Prostaglandin E1 Inhibits GLI2 Amplification-Associated Activation of the Hedgehog Pathway and Drug Refractory Tumor Growth.

Aberrant activation of the Hedgehog (HH) signaling pathway underlines the initiation and progression of a multitude of cancers. The effectiveness of the leading drugs vismodegib (GDC-0449) and sonidegib (LDE225), both Smoothened (SMO) antagonists, is compromised by acquisition of mutations that alter pathway components, notably secondary mutations in SMO and amplification of GLI2, a transcriptional mediator at the end of the pathway. Pharmacologic blockade of GLI2 activity could ultimately overcome these diversified refractory mechanisms, which would also be effective in a broader spectrum of primary tumors than current SMO antagonists. To this end, we conducted a high-content screening directly analyzing the ciliary translocation of GLI2, a key event for GLI2 activation in HH signal transduction. Several prostaglandin compounds were shown to inhibit accumulation of GLI2 within the primary cilium (PC). In particular, prostaglandin E1 (PGE1), an FDA-approved drug, is a potent GLI2 antagonist that overcame resistance mechanisms of both SMO mutagenesis and GLI2 amplification. Consistent with a role in HH pathway regulation, EP4 receptor localized to the PC. Mechanistically, PGE1 inhibited HH signaling through the EP4 receptor, enhancing cAMP-PKA activity, which promoted phosphorylation and degradation of GLI2 via the ubiquitination pathway. PGE1 also effectively inhibited the growth of drug refractory human medulloblastoma xenografts. Together, these results identify PGE1 and other prostaglandins as potential templates for complementary therapeutic development to circumvent resistance to current generation SMO antagonists in use in the clinic. SIGNIFICANCE: These findings show that PGE1 exhibits pan-inhibition against multiple drug refractory activities for Hedgehog-targeted therapies and elicits significant antitumor effects in xenograft models of drug refractory human medulloblastoma mimicking GLI2 amplification.

miR-6539 is a novel mediator of somatic cell reprogramming that represses the translation of Dnmt3b.

Global DNA hypomethylation has been shown to be involved in the pluripotency of induced pluripotent stem (iPS) cells. Relatedly, DNA methyltransferases (DNMTs) are believed to be a substantial barrier to genome-wide demethylation. There are two distinct stages of DNMT expression during iPS cell generation. In the earlier stage of reprogramming, the expression of DNMTs is repressed to overcome epigenetic barriers. During the late stage, the expression of DNMTs is upregulated to ensure iPS cells obtain the full pluripotency required for further development. This fact is strongly reminiscent of microRNAs (miRNAs), critical regulators of precise gene expression, may be central to coordinate the expression of DNMTs during reprogramming. Using a secondary inducible system, we found that miR-6539 had a unique expression dynamic during iPS cell generation that inversely correlated with DNMT3B protein levels. Enforced upregulation of miR-6539 during the early stage of reprogramming increased the efficiency of iPS cell generation, while enforced downregulation impaired efficiency. Further analysis showed that Dnmt3b mRNA is the likely target of miR-6539. Notably, miR-6539 repressed Dnmt3b translation via a target site located in the coding sequence. Our study has therefore identified miR-6539 as a novel mediator of somatic cell reprogramming and, to the best of our knowledge, is the first to demonstrate miRNA-mediated translation inhibition in somatic cell reprogramming via targeting the coding sequence. Our study contributes to understand the mechanisms that underlie the miRNA-mediated epigenetic remodeling that occurs during somatic cell reprogramming.

Hedgehog Signaling: From Basic Biology to Cancer Therapy.

The Hedgehog (HH) signaling pathway was discovered originally as a key pathway in embryonic patterning and development. Since its discovery, it has become increasingly clear that the HH pathway also plays important roles in a multitude of cancers. Therefore, HH signaling has emerged as a therapeutic target of interest for cancer therapy. In this review, we provide a brief overview of HH signaling and the key molecular players involved and offer an up-to-date summary of our current knowledge of endogenous and exogenous small molecules that modulate HH signaling. We discuss experiences and lessons learned from the decades-long efforts toward the development of cancer therapies targeting the HH pathway. Challenges to develop next-generation cancer therapies are highlighted.

Generation of fully pluripotent female murine-induced pluripotent stem cells.

The high quality of induced pluripotent stem cells (iPSCs) has been determined to be high-grade chimeras that are competent for germline transmission, and viable mice can be generated through tetraploid complementation. Most of the high-quality iPSCs described to date have been male. Female iPSCs, especially fully pluripotent female iPSCs, are also essential for clinical applications and scientific research. Here, we show, for the first time, that a gender-mixed induction strategy could lead to a skewed sex ratio of iPSCs. After reprogramming, 50%, 70%, and 90% female initiating mouse embryonic fibroblasts at different male ratios resulted in 14.1 ± 6.8% (P < 0.05), 31.8 ± 5.4% (P < 0.05), and 80.1 ± 2.8% (P < 0.05) female iPSCs, respectively. Furthermore, these female iPSCs had pluripotent properties typical of embryonic stem cells. Importantly, these fully pluripotent female iPSCs could generate viable mice by tetraploid complementation. These findings indicate that high-quality female iPSCs could be derived effectively, and suggest that clinical application of female iPSCs is feasible.

Facilitated ignition in turbulence through differential diffusion.

Contrary to the belief that ignition of a combustible mixture by a high-energy kernel is more difficult in turbulence than in quiescence because of the increased dissipation rate of the deposited energy, we experimentally demonstrate that it can actually be facilitated by turbulence for mixtures whose thermal diffusivity sufficiently exceeds its mass diffusivity. In such cases, turbulence breaks the otherwise single spherical flame of positive curvature, and hence positive aerodynamics stretch, into a multitude of wrinkled flamelets subjected to either positive or negative stretch, such that the intensified burning of the latter constitutes local sources to facilitate ignition.

Scaling of turbulent flame speed for expanding flames with Markstein diffusion considerations.

In this paper we clarify the role of Markstein diffusivity, which is the product of the planar laminar flame speed and the Markstein length, on the turbulent flame speed and its scaling, based on experimental measurements on constant-pressure expanding turbulent flames. Turbulent flame propagation data are presented for premixed flames of mixtures of hydrogen, methane, ethylene, n-butane, and dimethyl ether with air, in near-isotropic turbulence in a dual-chamber, fan-stirred vessel. For each individual fuel-air mixture presented in this work and the recently published iso-octane data from Leeds, normalized turbulent flame speed data of individual fuel-air mixtures approximately follow a Re_{T,f}^{0.5} scaling, for which the average radius is the length scale and thermal diffusivity is the transport property of the turbulence Reynolds number. At a given Re_{T,f}^{}, it is experimentally observed that the normalized turbulent flame speed decreases with increasing Markstein number, which could be explained by considering Markstein diffusivity as the leading dissipation mechanism for the large wave number flame surface fluctuations. Consequently, by replacing thermal diffusivity with the Markstein diffusivity in the turbulence Reynolds number definition above, it is found that normalized turbulent flame speeds could be scaled by Re_{T,M}^{0.5} irrespective of the fuel, equivalence ratio, pressure, and turbulence intensity for positive Markstein number flames.

Melatonin improves the reprogramming efficiency of murine-induced pluripotent stem cells using a secondary inducible system.

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all-iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10(-6), 10(-7), 10(-8), 10(-9), or 10(-10 )m) or with vitamin C (50 µg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10(-8) (0.81 ± 0.04%), and 10(-9 )m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT2 was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT1 and MT2 receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis-related genes p53 and p21 was lower in the group treated with 10(-9) m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53-mediated apoptotic pathway.

Flame speed and self-similar propagation of expanding turbulent premixed flames.

In this Letter we present turbulent flame speeds and their scaling from experimental measurements on constant-pressure, unity Lewis number expanding turbulent flames, propagating in nearly homogeneous isotropic turbulence in a dual-chamber, fan-stirred vessel. It is found that the normalized turbulent flame speed as a function of the average radius scales as a turbulent Reynolds number to the one-half power, where the average radius is the length scale and the thermal diffusivity is the transport property, thus showing self-similar propagation. Utilizing this dependence it is found that the turbulent flame speeds from the present expanding flames and those from the Bunsen geometry in the literature can be unified by a turbulent Reynolds number based on flame length scales using recent theoretical results obtained by spectral closure of the transformed G equation.