Genetic and Functional Analyses Point to FAN1 as the Source of Multiple Huntington Disease Modifier Effects.

A recent genome-wide association study of Huntington disease (HD) implicated genes involved in DNA maintenance processes as modifiers of onset, including multiple genome-wide significant signals in a chr15 region containing the DNA repair gene Fanconi-Associated Nuclease 1 (FAN1). Here, we have carried out detailed genetic, molecular, and cellular investigation of the modifiers at this locus. We find that missense changes within or near the DNA-binding domain (p.Arg507His and p.Arg377Trp) reduce FAN1's DNA-binding activity and its capacity to rescue mitomycin C-induced cytotoxicity, accounting for two infrequent onset-hastening modifier signals. We also idenified a third onset-hastening modifier signal whose mechanism of action remains uncertain but does not involve an amino acid change in FAN1. We present additional evidence that a frequent onset-delaying modifier signal does not alter FAN1 coding sequence but is associated with increased FAN1 mRNA expression in the cerebral cortex. Consistent with these findings and other cellular overexpression and/or suppression studies, knockout of FAN1 increased CAG repeat expansion in HD-induced pluripotent stem cells. Together, these studies support the process of somatic CAG repeat expansion as a therapeutic target in HD, and they clearly indicate that multiple genetic variations act by different means through FAN1 to influence HD onset in a manner that is largely additive, except in the rare circumstance that two onset-hastening alleles are present. Thus, an individual's particular combination of FAN1 haplotypes may influence their suitability for HD clinical trials, particularly if the therapeutic agent aims to reduce CAG repeat instability.

Population-specific genetic modification of Huntington's disease in Venezuela.

Modifiers of Mendelian disorders can provide insights into disease mechanisms and guide therapeutic strategies. A recent genome-wide association (GWA) study discovered genetic modifiers of Huntington's disease (HD) onset in Europeans. Here, we performed whole genome sequencing and GWA analysis of a Venezuelan HD cluster whose families were crucial for the original mapping of the HD gene defect. The Venezuelan HD subjects develop motor symptoms earlier than their European counterparts, implying the potential for population-specific modifiers. The main Venezuelan HD family inherits HTT haplotype hap.03, which differs subtly at the sequence level from European HD hap.03, suggesting a different ancestral origin but not explaining the earlier age at onset in these Venezuelans. GWA analysis of the Venezuelan HD cluster suggests both population-specific and population-shared genetic modifiers. Genome-wide significant signals at 7p21.2-21.1 and suggestive association signals at 4p14 and 17q21.2 are evident only in Venezuelan HD, but genome-wide significant association signals at the established European chromosome 15 modifier locus are improved when Venezuelan HD data are included in the meta-analysis. Venezuelan-specific association signals on chromosome 7 center on SOSTDC1, which encodes a bone morphogenetic protein antagonist. The corresponding SNPs are associated with reduced expression of SOSTDC1 in non-Venezuelan tissue samples, suggesting that interaction of reduced SOSTDC1 expression with a population-specific genetic or environmental factor may be responsible for modification of HD onset in Venezuela. Detection of population-specific modification in Venezuelan HD supports the value of distinct disease populations in revealing novel aspects of a disease and population-relevant therapeutic strategies.

Novel allele-specific quantification methods reveal no effects of adult onset CAG repeats on HTT mRNA and protein levels.

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.

Full sequence of mutant huntingtin 3'-untranslated region and modulation of its gene regulatory activity by endogenous microRNA.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3'-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3'-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3'-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3'-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3'-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.

Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9.

A comprehensive genetics-based precision medicine strategy to selectively and permanently inactivate only mutant, not normal allele, could benefit many dominantly inherited disorders. Here, we demonstrate the power of our novel strategy of inactivating the mutant allele using haplotype-specific CRISPR/Cas9 target sites in Huntington's disease (HD), a late-onset neurodegenerative disorder due to a toxic dominant gain-of-function CAG expansion mutation. Focusing on improving allele specificity, we combined extensive knowledge of huntingtin (HTT) gene haplotype structure with a novel personalized allele-selective CRISPR/Cas9 strategy based on Protospacer Adjacent Motif (PAM)-altering SNPs to target patient-specific CRISPR/Cas9 sites, aiming at the mutant HTT allele-specific inactivation for a given diplotype. As proof-of-principle, simultaneously using two CRISPR/Cas9 guide RNAs (gRNAs) that depend on PAM sites generated by SNP alleles on the mutant chromosome, we selectively excised ∼44 kb DNA spanning promoter region, transcription start site, and the CAG expansion mutation of the mutant HTT gene, resulting in complete inactivation of the mutant allele without impacting the normal allele. This excision on the disease chromosome completely prevented the generation of mutant HTT mRNA and protein, unequivocally indicating permanent mutant allele-specific inactivation of the HD mutant allele. The perfect allele selectivity with broad applicability of our strategy in disorders with diverse disease haplotypes should also support precision medicine through inactivation of many other gain-of-function mutations.

Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6.

Myofibrillar myopathy 6 (MFM6) is a rare childhood-onset myopathy characterized by myofibrillar disintegration, muscle weakness, and cardiomyopathy. The genetic cause of MFM6 is p.Pro209Leu mutation (rs121918312-T) in the BAG3 gene, which generates the disease outcomes in a dominant fashion. Since the consequences of the BAG3 mutation are strong and rapidly progressing, most MFM6 patients are due to de novo mutation. There are no effective treatments for MFM6 despite its well-known genetic cause. Given p.Pro209Leu mutation is dominant, regenerative medicine approaches employing orthologous stem cells in which mutant BAG3 is inactivated offer a promising avenue. Here, we developed personalized allele-specific CRISPR-Cas9 strategies capitalizing on PAM-altering SNP and PAM-proximal SNP. In order to identify the disease chromosome carrying the de novo mutation in our two affected individuals, haplotype phasing through cloning-sequencing was performed. Based on the sequence differences between mutant and normal BAG3, we developed personalized allele-specific CRISPR-Cas9 strategies to selectively inactivate the mutant allele 1) by preventing the transcription of the mutant BAG3 and 2) by inducing nonsense-mediated decay (NMD) of mutant BAG3 mRNA. Subsequent experimental validation in patient-derived induced pluripotent stem cell (iPSC) lines showed complete allele specificities of our CRISPR-Cas9 strategies and molecular consequences attributable to inactivated mutant BAG3. In addition, mutant allele-specific CRISPR-Cas9 targeting did not alter the characteristics of iPSC or the capacity to differentiate into cardiomyocytes. Together, our data demonstrate the feasibility and potential of personalized allele-specific CRISPR-Cas9 approaches to selectively inactivate the mutant BAG3 to generate cell resources for regenerative medicine approaches for MFM6.

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease.

An expanded CAG repeat in the huntingtin gene (HTT) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and guide RNAs (gRNAs) efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased in the liver by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion strategies in HD and potentially other repeat expansion disorders.

Genetic modifiers of somatic expansion and clinical phenotypes in Huntington's disease reveal shared and tissue-specific effects.

Huntington's disease (HD), due to expansion of a CAG repeat in HTT , is representative of a growing number of disorders involving somatically unstable short tandem repeats. We find that overlapping and distinct genetic modifiers of clinical landmarks and somatic expansion in blood DNA reveal an underlying complexity and cell-type specificity to the mismatch repair-related processes that influence disease timing. Differential capture of non-DNA-repair gene modifiers by multiple measures of cognitive and motor dysfunction argues additionally for cell-type specificity of pathogenic processes. Beyond trans modifiers, differential effects are also illustrated at HTT by a 5'-UTR variant that promotes somatic expansion in blood without influencing clinical HD, while, even after correcting for uninterrupted CAG length, a synonymous sequence change at the end of the CAG repeat dramatically hastens onset of motor signs without increasing somatic expansion. Our findings are directly relevant to therapeutic suppression of somatic expansion in HD and related disorders and provide a route to define the individual neuronal cell types that contribute to different HD clinical phenotypes.

Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo.

Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease.

An expanded CAG repeat in the huntingtin gene ( HTT ) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and gRNAs efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion base editing strategies in HD and potentially other repeat expansion disorders.

Haplotype-specific insertion-deletion variations for allele-specific targeting in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in huntingtin (HTT). Given an important role for HTT in development and significant neurodegeneration at the time of clinical manifestation in HD, early treatment of allele-specific drugs represents a promising strategy. The feasibility of an allele-specific antisense oligonucleotide (ASO) targeting single-nucleotide polymorphisms (SNPs) has been demonstrated in models of HD. Here, we constructed a map of haplotype-specific insertion-deletion variations (indels) to develop alternative mutant-HTT-specific strategies. We mapped indels annotated in the 1000 Genomes Project data on common HTT haplotypes, revealing candidate indels for mutant-specific HTT targeting. Subsequent sequencing of an HD family confirmed candidate sites and revealed additional allele-specific indels. Interestingly, the most common normal HTT haplotype carries indels of big allele length differences at many sites, further uncovering promising haplotype-specific targets. When patient-derived cells carrying the most common HTT diplotype were treated with ASOs targeting the mutant alleles of candidate indels (rs772629195 or rs72239206), complete mutant specificity was observed. In summary, our map of haplotype-specific indels permits the identification of allele-specific targets in HD subjects, potentially contributing to the development of safe HTT-lowering therapeutics that are suitable for early treatment in HD.

PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease.

Huntington's disease (HD) is caused by an expanded CAG repeat in huntingtin (HTT). Since HD is dominant and loss of HTT leads to neurological abnormalities, safe therapeutic strategies require selective inactivation of mutant HTT. Previously, we proposed a concept of CRISPR-Cas9 using mutant-specific PAM sites generated by SNPs to selectively inactivate mutant HTT. Aiming at revealing suitable targets for clinical development, we analyzed the largest HD genotype dataset to identify target PAM-altering SNPs (PAS) and subsequently evaluated their allele specificities. The gRNAs based on the PAM sites generated by rs2857935, rs16843804, and rs16843836 showed high levels of allele specificity in patient-derived cells. Simultaneous use of two gRNAs based on rs2857935-rs16843804 or rs2857935-rs16843836 produced selective genomic deletions in mutant HTT and prevented the transcription of mutant HTT mRNA without impacting the expression of normal counterpart or re-integration of the excised fragment elsewhere in the genome. RNA-seq and off-target analysis confirmed high levels of allele specificity and the lack of recurrent off-targeting. Approximately 60% of HD subjects are eligible for mutant-specific CRISPR-Cas9 strategies of targeting one of these three PAS in conjunction with one non-allele-specific site, supporting high applicability of PAS-based allele-specific CRISPR approaches in the HD patient population.

Allele-specific silencing of the gain-of-function mutation in Huntington's disease using CRISPR/Cas9.

Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest that selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic protospacer adjacent motif-altering (PAM-altering) SNP (PAS), we developed an allele-specific CRISPR/Cas9 strategy to permanently inactivate mutant HTT through nonsense-mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant-specific CRISPR/Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR/Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR/Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR/Cas9 strategy inactivates mutant HTT through NMD. In addition, GUIDE-Seq analysis and subsequent validation experiments support high levels of on-target gene specificity. Our data demonstrate a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR/Cas9 and supporting its therapeutic potential in HD.

Ultraviolet B radiation activates NF-κB and induces iNOS expression in HR-1 hairless mouse skin: role of IκB kinase-ß.

Exposure to ultraviolet B (UVB) radiation is known to cause inflammatory tissue damage and skin cancer. One of the molecular links between inflammation and cancer is the eukaryotic transcription factor nuclear factor-kappaB (NF-κB), which is known to regulate expression of various pro-inflammatory genes including inducible nitric oxide synthase (iNOS). The present study was aimed at elucidating the molecular mechanisms underlying UVB-induced NF-κB activation and iNOS expression in hairless mouse skin. Irradiation of male HR-1 hairless mouse skin with UVB (5 kJ/m(2) ) resulted in increased degradation of IκBα, nuclear translocation of p65 and p50, and the DNA binding of NF-κB. Exposure to UVB radiation induced the phosphorylation and the catalytic activity of an upstream kinase IκB kinase-ß (IKKß). Pharmacological inhibition of IKKß attenuated UVB-induced NF-κB activation in mouse skin. Irradiation of mouse skin with UVB also increased phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Pretreatment with SC-514, a specific inhibitor of IKKß, attenuated UVB-induced phosphorylation of ERK and p38 MAP kinase. A kinetic study showed that UVB significantly increased the expression of iNOS in mouse skin at 6 h postirradiation, which was abrogated by pretreatment with SC-514. In conclusion, the upstream kinase IKKß is involved in UVB-induced activation of MAP kinases and NF-κB, and expression of iNOS in mouse skin.

Curcumin induces stabilization of Nrf2 protein through Keap1 cysteine modification.

The present study was aimed to investigate the effects of curcumin, a representative chemopreventive phytochemical with pronounced antioxidant and anti-inflammatory properties, on activation of Nrf2 and expression of its target protein heme oxygenase-1 (HO-1) in mouse skin in vivo and in cultured murine epidermal cells. Treatment of mouse epidermal JB6 cells with curcumin resulted in the induction of HO-1 expression, and this was abrogated in cells transiently transfected with Nrf2 siRNA. While curcumin treatment increased protein expression of Nrf2, it did not alter the steady-state level of the Nrf2 mRNA transcript. Treatment of cells with curcumin stabilized Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of this transcription factor. Tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,ß-unsaturated carbonyl group, failed to induce HO-1 expression as well as nuclear translocation of Nrf2 and its binding to the antioxidant/electrophile response elements. Cells transfected with a mutant Keap1 protein in which cysteine 151 (Cys151) is replaced by serine exhibited marked reduction in curcumin-induced Nrf2 transactivation. Mass spectrometric analysis revealed that curcumin binds to Keap1 Cys151, supporting that this amino acid is a critical target for curcumin modification of Keap1, which facilitates the liberation of Nrf2. Thus, it is likely that the α,ß-unsaturated carbonyl moiety of curcumin is essential for its binding to Keap1 and stabilization of Nrf2 by hampering ubiquitination and proteasomal degradation.

The prospects of CRISPR-based genome engineering in the treatment of neurodegenerative disorders.

Over the past few decades, as gene discovery methods and sequencing technologies have evolved, many genetic variations that significantly increase the risk of or cause neurodegenerative diseases have been identified. However, knowledge of those pathogenic mutations and subsequent mechanism-focused studies has rarely yielded effective treatments, warranting alternative strategies for refining rational therapeutic targets. Nevertheless, with the evolution of gene targeting methods, it has been increasingly recognized that the disease-causing gene itself is the best therapeutic target even when we do not have a full understanding of its biological functions. Considering this, CRISPR/Cas gene editing technology offers the promise of permanently silencing or correcting the disease-causing mutations, potentially overcoming key limitations of RNA-targeting approaches. The versatile CRISPR/Cas-based strategies have the potential to become treatment options for challenging disorders such as neurodegenerative diseases. Here, we summarize recent reports of preclinical applications of CRISPR/Cas in models of neurodegenerative disorders to provide perspectives on therapeutic gene editing for diseases of the nervous system.

Endogenous ω-3 Fatty Acid Production by fat-1 Transgene and Topically Applied Docosahexaenoic Acid Protect against UVB-induced Mouse Skin Carcinogenesis.

The present study was intended to explore the effects of endogenously produced ω-3 polyunsaturated fatty acids (PUFAs) on ultraviolet B (UVB)-induced skin inflammation and photocarcinogenesis using hairless fat-1 transgenic mice harboring ω-3 desaturase gene capable of converting ω-6 to ω-3 PUFAs. Upon exposure to UVB irradiation, fat-1 transgenic mice exhibited a significantly reduced epidermal hyperplasia, oxidative skin damage, and photocarcinogenesis as compared to wild type mice. The transcription factor, Nrf2 is a master regulator of anti-inflammatory and antioxidant gene expression. While the protein expression of Nrf2 was markedly enhanced, the level of its mRNA transcript was barely changed in the fat-1 transgenic mouse skin. Topical application of docosahexaenoic acid (DHA), a representative ω-3 PUFA, in wild type hairless mice induced expression of the Nrf2 target protein, heme oxygenase-1 in the skin and protected against UVB-induced oxidative stress, inflammation and papillomagenesis. Furthermore, transient overexpression of fat-1 gene in mouse epidermal JB6 cells resulted in the enhanced accumulation of Nrf2 protein. Likewise, DHA treated to JB6 cells inhibited Nrf2 ubiquitination and stabilized it. Taken together, our results indicate that functional fat-1 and topically applied DHA potentiate cellular defense against UVB-induced skin inflammation and photocarcinogenesis through elevated activation of Nrf2 and upregulation of cytoprotective gene expression.

Protein tyrosine phosphatases PTP-1B, SHP-2, and PTEN facilitate Rb/E2F-associated apoptotic signaling.

To maintain tissue homeostasis, apoptosis is functionally linked to the cell cycle through the retinoblastoma (Rb)/E2F pathway. When the Rb tumor suppressor protein is functionally inactivated, E2F1 elicits an apoptotic response through both intrinsic (caspase-9 mediated) and extrinsic (caspase-8 mediated) apoptotic pathways in order to eliminate hyperproliferative cells. Rb/E2F-associated apoptosis has been demonstrated to be associated with the loss of constitutive transcriptional repression by Rb/E2F complexes and mediated by caspase-8. Protein tyrosine phosphatases (PTPs) PTP-1B and SHP-2 have been previously shown to be directly activated by loss of Rb/E2F repression during Rb/E2F-associated apoptosis. In this current study, we demonstrate that the PTEN tumor suppressor is also directly activated by loss of Rb/E2F repression. We also demonstrate that PTP-1B, SHP-2, and PTEN play a functional role in Rb/E2F-associated apoptosis. Knockdown of PTP1B, SHP2, or PTEN expression with small interfering RNA (siRNA) in apoptotic cells increases cell viability and rescues cells from the Rb/E2F-associated apoptotic response. Furthermore, rescue from apoptosis coincides with inhibition of caspase-8 and caspase-3 cleavage (activation). Our results indicate PTP-1B, SHP-2, and PTEN all play a functional role in Rb/E2F-associated apoptotic signal transduction and provide further evidence that PTP-1B, SHP-2, and PTEN can contribute to tumor suppression through an Rb/E2F-associated mechanism.

Phloretin inhibits phorbol ester-induced tumor promotion and expression of cyclooxygenase-2 in mouse skin: extracellular signal-regulated kinase and nuclear factor-κB as potential targets.

The present study investigated the effect of phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cyclooxygenase-2 (COX-2) expression and tumor promotion in mouse skin and explored the underlying molecular mechanisms. Topical application of phloretin significantly inhibited 7,12-dimethylbenz[a]anthracene-initiated and TPA-promoted mouse skin carcinogenesis. Pretreatment with phloretin on the dorsal skin of mice inhibited TPA-induced COX-2 expression in a dose-dependent manner. To elucidate the molecular mechanism underlying COX-2 inhibition by phloretin, we examined its effect on TPA-induced activation of nuclear factor-κB (NF-κB), a ubiquitous transcription factor responsible for TPA-induced COX-2 expression in mouse skin. Topically applied phloretin decreased the TPA-induced DNA binding of NF-κB. In addition, phloretin inhibited the phosphorylation as well as the catalytic activity of extracellular signal-regulated kinase (ERK), which was previously found to activate NF-κB and induce COX-2 expression in TPA-treated mouse skin. Taken together, the inhibitory effects of phloretin on TPA-induced NF-κB activation and COX-2 expression through the modulation of ERK signaling may partly account for its antitumor-promoting effect on mouse skin carcinogenesis.

Zerumbone induces heme oxygenase-1 expression in mouse skin and cultured murine epidermal cells through activation of Nrf2.

Zerumbone, a sesquiterpene derived from tropical ginger, contains an electrophilic α,ß-unsaturated carbonyl moiety and was found to suppress chemically induced papilloma formation in mouse skin. Here, we report that topical application of zerumbone onto dorsal skin of hairless mice induces activation of NF-E2-related factor 2 (Nrf2) and expression of heme oxygenase-1 (HO-1). We compared the levels of HO-1 protein in the skin of zerumbone-treated Nrf2 wild-type and Nrf2 knockout mice, and nrf2-deficient mice expressed HO-1 protein to a much lesser extent than the wild-type animals following topical application of zerumbone. Treatment of mouse epidermal JB6 cells with zerumbone caused a marked increase of Nrf2 nuclear translocation followed by the promoter activity of HO-1, and also enhanced direct binding of Nrf2 to the antioxidant response element. Moreover, knockdown of Nrf2 in JB6 cells diminished the zerumbone-induced upregulation of HO-1. Notably, α-humulene and 8-hydroxy-α-humulene, the structural analogues of zerumbone that lack the α,ß-unsaturated carbonyl group, failed to activate Nrf2 and were unable to increase HO-1 expression. Unlike zerumbone, these nonelectrophilic analogues could not suppress the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced JB6 cell transformation and the intracellular accumulation of reactive oxygen species (ROS). Interestingly, when JB6 cells were treated with carbon monoxide-releasing molecule that mimics the HO-1 activity, the TPA-induced ROS production was markedly reduced. Taken together, these findings suggest that upregulation of HO-1 expression by zerumbone is mediated through activation of Nrf2 signaling, which provides a mechanistic basis for the chemopreventive effects of this sesquiterpene on mouse skin carcinogenesis.