RNA circles with minimized immunogenicity as potent PKR inhibitors.

Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.

Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

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.

Thymidylate synthase disruption to limit cell proliferation in cell therapies.

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.

Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?

The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.

The Association Between Thymidylate Synthase Gene Polymorphisms and the Risk of Ischemic Stroke in Chinese Han Population.

Family history of hypertension, smoking, diabetes and alcohol consumption and atherosclerotic plaque were identified as common risk factors in IS. We aimed at investigating the relationship between Thymidylate Synthase (TS) gene polymorphisms and ischemic stroke (IS).This case-control research selected and genotyped three single nucleotide polymorphisms (SNPs)of TS( rs699517, rs2790, and rs151264360) with Sanger sequencing in Chinese Han population. We also adopted logistic regression analysis in genetic models for calculating odds ratios and 95% confidence intervals. Genotype-Tissue Expression(GTEx) database analyzed the tissue-specific expression and TS polymorphisms. The ischemic stroke patients showed higher low-density lipoprotein cholesterol and total homocysteine (tHcy). It was found that patients with the TT genotype of rs699517 and GG genotype of rs2790 had larger degrees of tHcy than those with CC + CT genotypes and AA + AG genotypes, respectively. The genotype distribution of the three SNPs did not deviate from Hardy-Weinberg equilibrium (HWE). Haplotype analysis showed that T-G-del was the major haplotype in IS, and C-A-ins was the major haplotype in controls. GTEx database indicated that the rs699517 and rs2790 increased the expression of TS in healthy human and associated with TS expression level in a single tissue. In conclusion: This study has shown that TS rs699517 and rs2790 were significantly related to ischemic stroke patients.

Sodium Butyrate Inhibits the Expression of Thymidylate Synthase and Induces Cell Death in Colorectal Cancer Cells.

The most commonly used chemotherapy for colorectal cancer (CRC) is the application of 5-fluorouracil (5-FU). Inhibition of thymidylate synthase (TYMS) expression appears to be a promising strategy to overcome the decreased sensitivity to 5-FU caused by high expression of TYMS, which can be induced by 5-FU treatment. Several compounds have been shown to potentially inhibit the expression of TYMS, but it is unclear whether short-chain fatty acids (SCFAs), which are naturally produced by bacteria in the human intestine, can regulate the expression of TYMS. Sodium butyrate (NaB) is the most widely known SCFA for its beneficial effects. Therefore, we investigated the enhancing effects on inhibition of cell viability and induction of apoptosis after co-treatment of NaB with 5-FU in two CRC cell lines, HCT116 and LoVo. This study suggests that the effect of NaB in improving therapeutic sensitivity to 5-FU in CRC cells may result from a mechanism that strongly inhibits the expression of TYMS. This study also shows that NaB inhibits the migration of CRC cells and can cause cell cycle arrest in the G2/M phase. These results suggest that NaB could be developed as a potential therapeutic adjuvant to improve the therapeutic effect of 5-FU in CRC.

Analysis of the TSER and G>C variants in the TYMS gene: a high frequency of low expression genotypes predicted in the Mexican population.

BACKGROUND: In the TYMS gene promoter, there is a repeat polymorphism (TSER) that affects the expression level of the thymidylate synthetase (TS) enzyme involved in the response to some anticancer drugs. The G>C transversion located in the TSER*3R allele decreases the expression level of the TS enzyme avoiding the upstream stimulatory factor (USF-1) binding site. Despite the biomedical impact of the SNP G>C, only TSER has been reported in most worldwide populations. Thus, we studied both TSER and SNP G>C variants in the Mexican population. SUBJECTS AND METHODS: A population sample (n = 156) was genotyped for the TSER and G>C variants by PCR and PCR-RFLPs, respectively, followed by PAGE and silver staining. RESULTS: For TSER, the most frequent allele was 2 R (52.56%), as well as the genotype 2 R/3R (42.3%). Comparison with Latin American, European, and American (USA) populations suggest a heterogeneous worldwide distribution (FST-value = 0.01564; p-value = 0.0000). When the G>C variant was included (2RG, 3RG, and 3RC), a high frequency of low expression genotypes was observed: 2RG/2RG, 2RG/3RC, and 3RC/3RC (84.6%). CONCLUSION: The high frequency of genotypes associated with low TS enzyme expression justifies obtaining the TYMS gene variant profile in Mexican patient's candidates to pharmaceutical treatments like 5'-Fluoracil, methotrexate, and pemetrex.

Association of Thymidylate Synthase (TS) Gene Polymorphisms with Incidence and Prognosis of Coronary Artery Disease.

Coronary artery disease (CAD) is a prevalent cardiovascular condition characterized by the accumulation of plaque within coronary arteries. While distinct features of CAD have been reported, the association between genetic factors and CAD in terms of biomarkers was insufficient. This study aimed to investigate the connection between genetic factors and CAD, focusing on the thymidylate synthase (TS) gene, a gene involved in DNA synthesis and one-carbon metabolism. TS plays a critical role in maintaining the deoxythymidine monophosphate (dTMP) pool, which is essential for DNA replication and repair. Therefore, our research targeted single nucleotide polymorphisms that could potentially impact TS gene expression and lead to dysfunction. Our findings strongly associate the TS 1100T>C and 1170A>G genotypes with CAD susceptibility. We observed that TS 1100T>C polymorphisms increased disease susceptibility in several groups, while the TS 1170A>G polymorphism displayed a decreasing trend for disease risk when interacting with clinical factors. Furthermore, our results demonstrate the potential contribution of the TS 1100/1170 haplotypes to disease susceptibility, indicating a synergistic interaction with clinical factors in disease occurrence. Based on these findings, we propose that polymorphisms in the TS gene had the possibility of clinically useful biomarkers for the prevention, prognosis, and management of CAD in the Korean population.

IncRNA TYMSOS Promotes Epithelial-Mesenchymal Transition and Metastasis in Thyroid Carcinoma through Regulating MARCKSL1 and Activating the PI3K/Akt Signaling Pathway.

Thyroid carcinoma (THCA) has been increasing in incidence greater than other cancers. Long noncoding RNAs (lncRNAs) were reported to play crucial roles in THCA development. Our study aimed to explore the underlying mechanism of lncRNA thymidylate synthetase opposite strand RNA (TYMSOS) in THCA. TYMSOS and myristoylated alanine rich protein kinase C substrate like 1 (MARCKSL1) were upregulated whereas miR-130a-5p was downregulated in THCA cells and tissues. The results of loss-of-function assays showed that TYMSOS knockdown inhibited cell metastasis and epithelial-mesenchymal transition (EMT) in THCA. TYMSOS was primarily distributed in the cytoplasm of THCA cells, as shown by FISH assay. RNA pulldown and luciferase reporter assay further showed that TYMSOS binds with miR-130a-5p. Luciferase reporter assay also revealed that MARCKSL1 is targeted by miR-130a-5p. Rescue assay showed that the suppression of TYMSOS downregulation on THCA cell malignant behaviors was reversed by MARCKSL1 overexpression. Additionally, overexpressing MARCKSL1 offset the inhibition of TYMSOS downregu-lation on the PI3K/Akt signaling pathway. TYMSOS knockdown inhibits the growth of THCA tumors, as in vivo assays showed. Collectively, TYMSOS facilitates THCA progression by sponging miR-130a-5p and upregulating MARCKSL1 to activate the PI3K/Akt signaling pathway, providing new avenues for THCA treatment.

MALAT1-miRNAs network regulate thymidylate synthase and affect 5FU-based chemotherapy.

BACKGROUND: The active metabolite of 5-Fluorouracil (5FU), used in the treatment of several types of cancer, acts by inhibiting the thymidylate synthase encoded by the TYMS gene, which catalyzes the rate-limiting step in DNA replication. The major failure of 5FU-based cancer therapy is the development of drug resistance. High levels of TYMS-encoded protein in cancerous tissues are predictive of poor response to 5FU treatment. Expression of TYMS is regulated by various mechanisms, including involving non-coding RNAs, both miRNAs and long non-coding RNAs (lncRNAs). AIM: To delineate the miRNAs and lncRNAs network regulating the level of TYMS-encoded protein. MAIN BODY: Several miRNAs targeting TYMS mRNA have been identified in colon cancers, the levels of which can be regulated to varying degrees by lncRNAs. Due to their regulation by the MALAT1 lncRNA, these miRNAs can be divided into three groups: (1) miR-197-3p, miR-203a-3p, miR-375-3p which are downregulated by MALAT1 as confirmed experimentally and the levels of these miRNAs are actually reduced in colon and gastric cancers; (2) miR-140-3p, miR-330-3p that could potentially interact with MALAT1, but not yet supported by experimental results; (3) miR-192-5p, miR-215-5p whose seed sequences do not recognize complementary response elements within MALAT1. Considering the putative MALAT1-miRNAs interaction network, attention is drawn to the potential positive feedback loop causing increased expression of MALAT1 in colon cancer and hepatocellular carcinoma, where YAP1 acts as a transcriptional co-factor which, by binding to the TCF4 transcription factor/ ß-catenin complex, may increase the activation of the MALAT1 gene whereas the MALAT1 lncRNA can inhibit miR-375-3p which in turn targets YAP1 mRNA. CONCLUSION: The network of non-coding RNAs may reduce the sensitivity of cancer cells to 5FU treatment by upregulating the level of thymidylate synthase.

Gastric cancer with microsatellite instability displays increased thymidylate synthase expression.

BACKGROUND: Gastric cancer (GC) with microsatellite instability (MSI) is a less aggressive disease and associated with resistance to 5-fluorouracil (5-FU)-based chemotherapy (CMT). Thymidylate synthase (TS) is inhibited by 5-FU, and another potential mediator of therapeutic resistance to 5-FU. Therefore, we aimed to analyze the association between MSI and TS expression in GC, and its impact on disease outcomes. METHODS: We retrospectively evaluated GC who underwent D2-gastrectomy. MSI and TS were analyzed by immunohistochemistry. We also investigated p53 expression, PD-L1 status, and tumor-infiltrating lymphocytes (CD4 and CD8). RESULTS: Out of 284 GC, 60 (21.1%) were MSI. Median TS-score for all cases was 16.5. TS expression was significantly higher in MSI compared to microsatellite-stable (MSS; p < 0.001). Considering both status, GC were classified in four groups: 167 (58.8%) MSS + TS-low; 57 (20.1%) MSS + TS-High; 24 (8.5%) MSI + TS-low; and 36 (12.7%) MSI + TS-high. MSI + TS-high group had less advanced pTNM stage, higher CD8+T cells levels (p < 0.001) and PD-L1 positivity (p < 0.001). Normal p53 expression was related to MSI GC (p < 0.001). Improved survival was observed in MSI + TS-high, but no survival benefit was seen with CMT. CONCLUSION: MSI GC was associated with high TS levels, which may explain therapeutic resistance to 5-FU. Additionally, MSI + TS-high showed better survival, but without improvement with CMT.

Dihydrofolate reductase, thymidylate synthase, and serine hydroxy methyltransferase: successful targets against some infectious diseases.

Parasitic diseases have a serious impact on the world in terms of health and economics and are responsible for worldwide mortality and morbidity. The present review features the hybrid targeting involving three main enzymes for the treatment of different parasitic diseases. The enzymes Dihydrofolate reductase, thymidylate synthase, and Serine hydroxy methyltransferase play an essential role in the folate pathway. The present review focuses on these enzymes, which can be targeted against several diseases. It shed light on the past, present, and future of these targets, and it can be assessed that these targets can play a significant role against several infectious diseases. For combating viral and protozoal infectious diseases, these targets in combination should be addressed.

Genetic Markers of Therapeutic Efficacy of Methotrexate in Patients with Psoriasis.

We studied the effect of C677T and A1298C polymorphisms of the MTHFR gene and 2R/3R polymorphisms of the TYMS gene on the sensitivity to methotrexate in patients with psoriasis (n=139). It was shown that genotype 3R/3R TYMS (OR 8.86, 95%CI 2.00-39.22) and complex genotypes MTHFR1298:A;TYMS:3R (OR 8.20, 95%CI 2.36-28.48) and MTHFR677:C;TYMS:3R (OR 5.40, 95%CI 1.95-14.94) were associated with sensitivity to methotrexate, while genotype 2R/2R TYMS (OR 8.20, 95%CI 2.36-28.48) and complex genotypes MTHFR1298:C;MTHFR677:T;TYMS:2R (OR 0.18, 95%CI 0.06-0.56) and MTHFR1298:C;MTHFR677:T (OR 0.23, 95%CI 0.09-0.59) were associated with resistance to methotrexate. The results can be used for preventive assessment of the effectiveness of methotrexate treatment in patients with psoriasis.

Immunohistochemistry with 3 different clones in anaplastic lymphoma kinase fluorescence in situ hybridization positive non-small-cell lung cancer with thymidylate synthase expression analysis: a multicentre, retrospective, Italian study.

Introduction: ALK rearrangement is the only druggable oncogenic driver detectable by immunohistochemistry (IHC) not requiring further confirmation of positivity in accessing first-line specific inhibitors. ALK-positive patients experience clinical benefit from pemetrexed-based chemotherapy possibly due to lower thymidylate synthase (TS) levels. This study assesses agreement with three different ALK IHC clones in 37 FISH-positive NSCLC. TS expression by real time (RT)-PCR was compared with ALK FISH-negative cases. Materials and methods: 37 ALK FISH-positive NSCLC cases diagnosed between 2010 and 2015 in 7 Italian centres were investigated with ICH using three different anti-ALK antibodies (ALK1, 5A4 and D5F3). Staining for ALK1 and 5A4 was graded as 0+,1+,2+, and 3+, while the scoring for D5F3 was recorded as negative or positive. Proportion agreement analysis was done using Cohen's unweighted kappa (k). TS and ß-actin expression levels were analysed by quantitative RT-PCR. Comparison between TS expression in ALK FISH-positive specimens and a control cohort of ALK FISH-negative ones was performed with the Mann-Whitney and Kruskal-Wallis tests. Results: Considering 2+ and 3+ as positive, the proportion of IHC agreement was 0.1691 (95% CI 0-0.4595) for ALK1/5A4, 0.1691 (95% CI 0-0.4595) for ALK1/D5F3, and 1 for D5F3/5A4. Considering 3+ as positive, it was 0.1543 (95% CI 0-0.4665) for ALK1/ 5A4, 0.0212 (95% CI 0-0.1736) for ALK1/D5F3, and 0.2269 (95% CI 0-0.5462) for 5A4/D5F3. Median TS expression was 6.07 (1.28-14.94) and ALK-positive cases had a significant lower TS expression than ALK-negative tumours (p = 0.002). Conclusions: IHC proved to be a reliable tool for the diagnosis of ALK-rearranged NSCLC. D5F3 and 5A4 clones have the highest percentage of agreement. TS levels are significantly lower in FISH-positive patients.

Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase.

The observables associated with protein intrinsic fluorescence - spectra, time decays, anisotropies - offer opportunities to monitor in real time and non-invasively a protein's functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.

The prevalence and clinical relevance of 2R/2R TYMS genotype in patients with gastrointestinal malignancies treated with fluoropyrimidine-based chemotherapy regimens.

INTRODUCTION: The prevalence of 2R/2R TYMS genotype is variable but estimated to be around 20-30% in Caucasians. The clinical relevance of TYMS 2R/2R genotype in predicting severe fluoropyrimidine-related adverse events (FrAE) is controversial. Here, we explored the prevalence and clinical relevance of 2R/2R TYMS genotype. METHODS: Between 2011 and 2018, 126 patients were genotyped for TYMS. FrAEs were graded according to CTCAE version 5.0. Fisher's exact test was used for statistical analysis. RESULTS: The prevalence of TYMS 2R/2R genotype was 24.6%. Among patients with TYMS genotypes (N = 71) that predict decreased TS expression, 2R/2R TYMS genotype was the most common TYMS genotype seen in female (57%) and African American (60%) patients. Among patients with genotypes that predict increased TS expression (N = 55), 12 patients had grade 3-4 FrAEs (22%), while among patients with genotypes that predict decreased TS expression (N = 71), 30 patients had grade 3-4 FrAEs (42%) (p = 0.0219). Compared to patients with genotypes predicting increased TS expression, 17 out of 31 patients (55%) with TYMS 2R/2R genotype had grade 3-4 FrAEs (p = 0.0039) and 15 out 40 patients (38%) with TYMS 2R/3RC and TYMS 3RC/3RC genotype had grade 3-4 FrAEs (p = 0.1108). CONCLUSION: The prevalence of TYMS 2R/2R genotype was 24.6%, and it had a unique sex and ethnic distribution. Polymorphism in the promoter region of TYMS gene that predicts decreased TS expression due to 2R/2R variant was associated with grade 3-4 FrAEs. These data suggest that genotyping patients who are not DPD deficient for TYMS might identify patients at risk of severe FrAEs.

TYMSOS drives the proliferation, migration, and invasion of gastric cancer cells by regulating ZNF703 via sponging miR-4739.

Gastric cancer (GC) is a kind of malignancy originating from the epithelium of gastric mucosa. Long noncoding RNAs (lncRNAs) are tightly related to the GC progression. Herein, our research was meant to investigate a novel lncRNA thymidylate synthetase opposite strand (TYMSOS) in GC. Quantitative real-time polymerase chain reaction was used to analyze TYMSOS expression in GC cells. 5-Ethynyl-2'-deoxyuridine, flow cytometry analysis, and transwell assay detected the influence of TYMSOS on GC cell proliferation, apoptosis, migration, and invasion. Subcellular fractionation and fluorescent in situ hybridization assays determined the cellular localization of TYMSOS in GC cells. Bioinformatics programs, RNA-binding protein immunoprecipitation, RNA pull-down, and luciferase reporter assays measured the molecular interplays of TYMSOS in GC cells. In brief, TYMSOS was highly expressed in GC cells, and TYMSOS silence inhibited GC cell proliferation, migration, and invasion while elevating cell apoptosis. Functionally, TYMSOS functioned as a competing endogenous RNA to posttranscriptionally modulate GC progression. TYMSOS interacted with miR-4739 to regulate its target gene zinc finger protein 703. Collectively, our study proved the tumor-promoting role of TYMSOS in GC cells, which might offer the utility value for GC treatment.

Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells.

5-Fluorouracil (5-FU) is a cornerstone drug used in the treatment of colorectal cancer (CRC). However, the development of resistance to 5-FU and its analogs remain an unsolved problem in CRC treatment. In this study, we investigated the molecular mechanisms and tumor biological aspects of 5-FU resistance in CRC HCT116 cells. We established an acquired 5-FU-resistant cell line, HCT116RF10. HCT116RF10 cells were cross-resistant to the 5-FU analog, fluorodeoxyuridine. In contrast, HCT116RF10 cells were collaterally sensitive to SN-38 and CDDP compared with the parental HCT16 cells. Whole-exome sequencing revealed that a cluster of genes associated with the 5-FU metabolic pathway were not significantly mutated in HCT116 or HCT116RF10 cells. Interestingly, HCT116RF10 cells were regulated by the function of thymidylate synthase (TS), a 5-FU active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) inhibiting enzyme. Half of the TS was in an active form, whereas the other half was in an inactive form. This finding indicates that 5-FU-resistant cells exhibited increased TS expression, and the TS enzyme is used to trap FdUMP, resulting in resistance to 5-FU and its analogs.

Development and validation of a five-gene model to predict postoperative brain metastasis in operable lung adenocarcinoma.

One of the most common sites of extra-thoracic distant metastasis of nonsmall-cell lung cancer is the brain. Our study was performed to discover genes associated with postoperative brain metastasis in operable lung adenocarcinoma (LUAD). RNA seq was performed in specimens of primary LUAD from seven patients with brain metastases and 45 patients without recurrence. Immunohistochemical (IHC) assays of the differentially expressed genes were conducted in 272 surgical-resected LUAD specimens. LASSO Cox regression was used to filter genes related to brain metastasis and construct brain metastasis score (BMS). GSE31210 and GSE50081 were used as validation datasets of the model. Gene Set Enrichment Analysis was performed in patients stratified by risk of brain metastasis in the TCGA database. Through the initial screening, eight genes (CDK1, KPNA2, KIF11, ASPM, CEP55, HJURP, TYMS and TTK) were selected for IHC analyses. The BMS based on protein expression levels of five genes (TYMS, CDK1, HJURP, CEP55 and KIF11) was highly predictive of brain metastasis in our cohort (12-month AUC: 0.791, 36-month AUC: 0.766, 60-month AUC: 0.812). The validation of BMS on overall survival of GSE31210 and GSE50081 also showed excellent predictive value (GSE31210, 12-month AUC: 0.682, 36-month AUC: 0.713, 60-month AUC: 0.762; GSE50081, 12-month AUC: 0.706, 36-month AUC: 0.700, 60-month AUC: 0.724). Further analyses showed high BMS was associated with pathways of cell cycle and DNA repair. A five-gene predictive model exhibits potential clinical utility for the prediction of postoperative brain metastasis and the individual management of patients with LUAD after radical resection.

MicroRNA-375-3p enhances chemosensitivity to 5-fluorouracil by targeting thymidylate synthase in colorectal cancer.

Resistance to chemotherapy is a major challenge for the treatment of patients with colorectal cancer (CRC). Previous studies have found that microRNAs (miRNAs) play key roles in drug resistance; however, the role of miRNA-373-3p (miR-375-3p) in CRC remains unclear. The current study aimed to explore the potential function of miR-375-3p in 5-fluorouracil (5-FU) resistance. MicroRNA-375-3p was found to be widely downregulated in human CRC cell lines and tissues and to promote the sensitivity of CRC cells to 5-FU by inducing colon cancer cell apoptosis and cycle arrest and by inhibiting cell growth, migration, and invasion in vitro. Thymidylate synthase (TYMS) was found to be a direct target of miR-375-3p, and TYMS knockdown exerted similar effects as miR-375-3p overexpression on the CRC cellular response to 5-FU. Lipid-coated calcium carbonate nanoparticles (NPs) were designed to cotransport 5-FU and miR-375-3p into cells efficiently and rapidly and to release the drugs in a weakly acidic tumor microenvironment. The therapeutic effect of combined miR-375 + 5-FU/NPs was significantly higher than that of the individual treatments in mouse s.c. xenografts derived from HCT116 cells. Our results suggest that restoring miR-375-3p levels could be a future novel therapeutic strategy to enhance chemosensitivity to 5-FU.