Blood FOLR3 methylation dysregulations and heterogeneity in non-small lung cancer highlight its strong associations with lung squamous carcinoma.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers. Early detection is crucial to reduce lung cancer-related mortality. Aberrant DNA methylation occurs early during carcinogenesis and can be detected in blood. It is essential to investigate the dysregulated blood methylation markers for early diagnosis of NSCLC. METHODS: NSCLC-associated methylation gene folate receptor gamma (FOLR3) was selected from an Illumina 850K array analysis of peripheral blood samples. Mass spectrometry was used for validation in two independent case-control studies (validation I: n = 2548; validation II: n = 3866). Patients with lung squamous carcinoma (LUSC) or lung adenocarcinoma (LUAD), normal controls (NCs) and benign pulmonary nodule (BPN) cases were included. FOLR3 methylations were compared among different populations. Their associations with NSCLC clinical features were investigated. Receiver operating characteristic analyses, Kruskal-Wallis test, Wilcoxon test, logistics regression analysis and nomogram analysis were performed. RESULTS: Two CpG sites (CpG_1 and CpG_2) of FOLR3 was significantly lower methylated in NSCLC patients than NCs in the discovery round. In the two validations, both LUSC and LUAD patients presented significant FOLR3 hypomethylations. LUSC patients were highlighted to have significantly lower methylation levels of CpG_1 and CpG_2 than BPN cases and LUAD patients. Both in the two validations, CpG_1 methylation and CpG_2 methylation could discriminate LUSC from NCs well, with areas under the curve (AUCs) of 0.818 and 0.832 in validation I, and 0.789 and 0.780 in validation II. They could also differentiate LUAD from NCs, but with lower efficiency. CpG_1 and CpG_2 methylations could also discriminate LUSC from BPNs well individually in the two validations. With the combined dataset of two validations, the independent associations of age, gender, and FOLR3 methylation with LUSC and LUAD risk were shown and the age-gender-CpG_1 signature could discriminate LUSC and LUAD from NCs and BPNs, with higher efficiency for LUSC. CONCLUSIONS: Blood-based FOLR3 hypomethylation was shown in LUSC and LUAD. FOLR3 methylation heterogeneity between LUSC and LUAD highlighted its stronger associations with LUSC. FOLR3 methylation and the age-gender-CpG_1 signature might be novel diagnostic markers for the early detection of NSCLC, especially for LUSC.

Purifying selection against gene conversions in the folate receptor genes of primates.

We characterized the gene conversions between the human folate receptor (FOLR) genes and those of five other primate species. We found 26 gene conversions having an average length of 534 nucleotides. The length of these conversions is correlated with sequence similarity, converted regions have a higher GC-content and the average size of converted regions from a functional donor to another functional donor is significantly smaller than the average size from a functional donor to a pseudogene. Furthermore, the few conversions observed in the FOLR1 and FOLR2 genes did not change any amino acids in their coding regions and did not affect their promoter regions. In contrast, the promoter and coding regions of the FOLR3 gene are frequently converted and these conversions changed many amino acids in marmoset. These results suggest that purifying selection is limiting the functional impact that frequent gene conversions have on functional folate receptor genes.

Characterization of soluble folate receptors (folate binding proteins) in humans. Biological roles and clinical potentials in infection and malignancy.

This review surveys soluble Folate Receptors (FOLRs) in humans. FOLR1 and FOLR2 are equipped with cellular glycosylphosphatidylinositol (GPI) anchors. FOLR1 is secreted from epithelia with or without a micelle-encapsulated GPI-anchor into milk and other body fluids/secretions, e.g. semen where its interaction with spermatozoa indicates a role in male fertility. FOLR1 and FOLR2 serve as serum biomarkers of various diseases. FOLR3 possesses no GPI-anchor and originates from secretory granules of neutrophil granulocytes; its concentration in serum correlates to the FOLR3 content in leukocytes and rises with increased leukocyte counts (infection, malignancy and pregnancy). FOLR3 exerts anti-microbial and anti-tumor effects by depriving bacteria and tumor cells of natural folates. Megalin receptors mediate reabsorption of ultrafiltered folate-bound FOLR into cells of proximal kidney tubules and of folate-bound FOLR uptake in growing embryos. Megalin receptors overexpressed in malignant tumors could be suitable therapeutic targets for folate-conjugated cytotoxic agents utilizing soluble FOLRs as vectors.

Time-course transcriptional profiling of human amniotic fluid-derived stem cells using microarray.

PURPOSE: To maintain the homeostasis of stem cells and prevent their ability to initiate tumorigenesis, it is important to identify and modify factors that prevent or accelerate stem cell senescence. We used microarrays to attempt to identify such factors in human amniotic fluid (HAF)-derived stem cells. MATERIALS AND METHODS: To identify gene expression changes over a time course, we compared gene expression profiles of HAF-derived stem cells in different passages (1(st), 2(nd), 4(th), 6(th), 8(th), and 10(th)) using a Sentrix Human illumina microarray. RESULTS: Of the 25,804 genes in the microarray chip, 1,970 showed an over 2-fold change relative to the control (the 1(st) passage)-either upregulated or downregulated. Quantitative real-time PCR validated the microarray data for selected genes: markedly increased genes were CXCL12, cadherin 6 (CDH6), and folate receptor 3 (FOLR3). Downregulated genes included cyclin D2, keratin 8, insulin-like growth factor 2 (IGF2), natriuretic peptide precursor B (NPPB) and cellular retinoic acid binding protein 2 (CRABP2). The expression pattern of the selected genes was consistent with the microarray data except for CXCL12 and IGF2. Interestingly, the expression of NPPB was dramatically downregulated along the time course; it was almost completely shut-down by the 10(th) passage. In contrast, FOLR3 mRNA expression was dramatically increased. CONCLUSION: Taken together, although a function for NPPB and FOLR3 in stem cell senescence has not been reported, our results strongly suggest that NPPB and/or FOLR3 play a significant role in the regulation of stem cell senescence.