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Folate deficient tumor microenvironment promotes epithelial-to-mesenchymal transition and cancer stem-like phenotypes.

ABSTRACT: Clinically, serum level of folate has been negatively correlated to the stage and progression of liver cancer. Nevertheless, the functional consequence of folate deficiency (FD) in malignancy has not been fully investigated. Human hepatocellular carcinoma (HCC) cells (as study model) and other cancer types such as lung and glioma were cultured under folate deficient (FD) and folate complete (FD) conditions. Molecular characterization including intracellular ROS/RNS (reactive oxygen/nitrogen species), viability, colony formation, cancer stem-like cell (CSC) phenotype analyses were performed. In vivo tumorigenesis under FD and FC conditions were also examined. FD induced a significant increase in ROS and RNS, suppressing proliferative ability but inducing metastatic potential. Mesenchymal markers such as Snail, ZEB2, and Vimentin were significantly up-regulated while E-cadherin down-regulated. Importantly, CSC markers such as Oct4, β-catenin, CD133 were induced while PRRX1 decreased under FD condition. Furthermore, FD-conditioned HCC cells showed a decreased miR-22 level, leading to the increased expression of its target genes including HDAC4, ZEB2 and Oct4. Finally, xenograft mouse model demonstrated that FD diet promoted tumorigenesis and metastasis as compared to their FC counterparts. Our data provides rationales for the consideration of folate supplement as a metastasis preventive measure.

SUBMITTER: Su YH

PROVIDER: S-EPMC5078091 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.

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Folate deficient tumor microenvironment promotes epithelial-to-mesenchymal transition and cancer stem-like phenotypes.

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