Thymidylate Synthase Expression Research Articles

NEK2 promotes the migration, invasion, proliferation of ESCC and mediates ESCC immunotherapy

PurposeEsophageal squamous cell carcinoma (ESCC) is a disease with a high incidence rate and high mortality worldwide. The Never in Mitosis A (NIMA) family member NIMA-related kinase 2 (NEK2) plays an important role in mitosis. However, the role of NEK2 in the pathogenesis of ESCC remains unclear. Patients and methodsThe expression and function of NEK2 in TCGA and GEO data sets were analyzed by bioinformatics. We verified the expression of NEK2 in ESCC tissues and cell lines by Western blotting and immunohistochemical methods and further explored the relationship between tumor stage and NEK2 expression. The differences in NEK2 expression and survival in patients with EC were verified by bioinformatics analysis. ESCC cell lines with stable knockdown of NEK2 were established by lentivirus-mediated shRNA delivery. The effects of NEK2 on ESCC cells were analyzed on the cytological level with assays including CCK-8, EdU, cell scratch, Transwell migration and invasion, colony formation, flow cytometry and apoptosis assays. Tumor growth was measured in a mouse xenograft model. ResultsWe found that NEK2 is highly expressed in ESCC tissues and ESCC cells and that the high expression of NEK2 is associated with poor tumor healing. Knockdown of the NEK2 gene inhibits the migration, proliferation, invasion and cell cycle of ESCC cells. Biologic analysis shows that NEK2 is involved in biological processes such as progression and apoptosis of esophageal cancer, and is related to E2F.Mechanistically, NEK2 knockdown decreases the expression levels of E2F1 and IGF2. NEK2 competes with the transcription factor E2F1 to bind CDC20, resulting in decreased degradation and increased expression of E2F1. IGF2 expression is also increased, which promotes the expression of thymidylate synthase, further promoting the drug resistance of ESCC cells. NEK2 is associated with immune infiltration in esophageal cancer. ConclusionNEK2 is highly expressed in ESCC and can promote the migration, proliferation and invasion of ESCC cells. NEK2 mediates ESCC immunotherapy.

Image-guided metabolomics and transcriptomics reveal tumour heterogeneity in luminal A and B human breast cancer beyond glucose tracer uptake.

Breast cancer is a metabolically heterogeneous disease, and although the concept of heterogeneous cancer metabolism is known, its precise role in human breast cancer is yet to be fully elucidated. We investigated in an explorative approach a cohort of 42 primary mamma carcinoma patients with positron emission tomography/magnetic resonance imaging (PET/MR) prior to surgery, followed by histopathology and molecular diagnosis. From a subset of patients, which showed high metabolic heterogeneity based on tracer uptake and pathology classification, tumour centre and periphery specimen tissue samples were further investigated by a targeted breast cancer gene expression panel and quantitative metabolomics by nuclear magnetic resonance (NMR) spectroscopy. All data were analysed in a combinatory approach. [18 F]FDG (2-deoxy-2-[fluorine-18]fluoro-d-glucose) tracer uptake confirmed dominance of glucose metabolism in the breast tumour centre, with lower levels in the periphery. Additionally, we observed differences in lipid and proliferation related genes between luminal A and B subtypes in the centre and periphery. Tumour periphery showed elevated acetate levels and enrichment in lipid metabolic pathways genes especially in luminal B. Furthermore, serine was increased in the periphery and higher expression of thymidylate synthase (TYMS) indicated one-carbon metabolism increased in tumour periphery. The overall metabolic activity based on [18 F]FDG uptake of luminal B subtype was higher than that of luminal A and the difference between the periphery and centre increased with tumour grade. Our analysis indicates variations in metabolism among different breast cancer subtypes and sampling locations which details the heterogeneity of the breast tumours. Correlation analysis of [18 F]FDG tracer uptake, transcriptome and tumour metabolites like acetate and serine facilitate the search for new candidates for metabolic tracers and permit distinguishing luminal A and B. This knowledge may help to differentiate subtypes preclinically or to provide patients guide for neoadjuvant therapy and optimised surgical protocols based on individual tumour metabolism.

563 - The effect of tumor microenvironment and thymidylate synthase expression levels on growth and metastasis of colorectal cancer

563 - The effect of tumor microenvironment and thymidylate synthase expression levels on growth and metastasis of colorectal cancer

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.

IMMUNOHISTOCHEMICAL EXPRESSION OF THYMIDYLATE SYNTHASE IN DIFFERENT GRADES OF COLORECTAL CARCINOMA

Objective: To Evaluate the Immunohistochemical Expression of Thymidylate Synthase in different grades and histological types of Colorectal Carcinoma.
 Study Design: Cross sectional, Descriptive study
 Place and duration of study: This study was conducted from June 2019 to November 2020 in the Department of Pathology at Postgraduate Medical Institute Lahore / Lahore General Hospital. 
 Methodology: Forty resected gut specimens diagnosed on light microscopy as Colorectal adenocarcinoma were included on the basis of non-probability, consecutive sampling technique. Cases were reviewed and histological typing and grading was done separately by two competent Histopathologists. Immunohistochemical staining for Thymidylate Synthase was performed. Results of Thymidylate Synthase immunostaining were interpreted by the two observers based on the cytoplasmic staining of tumor cells. Thymidylate Synthase expression was quantified through a visual grading system depending upon the staining intensity and percentage of stained cells. 
 Results: Out of 40 cases of adenocarcinoma, majority of the patients (45%) belong to the age range of 51-60 years. There were 31(77.5%) male patients while 9(22.5%) were females. 28 (22%) cases were classified as low grade while 12 (11%) cases were reported as high-grade carcinomas. Amongst these Thymidylate Synthase expression was seen in 78.6% of low-grade carcinoma and 91.6% of high-grade carcinomas. Regarding histological subtyping, 23(57.5 %) cases were identified as non-mucinous adenocarcinomas, (9)22.5% as mucinous adenocarcinoma and (8) 20% as signet ring adenocarcinoma. Thymidylate Synthase expression was found to be similar i.e., 78% in non- mucinous as well as mucinous carcinomas however, 100% positivity was noticed in signet ring carcinomas. The overall positivity for Thymidylate Synthase was found to be 82.5%. 
 Conclusion: Colorectal carcinoma was prevalent among males of 51-60 years of age. 82.5% cases of colorectal carcinoma expressed Thymidylate synthase receptors and its expression was found to be higher in high grade tumors.

Pilot Study of High-Dose Pemetrexed in Patients with Progressive Chordoma.

Chordomas are ultrarare tumors of the axial spine and skull-base without approved systemic therapy. Most chordomas have negative expression of thymidylate synthase (TS), suggesting a potential for responding to the antifolate agent pemetrexed, which inhibits TS and other enzymes involved in nucleotide biosynthesis. We evaluated the therapeutic activity and safety of high-dose pemetrexed in progressive chordoma. Adult patients with previously treated, progressive chordoma participated in an open-label, single-institution, single-arm, pilot clinical trial of intravenous pemetrexed 900 mg/m2 every 3 weeks and supportive medications of folic acid, vitamin B12, and dexamethasone. The primary endpoint was objective response rate according to RECIST v1.1. Secondary endpoints included adverse events, progression-free survival (PFS), tumor molecular profiles, and alterations in tissue and blood-based biomarkers. Fifteen patients were enrolled and the median number of doses administered was 15 (range, 4-31). One patient discontinued treatment due to psychosocial issues after four cycles and one contracted COVID-19 after 13 cycles. Of the 14 response-evaluable patients, 2 (14%) achieved a partial response and 10 (71%) demonstrated stable disease. Median PFS was 10.5 months (95% confidence interval: 9 months-undetermined) and 6-month PFS was 67%. Adverse events were expected and relatively mild, with one grade 3 creatinine increased, and one each of grade 3 and 4 lymphopenia. No grade 5 adverse events, unexpected toxicities, or dose-limiting toxicities were observed. Several patients reported clinical improvement in disease-related symptoms. High-dose pemetrexed appears tolerable and shows objective antitumor activity in patients with chordoma. Phase II studies of high-dose pemetrexed are warranted.

CTNI-15. PILOT STUDY OF HIGH-DOSE PEMETREXED FOR PROGRESSIVE CHORDOMA

Abstract INTRODUCTION Chordomas are ultra-rare tumors of the spine, and no systemic therapy is approved for refractory disease. Pemetrexed is a multitargeted antifolate that inhibits thymidylate synthase (TS) and other enzymes involved in nucleotide biosynthesis. Considering the majority of chordomas do not express TS, we sought to evaluate the therapeutic activity and safety of high-dose pemetrexed. METHODS Adults with previously-treated chordoma were enrolled on an open-label, single-institution, single-arm, pilot clinical trial. Pemetrexed 900 mg/m2 was administered intravenously every 3 weeks, with supportive medications of folic acid, vitamin B12, and dexamethasone. The primary endpoint was objective response rate according to RECIST v1.1. Secondary and exploratory endpoints included adverse events, progression-free survival (PFS), tumor molecular profiles, and alterations in potential tissue and blood-based biomarkers before and after treatment. RESULTS Fifteen patients with chordoma received a median of 15 (range 4-31) doses of pemetrexed. Median PFS was 10.5 months (95% CI 9 months-undetermined) and 6-month PFS was 67%. Out of 14 response-evaluable patients, two (14%) achieved a partial response and ten (71%) demonstrated stable disease. Adverse events were generally mild at grades 1-2, the most common being fatigue, rash, nausea, constipation, diarrhea, mucositis, vomiting, alopecia, pruritus, lower extremity edema, and alanine transaminase increased. One Grade 3 creatinine increase, one Grade 3 lymphopenia, and one Grade 4 lymphopenia occurred and there were no Grade 5 adverse events, unexpected toxicities, or dose-limiting toxicities. Clinical improvement in disease-related symptoms occurred in several patients. Tumor molecular profiling revealed low mutational burden and though not statistically significant, positive TS expression trended negatively with disease progression. Cell-free microRNAs (cfmiRs) were concordantly detected in plasma and tissues and a blood cfmiR signature was identified that distinguished chordoma from health donors. CONCLUSION High-dose pemetrexed is safe and well-tolerated in chordoma patients. A phase II study is in development. (NCT03955042)

Thymidylate synthase promotes esophageal squamous cell carcinoma growth by relieving oxidative stress through activating nuclear factor erythroid 2-related factor 2 expression.

Thymidylate synthase (TYMS) is involved in the malignant process of multiple cancers, and has gained much attention as a cancer treatment target. However, the mechanism in carcinogenesis of esophageal squamous cell cancer (ESCC) is little reported. The present study was to clear the biological roles and carcinogenic mechanism of TYMS in ESCC, and explored the possibility to use TYMS as a tumor marker in diagnosis and a drug target for the treatment of ESCC. Stably TYMS-overexpression cells established by lentivirus transduction were used for the analysis of cell proliferation. RNA sequencing was performed to explore the possible carcinogenic mechanisms. GEPIA databases analysis showed that TYMS expression in esophageal cancer tissues was higher than that in normal tissues. The MTT assay, colony formation assay, and nude mouse subcutaneous tumor model found that the overexpression of TYMS increased cell proliferation. Transcriptome sequencing analysis revealed that the promoted cell proliferation in TYMS-overexpression ESCC cells were mediated through activating genes expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 dependent antioxidant enzymes to relieve oxidative stress, which was confirmed by increased glutathione (GSH), glutathione peroxidase (GPX) activities, and reduced reactive oxygen species. Nrf2 active inhibitors (ML385) used in TYMS-overexpression cells inhibited the expression of Nrf2-dependent antioxidant enzyme genes, thereby increasing oxidative stress and blocking cell proliferation. Our study indicated a novel and effective regulatory capacity of TYMS in the cell proliferation of ESCC by relieving oxidative stress through activating expression of Nrf2 and Nrf2-dependent antioxidant enzymes genes. These properties make TYMS and Nrf2 as appealing targets for ESCC clinical chemotherapy.

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.

Rapid and Cost-Efficient Detection of RET Rearrangements in a Large Consecutive Series of Lung Carcinomas.

RET-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5'/3'-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations.

Activity of pemetrexed in pre-clinical chordoma models and humans

Chordomas are rare slow growing tumors, arising from embryonic remnants of notochord with a close predilection for the axial skeleton. Recurrence is common and no effective standard medical therapy exists. Thymidylate synthase (TS), an intracellular enzyme, is a key rate-limiting enzyme of DNA biosynthesis and repair which is primarily active in proliferating and metabolically active cells. Eighty-four percent of chordoma samples had loss of TS expression which may predict response to anti-folates. Pemetrexed suppresses tumor growth by inhibiting enzymes involved in folate metabolism, resulting in decreased availability of thymidine which is necessary for DNA synthesis. Pemetrexed inhibited growth in a preclinical mouse xenograft model of human chordoma. We report three cases of metastatic chordoma that had been heavily treated previously with a variety of standard therapies with poor response. In two cases, pemetrexed was added and objective responses were observed on imaging with one patient on continuous treatment for > 2 years with continued shrinkage. One case demonstrated tumor growth after treatment with pemetrexed. The two cases which had a favorable response had a loss of TS expression, whereas the one case with progressive disease had TS present. These results demonstrate the activity of pemetrexed in recurrent chordoma and warrant a prospective clinical trial which is ongoing (NCT03955042).

Abstract 6178: A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance

Abstract Colorectal cancer (CRC) is the 3rd leading cause of cancer-related mortality. The mortality associated with colon cancer results notably from metastatic disease (mCRC), with a 5-year survival rate for patients with stage IV CRC being &amp;lt; 14%. Despite extensive efforts towards developing personalized therapies, limited success has been achieved and chemotherapeutic regimens that include the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) are central to patient management. While using 5-FU-based regimens such as FOLFOX and FOLFIRI in combination with biologics confer a survival benefit, their ineffectiveness at promoting long-term survival in a substantial fraction of mCRC patients and associated toxicities limits their use. This underscores the pressing need to develop improved FP drugs. To overcome the limitations of 5-FU that include resistance due to elevated expression of thymidylate synthase (TS) and inefficient anabolic metabolism to FdUMP, the TS-inhibitory/active metabolite, we are developing FP polymers consisting of single-stranded DNA with FdUMP (only active metabolite) as the repeating nucleotide unit. Our therapeutic objective is to initiate a Phase I clinical trial with a new 2nd generation nanoscale FP polymer (CF10) that demonstrates promising anticancer effects in several preclinical models and significantly lower systemic toxicity. We observed that CF10 displayed significantly higher anti-tumor activity than 5-FU in flank and syngeneic tumor models. Additionally, CF10 treatment improved survival (84.5 days vs 32 days; P &amp;lt; 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer mice model that spontaneously metastasized to the liver. Interestingly, a reduction in metastatic tumor burden in the CF10 treatment group led us to hypothesize that CF10 could inhibit the TS-mediated EMT phenotype and pro-metastatic activities in colorectal cancer cells (CRCs). This was supported by increased vimentin, decreased E-cadherin expression, and increased cell migration and invasion in TS-overexpressing CRC cells (“Tet-on” system). Alkaline comet assay, immunofluorescence imaging, and immunoblot identified significantly higher replication stress with CF10 treatment than 5-FU in mouse, rat, and human-established CRC cell lines and primary CRC cells. Strikingly, we also discovered that CF10 attenuates the growth of TS-overexpressing primary colonoids. In conclusion, CF10 induces increased replication stress compared to a similar concentration of conventional drugs. Our findings suggest the significance of elevated TS in CRC metastatic progression and 5-FU resistance and demonstrate that CF10 may be effective at inhibiting CRC metastatic progression. CF10 may be a successful candidate for early-phase clinical trials to treat mCRC. Citation Format: Naresh Sah, Pamela Luna, Chinnadurai Mani, William Gmeiner, Komaraiah Palle. A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6178.

Synthesis and Biological Evaluation of Novel Uracil Derivatives as Thymidylate Synthase Inhibitors.

Cell division is driven by nucleic acid metabolism, and thymidylate synthase (TYMS) catalyzes a rate-limiting step in nucleotide synthesis. As a result, thymidylate synthase has emerged as a critical target in chemotherapy. 5-Fluorouracil (5-FU) is currently being used to treat a wide range of cancers, including breast, pancreatic, head and neck, colorectal, ovarian, and gastric cancers The objective of this study was to establish a new methodology for the low-cost, one-pot synthesis of uracil derivatives (UD-1 to UD-5) and to evaluate their therapeutic potential in BC cells. One-pot organic synthesis processes using a single solvent were used for the synthesis of drug analogues of Uracil. Integrated bioinformatics using GEPIA2, UALCAN, and KM plotter were utilized to study the expression pattern and prognostic significance of TYMS, the key target gene of 5-fluorouracil in breast cancer patients. Cell viability, cell proliferation, and colony formation assays were used as in vitro methods to validate the in silico lead obtained. BC patients showed high levels of thymidylate synthase, and high expression of thymidylate synthase was found associated with poor prognosis. In silico studies indicated that synthesized uracil derivatives have a high affinity for thymidylate synthase. Notably, the uracil derivatives dramatically inhibited the proliferation and colonization potential of BC cells in vitro. In conclusion, our study identified novel uracil derivatives as promising therapeutic options for breast cancer patients expressing the augmented levels of thymidylate synthase.

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing.

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.

The Predictive Value of ERCC1, RRM1, and Thymidylate Synthase in Advanced Malignant Pleural Mesothelioma Patients Treated with Platinum-Based Chemotherapy.

A rare yet severe neoplasia called malignant pleural mesothelioma (MPM) typically manifests itself in advanced stages. Despite some improvements in the treatment of patients with MPM, this malignancy continues to have a detrimental prognosis . The primary goal of the present study was to assess the associatin between ERCC1, RRM1, and thymidylate synthase (TS) expression and disease outcome in patients with malignant pleural mesothelioma (MPM) treated with either pemetrexed plus cisplatin or gemcitabine plus cisplatin. This prospective cohort study was done on ninety-one advanced MPM patients. The patients received either pemetrexed plus platinum (55 of 91) or gemcitabine plus platinum chemotherapy (36 of 91). Tissue biopsy was taken at time of diagnosis. Immunohistochemistry was used to assess the levels of ERCC1, RRM1, and TS transcription in tissue biopsies (paraffin embedded). Based on the findings, 70% of patients with low expression of TS had low expression of ERCC1, and 68% of patients with high expression of TS had high expression of ERCC1, suggesting a significat association between ERCC1 expression and TS expression (p=0.005). However, there was no relation between ERCC1 and RRM1 expression patterns (p= 0.296). In patients underwen platinum-based theraphy (n 91), a significant correlation was detected between low ERCC1 median High-scoring and longer progression time (TTP;9.6 v 5.3 months;P< .001) or overall survival (OS) (OS;18.1 v 9.1 months; P<0.001). A significant correlation was found between low TS protein expression and longer time progression (TTP; 11.8 v 5.4 months; P< .001) or OS (OS; 19.8 v 8.5 months; P <0.001) in patients undergoing pemetrexed plus platinum chemotherapy (n 55). Low RRM1 expression was accompanied by high progression free survival (TTP; 10.6 v 3.8 months) and OS (OS; 20.6 v 8.6 months) in patients receiving gemcitabine plus platinum chemotherapy. Based on the multivariate test results, the independent variables significantly affecting OS were tumor stage (HR: 2.3; 95% CI: 1.1-4.9; p= 0.021) and ERCC1 (HR: 2.7; 95% CI: 1.7-4.3; p < 0.001). Decreased TS protein expression can be indicative of greater responsivness to pemtrexed and of longer TTP and OS in individuals with advanced MPM (locally progressed or metastatic) who are receiving pemetrexed-based chemotheraphy. Low ERCC1 expressions in individuals with advanced MPM can predict increased PFS and OS, as well as a better responsivness to platinum-based chemotherapy. In patients with progressed MPM receiving gemcitabine plus cisplatin chemotherapy, lower RRM1 expression was associated with a better prognosis, longer PFS, and longer OS.

Review of 5-FU resistance mechanisms in colorectal cancer: clinical significance of attenuated on-target effects.

The emergence of chemoresistant disease during chemotherapy with 5-Fluorouracil-based (5-FU-based) regimens is an important factor in the mortality of metastatic CRC (mCRC). The causes of 5-FU resistance are multi-factorial, and besides DNA mismatch repair deficiency (MMR-D), there are no widely accepted criteria for determining which CRC patients are not likely to be responsive to 5-FU-based therapy. Thus, there is a need to systematically understand the mechanistic basis for 5-FU treatment failure and an urgent need to develop new approaches for circumventing the major causes of 5-FU resistance. In this manuscript, we review mechanisms of 5-FU resistance with an emphasis on: (1) altered anabolic metabolism limiting the formation of the primary active metabolite Fluorodeoxyuridylate (5-Fluoro-2'-deoxyuridine-5'-O-monophosphate; FdUMP); (2) elevated expression or activity of the primary enzymatic target thymidylate synthase (TS); and (3) dysregulated programmed cell death as important causes of 5-FU resistance. Importantly, these causes of 5-FU resistance can potentially be overcome through the use of next-generation fluoropyrimidine (FP) polymers (e.g., CF10) that display reduced dependence on anabolic metabolism and more potent TS inhibitory activity.

Targeting Thymidylate Synthase and tRNA-Derived Non-Coding RNAs Improves Therapeutic Sensitivity in Colorectal Cancer.

Some colorectal cancer (CRC) patients are resistant to 5-fluorouracil (5-FU), and high expression levels of thymidylate synthase (TS) contribute to this resistance. This study investigated whether quercetin, a representative polyphenol compound, could enhance the effect of 5-FU in CRC cells. Quercetin suppressed TS levels that were increased by 5-FU in CRC cells and promoted the expression of p53. Quercetin also induced intracellular and mitochondrial reactive oxygen species (ROS) production and Ca2+ dysregulation in a 5-FU-independent pathway in CRC cells. Furthermore, quercetin decreased mitochondrial membrane potential in CRC cells and inhibited mitochondrial respiration. Moreover, quercetin regulated the expression of specific tiRNAs, including tiRNAHisGTG, and transfection of a tiRNAHisGTG mimic further enhanced the apoptotic effect of quercetin in CRC cells. An enhanced sensitivity to 5-FU was also confirmed in colitis-associated CRC mice treated with quercetin. The treatment of quercetin decreased survival rates of the CRC mouse model, with reductions in the number of tumors and in the disease activity index. Also, quercetin suppressed TS and PCNA protein expression in the distal colon tissue of CRC mice. These results suggest that quercetin has the potential to be used as an adjuvant with 5-FU for the treatment of CRC.

Gene therapy using genome-edited iPS cells for targeting malignant glioma.

Glioblastoma is characterized by diffuse infiltration into the normal brain. Invasive glioma stem cells (GSCs) are an underlying cause of treatment failure. Despite the use of multimodal therapies, the prognosis remains dismal. New therapeutic approach targeting invasive GSCs is required. Here, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited human-induced pluripotent stem cell (hiPSC) expressing a suicide gene had higher tumor-trophic migratory capacity compared with mesenchymal stem cells (MSCs), leading to marked in vivo antitumor effects. High migratory capacity in iPSC-NSCs was related to self-repulsive action and pathotropism involved in EphB-ephrinB and CXCL12-CXCR4 signaling. The gene insertion to ACTB provided higher and stable transgene expression than other common insertion sites, such as GAPDH or AAVS1. Ferroptosis was associated with enhanced antitumor immune responses. The thymidylate synthase and dihydroprimidine dehydrogenase expressions predicted the treatment efficacy of therapeutic hiPSC-NSCs. Our results indicate the potential benefit of genome-edited iPS cells based gene therapy for invasive GSCs. Furthermore, the present research concept may become a platform to promote clinical studies using hiPSC.

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.

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. 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 β-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. 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). 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.

Does Molecular Profiling of KRAS-Mutant Non-Squamous Non-Small Cell Lung Cancer (NSCLC) Help in Treatment Strategy Planning?

Background: Several studies suggest that patients with KRAS-mutant NSCLC fail to benefit from standard systemic therapies and do not respond to EGFR inhibitors. Most recently, KRAS 12c data suggest specific treatment for improving ORR and OS. There is a clear need for therapies specifically developed for these patients. Moreover, data that might be suggestive of a response to specific therapies, such as BRCA1, are needed, and two mutations that were studied in other malignancies show more response to PARP inhibitors. Molecular profiling has the potential to identify other potential targets that may provide better treatment and novel targeted therapy for KRAS-mutated NSCLC. Methods: We purified RNA from archived tissues of patients with stage I and II NSCLC with wild-type (wt) and mutant (mt) KRAS tumors; paired normal tissue adjacent to the tumor from 20 and 17 patients, respectively, and assessed, using real-time reverse transcriptase–polymerase chain reaction (RT-PCR), the expression of four genes involved in DNA synthesis and repair, including thymidylate synthase (TS), BRCA1, ECCR1, RAP80, and the proto-oncogene SRC. Additionally, we assessed the expression of PD-L1 in mt KRAS tumors with immunohistochemistry using an antibody against PD-L1. Results: Our results show that in mt KRAS tumors, the level of expression of ERCC1, TS, and SRC was significantly increased in comparison to paired normal lung tissue (p ≤ 0.04). The expression of BRCA1 and RAP80 was similar in both mt KRAS tumors and paired normal tissue. Furthermore, the expression of BRCA1, TS, and SRC was significantly increased in wt KRAS tumors relative to their expression in the normal lung tissue (p < 0.044). The expression of ERCC1 and RAP80 was similar in wt KRAS tumors and paired normal tissue. Interestingly, SRC expression in mt KRAS tumors was decreased in comparison to wt KRAS tumors. Notably, there was an expression of PD-L1 in the tumor and stromal cells in a few (5 out of 20) mt KRAS tumors. Our results suggest that a greater ERCC1 expression in mt KRAS tumors might increase platinum resistance in this group of patients, whereas the greater expression of BRCA1 in wt KRAS tumor might be suggestive of the sensitivity of taxanes. Our data also suggest that the combination of an SRC inhibitor with a TS inhibitor, such as pemetrexed, might improve the outcome of patients with NSCLC and in particular, patients with wt KRAS tumors. PD-L1 expression in tumors, and especially stromal cells, suggests a better outcome. Conclusion: mt KRAS NSCLC patients might benefit from a treatment strategy that targets KRAS in combination with therapeutic agents based on pharmacogenomic markers, such as SRC and BRCA1. mt KRAS tumors are likely to be platinum-, taxane-, and pemetrexed-resistant, as well as having a low level of PD-L1 expression; thus, they are less likely to receive single-agent immunotherapy, such as pembrolizumab, as the first-line therapy. wt KRAS tumors with BRCA1 positivity tend to be sensitive to taxane therapy and, potentially, platinum. Our results suggest the need to develop targeted therapies for KRAS-mutant NSCLC or combine the targeting of oncogenic KRAS in addition to other therapeutic agents specific to the molecular profile of the tumor.