Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling.

Recent studies reveal that the mechanical environment influences the behavior and function of various types of cells, including stem cells. However, signaling pathways involved in the mechanical regulation of stem cell properties remain largely unknown. Using polyacrylamide gels with varying Young's moduli as substrates, we demonstrate that mouse embryonic stem cells (mESCs) are induced to differentiate on substrates with defined elasticity, involving the Src-ShcA-MAP kinase pathway. While the dual inhibition of mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), termed "2i," was reported to sustain the pluripotency of mESCs, we find it to be substrate elasticity dependent. In contrast, Src inhibition in addition to 2i allows mESCs to retain their pluripotency independent of substrate elasticity. The alternative dual inhibition of Src and GSK3 ("alternative 2i") retains the pluripotency and self-renewal of mESCs in vitro and is instrumental in efficiently deriving mESCs from preimplantation mouse embryos. In addition, the transplantation of mESCs, maintained under the alternative 2i condition, to immunodeficient mice leads to the formation of teratomas that include differentiation into three germ layers. Furthermore, mESCs established with alternative 2i contributed to chimeric mice production and transmitted to the germline. These results reveal a role for Src-ShcA-MAP kinase signaling in the mechanical regulation of mESC properties and indicate that alternative 2i is a versatile tool for the maintenance of mESCs in serum-free conditions as well as for the derivation of mESCs.

A KDM6 inhibitor potently induces ATF4 and its target gene expression through HRI activation and by UTX inhibition.

UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.

Structural insights into trans-histone regulation of H3K4 methylation by unique histone H4 binding of MLL3/4.

MLL3 and MLL4 are two closely related members of the SET1/MLL family of histone H3K4 methyltransferases and are responsible for monomethylating histone H3K4 on enhancers, which are essential in regulating cell-type-specific gene expression. Mutations of MLL3 or MLL4 have been reported in different types of cancer. Recently, the PHD domains of MLL3/4 have been reported to recruit the MLL3/4 complexes to their target genes by binding to histone H4 during the NT2/D1 stem cell differentiation. Here we show that an extended PHD domain (ePHD6) involving the sixth PHD domain and its preceding zinc finger in MLL3 and MLL4 specifically recognizes an H4H18-containing histone H4 fragment and that modifications of residues surrounding H4H18 modulate H4 binding to MLL3/4. Our in vitro methyltransferase assays and cellular experiments further reveal that the interaction between ePHD6 of MLL3/4 and histone H4 is required for their nucleosomal methylation activity and MLL4-mediated neuronal differentiation of NT2/D1 cells.

A Molecular Mechanism To Switch the Aryl Hydrocarbon Receptor from a Transcription Factor to an E3 Ubiquitin Ligase.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, we also observed lower ERα protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors.

Hypoxia is a Key Driver of Alternative Splicing in Human Breast Cancer Cells.

Adaptation to hypoxia, a hallmark feature of many tumors, is an important driver of cancer cell survival, proliferation and the development of resistance to chemotherapy. Hypoxia-induced stabilization of hypoxia-inducible factors (HIFs) leads to transcriptional activation of a network of hypoxia target genes involved in angiogenesis, cell growth, glycolysis, DNA damage repair and apoptosis. Although the transcriptional targets of hypoxia have been characterized, the alternative splicing of transcripts that occurs during hypoxia and the roles they play in oncogenesis are much less understood. To identify and quantify hypoxia-induced alternative splicing events in human cancer cells, we performed whole transcriptome RNA-Seq in breast cancer cells that are known to provide robust transcriptional response to hypoxia. We found 2005 and 1684 alternative splicing events including intron retention, exon skipping and alternative first exon usage that were regulated by acute and chronic hypoxia where intron retention was the most dominant type of hypoxia-induced alternative splicing. Many of these genes are involved in cellular metabolism, transcriptional regulation, actin cytoskeleton organisation, cancer cell proliferation, migration and invasion, suggesting they may modulate or be involved in additional features of tumorigenic development that extend beyond the known functions of canonical full-length transcripts.

Inhibition of the H3K9 methyltransferase G9A attenuates oncogenicity and activates the hypoxia signaling pathway.

Epigenetic mechanisms play important roles in the regulation of tumorigenesis, and hypoxia-induced epigenetic changes may be critical for the adaptation of cancer cells to the hypoxic microenvironment of solid tumors. Previously, we showed that loss-of-function of the hypoxia-regulated H3K9 methyltransferase G9A attenuates tumor growth. However, the mechanisms by which blockade of G9A leads to a tumor suppressive effect remain poorly understood. We show that G9A is highly expressed in breast cancer and is associated with poor patient prognosis, where it may function as a potent oncogenic driver. In agreement with this, G9A inhibition by the small molecule inhibitor, BIX-01294, leads to increased cell death and impaired cell migration, cell cycle and anchorage-independent growth. Interestingly, whole transcriptome analysis revealed that genes involved in diverse cancer cell functions become hypoxia-responsive upon G9A inhibition. This was accompanied by the upregulation of the hypoxia inducible factors HIF1α and HIF2α during BIX-01294 treatment even in normoxia that may facilitate the tumor suppressive effects of BIX-01294. HIF inhibition was able to reverse some of the transcriptional changes induced by BIX-01294 in hypoxia, indicating that the HIFs may be important drivers of these derepressed target genes. Therefore, we show that G9A is a key mediator of oncogenic processes in breast cancer cells and G9A inhibition by BIX-01294 can successfully attenuate oncogenicity even in hypoxia.

The impact of mechanical stress on stem cell properties: The link between cell shape and pluripotency.

Embryonic development and differentiation are controlled largely by external stimuli. Mechanical forces, such as those exerted by the surrounding cells and tissues, gravity and substrate rigidity, have been shown to affect cell morphology and spreading, thus triggering signaling pathways that dictate their development. These mechanosignaling pathways play important roles in cellular differentiation and the determination of cell fate. In this review, we discuss the effects of external environmental stimuli on cell differentiation and how this affects pluripotency, as well as the key molecules and pathways involved in mechanosignaling, particularly in relation to embryonic stem cells. Advances in experimental techniques and devices used to study the different aspects of mechanobiology are also examined. Finally, the effects of mechanical stress on the initiation and maintenance of pathological processes such as cancer, as well as their implications for prognosis and possible therapies are discussed.

The hypoxia-inducible epigenetic regulators Jmjd1a and G9a provide a mechanistic link between angiogenesis and tumor growth.

Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression.