Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element.

Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.

N6-Methyladenosine Modification Controls Circular RNA Immunity.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.

Aldehyde dehydrogenase 2*2 knock-in mice show increased reactive oxygen species production in response to cisplatin treatment.

BACKGROUND: The aldehyde dehydrogenase (ALDH) enzyme family metabolizes and detoxifies both exogenous and endogenous aldehydes. Since chemotherapeutic agents, such as cisplatin, generate cytotoxic aldehydes and oxidative stress, and chemoresistant cancer cells express high levels of ALDH enzymes, we hypothesized that different ALDH expression within cells may show different chemosensitivity. ALDH2 has the lowest Km for acetaldehyde among ALDH isozymes and detoxifies acetaldehydes in addition to other reactive aldehydes, such as 4-hydroxy-nonenal, malondialdehyde and acrolein produced from lipid peroxidation by reactive oxygen species (ROS). Thus, cells with an ALDH2 variant may sensitize them to these ROS-inducing chemotherapy drugs. METHODS: Here, we used wild type C57BL/6 mice and ALDH2*2 knock-in mutant mice and compared the basal level of ROS in different tissues. Then, we treated the mice with cisplatin, isolated cells from organs and fractionated them into lysates containing mitochondrial and cytosolic fractions, treated with cisplatin again in vitro, and compared the level of ROS generated. RESULTS: We show that overall ROS production increases with cisplatin treatment in cells with ALDH2 mutation. The treatment of cisplatin in the wild type mice did not change the level of ROS compared to PBS treated controls. In contrast, ALDH2*2 knock-in mutant mice showed a significantly increased level of ROS compared to wild type mice in tongue, lung, kidney and brain tissues without any treatment. ALDH2*2 mutant mice showed 20% of the ALDH2 activity in the kidney compared to wild type mice. Treatment of ALDH2*2 mutant mice with cisplatin showed increased ROS levels in the mitochondrial fraction of kidney. In the cytosolic fraction, treatment of mutant mice with cisplatin increased ROS levels in lung and brain compared to PBS treated controls. Furthermore, ALDH2*2 mutant mice treated with cisplatin showed increased cytotoxicity in the kidney cells compared to PBS treated mutant controls. CONCLUSIONS: These data indicate that deficiency in ALDH2 activity may contribute to increased cisplatin sensitivity and cytotoxicity by producing more ROS by the treatment. Based on these data, the amount of cisplatin used in patients may need to be adjusted based on their ALDH2 variant profile.

Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.

Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodelling activities. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumours and metastases, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

A noise robust image reconstruction using slice aware cycle interpolator network for parallel imaging in MRI.

BACKGROUND: Reducing Magnetic resonance imaging (MRI) scan time has been an important issue for clinical applications. In order to reduce MRI scan time, imaging acceleration was made possible by undersampling k-space data. This is achieved by leveraging additional spatial information from multiple, independent receiver coils, thereby reducing the number of sampled k-space lines. PURPOSE: The aim of this study is to develop a deep-learning method for parallel imaging with a reduced number of auto-calibration signals (ACS) lines in noisy environments. METHODS: A cycle interpolator network is developed for robust reconstruction of parallel MRI with a small number of ACS lines in noisy environments. The network estimates missing (unsampled) lines of each coil data, and these estimated missing lines are then utilized to re-estimate the sampled k-space lines. In addition, a slice aware reconstruction technique is developed for noise-robust reconstruction while reducing the number of ACS lines. We conducted an evaluation study using retrospectively subsampled data obtained from three healthy volunteers at 3T MRI, involving three different slice thicknesses (1.5, 3.0, and 4.5 mm) and three different image contrasts (T1w, T2w, and FLAIR). RESULTS: Despite the challenges posed by substantial noise in cases with a limited number of ACS lines and thinner slices, the slice aware cycle interpolator network reconstructs the enhanced parallel images. It outperforms RAKI, effectively eliminating aliasing artifacts. Moreover, the proposed network outperforms GRAPPA and demonstrates the ability to successfully reconstruct brain images even under severe noisy conditions. CONCLUSIONS: The slice aware cycle interpolator network has the potential to improve reconstruction accuracy for a reduced number of ACS lines in noisy environments.

Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases.

Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated ß-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.

PICASSO allows ultra-multiplexed fluorescence imaging of spatially overlapping proteins without reference spectra measurements.

Ultra-multiplexed fluorescence imaging requires the use of spectrally overlapping fluorophores to label proteins and then to unmix the images of the fluorophores. However, doing this remains a challenge, especially in highly heterogeneous specimens, such as the brain, owing to the high degree of variation in the emission spectra of fluorophores in such specimens. Here, we propose PICASSO, which enables more than 15-color imaging of spatially overlapping proteins in a single imaging round without using any reference emission spectra. PICASSO requires an equal number of images and fluorophores, which enables such advanced multiplexed imaging, even with bandpass filter-based microscopy. We show that PICASSO can be used to achieve strong multiplexing capability in diverse applications. By combining PICASSO with cyclic immunofluorescence staining, we achieve 45-color imaging of the mouse brain in three cycles. PICASSO provides a tool for multiplexed imaging with high accessibility and accuracy for a broad range of researchers.

PKCδ activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells.

BACKGROUND: PKCδ is generally known as a pro-apoptotic and anti-proliferative enzyme in human prostate cancer cells. METHODS: Here, we investigated the role of PKCδ on the growth of PC-3 human prostate cancer cells in vivo and in vitro. RESULTS: We found that sustained treatment with a specific PKCδ activator (ψδ receptor for active C kinase, ψδRACK) increased growth of PC-3 xenografts. There was increased levels of HIF-1α, vascular endothelial growth factor and CD31-positive cells in PC-3 xenografts, representative of increased tumor angiogenesis. Mechanistically, PKCδ activation increased the levels of reactive oxygen species (ROS) by binding to and phosphorylating NADPH oxidase, which induced its activity. Also, PKCδ-induced activation of NADPH oxidase increased the level of HIF-1α. CONCLUSIONS: Our results using tumors from the PC-3 xenograft model suggest that PKCδ activation increases angiogenic activity in androgen-independent PC-3 prostate cancer cells by increasing NADPH oxidase activity and HIF-1α levels and thus may partly be responsible for increased angiogenesis in advanced prostate cancer.

Overcoming anticancer resistance by photodynamic therapy-related efflux pump deactivation and ultrasound-mediated improved drug delivery efficiency.

One of the major obstacles to successful chemotherapy is multi-drug resistance (MDR). A multi-drug resistant cancerous cell abnormally overexpresses membrane transporters that pump anticancer drugs out of the cell, resulting in low anticancer drug delivery efficiency. To overcome the limitation, many attempts have been performed to inhibit the abilities of efflux receptors chemically or genetically or to increase the delivery efficiency of anticancer drugs. However, the results have not yet been satisfactory. In this study, we developed nanoparticle-microbubble complexes (DOX-NPs/Ce6-MBs) by conjugating doxorubicin loaded human serum albumin nanoparticles (DOX-NPs) onto the surface of Chlorin e6 encapsulated microbubbles (Ce6-MBs) in order to maximize anticancer efficiency by overcoming MDR. Under the ultrasound irradiation, DOX-NPs and Ce6 encapsulating self-assembled liposomes or micelles were effectively delivered into the cells due to the sonoporation effect caused by the microbubble cavitation. At the same time, reactive oxygen (ROS) generated from intracellularly delivered Ce6 by laser irradiation arrested the activity of ABCG2 efflux receptor overexpressed in doxorubicin-resistant breast cancer cells (MCF-7/ADR), resulting in increased the chemotherapy efficacy. In addition, the total number of side population cells that exhibit the properties of cancer stem-like cells were also reduced by the combination of photodynamic therapy and chemotherapy. In conclusion, DOX-NPs/Ce6-MBs will provide a platform for simultaneously overcoming MDR and increasing drug delivery and therefore, treatment efficiency, under ultrasound irradiation.

Replication study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Lesnik et al., 2016) that described how we intended to replicate selected experiments from the paper 'Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET' (Peinado et al., 2012). Here we report the results. We regenerated tumor cells stably expressing a short hairpin to reduce Met expression (shMet) using the same highly metastatic mouse melanoma cell line (B16-F10) as the original study, which efficiently downregulated Met in B16F10 cells similar to the original study (Supplementary Figure 5A; Peinado et al., 2012). Exosomes from control cells expressed Met, which was reduced in exosomes from shMet cells; however, we were unable to reliably detect phosphorylated Met in exosomes. We tested the effect of exosome-dependent Met signaling on primary tumor growth and metastasis. Similar to the results in the original study, we did not find a statistically significant change in primary tumor growth. Measuring lung and femur metastases, we found a small increase in metastatic burden with exosomes from control cells that was diminished when Met expression was reduced; however, while the effects were in the same direction as the original study (Figure 4E; Peinado et al., 2012), they were not statistically significant. Differences between the original study and this replication attempt, such as level of knockdown efficiency, cell line genetic drift, sample sizes, study endpoints, and variability of observed metastatic burden, are factors that might have influenced the outcomes. Finally, we report meta-analyses for each result.

Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor.

Chemoresistant cancer cells express high levels of aldehyde dehydrogenases (ALDHs), particularly in head and neck squamous cell carcinoma (HNSCC). The ALDH family of enzymes detoxify both exogenous and endogenous aldehydes. Since many chemotherapeutic agents, such as cisplatin, result in the generation of cytotoxic aldehydes and oxidative stress, we hypothesized that cells expressing high levels of ALDH may be more chemoresistant due to their increased detoxifying capacity and that inhibitors of ALDHs may sensitize them to these drugs. Here, we show that overall ALDH activity is increased with cisplatin treatment of HNSCC and that ALDH3A1 protein expression is particularly enriched in cells treated with cisplatin. Activation of ALDH3A1 by a small molecule activator (Alda-89) increased survival of HNSCC cells treated with cisplatin. Conversely, treatment with a novel small molecule ALDH inhibitor (Aldi-6) resulted in a marked decrease in cell viability, and the combination of Aldi-6 and cisplatin resulted in a more pronounced reduction of cell viability and a greater reduction in tumor burden in vivo than what was observed with cisplatin alone. These data indicate that ALDH3A1 contributes to cisplatin resistance in HNSCC and that the targeting of ALDH, specifically, ALDH3A1, appears to be a promising strategy in this disease.

Registered report: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET" by Peinado and colleagues, published in Nature Medicine in 2012 (Peinado et al., 2012). The key experiments being replicated are from Figures 4E, as well as Supplementary Figures 1C and 5A. In these experiments, Peinado and colleagues show tumor exosomes enhance metastasis to bones and lungs, which is diminished by reducing Met expression in exosomes (Peinado et al., 2012). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Boosting Cancer Immunotherapy with Anti-CD137 Antibody Therapy.

In the past 5 years, immunomodulatory antibodies have revolutionized cancer immunotherapy. CD137, a member of the tumor necrosis factor receptor superfamily, represents a promising target for enhancing antitumor immune responses. CD137 helps regulate the activation of many immune cells, including CD4(+) T cells, CD8(+) T cells, dendritic cells, and natural killer cells. Recent studies indicate that the antitumor efficacy of therapeutic tumor-targeting antibodies can be augmented by the addition of agonistic antibodies targeting CD137. As ligation of CD137 provides a costimulatory signal in multiple immune cell subsets, combination therapy of CD137 antibody with therapeutic antibodies and/or vaccination has the potential to improve cancer treatment. Recently, clinical trials of combination therapies with agonistic anti-CD137 mAbs have been launched. In this review, we discuss the recent advances and clinical promise of agonistic anti-CD137 monoclonal antibody therapy.

In vivo demonstration of enhanced radiotherapy using rare earth doped titania nanoparticles.

Radiation therapy is often limited by damage to healthy tissue and associated side-effects; restricting radiation to ineffective doses. Preferential incorporation of materials into tumour tissue can enhance the effect of radiation. Titania has precedent for use in photodynamic therapy (PDT), generating reactive oxygen species (ROS) upon photoexcitation, but is limited by the penetration depth of UV light. Optimization of a nanomaterial for interaction with X-rays could be used for deep tumour treatment. As such, titania nanoparticles were doped with gadolinium to optimize the localized energy absorption from a conventional medical X-ray, and further optimized by the addition of other rare earth (RE) elements. These elements were selected due to their large X-ray photon interaction cross-section, and potential for integration into the titania crystal structure. Specific activation of the nanoparticles by X-ray can result in generation of ROS leading to cell death in a tumour-localized manner. We show here that intratumoural injection of RE doped titania nanoparticles can enhance the efficacy of radiotherapy in vivo.

Discovery and preclinical validation of drug indications using compendia of public gene expression data.

The application of established drug compounds to new therapeutic indications, known as drug repositioning, offers several advantages over traditional drug development, including reduced development costs and shorter paths to approval. Recent approaches to drug repositioning use high-throughput experimental approaches to assess a compound's potential therapeutic qualities. Here, we present a systematic computational approach to predict novel therapeutic indications on the basis of comprehensive testing of molecular signatures in drug-disease pairs. We integrated gene expression measurements from 100 diseases and gene expression measurements on 164 drug compounds, yielding predicted therapeutic potentials for these drugs. We recovered many known drug and disease relationships using computationally derived therapeutic potentials and also predict many new indications for these 164 drugs. We experimentally validated a prediction for the antiulcer drug cimetidine as a candidate therapeutic in the treatment of lung adenocarcinoma, and demonstrate its efficacy both in vitro and in vivo using mouse xenograft models. This computational method provides a systematic approach for repositioning established drugs to treat a wide range of human diseases.

Dehydroepiandrosterone supplement increases malate dehydrogenase activity and decreases NADPH-dependent antioxidant enzyme activity in rat hepatocellular carcinogenesis.

Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has antioxidant effects. Here we aimed to determine the mechanism of actions of DHEA, in comparison to vitamin E, in a chemically-induced hepatocellular carcinoma model in rats. Sprague-Dawley rats were administered with control diet without a carcinogen, diets with 1.5% vitamin E, 0.5% DHEA and both of the compounds with a carcinogen for 6 weeks. The doses were previously reported to have anti-cancer effects in animals without known toxicities. With DHEA treatment, cytosolic malate dehydrogenase activities were significantly increased by ~5 fold and glucose 6-phosphate dehydrogenase activities were decreased by ~25% compared to carcinogen treated group. Activities of Se-glutathione peroxidase in the cytotol was decreased significantly with DHEA treatment, confirming its antioxidative effect. However, liver microsomal cytochrome P-450 content and NADPH-dependent cytochrome P-450 reductase activities were not altered with DHEA treatment. Vitamin E treatment decreased cytosolic Se-glutathione peroxidase activities in accordance with our previous reports. However, vitamin E did not alter glucose 6-phosphate dehydrogenase or malate dehydrogenase activities. Our results suggest that DHEA may have decreased tumor nodule formation and reduced lipid peroxidation as previously reported, possibly by increasing the production of NADPH, a reducing equivalent for NADPH-dependent antioxidant enzymes. DHEA treatment tended to reduce glucose 6-phosphate dehydrogenase activities, which may have resulted in limited supply for de novo synthesis of DNA via inhibiting the hexose monophophaste pathway. Although both DHEA and vitamin E effectively reduced preneoplastic foci in this model, they seemed to function in different mechanisms. In conclusion, DHEA may be used to reduce hepatocellular carcinoma growth by targeting NADPH synthesis, cell proliferation and anti-oxidant enzyme activities during tumor growth.

Centrosomal PKCbetaII and pericentrin are critical for human prostate cancer growth and angiogenesis.

Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth, but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCbetaII was activated during increased angiogenesis and that PKCbetaII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCbetaII-selective inhibitor, betaIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCbetaII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCbetaII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be up-regulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is up-regulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCbetaII inhibitor such as betaIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.