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Transcription factors are key determinants of lineage commitment during mammalian development. However, the function and molecular mechanism for the transcription factors in the formation of three primary germ layers during human embryonic development are not fully elucidated. Here, we report that homeobox-containing transcription factor HESX1 plays a critical role in mesendodermal (ME) commitment of human embryonic stem cells (hESCs). Our results show that expression of HESX1 in hESCs is regulated by OCT4 and NANOG, and that its expression level changes with hESC differentiation. We find that knockdown of HESX1 does not disrupt the undifferentiated state of hESCs, in terms of cell morphology and expression levels of pluripotency-associated genes. However, HESX1 deficiency in hESCs impairs their ME commitment, whereas forced expression of HESX1 significantly enhances ME marker expression during ME commitment. Interestingly, HESX1 knockdown in hESCs represses ERK1/2 signaling activated by ME induction, while overexpression of HESX1 markedly enhances ERK1/2 activity during ME commitment of hESCs. Of note, MEK inhibitor PD0325901 weakens or even eliminates HESX1 overexpression-mediated promotive effects on ME induction in a dosage-dependent manner. Together, this study identifies a novel role of HESX1 in hESC commitment to ME cells and establishes the functional link between a transcription factor and lineage-associated signaling. These findings would help to better understand early human development and develop more efficient protocols to induce hESC differentiation to desired lineages. • Expression of HESX1 in hESCs is regulated by OCT4 and NANOG. • HESX1 deficiency impairs induction of mesendodermal (ME) transcription program of hESCs. • HESX1 overexpression (OE) enhances ME commitment of hESCs. • HESX1 modulates ERK1/2 signaling during hESC commitment to ME cells. • ERK1/2 signaling inhibition abrogates the enhancement of HESX1 OE on ME induction. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) have the unique feature of unlimited self-renewal and differentiation into derivatives of all three germ layers in human body, providing a powerful in vitro model for studying cell differentiation. FGF2, BMP4 and TGF-β signaling have been shown to play crucial roles in mesendodermal differentiation of hESCs. However, their underlying molecular mechanisms and other signaling pathways potentially involved in mesendodermal differentiation of hESCs remain to be further investigated. In this study, we uncover that VEGF signaling pathway plays a critical role in the mesendodermal induction of hESCs. Treating hESCs with Lenvatinib, a pan-inhibitor of VEGF receptors (VEGFRs), impedes their mesendodermal induction. Conversely, overexpression of VEGFA165, a major human VEGF isoform, promotes the mesendodermal differentiation. Similar to the VEGFR inhibitor, MEK inhibitor PD0325901 hinders mesendodermal induction of hESCs. In contrast, overexpression of ERK2GOF, an intrinsically active ERK2 mutant, markedly reduces the inhibitory effect of the VEGFR inhibitor. Thus, the MEK-ERK cascade plays an important role for the function of VEGF signaling pathway in the mesendodermal induction of hESCs. All together, this study identifies the critical role of VEGF signaling pathway as well as potential crosstalk of VEGF signaling pathway with other known signaling pathways in mesendodermal differentiation of hESCs. • Inhibition of VEGF receptors suppresses mesendodermal induction of hESCs. • Inhibition of VEGF receptors reduces levels of pERK1/2, pSMAD1/5 and pSMAD2/3. • Forced expression of VEGFA165 promotes mesendodermal induction. • Inhibition of MEK-ERK cascade hinders mesendodermal induction of hESCs. • MEK-ERK cascade is implicated in the role of VEGF pathway in mesendodermal induction. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) can self-renew infinitely and differentiate into the cell types of all lineages of our body, holding great promise for investigating early human embryo development and providing functional cells for disease treatment. For the full application of hESCs, it is necessary to elucidate how hESCs maintain their identity. Recent studies have shown that glycolysis and mitochondrial respiration are linked to pluripotency states. However, the function of mitochondrial respiration in hESCs has not been fully understood. Herein, we report that the adenosine triphosphate (ATP) production rate is comparable between mitochondrial respiration and glycolysis, suggesting an important contribution of mitochondrial respiration to ATP production in conventionally cultured hESCs. To investigate the function of mitochondrial respiration, we silence OGDH expression in hESCs by the inducible CRISPRi method, and find that OGDH knockdown (KD) results in disrupted TCA (tricarboxylic acid) cycle, and diminished mitochondrial respiration activity and total ATP level. Moreover, OGDH KD leads to hESC death and aberrant transcriptional program. Interestingly, blockage of the electron transport chain (ETC) by small molecule inhibitors gives rise to the phenotype similar to that observed in OGDH deficient hESCs. Therefore, genetic and pharmacological perturbations of the mitochondrial respiration impair identity of hESCs. Collectively, our study highlights the pivotal role of the mitochondrial respiration activity for the stemness maintenance of primed hESCs, and unveils OGDH as a key regulator for the proper production of ATP and TCA cycle metabolites in primed hESCs. • Mitochondrial respiration contributes to ATP production in primed hESCs. • Inducible knockdown of OGDH provides a useful model to study TCA cycle. • OGDH deficiency reduces maximal mitochondrial respiration and ATP levels. • OGDH deficiency leads to hESC death and an aberrant transcriptomic profile. • Electron transport chain inhibition causes hESC death and aberrant gene expression. [ABSTRACT FROM AUTHOR]

Keratinocyte hyperproliferation is an essential link in skin cancer pathogenesis. Peroxiredoxin I (Prx I) is known to regulate cancer cell proliferation, differentiation, and apoptosis, but its role in skin cancer remains unclear. This study aimed to elucidate the role and mechanism of Prx I in skin cancer pathogenesis. Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were used to create a skin tumor model of the initiation/promotion stage of cancer. The role of Prx I in H 2 O 2 -induced keratinocyte apoptosis was also investigated. After DMBA/TPA treatment, Prx I deficiency was significantly associated with less skin tumors, lower Bcl-2 expression, and higher p-p38 and cleaved caspase-3 expressions in Prx I knockout tumors than in wild-type controls. H 2 O 2 stimulation caused more cellular apoptosis in Prx I knockdown HaCaT cells than in normal HaCaT cells. The signaling study revealed that Bcl-2, p-p38, and cleaved caspase-3 expressions were consistent with the results in the tumors. In conclusion, the deletion of Prx I triggered the DMBA/TPA-induced skin tumor formation in vivo and in vitro by regulating the reactive oxygen species (ROS)-p38 mitogen-activated protein kinase (MAPK) pathway. These findings provide a theoretical basis for treating skin cancer. • Peroxiredoxin I deficiency was associated with a smaller number of skin tumors. • Peroxiredoxin I deficiency was related to lower Bcl-2 and higher p-p38 and c-C3. • Peroxiredoxin I had no significant effect on the later stage of tumor development. • H 2 O 2 stimulation caused more cellular apoptosis in peroxiredoxin I-deficient mice. [ABSTRACT FROM AUTHOR]

A wealth of studies illustrate the powerful antioxidant activities and health-promoting functions of dietary phenolic compounds, e.g., anthocyanins, flavonoids, and phenolic compounds. Ferulate is methylated from caffeoyl CoA using S-adenosyl-L-methionine (SAM) as methyl donor catalyzed by caffeoyl CoA methyltransferase (CCoAOMT). Here we show that Arabidopsis CCoAOMT7 contributes to ferulate content in the stem cell wall. CCoAOMT7 was further shown to bind S-adenosyl-L-homocysteine hydrolase (SAHH), a critical step in SAM synthesis to release feedback suppression on CCoAOMT. CCoAOMT7 also bound S-adenosyl-L-methionine synthases (SAMSs) in vivo , which were mediated by SAHH1. Interruptions of endogenous SAHH1 by artificial miRNA or SAMSs by T-DNA insertion significantly reduced ferulate contents in the stem cell wall. This data reveals a novel protein complex of SAM synthesis cycle associated with O-methyltransferase and provides new insights into cellular methylation processes. Image 1 • CCoAOMT7 contributes to ferulate accumulation in cell wall. • SAHH1 and SAHH2 protein directly interact with CCoAOMT7. • SAMSs associate with CCoAOMT7-SAHH complex mediated by SAHH1. [ABSTRACT FROM AUTHOR]

N 6 -Methyladenosine (m 6 A) represents the most prevalent internal modification in mammalian mRNAs. Emerging evidences suggest that m 6 A modification is profoundly implicated in many biological processes, including cancer development. However, limited knowledge is available about the functional importance of m 6 A in lung cancer. In this study, by data mining The Cancer Genome Atlas (TCGA) database, we first identified fat mass- and obesity-associated protein (FTO) as a prognostic factor for lung squamous cell carcinoma (LUSC). Then we showed that FTO, but not other m 6 A modification genes including METTL3, METTL14 and ALKBH5, was the major dysregulated factor responsible for aberrant m 6 A modification in LUSC. Loss-of-function studies suggested that FTO knockdown effectively inhibited cell proliferation and invasion, while promoted cell apoptosis of L78 and NCI-H520 cells. Furthermore, overexpression of FTO, but not its mutant form, facilitated the malignant phenotypes of CHLH-1 cells. Mechanistically, FTO enhanced MZF1 expression by reducing m 6 A levels and mRNA stability in MZF1 mRNA transcript, leading to oncogenic functions. Taken together, our study demonstrates the functional importance of FTO in the tumor progression of LUSC and provides a potential therapeutic target for LUSC treatment. [ABSTRACT FROM AUTHOR]