Cell Fate In Response Research Articles

SALL4 controls cell fate in response to DNA base composition

SummaryMammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.

Improved epithelial cell-cell adhesion using molecular mobility of supramolecular surfaces.

Cells can sense the surrounding microenvironmental properties including contact with biomaterials. Although in vitro cell fates in response to the physical properties of cell-adhesive materials have been widely reported, their influence on cell-cell adhesion is unclear. Here, we investigated the role of molecular mobility on polyrotaxane surfaces in epithelial cell-cell adhesion. Polyrotaxane surfaces with high mobility induced cytoplasmic yes-associated protein (YAP) localization in epithelial cells, whereas those with low mobility induced nuclear YAP localization, suggesting that YAP localization is switched by the mobility of the polyrotaxane surface. The cytoplasmic YAP localization increased the expression of tight junction-associated genes. A scratch assay revealed that although the epithelial cells on the low mobile surface rapidly initiated their migration, the cells on the highly mobile surface delayed their migration. Thus, this finding suggests that polyrotaxane surfaces with higher mobility induce cytoplasmic YAP localization, leading to stronger cell-cell adhesion. The polyrotaxane biointerface is promising as a powerful tool to improve the physical immune system and repair biological tissues.

Making Connections: p53 and the Cathepsin Proteases as Co-Regulators of Cancer and Apoptosis.

Simple SummaryThis article describes an emerging area of significant interest in cancer and cell death and the relationships shared by these through the p53 and cathepsin proteins. While it has been demonstrated that the p53 protein can directly induce the leakage of cathepsin proteases from the lysosome, directly triggering cell death, little is known about what factors set the threshold at which the lysosome can become permeabilized. It appears that the expression levels of cathepsin proteases may be central to this process, with some of them being transcriptionally regulated by p53. The consequences of such a mechanism have serious implications for lysosomal-mediated apoptosis and have significant input into the design of therapeutics and their strategic use. In this review, we highlight the importance of extending such findings to other cathepsin family members and the need to assess the roles of p53 isoforms and mutants in furthering this mechanism.While viewed as the “guardian of the genome”, the importance of the tumor suppressor p53 protein has increasingly gained ever more recognition in modulating additional modes of action related to cell death. Slowly but surely, its importance has evolved from a mutated genetic locus heavily implicated in a wide array of cancer types to modulating lysosomal-mediated cell death either directly or indirectly through the transcriptional regulation of the key signal transduction pathway intermediates involved in this. As an important step in determining the fate of cells in response to cytotoxicity or during stress response, lysosomal-mediated cell death has also become strongly interwoven with the key components that give the lysosome functionality in the form of the cathepsin proteases. While a number of articles have been published highlighting the independent input of p53 or cathepsins to cellular homeostasis and disease progression, one key area that warrants further focus is the regulatory relationship that p53 and its isoforms share with such proteases in regulating lysosomal-mediated cell death. Herein, we review recent developments that have shaped this relationship and highlight key areas that need further exploration to aid novel therapeutic design and intervention strategies.

Integrating Quantitative Assays with Biologically Based Mathematical Modeling for Predictive Oncology.

SummaryWe provide an overview on the use of biological assays to calibrate and initialize mechanism-based models of cancer phenomena. Although artificial intelligence methods currently dominate the landscape in computational oncology, mathematical models that seek to explicitly incorporate biological mechanisms into their formalism are of increasing interest. These models can guide experimental design and provide insights into the underlying mechanisms of cancer progression. Historically, these models have included a myriad of parameters that have been difficult to quantify in biologically relevant systems, limiting their practical insights. Recently, however, there has been much interest calibrating biologically based models with the quantitative measurements available from (for example) RNA sequencing, time-resolved microscopy, and in vivo imaging. In this contribution, we summarize how a variety of experimental methods quantify tumor characteristics from the molecular to tissue scales and describe how such data can be directly integrated with mechanism-based models to improve predictions of tumor growth and treatment response.

Energy expenditure during cell spreading influences the cellular response to matrix stiffness

Cells respond to the mechanical properties of the extracellular matrix (ECM) through formation of focal adhesions (FAs), re-organization of the actin cytoskeleton and adjustment of cell contractility. These are energy-demanding processes, but a potential causality between mechanical cues (matrix stiffness) and cellular (energy) metabolism remains largely unexplored. Here, we cultured human mesenchymal stem cells (hMSCs) on stiff (20 kPa) or soft (1 kPa) substrate and demonstrate that cytoskeletal reorganization and FA formation spreading on stiff substrates lead to a drop in intracellular ATP levels, correlating with activation of AMP-activated protein kinase (AMPK). The resulting increase in ATP levels further facilitates cell spreading and reinforces cell tension of the steady state, and coincides with nuclear localization of YAP/TAZ and Runx2. While on soft substrates (1 kPa), lowered ATP levels limit these cellular mechanoresponses. Furthermore, genetic ablation of AMPK lowered cellular ATP levels on stiff substrate and strongly reduced responses to substrate stiffness. Together, these findings reveal a hitherto unidentified relationship between energy expenditure and the cellular mechanoresponse, and point to AMPK as a key mediator of stem cell fate in response to ECM mechanics.

A p53-Dependent Checkpoint Induced upon DNA Damage Alters Cell Fate during hiPSC Differentiation.

SummaryThe ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important model of early human development and a tool for tissue engineering. However, the factors that disturb the intricate transcriptional choreography of differentiation remain incompletely understood. Here, we uncover a critical time window during which DNA damage significantly reduces the efficiency and fidelity with which hiPSCs differentiate to definitive endoderm. DNA damage prevents the normal reduction of p53 levels as cells pass through the epithelial-to-mesenchymal transition, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a “differentiation checkpoint” in early endoderm differentiation that alters cell fate in response to DNA damage.

103 ATF6-MARCH5-MFN2 axis regulates melanoma cell survival upon ER stress through mitochondrial dynamics

Solid tumor microenvironment typically characterizes by hypoxia and nutrients deprivation, which generally results in severe endoplasmic reticulum (ER) stress in tumor cells. It has been shown that cancer cells are relatively resistant to environmental and pharmacological ER stress-induced cell death. However, the underlying mechanism is far from understood. Mitochondrial dynamics have been greatly implicated in determining cell fate in response to stress. Herein, we demonstrate that ATF6-MARCH5-MFN2 axis-mediated mitochondrial dynamics remodeling functions as a pro-survival mechanism in melanoma cells upon ER stress through the activation of mitophagy.

CRISPR-mediated knockdown of miR-214 modulates cell fate in response to anti-cancer drugs in HPV-negative and HPV-positive cervical cancer cells

Cervical cancer is the fourth most common cause of mortality in women worldwide. In this study we investigated the effect of a tumour suppressor microRNA miR-214 in modulating the cell death against chemotherapeutic drugs like Doxorubicin, Cisplatin and Paclitaxel. CRISPR-facilitated knockdown and plasmid-based overexpression of miR-214 was performed in cervical cancer cell lines HeLa, C33A and CaSki. It was observed that knocking out miR-214 resulted in reduced apoptosis and cell migration upon drug treatments; while overexpression of miR-214 resulted in marginal increase in apoptosis and cell migration when treated with drugs. However, miR-214 had very little effect on production of reactive oxygen species. Our results also indicate that Doxorubicin was least effective and Paclitaxel most effective in inducing cell death. A combination of miR-214 overexpression and Paclitaxel treatment was found to be most effective in inducing cell death in cervical cancer cells. Analysis of cell cycle phases followed by apoptotic markers also showed that miR-214 overexpression along with Paclitaxel treatment caused an increase in PARP and decline of PI-3 kinase/Akt levels. Therefore, miR-214 levels determine the fate of the cancer cell during chemotherapeutic treatment.

A20 Restricts Inflammatory Response and Desensitizes Gingival Keratinocytes to Apoptosis.

The pathophysiology of periodontal disease involves a perturbed immune system to a dysbiotic microflora leading to unrestrained inflammation, collateral tissue damage, and various systemic complications. Gingival epithelial cells function as an important part of immunity to restrict microbial invasion and orchestrate the subsequent innate responses. A20 (TNFAIP3), an ubiquitin-editing enzyme, is one of the key regulators of inflammation and cell death in numerous tissues including gastrointestinal tract, skin, and lungs. Emerging evidence indicates A20 as an essential molecule in the oral mucosa as well. In this study, we characterized the role of A20 in human telomerase immortalized gingival keratinocytes (TIGKs) through loss and gain of function assays in preclinical models of periodontitis. Depletion of A20 through gene editing in TIGKs significantly increased IL-6 and IL-8 secretion in response to Porphyromonas gingivalis infection while A20 over-expression dampened the cytokine production compared to A20 competent cells through modulating NF-κB signaling pathway. In the subsequent experiments which assessed apoptosis, A20 depleted TIGKs displayed increased levels of cleaved caspase 3 and DNA fragmentation following P. gingivalis infection and TNF/CHX challenge compared to A20 competent cells. Consistently, there was reduced apoptosis in the cells overexpressing A20 compared to the control cells expressing GFP further substantiating the role of A20 in regulating gingival epithelial cell fate in response to exogenous insult. Collectively, our findings reveal first systematic evidence and demonstrate that A20 acts as a regulator of inflammatory response in gingival keratinocytes through its effect on NF-κB signaling and desensitizes cells to bacteria and cytokine induced apoptosis in the oral mucosa. As altered A20 levels can have profound effect on different cellular responses, future studies will determine whether A20-targeted therapies can be exploited to restrain periodontal inflammation and maintain oral mucosa tissue homeostasis.

A previously identified apoptosis inhibitor iASPP confers resistance to chemotherapeutic drugs by suppressing senescence in cancer cells

Cellular senescence is terminal cell cycle arrest that represents a prominent response to numerous anticancer therapies. The oncogene inhibitor of the apoptosis-stimulating protein of p53 (iASPP) plays essential roles in regulating cellular drug response by inhibiting apoptosis. However, whether or not it regulates chemotherapy-induced senescence (TIS) in cancer cells remains unclear. Here, using two commonly used cancer cell lines, HCT 116 and MCF-7, along with the xenograft mouse model, we found that iASPP inhibits senescence and also influences the senescence-associated secretory phenotype (SASP), which confers anticancer drug resistance independently of apoptosis. Mechanistically, iASPP is transcriptionally elevated by the p65 subunit of NF-κB in senescent cells and then translocates to the nucleus, where it binds p53 and NF-κBp65. This binding inhibits their transcriptional activities toward p21 and the key SASP factors interleukin (IL)-6/IL-8, respectively, and subsequently prevents senescence. Of note, we observed that iASPP knockdown sensitizes apoptosis-resistant cancers to doxorubicin treatment by promoting senescence both in vitro and in vivo We conclude that iASPP integrates the NF-κBp65- and p53-signaling pathways and thereby regulates cell fate in response to TIS, leading to chemotherapy resistance. These findings suggest that iASPP inhibition might be a strategy that could help restore senescence in cancer cells and improve outcomes of chemotherapy-based therapies.

Ribosome and Translational Control in Stem Cells.

Embryonic stem cells (ESCs) and adult stem cells (ASCs) possess the remarkable capacity to self-renew while remaining poised to differentiate into multiple progenies in the context of a rapidly developing embryo or in steady-state tissues, respectively. This ability is controlled by complex genetic programs, which are dynamically orchestrated at different steps of gene expression, including chromatin remodeling, mRNA transcription, processing, and stability. In addition to maintaining stem cell homeostasis, these molecular processes need to be rapidly rewired to coordinate complex physiological modifications required to redirect cell fate in response to environmental clues, such as differentiation signals or tissue injuries. Although chromatin remodeling and mRNA expression have been extensively studied in stem cells, accumulating evidence suggests that stem cell transcriptomes and proteomes are poorly correlated and that stem cell properties require finely tuned protein synthesis. In addition, many studies have shown that the biogenesis of the translation machinery, the ribosome, is decisive for sustaining ESC and ASC properties. Therefore, these observations emphasize the importance of translational control in stem cell homeostasis and fate decisions. In this review, we will provide the most recent literature describing how ribosome biogenesis and translational control regulate stem cell functions and are crucial for accommodating proteome remodeling in response to changes in stem cell fate.

Identifying fates of cancer cells exposed to mitotic inhibitors by quantitative phase imaging.

Cell cycle deregulation is a cancer hallmark that has stimulated the development of mitotic inhibitors with differing mechanisms of action. Quantitative phase imaging (QPI) is an emerging approach for determining cancer cell sensitivities to chemotherapies in vitro. Cancer cell fates in response to mitotic inhibitors are agent- and dose-dependent. Fates that lead to chromosomal instabilities may result in a survival advantage and drug resistance. Conventional techniques for quantifying cell fates are incompatible with growth inhibition assays that produce binary live/dead results. Therefore, we used QPI to quantify post-mitotic fates of G0/G1 synchronized HeLa cervical adenocarcinoma and M202 melanoma cells during 24 h of escalating-dose exposures to mitotic inhibitors, including microtubule inhibitors paclitaxel and colchicine, and an Aurora kinase A inhibitor, VX-680. QPI determined cell fates by measuring changes in cell biomass, morphology, and mean phase-shift. Cell fates fell into three groups: (1) bipolar division from drug failure; (2) cell death or sustained mitotic arrest; and (3) aberrant endocycling or multipolar division. In this proof-of-concept study, colchicine was most effective in producing desirable outcomes of sustained mitotic arrest or death throughout its dosing range, whereas both paclitaxel and VX-680 yielded dose-dependent multipolar divisions or endocycling, respectively. Furthermore, rapid completion of mitosis associated with bipolar divisions whereas prolonged mitosis associated with multipolar divisions or cell death. Overall, QPI measurement of drug-induced cancer cell fates provides a tool to inform the development of candidate agents by quantifying the dosing ranges over which suboptimal inhibitor choices lead to undesirable, aberrant cancer cell fates.

RITA requires eIF2\u03b1-dependent modulation of mRNA translation for its anti-cancer activity

Tumor protein 53 (p53, encoded by the TP53 gene) is a key tumor suppressor regulating cell fates in response to internal and external stresses. As TP53 is mutated or silenced in a majority of tumors, reactivation of p53 by small molecules represents a promising strategy in cancer therapeutics. One such agent is RITA (reactivation of p53 and induction of tumor cell apoptosis), which restores p53 expression in cells with hyperactive HDM2 and induces apoptosis. Yet, mechanisms underlying the anticancer activity of RITA are incompletely understood. Here we show that RITA suppresses mRNA translation independently of p53 by inducing eIF2α phosphorylation. Surprisingly, reactivation of p53 following RITA treatment is critically dependent on eIF2α phosphorylation. Moreover, inhibition of eIF2α phosphorylation attenuates pro-apoptotic and anti-neoplastic effects of RITA, while inducing phosphorylation of eIF2α enhances the anticancer activity of RITA. Collectively, these findings demonstrate that the translational machinery plays a major role in determining the antineoplastic activity of RITA, and suggest that combining p53 activators and translation modulators may be beneficial.

Metabolic Regulation of Hippocampal Neuroprogenitor Apoptosis After Irradiation.

The tumor suppressor p53 is an important regulator of cell fate response after DNA damage. Cell fate response following metabolic stresses has also been linked to p53-dependent pathways. In this study, we asked if 5'-adenosine monophosphate-activated protein kinase (AMPK), the master sensor of cellular energy balance, played a role in p53-dependent apoptosis of neural progenitor cells (NPCs) in the hippocampus after irradiation. Adult mice with targeted disruption of p53 or prkaa2 (gene that encodes AMPKα) in the brain were used to determine the role of p53 and AMPK, respectively, in radiation-induced apoptosis of NPCs in the hippocampus. The p53-dependent apoptosis of NPCs was associated with an increase in phospho-AMPK expression in the dentate gyrus at 8 hours after irradiation. Activation of AMPK was seen in granule neurons and subgranular NPCs. Compared with wildtype mice, apoptosis of NPCs was significantly attenuated in AMPK deficient (nestinCre: prkaa2fl/fl) mice after irradiation. AMPK deficiency did not however alter p53 activation in NPCs after irradiation. We conclude that AMPK may regulate apoptosis of hippocampal NPCs after irradiation. These findings suggest that cellular metabolism may play a role in determining cell fate response such as apoptosis after DNA damage in NPCs.

Effect of oxidative stress and 3-hydroxytyrosol on DNA methylation levels of miR-9 promoters.

Effect of oxidative stress and 3-hydroxytyrosol on DNA methylation levels of miR-9 promoters.

NF-κB/NKILA signaling modulates the anti-cancerous effects of EZH2 inhibition.

A wealth of evidence supports the broad therapeutic potential of NF‐κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF‐κB and the NF‐κB interacting long non‐coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non‐canonical actions of EZH2 on the regulation of NF‐κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non‐transformed breast epithelial cells, triple negative MDA‐MB‐231 cells and hormone responsive MCF‐7 cells, and measured changes in EZH2/NF‐κB/NKILA levels to confirm their interdependence. We demonstrate cell line‐specific fluctuations in these factors that functionally contribute to epithelial‐to‐mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA‐MB‐231 cell motility and CDK4‐mediated MCF‐7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non‐transformed cells. Notably, these events are mediated by a cell‐context dependent gain or loss of NKILA and NF‐κB. Depletion of NF‐κB in non‐transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF‐κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.

Scaling laws of cell-fate responses to transient stress

Analysis and modelling of dose-survival curves of cells and tissues are often used to assess therapeutic efficacy or environmental risks, much less to infer the intracellular regulatory mechanisms of cellular stress response. However, systematic measurements of how cell survival depends on the time profile of stress, such as exposure duration, provide practical means to decipher the homeostatic dynamics of stress-response regulatory networks. In this paper, we propose a dynamical framework to theoretically address the relationship between cell fate response to a transient stress and the underlying regulatory feedback mechanisms. A simple network topology that couples a homeostatic negative feedback and a death-triggering positive feedback is shown to display four response regimes for which the iso-effect relationships between duration and intensity are captured by specific power laws. These distinct response regimes define several windows of stress duration for which lethality is not merely proportional to the product of intensity and duration, and, thus, for which cells are either more tolerant or more vulnerable to a given dose. Overall, this study highlights the differential roles of feedback strength, timescale and nonlinearity in promoting survivability to particular stress profiles, providing a valuable framework for a comparative analysis of diverse stress-specific regulatory networks.

The histone demethylase JMJD2B is critical for p53-mediated autophagy and survival in Nutlin-treated cancer cells

Autophagy promotes cancer cell survival in response to p53 activation by the anticancer agent Nutlin-3a (Nutlin). We reported previously that Nutlin kills MDM2-amplified cancer cells and that this killing is associated with an inhibition of glucose metabolism, reduced α-ketoglutarate (α-KG) levels, and reduced autophagy. In the current report, using SJSA1, U2OS, A549, and MHM cells, we found that Nutlin alters histone methylation in an MDM2 proto-oncogene-dependent manner and that this, in turn, regulates autophagy-related gene (ATG) expression and cell death. In MDM2-amplified cells, Nutlin increased histone (H) 3 lysine (K) 9 and K36 trimethylation (me3) coincident with reduced autophagy and increased apoptosis. Blocking histone methylation restored autophagy and rescued these cells from Nutlin-induced killing. In MDM2-nonamplified cells, H3K9me3 and H3K36me3 levels were either reduced or not changed by the Nutlin treatment, and this coincided with increased autophagy and cell survival. Blocking histone demethylation reduced autophagy and sensitized these cells to Nutlin-induced killing. Further experiments suggested that MDM2 amplification increases histone methylation in Nutlin-treated cells by causing depletion of the histone demethylase Jumonji domain-containing protein 2B (JMJD2B). Finally, JMJD2B knockdown or inhibition increased H3K9/K36me3 levels, decreased ATG gene expression and autophagy, and sensitized MDM2-nonamplified cells to apoptosis. Together, these results support a model in which MDM2- and JMJD2B-regulated histone methylation levels modulate ATG gene expression, autophagy, and cell fate in response to the MDM2 antagonist Nutlin-3a.

P53 Binds Preferentially to Non-B DNA Structures Formed by the Pyrimidine-Rich Strands of GAA·TTC Trinucleotide Repeats Associated with Friedreich's Ataxia.

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich’s ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich’s ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs’ non-B DNA structures.

Sialylation of MUC4β N-glycans by ST6GAL1 orchestrates human airway epithelial cell differentiation associated with type-2 inflammation.

Although type-2-induced (T2-induced) epithelial dysfunction is likely to profoundly alter epithelial differentiation and repair in asthma, the mechanisms for these effects are poorly understood. A role for specific mucins, heavily N-glycosylated epithelial glycoproteins, in orchestrating epithelial cell fate in response to T2 stimuli has not previously been investigated. Levels of a sialylated MUC4β isoform were found to be increased in airway specimens from asthmatic patients in association with T2 inflammation. We hypothesized that IL-13 would increase sialylation of MUC4β, thereby altering its function and that the β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) would regulate the sialylation. Using human biologic specimens and cultured primary human airway epithelial cells (HAECs),we demonstrated that IL-13 increases ST6GAL1-mediated sialylation of MUC4β and that both were increased in asthma, particularly in sputum supernatant and/or fresh isolated HAECs with elevated T2 biomarkers. ST6GAL1-induced sialylation of MUC4β altered its lectin binding and secretion. Both ST6GAL1 and MUC4β inhibited epithelial cell proliferation while promoting goblet cell differentiation. These in vivo and in vitro data provide strong evidence for a critical role for ST6GAL1-induced sialylation of MUC4β in epithelial dysfunction associated with T2-high asthma, thereby identifying specific sialylation pathways as potential targets in asthma.