Crosstalk between the vitamin D receptor (VDR) and miR-214 in regulating SuFu, a hedgehog pathway inhibitor in breast cancer cells.

The vitamin D receptor (VDR), and its ligand 1α,25-dihydroxyvitamin D3 (1,25D3) prevent breast cancer development and progression, yet the molecular mechanisms governing this are unclear. MicroRNAs (miRNAs) on the other hand, promote or inhibit breast cancer growth. To understand how VDR regulates miRNAs, we compared miRNA expression of wild-type (WT) and VDR knockout (VDRKO) breast cancer cells by a Mouse Breast Cancer miRNA PCR array. Compared to VDR WT cells, expressions of miR-214, miR-199a-3p and miR-199a-5p of the miR-199a/miR-214 cluster were 42, 15, and 10 fold higher in VDRKO cells respectively. Overexpression of VDR in breast cancer cells reduced the miR-199a/miR-214 cluster expression by 30%. VDR status also negatively correlated with Dnm3os expression, a non-coding RNA transcript of the dynamin-3 gene encoding the miR-199a/miR-214 cluster, suggesting that VDR represses this cluster through Dnm3os. Conversely, overexpression of miR-214 in MCF-7 and T47D cells antagonized VDR mediated signaling. Furthermore, there was a positive correlation between VDR status and the expression of Suppressor of fused gene (SuFu), a hedgehog pathway inhibitor. miR-214 on the other hand suppressed SuFu protein expression. These findings suggest a crosstalk between VDR and miR-214 in regulating hedgehog signaling in breast cancer cells, providing new therapies for breast cancer.

Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux.

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.

Effect of Error Propagation in Stable Isotope Tracer Studies: An Approach for Estimating Impact on Apparent Biochemical Flux.

Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e., the precursor and product labeling pre- and posttracer administration. This seemingly simple problem has 1 correct solution and 80 erroneous outcomes. In this report, we outline a methodology for evaluating the effect of error propagation on apparent physiological endpoints. We demonstrate examples of how to evaluate the influence of analytical error in case studies concerning lipid and protein synthesis; we have focused on (2)H2O as a tracer and contrast different mass spectrometry platforms including GC-quadrupole-MS, GC-pyrolysis-IRMS, LC-quadrupole-MS, and high-resolution FT-ICR-MS. The method outlined herein can be used to determine how to minimize variations in the apparent biology by altering the dose and/or the type of tracer. Likewise, one can facilitate biological studies by estimating the reduction in the noise of an outcome that is expected for a given increase in the number of replicate injections.

In vivo isotopically labeled atherosclerotic aorta plaques in ApoE KO mice and molecular profiling by matrix-assisted laser desorption/ionization mass spectrometric imaging.

RATIONALE: The ability to quantify rates of formation, regression and/or remodeling of atherosclerotic plaque should facilitate a better understanding of the pathogenesis and management of cardiovascular disease. In the current study, we coupled a stable isotope labeled tracer protocol with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine spatial and temporal lipid dynamics in atherosclerotic plaque. METHODS: To promote plaque formation in the aorta region, ApoE KO mice were fed a high cholesterol diet (0.15% cholesterol) and orally dosed with (2,2,3,4,4,6-d(6))-cholesterol over several weeks. Tissue sections of ~10 µm thickness were analyzed by MALDI-MSI using matrix deposition by either chemical sublimation or acoustic droplet ejection. RESULTS: MALDI-MSI yielded distinct spatial distribution information for a variety of lipid classes including specific lysophosphatidylcholines typically associated with atherosclerosis-related tissue damage such as phospholipase 2 (Lp-PLA(2)) that mediate chemotactic responses to inflammation (e.g. LPC 16:0, LPC 18:0 and LPC 18:1) as well as free cholesterol and cholesteryl esters that contribute to atheroma formation. MALDI mass spectra acquired from aorta tissue sections clearly distinguished non-esterified and esterified versions of (2,2,3,4,4,6-d(6))-cholesterol within aortic plaque regions and showed distinct spatial accumulation of the cholesterol tracer. CONCLUSIONS: The ability to couple stable isotope based protocols with MALDI-MSI enables a novel strategy to characterize the effects of therapeutic treatments on atherosclerotic plaque formation, regression and potential remodeling of the complex lipid components with high chemical specificity and spatiotemporal information.

Downregulation of MHC-I expression is prevalent but reversible in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (P < 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC.

Effects of anacetrapib on plasma lipids, apolipoproteins and PCSK9 in healthy, lean rhesus macaques.

Inhibition of cholesteryl ester transfer protein (CETP) has been vigorously pursued as a potential therapy to treat patients who are at an elevated risk for coronary artery disease. Anacetrapib, a novel CETP inhibitor, has been shown clinically to raise HDL cholesterol and reduce LDL cholesterol when provided as monotherapy or when co-administered with a statin. Preclinically, the effects of anacetrapib on the functionality and composition of HDL have been extensively studied. In contrast, the effects of anacetrapib on other parameters related to lipoprotein metabolism and cardiovascular risk have been difficult to explore. The aim of the present investigation was to evaluate the effects of anacetrapib in rhesus macaques and to compare these to effects reported in dyslipidemic humans. Our results from two separate studies show that administration of anacetrapib (150 mg/kg q.d. for 10 days) to rhesus macaques results in alterations in CETP activity (reduced by more than 70%) and HDL cholesterol (increased by more than 110%) which are similar to those reported in dyslipidemic humans. Levels of LDL cholesterol were reduced by more than 60%, an effect slightly greater than what has been observed clinically. Treatment with anacetrapib in this model was also found to lead to statistically significant reductions in plasma PCSK9 and to reduce cholesterol excursion in the combined chylomicron and remnant lipoprotein fraction isolated from plasma by fast protein liquid chromatography. Collectively, these data suggest that rhesus macaques may be a useful translational model to study the mechanistic effects of CETP inhibition.

Static and turnover kinetic measurement of protein biomarkers involved in triglyceride metabolism including apoB48 and apoA5 by LC/MS/MS.

LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid trafficking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantified from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the first time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates.

Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

BACKGROUND: Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. RESULTS: We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. CONCLUSION: Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

New methodologies for studying lipid synthesis and turnover: looking backwards to enable moving forwards.

Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Determination of low levels of 2H-labeling using high-resolution mass spectrometry: application in studies of lipid flux and beyond.

RATIONALE: The ability to measure low levels of (2)H-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering (2)H2O and then measuring the incorporation of (2)H into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. (13)C. Conventional approaches rely on coupling gas chromatographic separation of lipids with either quadrupole-mass spectrometry (q-MS) and/or pyrolysis-isotope ratio mass spectrometry (IRMS). The former is limited by high background labeling (primarily from (13)C) whereas the latter is not suitable for routine high-throughput analyses. METHODS: We have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the (2)H-enrichment of fatty acids and peptides. RESULTS: In contrast to IRMS, which requires ~30 min per analysis, it is possible to measure the (2)H-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in ~3 min per sample. In addition, Qq-FT-ICR MS enabled measurements of the (2)H-enrichment of peptides (which is not possible using IRMS). CONCLUSIONS: High-resolution mass spectrometry can be used to measure low levels of (2)H-labeling so we expect that this approach will enhance studies of metabolic flux that rely on (2)H-labeled tracers, e.g. (2)H2O. However, since the high-resolution analyses require greater amounts of a given analyte one potential limitation centers on the overall sensitivity. Presumably, future advances can overcome this barrier.

Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.

RATIONALE: Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo(a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects. METHODS: Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [(2)H3]-leucine tracer into protein-derived peptides. RESULTS: We demonstrated that it is feasible to quantify the incorporation of [(2)H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray. CONCLUSIONS: The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [(2)H3]-leucine.

Use of [13C18] oleic acid and mass isotopomer distribution analysis to study synthesis of plasma triglycerides in vivo: analytical and experimental considerations.

We have previously reported on a liquid chromatography-mass spectrometry method to determine the disposition of [(13)C18]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision, and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted, and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail, and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue; however, the present report provides investigators with the tools necessary to conduct such studies.

FAM129B is a novel regulator of Wnt/ß-catenin signal transduction in melanoma cells.

The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/ß-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/ß-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of ß-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/ß-catenin signaling.  Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a ß-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/ß-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/ß-catenin signaling to apoptosis in melanoma.

Rapid detection and quantification of apolipoprotein L1 genetic variants and total levels in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Human genetics studies in African Americans have shown a strong correlation between polymorphisms in the ApoL1 gene and chronic kidney disease (CKD). To gain further insight into the etiology of ApoL1-associated kidney diseases, the determination of circulating levels of both wild type as well as ApoL1 variants could be of significant use. To date, antibodies that discriminate between all three ApoL1 variant forms (wild type, G1 and G2) are not available. We aimed to develop a rapid method for detecting and quantifying ApoL1 variants and total levels in plasma. METHODS: Ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) in multiple-reaction monitoring acquisition mode was used to quantify ApoL1. RESULTS: We demonstrated that it is feasible to detect and quantify ApoL1 variants (wild type, G1 and G2), and total ApoL1 concentrations in plasma. ApoL1 genotypes determined by LC/MS agreed perfectly with the traditional method DNA sequencing for 74 human subjects. The method exhibited at least three orders of linearity with a lower limit of quantification of 10 nM. Moreover, the method can readily be multiplexed for the quantification of a panel of protein markers in a single sample. CONCLUSIONS: The method reported herein obviates the need to perform DNA genotyping of ApoL1 variants, which is of significant value in cases where stored samples are unsuitable for DNA analysis. More importantly, the method could potentially be of use in the early identification of individuals at risk of developing CKD, and for the stratification of patients for treatment with future ApoL1-modifying therapies.

A distinct Smoothened mutation causes severe cerebellar developmental defects and medulloblastoma in a novel transgenic mouse model.

Deregulated developmental processes in the cerebellum cause medulloblastoma, the most common pediatric brain malignancy. About 25 to 30% of cases are caused by mutations increasing the activity of the Sonic hedgehog (Shh) pathway, a critical mitogen in cerebellar development. The proto-oncogene Smoothened (Smo) is a key transducer of the Shh pathway. Activating mutations in Smo that lead to constitutive activity of the Shh pathway have been identified in human medulloblastoma. To understand the developmental and oncogenic effects of two closely positioned point mutations in Smo, we characterized NeuroD2-SmoA2 mice and compared them to NeuroD2-SmoA1 mice. While both SmoA1 and SmoA2 transgenes cause medulloblastoma with similar frequencies and timing, SmoA2 mice have severe aberrations in cerebellar development, whereas SmoA1 mice are largely normal during development. Intriguingly, neurologic function, as measured by specific tests, is normal in the SmoA2 mice despite extensive cerebellar dysplasia. We demonstrate how two nearly contiguous point mutations in the same domain of the encoded Smo protein can produce striking phenotypic differences in cerebellar development and organization in mice.

A multiplexed siRNA screening strategy to identify genes in the PARP pathway.

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).

Quantification of circulating D-dimer by peptide immunoaffinity enrichment and tandem mass spectrometry.

D-dimer is a product of the coagulation cascade and is associated with venous thromboembolism, disseminated intravascular coagulation, and additional clinical conditions. Despite its importance, D-dimer measurement has limited clinical utility due in part to the lack of reliable assays. The difficulty in developing an immunoassay that is specific for D-dimer arises from the inherent heterogeneity in its structure. In this report, we describe a highly specific method for the quantification of D-dimer level in human plasma. In our method, the reciprocally cross-linked peptide resulting from factor XIIIa-catalyzed dimerization of fibrin γ chains was selected to represent the D-dimer antigen. Using an antipeptide antibody, we enriched the cross-linked peptide from trypsin-digested plasma prior to quantitative analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The assay has a quantitative range of 500 pmol/L to 100 nmol/L in human plasma. In further characterization of the assay, we found that it exhibited good correlation with fibrinolytic activity in human donors and with thrombin generation and clot strength in an in vitro thromboelastography assay. These observations thus establish the biological relevance of the assay and suggest it may be a valuable biomarker in characterization and treatment of blood coagulation disorders.

Functional genomics identifies therapeutic targets for MYC-driven cancer.

MYC oncogene family members are broadly implicated in human cancers, yet are considered "undruggable" as they encode transcription factors. MYC also carries out essential functions in proliferative tissues, suggesting that its inhibition could cause severe side effects. We elected to identify synthetic lethal interactions with c-MYC overexpression (MYC-SL) in a collection of ~3,300 druggable genes, using high-throughput siRNA screening. Of 49 genes selected for follow-up, 48 were confirmed by independent retesting and approximately one-third selectively induced accumulation of DNA damage, consistent with enrichment in DNA-repair genes by functional annotation. In addition, genes involved in histone acetylation and transcriptional elongation, such as TRRAP and BRD4, were identified, indicating that the screen revealed known MYC-associated pathways. For in vivo validation we selected CSNK1e, a kinase whose expression correlated with MYCN amplification in neuroblastoma (an established MYC-driven cancer). Using RNAi and available small-molecule inhibitors, we confirmed that inhibition of CSNK1e halted growth of MYCN-amplified neuroblastoma xenografts. CSNK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms, whereas our data provide a previously unknown link with oncogenic MYC. Furthermore, expression of CSNK1e correlated with c-MYC and its transcriptional signature in other human cancers, indicating potential broad therapeutic implications of targeting CSNK1e function. In summary, through a functional genomics approach, pathways essential in the context of oncogenic MYC but not to normal cells were identified, thus revealing a rich therapeutic space linked to a previously "undruggable" oncogene.

Synthetic lethality of PARP inhibition in BRCA-network disrupted tumor cells is associated with interferon pathway activation and enhanced by interferon-γ.

Tumor suppressor genes BRCA1 and BRCA2 function in a complex gene network that regulates homologous recombination and DNA double-strand break repair. Disruption of the BRCA-network through gene mutation, deletion, or RNAi-mediated silencing can sensitize cells to small molecule inhibitors of poly (ADP-ribose) polymerase (PARPi). Here, we demonstrate that BRCA-network disruption in the presence of PARPi leads to the selective induction and enhancement of interferon pathway and apoptotic gene expression in cultured tumor cells. In addition, we report PARPi cytotoxicity in BRCA1-deficient tumor cells is enhanced >10-fold when combined with interferon-γ. These findings establish a link between synthetic lethality of PARPi in BRCA-network disrupted cells and interferon pathway activation triggered by genetic instability.

p53-responsive miR-194 inhibits thrombospondin-1 and promotes angiogenesis in colon cancers.

Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis encoded by the THBS1 gene, whose promoter is activated by p53. In advanced colorectal cancers (CRC), its expression is sustained or even slightly increased despite frequent loss of p53. Here, we determined that in HCT116 CRC cells, p53 activates the THBS1 primary transcript, but fails to boost THBS1 mRNA or protein levels, implying posttranscriptional regulation by microRNAs (miRNA). In a global miRNA gain-of-function screen done in the Dicer-deficient HCT116 variant, several miRNAs negatively regulated THBS1 mRNA and protein levels, one of them being miR-194. Notably, in agreement with published data, p53 upregulated miR-194 expression in THBS1 retrovirus-transduced HCT116 cells, leading to decreased TSP-1 levels. This negative effect was mediated by a single miR-194 complementary site in the THBS1 3'-untranslated region, and its elimination resulted in TSP-1 reactivation, impaired angiogenesis in Matrigel plugs, and reduced growth of HCT116 xenografts. Conversely, transient overexpression of miR-194 in HCT116/THBS1 cells boosted Matrigel angiogenesis, and its stable overexpression in Ras-induced murine colon carcinomas increased microvascular densities and vessel sizes. Although the overall contribution of miR-194 to neoplastic growth is context dependent, p53-induced activation of this GI tract-specific miRNA during ischemia could promote angiogenesis and facilitate tissue repair.