Genetic influence on vascular smooth muscle cell apoptosis.

Vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis play important roles in many physiological processes and pathological conditions. To identify genetic influences on VSMC behavior, we measured these traits and undertook genome-wide association studies in primary umbilical artery-derived VSMCs from >2000 individuals. Although there were no genome-wide significant associations for VSMC proliferation or migration, genetic variants at two genomic loci (7p15.3 and 7q32.3) showed highly significant associations with VSMC apoptosis (P = 1.95 × 10-13 and P = 7.47 × 10-9, respectively). The lead variant at the 7p51.3 locus was associated with increased expression of the GSDME and PALS2 genes in VSMCs. Knockdown of GSDME or PALS2 in VSMCs attenuated apoptotic cell death. A protein co-immunoprecipitation assay indicated that GSDME complexed with PALS2. PALS2 knockdown attenuated activated caspase-3 and GSDME fragmentation, whilst GSDME knockdown also reduced activated caspase-3. These findings provide new insights into the genetic regulation of VSMC apoptosis, with potential utility for therapeutic development.

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism.

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Effects of Coronary Artery Disease-Associated Variants on Vascular Smooth Muscle Cells.

BACKGROUND: Genome-wide association studies have identified many genetic loci that are robustly associated with coronary artery disease (CAD). However, the underlying biological mechanisms are still unknown for most of these loci, hindering the progress to medical translation. Evidence suggests that the genetic influence on CAD susceptibility may act partly through vascular smooth muscle cells (VSMCs). METHODS: We undertook genotyping, RNA sequencing, and cell behavior assays on a large bank of VSMCs (n>1499). Expression quantitative trait locus and splicing quantitative trait locus analyses were performed to identify genes with an expression that was influenced by CAD-associated variants. To identify candidate causal genes for CAD, we ascertained colocalizations of VSMC expression quantitative trait locus signals with CAD association signals by performing causal variants identification in associated regions analysis and the summary data-based mendelian randomization test. Druggability analysis was then performed on the candidate causal genes. CAD risk variants were tested for associations with VSMC proliferation, migration, and apoptosis. Collective effects of multiple CAD-associated variants on VSMC behavior were estimated by polygenic scores. RESULTS: Approximately 60% of the known CAD-associated variants showed statistically significant expression quantitative trait locus or splicing quantitative trait locus effects in VSMCs. Colocalization analyses identified 84 genes with expression quantitative trait locus signals that significantly colocalized with CAD association signals, identifying them as candidate causal genes. Druggability analysis indicated that 38 of the candidate causal genes were druggable, and 13 had evidence of drug-gene interactions. Of the CAD-associated variants tested, 139 showed suggestive associations with VSMC proliferation, migration, or apoptosis. A polygenic score model explained up to 5.94% of variation in several VSMC behavior parameters, consistent with polygenic influences on VSMC behavior. CONCLUSIONS: This comprehensive analysis shows that a large percentage of CAD loci can modulate gene expression in VSMCs and influence VSMC behavior. Several candidate causal genes identified are likely to be druggable and thus represent potential therapeutic targets.

Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes.

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Influence of a Coronary Artery Disease-Associated Genetic Variant on FURIN Expression and Effect of Furin on Macrophage Behavior.

Objective- Genome-wide association studies have revealed a robust association between genetic variation on chromosome 15q26.1 and coronary artery disease (CAD) susceptibility; however, the underlying biological mechanism is still unknown. The lead CAD-associated genetic variant (rs17514846) at this locus resides in the FURIN gene. In advanced atherosclerotic plaques, furin is expressed primarily in macrophages. We investigated whether this CAD-associated variant alters FURIN expression and whether furin affects monocyte/macrophage behavior. Approach and Results- A quantitative reverse transcription polymerase chain reaction analysis showed that leukocytes from individuals carrying the CAD risk allele (A) of rs17514846 had increased FURIN expression. A chromatin immunoprecipitation assay revealed higher RNA polymerase II occupancy in the FURIN gene in mononuclear cells of individuals carrying this allele. A reporter gene assay in transiently transfected monocytes/macrophages indicated that the CAD risk allele had higher transcriptional activity than the nonrisk allele (C). An analysis of isogenic monocyte cell lines created by CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing showed that isogenic cells with the A/A genotype for rs17514846 had higher FURIN expression levels than the isogenic cells with the C/C genotype. An electrophoretic mobility shift assay exhibited preferential binding of a nuclear protein to the risk allele. Studies of monocytes/macrophages with lentivirus-mediated furin overexpression or shRNA (short hairpin RNA)-induced furin knockdown showed that furin overexpression promoted monocyte/macrophage migration, increased proliferation, and reduced apoptosis whereas furin knockdown had the opposite effects. Conclusions- Our study shows that the CAD-associated genetic variant increases FURIN expression and that furin promotes monocyte/macrophage migration and proliferation while inhibiting apoptosis, providing a biological mechanism for the association between variation at the chromosome 15q26.1 locus and CAD risk.

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.

Cancer chemoprevention: Evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice.

Resveratrol is widely promoted as a potential cancer chemopreventive agent, but a lack of information on the optimal dose prohibits rationally designed trials to assess efficacy. To challenge the assumption that "more is better," we compared the pharmacokinetics and activity of a dietary dose with an intake 200 times higher. The dose-response relationship for concentrations generated and the metabolite profile of [(14)C]-resveratrol in colorectal tissue of cancer patients helped us to define clinically achievable levels. In Apc(Min) mice (a model of colorectal carcinogenesis) that received a high-fat diet, the low resveratrol dose suppressed intestinal adenoma development more potently than did the higher dose. Efficacy correlated with activation of adenosine monophosphate-activated protein kinase (AMPK) and increased expression of the senescence marker p21. Nonlinear dose responses were observed for AMPK and mechanistic target of rapamycin (mTOR) signaling in mouse adenoma cells, culminating in autophagy and senescence. In human colorectal tissues exposed to low dietary concentrations of resveratrol ex vivo, we measured enhanced AMPK phosphorylation and autophagy. The expression of the cytoprotective NAD(P)H dehydrogenase, quinone 1 (NQO1) enzyme was also increased in tissues from cancer patients participating in our [(14)C]-resveratrol trial. These findings warrant a revision of developmental strategies for diet-derived agents designed to achieve cancer chemoprevention.

Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells.

Resveratrol has many proposed health benefits, including the prevention of cancers, but its low bioavailability is considered a limiting factor in translating these effects to humans. Based on in vivo and clinical studies we have shown that resveratrol is indeed rapidly metabolized by phase II enzymes, and that resveratrol sulfates are deconjugated by steroid sulfatases to afford free resveratrol in vitro and in vivo and hence act as an intracellular reservoir for resveratrol. Further, we have demonstrated that at clinically achievable concentrations of resveratrol sulfate, parent resveratrol is regenerated within human colorectal cancer, but not normal epithelial cells, and is responsible for inducing autophagy with senescence selectively in cancer cells.

Sulfate metabolites provide an intracellular pool for resveratrol generation and induce autophagy with senescence.

The phytochemical resveratrol has been shown to exert numerous health benefits in preclinical studies, but its rapid metabolism and resulting poor bioavailability may limit translation of these effects to humans. Resveratrol metabolites might contribute to in vivo activity through regeneration of the parent compound. We present quantitation of sulfate and glucuronide conjugates of resveratrol in human plasma and tissue after repeated ingestion of resveratrol by volunteers and cancer patients, respectively. Subsequent pharmacokinetic characterization of a mixture of resveratrol-3-O-sulfate and resveratrol-4'-O-sulfate in mice showed that these metabolites are absorbed orally but have low bioavailabilities of ~14 and 3%, respectively. Sulfate hydrolysis in vivo liberated free resveratrol, which accounted for ~2% of the total resveratrol species present in mouse plasma. Monosulfate metabolites were also converted to the parent in human colorectal cells. The extent of cellular uptake was dependent on specific membrane transporters and dictated antiproliferative activity. Sulfate metabolites induced autophagy and senescence in human cancer cells; these effects were abrogated by inclusion of a sulfatase inhibitor, which reduced intracellular resveratrol. Together, our findings suggest that resveratrol is delivered to target tissues in a stable sulfate-conjugated form and that the parent compound is gradually regenerated in selected cells and may give rise to the beneficial effects in vivo. At doses considered to be safe in humans, resveratrol generated via this route may be of greater importance than the unmetabolized form.

The intermediate-activity (L597V)BRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway.

(L597V)BRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline (L597V)BRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of (L597V)Braf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined (L597V)Braf with (G12D)Kras and found that (L597V)Braf modified (G12D)Kras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity (V600E)Braf mutant. Mek/Erk activation levels were comparable with those driven by (V600E)Braf in the double-mutant cells, and the gene expression signature was more similar to that induced by (V600E)Braf than (G12D)Kras. However, unlike (V600E)Braf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, (L597V)Braf epistatically modifies the transforming effects of driver oncogenes.

Regulation of MEK/ERK pathway output by subcellular localization of B-Raf.

The strength and duration of intracellular signalling pathway activation is a key determinant of the biological outcome of cells in response to extracellular cues. This has been particularly elucidated for the Ras/Raf/MEK [mitogen-activated growth factor/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling pathway with a number of studies in fibroblasts showing that sustained ERK signalling is a requirement for S-phase entry, whereas transient ERK signalling does not have this capability. A major unanswered question, however, is how a cell can sustain ERK activation, particularly when ERK-specific phosphatases are transcriptionally up-regulated by the pathway itself. A major point of ERK regulation is at the level of Raf, and, to sustain ERK activation in the presence of ERK phosphatases, sustained Raf activation is a requirement. Three Raf proteins exist in mammals, and the activity of all three is induced following growth factor stimulation of cells, but only B-Raf activity is maintained at later time points. This observation points to B-Raf as a regulator of sustained ERK activation. In the present review, we consider evidence for a link between B-Raf and sustained ERK activation, focusing on a potential role for the subcellular localization of B-Raf in this key physiological event.

Predicting the physiological relevance of in vitro cancer preventive activities of phytochemicals.

There is growing interest in the ability of phytochemicals to prevent chronic diseases, such as cancer and heart disease. However, some of these agents have poor bioavailability and many of the in-depth studies into their mechanisms of action have been carried out in vitro using doses which are unachievable in humans. In order to optimize the design of chemopreventive treatment, it is important to determine which of the many reported mechanisms of action are clinically relevant. In this review we consider the physiologically achievable doses for a few of the best studied agents (indole-3-carbinol, diindolylmethane, curcumin, epigallocatechin-3-gallate and resveratrol) and summarize the data derived from studies using these low concentrations in cell culture. We then cite examples of in vitro effects which have been observed in vivo. Finally, the ability of agent combinations to act synergistically or antagonistically is considered. We conclude that each of the compounds shows an encouraging range of activities in vitro at concentrations which are likely to be physiologically relevant. There are also many examples of in vivo studies which validate in vitro observations. An important consideration is that combinations of agents can result in significant activity at concentrations where any single agent is inactive. Thus, for each of the compounds reviewed here, in vitro studies have provided useful insights into their mechanisms of action in humans. However, data are lacking on the full range of activities at low doses in vitro and the benefits or otherwise of combinations in vivo.

Involvement of Nrf2, p38, B-Raf, and nuclear factor-kappaB, but not phosphatidylinositol 3-kinase, in induction of hemeoxygenase-1 by dietary polyphenols.

The highly inducible enzyme, hemeoxygenase-1 (HO-1), metabolizes heme, thereby protecting a variety of cells against oxidative stress and apoptosis. Up-regulation by cancer chemopreventive agents has been reported, but its regulation and function in transformed cells are unclear. We compared induction by two dietary polyphenols, curcumin and epigallocatechin-3-gallate (EGCG), with that by the endogenous substrate hemin in epithelial and endothelial cells and examined the relevance to apoptosis. Curcumin or hemin (20 microM) induced HO-1 in breast cells from 5 to 24 h. Curcumin (5-40 microM) or hemin (5-100 microM) induced HO-1 and nuclear levels of nuclear factor (erythroid-derived 2)-related factor (Nrf2) in a dose-dependent manner. EGCG had no effect in breast cells, but at 30 microM, it induced nuclear translocation of Nrf2 and HO-1 expression in B-lymphoblasts. In all cases, induction was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580). The nuclear factor-kappaB (NF-kappaB)-DNA binding inhibitor helenalin (20 microM) also prevented induction. However, wortmannin had no effect, suggesting that PI3K was not involved. Curcumin and hemin also induced nuclear Nrf2 and HO-1 effectively in wild-type mouse embryo fibroblasts (wt MEFs) and in B-Raf(-/-) MEFs but not in Nrf2(-/-) MEFs. However, EGCG (5-20 microM) induced HO-1 only in wt MEFs. Results suggest that signaling through p38 mitogen-activated protein kinase, NF-kappaB, and Nrf2 as well as other unidentified molecules is involved in HO-1 induction by hemin and both polyphenols, but cell-specific factors also play a role, particularly with respect to EGCG. Induction of HO-1 by curcumin, EGCG, or low concentrations (5-10 microM) of helenalin did not protect MDA-MB468 breast cells or B-lymphoblasts from apoptosis.