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Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS-/--pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic ß cells. Our results suggest that supplementation with exogenous thymidine affects stem cell proliferation, but not the function of stem cell-derived cells. After differentiation, postmitotic cells do not require thymidine in vitro or in vivo, as shown by the production of functional human insulin in mice up to 5 months after implantation of stem cell-derived pancreatic tissue.

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Introduction: One of the primary obstacles faced by individuals with advanced colorectal cancer (CRC) is the potential development of acquired chemoresistance as the disease advances. Studies have indicated a direct association between elevated levels of miR-92a-3p and the progression, metastasis, and chemoresistance observed in CRC. We proposed that miR-92a-3p impairs FOLFOX (fluorouracil/oxaliplatin) chemotherapy response by upregulating the expression of chemoresistance biomarker genes through the activation of ß-catenin and epithelial-mesenchymal transition (EMT). These FOLFOX biomarker genes include the pyrimidine biosynthesis pathway genes dihydropyrimidine dehydrogenase (DPYD), thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), and the genes encoding the DNA repair complexes subunits ERCC1 and ERCC2, and XRCC1. Methods: To assess this, we transfected SW480 and SW620 colon cancer cell lines with miR-92a-3p mimics and then quantified the expression of DPYD, TYMS, MTHFR, ERCC1, ERCC2, and XRCC1, the expression of EMT markers and transcription factors, and activation of ß-catenin. Results and discussion: Our results reveal that miR-92a-3p does not affect the expression of DPYD, TYMS, MTHFR, and ERCC1. Furthermore, even though miR-92a-3p affects ERCC2, XRCC1, E-cadherin, and ß-catenin mRNA levels, it has no influence on their protein expression. Conclusion: We found that miR-92a-3p does not upregulate the expression of proteins of DNA-repair pathways and other genes involved in FOLFOX chemotherapy resistance.

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Background: Acute Respiratory Distress Syndrome (ARDS) is a common condition in the intensive care unit (ICU) with a high mortality rate, yet the diagnosis rate remains low. Recent studies have increasingly highlighted the role of aging in the occurrence and progression of ARDS. This study is committed to investigating the pathogenic mechanisms of cellular and genetic changes in elderly ARDS patients, providing theoretical support for the precise treatment of ARDS. Methods: Gene expression profiles for control and ARDS samples were obtained from the Gene Expression Omnibus (GEO) database, while aging-related genes (ARGs) were sourced from the Human Aging Genomic Resources (HAGR) database. Differentially expressed genes (DEGs) were subjected to functional enrichment analysis to understand their roles in ARDS and aging. The Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning pinpointed key modules and marker genes, with ROC curves illustrating their significance. The expression of four ARDS-ARDEGs was validated in lung samples from aged mice with ARDS using qRT-PCR. Gene set enrichment analysis (GSEA) investigated the signaling pathways and immune cell infiltration associated with TYMS expression. Single-nucleus RNA sequencing (snRNA-Seq) explored gene-level differences among cells to investigate intercellular communication during ARDS onset and progression. Results: ARDEGs are involved in cellular responses to DNA damage stimuli, inflammatory reactions, and cellular senescence pathways. The MEmagenta module exhibited a significant correlation with elderly ARDS patients. The LASSO, RRF, and XGBoost algorithms were employed to screen for signature genes, including CKAP2, P2RY14, RBP2, and TYMS. Further validation emphasized the potential role of TYMS in the onset and progression of ARDS. Immune cell infiltration indicated differential proportion and correlations with TYMS expression. SnRNA-Seq and cell-cell communication analysis revealed that TYMS is highly expressed in endothelial cells, and the SEMA3 signaling pathway primarily mediates cell communication between endothelial cells and other cells. Conclusion: Endothelial cell damage associated with aging could contribute to ARDS progression by triggering inflammation. TYMS emerges as a promising diagnostic biomarker and potential therapeutic target for ARDS.

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Human high-mobility group-B (HMGB) proteins regulate gene expression in prostate cancer (PCa), a leading cause of oncological death in men. Their role in aggressive PCa cancers, which do not respond to hormonal treatment, was analyzed. The effects of HMGB1 and HMGB2 silencing upon the expression of genes previously related to PCa were studied in the PCa cell line PC-3 (selected as a small cell neuroendocrine carcinoma, SCNC, PCa model not responding to hormonal treatment). A total of 72% of genes analyzed, using pre-designed primer panels, were affected. HMGB1 behaved mostly as a repressor, but HMGB2 as an activator. Changes in SERPINE1, CDK1, ZWINT, and FN1 expression were validated using qRT-PCR after HMGB1 silencing or overexpression in PC-3 and LNCaP (selected as an adenocarcinoma model of PCa responding to hormonal treatment) cell lines. Similarly, the regulatory role of HMGB2 upon SERPINE1, ZWINT, FN1, IGFPB3, and TYMS expression was validated, finding differences between cell lines. The correlation between the expression of HMGB1, HMGB2, and their targets was analyzed in PCa patient samples and also in PCa subgroups, classified as neuroendocrine positive or negative, in public databases. These results allow a better understanding of the role of HMGB proteins in PCa and contribute to find specific biomarkers for aggressive PCa.

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BACKGROUND: There are currently no validated criteria that entirely explain or predict response to methotrexate (MTX) treatment in rheumatoid arthritis (RA). We tried to identify the connection between three variants (RFC1 G80A (rs1051266), TYMS 2R/3R (rs34743033), and ATIC C347G (rs2372536)) in the folate pathway of MTX metabolism and the response to MTX monotherapy in a cohort of RA cases. METHODS: A prospective study on 100 RA patients on MTX monotherapy was performed. Disease activity was measured at the start of treatment and 6 months after treatment with MTX. The patients were then split into two groups: those who responded to the treatment and those who did not. The molecular genetic study for the RFC1 (G80A) variant was employed via the PCR-restriction fragment length polymorphism (PCR-RFLP) technique, the ATIC (C347G) variant was performed using TaqMan allelic discrimination real-time PCR, and the tandem repeat sequences of TYMS (2R/3R) were amplified by conventional PCR and detected by agarose gel electrophoresis. RESULTS: The genotype distribution of RFC-1 (G80A) showed significant variations among non-responders and responders in the recessive genetic model. A significant difference was found in TYMS (2R/3R) in the dominant and heterozygous genetic models. However, ATIC (C347G) genotype frequency did not exhibit substantial link with drug response in all genetic models. Furthermore, the genotype and allele rates of the analyzed variants did not show any significant association with adverse events in all genetic models. CONCLUSION: The 80AA genotype of RFC-1 G80A and the 2R/3R or 3R/3R genotypes of TYMS 2R/3R are more vulnerable to the good consequences of MTX therapy. Key Points • Current recommendations support the gold standard role of MTX as a first-line monotherapy for RA patients. However, up to 40% of RA patients do not respond or exhibit partial response to MTX. • Persistent disease activity due to treatment unresponsiveness will affect the long-term outcomes in RA patients. • We aimed, through molecular genetic study, to identify the connection between three variants in the folate pathway of MTX metabolism and the response to methotrexate monotherapy in a cohort of RA patients.

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Background: Colorectal cancer, is one of most prevalent the cancer in the world. 5-Fluorouracil is a standard chemotherapeutic drug while the acquisition of resistance to 5-Fluorouracil is one of the problems during treatment. In this study, we aimed to find the miRNAs that modulate the expression of Tyms and Abcg2 as resistance-inducing genes in the resistant cell lines to 5-Fluorouracil. Methods: 5-Fluorouracil-resistant HCT116 and SW480 cell lines were generated by consecutive treatment of cells with 5-Fluorouracil. This resistance induction was validated by MTT assays. The expression of the Tyms and Abcg2 gene and miR-548c-3p were studied by quantitative real-time PCR in the cell lines. Results: We hypothesized that miR-548c-3p is targeting Tyms and Abcg2 simultaneously. Increased expression Tyms gene in the two most resistant cell lines derived from HCT116 and all resistant cell lines derived from SW480 except one were seen. Increased expression of Abcg2 was observed in the most resistant HCT116-derived cell line and all resistant cell lines, derived from SW480. In all resistant cell lines, the expression of miR-548c-3p was decreased. Conclusion: It can be concluded downregulation of miR548c-3p is in line with Tyms and Abcg2 overexpression in resistant cell lines to 5-Fluorouracil.

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Introduction: Dihydropyrimidine dehydrogenase (DPD), encoded by DPYD gene, is the rate-limiting enzyme responsible for fluoropyrimidine (FP) catabolism. DPYD gene variants seriously affect DPD activity and are well validated predictors of FP-associated toxicity. DPYD variants rs3918290, rs55886062, rs67376798, and rs75017182 are currently included in FP genetic-based dosing guidelines and are recommended for genotyping by the European Medicines Agency (EMA) before treatment initiation. In Greece, however, no data exist on DPYD genotyping. The aim of the present study was to analyze prevalence of DPYD rs3918290, rs55886062, rs67376798, rs75017182, and, additionally, rs1801160 variants, and assess their association with FP-induced toxicity in Greek cancer patients. Methods: Study group consisted of 313 FP-treated cancer patients. DPYD genotyping was conducted on QuantStudio ™ 12K Flex Real-Time PCR System (ThermoFisher Scientific) using the TaqMan® assays C__30633851_20 (rs3918290), C__11985548_10 (rs55886062), C__27530948_10 (rs67376798), C_104846637_10 (rs75017182) and C__11372171_10 (rs1801160). Results: Any grade toxicity (1-4) was recorded in 208 patients (66.5%). Out of them, 25 patients (12%) experienced grade 3-4 toxicity. DPYD EMA recommended variants were detected in 9 patients (2.9%), all experiencing toxicity (p = 0.031, 100% specificity). This frequency was found increased in grade 3-4 toxicity cases (12%, p = 0.004, 97.9% specificity). DPYD deficiency increased the odds of grade 3-4 toxicity (OR: 6.493, p = 0.014) and of grade 1-4 gastrointestinal (OR: 13.990, p = 0.014), neurological (OR: 4.134, p = 0.040) and nutrition/metabolism (OR: 4.821, p = 0.035) toxicities. FP dose intensity was significantly reduced in DPYD deficient patients (ß = -0.060, p <0.001). DPYD rs1801160 variant was not associated with FP-induced toxicity or dose intensity. Triple interaction of DPYD*TYMS*MTHFR was associated with grade 3-4 toxicity (OR: 3.725, p = 0.007). Conclusion: Our findings confirm the clinical validity of DPYD reduced function alleles as risk factors for development of FP-associated toxicity in the Greek population. Pre-treatment DPYD genotyping should be implemented in clinical practice and guide FP dosing. DPYD*gene interactions merit further investigation as to their potential to increase the prognostic value of DPYD genotyping and improve safety of FP-based chemotherapy.

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Enzyme abundance, catalytic activity, and ultimately sequence are all shaped by the need of growing cells to maintain metabolic flux while minimizing accumulation of deleterious intermediates. While much prior work has explored the constraints on protein sequence and evolution induced by physical protein-protein interactions, the sequence-level constraints emerging from non-binding functional interactions in metabolism remain unclear. To quantify how variation in the activity of one enzyme constrains the biochemical parameters and sequence of another, we focused on dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), a pair of enzymes catalyzing consecutive reactions in folate metabolism. We used deep mutational scanning to quantify the growth rate effect of 2,696 DHFR single mutations in 3 TYMS backgrounds under conditions selected to emphasize biochemical epistasis. Our data are well-described by a relatively simple enzyme velocity to growth rate model that quantifies how metabolic context tunes enzyme mutational tolerance. Together our results reveal the structural distribution of epistasis in a metabolic enzyme and establish a foundation for the design of multi-enzyme systems.

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Colorectal cancer (CRC) is in the forefront of malignancies for its high incidence and mortality. 5-Fluorouracil (5-FU) is one of the most widely used effective drugs for the treatment of CRC. However, there is an urgent need in reducing its systemic side effects and chemoresistance, in order to make 5-FU-based chemotherapy more effective in the treatment of CRC. In this study, engineered CRC cells were established to overexpress miR-323a-3p, which was a tumor suppressor that targeted both EGFR and TYMS. Then miR-323a-3p-loaded exosomes (miR-Exo) were obtained with suitable methods of collection and purification. We found that miR-Exo significantly inhibited CRC cell proliferation and induced apoptosis by the way of targeting EGFR directly in the cells, which eventually led to desirable tumor regression in the cell derived xenograft (CDX) and patient derived xenograft (PDX) tumor mice models. Moreover, we discovered that miR-323a-3p released from miR-Exo directly inhibited the upregulation of thymidylate synthase (TYMS) induced by 5-FU-resistence in CRC cells, resulting in the revival of tumor cytotoxicity from 5-FU. MiR-Exo could effectively induce the CRC cell apoptosis by targeting EGFR and TYMS, and enhance the therapeutic effects of 5-FU on CRC. Our work demonstrates the potency of miR-Exo for advanced CRC biotherapy.

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Uterine leiomyosarcomas (uLMS) have a poor prognosis and a high percentage of recurrent disease. Bioinformatics has become an integral element in rare cancer studies by overcoming the inability to collect a large enough study population. This study aimed to investigate and highlight crucial genes, pathways, miRNAs, and transcriptional factors (TF) on uLMS samples from five Gene Expression Omnibus datasets and The Cancer Genome Atlas Sarcoma study. Forty-one common differentially expressed genes (DEGs) were enriched and annotated by the DAVID software. With protein-protein interaction (PPI) network analysis, we selected ten hub genes that were validated with the TNMplotter web tool. We used the USCS Xena browser for survival analysis. We also predicted TF-gene and miRNA-gene regulatory networks along with potential drug molecules. TYMS and TK1 correlated with overall survival in uLMS patients. Finally, our results propose further validation of hub genes (TYMS and TK1), miR-26b-5p, and Sp1 as biomarkers of pathogenesis, prognosis, and differentiation of uLMS. Regarding the aggressive behavior and poor prognosis of uLMS, with the lack of standard therapeutic regimens, in our opinion, the results of our study provide enough evidence for further investigation of the molecular basis of uLMS occurrence and its implication in the diagnosis and therapy of this rare gynecological malignancy.

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Probiotic roles of Clostridium butyricum (C.B) are involved in regulating disease and cancers, yet the mechanistic basis for these regulatory roles remains largely unknown. Here, we demonstrate that C.B reprograms the proliferation, migration, stemness, and tumor growth in CRC by regulating pivotal signal molecules including MYC. Destabilization of MYC by C.B supplementation suppresses cancer cell proliferation/metastasis, sensitizes 5-FU treatment, and boosts responsiveness of anti-PD1 therapy. MYC is a transcriptional regulator of Thymidylate synthase (TYMS), a key target of the 5-FU. Also MYC is known to impact on PD-1 expression. Mechanistically, C.B treatment of CRC cells results in MYC degradation by enhancing proteasome-mediated ubiquitination, thereby mitigating MYC-mediated 5-FU resistance and boosting anti-PD1 immunotherapeutic efficacy. Together, our findings uncover previously unappreciated links between C.B and CRC cell signaling, providing insight into the tumorigenesis modulating mechanisms of C.B in boosting chemo/immune therapies.

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Mammalian metabolism comprises a series of interlinking pathways that include two major cycles: the folate and methionine cycles. The folate-mediated metabolic cycle uses several oxidation states of tetrahydrofolate to carry activated one-carbon units to be readily used and interconverted within the cell. They are required for nucleotide synthesis, methylation and metabolism, and particularly for proliferation of cancer cells. Based on the latest progress in genome-wide CRISPR loss-of-function viability screening of 789 cell lines, we focus on the most cancer-dependent enzymes in this pathway, especially those that are hyperactivated in cancer, to provide new insight into the chemical basis for cancer drug development. Since the complete 3D structure of several of these enzymes of the one-carbon pathway in their active form are not available, we used homology modelling integrated with the interpretation of the reaction mechanism. In addition, have reconstructed the most likely scenario for the reactions taking place paired with their catalytic competence that provides a testable framework for this pathway.

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In the present study, we found that EZH1 depletion in MYCN-amplified neuroblastoma cells resulted in significant cell death as well as xenograft inhibition. EZH1 depletion decreased the level of H3K27me1; the interaction and protein stabilization of MYCN and EZH1 appear to play roles in epigenetic transcriptional regulation. Transcriptome analysis of EZH1-depleted cells resulted in downregulation of the cell cycle progression-related pathway. In particular, Gene Set Enrichment Analysis revealed downregulation of reactome E2F-mediated regulation of DNA replication along with key genes of this process, TYMS, POLA2, and CCNA1. TYMS and POLA2 were transcriptionally activated by MYCN and EZH1-related epigenetic modification. Treatment with the EZH1/2 inhibitor UNC1999 also induced cell death, decreased H3K27 methylation, and reduced the levels of TYMS in neuroblastoma cells. Previous reports indicated neuroblastoma cells are resistant to 5-fluorouracil (5-FU) and TYMS (encoding thymidylate synthetase) has been considered the primary site of action for folate analogues. Intriguingly, UNC1999 treatment significantly sensitized MYCN-amplified neuroblastoma cells to 5-FU treatment, suggesting that EZH inhibition could be an effective strategy for development of a new epigenetic treatment for neuroblastoma.

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Dyskeratosis congenita (DC) is an inherited bone-marrow-failure disorder characterized by a triad of mucocutaneous features that include abnormal skin pigmentation, nail dystrophy, and oral leucoplakia. Despite the identification of several genetic variants that cause DC, a significant proportion of probands remain without a molecular diagnosis. In a cohort of eight independent DC-affected families, we have identified a remarkable series of heterozygous germline variants in the gene encoding thymidylate synthase (TYMS). Although the inheritance appeared to be autosomal recessive, one parent in each family had a wild-type TYMS coding sequence. Targeted genomic sequencing identified a specific haplotype and rare variants in the naturally occurring TYMS antisense regulator ENOSF1 (enolase super family 1) inherited from the other parent. Lymphoblastoid cells from affected probands have severe TYMS deficiency, altered cellular deoxyribonucleotide triphosphate pools, and hypersensitivity to the TYMS-specific inhibitor 5-fluorouracil. These defects in the nucleotide metabolism pathway resulted in genotoxic stress, defective transcription, and abnormal telomere maintenance. Gene-rescue studies in cells from affected probands revealed that post-transcriptional epistatic silencing of TYMS is occurring via elevated ENOSF1. These cell and molecular abnormalities generated by the combination of germline digenic variants at the TYMS-ENOSF1 locus represent a unique pathogenetic pathway for DC causation in these affected individuals, whereas the parents who are carriers of either of these variants in a singular fashion remain unaffected.

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Aim: To investigate the association of DPYD, MTHFR and TYMS polymorphisms on 5-fluorouracil (5-FU) related toxicities and patient survival. Materials & methods: A total of 103 colorectal cancer patients prescribed 5-FU were included in the study. Genotyping was conducted for several DPYD, MTHFR and TYMS polymorphisms using a microarray analyzer. Results: DPYD 496A>G polymorphism was found to be significantly associated with 5-FU related grade 0-2, but not severe toxicities (p = 0.02). Furthermore, patients with DPYD 85TC and CC genotypes had longer progression and overall survival times compared to TT genotypes in our study group (log rank = 6.60; p = 0.01 and log rank = 4.40; p = 0.04, respectively). Conclusion: According to our results, DPYD 496AG and GG genotypes might be protective against severe adverse events compared to the AA genotype. Another DPYD polymorphism, 85T>C, may be useful in colorectal cancer prognosis. Further studies for both polymorphisms should be conducted in larger populations to achieve accurate results.

5-fluorouracil (5-FU) is a widely used drug for chemotherapy in colorectal cancer. In this study, we investigated the relationship between the severity of 5-FU induced adverse events and several variations in DPYD, MTHFR and TYMS genes, which encode the enzymes involved in 5-FU metabolism in a total of 103 colorectal patients. We also examined the relationship between the polymorphisms and progression-free and overall survival times of the patients in our study group. Among the variations, DPDY 496A>G polymorphism was found to be associated with 5-FU induced adverse events. Also, the DPYD 85T>C polymorphism was detected to be associated with longer progression-free and overall survival times.

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Thymidylate synthase (TYMS) is the crucial enzymatic precursor for DNA biosynthesis and, therefore, the critical target for numerous types of chemotherapy, including the most frequently applied agent in colon cancer treatment 5-fluorouracil (5-FU). TYMS also seems to be associated with cancer metastasis and acquiring mesenchymal character by tumor cells during epithelial-mesenchymal transition (EMT). Based on that knowledge, we decided to investigate the role of TYMS in the modulation of invasive ability in colon cancer cells, where its effect on cancer metastasis has not been studied in detail before. We employed colon cancer cells isolated from different stages of tumor development, cells undergoing EMT, and TYMS overexpressing cells. The elongation ratio, cell migration, invasion assay, and MMP-7 secretion were applied to analyze the cell behavior. Important epithelial and mesenchymal markers characteristic of EMT were examined at the protein level by Western blot assay. Overall, our study showed a correlation between TYMS level and invasion ability in colon cancer cells and, above all, a crucial role of TYMS in the EMT regulation. We postulate that chemotherapeutics that decrease or inhibit TYMS expression could increase the effectiveness of the therapy in patients with colon cancer, especially in the metastatic stage.

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Background: To investigate relationships between five single nucleotide polymorphisms (SNP) in four maternal genes involved in one carbon metabolism and adverse pregnancy outcomes, including preterm birth (PTB), low birth weight (LBW), and small-for-gestational-age (SGA). Methods: This was a prospective mother and child cohort study in Wuqiang, China. Pregnant women (n = 939) were recruited from Jun 2016 to Oct 2018. Pregnancy outcomes (PTB, LBW, and SGA) were extracted from medical records and other information including age at childbearing, maternal education level, gravidity, parity, pre-pregnancy weight and height was collected by using a structured questionnaire. The maternal serum folate concentration was measured by using Abbott Architect i2000SR chemiluminescence analyzer in the first prenatal care visit. DNA genotyping of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G, and thymidylate synthetase (TYMS) rs3819102 was processed by Sequenom MassARRAY iPLEX Platform. Univariate and multivariate logistics regression analysis were used to test the relationships between 5 SNPs and PTB, LBW, SGA. Results: Totally, 849 dyads of women and infants were included in the analysis. The prevalence of PTD, LBW, and SGA were 3.76%, 1.58%, and 5.31% respectively. The homozygote frequencies of MTHFR C677T, MTHFR A1298C, MTRR A66G, MTR A2756G, and TYMS rs3819102 were 44.2%, 1.4%, 6.7%, 1.3%, and 3.2%, and the alt allele frequencies were 66.1%, 10.8%, 24.9%, 10.5%, and 20.5% respectively. The average serum folate concentration was 11.95 ng/mL and the folate deficiency rate was 0.47%. There were no significant associations between MTHFR C677T, MTHFR A1298C, MTRR A66G, MTR A2756G, TYMS rs3819102 alleles and PTD, LBW, SGA (p > 0.05). Conclusions: In the population with adequate folate status and low prevalence of adverse pregnancy outcomes, MTHFR C677T, MTHFR A1298C, MTRR A66G, MTR A2756G, TYMS rs3819102 alleles may not be related to PTD, LBW, and SGA.

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BACKGROUND: The expression of thymidylate synthase (TYMS) is significantly up-regulated in various cancers and associated with the poor prognosis of patients. However, the role of TYMS in the progression of colorectal cancer (CRC) is unclear. METHODS: Cell function assay, biology information analysis, and RNA sequencing were used to investigate the role of TYMS in the progression of CRC and underlining molecular mechanism. SPSS22.0 statistical software and GraphPad Prism 5 (Graphpad software) were used for statistical analysis. RESULTS: Our results showed that TYMS expression was higher in CRC tissues than that in non-tumor colorectal mucosa tissues. TYMS knockdown inhibited the proliferation, migration and invasion of HCT116 and HT29 cells, and the spheroid formation of HCT116 cells. The underling mechanism demonstrated that TYMS promoted the progression of CRC by regulating EMT-related proteins including E-cadherin, Vimentin, MMP-9 and stem cell biomarkers including CD133 and CD44. Furthermore, DEG sequencing showed that TYMS knockdown enriched the pathways of metastasis and metabolism by GO and KEGG analysis. We identified TM4SF4 was the downstream target of TYMS in CRC cells. TM4SF4 overexpression increased migration and invasion of CRC cells by regulating EMT and CD133 expression. CONCLUSIONS: Our findings suggest that TYMS-TM4SF4 axis may promote the progression of CRC by EMT and upregulating stem cell markers.

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OBJECTIVES: The fluoropyrimidine derivatives 5-Fluorouracil and Capecitabine are widely used for the treatment of solid tumors. Fluoropyrimidine metabolism involves a cascade of different enzymes, including MTHFR enzyme. MTHFR c.665C>T polymorphism, leading to decreased MTHFR activity, is a potential pharmacogenomic marker for fluoropyrimidine drug response. The aim of the present study was to analyze the association of MTHFR c.665C>T polymorphism with fluoropyrimidine response in terms of therapy induced adverse events (AEs), requirement of dose reduction and delayed drug administration or therapy discontinuation. METHODS: The study group consisted of 313 fluoropyrimidine-treated cancer patients. PCR-RFLP was used to analyze MTHFR c.665C>T polymorphism. RESULTS: In female patients, MTHFR c.665 CT and TT genotypes were associated with dose reduction (p=0.029). In gender stratification, regression analysis adjusted for age of disease onset, body surface area and AE incidence, showed that MTHFR CT and TT genotypes increased both need for fluoropyrimidine dose reduction (OR 5.050, 95% CI 1.346-18.948, p=0.016) and percentage of dose reduction (ß=3.318, 95% C.I. 1.056-5.580, p=0.004) in female patients. Such differences were not present in male patients. No other associations were found. CONCLUSIONS: MTHFR c.665C>T polymorphism was associated with fluoropyrimidine dose reduction in female cancer patients. This gender*MTHFR interaction merits further investigation.

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BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and one of the major causes of cancer-related death. Thymidylate synthase (TYMS) catalyzes the methylation of deoxy guanosine to deoxy thymidylate, which is a crucial gene for DNA repair and replication. Thus, TYMS was reported to be closely associated with developing a variety of tumors, but it has been poorly studied in HCC. MATERIALS AND METHODS: We used the cell counting kit-8 (CCK-8), BrdU, and CFSE assay to measure cell proliferation. The flow cytometry assay and the TUNEL assay were used for assessing cell apoptosis. The flow cytometry assay was used to analyze the cell cycle. The Transwell invasion assay and the wound healing assay were conducted to determine the invasive ability of the cells. RT-qPCR and Western blot analyses were performed to evaluate the mRNA and protein expression levels of specific genes, respectively. RESULTS: TYMS was found to be upregulated in both HCC cells and patient samples. High expression of TYMS was associated with an unfavorable prognosis in HCC patients based on the TCGA-LIHC dataset. Cell proliferation, apoptosis, and invasion assays revealed that TYMS promoted the proliferation and invasion of HCC cells as well as inhibited apoptosis. In addition, TYMS is a downstream target of FOXM1. TYMS knockdown reversed the 5-FU resistance caused by FOXM1 overexpression and re-sensitized HCC cells to 5-FU treatment. CONCLUSION: This study suggested that TYMS serves as an oncogene in HCC, and targeting the FOXM1-TYMS axis may help improve the survival of HCC patients as well as provide new insights for treating advanced HCC patients.