50 results on '"Rajasekhar VK"'
Search Results
2. Augmenting CAR T-cell Functions with LIGHT.
- Author
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Cai W, Tanaka K, Mi X, Rajasekhar VK, Khan JF, Yoo S, de Stanchina E, Rahman J, Mathew S, Abrahimi P, Souness S, Purdon TJ, McDowell JR, Meyerberg J, Fujino T, Healey JH, Abdel-Wahab O, Scheinberg DA, Brentjens RJ, and Daniyan AF
- Subjects
- Humans, Animals, Cell Line, Tumor, Cytotoxicity, Immunologic, Mice, Lymphotoxin beta Receptor immunology, Lymphotoxin beta Receptor metabolism, Antigens, Neoplasm immunology, Xenograft Model Antitumor Assays, Neoplasms immunology, Neoplasms therapy, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen metabolism, Immunotherapy, Adoptive methods, Tumor Necrosis Factor Ligand Superfamily Member 14 metabolism, T-Lymphocytes immunology
- Abstract
Chimeric antigen receptor (CAR) T-cell therapy has resulted in remarkable clinical success in the treatment of B-cell malignancies. However, its clinical efficacy in solid tumors is limited, primarily by target antigen heterogeneity. To overcome antigen heterogeneity, we developed CAR T cells that overexpress LIGHT, a ligand of both lymphotoxin-β receptor on cancer cells and herpes virus entry mediator on immune cells. LIGHT-expressing CAR T cells displayed both antigen-directed cytotoxicity mediated by the CAR and antigen-independent killing mediated through the interaction of LIGHT with lymphotoxin-β receptor on cancer cells. Moreover, CAR T cells expressing LIGHT had immunostimulatory properties that improved the cells' proliferation and cytolytic profile. These data indicate that LIGHT-expressing CAR T cells may provide a way to eliminate antigen-negative tumor cells to prevent antigen-negative disease relapse., (©2024 American Association for Cancer Research.)
- Published
- 2024
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3. Multi-parametric atlas of the pre-metastatic liver for prediction of metastatic outcome in early-stage pancreatic cancer.
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Bojmar L, Zambirinis CP, Hernandez JM, Chakraborty J, Shaashua L, Kim J, Johnson KE, Hanna S, Askan G, Burman J, Ravichandran H, Zheng J, Jolissaint JS, Srouji R, Song Y, Choubey A, Kim HS, Cioffi M, van Beek E, Sigel C, Jessurun J, Velasco Riestra P, Blomstrand H, Jönsson C, Jönsson A, Lauritzen P, Buehring W, Ararso Y, Hernandez D, Vinagolu-Baur JP, Friedman M, Glidden C, Firmenich L, Lieberman G, Mejia DL, Nasar N, Mutvei AP, Paul DM, Bram Y, Costa-Silva B, Basturk O, Boudreau N, Zhang H, Matei IR, Hoshino A, Kelsen D, Sagi I, Scherz A, Scherz-Shouval R, Yarden Y, Oren M, Egeblad M, Lewis JS, Keshari K, Grandgenett PM, Hollingsworth MA, Rajasekhar VK, Healey JH, Björnsson B, Simeone DM, Tuveson DA, Iacobuzio-Donahue CA, Bromberg J, Vincent CT, O'Reilly EM, DeMatteo RP, Balachandran VP, D'Angelica MI, Kingham TP, Allen PJ, Simpson AL, Elemento O, Sandström P, Schwartz RE, Jarnagin WR, and Lyden D
- Subjects
- Humans, Male, Female, Middle Aged, Aged, Biopsy, Neoplasm Staging, Pancreatectomy, Extracellular Traps metabolism, Prognosis, Pancreatic Neoplasms pathology, Pancreatic Neoplasms genetics, Pancreatic Neoplasms surgery, Liver Neoplasms secondary, Liver Neoplasms pathology, Liver Neoplasms genetics, Liver pathology, Liver metabolism
- Abstract
Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B
+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)- Published
- 2024
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4. GLUT1 overexpression enhances CAR T cell metabolic fitness and anti-tumor efficacy.
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Shi Y, Kotchetkov IS, Dobrin A, Hanina SA, Rajasekhar VK, Healey JH, and Sadelain M
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- Animals, Humans, Mice, Cell Line, Tumor, Disease Models, Animal, Precursor Cell Lymphoblastic Leukemia-Lymphoma therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics, T-Lymphocytes immunology, T-Lymphocytes metabolism, Tumor Microenvironment immunology, Glucose Transporter Type 1 genetics, Glucose Transporter Type 1 metabolism, Immunotherapy, Adoptive methods, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen metabolism, Receptors, Chimeric Antigen immunology, Xenograft Model Antitumor Assays
- Abstract
The tumor microenvironment presents many obstacles to effective chimeric antigen receptor (CAR) T cell therapy, including glucose competition from tumor and myeloid cells. Using mouse models of acute lymphoblastic leukemia (ALL), renal cell carcinoma (RCC), and glioblastoma (GBM), we show that enforced expression of the glucose transporter GLUT1 enhances anti-tumor efficacy and promotes favorable CAR-T cell phenotypes for two clinically relevant CAR designs, 19-28z and IL13Rα2-BBz. In the NALM6 ALL model, 19-28z-GLUT1 promotes T stem cell-like memory formation and prolongs survival. RNA sequencing of these CAR-T cells reveals that the overexpression of GLUT1, but not GLUT3, enriches for genes involved in glycolysis, mitochondrial respiration, and memory precursor phenotypes. Extending these data, 19-28z-GLUT1 CAR-T cells improve tumor control and response to rechallenge in an RCC patient-derived xenograft model. Furthermore, IL13Rα2-BBz CAR-T cells overexpressing GLUT1 prolong the survival of mice bearing orthotopic GBMs and exhibit decreased exhaustion markers. This novel engineering approach can offer a competitive advantage to CAR-T cells in harsh tumor environments where glucose is limiting., Competing Interests: Declaration of interests The authors declare no financial competing interests. M.S. reports research funding from Takeda Pharmaceuticals, Atara Biotherapeutics, Fate Therapeutics, and Mnemo Therapeutics, all of which are unrelated to the present research. M.S. is a scientific cofounder of Mnemo Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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5. Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization.
- Author
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Ben-Chetrit N, Niu X, Sotelo J, Swett AD, Rajasekhar VK, Jiao MS, Stewart CM, Bhardwaj P, Kottapalli S, Ganesan S, Loyher PL, Potenski C, Hannuna A, Brown KA, Iyengar NM, Giri DD, Lowe SW, Healey JH, Geissmann F, Sagi I, Joyce JA, and Landau DA
- Abstract
Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80
high Sca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo , as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy., Competing Interests: The authors. D.A.L and N.B.-C are co-inventors on a patent application filed by Weill Cornell Medicine related to the findings in this manuscript. DAL has served as a consultant for Abbvie, AstraZeneca and Illumina and is on the Scientific Advisory Board of Mission Bio, Pangea, Alethiomics, and C2i Genomics; DAL has received prior research funding from BMS, 10x Genomics, Ultima Genomics, and Illumina unrelated to the current manuscript.- Published
- 2023
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6. Tumour extracellular vesicles and particles induce liver metabolic dysfunction.
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Wang G, Li J, Bojmar L, Chen H, Li Z, Tobias GC, Hu M, Homan EA, Lucotti S, Zhao F, Posada V, Oxley PR, Cioffi M, Kim HS, Wang H, Lauritzen P, Boudreau N, Shi Z, Burd CE, Zippin JH, Lo JC, Pitt GS, Hernandez J, Zambirinis CP, Hollingsworth MA, Grandgenett PM, Jain M, Batra SK, DiMaio DJ, Grem JL, Klute KA, Trippett TM, Egeblad M, Paul D, Bromberg J, Kelsen D, Rajasekhar VK, Healey JH, Matei IR, Jarnagin WR, Schwartz RE, Zhang H, and Lyden D
- Subjects
- Animals, Mice, Cytochrome P-450 Enzyme System genetics, Tumor Microenvironment, Tumor Necrosis Factor-alpha antagonists & inhibitors, Tumor Necrosis Factor-alpha metabolism, Liver Neoplasms secondary, Humans, Inflammation metabolism, Palmitic Acid metabolism, Kupffer Cells, Oxidative Phosphorylation, rab27 GTP-Binding Proteins deficiency, Extracellular Vesicles metabolism, Fatty Acids metabolism, Fatty Liver drug therapy, Fatty Liver etiology, Fatty Liver metabolism, Fatty Liver prevention & control, Liver metabolism, Liver pathology, Liver physiopathology, Pancreatic Neoplasms metabolism, Pancreatic Neoplasms pathology
- Abstract
Cancer alters the function of multiple organs beyond those targeted by metastasis
1,2 . Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
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7. ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance.
- Author
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Strillacci A, Sansone P, Rajasekhar VK, Turkekul M, Boyko V, Meng F, Houck-Loomis B, Brown D, Berger MF, Hendrickson RC, Chang Q, de Stanchina E, Pareja F, Reis-Filho JS, Rajappachetty RS, Del Priore I, Liu B, Cai Y, Penson A, Mastroleo C, Berishaj M, Borsetti F, Spisni E, Lyden D, Chandarlapaty S, and Bromberg J
- Abstract
Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(-) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance., (© 2022. The Author(s).)
- Published
- 2022
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8. Pharmacologically controlling protein-protein interactions through epichaperomes for therapeutic vulnerability in cancer.
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Joshi S, Gomes ED, Wang T, Corben A, Taldone T, Gandu S, Xu C, Sharma S, Buddaseth S, Yan P, Chan LYL, Gokce A, Rajasekhar VK, Shrestha L, Panchal P, Almodovar J, Digwal CS, Rodina A, Merugu S, Pillarsetty N, Miclea V, Peter RI, Wang W, Ginsberg SD, Tang L, Mattar M, de Stanchina E, Yu KH, Lowery M, Grbovic-Huezo O, O'Reilly EM, Janjigian Y, Healey JH, Jarnagin WR, Allen PJ, Sander C, Erdjument-Bromage H, Neubert TA, Leach SD, and Chiosis G
- Subjects
- Animals, Female, Heterografts, Humans, Mice, Signal Transduction, Epigenesis, Genetic, Genome, Molecular Chaperones genetics, Neoplasms genetics, Protein Interaction Mapping, Protein Interaction Maps
- Abstract
Cancer cell plasticity due to the dynamic architecture of interactome networks provides a vexing outlet for therapy evasion. Here, through chemical biology approaches for systems level exploration of protein connectivity changes applied to pancreatic cancer cell lines, patient biospecimens, and cell- and patient-derived xenografts in mice, we demonstrate interactomes can be re-engineered for vulnerability. By manipulating epichaperomes pharmacologically, we control and anticipate how thousands of proteins interact in real-time within tumours. Further, we can essentially force tumours into interactome hyperconnectivity and maximal protein-protein interaction capacity, a state whereby no rebound pathways can be deployed and where alternative signalling is supressed. This approach therefore primes interactomes to enhance vulnerability and improve treatment efficacy, enabling therapeutics with traditionally poor performance to become highly efficacious. These findings provide proof-of-principle for a paradigm to overcome drug resistance through pharmacologic manipulation of proteome-wide protein-protein interaction networks., (© 2021. The Author(s).)
- Published
- 2021
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9. CSF1/CSF1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment.
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Fujiwara T, Yakoub MA, Chandler A, Christ AB, Yang G, Ouerfelli O, Rajasekhar VK, Yoshida A, Kondo H, Hata T, Tazawa H, Dogan Y, Moore MAS, Fujiwara T, Ozaki T, Purdue E, and Healey JH
- Subjects
- Animals, Apoptosis, Bone Neoplasms drug therapy, Bone Neoplasms immunology, Bone Neoplasms metabolism, Bone Neoplasms pathology, CD8-Positive T-Lymphocytes immunology, Cell Proliferation, Female, Gene Expression Regulation, Neoplastic, Humans, Lung Neoplasms drug therapy, Lung Neoplasms immunology, Lung Neoplasms metabolism, Lung Neoplasms secondary, Mice, Mice, Inbred C3H, Osteosarcoma drug therapy, Osteosarcoma metabolism, Osteosarcoma pathology, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Aminopyridines pharmacology, Lymphocytes, Tumor-Infiltrating immunology, Macrophage Colony-Stimulating Factor antagonists & inhibitors, Osteosarcoma immunology, Pyrroles pharmacology, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor antagonists & inhibitors, Tumor Microenvironment, Tumor-Associated Macrophages immunology
- Abstract
Colony-stimulating factor 1 (CSF1) is a primary regulator of the survival, proliferation, and differentiation of monocyte/macrophage that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). Considering current advances in understanding the role of the inflammatory tumor microenvironment, targeting the components of the sarcoma microenvironment, such as TAMs, is a viable strategy. Here, we investigated the effect of PLX3397 (pexidartinib) as a potent inhibitor of the CSF1 receptor (CSF1R). PLX3397 was recently approved by the Food and Drug Administration (FDA) to treat tenosynovial giant cell tumor and reprogram TAMs whose infiltration correlates with unfavorable prognosis of sarcomas. First, we confirmed by cytokine arrays of tumor-conditioned media (TCM) that cytokines including CSF1 are secreted from LM8 osteosarcoma cells and NFSa fibrosarcoma cells. The TCM, like CSF1, stimulated ERK1/2 phosphorylation in bone marrow-derived macrophages (BMDMs), polarized BMDMs toward an M2 (TAM-like) phenotype, and strikingly promoted BMDM chemotaxis. In vitro administration of PLX3397 suppressed pERK1/2 stimulation by CSF1 or TCM, and reduced M2 polarization, survival, and chemotaxis in BMDMs. Systemic administration of PLX3397 to the osteosarcoma orthotopic xenograft model significantly suppressed the primary tumor growth and lung metastasis, and thus improved metastasis-free survival. PLX3397 treatment concurrently depleted TAMs and FOXP3
+ regulatory T cells and, surprisingly, enhanced infiltration of CD8+ T cells into the microenvironments of both primary and metastatic osteosarcoma sites. Our preclinical results show that PLX3397 has strong macrophage- and T-cell-modulating effects that may translate into cancer immunotherapy for bone and soft-tissue sarcomas., (©2021 American Association for Cancer Research.)- Published
- 2021
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10. Anti-tumor effects of an ID antagonist with no observed acquired resistance.
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Wojnarowicz PM, Escolano MG, Huang YH, Desai B, Chin Y, Shah R, Xu S, Yadav S, Yaklichkin S, Ouerfelli O, Soni RK, Philip J, Montrose DC, Healey JH, Rajasekhar VK, Garland WA, Ratiu J, Zhuang Y, Norton L, Rosen N, Hendrickson RC, Zhou XK, Iavarone A, Massague J, Dannenberg AJ, Lasorella A, and Benezra R
- Abstract
ID proteins are helix-loop-helix (HLH) transcriptional regulators frequently overexpressed in cancer. ID proteins inhibit basic-HLH transcription factors often blocking differentiation and sustaining proliferation. A small-molecule, AGX51, targets ID proteins for degradation and impairs ocular neovascularization in mouse models. Here we show that AGX51 treatment of cancer cell lines impairs cell growth and viability that results from an increase in reactive oxygen species (ROS) production upon ID degradation. In mouse models, AGX51 treatment suppresses breast cancer colonization in the lung, regresses the growth of paclitaxel-resistant breast tumors when combined with paclitaxel and reduces tumor burden in sporadic colorectal neoplasia. Furthermore, in cells and mice, we fail to observe acquired resistance to AGX51 likely the result of the inability to mutate the binding pocket without loss of ID function and efficient degradation of the ID proteins. Thus, AGX51 is a first-in-class compound that antagonizes ID proteins, shows strong anti-tumor effects and may be further developed for the management of multiple cancers.
- Published
- 2021
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11. Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to PHGDH Inhibition.
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Ngo B, Kim E, Osorio-Vasquez V, Doll S, Bustraan S, Liang RJ, Luengo A, Davidson SM, Ali A, Ferraro GB, Fischer GM, Eskandari R, Kang DS, Ni J, Plasger A, Rajasekhar VK, Kastenhuber ER, Bacha S, Sriram RK, Stein BD, Bakhoum SF, Snuderl M, Cotzia P, Healey JH, Mainolfi N, Suri V, Friedman A, Manfredi M, Sabatini DM, Jones DR, Yu M, Zhao JJ, Jain RK, Keshari KR, Davies MA, Vander Heiden MG, Hernando E, Mann M, Cantley LC, and Pacold ME
- Subjects
- Animals, Antineoplastic Agents pharmacology, Brain metabolism, Brain Neoplasms secondary, Cell Line, Tumor, Datasets as Topic, Drug Resistance, Neoplasm, Female, Gene Knockdown Techniques, Glycine analysis, Glycine metabolism, Humans, Metabolomics, Mice, Phosphoglycerate Dehydrogenase genetics, Phosphoglycerate Dehydrogenase metabolism, Proteomics, RNA-Seq, Serine analysis, Serine metabolism, Tumor Microenvironment drug effects, Xenograft Model Antitumor Assays, Antineoplastic Agents therapeutic use, Brain pathology, Brain Neoplasms drug therapy, Phosphoglycerate Dehydrogenase antagonists & inhibitors
- Abstract
A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo , genetic suppression and pharmacologic inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggest that PHGDH inhibitors may be useful in the treatment of brain metastasis. SIGNIFICANCE: Using proteomics, metabolomics, and multiple brain metastasis models, we demonstrate that the nutrient-limited environment of the brain potentiates brain metastasis susceptibility to serine synthesis inhibition. These findings underscore the importance of studying cancer metabolism in physiologically relevant contexts, and provide a rationale for using PHGDH inhibitors to treat brain metastasis. This article is highlighted in the In This Issue feature, p. 1241 ., (©2020 American Association for Cancer Research.)
- Published
- 2020
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12. Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.
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Hoshino A, Kim HS, Bojmar L, Gyan KE, Cioffi M, Hernandez J, Zambirinis CP, Rodrigues G, Molina H, Heissel S, Mark MT, Steiner L, Benito-Martin A, Lucotti S, Di Giannatale A, Offer K, Nakajima M, Williams C, Nogués L, Pelissier Vatter FA, Hashimoto A, Davies AE, Freitas D, Kenific CM, Ararso Y, Buehring W, Lauritzen P, Ogitani Y, Sugiura K, Takahashi N, Alečković M, Bailey KA, Jolissant JS, Wang H, Harris A, Schaeffer LM, García-Santos G, Posner Z, Balachandran VP, Khakoo Y, Raju GP, Scherz A, Sagi I, Scherz-Shouval R, Yarden Y, Oren M, Malladi M, Petriccione M, De Braganca KC, Donzelli M, Fischer C, Vitolano S, Wright GP, Ganshaw L, Marrano M, Ahmed A, DeStefano J, Danzer E, Roehrl MHA, Lacayo NJ, Vincent TC, Weiser MR, Brady MS, Meyers PA, Wexler LH, Ambati SR, Chou AJ, Slotkin EK, Modak S, Roberts SS, Basu EM, Diolaiti D, Krantz BA, Cardoso F, Simpson AL, Berger M, Rudin CM, Simeone DM, Jain M, Ghajar CM, Batra SK, Stanger BZ, Bui J, Brown KA, Rajasekhar VK, Healey JH, de Sousa M, Kramer K, Sheth S, Baisch J, Pascual V, Heaton TE, La Quaglia MP, Pisapia DJ, Schwartz R, Zhang H, Liu Y, Shukla A, Blavier L, DeClerck YA, LaBarge M, Bissell MJ, Caffrey TC, Grandgenett PM, Hollingsworth MA, Bromberg J, Costa-Silva B, Peinado H, Kang Y, Garcia BA, O'Reilly EM, Kelsen D, Trippett TM, Jones DR, Matei IR, Jarnagin WR, and Lyden D
- Subjects
- Animals, Biomarkers, Tumor blood, Cell Line, HSC70 Heat-Shock Proteins metabolism, Humans, Machine Learning, Mice, Mice, Inbred C57BL, Microfilament Proteins metabolism, Neoplasms metabolism, Proteome analysis, Proteome metabolism, Proteomics methods, Sensitivity and Specificity, Tetraspanin 29 metabolism, rap GTP-Binding Proteins metabolism, Biomarkers, Tumor metabolism, Extracellular Vesicles metabolism, Neoplasms diagnosis
- Abstract
There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type., Competing Interests: Declaration of Interests D.L., A.H., H.S.K., and L.B. have filed a U.S. patent application related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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13. Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer.
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Yang J, Wang T, Zhao L, Rajasekhar VK, Joshi S, Andreou C, Pal S, Hsu HT, Zhang H, Cohen IJ, Huang R, Hendrickson RC, Miele MM, Pei W, Brendel MB, Healey JH, Chiosis G, and Kircher MF
- Subjects
- Animals, Apoptosis, Breast Neoplasms pathology, Cell Death, Female, Heterografts, Lipoproteins, Magnetic Resonance Imaging methods, Mice, Mice, Inbred BALB C, Mitogen-Activated Protein Kinase Kinases drug effects, Nanotechnology methods, Optical Imaging, Phosphatidylinositol 3-Kinases drug effects, Proteomics, Theranostic Nanomedicine methods, Breast Neoplasms diagnostic imaging, Breast Neoplasms drug therapy, Gold chemistry, Gold pharmacology, Lactalbumin chemistry, Lactalbumin pharmacology
- Abstract
Theranostic agents should ideally be renally cleared and biodegradable. Here, we report the synthesis, characterization and theranostic applications of fluorescent ultrasmall gold quantum clusters that are stabilized by the milk metalloprotein alpha-lactalbumin. We synthesized three types of these nanoprobes that together display fluorescence across the visible and near-infrared spectra when excited at a single wavelength through optical colour coding. In live tumour-bearing mice, the near-infrared nanoprobe generates contrast for fluorescence, X-ray computed tomography and magnetic resonance imaging, and exhibits long circulation times, low accumulation in the reticuloendothelial system, sustained tumour retention, insignificant toxicity and renal clearance. An intravenously administrated near-infrared nanoprobe with a large Stokes shift facilitated the detection and image-guided resection of breast tumours in vivo using a smartphone with modified optics. Moreover, the partially unfolded structure of alpha-lactalbumin in the nanoprobe helps with the formation of an anti-cancer lipoprotein complex with oleic acid that triggers the inhibition of the MAPK and PI3K-AKT pathways, immunogenic cell death and the recruitment of infiltrating macrophages. The biodegradability and safety profile of the nanoprobes make them suitable for the systemic detection and localized treatment of cancer.
- Published
- 2020
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14. Photobiomodulation effects on head and neck squamous cell carcinoma (HNSCC) in an orthotopic animal model.
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Barasch A, Li H, Rajasekhar VK, Raber-Durlacher J, Epstein JB, Carroll J, and Haimovitz-Friedman A
- Subjects
- Animals, Cell Line, Tumor, Dermatitis pathology, Disease Models, Animal, Humans, Mice, Mice, Nude, Mice, SCID, Neoplasm Transplantation, Stomatitis pathology, Transplantation, Heterologous, Low-Level Light Therapy adverse effects, Low-Level Light Therapy methods, Mucositis pathology, Radiotherapy adverse effects, Squamous Cell Carcinoma of Head and Neck radiotherapy
- Abstract
Background: Photobiomodulation (PBM) has shown efficacy in preventing and treating cancer therapy-induced mucositis and dermatitis. However, there is contradictory information regarding the effect of PBM on (pre)malignant cells, which has led to questions regarding the safety of this technique. We address this issue using an orthotopic mouse model (Cal-33) with human squamous cell carcinoma of the oral cavity., Methods: Mice with actively growing orthotopic Cal-33 head and neck carcinoma tumors were divided into 4 groups: control, PBM only, radiation therapy (RT) only, and PBM + RT. We performed three experiments: (1) PBM at 660 nm, 18.4 J/cm
2 , and 5 RT × 4 Gy doses delivered daily; (2) PBM at 660 nm, 18.4 J/cm2 , and 1 × 15 Gy RT; and (3) PBM at 660 nm + 850 nm, 45 mW/cm2 , 3.4 J/cm2 , and 1 × 15 Gy RT. Mice were weighed daily and tumor volumes were evaluated by IVIS. Survival time was also evaluated., Results: Animals treated with RT survived significantly longer and had significantly smaller tumor volume when compared with the control and PBM-only treatment groups. No significant differences were noted between the RT alone and PBM + RT groups in any of the experiments., Conclusion: Our results suggest that PBM at the utilized parameters does not provide protection to the tumor from the killing effects of RT.- Published
- 2020
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15. Hematopoietic Stem and Progenitor Cells Exhibit Stage-Specific Translational Programs via mTOR- and CDK1-Dependent Mechanisms.
- Author
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Spevak CC, Elias HK, Kannan L, Ali MAE, Martin GH, Selvaraj S, Eng WS, Ernlund A, Rajasekhar VK, Woolthuis CM, Zhao G, Ha CJ, Schneider RJ, and Park CY
- Subjects
- Hematopoietic Stem Cells, Signal Transduction, TOR Serine-Threonine Kinases, Hematopoietic Stem Cell Transplantation, Proteomics
- Abstract
Hematopoietic stem cells (HSCs) require highly regulated rates of protein synthesis, but it is unclear if they or lineage-committed progenitors preferentially recruit transcripts to translating ribosomes. We utilized polysome profiling, RNA sequencing, and whole-proteomic approaches to examine the translatome in LSK (Lin
- Sca-1+ c-Kit+ ) and myeloid progenitor (MP; Lin- Sca-1- c-Kit+ ) cells. Our studies show that LSKs exhibit low global translation but high translational efficiencies (TEs) of mRNAs required for HSC maintenance. In contrast, MPs activate translation in an mTOR-independent manner due, at least in part, to proteasomal degradation of mTOR by the E3 ubiquitin ligase c-Cbl. In the near absence of mTOR, CDK1 activates eIF4E-dependent translation in MPs through phosphorylation of 4E-BP1. Aberrant activation of mTOR expression and signaling in c-Cbl-deficient MPs results in increased mature myeloid lineage output. Overall, our data demonstrate that hematopoietic stem and progenitor cells (HSPCs) undergo translational reprogramming mediated by previously uncharacterized mechanisms of translational regulation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2020
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16. Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.
- Author
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Rodrigues G, Hoshino A, Kenific CM, Matei IR, Steiner L, Freitas D, Kim HS, Oxley PR, Scandariato I, Casanova-Salas I, Dai J, Badwe CR, Gril B, Tešić Mark M, Dill BD, Molina H, Zhang H, Benito-Martin A, Bojmar L, Ararso Y, Offer K, LaPlant Q, Buehring W, Wang H, Jiang X, Lu TM, Liu Y, Sabari JK, Shin SJ, Narula N, Ginter PS, Rajasekhar VK, Healey JH, Meylan E, Costa-Silva B, Wang SE, Rafii S, Altorki NK, Rudin CM, Jones DR, Steeg PS, Peinado H, Ghajar CM, Bromberg J, de Sousa M, Pisapia D, and Lyden D
- Subjects
- Animals, Brain metabolism, Brain pathology, Brain Neoplasms metabolism, Brain Neoplasms mortality, Brain Neoplasms pathology, Cell Line, Tumor, Cell Movement, Cell Proliferation, Chemokine CCL1 genetics, Chemokine CCL1 metabolism, Chemokine CXCL1 genetics, Chemokine CXCL1 metabolism, Cyclooxygenase 2 genetics, Cyclooxygenase 2 metabolism, Endothelial Cells metabolism, Endothelial Cells pathology, Exosomes pathology, Humans, Hyaluronoglucosaminidase metabolism, Mice, Mice, Inbred C57BL, Mice, Nude, Neoplasm Metastasis, Neovascularization, Pathologic metabolism, Neovascularization, Pathologic mortality, Neovascularization, Pathologic pathology, Signal Transduction, Survival Analysis, Tumor Burden, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha metabolism, Xenograft Model Antitumor Assays, Brain Neoplasms genetics, Exosomes metabolism, Gene Expression Regulation, Neoplastic, Hyaluronoglucosaminidase genetics, Neovascularization, Pathologic genetics, Tumor Microenvironment genetics
- Abstract
The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP
+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.- Published
- 2019
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17. Low-Dose Radiation Conditioning Enables CAR T Cells to Mitigate Antigen Escape.
- Author
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DeSelm C, Palomba ML, Yahalom J, Hamieh M, Eyquem J, Rajasekhar VK, and Sadelain M
- Subjects
- Animals, Antigens, CD19 immunology, Antigens, Neoplasm chemistry, Antigens, Neoplasm immunology, Antigens, Neoplasm radiation effects, CA-19-9 Antigen, Combined Modality Therapy, Disease Models, Animal, Humans, Insulin-Secreting Cells immunology, Insulin-Secreting Cells radiation effects, Mice, Oligosaccharides chemistry, Oligosaccharides immunology, Oligosaccharides therapeutic use, Pancreatic Neoplasms drug therapy, Pancreatic Neoplasms genetics, Radiation, Radiation Dosage, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen therapeutic use, Sequence Analysis, RNA, TNF-Related Apoptosis-Inducing Ligand immunology, Antigens, CD19 therapeutic use, Immunotherapy, Adoptive, Pancreatic Neoplasms immunology, Pancreatic Neoplasms radiotherapy, TNF-Related Apoptosis-Inducing Ligand genetics
- Abstract
CD19 chimeric antigen receptors (CARs) have demonstrated great efficacy against a range of B cell malignancies. However, antigen escape and, more generally, heterogeneous antigen expression pose a challenge to applying CAR therapy to a wide range of cancers. We find that low-dose radiation sensitizes tumor cells to immune rejection by locally activated CAR T cells. In a model of pancreatic adenocarcinoma heterogeneously expressing sialyl Lewis-A (sLeA), we show that not only sLeA
+ but also sLeA- tumor cells exposed to low-dose radiation become susceptible to CAR therapy, reducing antigen-negative tumor relapse. RNA sequencing analysis of low-dose radiation-exposed tumors reveals the transcriptional signature of cells highly sensitive to TRAIL-mediated death. We find that sLeA-targeted CAR T cells produce TRAIL upon engaging sLeA+ tumor cells, and eliminate sLeA- tumor cells previously exposed to systemic or local low-dose radiation in a TRAIL-dependent manner. These findings enhance the prospects for successfully applying CAR therapy to heterogeneous solid tumors. Local radiation is integral to many tumors' standard of care and can be easily implemented as a CAR conditioning regimen., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2018
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18. Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.
- Author
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Verginelli F, Perconti S, Vespa S, Schiavi F, Prasad SC, Lanuti P, Cama A, Tramontana L, Esposito DL, Guarnieri S, Sheu A, Pantalone MR, Florio R, Morgano A, Rossi C, Bologna G, Marchisio M, D'Argenio A, Taschin E, Visone R, Opocher G, Veronese A, Paties CT, Rajasekhar VK, Söderberg-Nauclér C, Sanna M, Lotti LV, and Mariani-Costantini R
- Subjects
- Animals, Antineoplastic Agents pharmacology, Cell Line, Head and Neck Neoplasms genetics, Head and Neck Neoplasms pathology, Humans, Imatinib Mesylate pharmacology, Mice, Inbred NOD, Mice, SCID, MicroRNAs metabolism, Neural Stem Cells drug effects, Neural Stem Cells metabolism, Neural Stem Cells pathology, Organogenesis drug effects, Organogenesis physiology, Paraganglioma genetics, Paraganglioma pathology, Primary Cell Culture, Tumor Microenvironment drug effects, Tumor Microenvironment physiology, Xenograft Model Antitumor Assays, Antineoplastic Agents therapeutic use, Head and Neck Neoplasms drug therapy, Head and Neck Neoplasms physiopathology, Imatinib Mesylate therapeutic use, Paraganglioma drug therapy, Paraganglioma physiopathology
- Abstract
Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.
- Published
- 2018
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19. Correction to: Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.
- Author
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Verginelli F, Perconti S, Vespa S, Schiavi F, Prasad SC, Lanuti P, Cama A, Tramontana L, Esposito DL, Guarnieri S, Sheu A, Pantalone MR, Florio R, Morgano A, Rossi C, Bologna G, Marchisio M, D'Argenio A, Taschin E, Visone R, Opocher G, Veronese A, Paties CT, Rajasekhar VK, Söderberg-Nauclér C, Sanna M, Lotti LV, and Mariani-Costantini R
- Abstract
The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.
- Published
- 2018
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20. Quantitative self-assembly prediction yields targeted nanomedicines.
- Author
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Shamay Y, Shah J, Işık M, Mizrachi A, Leibold J, Tschaharganeh DF, Roxbury D, Budhathoki-Uprety J, Nawaly K, Sugarman JL, Baut E, Neiman MR, Dacek M, Ganesh KS, Johnson DC, Sridharan R, Chu EL, Rajasekhar VK, Lowe SW, Chodera JD, and Heller DA
- Subjects
- Animals, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Drug Carriers metabolism, Drug Carriers pharmacokinetics, Endocytosis, Indoles chemistry, Mice, Nanoparticles chemistry, Particle Size, Tissue Distribution, Drug Carriers chemistry, Nanomedicine methods
- Abstract
Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection.
- Published
- 2018
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21. Chromosomal instability drives metastasis through a cytosolic DNA response.
- Author
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Bakhoum SF, Ngo B, Laughney AM, Cavallo JA, Murphy CJ, Ly P, Shah P, Sriram RK, Watkins TBK, Taunk NK, Duran M, Pauli C, Shaw C, Chadalavada K, Rajasekhar VK, Genovese G, Venkatesan S, Birkbak NJ, McGranahan N, Lundquist M, LaPlant Q, Healey JH, Elemento O, Chung CH, Lee NY, Imielenski M, Nanjangud G, Pe'er D, Cleveland DW, Powell SN, Lammerding J, Swanton C, and Cantley LC
- Subjects
- Animals, Brain Neoplasms genetics, Brain Neoplasms pathology, Brain Neoplasms secondary, Breast Neoplasms genetics, Breast Neoplasms pathology, Breast Neoplasms secondary, Carcinoma, Squamous Cell genetics, Carcinoma, Squamous Cell pathology, Cell Line, Chromosome Segregation, Cytosol enzymology, Female, Head and Neck Neoplasms genetics, Head and Neck Neoplasms pathology, Humans, Inflammation genetics, Inflammation metabolism, Membrane Proteins metabolism, Mesoderm metabolism, Mice, Micronuclei, Chromosome-Defective, NF-kappa B metabolism, Nucleotidyltransferases metabolism, Xenograft Model Antitumor Assays, Chromosomal Instability genetics, Cytosol metabolism, DNA, Neoplasm metabolism, Neoplasm Metastasis genetics
- Abstract
Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.
- Published
- 2018
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22. HPMA-Copolymer Nanocarrier Targets Tumor-Associated Macrophages in Primary and Metastatic Breast Cancer.
- Author
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Zimel MN, Horowitz CB, Rajasekhar VK, Christ AB, Wei X, Wu J, Wojnarowicz PM, Wang D, Goldring SR, Purdue PE, and Healey JH
- Subjects
- Animals, Breast Neoplasms metabolism, Disease Models, Animal, Female, Humans, Mice, Neoplasm Metastasis, Polymers, Breast Neoplasms genetics, Macrophages metabolism, Methacrylates metabolism
- Abstract
Polymeric nanocarriers such as N -(2-hydroxypropyl) methacrylamide (HPMA) copolymers deliver drugs to solid tumors and avoid the systemic toxicity of conventional chemotherapy. Because HPMA copolymers can target sites of inflammation and accumulate within innate immune cells, we hypothesized that HPMA copolymers could target tumor-associated macrophages (TAM) in both primary and metastatic tumor microenvironments. We verified this hypothesis, first in preliminary experiments with isolated bone marrow macrophage cultures in vitro and subsequently in a spontaneously metastatic murine breast cancer model generated from a well-established, cytogenetically characterized 4T1 breast cancer cell line. Using our standardized experimental conditions, we detected primary orthotopic tumor growth at 7 days and metastatic tumors at 28 days after orthotopic transplantation of 4T1 cells into the mammary fat pad. We investigated the uptake of HPMA copolymer conjugated with Alexa Fluor 647 and folic acid (P-Alexa647-FA) and HPMA copolymer conjugated with IRDye 800CW (P-IRDye), following their retroorbital injection into the primary and metastatic tumor-bearing mice. A significant uptake of P-IRDye was observed at all primary and metastatic tumor sites in these mice, and the P-Alexa647-FA signal was found specifically within CD11b
+ TAMs costained with pan-macrophage marker CD68. These findings demonstrate, for the first time, a novel capacity of a P-Alexa647-FA conjugate to colocalize to CD11b+ CD68+ TAMs in both primary and metastatic breast tumors. This underscores the potential of this HPMA nanocarrier to deliver functional therapeutics that specifically target tumor-promoting macrophage activation and/or polarization during tumor development. Mol Cancer Ther; 16(12); 2701-10. ©2017 AACR ., (©2017 American Association for Cancer Research.)- Published
- 2017
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23. An Integrated Systems Biology Approach Identifies TRIM25 as a Key Determinant of Breast Cancer Metastasis.
- Author
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Walsh LA, Alvarez MJ, Sabio EY, Reyngold M, Makarov V, Mukherjee S, Lee KW, Desrichard A, Turcan Ş, Dalin MG, Rajasekhar VK, Chen S, Vahdat LT, Califano A, and Chan TA
- Subjects
- Animals, Breast Neoplasms metabolism, Breast Neoplasms mortality, Breast Neoplasms pathology, Female, Gene Regulatory Networks, Genes, Reporter, Heterografts, Humans, Luciferases genetics, Luciferases metabolism, Lung Neoplasms metabolism, Lung Neoplasms mortality, Lung Neoplasms secondary, Mice, Mice, Nude, Neoplasm Proteins metabolism, Receptors, Estrogen genetics, Receptors, Estrogen metabolism, Signal Transduction, Survival Analysis, Systems Biology, Transcription Factors metabolism, Tripartite Motif Proteins metabolism, Ubiquitin-Protein Ligases metabolism, Breast Neoplasms genetics, Gene Expression Regulation, Neoplastic, Lung Neoplasms genetics, Neoplasm Proteins genetics, Transcription Factors genetics, Tripartite Motif Proteins genetics, Ubiquitin-Protein Ligases genetics
- Abstract
At the root of most fatal malignancies are aberrantly activated transcriptional networks that drive metastatic dissemination. Although individual metastasis-associated genes have been described, the complex regulatory networks presiding over the initiation and maintenance of metastatic tumors are still poorly understood. There is untapped value in identifying therapeutic targets that broadly govern coordinated transcriptional modules dictating metastatic progression. Here, we reverse engineered and interrogated a breast cancer-specific transcriptional interaction network (interactome) to define transcriptional control structures causally responsible for regulating genetic programs underlying breast cancer metastasis in individual patients. Our analyses confirmed established pro-metastatic transcription factors, and they uncovered TRIM25 as a key regulator of metastasis-related transcriptional programs. Further, in vivo analyses established TRIM25 as a potent regulator of metastatic disease and poor survival outcome. Our findings suggest that identifying and targeting keystone proteins, like TRIM25, can effectively collapse transcriptional hierarchies necessary for metastasis formation, thus representing an innovative cancer intervention strategy., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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24. Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.
- Author
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Sansone P, Berishaj M, Rajasekhar VK, Ceccarelli C, Chang Q, Strillacci A, Savini C, Shapiro L, Bowman RL, Mastroleo C, De Carolis S, Daly L, Benito-Martin A, Perna F, Fabbri N, Healey JH, Spisni E, Cricca M, Lyden D, Bonafé M, and Bromberg J
- Subjects
- Animals, Breast Neoplasms genetics, Breast Neoplasms metabolism, Cancer-Associated Fibroblasts, Case-Control Studies, Cell-Derived Microparticles genetics, Cell-Derived Microparticles metabolism, Drug Resistance, Neoplasm, Estradiol analogs & derivatives, Estradiol pharmacology, Female, Fulvestrant, HeLa Cells, Heterografts, Humans, Interleukin-6 metabolism, MCF-7 Cells, Mice, Mice, Inbred NOD, Mice, SCID, MicroRNAs administration & dosage, MicroRNAs genetics, Neoplasms, Hormone-Dependent genetics, Neoplasms, Hormone-Dependent metabolism, Neoplasms, Hormone-Dependent pathology, Neoplastic Stem Cells metabolism, STAT3 Transcription Factor metabolism, Stromal Cells metabolism, Antineoplastic Agents, Hormonal pharmacology, Breast Neoplasms drug therapy, Breast Neoplasms pathology, Cell-Derived Microparticles pathology, Neoplastic Stem Cells pathology, Stromal Cells pathology
- Abstract
The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ER
lo /Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg , HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR ., (©2017 American Association for Cancer Research.)- Published
- 2017
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25. Tumour-specific PI3K inhibition via nanoparticle-targeted delivery in head and neck squamous cell carcinoma.
- Author
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Mizrachi A, Shamay Y, Shah J, Brook S, Soong J, Rajasekhar VK, Humm JL, Healey JH, Powell SN, Baselga J, Heller DA, Haimovitz-Friedman A, and Scaltriti M
- Subjects
- Animals, Carcinoma, Squamous Cell metabolism, Cell Line, Tumor, Class I Phosphatidylinositol 3-Kinases metabolism, Drug Delivery Systems methods, Head and Neck Neoplasms metabolism, Humans, Mice, Nude, Microscopy, Electron, Scanning, Nanoparticles ultrastructure, Protein Kinase Inhibitors administration & dosage, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacology, Thiazoles administration & dosage, Thiazoles chemistry, Treatment Outcome, Carcinoma, Squamous Cell drug therapy, Class I Phosphatidylinositol 3-Kinases antagonists & inhibitors, Head and Neck Neoplasms drug therapy, Nanoparticles chemistry, Thiazoles pharmacology, Xenograft Model Antitumor Assays
- Abstract
Alterations in PIK3CA, the gene encoding the p110α subunit of phosphatidylinositol 3-kinase (PI3Kα), are frequent in head and neck squamous cell carcinomas. Inhibitors of PI3Kα show promising activity in various cancer types, but their use is curtailed by dose-limiting side effects such as hyperglycaemia. In the present study, we explore the efficacy, specificity and safety of the targeted delivery of BYL719, a PI3Kα inhibitor currently in clinical development in solid tumours. By encapsulating BYL719 into P-selectin-targeted nanoparticles, we achieve specific accumulation of BYL719 in the tumour milieu. This results in tumour growth inhibition and radiosensitization despite the use of a sevenfold lower dose of BYL719 compared with oral administration. Furthermore, the nanoparticles abrogate acute and chronic metabolic side effects normally observed after BYL719 treatment. These findings offer a novel strategy that could potentially enhance the efficacy of PI3Kα inhibitors while mitigating dose-limiting toxicity in patients with head and neck squamous cell carcinomas.
- Published
- 2017
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26. Self-renewal of CD133(hi) cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer.
- Author
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Sansone P, Ceccarelli C, Berishaj M, Chang Q, Rajasekhar VK, Perna F, Bowman RL, Vidone M, Daly L, Nnoli J, Santini D, Taffurelli M, Shih NN, Feldman M, Mao JJ, Colameco C, Chen J, DeMichele A, Fabbri N, Healey JH, Cricca M, Gasparre G, Lyden D, Bonafé M, and Bromberg J
- Subjects
- AC133 Antigen, Anastrozole, Androstadienes, Animals, Antigens, CD metabolism, Bone Neoplasms secondary, Breast Neoplasms pathology, Carcinoma, Ductal, Breast secondary, Carcinoma, Lobular secondary, Cell Line, Tumor, Estradiol analogs & derivatives, Female, Flow Cytometry, Fulvestrant, Glycoproteins metabolism, Humans, In Vitro Techniques, Interleukin-6 genetics, Letrozole, Leuprolide, MCF-7 Cells, Mice, Inbred NOD, Mice, SCID, Neoplasm Metastasis, Neoplasm Transplantation, Nitriles, Oxidative Phosphorylation, Peptides metabolism, Real-Time Polymerase Chain Reaction, Receptor, Notch3, Receptors, Estrogen metabolism, Receptors, Notch genetics, Signal Transduction genetics, Tamoxifen, Triazoles, Antineoplastic Agents, Hormonal, Bone Neoplasms genetics, Breast Neoplasms genetics, Carcinoma, Ductal, Breast genetics, Carcinoma, Lobular genetics, Cell Self Renewal genetics, Drug Resistance, Neoplasm genetics, Gene Expression Regulation, Neoplastic, Neoplastic Stem Cells metabolism
- Abstract
The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133(hi)/ER(lo) cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133(hi)/ER(lo)/IL6(hi) cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133(hi)/ER(lo)/OXPHOS(lo). These cells exit metabolic dormancy via an IL6-driven feed-forward ER(lo)-IL6(hi)-Notch(hi) loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133(hi) CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133(hi)/ER(lo) cells mediating metastatic progression, which is sensitive to dual targeted therapy.
- Published
- 2016
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27. Tumour exosome integrins determine organotropic metastasis.
- Author
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Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, Molina H, Kohsaka S, Di Giannatale A, Ceder S, Singh S, Williams C, Soplop N, Uryu K, Pharmer L, King T, Bojmar L, Davies AE, Ararso Y, Zhang T, Zhang H, Hernandez J, Weiss JM, Dumont-Cole VD, Kramer K, Wexler LH, Narendran A, Schwartz GK, Healey JH, Sandstrom P, Labori KJ, Kure EH, Grandgenett PM, Hollingsworth MA, de Sousa M, Kaur S, Jain M, Mallya K, Batra SK, Jarnagin WR, Brady MS, Fodstad O, Muller V, Pantel K, Minn AJ, Bissell MJ, Garcia BA, Kang Y, Rajasekhar VK, Ghajar CM, Matei I, Peinado H, Bromberg J, and Lyden D
- Subjects
- Animals, Biomarkers metabolism, Brain cytology, Cell Line, Tumor, Endothelial Cells cytology, Endothelial Cells metabolism, Epithelial Cells cytology, Epithelial Cells metabolism, Female, Fibroblasts cytology, Fibroblasts metabolism, Genes, src, Humans, Integrin alpha6beta1 metabolism, Integrin alpha6beta4 antagonists & inhibitors, Integrin alpha6beta4 metabolism, Integrin beta Chains metabolism, Integrin beta4 metabolism, Integrins antagonists & inhibitors, Kupffer Cells cytology, Kupffer Cells metabolism, Liver cytology, Lung cytology, Mice, Mice, Inbred C57BL, Organ Specificity, Phosphorylation, Receptors, Vitronectin antagonists & inhibitors, Receptors, Vitronectin metabolism, S100 Proteins genetics, Brain metabolism, Exosomes metabolism, Integrins metabolism, Liver metabolism, Lung metabolism, Neoplasm Metastasis pathology, Neoplasm Metastasis prevention & control, Tropism
- Abstract
Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.
- Published
- 2015
- Full Text
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28. Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium.
- Author
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Navaratnam J, Utheim TP, Rajasekhar VK, and Shahdadfar A
- Abstract
Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented.
- Published
- 2015
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29. RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.
- Author
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Wolfe AL, Singh K, Zhong Y, Drewe P, Rajasekhar VK, Sanghvi VR, Mavrakis KJ, Jiang M, Roderick JE, Van der Meulen J, Schatz JH, Rodrigo CM, Zhao C, Rondou P, de Stanchina E, Teruya-Feldstein J, Kelliher MA, Speleman F, Porco JA Jr, Pelletier J, Rätsch G, and Wendel HG
- Subjects
- Animals, Antineoplastic Agents, Phytogenic pharmacology, Antineoplastic Agents, Phytogenic therapeutic use, Base Sequence, Cell Line, Tumor, Epigenesis, Genetic, Female, Humans, Mice, Mice, Inbred C57BL, Nucleotide Motifs, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma drug therapy, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma genetics, Ribosomes metabolism, Transcription Factors metabolism, Transcription, Genetic drug effects, Transcription, Genetic genetics, Triterpenes pharmacology, 5' Untranslated Regions genetics, Eukaryotic Initiation Factor-4A metabolism, G-Quadruplexes, Oncogene Proteins biosynthesis, Oncogene Proteins genetics, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma metabolism, Protein Biosynthesis drug effects
- Abstract
The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.
- Published
- 2014
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30. Multifaceted regulation of somatic cell reprogramming by mRNA translational control.
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Tahmasebi S, Alain T, Rajasekhar VK, Zhang JP, Prager-Khoutorsky M, Khoutorsky A, Dogan Y, Gkogkas CG, Petroulakis E, Sylvestre A, Ghorbani M, Assadian S, Yamanaka Y, Vinagolu-Baur JR, Teodoro JG, Kim K, Yang XJ, and Sonenberg N
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Cycle Proteins, Cells, Cultured, Cellular Reprogramming genetics, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Eukaryotic Initiation Factors genetics, Eukaryotic Initiation Factors metabolism, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Mice, Models, Biological, Phosphoproteins genetics, Phosphoproteins metabolism, Proto-Oncogene Proteins c-myc genetics, Proto-Oncogene Proteins c-myc metabolism, SOXB1 Transcription Factors genetics, SOXB1 Transcription Factors metabolism, Cellular Reprogramming physiology
- Abstract
Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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31. Crizotinib, a c-Met inhibitor, prevents metastasis in a metastatic uveal melanoma model.
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Surriga O, Rajasekhar VK, Ambrosini G, Dogan Y, Huang R, and Schwartz GK
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- Animals, Antineoplastic Agents administration & dosage, Cell Line, Tumor, Cell Movement drug effects, Cell Movement genetics, Crizotinib, Disease Models, Animal, Gene Expression, Gene Knockdown Techniques, Hepatocyte Growth Factor biosynthesis, Humans, Male, Melanoma drug therapy, Melanoma genetics, Mice, Neoplasm Metastasis, Protein Kinase Inhibitors administration & dosage, Proto-Oncogene Proteins c-met genetics, Proto-Oncogene Proteins c-met metabolism, Pyrazoles administration & dosage, Pyridines administration & dosage, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Tumor Burden drug effects, Uveal Neoplasms drug therapy, Uveal Neoplasms genetics, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Melanoma metabolism, Melanoma pathology, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-met antagonists & inhibitors, Pyrazoles pharmacology, Pyridines pharmacology, Uveal Neoplasms metabolism, Uveal Neoplasms pathology
- Abstract
Uveal melanoma is the most common primary intraocular malignant tumor in adults and half of the primary tumors will develop fatal metastatic disease to the liver and the lung. Crizotinib, an inhibitor of c-Met, anaplastic lymphoma kinase (ALK), and ROS1, inhibited the phosphorylation of the c-Met receptor but not of ALK or ROS1 in uveal melanoma cells and tumor tissue. Consequently, migration of uveal melanoma cells was suppressed in vitro at a concentration associated with the specific inhibition of c-Met phosphorylation. This effect on cell migration could be recapitulated with siRNA specific to c-Met but not to ALK or ROS1. Therefore, we developed a uveal melanoma metastatic mouse model with EGFP-luciferase-labeled uveal melanoma cells transplanted by retro-orbital injections to test the effect of crizotinib on metastasis. In this model, there was development of melanoma within the eye and also metastases to the liver and lung at 7 weeks after the initial transplantation. When mice were treated with crizotinib starting 1 week after the transplantation, we observed a significant reduction in the development of metastases as compared with untreated control sets. These results indicate that the inhibition of c-Met activity alone may be sufficient to strongly inhibit metastasis of uveal melanoma from forming, suggesting crizotinib as a potential adjuvant therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease., (©2013 AACR.)
- Published
- 2013
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32. Targeting cap-dependent translation blocks converging survival signals by AKT and PIM kinases in lymphoma.
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Schatz JH, Oricchio E, Wolfe AL, Jiang M, Linkov I, Maragulia J, Shi W, Zhang Z, Rajasekhar VK, Pagano NC, Porco JA Jr, Teruya-Feldstein J, Rosen N, Zelenetz AD, Pelletier J, and Wendel HG
- Subjects
- Animals, Antibiotics, Antineoplastic pharmacology, Drug Resistance, Neoplasm drug effects, Drug Resistance, Neoplasm genetics, Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Enzymologic genetics, Gene Expression Regulation, Neoplastic drug effects, Gene Expression Regulation, Neoplastic genetics, Humans, Lymphoma drug therapy, Lymphoma genetics, Mechanistic Target of Rapamycin Complex 1, Mice, Multiprotein Complexes, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases genetics, Proteins genetics, Proteins metabolism, Proto-Oncogene Proteins antagonists & inhibitors, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-pim-1 antagonists & inhibitors, Proto-Oncogene Proteins c-pim-1 genetics, RNA Caps genetics, Sirolimus pharmacology, TOR Serine-Threonine Kinases, Transcription Factors genetics, Transcription Factors metabolism, Tumor Cells, Cultured, Lymphoma metabolism, Protein Biosynthesis, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt metabolism, Proto-Oncogene Proteins c-pim-1 metabolism, RNA Caps metabolism, Signal Transduction
- Abstract
New anticancer drugs that target oncogenic signaling molecules have greatly improved the treatment of certain cancers. However, resistance to targeted therapeutics is a major clinical problem and the redundancy of oncogenic signaling pathways provides back-up mechanisms that allow cancer cells to escape. For example, the AKT and PIM kinases produce parallel oncogenic signals and share many molecular targets, including activators of cap-dependent translation. Here, we show that PIM kinase expression can affect the clinical outcome of lymphoma chemotherapy. We observe the same in animal lymphoma models. Whereas chemoresistance caused by AKT is readily reversed with rapamycin, PIM-mediated resistance is refractory to mTORC1 inhibition. However, both PIM- and AKT-expressing lymphomas depend on cap-dependent translation, and genetic or pharmacological blockade of the translation initiation complex is highly effective against these tumors. The therapeutic effect of blocking cap-dependent translation is mediated, at least in part, by decreased production of short-lived oncoproteins including c-MYC, Cyclin D1, MCL1, and the PIM1/2 kinases themselves. Hence, targeting the convergence of oncogenic survival signals on translation initiation is an effective alternative to combinations of kinase inhibitors., (© 2011 Schatz et al.)
- Published
- 2011
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33. Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-κB signalling.
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Rajasekhar VK, Studer L, Gerald W, Socci ND, and Scher HI
- Abstract
Androgen depletion is a key strategy for treating human prostate cancer, but the presence of hormone-independent cells escaping treatment remains a major therapeutic challenge. Here, we identify a minor subset of stem-like human prostate tumour-initiating cells (TICs) that do not express prostate cancer markers, such as androgen receptor or prostate specific antigen. These TICs possess stem cell characteristics and multipotency as demonstrated by in vitro sphere-formation and in vivo tumour-initiation, respectively. The cells represent an undifferentiated subtype of basal cells and can be purified from prostate tumours based on coexpression of the human pluripotent stem cell marker TRA-1-60 with CD151 and CD166. Such triple-marker-positive TICs recapitulate the original parent tumour heterogeneity in serial xeno-transplantations indicating a tumour cell hierarchy in human prostate cancer development. These TICs exhibit increased nuclear factor-κB activity. These findings are important in understanding the molecular basis of human prostate cancer.
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- 2011
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34. Role of 3'UTRs in the translation of mRNAs regulated by oncogenic eIF4E--a computational inference.
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Santhanam AN, Bindewald E, Rajasekhar VK, Larsson O, Sonenberg N, Colburn NH, and Shapiro BA
- Subjects
- Codon, Initiator, Codon, Terminator, MicroRNAs genetics, Nucleic Acid Conformation, RNA, Messenger chemistry, 3' Untranslated Regions, Eukaryotic Initiation Factor-4E physiology, Oncogene Proteins physiology, Protein Biosynthesis genetics, RNA, Messenger genetics
- Abstract
Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3'UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3'UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5'UTR and microRNA binding at the 3'UTR.
- Published
- 2009
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35. The 5th International Society for Stem Cell Research (ISSCR) Annual Meeting, June 2007.
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Rajasekhar VK, Dalerba P, Passegué E, Lagasse E, and Najbauer J
- Subjects
- Animals, Epigenesis, Genetic, Humans, Adult Stem Cells, Neoplastic Stem Cells
- Abstract
This report presents highlights of discussions that focused on the biology of cancer stem cells as conducted at the fifth Annual Meeting of the International Society for Stem Cell Research, held in Cairns, Australia, June 17-20, 2007. The function of adult stem cells is believed to depend on their niches, that is, the microenvironment in which these stem cells reside. A similar concept applies to understanding the development of cancer, as it is becoming increasingly clear that only a small subset of cancer cell populations is capable of initiating/sustaining tumor formation. These tumorigenic cells, commonly referred to as cancer stem cells, also appear to reside in particular niches, and they bear the known, albeit dysfunctional, stem cell characteristics of self-renewal and differentiation. Dysregulation of stem cell niches is thought to contribute to tumorigenesis by affecting the complex network of signaling interactions that occur between stem cells and their neighboring cells, thus imbalancing the physiological controls on self-renewal and differentiation processes. This hypothesis was widely explored at the conference to shed new light on the mechanisms of tumor origin and progression and to unveil novel antitumor therapeutic approaches.
- Published
- 2008
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36. Concise review: roles of polycomb group proteins in development and disease: a stem cell perspective.
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Rajasekhar VK and Begemann M
- Subjects
- Animals, Cell Differentiation genetics, Cell Lineage genetics, Chromatin physiology, Embryonic Development genetics, Gene Expression Profiling, Genes, Homeobox genetics, Humans, Mice, Neoplastic Stem Cells cytology, Polycomb-Group Proteins, Promoter Regions, Genetic genetics, Regulatory Elements, Transcriptional genetics, Repressor Proteins genetics, Cell Differentiation physiology, Cell Lineage physiology, Cell Transformation, Neoplastic genetics, Gene Expression Regulation, Developmental physiology, Gene Silencing physiology, Repressor Proteins physiology, Stem Cells cytology
- Abstract
The acquisition and maintenance of cell fate are essential for metazoan growth and development. A strict coordination between genetic and epigenetic programs regulates cell fate determination and maintenance. Polycomb group (PcG) genes are identified as essential in these epigenetic developmental processes. These genes encode components of multimeric transcriptional repressor complexes that are crucial in maintaining cell fate. PcG proteins have also been shown to play a central role in stem cell maintenance and lineage specification. PcG proteins, together with a battery of components including sequence-specific DNA binding/accessory factors, chromatin remodeling factors, signaling pathway intermediates, noncoding small RNAs, and RNA interference machinery, generally define a dynamic cellular identity through tight regulation of specific gene expression patterns. Epigenetic modification of chromatin structure that results in expression silencing of specific genes is now emerging as an important molecular mechanism in this process. In embryonic stem (ES) cells and adult stem cells, such specific genes represent those associated with differentiation and development, and silencing of these genes in a PcG protein-dependent manner confers stemness. ES cells also contain novel chromatin motifs enriched in epigenetic modifications associated with both activation and repression of genes, suggesting that certain genes are poised for activation or repression. Interestingly, these chromatin domains are highly coincident with the promoters of developmental regulators, which are also found to be occupied by PcG proteins. The epigenetic integrity is compromised, however, by mutations or other alterations that affect the function of PcG proteins in stem cells leading to aberrant cell proliferation and tissue transformation, a hallmark of cancer. Disclosure of potential conflicts of interest is found at the end of this article.
- Published
- 2007
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37. Epigenetic activation of a subset of mRNAs by eIF4E explains its effects on cell proliferation.
- Author
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Mamane Y, Petroulakis E, Martineau Y, Sato TA, Larsson O, Rajasekhar VK, and Sonenberg N
- Subjects
- 5' Untranslated Regions genetics, Animals, Apoptosis, Apoptosis Regulatory Proteins biosynthesis, Apoptosis Regulatory Proteins genetics, Cell Division genetics, Eukaryotic Initiation Factor-4E genetics, Intercellular Signaling Peptides and Proteins biosynthesis, Intercellular Signaling Peptides and Proteins genetics, Mice, NIH 3T3 Cells cytology, Oligonucleotide Array Sequence Analysis, Polyribosomes metabolism, Protein Biosynthesis, RNA, Messenger genetics, Recombinant Fusion Proteins physiology, Regulatory Sequences, Ribonucleic Acid genetics, Ribosomal Proteins biosynthesis, Ribosomal Proteins genetics, Epigenesis, Genetic, Eukaryotic Initiation Factor-4E physiology, RNA, Messenger biosynthesis
- Abstract
Background: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5' cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E., Methodology/principal Findings: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis., Conclusions/significance: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs.
- Published
- 2007
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38. Analytical methods for cancer stem cells.
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Rajasekhar VK
- Subjects
- Cell Differentiation, Cell Proliferation, Humans, Adult Stem Cells cytology, Cell Lineage, Cell Separation methods, Flow Cytometry methods, Fluorescent Antibody Technique methods, Neoplastic Stem Cells cytology, Regeneration physiology
- Abstract
The primary characteristics of adult stem cells are maintaining prolonged quiescence, ability to self-renew and plasticity to differentiate into multiple cell types. These properties are evolutionarily conserved from fruit fly to humans. Similar to normal tissue repair in organs, the stem cell concept is inherently impregnated in the etiology of cancer. Tumors contain a minor population of tumor-initiating cells, called "cancer stem cells". The cancer stem cells maintain some similarities in self-renewal and differentiation features of normal adult stem cells. Therefore, various methods developed originally for the analysis and characterization of adult stem cells are being extended to evaluate cancer stem cells. Relevant methods that are used generally across normal stem cells as well as cancer stem cells are summarized. Combination of two or more of these methods for validation of cancer stem cells appears to be a promising approach for the precise isolation and analysis of cancer stem cells.
- Published
- 2007
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39. Molecular insights into the function, fate, and prospects of stem cells.
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Rajasekhar VK and Vemuri MC
- Subjects
- Adult, Animals, Cell Differentiation, Embryo, Mammalian cytology, Forecasting, Genetic Therapy, Humans, Neoplasms pathology, Pluripotent Stem Cells cytology, Tissue Engineering, Stem Cells cytology, Stem Cells physiology
- Abstract
This article forms a review and an appraisal of the third annual meeting of the International Society for Stem Cell Research (http://www.isscr.org), held in San Francisco on June 23-25, 2005. The focus of the meeting was recent advances in stem cell biology. More than 2,000 scientists from around the world met to discuss stem cell research, clinical applications, and the ethical hurdles facing the field. Major topics highlighted during the meeting included the self-renewal and differentiation of embryonic stem cells as well as adult stem cells. Presentations included diverse topics such as cancer stem cells, tissue-specific stem cells, technology development, and clinical aspects of stem cells. Given the excitement the field has generated, linking basic stem cell research and clinical applications was paramount for discussion at the meeting. With the current resources in molecular biology research, improvements in genetic engineering, postgenomic capabilities, and biotechnological advances, it appears timely that stem cell biology research is headed toward making a major therapeutic contribution to human health.
- Published
- 2005
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40. Postgenomic global analysis of translational control induced by oncogenic signaling.
- Author
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Rajasekhar VK and Holland EC
- Subjects
- 3' Untranslated Regions metabolism, Animals, Eukaryotic Initiation Factor-4E physiology, Gene Expression Profiling, Humans, Neoplasms genetics, Oligonucleotide Array Sequence Analysis, Phosphatidylinositol 3-Kinases physiology, Proto-Oncogene Proteins physiology, Proto-Oncogene Proteins c-akt, RNA, Messenger chemistry, RNA, Messenger metabolism, Ribosomes metabolism, Transforming Growth Factor beta physiology, ras Proteins physiology, Neoplasms etiology, Protein Biosynthesis, Protein Serine-Threonine Kinases, Signal Transduction physiology
- Abstract
It is commonly assumed that developmental and oncogenic signaling achieve their phenotypic effects primarily by directly regulating the transcriptional profile of cells. However, there is growing evidence that the direct effect on transcription may be overshadowed by differential effects on the translational efficiency of specific existing mRNA species. Global analysis of this effect using microarrays indicates that this mechanism of controlling protein production provides a highly specific, robust, and rapid response to oncogenic and developmental stimuli. The mRNAs so affected encode proteins involved in cell-cell interaction, signal transduction, and growth control. Furthermore, a large number of transcription factors capable of secondarily rearranging the transcriptional profile of the cell are controlled at this level as well. To what degree this translational control is either necessary or sufficient for tumor formation or maintenance remains to be determined.
- Published
- 2004
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41. Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes.
- Author
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Rajasekhar VK, Viale A, Socci ND, Wiedmann M, Hu X, and Holland EC
- Subjects
- Animals, Brain Neoplasms metabolism, Cell Cycle Proteins metabolism, Cell Transformation, Neoplastic drug effects, Cluster Analysis, Enzyme Inhibitors pharmacology, Gene Expression Regulation, Neoplastic genetics, Glioblastoma metabolism, Humans, Mice, Mice, Transgenic, Oligonucleotide Array Sequence Analysis, Polyribosomes genetics, Protein Biosynthesis drug effects, Protein Biosynthesis genetics, Proto-Oncogene Proteins antagonists & inhibitors, Proto-Oncogene Proteins c-akt, RNA, Messenger analysis, RNA, Messenger genetics, Signal Transduction drug effects, Signal Transduction genetics, Tumor Cells, Cultured, ras Proteins antagonists & inhibitors, Brain Neoplasms genetics, Cell Transformation, Neoplastic genetics, Glioblastoma genetics, Polyribosomes metabolism, Protein Serine-Threonine Kinases, Proto-Oncogene Proteins genetics, ras Proteins genetics
- Abstract
In order to determine the global effects of oncogenic Ras and Akt signaling pathways on translational efficiencies, we compared the gene expression profiles of total cellular mRNA and mRNA associated with polysomes. We found that the immediate effect of Ras and Akt signaling blockade on transcription was relatively modest; however, the profile of mRNA associated with polysomes was substantially altered. These observations indicate that the immediate effect of Ras and Akt signaling regulates the recruitment of specific mRNAs to ribosomes to a far greater extent than they regulate the production of mRNAs by transcriptional effects. The mRNAs most affected are those encoding proteins that regulate growth, transcription regulation, cell to cell interactions, and morphology. These data support a model whereby Ras and Akt signaling primarily lead to cellular transformation by altering the transcriptome and producing a radical shift in the composition of mRNAs associated with actively translating polysomes.
- Published
- 2003
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42. Early events in the signal pathway for the oxidative burst in soybean cells exposed to avirulent pseudomonas syringae pv glycinea
- Author
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Rajasekhar VK, Lamb C, and Dixon RA
- Abstract
Soybean (Glycine max) cv Williams 82 suspension cultures exhibit an oxidative burst approximately 3 h after challenge with Pseudomonas syringae pv glycinea (Psg) harboring the avrA (avirulence) gene. Pretreatment with the tyrosine (Tyr) kinase inhibitor herbimycin A or the serine/threonine kinase inhibitor K252a abolished the burst and subsequent induction of glutathione S-transferase. However, imposition of a 45-min rest period between pathogen challenge and subsequent addition of the kinase inhibitors resulted in escape from inhibition by herbimycin A, whereas inhibition by K252a persisted. Suramin, a G-protein inhibitor, inhibited the burst if added up to 90 min after pathogen challenge. The burst was also induced by the ion channel generator amphotericin B, and this induction was sensitive to suramin and K252a. Conversely, the ion channel blocker anthracene-9-carboxylate inhibited the Psg:avrA-induced burst. Psg:avrA rapidly induced Tyr phosphorylation of several proteins, and this was inhibited by herbimycin A or anthracene 9-carboxylic acid. These data suggest that the activation of ion channels is followed by an upstream Tyr kinase before the serine/threonine kinase-dependent steps in the signal pathway leading to the oxidative burst. Psg:avrA-dependent induction of phenylalanine ammonia-lyase was not inhibited by herbimycin or suramin, suggesting the operation of different signal pathways for the oxidative burst and phenylpropanoid-derived defense responses.
- Published
- 1999
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43. Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms.
- Author
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Shirasu K, Nakajima H, Rajasekhar VK, Dixon RA, and Lamb C
- Subjects
- Cell Survival drug effects, Hydrogen Peroxide metabolism, Phenylalanine analogs & derivatives, Phenylalanine pharmacology, Plants, Genetically Modified, Respiratory Burst, Salicylates chemistry, Salicylic Acid, Plant Diseases, Plant Growth Regulators pharmacology, Salicylates pharmacology, Signal Transduction
- Abstract
The phenylpropanoid-derived natural product salicylic acid (SA) plays a key role in disease resistance. However, SA administered in the absence of a pathogen is a paradoxically weak inductive signal, often requiring concentrations of 0.5 to 5 mM to induce acquired resistance or related defense mechanisms or to precondition signal systems. In contrast, endogenous SA accumulates to concentrations of < 70 microM at the site of attempted infection. Here, we show that although 10 to 100 microM SA had negligible effects when administered to soybean cell suspensions in the absence of a pathogen, physiological concentrations of SA markedly enhanced the induction of defense gene transcripts, H2O2 accumulation, and hypersensitive cell death by an avirulent strain of Pseudomonas syringae pv glycinea, with optimal effects being at approximately 50 microM. SA also synergistically enhanced H2O2 accumulation in response to the protein phosphatase type 2A inhibitor cantharidin in the absence of a pathogen. The synergistic effect of SA was potent, rapid, and insensitive to the protein synthesis inhibitor cycloheximide, and we conclude that SA stimulates an agonist-dependent gain control operating at an early step in the signal pathway for induction of the hypersensitive response. This fine control mechanism differs from previously described time-dependent, inductive coarse control mechanisms for SA action in the absence of a pathogen. Induction of H2O2 accumulation and hypersensitive cell death by avirulent P. s. glycinea was blocked by the phenylpropanoid synthesis inhibitor alpha-aminooxy-beta-phenylpropionic acid, and these responses could be rescued by exogenous SA. Because the agonist-dependent gain control operates at physiological levels of SA, we propose that rapid fine control signal amplification makes an important contribution to SA function in the induction of disease resistance mechanisms.
- Published
- 1997
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44. RNA Editing in Plant Mitochondria: [alpha]-Phosphate Is Retained during C-to-U Conversion in mRNAs.
- Author
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Rajasekhar VK and Mulligan RM
- Abstract
RNA editing in higher plant mitochondria frequently results in the post-transcriptional conversion of specific cytidine residues to uridine residues and infrequently results in the reverse conversion. The mechanisms by which this transition could occur are deamination or transamination of the amide at C-4 of cytosine, transglycosylation of the ribosyl residue, or deletion of a CMP residue and insertion of a UMP residue. Intact maize or petunia mitochondria were supplied with [alpha]-32P-CTP to radiolabel CMP residues in the nascent transcripts, and the fate of the [alpha]-phosphate was examined by digestion of the RNA to nucleotide monophosphates and analysis by two-dimensional chromatography. A small fraction of radioactivity comigrated with UMP on two different two-dimensional thin-layer chromatography systems, and the amount of radiolabeled UMP increased between l0-min and 2-hr incubations. The conversion of cytidine-to-uridine residues was detected in the highly edited mRNA fraction but was not detected in the rRNA fraction. Recovery of radiolabeled UMP residues suggests that the [alpha]-phosphate is retained during the editing reaction. These results are consistent with either deamination or transamination, or transglycosylation mechanisms for RNA editing.
- Published
- 1993
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45. Pea chloroplast DNA primase: characterization and role in initiation of replication.
- Author
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Nielsen BL, Rajasekhar VK, and Tewari KK
- Subjects
- Antibodies metabolism, Chloroplasts metabolism, Chromatography, DNA Primase, DNA Replication physiology, DNA, Single-Stranded metabolism, Dicarboxylic Acids pharmacology, Fabaceae metabolism, Kinetics, Organophosphorus Compounds pharmacology, RNA Nucleotidyltransferases antagonists & inhibitors, RNA Nucleotidyltransferases chemistry, RNA Nucleotidyltransferases isolation & purification, Chloroplasts enzymology, Fabaceae enzymology, Plants, Medicinal, RNA Nucleotidyltransferases metabolism
- Abstract
A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
- Published
- 1991
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46. Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes.
- Author
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Rajasekhar VK, Sun E, Meeker R, Wu BW, and Tewari KK
- Subjects
- Bacterial Toxins pharmacology, Base Sequence, Chromatography, Affinity, DNA, Ribosomal genetics, DNA, Ribosomal ultrastructure, DNA-Directed RNA Polymerases antagonists & inhibitors, DNA-Directed RNA Polymerases isolation & purification, DNA-Directed RNA Polymerases ultrastructure, Dicarboxylic Acids pharmacology, Fabaceae enzymology, Fluorescent Antibody Technique, Microscopy, Electron, Molecular Sequence Data, Organophosphorus Compounds pharmacology, Promoter Regions, Genetic, Protein Binding, RNA, Ribosomal, 16S genetics, Restriction Mapping, Chloroplasts enzymology, DNA-Directed RNA Polymerases metabolism, Fabaceae genetics, Genes, Plant, Plants, Medicinal, RNA, Messenger genetics, RNA, Ribosomal genetics, Transcription, Genetic drug effects
- Abstract
Pea chloroplast RNA polymerase has been obtained with about 2000-fold purification using DEAE-cellulose and phosphocellulose chromatography. The purified enzyme contained ten prominent polypeptides of 150, 130, 115, 110, 95, 85, 75, 48, 44 and 39 kDa and four other minor polypeptides of 90, 34, 32 and 27 kDa. Purification of this enzyme using chloroplast 16S rDNA promoter affinity column chromatography also yielded an enzyme with similar polypeptides. Purified polyclonal antibodies against the purified chloroplast RNA polymerase were found to recognize most of the polypeptides of the enzyme in Western blot experiments. Primary mobility shift of the 16S rRNA gene and ribulose-1,5-bisphosphate carboxylase large subunit (rbc-L) gene promoters observed with the chloroplast RNA polymerase was abolished by these antibodies. The specific in vitro transcription of these rRNA and mRNA genes was also inhibited by these antibodies. The transcription of the rRNA and mRNA genes was also abolished by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase. The chloroplast RNA polymerase was found to bind specifically to the chloroplast 16S rRNA gene promoter region as visualized in electron microscopy. The presence of the polypeptides of 130, 110, 75-95 and 48 kDa in the DNA-enzyme complex was confirmed by a novel approach using immunogold labeling with the respective antibodies. The polypeptides of this purified RNA polymerase were found to be localized in chloroplasts by an indirect immunofluorescence.
- Published
- 1991
- Full Text
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47. Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons.
- Author
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Rajasekhar VK, Gowri G, and Campbell WH
- Abstract
In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome.
- Published
- 1988
- Full Text
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48. Expression of nuclear genes as affected by treatments acting on the plastids.
- Author
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Oelmüller R, Levitan I, Bergfeld R, Rajasekhar VK, and Mohr H
- Abstract
Unlabelled: In a preceding paper (Oelmüller and Mohr 1986, Planta 167, 106-113) it was shown that in the cotyledons of the mustard (Sinapis alba L.) seedling the integrity of the plastid is a necessary prerequisite for phytochrome-controlled appearance of translatable mRNA for the nuclear-encoded small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCP). It was concluded that a signal from the plastid is essential for the expression of nuclear genes involved in plastidogenesis. The present study was undertaken to characterize this postulated signal. Chloramphenicol, an inhibitor of intraplastidic protein synthesis and Norflurazon, an inhibitor of carotenoid synthesis (to bring about photooxidative sensitivity of the plastids) were applied. We obtained the following major results. (i) After a brief period of photooxidative damage a rapid decrease of the above translatable mRNAs was observed., Conclusion: the signal is short-lived and thus required continually. (ii) Once the plastids became damaged by photooxidation, no recovery with regard to nuclear gene expression was observed after a transfer to non-damaging light conditions., Conclusion: even a brief period of damage suffices to prevent production of the signal. (iii) Chloramphenicol inhibited nuclear gene expression (SSU, LHCP) and plastidic development when applied during the early stages of plastidogenesis. Once a certain stage had been reached (between 36-48 h after sowing at 25° C) nuclear gene expression became remarkably insensitive toward inhibition of intraplastidic translation., Conclusion: a certain developmental stage of the plastid must be reached before the signal is released by the plastid. (iv) Under the growth conditions we adopted in our experiments the plastids in the mesophyll cells of mustard cotyledons developed essentially between 36 and 120 (-144) h after sowing. Only during this period could translatable mRNAs for SSU and LHCP be detected., Conclusion: the signal is released by the plastids only during this time span.
- Published
- 1986
- Full Text
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49. Effect of ammonium and nitrate on growth and appearance of nitrate reductase and nitrite reductase in dark- and light-grown mustard seedlings.
- Author
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Rajasekhar VK and Mohr H
- Abstract
Nitrate-induced and phytochrome-modulated appearance of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by externally supplied ammonium (NH 4 (+) ). In short-term experiments between 60 and 78 h after sowing it was found that in darkness NH 4 (+) -simultaneously given with NO 3 (-) -strongly inhibits appearance of nitrate-inducible NR and NIR whereas in continuous far-red light-which operates exclusively via phytochrome without significant chlorophyll formation -NH 4 (+) (simultaneously given with NO 3 (-) ) strongly stimulates appearance of NR. The NIR levels are not affected. This indicates that NR and NIR levels are regulated differently. In the absence of external NO 3 (-) appearance of NR is induced by NH4 in darkness as well as in continuous far-red light whereas NIR levels are not affected. On the other hand, in the absence of external NO 3 (-) , exogenous NH 4 (+) strongly inhibits growth of the mustard seedling in darkness as well as in continuous far-red light. This effect can be abolished by simultaneously supplying NO 3 (-) . The adverse effect of NH 4 (+) on growth ('NH 4 (+) -toxicity') cannot be attributed to pH-changes in the medium since it was shown that neither the growth responses nor the changes of the enzyme levels are related to pH changes in the medium. Non-specific osmotic effects are not involved either.
- Published
- 1986
- Full Text
- View/download PDF
50. Appearance of nitrite reductase in cotyledons of the mustard (Sinapis alba L.) seedling as affected by nitrate, phytochrome and photooxidative damage of plastids.
- Author
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Rajasekhar VK and Mohr H
- Abstract
Nitrite reductase (NIR; EC 1.7.7.1) is a central enzyme in nitrate assimilation and is localized in plastids. The present study concerns the regulation of the appearance of NIR in cotyledons of the mustard (Sinapis alba L.) seedling. It was shown that light exerts its positive control over the nitrate-mediated induction of NIR via the farred-absorbing form of phytochrome. Without nitrate the light effect cannot express itself; even though the light signal is accumulated in the cotyledons it remains totally cryptic in the absence of nitrate. Moreover, it was recognised that 'intact plastids' are important in the control of the appearance of NIR. If the plastids are damaged by photooxidation the action of nitrate and phytochrome on NIR appearance is abolished. The appearance of nitrate reductase (NR; EC 1.6.6.1) responds similarly to photooxidative damage even though this enzyme is cytosolic. While the data strongly indicate that some 'plastidic signal' is a prerequisite for the nitrate-induced and phytochrome-modulated appearance of NIR and NR, the possibility could not be ruled out that photooxidative damage affects the accumulation of NIR in the organelle.
- Published
- 1986
- Full Text
- View/download PDF
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