m6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-ß-induced epithelial-mesenchymal transition of lung cancer cells.

N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta-induced EMT of lung cancer cells.

ASH2L, a COMPASS core subunit, is involved in the cell invasion and migration of triple-negative breast cancer cells through the epigenetic control of histone H3 lysine 4 methylation.

ASH2L (Absent-Small-Homeotic-2-Like protein) is a core subunit of the COMPASS (COMplex of Proteins ASsociated with Set1) complex, the most notable writer of the methylation of histone H3 lysine 4 (H3K4). The COMPASS complex regulates active promoters or enhancers for gene expression, and its dysfunction is associated with aberrant development and disease. Here, we demonstrated that ASH2L mediated the cell invasion and migration activity of triple-negative breast cancer cells through the interaction with the COMPASS components and the target genomic regions. Transcriptome analysis indicated a potential correlation between ASH2L and the genes involved in inflammatory/immune responses. Among them, we found that the intrinsic expression of IL1B (interleukin 1 beta), an essential proinflammatory gene, was directly regulated by ASH2L. These results revealed a novel role of ASH2L on the maintenance of breast cancer malignancy possibly through H3K4 methylation of the target inflammatory/immune responsive genes.

KDM2B is involved in the epigenetic regulation of TGF-ß-induced epithelial-mesenchymal transition in lung and pancreatic cancer cell lines.

Polycomb repressive complex-1 (PRC1) induces transcriptional repression by regulating monoubiquitination of lysine 119 of histone H2A (H2AK119) and as such is involved in a number of biological and pathological processes including cancer development. Previously we demonstrated that PRC2, which catalyzes the methylation of histone H3K27, has an essential function in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung and pancreatic cancer cell lines. Since the cooperative activities of PRC1 and PRC2 are thought to be important for transcriptional repression in EMT program, we investigated the role of KDM2B, a member of PRC1 complex, on TGF-ß-induced EMT in this study. Knockdown of KDM2B inhibited TGF-ß-induced morphological conversion of the cells and enhanced cell migration and invasion potentials as well as the expression changes of EMT-related marker genes. Overexpression of KDM2B influenced the expression of several epithelial marker genes such as CDH1, miR200a, and CGN and enhanced the effects of TGF-ß. Mechanistic investigations revealed that KDM2B specifically recognized the regulatory regions of CDH1, miR200a, and CGN genes and induced histone H2AK119 monoubiquitination as a component of PRC1 complex, thereby mediating the subsequent EZH2 recruitment and histone H3K27 methylation process required for gene repression. Studies using KDM2B mutants confirmed that its DNA recognition property but not its histone H3 demethylase activity was indispensable for its function during EMT. This study demonstrated the significance of the regulation of histone H2A ubiquitination in EMT process and provided the possibility to develop novel therapeutic strategies for the treatment of cancer metastasis.

The m6A methyltransferase METTL3 contributes to Transforming Growth Factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB.

N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-ß-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-ß-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-ß-induced EMT of lung cancer cells through the regulation of JUNB.

MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells.

Long noncoding RNAs (lncRNAs) are important regulatory molecules in various biological and pathological processes, including cancer development. We have previously shown that the MEG3 lncRNA plays an essential role in transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of human lung cancer cells. In this study, we investigated the function of another lncRNA, MEG8, which shares the DLK1-DIO3 locus with MEG3, in the regulation of EMT. MEG8 lncRNA expression was immediately induced during TGF-ß-mediated EMT of A549 and LC2/ad lung cancer and Panc1 pancreatic cancer cell lines. MEG8 overexpression specifically suppressed the expression of microRNA-34a and microRNA-203 genes, resulting in up-regulation of SNAIL family transcriptional repressor 1 (SNAI1) and SNAI2 transcription factors, which repressed expression of cadherin 1 (CDH1)/E-cadherin. Mechanistic investigations revealed that MEG8 associates with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) protein and induces its recruitment to the regulatory regions of the two microRNA genes for histone H3 methylation and transcriptional repression. Interestingly, expression of both MEG8 and MEG3, but not each individually, could induce EMT-related cell morphological changes and increased cell motility in the absence of TGF-ß by activating the gene expression program required for EMT. MEG8 knockdown indicated that endogenous MEG8 lncRNA is indispensable for TGF-ß-induced EMT in A549 lung cancer and Panc1 pancreatic cancer cells. Our findings indicate that MEG8 lncRNA significantly contributes to epigenetic EMT induction and increase our understanding of the lncRNA-mediated regulatory mechanisms involved in malignant progression of cancer.

Epigenetic regulation of epithelial-mesenchymal transition by KDM6A histone demethylase in lung cancer cells.

Histone methylation is associated with various biological and pathological processes including cancer development. KDM6A is a candidate tumor suppressor gene that encodes a histone H3 lysine 27 (H3K27) demethylase. In this study, we discovered that ectopic expression of KDM6A antagonized TGF-ß-induced epithelial-mesenchymal transition (EMT) and cell migration of lung cancer cell lines through its demethylase activity. KDM6A counteracted TGF-ß-dependent changes in the expression of EMT-related genes such as CDH1/E-cadherin, FN1/Fibronectin, ZEB family and microRNA-200 family. Mechanistic investigations revealed that KDM6A inhibited the recruitment of EZH2 histone H3K27 methyltransferase and H3K27 methylation on the regulatory regions of the target genes such as CDH1 and microRNA-200 family. Knockdown of KDM6A did not proceed EMT by itself, but influenced the expression of specific target genes critical for EMT, suggesting that endogenous KDM6A was involved in EMT-inducing transcriptional program. This study demonstrated a novel regulatory role of KDM6A histone demethylase in the epigenetic control of EMT process in lung cancer cells.

Proteomic insights into the regulatory function of ARID1A in colon cancer cells.

The AT-rich interacting domain-containing protein 1A (ARID1A) is a tumor suppressor gene that has been implicated in several cancers, including colorectal cancer (CRC). The present study used a proteomic approach to elucidate the molecular mechanisms of ARID1A in CRC carcinogenesis. Stable ARID1A-overexpressing SW48 colon cancer cells were established using lentivirus transduction and the successful overexpression of ARID1A was confirmed by western blotting. Label-free quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry identified 705 differentially altered proteins in the ARID1A-overexpressing cells, with 310 proteins significantly increased and 395 significantly decreased compared with empty vector control cells. Gene Ontology enrichment analysis highlighted the involvement of the altered proteins mainly in the Wnt signaling pathway. Western blotting supported these findings, as a decreased protein expression of Wnt target genes, including c-Myc, transcription factor T cell factor-1/7 and cyclin D1, were observed in ARID1A-overexpressing cells. Among the altered proteins involved in the Wnt signaling pathway, the interaction network analysis revealed that ARID1A exhibited a direct interaction with E3 ubiquitin-protein ligase zinc and ring finger 3 (ZNRF3), a negative regulator of the Wnt signaling pathway. Further analyses using the The Cancer Genome Atlas colon adenocarcinoma public dataset revealed that ZNRF3 expression significantly impacted the overall survival of patients with CRC and was positively correlated with ARID1A expression. Finally, an increased level of ZNRF3 in ARID1A-overexpressing cells was confirmed by western blotting. In conclusion, the findings of the present study suggest that ARID1A negatively regulates the Wnt signaling pathway through ZNRF3, which may contribute to CRC carcinogenesis.

High phosphate intake induces bone loss in nephrectomized thalassemic mice.

Although patients with either ß-thalassemia or chronic kidney disease (CKD) clinically correlate with severe osteoporosis, the mechanism by which CKD exposed to high phosphate affects bone turnover has not been characterized in ß-thalassemia. We aimed to determine the effects of renal insufficiency on high phosphate intake induced changes in bone metabolism after 5/6th nephrectomy in hemizygous ß-globin knockout (BKO) mice. Male BKO mice manifested severe anemia and osteopenia. Nephrectomy induced renal fibrosis and reduced renal function as assessed by increased serum urea nitrogen levels. Moreover, nephrectomy increased bone turnover leading to bone loss in wild type (WT) but not BKO mice. In nephrectomized BKO, PBS in drinking water induced hyperphosphatemia, and hypercalcemia along with osteopenia in both cancellous and cortical bone. Histomorphometric analysis confirmed reduced cancellous bone volume due to decreased bone formation rate, osteoblast number and osteoclast number. The mRNA levels for Alpl, Sp7, Kl, Tnfsf11, and Tnfsf11/Tnfrsf11b were decreased in nephrectomized BKO mice drinking PBS. Interestingly, Fgf23, a bone-derived hormone produced by osteocytes and osteoblasts in response to hyperphosphatemia, were remarkably increased in nephrectomized BKO mice following PBS intake. Serum FGF23 and erythropoietin levels were markedly elevated in BKO mice. Nephrectomy decreased serum erythropoietin but not FGF23 levels. Hyperphosphatemia in BKO mice increased serum erythropoietin, FGF23, and PTH levels, nominating these factors as candidate mediators of bone loss in thalassemic mice with CKD during phosphate retention.

Identification of candidate regulators of mandibular bone loss in FcγRIIB-/- Mice.

Patients with systemic lupus erythematosus (SLE) have increased inflammatory cytokines, leading to periodontitis and alveolar bone loss. However, the mechanisms driving this phenomenon are still unknown. Here, we have identified novel therapeutic targets for and mediators of lupus-mediated bone loss using RNA-sequencing (RNA-seq) in a FcγRIIB-/- mouse model of lupus associated osteopenia. A total of 2,710 upregulated and 3,252 downregulated DEGs were identified. The GO and KEGG annotations revealed that osteoclast differentiation, bone mineralization, ossification, and myeloid cell development were downregulated. WikiPathways indicated that Hedgehog, TNFα NF-κB and Notch signaling pathway were also decreased. We identified downregulated targets, Sufu and Serpina12, that have important roles in bone homeostasis. Sufu and Serpina12 were related to Hedgehog signaling proteins, including Gli1, Gli2, Gli3, Ptch1, and Ptch2. Gene knockdown analysis demonstrated that Sufu, and Serpina12 contributed to osteoclastogenesis and osteoblastogenesis, respectively. Osteoclast and osteoblast marker genes were significantly decreased in Sufu-deficient and Serpina12-deficient cells, respectively. Our results suggest that alterations in Hedgehog signaling play an important role in the pathogenesis of osteopenia in FcγRIIB-/- mice. The novel DEGs and pathways identified in this study provide new insight into the underlying mechanisms of mandibular bone loss during lupus development.