a mainstay drug of colorectal cancer. The anti-cancer drugs of pyrimidine antagonists are inserted into DNA and RNA or inhibit DNA synthesis pathway. Therefore, DNA damage is occurred and cancer cells become arrested or enter the apoptotic pathway. 5-FU is basically a pro-drug, and it is converted to an active drug by the several metabolic enzymes. Fluorodeoxyuridylate (5-FdUMP), which is the main metabolite of 5-FU, inhibits DNA synthesis through the binding with thymidylate synthase and induces apoptosis of cell. Another metabolite of 5-FU, 5-fluorouridine monophosphate (5-FUMP) is inserted into RNA and inhibits processing and function of RNA. One of the most critical points of chemotherapy is resistance of cancer cells against anti-cancer drugs. To study the resistant mechanism of cancer cells, we used C. elegans as a model system. We treated 5-FU to C. elegans and observed germ-line cell death and larval growth inhibition. Next, we studied C. elegans homologous genes of two main enzymes in the 5-FU metabolic pathway. They are dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS). DPD converts 5-FU to inactive molecule, and TS is a major target of 5-FU. Both two genes seem to be related 5-FU resistance, although there are several controversial reports. DPD and TS of C. elegans are identical over 60% to their human homologues. Overexpression of them inhibited germ cell death caused by 5-FU treatment. From these results, we concluded that C. elegans and human have similar conserved pathways of 5-FU metabolism. From these results, we started mutant screen to elucidate the 5-FU resistant mechanism. We selected 9 mutants from 72,000 F<SUB>1</SUB> screening, and they belonged to 2 complementation groups. Now, we are trying to clone these two mutants. We expect that one mutant should be cloned in a month, because we are doing rescue experiment with cosmids.

Maria Martinez-Iniesta et al. (2008) European Worm Meeting "Identification of response and resistance genes to 5-fluroracil (5-FU) in Caenorhabditis elegans."

Maria Martinez-Iniesta, Xavi Sole, Nuria Baixeras, Alberto Villanueva, Laura Padulles, Ramon Salazar, Victor Moreno, J Ramon Germa, Gabriel Capella

[European Worm Meeting, 2008]

Introduction: Colorectal cancer (CRC) is one of the leading causes of. cancer-related mortality in the Western world. In Spain is the second type. of cancer in incidence and mortality in both sexes and 50% of total. diagnostic cases death. Despite the improvement of diagnostic and treatment. only a few decreases have been observed. About 60% of all patients. diagnosed with CRC will present localized disease, being the surgery the. mainstay therapy. The rest of patients (Duke''s C and D) are diagnosed with. distal metastasis. 5-FU and oxaliplatin based chemotherapy (QT) are the. base of the standard treatment of colorectal cancer patients in both. adjuvant and metastasic setting. Initially, more patients did not respond. to QT treatments and others relapse after a short period of response. In. spite of the clinical relevance of 5-FU, little is known about the. mechanisms of resistance to this drug commonly observed in colorectal. cancer patients. Aim: To identify the genes involved in the mechanism of. resistance to 5-FU in the pluricellular model organisms Caenorhabditis. elegans (C.elegans), and posterior validation of these genes as a markers. of response/sensitivity to 5-FU chemotherapy treatments in tumor series.. Methods and Results: A 5-FU plate toxicity assay has been established. We. are characterizing the ceTS-1 gene, C.elegans homolog of human thymidylate. synthase gene, and temporal and cellular expression patterns during. development are studying in GFP transgenic worms. Different alternative. splicing isoforms are generating in CeTS-1 with unknown biological. implications that we are evaluating. Different phenotypes have been. associated with the worm 5-FU treatment. N2 wild-type worms and cib-1(e300). mutant, harboring a point mutation in Thymidilate Synthase gene (Schnabel. R), were synchronized, and both strains of worms were growth at different. drug concentrations and at different times. Profiles of 5-FU response are. being determined in C.elegans Affymetrix microarrays. To identify response. genes (we expected around 100-150 genes) we are performing a feeding double-. stranded RNA-mediated interference (dsRNAi) screening for response genes.. Conclusions: Identification in C. elegans of genes involved in the. resistance/sensitivity to 5-FU drug and later validation of their. predictive value of response in colorectal cancer series may permit to. dispose of the some genetic markers that finally can improve the CRC. patient treatment.. avillanueva@iconcologia.net

Quintaneiro, L. et al. (2017) International Worm Meeting "Host-microbe co-metabolism dictates cancer drug efficacy in C. elegans."

Bryson, K., Cabreiro, F., Greene, N. D. E., Norvaisas, P., Typas, A., Quintaneiro, L., Lui, P. P., Herrera-Dominguez, L., Scott, T. A.

[International Worm Meeting, 2017]

Fluorouracil (5-FU) is a fluoropyrimidine and uracil analogue commonly used in the treatment of colorectal cancer. 5-FU exerts cytotoxicity by inhibiting nucleotide synthesis and by misincoorporating into RNA and DNA, leading to cancer cell damage and death1. Despite the widespread use of this drug, new approaches are needed to boost efficacy and decrease toxicity. Furthermore, 5-FU treatment outcome is variable between patients and cannot be completely explained by genetic factors, suggesting that environmental factors could contribute to its efficacy. The gut microbiota is defined as the collective community of microbes that reside a host gastrointestinal tract, and have recently been linked to other elements of host fitness, such as immunity2 and metabolism3. However, not much is known about the interplay between drug, gut microbes and the host. To unravel the mechanism by which the microbiota is affecting the metabolism of 5-FU, we used the nematode C. elegans as a host model and microbe E. coli as a gut microbe model. Via high-throughput screens and metabolomic approaches we found that microbes can affect drug conversion and efficacy in the host. We discovered this effect to be influenced by bacterial metabolism and synthesis of ribonucleotides and vitamins B9 and B6. Additionally, we find deoxynucleotide pools imbalances in bacteria increased autophagy and cell death on host cells upon 5-FU treatment. Interestingly, this effect was found to be dependent and regulated by the host gene nucleoside diphosphate kinase ndk-1. In summary, our data suggests bacteria can contribute to drug efficacy on host metabolism through two distinct but complementary processes4. These findings emphasize the importance of the gut microbiome in the co-metabolism of host-targeted drugs. Moreover, this study reveals the potential therapeutic power of manipulating gut microbial communities to help treat disease and improve treatment outcomes. References: 1. Longley, D.B., et al. Nat. Rev. Cancer (2003) 2. Nicholson, J.K., et al. Science (2012) 3. Cabreiro, F., et al. Cell (2013) 4. Scott, A.T., Quintaneiro, L. M., et al. Cell (in press)

Peres, Tanara et al. (2021) International Worm Meeting "Nutrient-induced rewiring of microbial metabolic pathways modulate 5-fluorouracil efficacy in C. elegans"

Zimmermman, Johannes, Kaleta, Christoph, Chrysostomou, Despoina, Marchesi, Julian, W. Zatorska, Michalina, Barr, Alexis, Quintaneiro, Leonor, Cabreiro, Filipe, Martinez-Martinez, Daniel, Peres, Tanara, Kramer, Holger, Best, Lena, Marinos, Georgios, B. Mokochinski, Joao

[International Worm Meeting, 2021]

Microbiota derived metabolites have been shown to influence cancer susceptibility, tumour progression and therapy outcome. Diet is a major environmental factor influencing gut microbiota composition, dictating the abundance of potential oncometabolites. The complex interactions between diet and the microbiota need to be clarified so that dietary intake may be shaped to influence disease outcome. Having previously demonstrated the impact of bacterial metabolism in 5-fluorouracil (5-FU) cancer drug efficacy, we set out to investigate the role of nutrition in host-microbe responses to this drug, an antimetabolite widely used for colorectal cancer (CRC) chemotherapy. Host-microbe-drug-nutrient interaction and its influence on drug efficacy were assessed using a high-throughput 4-way screening approach. To understand gene-nutrient interactions at the bacterial level that regulate the effect of a drug on host physiology, genetically modified E. coli from the Keio deletion library were used to probe the role of bacterial metabolic pathways in 5-FU effects in a nutrient-dependent manner. We found that glycolytic nutrients, including sugars, importantly antagonise the toxic effects of 5-FU action on the host. Sugars induce a shift in metabolic pathways that favour the production of nucleotides, specifically UMP and UTP derived from the activation of pyrimidine de novo biosynthesis pathway in bacteria, counteracting pro-drug activation through the salvage pathway. We also found that bacterial mutations in the TCA cycle and pyruvate metabolism decrease drug efficacy in amino acid-based media. A combination of bacterial genetic work and metabolomics has revealed the presence of a novel bacterial metabolite with the capacity to increase 5-FU drug efficacy in both C. elegans and human cancer cells. Importantly, flux balance analysis of the cancer associated microbiota predicts an increased production of this metabolite in several organs. In particular, investigations of the gut microbiome from four independent human CRC cohorts shows an increased production of this metabolite, compared to healthy patients, further implicating this metabolite in drug cancer therapy. These findings highlight the potential of manipulating gut microbiota through diet to improve cancer therapy outcome.

Shih, Pei-Yin et al. (2013) International Worm Meeting "Transcription factors involved in dauer recovery."

Sternberg, Paul W., Shih, Pei-Yin

[International Worm Meeting, 2013]

In an adverse environment, the free-living nematode Caenorhabditis elegans becomes a dauer larva, a developmentally arrested stage. When the environment becomes favorable again, dauers recover and resume development. This post dauer is a dynamic period in that there are multiple structural, developmental and behavior modulations that occur. The analogous dauer states in some parasitic nematodes are called infective juveniles (IJ)s and dispersal juveniles (DJ)s. It has been shown that conserved neuron and signaling molecules control dauer/IJ recovery in C. elegans and parasitic nematodes Ancylostoma caninum and Heterorhabditis bacteriophora. However, how perception of the environmental change is converted to developmental changes is not well understood. In this study, we analyzed gene expression changes during dauer and dauer exit in C. elegans and found some transcription factors involved in the recovery process. We are now analyzing the transcription factors in more detail, including their roles in other parasitic nematodes. In addition, we are looking at the neural circuits that mediate the process of exiting dauer. Better understanding of the mechanism may help develop an evolutionary framework within the nematode phylum and potential novel treatment strategies by blocking IJ or DJ recovery.

Li, Xiao et al. (2019) International Worm Meeting "Cytosolic iron-sulfur protein assembly (CIA) is important for pyrimidine metabolism and immortal germline in C. elegans."

Tarailo-Graovac, Maja, Li, Xiao

[International Worm Meeting, 2019]

Proper integration of iron-sulfur (Fe-S) clusters into corresponding apoproteins is crucial for normal function of many DNA metabolism proteins including FANCJ/dog-1, RTEL1/rtel-1 and ERCC2/xpd-1 [1]. Biogenesis of Fe-S proteins is a multistep process regulated by partnership between mitochondria iron-sulfur cluster (ISC) assembly machinery, the ISC export machinery and the cytoplasm iron-sulfur protein assembly (CIA) system. The CIA targeting complex, consisting of CIAO1, MMS19 and CIAO2B, facilitates the insertion of Fe-S clusters to selected apoproteins [2]. To study the CIA targeting complex, we obtained a knockout allele for the highly conserved orthologous gene mms-19/MMS19, and observed that absence of MMS-19 did not result in an obvious phenotype unless exposed to 5-Fluorouracil (5-FU), a pyrimidine antagonist widely prescribed to treat cancers. Adverse reactions to 5-FU have been well-documented in cancer patients with germline variants in the DPYD gene encoding dihydropyrimidine dehydrogenase (DPD), a cytosolic Fe-S protein known for breaking down pyrimidines [3]. Our analyses of a dpyd-1/DPYD knockout allele revealed similarly strong sensitivity to 5-FU, suggesting that MMS-19 is important for DPD biogenesis in worms. Importantly, we then studied Y18D10A.9/CIAO1 gene and found that it is an essential gene as its knockout results in complete sterility. Further, we also created strains with missense alleles in highly conserved amino acids (invariant from yeast to human), which resulted in either complete sterility similar to the knockout allele or in a heat-triggered mortal germ line (Mrt) phenotype [4]. The Mrt phenotype was described almost two decades ago as a multigenerational phenotype where worms reproduce for several healthy generations but eventually become sterile. The CIAO1 hypomorphic Mrt mutant becomes progressively sterile in a few generations at 25 deg C. As some of the previously described Mrt mutants are in genes important for genome stability (e.g. mrt-2/RAD1) [4], our results suggest that the germline mortality, when CIAO1 is disrupted, may be explained by defective biogenesis of factors important for genome integrity, such as dog-1, rtel-1 and xpd-1. [1] PMID:22678362; [2] PMID: 25583461; [3] PMID: 10071185; [4] PMID: 10646593

Enrico Schmidt et al. (2008) C.elegans Neuronal Development Meeting "The Parkinson's Disease Related Kinases LRK-1 and PINK-1 are Functionally Connected via Rho family GTPases in C.elegans"

Ursula Schaffer, Erika Gromoff, Natalja Tscherwinski, Ralf Baumeister, Enrico Schmidt, Julia Samann, Monika Messerschmid

[C.elegans Neuronal Development Meeting, 2008]

Parkinson''s Disease (PD) is characterized by the loss of projection neurons in the substantia nigra. Dominant mutations in alpha-synuclein and LRRK2 (leucine-rich-repeat kinase) and recessive mutations in Parkin, DJ-1 and PINK1 (PTEN-induced kinase) segregate with the familial variants of PD. Mutants in these genes have been linked to mitochondrial dysfunction and oxidative as well as ER stress vulnerability. However, a contribution of these mutants to the pathological processes, the first manifestations of which are cytoskeletal defects, is not known at present. Studies in mammalian support the idea that PINK1 forms a protein complex with DJ-1 and acts in the same pathway as Parkin. Furthermore, a biochemical interaction between LRRK2 and Parkin was suggested recently. Taking these multiple functional interactions into account, we reasoned that LRK-1 and PINK-1 could contribute to overlapping biological functions. In order to identify common targets for the kinases we created a protein interaction network of PD-related proteins based on genetic and biochemical approaches. Bioinformatical analyses of the interaction network suggested a possible connection of the PD-related kinases to members of the family of Rho GTPases and their targets and regulators, which are key players of the cytoskeletal regulation. Using combined pharmacological and genetic methods we were able to confirm the bioinformatical prediction. We show that both LRK-1 and PINK-1 affect axon guidance and cell migration in C. elegans via the small GTPases of the Rho family.

Ralf Baumeister et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Parkinson's Disease associated factors LRK-1 and PINK-1 interact to regulate axon guidance and cell migration in C.elegans"

Julia Saemann, Enrico Schmidt, Ralf Baumeister, Jan Hegerman, Stefan Eimer, Ursula Schaeffer

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Parkinson''s Disease is characterized by the preferential loss of projection neurons in the substantia nigra. Dominant mutations in a-synuclein and LRRK2 (leucine-rich-repeat kinase) and recessive mutations in Parkin, DJ-1, and PINK1 (PTEN-induced kinase) segregate with the familial variants of PD. Mutations in these genes have been linked to mitochondrial dysfunction and increased vulnerability to oxidative, as well as ER, stress. However, how these mutations promote the pathological processes, the first manifestations of which are cytoskeletal defects in the affected neurons, is not known at present. Studies in mammalian support the idea that PINK1 forms a protein complex with DJ-1 and acts in the same pathway as Parkin. Furthermore, a biochemical interaction between LRRK2 and Parkin was suggested recently. Taking these multiple functional interactions into account, we reasoned that the C. elegans homologues, LRK-1 and PINK-1, could contribute to overlapping biological functions. In order to identify common targets for the kinases we created a protein interaction network of PD-related proteins based on genetic and biochemical approaches. We identified an interaction network linking PINK-1 and LRK-1 to Rho GTPases and their targets and regulators, which are key players of cytoskeletal regulation. We show that antagonistic activities of PINK-1 and LRK-1 regulate the activities of two members of the C. elegans Rho family to affect neuronal migrations and neurite pathfinding. Our data suggest that the combinatorial activity of both kinases is involved in controlling actin cytoskeleton dynamics in order to regulate neuronal integrity.

Garcia Gonzalez, Aurian P. et al. (2017) International Worm Meeting "Bacterial metabolism affects the C. elegans response to cancer chemotherapeutics."

Andersen, Erik C., Garcia Gonzalez, Aurian P., Walhout, Albertha J.M., Yilmaz, L. Safak, Shrestha, Shaleen, Ritter, Ashlyn D.

[International Worm Meeting, 2017]

The human microbiota greatly affects physiology and disease. However, the contribution of bacteria to the response to chemotherapeutic drugs remains poorly understood. Caenorhabditis elegans and its bacterial diet provide a powerful system to study host-bacteria interactions. Our group previously demonstrated that two bacterial diets, E. coli OP50 (E. coli) and C. aquatica DA1877 (Comamonas) elicit differences in life history traits: C. elegans fed a diet of Comamonas develop faster, have fewer offspring and have reduced lifespan than animals fed E. coli. Furthermore, we used genetics in both bacteria and C. elegans to identify vitamin B12 as a key nutrient provided by Comamonas, but not E. coli, which explains several of the differences in life history traits. Here, we use this system to study how E. coli and Comamonas affect the C. elegans response to chemotherapeutics. We find that different bacteria can increase the response to one drug yet decrease the effect of another. For example, animals fed Comamonas require ~100-fold more 5-fluoro-2'-deoxyuridine (FUDR) than those fed E. coli to elicit embryonic lethality. In contrast, animals fed E. coli require ~ 1.5-fold more camptothecin (CPT) than animals fed Comamonas to elicit embryonic lethality. Interestingly, we find that bacterial metabolism is required for the effect on drug efficacy of both FUDR and CPT. To identify the mechanisms in bacteria that affect C. elegans drug efficacy, we perform genetic screens in the two bacterial species using three chemotherapeutic drugs, 5-fluorouracil (5-FU), FUDR and CPT. We find numerous bacterial nucleotide metabolism genes that affect drug efficacy in C. elegans. Surprisingly, we find that 5-FU and FUDR act through bacterial ribonucleotide metabolism to elicit their cytotoxic effects in C. elegans, rather than by thymineless-death or DNA damage. Our study provides a blueprint for characterizing the role of bacteria in the host response to chemotherapeutics.