36 results on '"Samrat T. Kundu"'
Search Results
2. TMEM106B drives lung cancer metastasis by inducing TFEB-dependent lysosome synthesis and secretion of cathepsins
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Samrat T. Kundu, Caitlin L. Grzeskowiak, Jared J. Fradette, Laura A. Gibson, Leticia B. Rodriguez, Chad J. Creighton, Kenneth L. Scott, and Don L. Gibbons
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Science - Abstract
One of the major causes of cancer-related mortality is represented by metastatic lung cancer. Here the authors characterize the role of TMEM106B in driving metastatic lung adenocarcinoma and suggest that TMEM106B-mediated secretion of cathespin impacts cell migration and invasion of lung cancer cells, increasing metastatic spreading.
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- 2018
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- View/download PDF
3. In vivo screening identifies GATAD2B as a metastasis driver in KRAS-driven lung cancer
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Caitlin L. Grzeskowiak, Samrat T. Kundu, Xiulei Mo, Andrei A. Ivanov, Oksana Zagorodna, Hengyu Lu, Richard H. Chapple, Yiu Huen Tsang, Daniela Moreno, Maribel Mosqueda, Karina Eterovic, Jared J. Fradette, Sumreen Ahmad, Fengju Chen, Zechen Chong, Ken Chen, Chad J. Creighton, Haian Fu, Gordon B. Mills, Don L. Gibbons, and Kenneth L. Scott
- Subjects
Science - Abstract
KRAS-driven lung cancers represent an aggressive form of NSCLC. In this study the authors perform an in vivo functional screening and identify GATAD2B as a driver of tumor growth and metastasis in KRAS-driven lung cancer.
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- 2018
- Full Text
- View/download PDF
4. ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Mabel Perez-Oquendo, Roxsan Manshouri, Yanhua Tian, Jared J. Fradette, B. Leticia Rodriguez, Samrat T. Kundu, and Don L. Gibbons
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Cancer Research ,Oncology ,Molecular Biology - Abstract
Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non–small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. Implications: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.
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- 2023
5. Supplementary Table 2 from ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Don L. Gibbons, Samrat T. Kundu, B. Leticia Rodriguez, Jared J. Fradette, Yanhua Tian, Roxsan Manshouri, and Mabel Perez-Oquendo
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Potential ZEB1 acetylation-dependent target genes.
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- 2023
6. Supplementary Table 4 from ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Don L. Gibbons, Samrat T. Kundu, B. Leticia Rodriguez, Jared J. Fradette, Yanhua Tian, Roxsan Manshouri, and Mabel Perez-Oquendo
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qPCR Primer Sequences
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- 2023
7. Supplementary Table 3 from ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Don L. Gibbons, Samrat T. Kundu, B. Leticia Rodriguez, Jared J. Fradette, Yanhua Tian, Roxsan Manshouri, and Mabel Perez-Oquendo
- Abstract
Antibody information
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- 2023
8. Supplementary Figures 1-8 from ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Don L. Gibbons, Samrat T. Kundu, B. Leticia Rodriguez, Jared J. Fradette, Yanhua Tian, Roxsan Manshouri, and Mabel Perez-Oquendo
- Abstract
S1. K811 acetylation is integral to ZEB1 molecular weight. S2. &tilde225 kDa ZEB1 band regulates protein stability. S3. K811 acetylation facilitates ZEB1/NuRD interaction. S4. K811 acetylation directs the ZEB1/NuRD complex to bind at genomic promoters. S5. K811 acetylation decreased epithelial expression levels. S6. RNA-seq reveals K811 acetylation downregulates the expression of epithelial genes. S7. K811 acetylation in ZEB1 KO cells promotes lung adenocarcinoma migration and invasion. S8. K811 acetylation enhances metastatic potential of murine NSCLC cells.
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- 2023
9. Data from ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Don L. Gibbons, Samrat T. Kundu, B. Leticia Rodriguez, Jared J. Fradette, Yanhua Tian, Roxsan Manshouri, and Mabel Perez-Oquendo
- Abstract
Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non–small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition.Implications:The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.
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- 2023
10. Supplementary Table 6 from ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Don L. Gibbons, Samrat T. Kundu, B. Leticia Rodriguez, Jared J. Fradette, Yanhua Tian, Roxsan Manshouri, and Mabel Perez-Oquendo
- Abstract
Protein expression values of the immunoprecipitated samples
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- 2023
11. Supplementary Table 5 from ZEB1 is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis
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Don L. Gibbons, Samrat T. Kundu, B. Leticia Rodriguez, Jared J. Fradette, Yanhua Tian, Roxsan Manshouri, and Mabel Perez-Oquendo
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ChIP Primer Sequences
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- 2023
12. The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8+ cytotoxic T-cell response
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Samrat T. Kundu, B. Leticia Rodriguez, Laura A. Gibson, Amanda N. Warner, Mabel G. Perez, Rakhee Bajaj, Jared J. Fradette, Caleb A. Class, Luisa M. Solis, Frank R. Rojas Alvarez, Ignacio I. Wistuba, Lixia Diao, Fengju Chen, Mohit Sachdeva, Jing Wang, David G. Kirsch, Chad J. Creighton, and Don L. Gibbons
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Genetics ,Developmental Biology - Abstract
One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.
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- 2022
13. Lysosomal transmembrane protein TMEM106B alters TFEB signaling and the tumor immune microenvironment
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Amanda N. Warner, Samrat T. Kundu, Rakhee Bajaj, Bertha Leticia Rodriguez, and Don Gibbons
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- 2022
14. IMPAD1 and KDELR2 drive invasion and metastasis by enhancing Golgi-mediated secretion
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Samrat T. Kundu, Chad J. Creighton, Jared J. Fradette, Kenneth L. Scott, Caitlin L. Grzeskowiak, Rakhee Bajaj, and Don L. Gibbons
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0301 basic medicine ,Cancer Research ,Lung Neoplasms ,Vesicular Transport Proteins ,Fluorescent Antibody Technique ,Golgi Apparatus ,Matrix metalloproteinase ,Biology ,Article ,Metastasis ,03 medical and health sciences ,symbols.namesake ,0302 clinical medicine ,Cell Movement ,Cell Line, Tumor ,Genetics ,medicine ,Humans ,Secretion ,Neoplasm Invasiveness ,Lung cancer ,Molecular Biology ,Cell Proliferation ,Regulation of gene expression ,Cell growth ,Cancer ,Golgi apparatus ,medicine.disease ,Matrix Metalloproteinases ,Phosphoric Monoester Hydrolases ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,030220 oncology & carcinogenesis ,symbols ,Cancer research ,Disease Progression - Abstract
Summary Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide, due in part to its proclivity to metastasize. Identifying novel drivers of invasion and metastasis holds therapeutic potential for the disease. We conducted a gain-of-function invasion screen, which identified two separate hits, IMPAD1 and KDELR2, as robust, independent drivers of lung cancer invasion and metastasis. Given that IMPAD1 and KDELR2 are known to be localized to the ER-Golgi pathway, we studied their common mechanism of driving in vitro invasion and in vivo metastasis and demonstrated that they enhance Golgi-mediated function and secretion. Therapeutically inhibiting matrix metalloproteases (MMPs) suppressed both IMPAD1- and KDELR2-mediated invasion. The hits from this unbiased screen and the mechanistic validation highlight Golgi function as one of the key cellular features altered during invasion and metastasis.
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- 2020
15. The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8
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Samrat T, Kundu, B Leticia, Rodriguez, Laura A, Gibson, Amanda N, Warner, Mabel G, Perez, Rakhee, Bajaj, Jared J, Fradette, Caleb A, Class, Luisa M, Solis, Frank R, Rojas Alvarez, Ignacio I, Wistuba, Lixia, Diao, Fengju, Chen, Mohit, Sachdeva, Jing, Wang, David G, Kirsch, Chad J, Creighton, and Don L, Gibbons
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Mice ,MicroRNAs ,Lung Neoplasms ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,Tumor Microenvironment ,Animals ,Interleukin-2 ,CD8-Positive T-Lymphocytes ,T-Lymphocytes, Cytotoxic - Abstract
One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8
- Published
- 2021
16. Lung cancer models reveal SARS-CoV-2-induced EMT contributes to COVID-19 pathophysiology
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C. Allison Stewart, Carl M. Gay, Kavya Ramkumar, Kasey R. Cargill, Robert J. Cardnell, Monique B. Nilsson, Simon Heeke, Elizabeth M. Park, Samrat T. Kundu, Lixia Diao, Qi Wang, Li Shen, Yuanxin Xi, Bingnan Zhang, Carminia Maria Della Corte, Youhong Fan, Kiran Kundu, Boning Gao, Kimberley Avila, Curtis R. Pickering, Faye M. Johnson, Jianjun Zhang, Humam Kadara, John D. Minna, Don L. Gibbons, Jing Wang, John V. Heymach, Lauren Averett Byers, Stewart, C. A., Gay, C. M., Ramkumar, K., Cargill, K. R., Cardnell, R. J., Nilsson, M. B., Heeke, S., Park, E. M., Kundu, S. T., Diao, L., Wang, Q., Shen, L., Xi, Y., Zhang, B., Della Corte, C. M., Fan, Y., Kundu, K., Gao, B., Avila, K., Pickering, C. R., Johnson, F. M., Zhang, J., Kadara, H., Minna, J. D., Gibbons, D. L., Wang, J., Heymach, J. V., and Byers, L. A.
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Pulmonary and Respiratory Medicine ,Oncology ,medicine.medical_specialty ,Lung Neoplasms ,Coronavirus disease 2019 (COVID-19) ,viruses ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Population ,ACE2 ,Bronchi ,Peptidyl-Dipeptidase A ,NSCLC ,Pathogenesis ,Internal medicine ,medicine ,Humans ,ZEB1 ,Epithelial–mesenchymal transition ,Respiratory system ,education ,Lung cancer ,Lung ,Psychological repression ,Cell entry ,education.field_of_study ,SARS-CoV-2 ,business.industry ,Mesenchymal stem cell ,EMT ,AXL ,COVID-19 ,Cancer ,medicine.disease ,Editorial ,medicine.anatomical_structure ,Viral Receptor ,Cancer research ,Original Article ,business ,Bristol-Myers ,Human - Abstract
Background: SARS-CoV-2 infection is the cause of the respiratory illness COVID-19, which presents most frequently with respiratory symptoms SARS-CoV-2 cell entry requires interactions with ACE2 and TMPRSS2 on the surface of the host cell Cancer patients and, specifically, those with thoracic malignancies seem to experience poorer clinical outcomes Methods: We utilized bulk and single-cell transcriptional data from a combination of normal and malignant tissues and cells from aerodigestive and respiratory tracts to explore mechanisms governing the expression of ACE2 and TMPRSS2 Additionally, we determined the effect of EMT induction, ZEB1 modulation, and SARS-CoV-2 infection on ACE2 expression Results: Our bulk data suggests that aerodigestive and lung cancer models express a broad range of ACE2 and TMRPSS2, particularly in epithelial cells, and would serve as good models for studying SARS-CoV-2 infection We assessed the relationship between ACE2 and epithelial differentiation in numerous datasets, and found consistent positive correlations with transcriptional and microRNA signifiers of epithelial differentiation The miR-200 family – zinc finger E-box-binding homeobox 1 (ZEB1) pathway, which is an established regulator of EMT, also directly regulates ACE2 expression, likely via putative ZEB1 repressor sites located in the ACE2 promoter Furthermore, SARS-CoV-2 infection reduces ACE2 expression and shifts cells to a more mesenchymal phenotype with loss of EPCAM and upregulation of ZEB1 and other EMT-associated genes Conclusions: ACE2-positive cells are almost exclusively epithelial and unexpectedly rare, considering the devastating impact of this infection Following viral entry, SARS-CoV-2 infection induces molecular changes within the cells that are reminiscent of EMT, including increased ZEB1 ZEB1, in turn, appears to directly repress the expression of ACE2 This SARS-CoV-2-induced ACE2 deficiency, compounded by the downregulation of genes, including claudins, which play a critical role in restricting epithelial and endothelial permeability, exposes respiratory cells to increased risk of edema and acute respiratory distress syndrome (ARDS) Legal entity responsible for the study: The authors Funding: NIH/NCI R01-CA207295 (L A B ), NIH/NCI U01-CA213273 (L A B , J V H ), CCSG P30-CA01667 (L A B ), University of Texas SPORE in Lung Cancer P5-CA070907 (L A B , D L G , J V H , C M G ), the Department of Defense (LC170171;L A B ), Khalifa Bin Zayed Al Nahyan Foundation (C M G ), RP170067 (EMP), through generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shot Program and Andrew Sabin Family Fellowship, and The Rexanna Foundation for Fighting Lung Cancer Disclosure: C Gay: Research grant/Funding (self): Astra Zeneca J V Heymach: Advisory/Consultancy: AstraZeneca;Advisory/Consultancy: Boehringer Ingelheim;Advisory/Consultancy: Exelixis;Advisory/Consultancy: Genentech;Advisory/Consultancy: GlaxoSmithKline;Advisory/Consultancy: Guardant Health;Advisory/Consultancy: Hengrui;Advisory/Consultancy: Spectrum L A Byers: Advisory/Consultancy, Research grant/Funding (self): AstraZeneca;Advisory/Consultancy, Research grant/Funding (self): AbbVie;Advisory/Consultancy, Research grant/Funding (self): GenMab;Advisory/Consultancy: BergenBio;Advisory/Consultancy: Pharma Mar SA;Advisory/Consultancy, Research grant/Funding (self): Sierra Oncology;Advisory/Consultancy: Merck;Advisory/Consultancy: Bristol Myers Squibb;Advisory/Consultancy: Genentech;Advisory/Consultancy: Pfizer;Research grant/Funding (self): Tolero Pharmaceuticals All other authors have declared no conflicts of interest
- Published
- 2021
17. Impad1 and Syt11 work in an epistatic pathway that regulates EMT-mediated vesicular trafficking to drive lung cancer invasion and metastasis
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Rakhee Bajaj, B. Leticia Rodriguez, William K. Russell, Amanda N. Warner, Lixia Diao, Jing Wang, Maria G. Raso, Wei Lu, Khaja Khan, Luisa S. Solis, Harsh Batra, Ximing Tang, Jared F. Fradette, Samrat T. Kundu, and Don L. Gibbons
- Subjects
Gene Expression Regulation, Neoplastic ,MicroRNAs ,Synaptotagmins ,Epithelial-Mesenchymal Transition ,Lung Neoplasms ,Cell Movement ,Cell Line, Tumor ,Tumor Microenvironment ,Humans ,Neoplasm Invasiveness ,Neoplasm Metastasis ,General Biochemistry, Genetics and Molecular Biology - Abstract
Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput in vitro and in vivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression.
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- 2022
18. Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.
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Nileema Khapare, Samrat T Kundu, Lalit Sehgal, Mugdha Sawant, Rashmi Priya, Prajakta Gosavi, Neha Gupta, Hunain Alam, Madhura Karkhanis, Nishigandha Naik, Milind M Vaidya, and Sorab N Dalal
- Subjects
Medicine ,Science - Abstract
The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.
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- 2012
- Full Text
- View/download PDF
19. MBIP (MAP3K12 binding inhibitory protein) drives NSCLC metastasis by JNK-dependent activation of MMPs
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Jared J. Fradette, Rakhee Bajaj, Don L. Gibbons, Joshua K. Ochieng, Samrat T. Kundu, and B. Leticia Rodriguez
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0301 basic medicine ,Male ,Cancer Research ,Lung Neoplasms ,Matrix metalloproteinase ,Biology ,Article ,Metastasis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,In vivo ,Cell Movement ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,Genetics ,Carcinoma ,medicine ,Animals ,Humans ,Neoplasm Invasiveness ,Neoplasm Metastasis ,Molecular Biology ,Gene ,Cell Proliferation ,Regulation of gene expression ,Intracellular Signaling Peptides and Proteins ,medicine.disease ,Xenograft Model Antitumor Assays ,In vitro ,Matrix Metalloproteinases ,Gene Expression Regulation, Neoplastic ,Disease Models, Animal ,030104 developmental biology ,Cell culture ,030220 oncology & carcinogenesis ,Gain of Function Mutation ,Gene Knockdown Techniques ,Cancer research ,Disease Progression ,Female - Abstract
Metastasis is the cause for 90% of cancer-related mortalities. Identification of genetic drivers promoting dissemination of tumor cells may provide opportunities for novel therapeutic strategies. We previously reported an in vivo gain-of-function screen that identified ∼30 genes with a functional role in metastasis promotion and characterized detailed mechanistic functions of two hits. In the present study, we characterized the contribution of one of the identified genes, MBIP (MAP3K12 binding inhibitory protein), towards driving tumor invasion and metastasis. We demonstrate that expression of MBIP significantly enhances the cellular proliferation, migration and invasion of NSCLC cells in vitro and metastasis in vivo. We functionally characterized that MBIP mediates activation of the JNK pathway and induces expression of matrix metalloproteinases (MMPs), which are necessary for the invasive and metastatic phenotype. Our findings establish a novel mechanistic role of MBIP as a driver of NSCLC progression and metastasis.
- Published
- 2019
20. In vivo screening identifies GATAD2B as a metastasis driver in KRAS-driven lung cancer
- Author
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Daniela Moreno, Gordan B. Mills, Samrat T. Kundu, Andrey A. Ivanov, Karina Eterovic, Caitlin L. Grzeskowiak, Kenneth L. Scott, Haian Fu, Femgju Chen, Chad J. Creighton, Don L. Gibbons, Jared J. Fradette, Hengyu Lu, Yiu Huen Tsang, Zechen Chong, Xiulei Mo, Sumreen Ahmad, Ken Chen, Richard H. Chapple, Maribel Mosqueda, and Oksana Zagorodna
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0301 basic medicine ,Lung Neoplasms ,endocrine system diseases ,Mutant ,General Physics and Astronomy ,medicine.disease_cause ,GATA Transcription Factors ,Metastasis ,Mice ,Neoplasm Metastasis ,RNA, Small Interfering ,lcsh:Science ,Regulation of gene expression ,Multidisciplinary ,respiratory system ,3. Good health ,Gene Expression Regulation, Neoplastic ,Adenocarcinoma ,Female ,KRAS ,Signal Transduction ,Science ,Genetic Vectors ,Mice, Nude ,Cre recombinase ,Adenocarcinoma of Lung ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Proto-Oncogene Proteins c-myc ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Allele ,Lung cancer ,neoplasms ,Integrases ,Lentivirus ,General Chemistry ,medicine.disease ,Survival Analysis ,Xenograft Model Antitumor Assays ,digestive system diseases ,High-Throughput Screening Assays ,respiratory tract diseases ,Repressor Proteins ,030104 developmental biology ,Cancer research ,lcsh:Q - Abstract
Genetic aberrations driving pro-oncogenic and pro-metastatic activity remain an elusive target in the quest of precision oncology. To identify such drivers, we use an animal model of KRAS-mutant lung adenocarcinoma to perform an in vivo functional screen of 217 genetic aberrations selected from lung cancer genomics datasets. We identify 28 genes whose expression promoted tumor metastasis to the lung in mice. We employ two tools for examining the KRAS-dependence of genes identified from our screen: 1) a human lung cell model containing a regulatable mutant KRAS allele and 2) a lentiviral system permitting co-expression of DNA-barcoded cDNAs with Cre recombinase to activate a mutant KRAS allele in the lungs of mice. Mechanistic evaluation of one gene, GATAD2B, illuminates its role as a dual activity gene, promoting both pro-tumorigenic and pro-metastatic activities in KRAS-mutant lung cancer through interaction with c-MYC and hyperactivation of the c-MYC pathway., KRAS-driven lung cancers represent an aggressive form of NSCLC. In this study the authors perform an in vivo functional screening and identify GATAD2B as a driver of tumor growth and metastasis in KRAS-driven lung cancer.
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- 2018
21. Dynamic variations in epithelial-to-mesenchymal transition (EMT), ATM, and SLFN11 govern response to PARP inhibitors and cisplatin in small cell lung cancer
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C. Allison Stewart, Pan Tong, Robert J. Cardnell, Triparna Sen, Lerong Li, Carl M. Gay, Fatemah Masrorpour, You Fan, Rasha O. Bara, Ying Feng, Yuanbin Ru, Junya Fujimoto, Samrat T. Kundu, Leonard E. Post, Karen Yu, Yuqiao Shen, Bonnie S. Glisson, Ignacio Wistuba, John V. Heymach, Don L. Gibbons, Jing Wang, and Lauren Averett Byers
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0301 basic medicine ,Gerontology ,Epithelial-Mesenchymal Transition ,Lung Neoplasms ,SLFN11 ,Gene Expression ,Ataxia Telangiectasia Mutated Proteins ,Poly(ADP-ribose) Polymerase Inhibitors ,Gene Knockout Techniques ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Humans ,Gene silencing ,Medicine ,Epithelial–mesenchymal transition ,Cisplatin ,biology ,business.industry ,Molecular pathology ,EMT ,Nuclear Proteins ,SCLC ,Cancer ,Cadherins ,medicine.disease ,Immunohistochemistry ,Small Cell Lung Carcinoma ,humanities ,respiratory tract diseases ,3. Good health ,ALDH1A1 ,PARP inhibitor ,030104 developmental biology ,Oncology ,Drug Resistance, Neoplasm ,Gene Knockdown Techniques ,ATM ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,business ,Ovarian cancer ,Biomarkers ,DNA Damage ,Research Paper ,medicine.drug - Abstract
// C. Allison Stewart 1 , Pan Tong 2 , Robert J. Cardnell 1 , Triparna Sen 1 , Lerong Li 2 , Carl M. Gay 1 , Fatemah Masrorpour 1 , You Fan 1 , Rasha O. Bara 1 , Ying Feng 3 , Yuanbin Ru 3 , Junya Fujimoto 4 , Samrat T. Kundu 1 , Leonard E. Post 3 , Karen Yu 3 , Yuqiao Shen 3 , Bonnie S. Glisson 1 , Ignacio Wistuba 4 , John V. Heymach 1 , Don L. Gibbons 1 , Jing Wang 2 and Lauren Averett Byers 1 1 Department of Thoracic Head & Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 3 BioMarin Pharmaceutical, San Rafael, CA 94901, USA 4 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence to: Lauren Averett Byers, email: lbyers@mdanderson.org Keywords: SCLC, SLFN11, ATM, EMT, PARP inhibitor Received: December 10, 2016 Accepted: January 19, 2017 Published: February 15, 2017 ABSTRACT Small cell lung cancer (SCLC) is one of the most aggressive forms of cancer, with a 5-year survival
- Published
- 2017
22. P1.04-26 EMT-Associated Response and Resistance to MEK Inhibitor and Immune Checkpoint Blockade Combinations in KRAS-Mutant NSCLC
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Limo Chen, Bertha Leticia Rodriguez, Jennifer A. Wargo, David H. Peng, Don L. Gibbons, Jared J. Fradette, Laura A. Gibson, Samrat T. Kundu, Pierre Olivier Gaudreau, Triparna Sen, and C.M. Della Corte
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Pulmonary and Respiratory Medicine ,Oncology ,business.industry ,MEK inhibitor ,Mutant ,Cancer research ,medicine ,KRAS ,medicine.disease_cause ,business ,Immune checkpoint ,Blockade - Published
- 2019
23. ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism
- Author
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Jing Wang, Timothy P. Heffernan, Barbara Mino, Jared J. Fradette, Don L. Gibbons, Lixia Diao, Christopher A. Bristow, Alessandro Carugo, Samrat T. Kundu, Ignacio I. Wistuba, Yanan Yang, Michael Peoples, David H. Peng, Jaime Rodriguez Canales, Pan Tong, Pamela Villalobos, Rosalba Minelli, and Lauren Averett Byers
- Subjects
MAPK/ERK pathway ,Lung Neoplasms ,Mocetinostat ,MAP Kinase Signaling System ,medicine.drug_class ,medicine.disease_cause ,Article ,Mesoderm ,Proto-Oncogene Proteins p21(ras) ,Small hairpin RNA ,Mice ,chemistry.chemical_compound ,Cell Line, Tumor ,Neoplasms ,medicine ,Animals ,Humans ,Phosphorylation ,Lung cancer ,Protein kinase A ,Protein Kinase Inhibitors ,Cell Proliferation ,Mitogen-Activated Protein Kinase Kinases ,Receptors, Interleukin-17 ,Kinase ,Histone deacetylase inhibitor ,Zinc Finger E-box-Binding Homeobox 1 ,Epithelial Cells ,General Medicine ,medicine.disease ,Disease Models, Animal ,MicroRNAs ,Pyrimidines ,chemistry ,Drug Resistance, Neoplasm ,Benzamides ,Mutation ,Cancer research ,KRAS - Abstract
Mitogen-activated protein kinase kinase (MEK) inhibitors have failed to show clinical benefit in Kirsten rat sarcoma (KRAS)-mutant lung cancer due to various resistance mechanisms. To identify differential therapeutic sensitivities between epithelial and mesenchymal lung tumors, we performed in vivo small hairpin RNA (shRNA) screens, proteomic profiling, and analysis of patient tumor datasets, which revealed an inverse correlation between mitogen-activated protein kinase (MAPK) signaling dependency and a zinc finger E-box-binding homeobox 1 (ZEB1)-regulated epithelial-to-mesenchymal transition (EMT). Mechanistic studies determined that MAPK signaling dependency in epithelial lung cancer cells is due to the scaffold protein interleukin 17 receptor D (IL17RD), which is directly repressed by ZEB1. Lung tumors in multiple Kras mutant murine models with increased ZEB1 displayed low IL17RD expression, accompanied by MAPK-independent tumor growth and therapeutic resistance to MEK inhibition. Suppression of ZEB1 function with miR-200 expression or the histone deacetylase (HDAC) inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and markedly reduced in vivo tumor growth, showing a promising combinatorial treatment strategy for KRAS-mutant cancers. In human lung tumor samples, high ZEB1 and low IL17RD expression correlated with low MAPK signaling, presenting potential markers that predict patient response to MEK inhibitors.
- Published
- 2019
24. TMEM106B drives lung cancer metastasis by inducing TFEB-dependent lysosome synthesis and secretion of cathepsins
- Author
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Kenneth L. Scott, Samrat T. Kundu, Laura A. Gibson, Leticia B. Rodriguez, Caitlin L. Grzeskowiak, Jared J. Fradette, Chad J. Creighton, and Don L. Gibbons
- Subjects
0301 basic medicine ,Lung Neoplasms ,General Physics and Astronomy ,Metastasis ,Mice ,Protein Isoforms ,Neoplasm Metastasis ,lcsh:Science ,Multidisciplinary ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,respiratory system ,Prognosis ,3. Good health ,Gene Expression Regulation, Neoplastic ,medicine.anatomical_structure ,Signal Transduction ,Science ,Adenocarcinoma of Lung ,Antineoplastic Agents ,Nerve Tissue Proteins ,Cysteine Proteinase Inhibitors ,General Biochemistry, Genetics and Molecular Biology ,Article ,Exocytosis ,03 medical and health sciences ,Leucine ,Lysosome ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Lung cancer ,Cathepsin ,business.industry ,Membrane Proteins ,General Chemistry ,medicine.disease ,Cathepsins ,Survival Analysis ,Xenograft Model Antitumor Assays ,respiratory tract diseases ,030104 developmental biology ,Cancer cell ,Proteolysis ,Cancer research ,TFEB ,Ectopic expression ,lcsh:Q ,Calcium ,business ,Lysosomes ,Genetic screen - Abstract
Metastatic lung cancer is the leading cause of cancer-associated mortality worldwide, therefore necessitating novel approaches to identify specific genetic drivers for lung cancer progression and metastasis. We recently performed an in vivo gain-of-function genetic screen to identify driver genes of lung cancer metastasis. In the study reported here, we identify TMEM106B as a primary robust driver of lung cancer metastasis. Ectopic expression of TMEM106B could significantly promote the synthesis of enlarged vesicular lysosomes that are laden with elevated levels of active cathepsins. In a TFEB-dependent manner, TMEM106B could modulate the expression of lysosomal genes of the coordinated lysosomal expression and regulation (CLEAR) pathway in lung cancer cells and patient samples. We also demonstrate that TMEM106B-induced lysosomes undergo calcium-dependent exocytosis, thereby releasing active lysosomal cathepsins necessary for TMEM106B-mediated cancer cell invasion and metastasis in vivo, which could be therapeutically prevented by pharmacological inhibition of cathepsins. Further, in TCGA LUAD data sets, 19% of patients show elevated expression of TMEM106B, which predicts for poor disease-free and overall-survival., One of the major causes of cancer-related mortality is represented by metastatic lung cancer. Here the authors characterize the role of TMEM106B in driving metastatic lung adenocarcinoma and suggest that TMEM106B-mediated secretion of cathespin impacts cell migration and invasion of lung cancer cells, increasing metastatic spreading.
- Published
- 2018
25. The miR-200 family and the miR-183~96~182 cluster target Foxf2 to inhibit invasion and metastasis in lung cancers
- Author
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Jing Wang, Don L. Gibbons, Lauren Averett Byers, Jonathon D. Roybal, Chad J. Creighton, Pan Tong, David H. Peng, Samrat T. Kundu, and Lixia Diao
- Subjects
0301 basic medicine ,Cancer Research ,Epithelial-Mesenchymal Transition ,Lung Neoplasms ,Mice, Inbred Strains ,Biology ,Article ,Metastasis ,03 medical and health sciences ,Cell Movement ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,microRNA ,Genetics ,medicine ,Animals ,Humans ,Epithelial–mesenchymal transition ,Lung cancer ,Molecular Biology ,Zeb1 ,Homeodomain Proteins ,Regulation of gene expression ,EMT ,Zinc Finger E-box-Binding Homeobox 1 ,Cancer ,Forkhead Transcription Factors ,miR-200 ,invasion ,Cadherins ,medicine.disease ,Xenograft Model Antitumor Assays ,Foxf2 ,3. Good health ,Gene Expression Regulation, Neoplastic ,lung cancer ,MicroRNAs ,030104 developmental biology ,Multigene Family ,Immunology ,Cancer cell ,Cancer research ,Ectopic expression ,miR-183-96-182 ,Transcription Factors - Abstract
Metastatic lung cancer is one of the most lethal forms of cancer and molecular pathways driving metastasis are still not clearly elucidated. Metastatic cancer cells undergo an epithelial-mesenchymal transition (EMT) where they lose their epithelial properties and acquire a migratory and invasive phenotype. Here we identify that expression of microRNAs from the miR-200 family and the miR-183~96~182 cluster are significantly co-repressed in non-small cell lung cancer (NSCLC) cell lines and primary tumors from multiple TCGA data sets with high EMT scores. Ectopic expression of the miR-183~96~182 cluster inhibited cancer cell migration and invasion, while its expression was tightly modulated by miR-200. We identified Foxf2 as a common, novel and direct target of both these microRNA families. Foxf2 expression tightly correlates with the transcription factor Zeb1 and is elevated in mesenchymal-like metastatic lung cancer cells. Foxf2 expression induced robust EMT, migration, invasion and metastasis in lung cancer cells, whereas Foxf2 inhibition significantly repressed these phenotypes. We also demonstrated that Foxf2 transcriptionally represses E-Cadherin and miR-200, independent of Zeb1, to form a double negative feedback loop. We therefore identified a novel mechanism whereby the miR-200 family and the miR-183~96~182 cluster inhibit lung cancer invasion and metastasis by targeting Foxf2.
- Published
- 2015
26. Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells
- Author
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Lalit Sehgal, Sorab N. Dalal, Milind M. Vaidya, Samrat T. Kundu, and Hunain Alam
- Subjects
Keratin 14 ,Cellular differentiation ,Mice, Nude ,Cell Cycle Proteins ,macromolecular substances ,Biology ,Mice ,Cell Line, Tumor ,Keratin ,Cell Adhesion ,Animals ,Humans ,Receptor, Notch1 ,Interphase ,Molecular Biology ,Involucrin ,Cytoskeleton ,Cell Proliferation ,chemistry.chemical_classification ,integumentary system ,Cell growth ,Keratin-14 ,Cell Differentiation ,Epithelial Cells ,Articles ,Cell Biology ,Antigens, Differentiation ,Molecular biology ,Extracellular Matrix ,Cell biology ,Enzyme Activation ,Keratin 5 ,HEK293 Cells ,Microscopy, Fluorescence ,chemistry ,Cell culture ,Gene Knockdown Techniques ,Keratin 7 ,Keratin-5 ,RNA Interference ,Proto-Oncogene Proteins c-akt ,Neoplasm Transplantation ,Signal Transduction - Abstract
Keratin expression in stratified epithelia is tightly regulated during squamous cell differentiation. Keratins 5 and 14 are expressed in mitotically active basal layer cells, but their function is not well defined. Reported here is the possible role of K14 in regulation of cell proliferation/differentiation in stratified epithelial cells., Keratins are cytoplasmic intermediate filament proteins preferentially expressed by epithelial tissues in a site-specific and differentiation-dependent manner. The complex network of keratin filaments in stratified epithelia is tightly regulated during squamous cell differentiation. Keratin 14 (K14) is expressed in mitotically active basal layer cells, along with its partner keratin 5 (K5), and their expression is down-regulated as cells differentiate. Apart from the cytoprotective functions of K14, very little is known about K14 regulatory functions, since the K14 knockout mice show postnatal lethality. In this study, K14 expression was inhibited using RNA interference in cell lines derived from stratified epithelia to study the K14 functions in epithelial homeostasis. The K14 knockdown clones demonstrated substantial decreases in the levels of the K14 partner K5. These cells showed reduction in cell proliferation and delay in cell cycle progression, along with decreased phosphorylated Akt levels. K14 knockdown cells also exhibited enhanced levels of activated Notch1, involucrin, and K1. In addition, K14 knockdown AW13516 cells showed significant reduction in tumorigenicity. Our results suggest that K5 and K14 may have a role in maintenance of cell proliferation potential in the basal layer of stratified epithelia, modulating phosphatidylinositol 3-kinase/Akt–mediated cell proliferation and/or Notch1-dependent cell differentiation.
- Published
- 2011
27. E-cadherin and plakoglobin recruit plakophilin3 to the cell border to initiate desmosome assembly
- Author
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Samrat T. Kundu, Nileema Khapare, Lalit Sehgal, Mansi S. Karkhanis, Sorab N. Dalal, and Prajakta Gosavi
- Subjects
Cell ,Fluorescent Antibody Technique ,Plakoglobin ,chemistry.chemical_element ,Biology ,Calcium ,Cell Line ,Cellular and Molecular Neuroscience ,Desmosome ,Cell Adhesion ,medicine ,Humans ,Molecular Biology ,Pharmacology ,Gene knockdown ,Microscopy, Confocal ,Cadherin ,Desmosomes ,Cell Biology ,Adhesion ,Cadherins ,HCT116 Cells ,Cell biology ,HEK293 Cells ,medicine.anatomical_structure ,chemistry ,Gene Knockdown Techniques ,Desmosome assembly ,Molecular Medicine ,gamma Catenin ,Plakophilins - Abstract
A decrease in the levels of the desmosomal plaque protein, plakophilin3 (PKP3), leads to a decrease in desmosome size and cell-cell adhesion. To test the hypothesis that PKP3 is required for desmosome formation, the recruitment of desmosomal components to the cell surface was studied in the PKP3 knockdown clones. The PKP3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared to vector controls, and did not form desmosomes in a calcium switch assay. Further analysis demonstrated that PKP3, plakoglobin (PG) and E-cadherin are present at the cell border at low concentrations of calcium. Loss of either PG or E-cadherin led to a decrease in the levels of PKP3 and other desmosomal proteins at the cell border. The results reported here are consistent with the model that PG and E-cadherin recruit PKP3 to the cell border to initiate desmosome formation.
- Published
- 2010
28. A novel pocket in 14-3-3ɛ is required to mediate specific complex formation with cdc25C and to inhibit cell cycle progression upon activation of checkpoint pathways
- Author
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Sorab N. Dalal, Prasanna Venkatraman, Amol S. Hosing, Samrat T. Kundu, and Elphine Telles
- Subjects
Models, Molecular ,Gene isoform ,Molecular Sequence Data ,Cell Cycle Proteins ,medicine.disease_cause ,Mice ,In vivo ,Dual-specificity phosphatase ,medicine ,Animals ,Humans ,cdc25 Phosphatases ,Amino Acid Sequence ,Structural motif ,Mitosis ,Phylogeny ,Mutation ,Base Sequence ,biology ,Cell Biology ,G2-M DNA damage checkpoint ,Cell cycle ,Protein Structure, Tertiary ,Rats ,Cell biology ,14-3-3 Proteins ,biology.protein ,Sequence Alignment ,Protein Binding ,Signal Transduction - Abstract
Mitotic progression requires the activity of the dual specificity phosphatase, cdc25C. Cdc25C function is inhibited by complex formation with two 14-3-3 isoforms, 14-3-3epsilon and 14-3-3gamma. To understand the molecular basis of specific complex formation between 14-3-3 proteins and their ligands, chimeric 14-3-3 proteins were tested for their ability to form a complex with cdc25C in vivo. Specific complex formation between cdc25C and 14-3-3epsilon in vivo requires a phenylalanine residue at position 135 (F135) in 14-3-3epsilon. Mutation of this residue to the corresponding residue present in other 14-3-3 isoforms (F135V) leads to reduced binding to cdc25C and a decrease in the ability to inhibit cdc25C function in vivo. Similarly, F135V failed to rescue the incomplete S phase and the G2 DNA damage checkpoint defects observed in cells lacking 14-3-3epsilon. A comparative analysis of the 14-3-3 structures present in the database suggested that the F135 in 14-3-3epsilon was required to maintain the integrity of a pocket that might be involved in secondary interactions with cdc25C. These results suggest that the specificity of the 14-3-3 ligand interaction may be dependent on structural motifs present in the individual 14-3-3 isoforms.
- Published
- 2009
29. Abstract 688: Identifying TMEM106B as a novel metastasis driver in non-small cell lung cancers through an in vivo gain-of-function screen
- Author
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Don L. Gibbons, Chad J. Creighton, Samrat T. Kundu, Caitlin L. Grzeskowiak, and Kenneth L. Scott
- Subjects
Cancer Research ,Cancer ,Biology ,medicine.disease ,medicine.disease_cause ,Bioinformatics ,Primary tumor ,Metastasis ,Transcriptome ,Oncology ,Gene duplication ,Cancer cell ,medicine ,Cancer research ,KRAS ,Lung cancer - Abstract
Metastatic lung cancer is the leading cause of cancer related death in the world and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Lung cancers commonly demonstrate genetic alteration of potent drivers like Kras, p53 or EGFR accompanied with hundreds of low frequency gene aberrations which are a mix of key driver events and nascent passenger mutations. We established a robust screening platform to selectively identify these functionally critical “driver” genes in lung cancers from a prioritized list of candidates selected by a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models and genomic data of human lung cancers from TCGA, focusing on elevated gene expression and/or gene amplification. We identified 225 putative candidate driver genes which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each gene. These candidate lines were used for a unique in vivo positive selection screen to identify functional metastasis drivers. We transplanted pools of 20 cDNA expressing lines into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences in the genomic DNA from metastatic lesions over primary tumors. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers. We validated our top hits for their individual in vivo metastatic potencies by gain-of-function/loss-of-function studies and identified TMEM106B as one of the primary drivers of metastasis. TMEM106B is a transmembrane protein localizing to the lysosomes and has been shown to drive expression of lysosomal genes. We hypothesize that elevated levels of TMEM106B is able to drive the expression and secretion of lysosomal enzymes thus making cancer cells hyper invasive and metastatic. We found strong correlation for expression of TMEM106B with several lysosomal genes in a panel of 1016 human lung cancers and are currently performing in depth studies to understand these mechanisms of TMEM106B driven metastasis. We are also analyzing the clinical relevance of TMEM106B based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Identification of such novel players will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for effective treatment options for lung cancer patients. Citation Format: Samrat Kundu, Caitlin Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons. Identifying TMEM106B as a novel metastasis driver in non-small cell lung cancers through an in vivo gain-of-function screen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 688.
- Published
- 2016
30. Abstract B25: A novel in vivo gain-of-function screen for metastasis drivers in non-small cell lung cancers
- Author
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Caitlin L. Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons, and Samrat T. Kundu
- Subjects
Genome instability ,Cancer Research ,Tissue microarray ,Biology ,Bioinformatics ,medicine.disease_cause ,medicine.disease ,Primary tumor ,Metastasis ,Transcriptome ,Oncology ,Gene duplication ,medicine ,Cancer research ,KRAS ,Lung cancer - Abstract
s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Metastatic lung cancer is the leading cause of cancer related mortality in the world. Insights on specific genetic causes are limited and novel approaches are necessary to elucidate specific genes that drive lung cancer progression and metastasis. Extensive genomic and transcriptomic profiling of human lung cancers by consortia like ‘The Cancer Genome Atlas’ (TCGA) and the ‘International Cancer Genome Consortium’ (ICGC) has revealed a complex genetic landscape marked by abundant aberrations. In addition to acquiring mutations or amplification of well documented drivers like Kras, p53 or EGFR, lung cancers also demonstrate an enrichment of hundreds of low frequency gene aberrations which are a mix of key driver mutations and nascent passenger alterations caused by progressive genomic instability that could have definite role in cancer progression and metastasis. Our goal was to establish a robust screening platform to selectively identify these functionally critical “driver” genes in lung cancers. To identify a prioritized list of potential candidates we performed a multi-level cross-species comparison of our published high confidence transcriptome data from genetically-engineered mouse models of lung cancers and human lung cancer genomic data from TCGA, focusing on elevated gene expression and/or gene amplification. We identified an enriched list of 225 putative driver genes in lung cancers which were used to construct a lentiviral based cDNA expression library with unique molecular barcoding of individual cDNAs. Using a non-metastatic syngeneic mouse lung cancer model we generated individually transduced stable over-expressing lines for each of the 225 genes. These candidate lines were then entered into parallel in vitro and in vivo screens. In vitro testing includes cell invasion assays in a 96-well format, with positive hits defined as those scoring above 3x standard deviations relative to the control cells expressing mCherry. Positive hits were revalidated using standard 24-well invasion assays and also studied for growth phenotypes in 3D cultures. To selectively identify genes with functional role in metastasis, we performed a unique in vivo positive selection screen to test individual and combinatorial effects of lung cancer driver aberrations. For this we used pools of 20 cDNA expressing lines transplanted into syngeneic mice and observed for primary tumor growth and occurrence of metastases in lungs and other organs. Metastatic lesions and primary tumors were collected and genomic DNA from these was used to identify the unique individual barcodes by NGS barcode enrichment analysis. Metastatic drivers were identified by the relative enrichment of the unique barcode sequences for all the respective pool cDNAs in the metastatic tissue. We have identified both known (e.g. MYC) and several novel (e.g. THRA, TMEM106B, GNAS) potential oncogenic and metastatic drivers from the pooled screen which are currently being revalidated for their individual in vivo metastatic potencies. We are performing gain-of-function/loss-of-function studies for the identified driver genes to determine the pathways which they regulate. We are also analyzing the clinical relevance of these genes based upon their expression pattern and prognostic utility across TCGA and other public datasets, along with in-house patient samples and tissue microarrays of resected and biopsy specimens. Our unique high through-put approach will significantly advance the field of cancer target discovery by identifying new drug targets and biomarkers, essential for novel effective treatment options for lung cancer patients. Citation Format: Samrat T. Kundu, Caitlin Grzeskowiak, Chad J. Creighton, Kenneth L. Scott, Don L. Gibbons. A novel in vivo gain-of-function screen for metastasis drivers in non-small cell lung cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B25.
- Published
- 2016
31. Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis
- Author
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Sorab N. Dalal, Samrat T. Kundu, Prajakta Gosavi, Mugdha Sawant, Madhura Karkhanis, Nishigandha Naik, Neha Gupta, Lalit Sehgal, Nileema Khapare, Milind M. Vaidya, Rashmi Priya, and Hunain Alam
- Subjects
Oligonucleotides ,lcsh:Medicine ,Protein tyrosine phosphatase ,Metastasis ,Mice ,0302 clinical medicine ,Neoplasms ,Keratin ,Molecular Cell Biology ,Basic Cancer Research ,Fluorescence Resonance Energy Transfer ,Electrophoresis, Gel, Two-Dimensional ,Neoplasm Metastasis ,Phosphorylation ,lcsh:Science ,Cytoskeleton ,chemistry.chemical_classification ,0303 health sciences ,Gene knockdown ,Multidisciplinary ,Microscopy, Confocal ,Reverse Transcriptase Polymerase Chain Reaction ,Desmosomes ,Immunohistochemistry ,Cellular Structures ,Cell biology ,Gene Expression Regulation, Neoplastic ,Cell Transformation, Neoplastic ,Oncology ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques ,Medicine ,Cellular Types ,Cell Division ,Research Article ,Phosphatase ,Blotting, Western ,Mice, Nude ,Biology ,Real-Time Polymerase Chain Reaction ,Immediate early protein ,Cell Growth ,Immediate-Early Proteins ,03 medical and health sciences ,Genetics ,Cancer Genetics ,Cell Adhesion ,Animals ,Humans ,Immunoprecipitation ,030304 developmental biology ,Keratin-8 ,lcsh:R ,Epithelial Cells ,HCT116 Cells ,Molecular biology ,chemistry ,Microscopy, Fluorescence ,Tumor progression ,Keratin 8 ,lcsh:Q ,Protein Tyrosine Phosphatases ,Plakophilins - Abstract
The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.
- Published
- 2012
32. KRAS alleles: the LCS6 3'UTR variant and KRAS coding sequence mutations in the NCI-60 panel
- Author
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Samrat T. Kundu, Marta Boeke, Sunitha Nallur, Trupti Paranjape, Frank J. Slack, and Joanne B. Weidhaas
- Subjects
Colorectal cancer ,Gene Expression ,Single-nucleotide polymorphism ,Biology ,medicine.disease_cause ,Polymorphism, Single Nucleotide ,Epigenesis, Genetic ,Proto-Oncogene Proteins p21(ras) ,Open Reading Frames ,Breast cancer ,Cell Line, Tumor ,Proto-Oncogene Proteins ,microRNA ,medicine ,Humans ,Allele ,neoplasms ,Molecular Biology ,3' Untranslated Regions ,Alleles ,Genetics ,Mutation ,Binding Sites ,Cell Biology ,medicine.disease ,digestive system diseases ,Gene Expression Regulation, Neoplastic ,MicroRNAs ,Cancer research ,ras Proteins ,KRAS ,Ovarian cancer ,Developmental Biology - Abstract
The KRAS-variant is a germline single nucleotide polymorphism (SNP) within the 3'UTR of the KRAS gene predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA (miRNA). The KRAS-variant is associated with increased risk of various cancers, including lung cancer, ovarian cancer and triple-negative breast cancer, and is associated with altered tumor biology in head and neck cancer, colon cancer and melanoma. To better understand the molecular pathways that may be regulated or affected by the presence of the KRAS-variant allele in cancer cells, we examined its prevalence in the NCI-60 panel of cell lines and sought to identify common features of the cell lines that carry the variant allele. This study provides a step forward towards understanding the molecular and pathological significance of the KRAS-variant.
- Published
- 2011
33. Loss of keratins 8 and 18 leads to alterations in α6β4-integrin-mediated signalling and decreased neoplastic progression in an oral-tumour-derived cell line
- Author
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Sorab N. Dalal, Hunain Alam, Milind M. Vaidya, and Samrat T. Kundu
- Subjects
Cell ,Motility ,Mice, Nude ,macromolecular substances ,Mice, SCID ,Biology ,Malignant transformation ,Mice ,Cell Movement ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Neoplasm Invasiveness ,Fascin ,Integrin alpha6beta4 ,Gene knockdown ,Keratin-18 ,Keratin-8 ,Cell Biology ,Actin cytoskeleton ,Cell biology ,stomatognathic diseases ,medicine.anatomical_structure ,Cell culture ,biology.protein ,Carcinoma, Squamous Cell ,Disease Progression ,Signal transduction ,Cell Division ,Signal Transduction - Abstract
Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression and invasion in squamous cell carcinomas (SCCs). To understand the molecular basis by which K8 promotes neoplastic progression in oral SCC (OSCC), K8 expression was inhibited in AW13516 cells. The K8-knockdown clones showed a significant reduction in tumorigenic potential, which was accompanied by a reduction in cell motility, cell invasion, decreased fascin levels, alterations in the organization of the actin cytoskeleton and changes in cell shape. Furthermore, K8 knockdown led to a decrease in α6β4 integrin levels and α6β4-integrin-dependent signalling events, which have been reported to play an important role in neoplastic progression in epithelial tissues. Therefore, modulation of α6β4 integrin signalling might be one of the mechanisms by which K8 and K18 promote malignant transformation and/or progression in OSCCs.
- Published
- 2011
34. Plakophilin3 downregulation leads to a decrease in cell adhesion and promotes metastasis
- Author
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James A. DeCaprio, Arvind Ingle, Sorab N. Dalal, Samrat T. Kundu, Prajakta Gosavi, Nileema Khapare, Girish B. Maru, Rachana Patel, and Amol S. Hosing
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,Blotting, Western ,Down-Regulation ,Mice, Nude ,Biology ,medicine.disease_cause ,Malignant transformation ,Metastasis ,Mice ,Downregulation and upregulation ,Desmosome ,medicine ,Cell Adhesion ,Animals ,Humans ,Neoplasm Metastasis ,Cell adhesion ,Wound Healing ,Reverse Transcriptase Polymerase Chain Reaction ,medicine.disease ,Blot ,HaCaT ,medicine.anatomical_structure ,Oncology ,Cancer research ,Carcinogenesis ,Plakophilins - Abstract
Plakophilin3 is a desmosomal plaque protein whose levels are reduced in poorly differentiated tumors of the oropharyngeal cavity and in invasive colon carcinomas. To test the hypothesis that plakophilin3 loss stimulates neoplastic progression, plakophilin3 expression was inhibited by DNA vector driven RNA interference in 3 epithelial cell lines, HCT116, HaCaT and fetal buccal mucosa. The plakophilin3-knockdown clones showed a decrease in cell-cell adhesion as assessed in a hanging drop assay, which was accompanied by an increase in cell migration. The HCT116 plakophilin3-knockdown clones showed a decrease in desmosome size as revealed by electron microscopy. These altered desmosomal properties were accompanied by colony formation in soft agar and growth to high density in culture. The HCT116-derived clones showed accelerated tumor formation in nude mice and increased metastasis to the lung, a phenotype consistent with the increased migration observed in vitro and is consistent with data from human tumors that suggests that plakophililn3 is lost in invasive and metastatic tumors. These data indicate that plakophilin3 loss leads to a decrease in cell-cell adhesion leading to the stimulation of neoplastic progression and metastasis.
- Published
- 2008
35. Abstract 955: High-throughput functional screening for metastasis drivers of lung cancer
- Author
-
Ping Wu, Kenneth L. Scott, Caitlin L. Grzeskowiak, Rosalba Minelli, Don L. Gibbons, and Samrat T. Kundu
- Subjects
Cancer Research ,Drug discovery ,Disease ,Biology ,medicine.disease_cause ,Bioinformatics ,medicine.disease ,Metastasis ,SNAI2 ,Oncology ,medicine ,Cancer research ,KRAS ,Lung cancer ,Survival rate ,Gene - Abstract
Large-scale profiling efforts by consortia such as The Cancer Genome Atlas (TCGA) are revealing the complexity of cancer genomes, which are comprised of causal “driver” aberrations and many biologically neutral “passengers”. Most cancers acquire one or more well-studied high frequency driver events that promote tumor growth (e.g., mutations/copy number changes in KRAS, TP53, EGFR, MYC). Much less is known about the thousands of low frequency gene aberrations and their contribution to cancer progression, particularly metastasis, which is the primary cause of cancer-related mortality. Comprehensive biological assessment of low frequency metastasis drivers is difficult given their large number and the fact that their activity may be influenced by the specific biological context of a given cancer such as tissue type, microenvironment, and the host immune system. We sought to address these challenges in the context of lung cancer, which presents as metastatic disease in approximately 65% of patients and carries a 5-year survival rate of 35,000 sequence verified human gene clones (2) a molecular barcoding strategy that permits simultaneous DNA tagging of gene clones through multi-fragment DNA recombineering for (3) pooled functional screening in vivo to identify metastasis drivers that work alone or in combination. We used these technologies to build gene libraries based on oncogenomics-guided integrations of mutant KRAS-specific gene signatures derived from mouse and human TCGA lung cancer datasets. The resulting barcoded libraries were delivered to non-metastatic lung cancer cells expressing oncogenic KRAS and then implanted into immune competent mice. Animals were sacrificed at maximal tumor burden and resulting primary tumors and metastases subjected to barcode enrichment analysis by next generation sequencing to identify gene aberrations enriched within metastatic lesions. Our screening approach has identified known (MYC and SNAI2) and many novel (e.g., MBIP and CCNE1) potent drivers of lung cancer growth and metastasis currently under mechanistic and pre-clinical evaluation. These efforts are revealing new pathways contributing to lung cancer aggression and our ultimate goal is to translate these findings into the care of metastatic lung cancer patients who have few treatment options. We have also scaled these efforts across other screening platforms, functionalizing thousands of aberrations across diverse cancer types. Together these systems reveal the highest priority targets to enroll in deep mechanistic biology studies and drug discovery and development programs. Citation Format: Caitlin L. Grzeskowiak, Rosalba Minelli, Ping Wu, Samrat Kundu, Don L. Gibbons, Kenneth L. Scott. High-throughput functional screening for metastasis drivers of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 955. doi:10.1158/1538-7445.AM2015-955
- Published
- 2015
36. Robust and specific inhibition of microRNAs in Caenorhabditis elegans
- Author
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Frank J. Slack and Samrat T. Kundu
- Subjects
Genetics ,Regulation of gene expression ,Genome, Helminth ,Gene knockdown ,biology ,Oligonucleotide ,Longevity ,RNA ,Cell Differentiation ,Computational biology ,Oligonucleotides, Antisense ,biology.organism_classification ,General Biochemistry, Genetics and Molecular Biology ,MicroRNAs ,Gene Expression Regulation ,microRNA ,Animals ,Minireview ,RNA, Small Interfering ,Caenorhabditis elegans ,General Agricultural and Biological Sciences ,Gene ,Conserved Sequence ,Function (biology) - Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of numerous target genes. Yet, while hundreds of miRNAs have been identified, little is known about their functions. In a recent report published in Silence, Zheng and colleagues demonstrate a technique for robust and specific knockdown of miRNA expression in Caenorhabditis elegans using modified antisense oligonucleotides, which could be utilized as a powerful tool for the study of regulation and function of miRNAs in vivo. See research article http://www.silencejournal.com/content/1/1/9
- Published
- 2010
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