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1.  Registered report: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET 
eLife  null;18:e07383.
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.
PMCID: PMC4749561  PMID: 26826285
Reproducibility Project: Cancer Biology; methodology; exosomes; metastasis; receptor tyrosine kinase; MET; Mouse
2.  Sustained inhibition of PKCα reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model 
Oncogene  2010;30(3):323-333.
Metastasis is the major reason for breast cancer-related deaths. Although there is a host of indirect evidence for a role of PKCα in primary breast cancer growth, its role in the molecular pathways leading to metastasis have not been comprehensively studied. By treating mice with αV5-3, a novel peptide inhibitor selective for PKCα, we were able to determine how PKCα regulates metastasis of mammary cancer cells using a syngeneic and orthotopic model. The primary tumor growth was not affected by αV5-3 treatment. However, the mortality rate was reduced and metastasis in the lung decreased by more than 90% in the αV5-3-treated mice relative to the control-treated mice. αV5-3 treatment reduced intravasation by reducing MMP-9 activities. αV5-3 treatment also reduced lung seeding of tumor cells and decreased cell migration, effects that were accompanied by a reduction in NFκB-activity and cell surface levels of the CXCL12 receptor, CXCR4. αV5-3 treatment caused no apparent toxicity in non-tumor bearing naïve mice. Rather, inhibiting PKCα protected against liver damage and increased the number of immune cells in tumor-bearing mice. Importantly, αV5-3 showed superior efficacy relative to anti-CXCR4 antibody in reducing metastasis, in vivo. Together, these data show that pharmacological inhibition of PKCα effectively reduces mammary cancer metastasis by targeting intravasation and lung seeding steps in the metastatic process and suggest that PKCα-specific inhibitors, such as αV5-3, can be used to study the mechanistic roles of PKCα specifically and may provide a safe and effective treatment for the prevention of lung metastasis of breast cancer patients.
PMCID: PMC3767436  PMID: 20856202
bioluminescence; mammary cancer; metastasis and protein kinase C
3.  Protein kinase C epsilon is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes 
Oncogene  2011;31(20):2593-2600.
Protein kinase C (PKC) ε, a member of the novel PKC family, plays key roles in mitogenesis and survival in normal and cancer cells. PKCε is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a shRNA approach, here we established that PKCε is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCε selective inhibitor peptide, εV1-2, reduced xenograft growth in mice. Both RNAi depletion and pharmacological inhibition of PKCε caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCε-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain (CARD)-containing proteins, and TNF ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis (GSEA) revealed that a vast majority of the genes changed in PKCε-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCε is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCε may represent an attractive therapeutic target for NSCLC.
PMCID: PMC3432976  PMID: 21996750
PKCε; non-small cell lung carcinoma; tumorigenesis; cell survival; apoptotic genes
4.  PKCδ activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells 
The Prostate  2010;71(9):946-954.
PKCδ is generally known as a pro-apoptotic and anti-proliferative enzyme in human prostate cancer cells.
Here, we investigated the role of PKCδ on the growth of PC-3 human prostate cancer cells in vivo and in vitro.
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.
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.
PMCID: PMC3544470  PMID: 21541971
angiogenesis; HIF-1α; NADPH oxidase; prostate cancer; protein kinase C
5.  Discovery and preclinical validation of drug indications using compendia of public gene expression data 
Science translational medicine  2011;3(96):96ra77.
The application of established drug compounds to novel 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 employ high-throughput experimental approaches to assess a compound’s potential therapeutic qualities. Here we present a systematic computational approach to predict novel therapeutic indications based on 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 demonstrate the ability to recover many known drug and disease relationships using computationally derived therapeutic potentials, and also predict many new indications for these drugs. We experimentally validated a prediction for the anti-ulcer drug cimetidine as a candidate therapeutic in the treatment of lung adenocarcinoma, and demonstrate both in vitro and in vivo using mouse xenograft models. This novel computational method provides a novel and systematic approach to reposition established drugs to treat a wide range of human diseases.
PMCID: PMC3502016  PMID: 21849665
6.  Long noncoding RNA HOTAIR reprograms chromatin state to promote cancer metastasis 
Nature  2010;464(7291):1071-1076.
Large intervening noncoding RNAs (lincRNAs) are pervasively transcribed in the genome1, 2, 3 yet their potential involvement in human disease is not well understood4,5. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodeling activities6,7,8. 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 tumors and metastases, and HOTAIR expression level in primary tumors 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 suggest that lincRNAs play active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.
PMCID: PMC3049919  PMID: 20393566
7.  Centrosomal PKCβII and pericentrin are critical for human prostate cancer growth and angiogenesis 
Cancer research  2008;68(16):6831-6839.
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 PKCβII was activated during increased angiogenesis and that PKCβII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCβII-selective inhibitor, βIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCβII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCβII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be upregulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is upregulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCβII inhibitor such as βIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.
PMCID: PMC2597632  PMID: 18701509
8.  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.
PMCID: PMC2815321  PMID: 20126370
Hepatocellular carcinoma; DHEA; malate dehydrogenase; NADPH and glucose 6-phosphate dehydrogenase

Results 1-8 (8)