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Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells.

ABSTRACT: Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs) derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maintain the ability to differentiate into the 3 germ layers and extraembryonic tissues, as well as behave normally when reintroduced into a healthy blastocyst. Retinoic acid signaling has been thoroughly interrogated in ECCs, especially in the F9 and P19 murine cell models, and while we have touched on this aspect, this review purposely highlights how some key transcription factors regulate pluripotency and cell stemness prior to this signaling. Another major focus is on the epigenetic regulation of ECCs and stem cells, and, towards that end, this review closes on what we see as a new frontier in combating aging and human disease, namely, how cellular metabolism shapes the epigenetic landscape and hence the pluripotency of all stem cells.

SUBMITTER: Kelly GM

PROVIDER: S-EPMC5360977 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells.

Kelly Gregory M GM Gatie Mohamed I MI

Stem cells international 20170308

Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs) derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maint ...[more]

PMID: 28373885

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Project description:Given the relatively long life of stem cells (SCs), efficient mechanisms of quality control to balance cell survival and resistance to external and internal stress are required. Our objective was to test the relevance of cell quality control mechanisms for SCs maintenance, differentiation and resistance to cell death. We compared cell quality control in P19 stem cells (P19SCs) before and after differentiation (P19dCs). Differentiation of P19SCs resulted in alterations in parameters involved in cell survival and protein homeostasis, including the redox system, cardiolipin and lipid profiles, unfolded protein response, ubiquitin-proteasome and lysosomal systems, and signaling pathways controlling cell growth. In addition, P19SCs pluripotency was correlated with stronger antioxidant protection, modulation of apoptosis, and activation of macroautophagy, which all contributed to preserve SCs quality by increasing the threshold for cell death activation. Furthermore, our findings identify critical roles for the PI3K-AKT-MTOR pathway, as well as autophagic flux and apoptosis regulation in the maintenance of P19SCs pluripotency and differentiation potential.Abbreviations: 3-MA: 3-methyladenine; AKT/protein kinase B: thymoma viral proto-oncogene; AKT1: thymoma viral proto-oncogene 1; ATG: AuTophaGy-related; ATF6: activating transcription factor 6; BAX: BCL2-associated X protein; BBC3/PUMA: BCL2 binding component 3; BCL2: B cell leukemia/lymphoma 2; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CL: cardiolipin; CTSB: cathepsin B; CTSD: cathepsin D; DDIT3/CHOP: DNA-damage inducible transcript 3; DNM1L/DRP1: dynamin 1-like; DRAM1: DNA-damage regulated autophagy modulator 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2, subunit alpha; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; ESCs: embryonic stem cells; KRT8/TROMA-1: cytokeratin 8; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NANOG: Nanog homeobox; NAO: 10-N-nonyl acridine orange; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; OPA1: OPA1, mitochondrial dynamin like GTPase; P19dCs: P19 differentiated cells; P19SCs: P19 stem cells; POU5F1/OCT4: POU domain, class 5, transcription factor 1; PtdIns3K: phosphatidylinositol 3-kinase; RA: retinoic acid; ROS: reactive oxygen species; RPS6KB1/p70S6K: ribosomal protein S6 kinase, polypeptide 1; SCs: stem cells; SOD: superoxide dismutase; SHC1-1/p66SHC: src homology 2 domain-containing transforming protein C1, 66 kDa isoform; SOX2: SRY (sex determining region Y)-box 2; SQSTM1/p62: sequestosome 1; SPTAN1/αII-spectrin: spectrin alpha, non-erythrocytic 1; TOMM20: translocase of outer mitochondrial membrane 20; TRP53/p53: transformation related protein 53; TUBB3/betaIII-tubulin: tubulin, beta 3 class III; UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

Project description:We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-β Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

Project description:Cancer cells with a less differentiated stem-like phenotype are more resistant to therapeutic manipulations than their differentiated counterparts, and are considered as one of the main causes of cancer persistence and relapse. As such, induction of differentiation in cancer stem-like cells (CSLCs) has emerged as an alternative strategy to enhance the efficacy of anticancer therapies. CSLCs are metabolically distinct from differentiated cells, and any aberration from the intrinsic metabolic state can induce differentiation of CSLCs. Therefore, metabolism-related molecular targets, with a capacity to promote differentiation within CSLCs, are of therapeutic importance. Here, we demonstrate that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme catalyzing the synthesis of amino acid serine, is important for maintaining the poorly differentiated, stem-like state of CSLCs. Our data shows that PHGDH deficiency impairs the tumorsphere formation capacity in embryonal carcinoma stem-like cells (ECSLCs), breast cancer stem-like cells (BCSLCs) and patient-derived brain tumor-initiating cells (BTICs), which is accompanied by the reduced expression of characteristic stemness-promoting factors, such as Oct4, Nanog, Sox-2, and Bmi-1. Mechanistically, PHGDH deficiency in ECSLCs promotes differentiation to various lineages via degradation of Oct4 and by increasing the stability of differentiation marker ?3-tubulin. Furthermore, PHGDH inhibition promotes p-mTOR independent but Beclin-1-dependent autophagy, independent of apoptosis. When studied in combination, the inhibition of both PHGDH and p-mTOR in ECSLCs causes further augmentation of autophagy, and additionally promotes apoptosis, demonstrating the clinical applicability of PHGDH-based manipulations in cancer therapies. Recapitulating these in vitro findings in CSLC models, the intratumoral PHGDH expression in patient-derived tumors is positively correlated with the mRNA levels of stemness factors, especially Oct4, and cancer patients co-expressing high levels of PHGDH and Oct4 display significantly lower survival than those with low PHGDH/Oct4 co-expression. Altogether, this study identifies a clinically-relevant role for PHGDH in the regulation of stemness-differentiation axis within CSLCs.

Dataset Information

Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells.

Publications

Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells.

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