Oxyberberine sensitizes liver cancer cells to sorafenib via inhibiting NOTCH1-USP7-c-Myc pathway.

BACKGROUND: Sorafenib is the first-line therapy for patients with advanced-stage HCC, but its clinical cure rate is unsatisfactory due to adverse reactions and drug resistance. Novel alternative strategies to overcome sorafenib resistance are urgently needed. Oxyberberine (OBB), a major metabolite of berberine in vivo, exhibits potential antitumor potency in various human malignancies, including liver cancer. However, it remains unknown whether and how OBB sensitizes liver cancer cells to sorafenib. METHODS: Cell viability, trypan blue staining and flow cytometry assays were employed to determine the synergistic effect of OBB and sorafenib on killing HCC cells. PCR, western blot, co-immunoprecipitation and RNA interference assays were used to decipher the mechanism by which OBB sensitizes sorafenib. HCC xenograft models and clinical HCC samples were utilized to consolidate our findings. RESULTS: We found for the first time that OBB sensitized liver cancer cells to sorafenib, enhancing its inhibitory effect on cell growth and induction of apoptosis in vitro. Interestingly, we observed that OBB enhanced the sensitivity of HCC cells to sorafenib by reducing ubiquitin-specific peptidase 7 (USP7) expression, a well-known tumor-promoting gene. Mechanistically, OBB inhibited notch homolog 1-mediated USP7 transcription, leading to the downregulation of V-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), which synergized with sorafenib to suppress liver cancer. Furthermore, animal results showed that cotreatment with OBB and sorafenib significantly inhibited the tumor growth of liver cancer xenografts in mice. CONCLUSIONS: These results indicate that OBB enhances the sensitivity of liver cancer cells to sorafenib through inhibiting notch homolog 1-USP7-c-Myc signaling pathway, which potentially provides a novel therapeutic strategy for liver cancer to improve the effectiveness of sorafenib.

Jiedu-Quyu-Ziyin Fang (JQZF) inhibits the proliferation and activation of B cells in MRL/lpr mice via modulating the AKT/mTOR/c-Myc signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiedu-Quyu-Ziyin Fang (JQZF) is a new herbal formula improved based on "Sheng Ma Bie Jia Tang" in the Golden Chamber, has been proved to be effective in the treatment of SLE. The ability of JQZF to prevent lymphocyte growth and survival has been demonstrated in earlier investigations. However, the specific mechanism of JQZF on SLE has not been fully investigated. AIM OF THE STUDY: To reveal the potential mechanisms of JQZF inhibiting B cell proliferation and activation in MRL/lpr mice. MATERIALS AND METHODS: MRL/lpr mice were treated with low-dose, high-dose JQZF and normal saline for 6 weeks. The effect of JQZF on disease improvement in MRL/lpr mice was studied using enzyme-linked immunosorbent assay (ELISA), histopathological staining, serum biochemical parameters and urinary protein levels. The changes of B lymphocyte subsets in the spleen were analyzed by flow cytometry. The contents of ATP and PA in B lymphocytes from the spleens of mice were determined by ATP content assay kit and PA assay kit. Raji cells (a B lymphocyte line) were selected as the cell model in vitro. The effects of JQZF on the proliferation and apoptosis of B cells were detected by flow cytometry and CCK8. The effect of JQZF on the AKT/mTOR/c-Myc signaling pathway in B cells were detected via western blot. RESULTS: JQZF, especially at high dose, significantly improved the disease development of MRL/lpr mice. Flow cytometry results showed that JQZF affected the proliferation and activation of B cells. In addition, JQZF inhibited the production of ATP and PA in B lymphocytes. In vitro cell experiments further confirmed that JQZF can inhibit Raji proliferation and promote cell apoptosis through AKT/mTOR/c-Myc signaling pathway. CONCLUSION: JQZF may affect the proliferation and activation of B cells by inhibiting the AKT/mTOR/c-Myc signaling pathway.

Protectin DX as a therapeutic strategy against frailty in mice.

Frailty in aging is driven by the dysregulation of multiple biological pathways. Protectin DX (PDX) is a docosahexaenoic acid (DHA)-derived molecule that alleviates many chronic inflammatory disorders, but its potential effects on frailty remain unknown. Our goal is to identify age-related impairments in metabolic systems and to evaluate the therapeutic potential of PDX on frailty, physical performance, and health parameters. A set of 22-month-old C57BL/6 male and female mice were assigned to vehicle (Old) or PDX daily gavage treatment for 9 weeks, whereas 6-month-old (Adult) mice received only vehicle. Forelimb and hindlimb strength, endurance, voluntary wheel activity and walking speed determined physical performance and were combined with a frailty index score and body weight loss to determine frailty status. Our data shows that old vehicle-treated mice from both sexes had body weight loss paralleling visceromegaly, and Old females also had impaired insulin clearance as compared to the Adult group. Aging was associated with physical performance decline together with higher odds of frailty development. There was also age-driven mesangial expansion and glomerular hypertrophy as well as bone mineral density loss. All of the in vivo and in vitro impairments observed with aging co-occurred with upregulation of inflammatory pathways and Myc signaling as well as downregulation of genes related to adipogenesis and oxidative phosphorylation in liver. PDX attenuated the age-driven physical performance (strength, exhaustion, walking speed) decline, promoted robustness, prevented bone losses and partially reversed changes in hepatic expression of Myc targets and metabolic genes. In conclusion, our data provides evidence of the beneficial therapeutic effect of PDX against features of frailty in mice. Further studies are warranted to investigate the mechanisms of action and the potential for human translation.

Sorafenib decreases glycemia by impairing hepatic glucose metabolism.

PURPOSE: Sorafenib has been reported to reduce blood glucose levels in diabetic and non-diabetic patients in previous retrospective studies. However, the mechanism of which the hypoglycemic effects of sorafenib is not clearly explored. In this study, we investigated the effect of sorafenib on blood glucose levels in diabetic and normal mice and explored the possible mechanism. METHODS: We established a mouse model of type 2 diabetes by a high-fat diet combined with a low-dose of streptozotocin (STZ), to identify the hypoglycemic effect of sorafenib in different mice. Glucose tolerance, insulin tolerance and pyruvate tolerance tests were done after daily gavage with sorafenib to diabetic and control mice. To explore the molecular mechanism by which sorafenib regulates blood glucose levels, hepatic glucose metabolism signaling was studied by a series of in vivo and in vitro experiments. RESULTS: Sorafenib reduced blood glucose levels in both control and diabetic mice, particularly in the latter. The diabetic mice exhibited improved glucose and insulin tolerance after sorafenib treatment. Further studies showed that the expressions of gluconeogenesis-related enzymes, such as PCK1, G6PC and PCB, were significantly decreased upon sorafenib treatment. Mechanistically, sorafenib downregulates the expression of c-MYC downstream targets PCK1, G6PC and PCB through blocking the ERK/c-MYC signaling pathway, thereby playing its hypoglycemic effect by impairing hepatic glucose metabolism. CONCLUSION: Sorafenib reduces blood glucose levels through downregulating gluconeogenic genes, especially in diabetic mice, suggesting the patients with T2DM when treated with sorafenib need more emphasis in monitoring blood glucose to avoid unnecessary hypoglycemia.

Carboxylesterase 2 induces mitochondrial dysfunction via disrupting lipid homeostasis in oral squamous cell carcinoma.

OBJECTIVE: Oral squamous cell carcinoma (OSCC) is characterized by high recurrence and metastasis and places a heavy burden on societies worldwide. Cancer cells thrive in a changing microenvironment by reprogramming lipidomic metabolic processes to provide nutrients and energy, activate oncogenic signaling pathways, and manage redox homeostasis to avoid lipotoxicity. The mechanism by which OSCC cells maintain lipid homeostasis during malignant progression is unclear. METHODS: The altered expression of fatty acid (FA) metabolism genes in OSCC, compared with that in normal tissues, and in OSCC patients with or without recurrence or metastasis were determined using public data from the TCGA and GEO databases. Immunohistochemistry was performed to examine the carboxylesterase 2 (CES2) protein level in our own cohort. CCK-8 and Transwell assays and an in vivo xenograft model were used to evaluate the biological functions of CES2. Mass spectrometry and RNA sequencing were performed to determine the lipidome and transcriptome alterations induced by CES2. Mitochondrial mass, mtDNA content, mitochondrial membrane potential, ROS levels, and oxygen consumption and apoptosis rates were evaluated to determine the effects of CES2 on mitochondrial function in OSCC. RESULTS: CES2 was downregulated in OSCC patients, especially those with recurrence or metastasis. CES2high OSCC patients showed better overall survival than CES2low OSCC patients. Restoring CES2 expression reduced OSCC cell viability and suppressed their migration and invasion in vitro, and it inhibited OSCC tumor growth in vivo. CES2 reprogrammed lipid metabolism in OSCC cells by hydrolyzing neutral lipid diacylglycerols (DGs) to release free fatty acids and reduce the membrane structure lipid phospholipids (PLs) synthesis. Free FAs were converted to acyl-carnitines (CARs) and transferred to mitochondria for oxidation, which induced reactive oxygen species (ROS) accumulation, mitochondrial damage, and apoptosis activation. Furthermore, the reduction in signaling lipids, e.g., DGs, PLs and substrates, suppressed PI3K/AKT/MYC signaling pathways. Restoring MYC rescued the diminished cell viability, suppressed migratory and invasive abilities, damaged mitochondria and reduced apoptosis rate induced by CES2. CONCLUSIONS: We demonstrated that CES2 downregulation plays an important role in OSCC by maintaining lipid homeostasis and reducing lipotoxicity during tumor progression and may provide a potential therapeutic target for OSCC.

Ginsenoside Rk1 regulates glutamine metabolism in hepatocellular carcinoma through inhibition of the ERK/c-Myc pathway.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers in the world. Recently, suppression of glutamine metabolism has become one of the hottest therapy targets for cancer treatment. There is a growing amount of research that indicates that ginsenosides possess good anti-tumor activity. However, the effect of ginsenoside Rk1 on glutamine metabolism in HCC is unclear. In this study, Rk1 was demonstrated to be effective at inhibiting the proliferation of HCC through the induction of cell cycle arrest and apoptosis. Especially, Rk1 was shown for the first time to inhibit glutamine metabolism in HCC. Rk1 downregulates GLS1 expression, and consequently decreases the GSH production, stimulating ROS accumulation to induce apoptosis. In addition, transcriptomic results showed that the ERK/c-Myc signaling pathway was enriched in HepG2. Rk1 exerts an inhibitory effect on glutamine metabolism in HCC by regulating the ERK/c-Myc signaling pathway, and inducing apoptosis in vitro and in vivo with less toxicity. Therefore, ginsenoside Rk1 could be a promising candidate for the clinical treatment of HCC.

Efficacy of pH-Sensitive Nanomedicines in Tumors with Different c-MYC Expression Depends on the Intratumoral Activation Profile.

Effective inhibition of the protein derived from cellular myelocytomatosis oncogene (c-Myc) is one of the most sought-after goals in cancer therapy. While several c-Myc inhibitors have demonstrated therapeutic potential, inhibiting c-Myc has proven challenging, since c-Myc is essential for normal tissues and tumors may present heterogeneous c-Myc levels demanding contrasting therapeutic strategies. Herein, we developed tumor-targeted nanomedicines capable of treating both tumors with high and low c-Myc levels by adjusting their ability to spatiotemporally control drug action. These nanomedicines loaded homologues of the bromodomain and extraterminal (BET) motif inhibitor JQ1 as epigenetic c-Myc inhibitors through pH-cleavable bonds engineered for fast or slow drug release at intratumoral pH. In tumors with high c-Myc expression, the fast-releasing (FR) nanomedicines suppressed tumor growth more effectively than the slow-releasing (SR) ones, whereas, in the low c-Myc tumors, the efficacy of the nanomedicines was the opposite. By studying the tumor distribution and intratumoral activation of the nanomedicines, we found that, despite SR nanomedicines achieved higher accumulation than the FR counterparts in both c-Myc high and low tumors, the antitumor activity profiles corresponded with the availability of activated drugs inside the tumors. These results indicate the potential of engineered nanomedicines for c-Myc inhibition and spur the idea of precision pH-sensitive nanomedicine based on cancer biomarker levels.

Potent antitumor effects of cell-penetrating peptides targeting STAT3 axis.

To date, there are no inhibitors that directly and specifically target activated STAT3 and c-Myc in the clinic. Although peptide-based inhibitors can selectively block activated targets, their clinical usage is limited because of low cell penetration and/or serum stability. Here, we generated cell-penetrating acetylated (acet.) STAT3, c-Myc, and Gp130 targeting peptides by attaching phosphorothioated (PS) polymer backbone to peptides. The cell-penetrating peptides efficiently penetrated cells and inhibited activation of the intended targets and their downstream genes. Locally or systemically treating tumor-bearing mice with PS-acet.-STAT3 peptide at low concentrations effectively blocked STAT3 in vivo, resulting in significant antitumor effects in 2 human xenograft models. Moreover, PS-acet.-STAT3 peptide penetrated and activated splenic CD8+ T cells in vitro. Treating immune-competent mice bearing mouse melanoma with PS-acet.-STAT3 peptide inhibited STAT3 in tumor-infiltrating T cells, downregulating tumor-infiltrating CD4+ T regulatory cells while activating CD8+ T effector cells. Similarly, systemic injections of the cell-penetrating c-Myc and Gp130 peptides prevented pancreatic tumor growth and induced antitumor immune responses. Taken together, we have developed therapeutic peptides that effectively and specifically block challenging cancer targets, resulting in antitumor effects through both direct tumor cell killing and indirectly through antitumor immune responses.

c-Myc Inhibition Using 10058-F4 Increased the Sensitivity of Acute Promyelocytic Leukemia Cells to Arsenic Trioxide Via Blunting PI3K/NF-κB Axis.

BACKGROUNDS: Although ATO is widely used to treat acute promelocytic leukemia (APL), the appropriate effects of the drug as a single agent are achieved in high doses which are not clinically achievable without the risk of side effects; highlighting the necessity of its application in a combined-modality. Herein, we aimed to investigate whether c-Myc inhibition could reinforce the anti-leukemic effect of ATO, while reducing its concentration in APL cells. METHODS: NB4 cells were treated with the relevant concentrations of 10058-F4 (c-Myc inhibitor) and ATO, and then the survival of the cells was evaluated using trypan blue, MTT and BrdU assays. Moreover, the mechanism of action of the agents were evaluated using Flow cytometry, qRT-PCR and western blot analysis. RESULTS: We found that the inhibition of c-Myc using 10058-F4 could enhance the anti-leukemic effect of ATO in APL cells through reducing the phosphorylation of IκB, decreasing the expression of the anti-apoptotic genes and in turn, inducing a caspase-3-dependent apoptotic cell death. Moreover, the combination of 10058-F4 and ATO abrogated the activation of the PI3K pathway, while neither agent had significant suppressive impact on this pathway; suggesting for the first time that probably the companionship of c-Myc inhibitor may be an appealing strategy for shifting the resistance condition toward a chemo-sensitive phenotype, without the necessity to elevate the effective dose of ATO. CONCLUSION: Given the efficacy of 10058-F4 in adjuvanting approaches, we suggest this small molecule inhibitor as an impressing agent to be used alongside ATO in the treatment of APL.

Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene.

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

Intrinsic cell-penetrating activity propels Omomyc from proof of concept to viable anti-MYC therapy.

Inhibiting MYC has long been considered unfeasible, although its key role in human cancers makes it a desirable target for therapeutic intervention. One reason for its perceived undruggability was the fear of catastrophic side effects in normal tissues. However, we previously designed a dominant-negative form of MYC called Omomyc and used its conditional transgenic expression to inhibit MYC function both in vitro and in vivo. MYC inhibition by Omomyc exerted a potent therapeutic impact in various mouse models of cancer, causing only mild, well-tolerated, and reversible side effects. Nevertheless, Omomyc has been so far considered only a proof of principle. In contrast with that preconceived notion, here, we show that the purified Omomyc mini-protein itself spontaneously penetrates into cancer cells and effectively interferes with MYC transcriptional activity therein. Efficacy of the Omomyc mini-protein in various experimental models of non-small cell lung cancer harboring different oncogenic mutation profiles establishes its therapeutic potential after both direct tissue delivery and systemic administration, providing evidence that the Omomyc mini-protein is an effective MYC inhibitor worthy of clinical development.

Functional Odontoblastic-Like Cells Derived from Human iPSCs.

The induced pluripotent stem cells (iPSCs) have an intrinsic capability for indefinite self-renewal and large-scale expansion and can differentiate into all types of cells. Here, we tested the potential of iPSCs from dental pulp stem cells (DPSCs) to differentiate into functional odontoblasts. DPSCs were reprogrammed into iPSCs via electroporation of reprogramming factors OCT-4, SOX2, KLF4, LIN28, and L-MYC. The iPSCs presented overexpression of the reprogramming genes and high protein expressions of alkaline phosphatase, OCT4, and TRA-1-60 in vitro and generated tissues from 3 germ layers in vivo. Dentin discs with poly-L-lactic acid scaffolds containing iPSCs were implanted subcutaneously into immunodeficient mice. After 28 d from implantation, the iPSCs generated a pulp-like tissue with the presence of tubular dentin in vivo. The differentiation potential after long-term expansion was assessed in vitro. iPSCs and DPSCs of passages 4 and 14 were treated with either odontogenic medium or extract of bioactive cement for 28 d. Regardless of the passage tested, iPSCs expressed putative markers of odontoblastic differentiation and kept the same mineralization potential, while DPSC P14 failed to do the same. Analysis of these data collectively demonstrates that human iPSCs can be a source to derive human odontoblasts for dental pulp research and test bioactivity of materials.

c-MYC inhibition impairs hypoxia response in glioblastoma multiforme.

The c-MYC oncoprotein is a DNA binding transcription factor that enhances the expression of many active genes. c-MYC transcriptional signatures vary according to the transcriptional program defined in each cell type during differentiation. Little is known on the involvement of c-MYC in regulation of gene expression programs that are induced by extracellular cues such as a changing microenvironment. Here we demonstrate that inhibition of c-MYC in glioblastoma multiforme cells blunts hypoxia-dependent glycolytic reprogramming and mitochondria fragmentation in hypoxia. This happens because c-MYC inhibition alters the cell transcriptional response to hypoxia and finely tunes the expression of a subset of Hypoxia Inducible Factor 1-regulated genes. We also show that genes whose expression in hypoxia is affected by c-MYC inhibition are able to distinguish the Proneural subtype of glioblastoma multiforme, thus potentially providing a molecular signature for this class of tumors that are the least tractable among glioblastomas.

Inhibidores BET. El singular compuesto (+)-JQ1 / BET inhibitors. The odd compound (+)-JQ1

El descubrimiento fortuito de los inhibidores BET (bromodomain extra-terminal), y en especial de los inhibidores del bromodominio BRD4, ha permitido modular con fármacos epigenéticos la transcripción del protooncogén c-MY C, entre otros genes implicados en la carcinogénesis y otras disfunciones. Estos fármacos, entre los que se encuentra el compuesto (+)-JQ1, han mostrado su potencial como anticancerosos, inmunomoduladores, anticonceptivos masculinos, antivirales, o agentes preventivos del fallo cardíaco. En esta revisión se analiza su descubrimiento, estructura, mecanismo de acción y sus perspectivas futuras

The serendipitous discovery of (bromodomain extra-terminal) (BET) inhibitors, especially of the BRD4 bromodomain inhibitors, has allowed modulation of gene transcription with epigenetic drugs. This is the case of protooncogen c- MY C and other genes involved in carcinogenesis and different disfunctions. These drugs, that include the compound (+)-JQ1, have shown their potencial as anticancer agents, immunomodulators, male contraceptives, antiviral, or heart failure chemopreventive agents, among others. In this review we analyze their discovery, structure, mechanism of action and future perspectives

Expansion of human and murine hematopoietic stem and progenitor cells ex vivo without genetic modification using MYC and Bcl-2 fusion proteins.

The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.

Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.

Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice.

The principal reason for failure of targeted cancer therapies is the emergence of resistant clones that regenerate the tumor. Therapeutic efficacy therefore depends on not only how effectively a drug inhibits its target, but also the innate or adaptive functional redundancy of that target and its attendant pathway. In this regard, the Myc transcription factors are intriguing therapeutic targets because they serve the unique and irreplaceable role of coordinating expression of the many diverse genes that, together, are required for somatic cell proliferation. Furthermore, Myc expression is deregulated in most-perhaps all-cancers, underscoring its irreplaceable role in proliferation. We previously showed in a preclinical mouse model of non-small-cell lung cancer that systemic Myc inhibition using the dominant-negative Myc mutant Omomyc exerts a dramatic therapeutic impact, triggering rapid regression of tumors with only mild and fully reversible side effects. Using protracted episodic expression of Omomyc, we now demonstrate that metronomic Myc inhibition not only contains Ras-driven lung tumors indefinitely, but also leads to their progressive eradication. Hence, Myc does indeed serve a unique and nondegenerate role in lung tumor maintenance that cannot be complemented by any adaptive mechanism, even in the most aggressive p53-deficient tumors. These data endorse Myc as a compelling cancer drug target.

MicroRNA-302 increases reprogramming efficiency via repression of NR2F2.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.

Differentiation of reprogrammed mouse cardiac fibroblasts into functional cardiomyocytes.

Fibroblasts can be reprogrammed by ectopic expression of reprogramming factors to yield induced pluripotent stem (iPS) cells that are capable of transdifferentiating into diverse types of somatic cell lines. In this study, we examined if functional cardiomyocytes (CMs) can be produced from mouse cardiac fibroblasts (CFs), using iPS cell factor-based reprogramming. CFs were isolated from Oct4-GFP-C57 mice and infected with a retrovirus expressing the Yamanaka reprogramming factors, Oct4, Sox2, Klf4, and c-Myc to reprogram the CFs into a CF-iPS cell line. Primary mouse embryonic fibroblast cells (MEFs) were used as a control. We found that the dedifferentiated CF-iPS cells showed similar biological characteristics (morphology, pluripotent factor expression, and methylation level) as embryonic stem cells (ESs) and MEF-iPS cells. We used the classical embryoid bodies (EBs)-based method and a transwell CM co-culture system to simulate the myocardial paracrine microenvironment for performing CF-iPS cell cardiogenic differentiation. Under this simulated myocardial microenvironment, CF-iPS cells formed spontaneously beating EBs. The transdifferentiated self-beating cells expressed cardiac-specific transcription and structural factors and also displayed typical myocardial morphology and electrophysiological characteristics. CFs can be dedifferentiated into iPS cells and further transdifferentiated into CMs. CFs hold great promise for CM regeneration as an autologous cell source for functional CM in situ without the need for exogenous cell transplantation in ischemic heart disease.

Generation of induced pluripotent stem cells from human Tenon's capsule fibroblasts.

PURPOSE: This study aimed to develop a feasible and efficient method for generating embryonic stem cell (ESC)-like induced pluripotent stem (iPS) cells from human Tenon's capsule fibroblasts (HTFs) through the expression of a defined set of transcription factors, which will have significant application value for ophthalmic personalized regenerative medicine. METHODS: HTFs were harvested from fresh samples, and reprogramming was induced by the exogenous expression of the four classic transcription factors, OCT-3/4, SOX-2, KLF-4, and C-MYC. The HTF-derived iPS (TiPS) cells were analyzed with phase contrast microscopy, real-time PCR, immunofluorescence, FACS analysis, alkaline phosphatase activity analysis, and a teratoma formation assay. Human ESC colonies were used as the positive control. RESULTS: The resulting HTF-derived iPS cell colonies were indistinguishable from human ESC colonies regarding morphology, gene expression levels, pluripotent gene expression, alkaline phosphatase activity, and the ability to generate all three embryonic germ layers. CONCLUSIONS: This study presents a simple, efficient, practical procedure for generating patient-tailored iPS cells from HTFs. These cells will serve as a valuable and preferred candidate donor cell population for ophthalmological regenerative medicine.