Fluoro-deoxyuridine monophosphate (FdUMP) is an active metabolite of 5-fluorouracil (5-FU) synthesized through two hypothesized pathways: The orotate phosphoribosyl transferase-ribonucleotide reductase (OPRT-RR) pathway and the thymidine phosphorylase-thymidine kinase (TP-TK) pathway. In the present study, the mechanism underlying 5-FU resistance was investigated, focusing on changes in 5-FU metabolism using MCF-7, 5-FU-resistant MCF-7/5-FUR, MDA-MB-231 and 5-FU-resistant MDA-MB-231/5-FUR breast cancer cells. The amount of FdUMP present following treatment with 5-FU was determined by the density of the upper band of thymidylate synthase detected by western blotting, and its changes were investigated. MCF-7/5-FUR cells exhibited 5-FU resistance (36.6-fold), and showed decreased OPRT (-69.3%) and TK (-42.6%) levels. MDA-MB-231/5-FUR cells also exhibited 5-FU resistance (15.8-fold), and showed decreased TP (-79.0%) and increased TK (+184%) levels. MCF-7/5-FUR and MDA-MB-231/5-FUR cells both showed decreased synthesis of FdUMP by 91 and 86%, respectively. In MCF-7 and MCF-7/5-FUR cells, the synthesis of FdUMP was decreased when 5-FU was combined with an RR inhibitor, indicating that FdUMP was synthesized through the OPRT-RR pathway. The synthesis of FdUMP was decreased when 5-FU was combined with a TP inhibitor in MDA-MB-231 cells and combined with an RR inhibitor in MDA-MB-231/5-FUR cells, indicating that the synthesis pathway of FdUMP was changed from the TP-TK pathway to the OPRT-RR pathway on acquiring resistance to 5-FU. Notably, the synthesis of FdUMP was increased and the resistance to 5-FU was reversed in MCF-7/5-FUR cells (half maximal inhibitory concentration (IC 50 ): 219.9 to 0.093 µM) and MDA-MB-231/5-FUR cells (IC 50 : 157.3 to 31.0 µM) when 5-FU was combined with a TP inhibitor. In conclusion, the metabolism of 5-FU and the mechanism underlying the resistance to 5-FU differed among cell lines, and inhibition of TP reversed resistance to 5-FU, thus suggesting that the combination of 5-FU and a TP inhibitor may be considered a promising cancer therapy.
Background: Epithelial-mesenchymal transition (EMT) is a well-known multistep process of cancer cell invasion and metastasis, as well as treatment resistance. Our group has been reporting the predictive role of scores generated using Gene Set Variation Analysis (GSVA) of Hallmark pathways in breast cancer. In this study, we hypothesized that EMT score high breast cancer is aggressive and is associated with poor clinical outcome. Material and Methods: The clinicopathological data and transcriptome data of breast cancer patients from three independent large publicly available databases, The Cancer Genome Atlas (TCGA, n = 1077), The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC, n = 1904), and GSE96058 (n=3069) were utilized. Survival analyses; Overall survival (OS), Disease-specific survival (DSS) and Disease-free survival (DFS) were performed by comparing the high and low score groups. Tumor immune microenvironment was analyzed utilizing the values reported by Thorsson et al. Also, single sample Gene Set Enrichment analysis (ssGSEA) was performed between EMT high and low expression groups utilizing singe cell sequence cohorts. Results: EMT score was generated by Gene Set Variation Analysis of a Hallmark gene set and we divided each cohort into EMT score high and low groups by utilizing median as the cutoff. To our surprise, EMT score of the primary tumor was not associated with metastasis (N and M categories of cancer staging), Nottingham histological grade, nor MKI67 expression levels consistently in TCGA, METABRIC, and GSE96058. EMT score high tumors were not associated with worse DFS, DSS, OS in TCGA and METABRIC and OS in GSE96058. Analyses using xCell demonstrated that EMT score high tumors were associated with high infiltration of stromal cells such as adipocyte (p< 0.001, p< 0.001 and p< 0.001, respectively) and fibroblasts (p< 0.001, p< 0.001 and p< 0.001, respectively) in all three cohorts, TCGA, METABRIC, and GSE96058. Also, myeloid cells such as macrophages (p< 0.001, p< 0.001 and p< 0.001, respectively) and dendritic cells (p< 0.001, p< 0.001 and p< 0.001, respectively) were highly infiltrated with EMT score high tumors. Result of ssGSEA of single cell sequence cohorts revealed that cancer associated fibroblasts demonstrated highest EMT scores compared with the other cell types such as cancer cells, T-cells, B-cells, or myeloid cells. In other words, EMT score of a bulk tumor may reflect the signature from fibroblasts rather than cancer cells. Conclusion: We found that cancer associated fibroblasts rather than cancer cells are the major source of the transcriptomic signatures of EMT in the bulk tumor, which cautions us to be careful with the interpretation of the results of EMT signature from a bulk tumor. Citation Format: Yoshihisa Tokumaru, Rongrong Wu, Junko Ukai, Masanori Oshi, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. Transcriptomic signature score of Epithelial-Mesenchymal Transition (EMT) of a bulk tumor may not reflect that of cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-03.
Background: Epithelial membrane protein 1 (EMP1) belongs to PMP22 gene family and is reported to be expressed low in numerous gastrointestinal cancers. However, little is known about its role in breast cancer. EMP1 functions to promote cell proliferation. In this study, we hypothesized that low EMP1 expressing breast cancer is associated with high proliferative characteristics and poor survival. Material and Methods: The clinicopathological data and transcriptome data of breast cancer patients from two independent large publicly available databases, The Cancer Genome Atlas (TCGA, n = 1090) and The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC, n = 1904), were utilized in the current study. Survival analyses; Overall survival (OS), Disease-specific survival (DSS) and Disease-free survival (DFS) were performed by comparing the high and low expression groups. CYT score, xCell, and other immunological factors were used to evaluate intratumoral immune cell composition. Also, gene set enrichment analysis (GSEA) was performed between EMP1 high and low expression groups. Results: We divided each cohort into EMP1 expression high and low groups by utilizing median cutoff. The expression levels of EMP1 were not associated with clinical stage in any subtypes. However, interestingly lower expression of EMP1 was significantly associated with more advanced grades in ER positive/HER2 negative (ER+/HER2) subtype in both TCGA and METABRIC cohorts (p< 0.001 and p< 0.001, respectively). Also, low EMP1 expressing tumors demonstrated higher MKI67 expression levels in ER+/HER2- subtype consistently in both TCGA (p< 0.001) and METABRIC cohorts (p< 0.001). Furthermore, GSEA demonstrated that low EMP1 expressing tumors enriched the gene sets associated with cell proliferation such as MYC Targets, E2F signaling, and G2M Checkpoint signaling, compared with low EMP1 expressing tumors in ER+/HER2- of both TCGA and METABRIC cohorts. On the contrary, high EMP1 expressing tumors enriched the immune related gene sets such as Coagulation, Inflammatory_Response, Complement, and IL-6_JAK_STAT3 Signaling in ER+/HER2- of both TCGA and METABRIC cohorts. To this end, we further hypothesized that high EMP1 tumors were associated with favorable tumor immune microenvironment (TIME) and analyzed TIME utilizing xCell. However, to our surprise high EMP1 expressing tumors were not associated with favorable TIME. Low EMP1 expressing tumors demonstrated worse DFS, DSS, and OS compared with high EMP1 expressing tumors in ER+/HER2- breast cancer patients (p=0.013, p=0.003, and p=0.006) which was not the case in the other subtypes in METABRIC cohort. Conclusion: Low EMP1 expressing tumors were associated with improved OS, DSS, DFS in ER-positive breast cancer patients. Also, low EMP1 expressing tumors were found to associate with advanced grades and enriched gene sets related to cell proliferation and cell cycle, which may explain the poor survival of patients with low EMP1 expressing ER+/HER2- breast cancer. Citation Format: Junko Ukai, Yoshihisa Tokumaru, Masanori Oshi, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. EMP1 low expressing tumor is associated with cell proliferation and poor overall survival of ER-positive breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-28.
vestibularis, PD peritonitis 〈Abstract〉 An 81 year old male, who was on continuous ambulatory peritoneal dialysis (CAPD), developed abdominal pain and cloudy peritoneal fluid. His white blood cell count was 17,510 cells/μL, his C reactive protein level was 1.36 mg/dL, and the white blood cell count of his peritoneal fluid was 16,670 cells/μL, suggesting acute peritonitis. Empiric therapy ameliorated his symptoms. A microbiological examination of the peritoneal fluid using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI TOF MS) revealed Streptococcus vestibularis. Herein, we present the first Japanese case report of peritonitis due to S. vestibularis related to CAPD.
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