Rebound pathway overactivation by cancer cells following discontinuation of PI3K or mTOR inhibition promotes cancer cell growth.

Whilst effects of anti-cancer drugs have been thoroughly explored, little is known about the repercussion of drug cessation. However, this has important clinical relevance since several clinical protocols such as intermittent drug scheduling lead to frequent drug discontinuation. In this study, we have thus investigated the consequences of withdrawal of agents that target the PI3K/AKT/mTOR signaling pathway in cancer cells. We report that washout of kinase inhibitors of mTOR or PI3K inhibitors led to a rapid and sustainable overactivation of AKT. Consequently, proliferation of tumor cells was significantly higher following drug washout in cancer cells that were pre-treated with mTOR or PI3K inhibitors compared to untreated cells. This effect was prevented by the addition of an AKT inhibitor following drug washout. Rebound AKT overactivation induced by mTOR or PI3K inhibitors discontinuation was mediated by IGF-1R, as demonstrated by its prevention in the presence of an IGF-1R inhibitor and by increased IGF-1R phosphorylation in treated cells versus control cells. Taken together, our results show that discontinuation of PI3K or mTOR inhibitors results in AKT overactivation that promotes tumor growth. They further highlight the benefit of adding an AKT inhibitor following cessation of PI3K or mTOR inhibitors

Advances in the therapy of Alzheimer's disease: Targeting amyloid beta and tau and perspectives for the future

Worldwide multidisciplinary translational research has led to a growing knowledge of the genetics and molecular pathogenesis of Alzheimer's disease (AD) indicating that pathophysiological brain alterations occur decades before clinical signs and symptoms of cognitive decline can be diagnosed. Consequently, therapeutic concepts and targets have been increasingly focused on early-stage illness before the onset of dementia; and distinct classes of compounds are now being tested in clinical trials. At present, there is a growing consensus that therapeutic progress in AD delaying disease progression would significantly decrease the expanding global burden. The evolving hypothesis- and evidence-based generation of new diagnostic research criteria for early-stage AD has positively impacted the development of clinical trial designs and the characterization of earlier and more specific target populations for trials in prodromal as well as in pre- and asymptomatic at-risk stages of AD

Diclofenac Potentiates Sorafenib-Based Treatments of Hepatocellular Carcinoma by Enhancing Oxidative Stress.

Sorafenib is the first developed systemic treatment for advanced forms of hepatocellular carcinoma, which constitutes the most frequent form of primary liver cancers and is a major global health burden. Although statistically significant, the positive effect of sorafenib on median survival remains modest, highlighting the need to develop novel therapeutic approaches. In this report, we introduce diclofenac, a nonsteroidal anti-inflammatory drug, as a potent catalyzer of sorafenib anticancer efficacy. Treatment of three different hepatocellular cancer cells (Huh-7, HepG2, and PLC-PRF-5) with sorafenib (5 µM, 24 h) and diclofenac (100 µM, 24 h) significantly increased cancer cell death compared to sorafenib or diclofenac alone. Anti-oxidant compounds, including N-acetyl-cysteine and ascorbic acid, reversed the deleterious effects of diclofenac/sorafenib co-therapy, suggesting that the generation of toxic levels of oxidative stress was responsible for cell death. Accordingly, whereas diclofenac increased production of mitochondrial oxygen reactive species, sorafenib decreased concentrations of glutathione. We further show that tumor burden was significantly diminished in mice bearing tumor xenografts following sorafenib/diclofenac co-therapy when compared to sorafenib or diclofenac alone. Taken together, these results highlight the anticancer benefits of sorafenib/diclofenac co-therapy in hepatocellular carcinoma. They further indicate that combining sorafenib with compounds that increase oxidative stress represents a valuable treatment strategy in hepatocellular carcinoma

Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rat's striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD

Efficacy and selectivity of the shSNP <i>in vitro</i>.

<p>(<b>A</b>) Quantitative real-time PCR analyses showing the silencing of <i>HTT</i> mRNA in transfected 293T cells co-expressing chimeric <i>HTT</i> and shSNP or the control shRNA. Levels of the chimeric <i>HTT</i> mRNA were normalized to β-Actin and are reported as mean percentages relative to the control condition (set at 100%) ± SEM (n = 3–5). One-way analyses of variance (ANOVA) were performed for each SNP. Newman-Keuls Post-hoc comparison between the shCtrl groups and shSNP groups indicated significant efficacy for sh39A, sh39C, sh50T, sh67T, sh67C (***P<0.001), sh60G (**P<0.01) and sh60A (*P<0.05) whereas the difference between the control condition and the sh50C condition was not statistically significant. The mismatched shRNA conditions were not significantly different from the control condition except for 50T+sh50C (**P<0.01), 67T+sh67C and 67C+sh67T (***P<0.001) and they were all significantly different from the matched shRNA conditions showing the selectivity of this approach <i>in vitro</i> (sh39A, sh39C, sh67C (***P<0.001), sh60G (**P<0.01), sh50C, sh50T, sh60A and sh67T (*P<0.05). (<b>B</b>) Representative western blot (n = 3) with anti-HA antibody illustrating production of the chimeric proteins. The efficacy test lanes (matched shRNA: M) evidence the decrease/absence of the corresponding chimeric proteins, whereas in selectivity lanes (mismatched shRNA: Ms) the mutant HTT is still present as in the control condition (c).</p

Allele specific knock-down of <i>HTT</i> mRNA in human HD NSC.

<p>Relative <i>HTT</i> mRNA levels normalized to controls (non-targeting siRNA) measured by RT-qPCR in HD-NSCs transfected with pan-allelic (siHtt6) or allele-specific <i>HTT</i>-targeting (si50T and si50C) siRNAs. HD-NSCs were derived from two HD-hESC lines, Huez2.3 (<b>A</b>) and SIVF018 (<b>B</b>), T/T homozygous and C/T heterozygous for SNP rs362331, respectively. n =  4. P-value by one-way ANOVA and Tukey's multiple comparison test; *P<0.05; **P<0.01; ***P<0.001; Error bars depict SEM.</p

Efficacy and selectivity of the GFP-shSNP <i>in vivo</i>.

<p>(<b>A</b>) and (<b>C</b>) DARPP-32 immunostaining and quantification 8 weeks after injection evidencing loss of this striatal marker in the control and mismatched injected areas but its preservation in the matched conditions, except for sh39 and sh67. The results are expressed as mean volumes in mm³ depleted of DARPP-32 ± SEM (n = 8). One-way ANOVAs were conducted for each SNP: 50C, 50T, 60A, 60G, 67T, and 67C: ***P<0.001. Newman-Keuls Post-hoc comparison between control conditions and test conditions showed a highly effective prevention of DARPP-32 loss for most of the matched shSNPs and also significant, although less so, prevention of loss for the mismatched sh50C and sh67. For the other mismatched conditions, the post-hoc test was not significant, indicating the selectivity of the shSNPs. (<b>B</b>) and (<b>D</b>) Ubiquitin immunostaining (low and high magnification pictures) and quantification showing large numbers of aggregates in the control and mismatched injected areas and fewer aggregates in the matched conditions. The results are expressed as mean numbers of ubiquitin-positive aggregates ± SEM (n = 8). One-way ANOVAs were conducted for each SNP: 39A, 39C, 50C, 50T, 60A, 60G, and 67T: ***P<0.001; 67C: **P<0.01. Newman-Keuls Post-hoc comparison between control and matched conditions showed highly significant prevention of aggregate formation for all matched shSNPs. For mismatched conditions, the post-hoc test was not significant except for the mismatched sh39 and sh67.</p

Effective and selective SNP-specific silencing in BACHD mice.

<p>(<b>A</b>) Four weeks after injection of constructs encoding fully matched shSNP in BACHD mice striatum, the GFP-positive area was dissected and <i>HTT</i> mRNA was assayed by RT-qPCR. Values for <i>HTT</i> mRNA were normalized to a reference peptidyl propyl isomerase A (<i>PPIA</i>) and are expressed as mean percentages relative to the control condition (set at 100%) ± SEM (n = 10). One-way ANOVAs were conducted for each matched SNP for <i>HTT</i> 39C, 50T, 60G, and 67C: ***P<0.001. Newman-Keuls Post-hoc comparison between the shCtrl and matched shRNA groups demonstrate significant silencing of targeted human <i>HTT</i> mRNA. (<b>B</b>) Samples all showed similar NeuN gene expression (One-way ANOVAs and Newman-Keuls Post-hoc comparison test). (<b>c</b>) Three weeks after injection of constructs encoding shCtrl, sh50T and sh50C in BACHD mice striatum, 1 mm³ punches of the GFP infected area were dissected and <i>HTT</i> mRNA was assayed by RT-qPCR with primers specific for the human <i>HTT</i>. Values for <i>HTT</i> mRNA were normalized to a reference <i>PPIA</i> and are expressed as mean percentages relative to the control condition (set at 100%) ± SEM (n = 4–6). One-way ANOVAs were conducted for each groups: *P<0.05. Newman-Keuls Post-hoc comparison between the shCtrl and the sh50T confirm the silencing of human mutant <i>HTT</i> mRNA, whereas no statistically significant difference was observed between shCtrl and sh50C groups, demonstrating the selectivity of <i>HTT</i> silencing.</p