Targeting NANOG and FAK via Cx26-derived Cell-penetrating Peptides in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of the interaction region of Cx26 attenuated tumor growth in preclinical models. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. Peptides with high affinity were engineered with a cell-penetrating antennapedia sequence and assessed in functional assays including cell proliferation, tumorsphere formation, and in vivo tumor growth, and downstream signaling changes were measured. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. Here, we provide proof-of-concept that a Cx26 peptide-based strategy inhibits growth and alters NANOG activity specifically in TNBC, indicating the therapeutic potential of this targeting approach.

siRNA-mediated silencing of Nanog reduces stemness properties and increases the sensitivity of HepG2 cells to cisplatin.

Liver cancer is one of the most lethal cancers having worldwide prevalence. Despite significant progress in cancer therapy, liver cancer-induced mortality is very high. Nanog, as an essential transcription factor modulating cellular multipotency, causes tumor progression, drug resistance, and preserves stemness properties in various tumors such as liver cancer. Thus, this research was conducted to evaluate the impact of combination therapy of Nanog siRNA/cisplatin on the sensitivity of liver cancer cells to this drug. HepG2 cells were transfected with Nanog siRNA and treated with cisplatin, individually and in combination. Then, it was observed that in transfected HepG2 cells, Nanog expression was significantly reduced at mRNA level and also these cells were sensitized to cisplatin. In addition, to assess the impact of Nanog siRNA and cisplatin individually and in combination on cells' viability, migration capacity, apoptosis, and cell cycle progression, the MTT, wound healing, colony formation assay, Annexin V/PI staining, and flow cytometry assays were applied on HepG2 cells, respectively. Also, the quantitive Real-Time PCR was used to check the expression of stemness-associated genes (CD44, CD133, and Sox2), and apoptosis-related genes (caspase-3, 8, 9, BAX and Bcl2) after combination therapy. It is indicated that the combination of Nanog siRNA and cisplatin significantly reduced proliferation, migration, and colony formation ability, as well as increased apoptosis rate, and cell cycle arrest. Also, it is found that the combination of Nanog siRNA and cisplatin down-regulated the expression of stemness-associated genes and up-regulated apoptosis-related genes in HepG2 cells. Hence, it can be suggested that Nanog inhibition in combination with cisplatin is a potential therapeutic strategy for developing new therapeutic approaches for liver cancer.

The pluripotency transcription factor OCT4 represses heme oxygenase-1 gene expression.

In embryonic stem (ES) cells, oxidative stress control is crucial for genomic stability, self-renewal, and cell differentiation. Heme oxygenase-1 (HO-1) is a key player of the antioxidant system and is also involved in stem cell differentiation and pluripotency acquisition. We found that the HO-1 gene is expressed in ES cells and induced after promoting differentiation. Moreover, downregulation of the pluripotency transcription factor (TF) OCT4 increased HO-1 mRNA levels in ES cells, and analysis of ChIP-seq public data revealed that this TF binds to the HO-1 gene locus in pluripotent cells. Finally, ectopic expression of OCT4 in heterologous systems repressed a reporter carrying the HO-1 gene promoter and the endogenous gene. Hence, this work highlights the connection between pluripotency and redox homeostasis.

Direct repression of Nanog and Oct4 by OTX2 modulates the contribution of epiblast-derived cells to germline and somatic lineage.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.

Suppression of Nanog inhibited cell migration and increased the sensitivity of colorectal cancer cells to 5-fluorouracil.

Nanog is a major transcription factor related to cellular multipotency that plays important roles in the development of tumor cells, drug resistance, migration, and stemness; indicating its great potential as a therapeutic target for various malignancies including colorectal cancer (CRC). Therefore, this study was aimed to evaluate the Nanog suppression effect using small interference RNA (siRNA) combined with 5-fluorouracil (5-FU) on CRC cells. Nanog-overexpressing SW-480 cells were transfected with Nanog si-RNA and treated with 5-FU, in combination or separately. Subsequently, it was observed that Nanog expression was significantly reduced after transfection of SW-480 cells using Nanog siRNA in mRNA and protein levels. Furthermore, Nanog knockdown significantly increased CRC cell sensitivity to 5-FU drug via modulating Bax and Bcl-2 mRNA expression. Also, Nanog knockdown and 5-FU treatment cooperatively decreased the migration and self-renewal ability of SW-480 cells by regulating the expression of relevant genes. Moreover, combination therapy led to cell cycle arrest at the sub-G1 phase in CRC cells. In conclusion, our results indicated that Nanog may play an important role in the drug sensitivity, migration, and self-renewal of CRC cells; suggesting Nanog as a promising target in combination with 5-FU for the development of new therapeutic approaches for CRC.

RNAi mediated silencing of Nanog expression suppresses the growth of human colorectal cancer stem cells.

Colorectal cancer (CRC) is the third most common cancer in the world known for its poor recurrence-free prognosis. Previous studies have shown that it is closely linked with cancer stem cells (CSCs), which have self-renewal potential and the capacity to differentiate into diverse populations. Nanog is an important transcription factor that functions to maintain the self-renewal and proliferation of embryonic stem cells; however, many recent studies have shown that Nanog is also highly expressed in many cancer stem cells. To investigate whether Nanog plays a crucial role in maintaining the stemness of colorectal CSCs, RNA interference was used to downregulate Nanog expression in the CRC stem cell line, EpCAM+CD44+HCT-116 cells (CCSCs). We examined the anti-tumor function of Nanog in vitro and in vivo, using small interfering RNA. Our results revealed that the Nanog mRNA expression level in CCSCs was higher than that in HCT-116 cells. We found that the depletion of Nanog inhibited proliferation and promoted apoptosis in CCSCs. In addition, the invasive ability of CCSCs was markedly restricted when Nanog was silenced by small interfering RNA. Furthermore, we found that the silencing of Nanog decreased tumor size and weight and improved the survival rate of tumor-bearing mice. In conclusion, these findings collectively demonstrate that Nanog, which is highly expressed in CRC stem cells, is a key factor in the development of tumor growth, and it may serve as a potential marker of prognosis and a novel and effective therapeutic target for the treatment of CRC.

Internal Oligoguanidinium Transporter: Mercury-Free Scalable Synthesis, Improvement of Cellular Localization, Endosomal Escape, Mitochondrial Localization, and Conjugation with Antisense Morpholino for NANOG Inhibition to Induce Chemosensitization of Taxol in MCF-7 Cells.

A nontoxic delivery vehicle is essential for the therapeutic applications of antisense phosphorodiamidate morpholino oligonucleotides (PMOs). Though guanidinium-rich or arginine-rich cellular transporter conjugated Vivo-PMO or PPMO has been developed for in vivo application, however, either their toxicity or stability has become an issue. Previously, we reported nonpeptidic internal guanidinium transporter (IGT) mediated delivery of PMO for gene silencing and got encouraging results. In this paper, we report the synthesis of IGT using a Hg-free method for scale up and N-terminal modification of IGT with a suitable hydrophobic or lipophilic group to improve the cell permeability, endosomal escape, and mitochondrial localization and to reduce toxicity in the MTT assay. For the delivery of PMO, IGT-PMO conjugate was synthesized to target NANOG in cells, a transcription factor required for cancer stem cell proliferation and embryonic development and is involved in many cancers. Our data shows IGT-PMO-facilitated NANOG inhibition, and thereby the prevention of EpCAM-N-Cadherin-Vimentin axis mediated epithelial to mesenchymal transition (EMT) in MCF-7 cells. Moreover, unlike taxol, NANOG inhibition influences the expression of stemness factor c-Myc, Hh-Gli signaling proteins, other cancer related factors, and their respective phenotypes in cancer cells. To the best of our knowledge, this is the first report to illustrate that the IGT-PMO-mediated NANOG inhibition increases the therapeutic potential of taxol and induces G0-G1 arrest in cancer cells to prevent cancer progression. However, it warrants further investigation in other cancer cells and preclinical platforms.

Modulating Pluripotency Network Genes with Omega-3 DHA is followed by Caspase- 3 Activation and Apoptosis in DNA Mismatch Repair-Deficient/KRAS-Mutant Colorectal Cancer Stem-Like Cells.

BACKGROUND: Targeting DNA mismatch repair-deficient/KRAS-mutant Colorectal Cancer Stem Cells (CRCSCs) with chemical compounds remains challenging. Modulating stemness factors Bmi-1, Sox-2, Oct-4 and Nanog in CRCSCs which are direct downstream targets of carcinogenesis pathways may lead to the reactivation of caspase-3 and apoptosis in these cells. Omega-3 DHA modulates different signaling pathways involved in carcinogenesis. However, little is known, whether in vitro concentrations of DHA equal to human plasma levels are able to modulate pluripotency genes expression, caspase-3 reactivation and apoptosis in DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells. METHODS: DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells (LS174T cells) were treated with DHA, after which, cell number and proliferation-rate, Bmi-1, Sox-2, Nanog and Oct-4 expression, caspase-3 activation and apoptosis were evaluated with different cellular and molecular techniques. RESULTS: DHA changed the morphology of cells to apoptotic forms and disrupted cell connections. After 48h treatment with 50- to 200µM DHA, cell numbers and proliferation-rates were measured to be 86%-35% and 93.6%-45.7% respectively. Treatment with 200 µM DHA dramatically decreased the expression of Bmi-1, Sox- 2, Oct-4 and Nanog by 69%, 70%, 97.5% and 53% respectively. Concurrently, DHA induced caspase-3 activation by 1.8-4.7-fold increases compared to untreated cells. An increase in the number of apoptotic cells ranging from 9.3%-38.4% was also observed with increasing DHA concentrations. CONCLUSIONS: DHA decreases the high expression level of pluripotency network genes suggesting Bmi-1, Sox-2, Oct-4 and Nanog as promising molecular targets of DHA. DHA reactivates caspase-3 and apoptosis in DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells, representing the high potential of this safe compound for therapeutic application in CRC.

NKL homeobox gene activities in normal and malignant myeloid cells.

Recently, we have documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis, which spotlights genes deregulated in lymphoid malignancies. Here, we enlarge this map to include normal NKL homeobox gene expressions in myelopoiesis by analyzing public expression profiling data and primary samples from developing and mature myeloid cells. We thus uncovered differential activities of six NKL homeobox genes, namely DLX2, HHEX, HLX, HMX1, NKX3-1 and VENTX. We further examined public expression profiling data of 251 acute myeloid leukemia (AML) and 183 myelodysplastic syndrome (MDS) patients, thereby identifying 24 deregulated genes. These results revealed frequent deregulation of NKL homeobox genes in myeloid malignancies. For detailed analysis we focused on NKL homeobox gene NANOG, which acts as a stem cell factor and is correspondingly expressed alone in hematopoietic progenitor cells. We detected aberrant expression of NANOG in a small subset of AML patients and in AML cell line NOMO-1, which served as a model. Karyotyping and genomic profiling discounted rearrangements of the NANOG locus at 12p13. But gene expression analyses of AML patients and AML cell lines after knockdown and overexpression of NANOG revealed regulators and target genes. Accordingly, NKL homeobox genes HHEX, DLX5 and DLX6, stem cell factors STAT3 and TET2, and the NOTCH-pathway were located upstream of NANOG while NKL homeobox genes HLX and VENTX, transcription factors KLF4 and MYB, and anti-apoptosis-factor MIR17HG represented target genes. In conclusion, we have extended the NKL-code to the myeloid lineage and thus identified several NKL homeobox genes deregulated in AML and MDS. These data indicate a common oncogenic role of NKL homeobox genes in both lymphoid and myeloid malignancies. For misexpressed NANOG we identified an aberrant regulatory network, which contributes to the understanding of the oncogenic activity of NKL homeobox genes.

The pluripotency transcription factor Nanog represses glutathione reductase gene expression in mouse embryonic stem cells.

OBJECTIVE: Redox homeostasis maintenance is essential to bring about cellular functions. Particularly, embryonic stem cells (ESCs) have high fidelity mechanisms for DNA repair, high activity of different antioxidant enzymes and low levels of oxidative stress. Although the expression and activity of antioxidant enzymes are reduced throughout the differentiation, the knowledge about the transcriptional regulation of genes involved in defense against oxidative stress is yet restricted. Since glutathione is a central component of a complex system involved in preserving cellular redox status, we aimed to study whether the expression of the glutathione reductase (Gsr) gene, which encodes an essential enzyme for cellular redox homeostasis, is modulated by the transcription factors critical for self-renewal and pluripotency of ESCs. RESULTS: We found that Gsr gene is expressed in ESCs during the pluripotent state and it was upregulated when these cells were induced to differentiate, concomitantly with Nanog decreased expression. Moreover, we found an increase in Gsr mRNA levels when Nanog was downregulated by a specific shRNA targeting this transcription factor in ESCs. Our results suggest that Nanog represses Gsr gene expression in ESCs, evidencing a role of this crucial pluripotency transcription factor in preservation of redox homeostasis in stem cells.

Dynamical Reorganization of Transcriptional Events Governs Robust Nanog Heterogeneity.

Nanog maintains the pluripotency of embryonic stem cells (ESCs), while demonstrating high expression heterogeneity. Intriguingly, the overall heterogeneity at the Nanog mRNA level under various culture conditions gets precisely partitioned into intrinsic and extrinsic fluctuations. However, the dynamical origin of such a robust transcriptional noise regulation still remains illusive. Herein, we propose a new stochastic simulation strategy that efficiently reconciles the strict apportioning of fluctuations observed in Nanog transcription, while predicting possible experimental scenarios to avoid such an exact noise segregation. Importantly, our model analyses reveal that different culture conditions essentially preserve the robust Nanog expression heterogeneity by altering the dynamics of transcriptional events. In the future, these insights will be useful to systematically maneuver cell-fate decision-making events of ESCs.

RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog.

Targeting cancer stem cells (CSCs) has been proposed as a new strategy to eradicate malignancies, including hepatocellular carcinoma (HCC). However, the mechanisms by which CSCs sustain their self-renewal and chemoresistance remain elusive. Nanog is a master transcriptional regulator of stemness, especially in CSCs. Its expression is tightly regulated by the ubiquitin-proteasome system in embryonic stem cells (ESCs). Whether the suppression of Nanog ubiquitination contributes to its over-expression in CSCs has not been explored. In addition, the role of receptor for activated C kinase 1 (RACK1), an adaptor protein implicated in HCC growth, in liver CSC-like traits remains to be determined. Methods: In vitro and in vivo assays were performed to investigate the role of RACK1 in liver CSC-like phenotype and murine ESC function. How RACK1 regulates Nanog expression was explored by immunoblotting and immunohistochemistry. The interaction of RACK1 with Nanog and the consequent effects on Nanog ubiquitination and stemness were then analyzed. Results: RACK1 promotes self-renewal and chemoresistance of human liver CSCs and maintains murine ESC function. Consistently, RACK1 enhances the expression of Nanog in human HCC cells and murine ESCs. The protein levels of RACK1 in clinical HCC tissues positively correlate with those of Nanog. Further exploration indicates that RACK1 directly binds to Nanog, which prevents its recruitment of E3 ubiquitin ligase FBXW8 and ubiquitin-dependent degradation. The interaction with Nanog is essential for RACK1 to promote stemness. Conclusions: Our data provide novel insights into the regulation of Nanog protein levels, as well the key role of RACK1 to enhance self-renewal and chemoresistance of CSCs in human HCC.

Inhibition of pancreatic cancer stem cell characteristics by α-Mangostin: Molecular mechanisms involving Sonic hedgehog and Nanog.

The current investigation was intended to elucidate the molecular mechanism of α-Mangostin in the regulation of pancreatic cancer stem cell (CSC) characteristics. Here, we demonstrate that α-Mangostin inhibited cell proliferation in pancreatic CSCs and cancer cell lines while it showed no effect on human pancreatic normal ductal epithelial cells. Also, α-Mangostin inhibited colony formation and induced apoptosis in these cells. Further, α-Mangostin inhibited the self-renewal capacity of CSCs isolated from human primary tumours and KrasG12D mice. Furthermore, α-Mangostin inhibited the invasive and metastatic ability of pancreatic CSCs by suppressing the epithelial-to-mesenchymal transition (EMT) via up-regulation of E-cadherin and down-regulation of mesenchymal phenotype by inhibiting N-cadherin, Snail and Slug expression. Interestingly, the pluripotency maintaining factors and CSC markers were inhibited by α-Mangostin thus suggesting that α-Mangostin can target CSCs to inhibit pancreatic cancer effectively. Gli signalling plays a crucial role in the self-renewal and pluripotency of CSCs. α-Mangostin inhibited the Gli transcription and the expression of Gli target genes (Nanog, Oct4, c-Myc, Sox-2 and KLF4) in CSCs. Using ChIP assay, we demonstrated that Nanog could directly bind to promoters of Cdk2, Cdk6, FGF4, c-Myc and α-Mangostin inhibited Nanog binding to these promoters. Conversely, the inhibitory effects of the α-Mangostin on CSC proliferation and Gli or Nanog transcription and their targets were abrogated by either enforced activation of sonic hedgehog (Shh) or by the overexpression of Nanog. Taken together, our studies suggest that α-Mangostin may act as Gli inhibitor and establishes the pre-clinical significance of α-Mangostin for the prevention and treatment of pancreatic cancer.

GATA5 inhibits hepatocellular carcinoma cells malignant behaviours by blocking expression of reprogramming genes.

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH-GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co-localization with ß-catenin in the cytoplasm, preventing ß-catenin from entering the nucleus. Treatment with the specific Wnt/ß-catenin pathway inhibitor salinomycin was able to reduce the expression of ß-catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA-GATA5 restored ß-catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of ß-catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/ß-catenin pathway and the reduction of reprogramming gene expression.

Reduction of NANOG Mediates the Inhibitory Effect of Aspirin on Tumor Growth and Stemness in Colorectal Cancer.

BACKGROUND/AIMS: Cancer stem cells (CSCs) are considered to be responsible for tumor relapse and metastasis, which serve as a potential therapeutic target for cancer. Aspirin has been shown to reduce cancer risk and mortality, particularly in colorectal cancer. However, the CSCs-suppressing effect of aspirin and its relevant mechanisms in colorectal cancer remain unclear. METHODS: CCK8 assay was employed to detect the cell viability. Sphere formation assay, colony formation assay, and ALDH1 assay were performed to identify the effects of aspirin on CSC properties. Western blotting was performed to detect the expression of the stemness factors. Xenograft model was employed to identify the anti-cancer effects of aspirin in vivo. Unpaired Student t test, ANOVA test and Kruskal-Wallis test were used for the statistical comparisons. RESULTS: Aspirin attenuated colonosphere formation and decreased the ALDH1 positive cell population of colorectal cancer cells. Aspirin inhibited xenograft tumor growth and reduced tumor cells stemness in nude mice. Consistently, aspirin decreased the protein expression of stemness-related transcription factors, including c-Myc, OCT4 and NANOG. Suppression of NANOG blocked the effect of aspirin on sphere formation. Conversely, ectopic expression of NANOG rescued the aspirin-repressed sphere formation, suggesting that NANOG is a key downstream target. Moreover, we found that aspirin repressed NANOG expression in protein level by decreasing its stability. CONCLUSION: We have provided new evidence that aspirin attenuates CSC properties through down-regulation of NANOG, suggesting aspirin as a promising therapeutic agent for colorectal cancer treatment.

Carboxyamidotriazole Synergizes with Sorafenib to Combat Non-Small Cell Lung Cancer through Inhibition of NANOG and Aggravation of Apoptosis.

Lung cancer is currently the leading cause of cancer-related deaths worldwide. In this study, we investigated the combination of carboxyamidotriazole (CAI) and sorafenib in non-small cell lung cancer (NSCLC) in vitro and in vivo to test whether CAI enhances the antitumor effects of sorafenib and reduces its side effects. The combination index (CI) showed that coadministration of CAI and sorafenib synergistically inhibited the proliferation of NSCLC cells (Lewis lung carcinoma, A549, and NCI-H1975 cells). Cell death as a result of the combination treatment was attributed to apoptosis, which was accompanied by activation of caspase-3 and poly(ADP-ribose) polymerase. In addition, combination therapy induced the accumulation of mitochondrial-associated reactive oxygen species, as well as depolarization of mitochondrial and reduced NANOG (homeobox protein NANOG) mRNA and protein expression. Basic fibroblast growth factor, a stimulator of NANOG, was applied to identify the possible mechanism. The addition of basic fibroblast growth factor followed by combined treatment may stimulate NANOG expression and synchronously rescue the accumulation of reactive oxygen species. C57BL/6J mice bearing Lewis lung carcinoma were randomized to receive vehicle (polyethylene glycol 400), CAI (30 mg/kg), low-dose sorafenib (SFB-L; 10 mg/kg), high-dose sorafenib (SFB-H; 30 mg/kg), or a CAI and SFB-L combination. Tumor growth was significantly suppressed in the combination group, and the efficacy of combination treatment was equivalent to that of the SFB-H monotherapy group. Furthermore, the combination group had reduced side effects compared with the SFB-H group, as indicated by weight preservation in mice. Our study illustrates that CAI enhances the antitumor activity of sorafenib in NSCLC and provides a novel strategy for NSCLC treatment.

Improvement of osteogenesis in dental pulp pluripotent-like stem cells by oligopeptide-modified poly(ß-amino ester)s.

Controlling pluripotent stem cell differentiation via genetic manipulation is a promising technique in regenerative medicine. However, the lack of safe and efficient delivery vehicles limits this application. Recently, a new family of poly(ß-amino ester)s (pBAEs) with oligopeptide-modified termini showing high transfection efficiency of both siRNA and DNA plasmid has been developed. In this study, oligopeptide-modified pBAEs were used to simultaneously deliver anti-OCT3/4 siRNA, anti-NANOG siRNA, and RUNX2 plasmid to cells from the dental pulp with pluripotent-like characteristics (DPPSC) in order to promote their osteogenic differentiation. Results indicate that transient inhibition of the pluripotency marker OCT3/4 and the overexpression of RUNX2 at day 7 of differentiation markedly increased and accelerated the expression of osteogenic markers. Furthermore, terminally-differentiated cells exhibited higher matrix mineralization and alkaline phosphatase activity. Finally, cell viability and genetic stability assays indicate that this co-delivery system has high chromosomal stability and minimal cytotoxicity. Therefore, we conclude that such co-delivery strategy is a safe and a quick option for the improvement of DPPSC osteogenic differentiation. STATEMENT OF SIGNIFICANCE: Controlling pluripotent stem cell differentiation via genetic manipulation is a promising technique in regenerative medicine. However, the lack of safe and efficient delivery vehicles limits this application. In this study, we propose the use of a new family of oligopeptide-modified pBAEs developed in our group to control the differentiation of dental pulp pluripotential stem cells (DPPSC). In order to promote their osteogenic differentiation. The strategy proposed markedly increased and accelerated the expression of osteogenic markers, cell mineralization and alkaline phosphatase activity. Finally, cell viability and genetic stability assays indicated that this co-delivery system has high chromosomal stability and minimal cytotoxicity. These findings open a new interesting path in the usage of non-viral gene delivery systems for the control of pluripotential stem cell differentiation.

CDK1-PDK1-PI3K/Akt signaling pathway regulates embryonic and induced pluripotency.

The mechanisms of how signaling pathways are coordinated and integrated for the maintenance of the self-renewal of human embryonic stem cells (hESCs) and the acquisition of pluripotency in reprogramming are still only partly understood. CDK1 is a key regulator of mitosis. Recently, CDK1 has been shown to be involved in regulating self-renewal of stem cells, even though the mechanistic role of how CDK1 regulates pluripotency is unknown. In this report, we aim to understand how CDK1 can control pluripotency by reducing CDK1 activity to a level that has no effect on cell cycle progression. We demonstrated that high levels of CDK1 is associated with the pluripotency stage of hESCs; and decreased CDK1 activity to a level without perturbing the cell cycle is sufficient to induce differentiation. CDK1 specifically targets the phosphorylation of PDK1 and consequently the activity of PI3K/Akt and its effectors ERK and GSK3ß. Evidence of the reversion of inactive CDK1-mediated differentiation by the inhibition of Akt signaling effectors suggests that the CDK1-PDK1-PI3K/Akt kinase cascade is a functional signaling pathway for the pluripotency of hESCs. Moreover, cyclin B1-CDK1 complexes promote somatic reprogramming efficiency, probably by regulating the maturation of induced pluripotent stem cells (iPSCs), as cyclin B1 stimulates a higher cellular level of LIN28A, suggesting that monitoring iPSC factors could be a new path for the enhancement of reprogramming efficiency. Together, we demonstrate an essential role for the CDK1-PDK1-PI3K/Akt kinase signaling pathway in the regulation of self-renewal, differentiation, and somatic reprogramming, which provides a novel kinase cascade mechanism for pluripotency control and acquisition.

CHD7, Oct3/4, Sox2, and Nanog control FoxD3 expression during mouse neural crest-derived stem cell formation.

Neural crest-derived stem cells (NCSCs) are tissue-specific stem cells derived from multipotent neural crest cells. NCSCs are present in some adult tissues such as dorsal root ganglia, sciatic nerve, and bone marrow. However, little is known about the formation mechanisms of these cells. We have shown that BMP2/Wnt3a signaling and a chromatin remodeler, CHD7, in mice help to maintain the multipotency of neural crest cells and lead to the formation of NCSCs. In the present study, we analyzed a regulatory gene cascade in the formation of mouse NCSCs. The inhibition of FoxD3 expression significantly suppressed the expression of Sox10, which is an indispensable transcription factor for mouse NCSC formation, in the presence of BMP2/Wnt3a. CHD7, Oct3/4, Sox2, and Nanog occupied multiple conserved regions of mouse FoxD3, mE1, mE2, and mE3, in a BMP2/Wnt3a-dependent manner. Furthermore, siRNA of CHD7, Oct3/4, Sox2, and Nanog significantly suppressed FoxD3 expression. The inhibition of histone H3K4 mono- or trimethylation also repressed FoxD3 expression. The present data suggest that CHD7, Oct3/4, Sox2, and Nanog directly induce FoxD3 expression when stimulated by BMP2/Wnt3a signaling, that FoxD3 promotes Sox10 expression, and that histone H3K4 methylation plays important roles in this process of mouse NCSC formation.

AT101 exerts a synergetic efficacy in gastric cancer patients with 5-FU based treatment through promoting apoptosis and autophagy.

Gastric cancer remains a disease with a high mortality rate despite of multiple therapeutic strategies. So far, it is very important to develop new treatment approaches to improve current therapeutic efficacy in gastric cancer. Apurinic/apyrimidinic endonuclease (APE1) involves in DNA base excision repair (BER) during DNA damage pathway. APE1 was found to be associated with poor overall survival with gastric cancer patients. In the in vitro experiment, we tested APE1 inhibitor-AT101 could potently inhibit gastric cancer cell growth and further induce cancer cell apoptosis and autophagy through p53-dependent pathway. Downregulation of APE1 by AT101 has ability to suppress gastric cancer cell migration and renewal through inhibition of CD133, Nanog and LC3expression. Based on findings that Her-2 positive expression cases has poor prognosis from our dataset and TCGA database, we investigated the role of AT101 in synergetic efficacy with 5-FU treatment in Her-2 overexpression gastric cancer in vivo, indicating that AT101 is able to enhance 5-FU in the shrinkage of xenograft mice tumor and induction of cell apoptosis. In summary, the data obtained from our study showed APE1 is guided as a potential therapeutic target for gastric cancer. AT101 could be regarded as a potent inhibitor to promote chemotherapeutic sensitivity in patients with gastric cancer.