Cells In Undifferentiated State Research Articles

EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) possess the intrinsic ability to differentiate into diverse cellular lineages, marking them as potent instruments in regenerative medicine. Nonetheless, the proclivity of these stem cells to generate teratomas post-transplantation presents a formidable obstacle to their therapeutic utility. In previous studies, we identified an array of cell surface proteins specifically expressed in the pluripotent state, as revealed through proteomic analysis. Here we focused on EPHA2, a protein found to be abundantly present on the surface of undifferentiated mouse ESCs and is diminished upon differentiation. Knock-down of Epha2 led to the spontaneous differentiation of mouse ESCs, underscoring a pivotal role of EPHA2 in maintaining an undifferentiated cell state. Further investigations revealed a strong correlation between EPHA2 and OCT4 expression during the differentiation of both mouse and human PSCs. Notably, removing EPHA2+ cells from mouse ESC-derived hepatic lineage reduced tumor formation after transplanting them into immune-deficient mice. Similarly, in human iPSCs, a larger proportion of EPHA2+ cells correlated with higher OCT4 expression, reflecting the pattern observed in mouse ESCs. Conclusively, EPHA2 emerges as a potential marker for selecting undifferentiated stem cells, providing a valuable method to decrease tumorigenesis risks after stem-cell transplantation in regenerative treatments.

PIWI pathway: bridging acute myeloid leukemia stemness and cellular differentiation.

PIWI proteins are stem cell-associated RNA-binding proteins crucial for survival of germ stem cells. In cancer, PIWI proteins are overexpressed. Specifically, PIWIL4 is highly expressed in multiple cancers with the highest levels found in acute myeloid leukemia (AML), an aggressive malignancy propagated by a population of leukemia stem cells (LSCs). Bamezai et al. (Blood Journal, blood, 2023, 142, 90-105) demonstrated that PIWIL4 supports AML blasts and LSCs but is not necessary for healthy human hematopoietic progenitor stem cells (HSPCs) function in vivo. PIWIL4 in AML acts by preventing the accumulation of R-loops in key genes for LSCs persistence implicated in: DNA damage, replicative stress, and transcription arrest. We report that PIWIL4 expression significantly decreases in THP-1 monocytes exposed to a differentiating agent, suggesting a potential role for PIWIL4 in maintaining the undifferentiated state of myeloid cells. PIWIL4 overexpression could lead to the emergence of LSCs, driving leukemia propagation and maintenance. Our findings correlate with the persistent overexpression of PIWIL4 in myeloid cancers as reported by Bamezai et al., and suggest that PIWIL4 may be involved in myeloid cell differentiation. In this perspective, we highlight recent findings on the implication of PIWI pathway in maintaining AML stemness. Additionally, we propose further investigation on the role of PIWI pathway in oncogenesis and cellular differentiation as a strategy to identify biomarkers and therapeutic targets for AML.

VAV2 orchestrates the interplay between regenerative proliferation and ribogenesis in both keratinocytes and oral squamous cell carcinoma

VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV−) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles’ heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation

ABSTRACT HOTAIRM1 is unlike most long non-coding RNAs in that its sequence is highly conserved across mammals. Such evolutionary conservation points to it having a role in key cellular processes. We previously reported that HOTAIRM1 is required to curb premature activation of downstream HOXA genes in a cell model recapitulating their sequential induction during development. We found that it regulates 3’ HOXA gene expression by a mechanism involving epigenetic and three-dimensional chromatin changes. Here we show that HOTAIRM1 participates in proper progression through the early stages of neuronal differentiation. We found that it can associate with the HOXA1 transcription factor and contributes to its downstream transcriptional program. Particularly, HOTAIRM1 affects the NANOG/POU5F1/SOX2 core pluripotency network maintaining an undifferentiated cell state. HOXA1 depletion similarly perturbed expression of these pluripotent factors, suggesting that HOTAIRM1 is a modulator of this transcription factor pathway. Also, given that binding of HOTAIRM1 to HOXA1 was observed in different cell types and species, our results point to this ribonucleoprotein complex as an integral part of a conserved HOTAIRM1-HOXA1 regulatory axis modulating the transition from a pluripotent to a differentiated neuronal state.

Single cell profiling of tumour biopsies and heterogeneity in response of dedifferentiated melanoma.

64 Background: Patients with advanced BRAFV600-mutant melanoma can be treated with immune checkpoint inhibitors or combination BRAF/MEK inhibitors. The sequence of these therapies can influence response to second-line treatment. Patients who progress on first-line PD1 blockade have poorer outcomes on BRAF/MEK inhibitors compared to patients who are treatment-naïve. Thus, defining the molecular features that are modified by therapy and that influence subsequent treatment responses require thorough characterization. Methods: In this study, tumor biopsies from five BRAF-mutant melanoma patients progressing on anti-PD1 (PROG) and five BRAF-mutant treatment-naïve (NAÏVE) patients were dissociated and treated ex vivo with BRAF/MEK inhibitors (dabrafenib+trametinib (DT)) before flow cytometry profiling and single cell transcriptome sequencing. Results: DT treatment did not alter tumor or immune cell proportions but effectively inhibited MAPK signaling by decreasing phosphorylated S6 levels in tumor cells within 8/10 melanoma samples. Single cell RNA-sequencing of the tumor dissociates identified four distinct melanoma differentiation states (e.g. melanocytic, transitory, neural crest-like and undifferentiated). The proportions of these cell states differed between tumor specimens, and were not affected by DT treatment. The neural crest-like and undifferentiated melanoma cells were enriched in PROG compared to NAIVE tumor samples and all differentiation states responded, albeit variably. For instance, MAPK inhibition after DT treatment was less pronounced in undifferentiated melanoma cells. Importantly, within the undifferentiated cell state, we identified a proportion of melanoma cells capable of responding to DT, and comparison of responding vs non-responding undifferentiated cells showed enrichment of pro-inflammatory IL-6 and TNFα signaling in the non-responding population. Elevated IL-6 and TNFα pathway activity correlated with decreased potency to MAPK inhibition, indicating that increased IL-6 and TNFα signaling in undifferentiated melanoma may contribute to resistance to MAPK inhibition. Conclusions: Ex vivo treatment of tumor dissociates coupled with single cell transcriptome sequencing identified cell state-specific responses to MAPK inhibition. Resistance to MAPK inhibition may be driven, in part, by tumor-intrinsic pro-inflammatory IL-6 and TNFα signalling.

Genetic predisposition to neuroblastoma results from a regulatory polymorphism that promotes the adrenergic cell state.

Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like mesenchymal cell state and a more differentiated sympathetic adrenergic cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional cofactor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism, G/T, that affects a GATA motif in the first intron of LMO1. Here, we showed that WT zebrafish with the GATA genotype developed adrenergic neuroblastoma, while knock-in of the protective TATA allele at this locus reduced the penetrance of MYCN-driven tumors, which were restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrated that TATA/TATA tumors also exhibited a mesenchymal cell state and were low risk at diagnosis. Thus, conversion of the regulatory GATA to a TATA allele in the first intron of LMO1 reduced the neuroblastoma-initiation rate by preventing formation of the adrenergic cell state. This mechanism was conserved over 400 million years of evolution, separating zebrafish and humans.

ANGPTL8 links inflammation and poor differentiation, which are characteristics of malignant renal cell carcinoma.

Inflammation is observed in many tumors, which affects metastasis, infiltration, and immune escape and causes poor differentiation of the cancer cells. However, the molecular basis underlying the relationship between inflammation and poor differentiation in tumors has not been identified. In this study, we demonstrate that angiopoietin-like protein-8 (ANGPTL8), which is induced by stress stimuli such as inflammation, is involved in the maintenance of the undifferentiated state of clear cell renal cell carcinoma (ccRCC) cells. ANGPTL8 is also involved in the production of chemokines that attract immune suppressor cells to the tumor microenvironment. ANGPTL8 sustains the continuous production of chemokines by activating the NF-κB signaling pathway and maintains the undifferentiated state of ccRCC cells. Finally, ANGPTL8 is induced by STAT3 signaling, which is activated by immune cells in the tumor microenvironment. These results support a role for ANGPTL8 in determining the properties of ccRCC by hampering tumor cell differentiation and establishing the tumor microenvironment.

Oxidative Stress and Its Modulation by Ladostigil Alter the Expression of Abundant Long Non-Coding RNAs in SH-SY5Y Cells.

Neurodegenerative disorders, brain injury, and the decline in cognitive function with aging are accompanied by a reduced capacity of cells in the brain to cope with oxidative stress and inflammation. In this study, we focused on the response to oxidative stress in SH-SY5Y, a human neuroblastoma cell line. We monitored the viability of the cells in the presence of oxidative stress. Such stress was induced by hydrogen peroxide or by Sin1 (3-morpholinosydnonimine) that generates reactive oxygen and nitrogen species (ROS and RNS). Both stressors caused significant cell death. Our results from the RNA-seq experiments show that SH-SY5Y cells treated with Sin1 for 24 h resulted in 94 differently expressed long non-coding RNAs (lncRNAs), including many abundant ones. Among the abundant lncRNAs that were upregulated by exposing the cells to Sin1 were those implicated in redox homeostasis, energy metabolism, and neurodegenerative diseases (e.g., MALAT1, MIAT, GABPB1-AS1, NEAT1, MIAT, GABPB1-AS1, and HAND2-AS1). Another group of abundant lncRNAs that were significantly altered under oxidative stress included cancer-related SNHG family members. We tested the impact of ladostigil, a bifunctional reagent with antioxidant and anti-inflammatory properties, on the lncRNA expression levels. Ladostigil was previously shown to enhance learning and memory in the brains of elderly rats. In SH-SY5Y cells, several lncRNAs involved in transcription regulation and the chromatin structure were significantly induced by ladostigil. We anticipate that these poorly studied lncRNAs may act as enhancers (eRNA), regulating transcription and splicing, and in competition for miRNA binding (ceRNA). We found that the induction of abundant lncRNAs, such as MALAT1, NEAT-1, MIAT, and SHNG12, by the Sin1 oxidative stress paradigm specifies only the undifferentiated cell state. We conclude that a global alteration in the lncRNA profiles upon stress in SH-SY5Y may shift cell homeostasis and is an attractive in vitro system to characterize drugs that impact the redox state of the cells and their viability.

Role of the JNK Pathway in Bladder Cancer.

Bladder cancer, one of the most frequently diagnosed cancers worldwide, is associated with high morbidity and mortality and a poor prognosis. The bladder cancer types include 1) non-muscle invasive bladder cancer (NMIBC) and 2) muscle invasive bladder cancer (MIBC). Metastases and chemoresistance in MIBC patients are the leading causes of the high death rate. c-Jun N-terminal kinase (JNK) is an important factor for the undifferentiated state of cancer cells. JNK belongs to the mitogen-activated protein kinases (MAPKs) family; it is activated by various extracellular stimuli, such as stress, radiation, and growth factors and mediates diverse cellular functions, such as apoptosis, autophagy, proliferation, invasion, and migration by mediating AKT (Ak strain transforming), ATG (Autophagy related), mTOR (Mammalian target of rapamycin), and caspases 3, 8, and 9. This review describes the JNK-related functions, mechanisms, and signaling in bladder cancer.

E-cadherin maintains the undifferentiated state of mouse spermatogonial progenitor cells via β-catenin

BackgroundCadherins play a pivotal role in facilitating intercellular interactions between spermatogonial progenitor cells (SPCs) and their surrounding microenvironment. Specifically, E-cadherin serves as a cellular marker of SPCs in many species. Depletion of E-cadherin in mouse SPCs showed no obvious effect on SPCs homing and spermatogenesis.ResultsHere, we investigated the regulatory role of E-cadherin in regulating SPCs fate. Specific deletion of E-cadherin in germ cells was shown to promote SPCs differentiation, evidencing by reduced PLZF+ population and increased c-Kit+ population in mouse testes. E-cadherin loss down-regulated the expression level of β-catenin, leading to the reduced β-catenin in nuclear localization for transcriptional activity. Remarkably, increasing expression level of Cadherin-22 (CDH22) appeared specifically after E-cadherin deletion, indicating CDH22 played a synergistic effect with E-cadherin in SPCs. By searching for the binding partners of β-catenin, Lymphoid enhancer-binding factor 1 (LEF1), T-cell factor (TCF3), histone deacetylase 4 (HDAC4) and signal transducer and activator 3 (STAT3) were identified as suppressors of SPCs differentiation by regulating acetylation of differentiation genes with PLZF.ConclusionsTwo surface markers of SPCs, E-cadherin and Cadherin-22, synergically maintain the undifferentiation of SPCs via the pivotal intermediate molecule β-catenin. LEF1, TCF3, STAT3 and HDAC4 were identified as co-regulatory factors of β-catenin in regulation of SPC fate. These observations revealed a novel regulatory pattern of cadherins on SPCs fate.

Polysialic Acid Sustains the Hypoxia-Induced Migration and Undifferentiated State of Human Glioblastoma Cells

Gliomas are the most common primary malignant brain tumors. Glioblastoma, IDH-wildtype (GBM, CNS WHO grade 4) is the most aggressive form of glioma and is characterized by extensive hypoxic areas that strongly correlate with tumor malignancy. Hypoxia promotes several processes, including stemness, migration, invasion, angiogenesis, and radio- and chemoresistance, that have direct impacts on treatment failure. Thus, there is still an increasing need to identify novel targets to limit GBM relapse. Polysialic acid (PSA) is a carbohydrate composed of a linear polymer of α2,8-linked sialic acids, primarily attached to the Neural Cell Adhesion Molecule (NCAM). It is considered an oncodevelopmental antigen that is re-expressed in various tumors. High levels of PSA-NCAM are associated with high-grade and poorly differentiated tumors. Here, we investigated the effect of PSA inhibition in GBM cells under low oxygen concentrations. Our main results highlight the way in which hypoxia stimulates polysialylation in U87-MG cells and in a GBM primary culture. By lowering PSA levels with the sialic acid analog, F-NANA, we also inhibited GBM cell migration and interfered with their differentiation influenced by the hypoxic microenvironment. Our findings suggest that PSA may represent a possible molecular target for the development of alternative pharmacological strategies to manage a devastating tumor like GBM.

The functional diversity of the POUV-class proteins across vertebrates.

POUV is a relatively newly emerged class of POU transcription factors present in jawed vertebrates (Gnathostomata). The function of POUV-class proteins is inextricably linked to zygotic genome activation (ZGA). A large body of evidence now extends the role of these proteins to subsequent developmental stages. While some functions resemble those of other POU-class proteins and are related to neuroectoderm development, others have emerged de novo. The most notable of the latter functions is pluripotency control by Oct4 in mammals. In this review, we focus on these de novo functions in the best-studied species harbouring POUV proteins-zebrafish, Xenopus (anamniotes) and mammals (amniotes). Despite the broad diversity of their biological functions in vertebrates, POUV proteins exert a common feature related to their role in safeguarding the undifferentiated state of cells. Here we summarize numerous pieces of evidence for these specific functions of the POUV-class proteins and recap available loss-of-function data.

Notch signaling in malignant gliomas: supporting tumor growth and the vascular environment.

Glioblastoma is the most malignant form of glioma, which is the most commonly occurring tumor of the central nervous system. Notch signaling in glioblastoma is considered to be a marker of an undifferentiated tumor cell state, associated with tumor stem cells. Notch is also known for facilitating tumor dormancy escape, recurrence and progression after treatment. Studies in vitro suggest that reducing, removing or blocking the expression of this gene triggers tumor cell differentiation, which shifts the phenotype away from stemness status and consequently facilitates treatment. In contrast, in the vasculature, Notch appears to also function as an important receptor that defines mature non-leaking vessels, and increasing its expression promotes tumor normalization in models of cancer in vivo. Failures in clinical trials with Notch inhibitors are potentially related to their opposing effects onthe tumor versus the tumor vasculature, which points to the need for a greater understanding of this signaling pathway.

PRD-Class Homeobox Genes in Bovine Early Embryos: Function, Evolution, and Overlapping Roles.

Eutherian Totipotent Cell Homeobox (ETCHbox) genes are mammalian-specific PRD-class homeobox genes with conserved expression in the preimplantation embryo but fast-evolving and highly divergent sequences. Here, we exploit an ectopic expression approach to examine the role of bovine ETCHbox genes and show that ARGFX and LEUTX homeodomain proteins upregulate genes normally expressed in the blastocyst; the identities of the regulated genes suggest that, in vivo, the ETCHbox genes play a role in coordinating the physical formation of the blastocyst structure. Both genes also downregulate genes expressed earlier during development and genes associated with an undifferentiated cell state, possibly via the JAK/STAT pathway. We find evidence that bovine ARGFX and LEUTX have overlapping functions, in contrast to their antagonistic roles in humans. Finally, we characterize a mutant bovine ARGFX allele which eliminates the homeodomain and show that homozygous mutants are viable. These data support the hypothesis of functional overlap between ETCHbox genes within a species, roles for ETCHbox genes in blastocyst formation and the change of their functions over evolutionary time.

Study of Stem Cells in Human Milk.

Stem cells are cells that have the ability to self-renew into an undifferentiated cell state, which can further delineate into distinct cell types. There are various sources of stem cells in the human body; some of them include cord blood, placental tissue, bone marrow, adipose tissue, dental pulp, etc. Breast milk could become an important source of stem cells in the near future because of its non-invasive isolation technique. Based on this nature, this study was conducted to isolate stem cells from breast milk and to show further potential implications of these cells. The total number of cells isolated from the milk ranged from 1.5 × 105 cells to 3 × 105 cells. As there was prolongation in the lactation period, the number of cells in the milk lowered significantly. There was no significant difference in the cell count in various gestational age groups. The cytochemistry analysis of these cells with their specific cell markers confirmed the presence of a homogenous population of mesenchymal stem cells. Further differentiation of these breast milk stem cell analyses showed transformation into adipocytes, chondrocytes, and osteoblasts in different culture mediums. So the presence of mesenchymal stem cells in human milk, which are multipotent in nature, makes it an important source of stem cells for further regenerative therapies, tissue culture techniques, and gene therapies. Due to this nature, these cells can be redirected to produce various tissues in the human body.

Negative Elongation Factor (NELF) Inhibits Premature Granulocytic Development in Zebrafish.

Gene expression is tightly regulated during hematopoiesis. Recent studies have suggested that RNA polymerase II (Pol II) promoter proximal pausing, a temporary stalling downstream of the promoter region after initiation, plays a critical role in regulating the expression of various genes in metazoans. However, the function of proximal pausing in hematopoietic gene regulation remains largely unknown. The negative elongation factor (NELF) complex is a key factor important for this proximal pausing. Previous studies have suggested that NELF regulates granulocytic differentiation in vitro, but its in vivo function during hematopoiesis remains uncharacterized. Here, we generated the zebrafish mutant for one NELF complex subunit Nelfb using the CRISPR-Cas9 technology. We found that the loss of nelfb selectively induced excessive granulocytic development during primitive and definitive hematopoiesis. The loss of nelfb reduced hematopoietic progenitor cell formation and did not affect erythroid development. Moreover, the accelerated granulocytic differentiation and reduced progenitor cell development could be reversed by inhibiting Pol II elongation. Further experiments demonstrated that the other NELF complex subunits (Nelfa and Nelfe) played similar roles in controlling granulocytic development. Together, our studies suggested that NELF is critical in controlling the proper granulocytic development in vivo, and that promoter proximal pausing might help maintain the undifferentiated state of hematopoietic progenitor cells.

Myc Supports Self-Renewal of Basal Cells in the Esophageal Epithelium.

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence.

An active RUNX1-ID1/ID3 axis governs differentiation and chemoresistance of cancer stem cell population in epithelial ovarian cancer cells

Progression, relapse, and therapy resistance are the most challenging features of cancer therapy that have been postulated to be driven by Cancer Stem Cell (CSC) population. This enigmatic subpopulation of cancer cells has therefore emerged as promising therapeutic candidate. We earlier reported enrichment of CSC-like side population (SP) with increasing resistance towards Cisplatin and Paclitaxel either alone or in combination in epithelial ovarian cancer (EOC) cells. This SP population is a small proportion of the total population of cancer cells characterised with high expression of drug transporters, a unique feature of stem cells and thereby can be isolated through their efflux properties of DNA binding dyes. While the bulk non-SP (NSP) population of the cancer cells lack overexpression of the drug transporters and thus can be identified as the dye containing population. In this study, we show that increased expression of Runt related transcription factor 1 (RUNX1) maintains undifferentiated state of CSC-like SP cells through upregulation of inhibitors of DNA binding/differentiation genes (ID1 and ID3) in late cisplatin-paclitaxel resistant cells. Higher RUNX1 expression was found to correlate with decreased median overall survival and disease-free survival in The Cancer Genome Atlas (TCGA) data set of high grade serous ovarian cancer (HGSOC) patients. The protein-protein interaction network analysis of 397 upregulated genes in RUNX1-high samples of TCGA data show significant enrichment of pathways known to negatively regulate CSC differentiation. Intriguingly RUNX1 inhibition not only induces CSC differentiation but also downregulates anti-apoptotic protein BCL2 in both SP and NSP cells and potentiates cytotoxic effects of Cisplatin-Paclitaxel in chemoresistant EOC cells. Inhibition of BCL2 through Venetoclax treatment, a small molecule BH3 mimic, sensitized these cells to platinum taxol treatment. Altogether, our data reveal new regulatory roles by RUNX1 to modulate CSC differentiation via ID1 and ID3 and to promote chemoresistance through BCL2 upregulation.

An Analysis of Structure-function Co-relation between GLI Oncoprotein and HLA Immune-gene Transcriptional Regulation through Molecular Docking

Background:GLI proteins play a significant role in the transduction of the Hedgehog (Hh) signaling pathway. A variety of human cancers, including the brain, gastrointestinal, lung, breast, and prostate cancers, demonstrate inappropriate activation of this pathway. GLI helps in proliferation and has an inhibitory role in the differentiation of hematopoietic stem cells. Malignancies may have a defect in differentiation. Different types of malignancies and undifferentiated cells have a low level of HLA expression on their cell surface.Objective:Human Leukocytic Antigen (HLA) downregulation is frequently observed in cancer cells. This work is aimed to hypothesize whether this downregulation of HLA molecules is GLI oncoprotein mediated or not. To understand the roles of different types of GLI oncoproteins on different classes of HLA transcriptional machinery was carried out through structure-based modeling and molecular docking studies.Methods:To investigate the role of GLI in HLA expression /downregulation is Hh-GLI mediated or not, molecular docking based computational interaction studies were performed between different GLI proteins (GLI1, GLI2, and GLI3) with TATA box binding protein (TBP) and compare the binding efficiencies of different HLA gene (both HLA class I and –II) regulating transcription factors (RelA, RFX5, RFXAP, RFXANK, CIITA, CREB1, and their combinations) with TBP. Due to unavailability of 3D protein structures of GLI2 and cyclin D2 (a natural ligand of GLI1) were modelled followed by structural validation by Ramachandran plot analysis.Results:GLI proteins especially, GLI1 and GLI2, have almost similar binding energy of RFX5-RFXANK- RFXAP and CIITA multi-protein complex to TBP but has lower binding energy between RelA to TBP.Conclusion:This study suggests that HLA class I may not be downregulated by GLI; however, over-expression of GLI1 is may be responsible for HLA class II downregulation. Thus this protein may be responsible for the maintenance of the undifferentiated state of malignant cells. This study also suggests the implicative role of GLI1 in the early definitive stage of hematopoiesis.

Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways

BackgroundStem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells.MethodsIn the present study, we used the previously constructed luciferase reporters driven by the promoter and 3’-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways.ResultsWe screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways.ConclusionsThese findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.