65 results on '"Dennis P.M. Hughes"'
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2. Data from Critical Role of Notch Signaling in Osteosarcoma Invasion and Metastasis
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Dennis P.M. Hughes, Patrick A. Zweidler-McKay, Yanwen Yang, and Pingyu Zhang
- Abstract
Purpose: Notch signaling is an important mediator of growth and survival in several cancer types, with Notch pathway genes functioning as oncogenes or tumor suppressors in different cancers. However, the role of Notch in osteosarcoma is unknown.Experimental Design: We assessed the expression of Notch pathway genes in human osteosarcoma cell lines and patient samples. We then used pharmacologic and retroviral manipulation of the Notch pathway and studied the effect on osteosarcoma cell proliferation, survival, anchorage-independent growth, invasion, and metastasis in vitro and in vivo.Results: Notch pathway genes, including Notch ligand DLL1, Notch1 and Notch2, and the Notch target gene HES1, were expressed in osteosarcoma cells, and expression of HES1 was associated with invasive and metastatic potential. Blockade of Notch pathway signaling with a small molecule inhibitor of γ secretase eliminated invasion in Matrigel without affecting cell proliferation, survival, or anchorage-independent growth. Manipulation of Notch and HES1 signaling showed a crucial role for HES1 in osteosarcoma invasiveness and metastasis in vivo.Conclusion: These studies identify a new invasion and metastasis-regulating pathway in osteosarcoma and define a novel function for the Notch pathway: regulation of metastasis. Because the Notch pathway can be inhibited pharmacologically, these findings point toward possible new treatments to reduce invasion and metastasis in osteosarcoma.
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- 2023
3. Supplementary Figure Legends 1-5, Table Legend from Critical Role of Notch Signaling in Osteosarcoma Invasion and Metastasis
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Dennis P.M. Hughes, Patrick A. Zweidler-McKay, Yanwen Yang, and Pingyu Zhang
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PDF file - 27K
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- 2023
4. Supplementary Figure 2 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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Heike Allgayer, Christian Manegold, Lothar R. Pilz, Erika Buchholz, Andrea Harms, Gordon B. Mills, Doris R. Siwak, Dennis P.M. Hughes, Laura D. Nelson, Irfan A. Asangani, and Dessislava A. Nikolova
- Abstract
Supplementary Figure 2 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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- 2023
5. Supplementary Figure 3 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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Heike Allgayer, Christian Manegold, Lothar R. Pilz, Erika Buchholz, Andrea Harms, Gordon B. Mills, Doris R. Siwak, Dennis P.M. Hughes, Laura D. Nelson, Irfan A. Asangani, and Dessislava A. Nikolova
- Abstract
Supplementary Figure 3 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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- 2023
6. Supplementary Figure 1 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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Heike Allgayer, Christian Manegold, Lothar R. Pilz, Erika Buchholz, Andrea Harms, Gordon B. Mills, Doris R. Siwak, Dennis P.M. Hughes, Laura D. Nelson, Irfan A. Asangani, and Dessislava A. Nikolova
- Abstract
Supplementary Figure 1 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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- 2023
7. Data from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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Heike Allgayer, Christian Manegold, Lothar R. Pilz, Erika Buchholz, Andrea Harms, Gordon B. Mills, Doris R. Siwak, Dennis P.M. Hughes, Laura D. Nelson, Irfan A. Asangani, and Dessislava A. Nikolova
- Abstract
Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non–small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif −190/−171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab. [Cancer Res 2009;69(6):2461–70]
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- 2023
8. Supplementary Figure 4 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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Heike Allgayer, Christian Manegold, Lothar R. Pilz, Erika Buchholz, Andrea Harms, Gordon B. Mills, Doris R. Siwak, Dennis P.M. Hughes, Laura D. Nelson, Irfan A. Asangani, and Dessislava A. Nikolova
- Abstract
Supplementary Figure 4 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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- 2023
9. Supplementary Figure 5 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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Heike Allgayer, Christian Manegold, Lothar R. Pilz, Erika Buchholz, Andrea Harms, Gordon B. Mills, Doris R. Siwak, Dennis P.M. Hughes, Laura D. Nelson, Irfan A. Asangani, and Dessislava A. Nikolova
- Abstract
Supplementary Figure 5 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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- 2023
10. Cardiovascular involvement by osteosarcoma: an analysis of 20 patients
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Wei Lien Wang, Sireesha Yedururi, Ajaykumar C. Morani, Najat C. Daw, Dennis P.M. Hughes, Peter M. Anderson, Gregory W. Gladish, and Srilakshmi Vallabhaneni
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Diagnostic Imaging ,Male ,medicine.medical_specialty ,Pathology ,Adolescent ,Bone Neoplasms ,Disease ,030204 cardiovascular system & hematology ,Sensitivity and Specificity ,Inferior vena cava ,Article ,Heart Neoplasms ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Radiology, Nuclear Medicine and imaging ,Thrombus ,Child ,Retrospective Studies ,Neuroradiology ,Observer Variation ,Osteosarcoma ,Lung ,medicine.diagnostic_test ,business.industry ,Ultrasound ,Reproducibility of Results ,Magnetic resonance imaging ,medicine.disease ,Vascular Neoplasms ,medicine.anatomical_structure ,medicine.vein ,Child, Preschool ,030220 oncology & carcinogenesis ,Pediatrics, Perinatology and Child Health ,Female ,Radiology ,business - Abstract
Although hematogenous spread of osteosarcoma is well known, the imaging findings of cardiovascular involvement by osteosarcoma are seldom reported and can be difficult to recognize. The enhanced resolution of modern CT and MRI scanners may lead to better detection of cardiovascular involvement. To describe the key imaging findings and clinical behavior of cardiovascular involvement by osteosarcoma. We retrospectively reviewed the imaging findings and clinical characteristics of 20 patients with cardiovascular involvement by osteosarcoma identified by two pediatric radiologists from a review of imaging studies at our institution from 2007 to 2013. At initial diagnosis, the median age of the patients was 15.1 years (range 4.8–24.6 years), and 7 (35%) patients had detectable metastases. Median time to detection of cardiovascular metastases was 1.8 years (range 0–7.3 years). Sixteen patients died of disease; 4 have survived a median of 7.4 years since initial diagnosis. The sites of cardiovascular involvement were the systemic veins draining the primary and metastatic osteosarcoma, pulmonary arteries, pulmonary veins draining the pulmonary metastases, and heart. A dilated and mineralized terminal pulmonary arteriole is an early sign of metastatic osteosarcoma in the lung. Unfamiliarity with the imaging features resulted in under-recognition and misinterpretation of intravascular tumor thrombus as bland thrombus. Knowledge of imaging findings in the era of modern imaging modalities has enhanced our ability to detect cardiovascular involvement and lung metastases early and avoid misinterpreting tumor thrombus in draining systemic veins or pulmonary arteries as bland thrombus.
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- 2015
11. Bone Sarcomas in Pediatrics: Progress in Our Understanding of Tumor Biology and Implications for Therapy
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Dennis P.M. Hughes, Rocio K. Rivera-Valentin, and Limin Zhu
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Bone Neoplasms ,Sarcoma, Ewing ,Leading Article ,Disease ,Bone Sarcoma ,Bioinformatics ,Pediatrics ,Pharmacotherapy ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Humans ,Pharmacology (medical) ,Pediatrics, Perinatology, and Child Health ,Child ,Osteosarcoma ,Tumor microenvironment ,business.industry ,Cancer ,medicine.disease ,3. Good health ,Clinical trial ,Pediatrics, Perinatology and Child Health ,Immunology ,Sarcoma ,business ,Signal Transduction - Abstract
The pediatric bone sarcomas osteosarcoma and Ewing sarcoma represent a tremendous challenge for the clinician. Though less common than acute lymphoblastic leukemia or brain tumors, these aggressive cancers account for a disproportionate amount of the cancer morbidity and mortality in children, and have seen few advances in survival in the past decade, despite many large, complicated, and expensive trials of various chemotherapy combinations. To improve the outcomes of children with bone sarcomas, a better understanding of the biology of these cancers is needed, together with informed use of targeted therapies that exploit the unique biology of each disease. Here we summarize the current state of knowledge regarding the contribution of receptor tyrosine kinases, intracellular signaling pathways, bone biology and physiology, the immune system, and the tumor microenvironment in promoting and maintaining the malignant phenotype. These observations are coupled with a review of the therapies that target each of these mechanisms, focusing on recent or ongoing clinical trials if such information is available. It is our hope that, by better understanding the biology of osteosarcoma and Ewing sarcoma, rational combination therapies can be designed and systematically tested, leading to improved outcomes for a group of children who desperately need them.
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- 2015
12. The Narrow-Spectrum HDAC Inhibitor Entinostat Enhances NKG2D Expression Without NK Cell Toxicity, Leading to Enhanced Recognition of Cancer Cells
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Cecele J. Denman, Shiguo Zhu, Dean A. Lee, Dennis P.M. Hughes, Eugenie S. Kleinerman, Ching C. Lau, Stephen Gottschalk, Zehra S. Cobanoglu, and Simin Kiany
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Time Factors ,Transcription, Genetic ,Pyridines ,Pharmaceutical Science ,Ligands ,Histones ,chemistry.chemical_compound ,Mice, Inbred NOD ,Pharmacology (medical) ,Promoter Regions, Genetic ,Entinostat ,Acetylation ,Sarcoma ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,Killer Cells, Natural ,NK Cell Lectin-Like Receptor Subfamily K ,Benzamides ,Colonic Neoplasms ,Intercellular Signaling Peptides and Proteins ,Molecular Medicine ,Biotechnology ,Antineoplastic Agents ,Mice, Transgenic ,chemical and pharmacologic phenomena ,Biology ,GPI-Linked Proteins ,Article ,Cell Line, Tumor ,MHC class I ,Animals ,Humans ,Pharmacology ,Binding Sites ,Lymphokine-activated killer cell ,Dose-Response Relationship, Drug ,Histocompatibility Antigens Class I ,Organic Chemistry ,NKG2D ,Xenograft Model Antitumor Assays ,Molecular biology ,Coculture Techniques ,Histone Deacetylase Inhibitors ,chemistry ,Cell culture ,Cancer cell ,Cancer research ,biology.protein ,Histone deacetylase - Abstract
Natural killer (NK) cell cytotoxicity correlates with the ligation of activating receptors (e.g., NKG2D) by their ligands (e.g., MHC class I-related chains [MIC] A and B) on target cells. Histone deacetylase inhibitors (HDACi) at high concentrations inhibit tumor growth and can increase NKG2D ligand expression on tumor targets, but are widely regarded as toxic to NK cells.We investigated the mechanism of entinostat, a benzamide-derivative narrow-spectrum HDACi, in augmenting the cytotoxicity of NK cells against human colon carcinoma and sarcoma by assessing gene and protein expression, histone acetylation, and cytotoxicity in in vitro and murine models.We observed that entinostat dose- and time-dependent increase in MIC expression in tumor targets and NKG2D in primary human NK cells, both correlating with increased acetylated histone 3 (AcH3) binding to associated promoters. Entinostat pretreatment of colon carcinoma and sarcoma cells, NK cells, or both led to enhanced overall cytotoxicity in vitro, which was reversed by NKG2D blockade, and inhibited growth of tumor xenografts. Lastly, we showed decreased expression of MICA and ULBP2 transcription in primary human osteosarcoma.Entinostat enhances NK cell killing of cancer cells through upregulation of both NKG2D and its ligands, suggesting an attractive approach for augmenting NK cell immunotherapy of solid tumors such as colon carcinoma and sarcomas.
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- 2013
13. Natural killer cell therapy and aerosol interleukin-2 for the treatment of osteosarcoma lung metastasis
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Dean A. Lee, Dennis P.M. Hughes, Nancy Gordon, Ling Yu, Eugenie S. Kleinerman, John Stewart, Sergei Guma, and Wei Lien Wang
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Interleukin 2 ,Lung ,business.industry ,NK Cell Lectin-Like Receptor Subfamily K ,medicine.medical_treatment ,Cell migration ,Hematology ,Immunotherapy ,medicine.disease ,medicine.anatomical_structure ,Oncology ,Apoptosis ,Pediatrics, Perinatology and Child Health ,Immunology ,Toxicity ,medicine ,Cancer research ,Osteosarcoma ,business ,medicine.drug - Abstract
PURPOSE Survival of patients with osteosarcoma lung metastases has not improved in 20 years. We evaluated the efficacy of combining natural killer [NK] cells with aerosol interleukin-2 [IL-2] to achieve organ-specific NK cell migration and expansion in the metastatic organ, and to decrease toxicity associated with systemic IL-2.
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- 2013
14. Complications in the surgical treatment of pediatric melanoma
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Paul E. Palmer, Cynthia E. Herzog, Andrea Hayes-Jordan, Dennis P.M. Hughes, Mary T. Austin, Kevin P. Lally, and Carla L. Warneke
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Male ,Risk ,medicine.medical_specialty ,Skin Neoplasms ,Adolescent ,medicine.medical_treatment ,Dermatologic Surgical Procedures ,Sentinel lymph node ,Inguinal Canal ,Postoperative Complications ,Biopsy ,medicine ,Humans ,Child ,Melanoma ,Lymph node ,Retrospective Studies ,medicine.diagnostic_test ,Sentinel Lymph Node Biopsy ,business.industry ,Wide local excision ,Infant ,General Medicine ,medicine.disease ,Surgery ,Dissection ,Logistic Models ,Treatment Outcome ,medicine.anatomical_structure ,Lymphedema ,Child, Preschool ,Axilla ,Pediatrics, Perinatology and Child Health ,Lymph Node Excision ,Female ,Complication ,business ,Follow-Up Studies - Abstract
Purpose The purpose of this study was to characterize the complications associated with surgical treatment of pediatric melanoma. Methods We retrospectively reviewed all pediatric patients who received surgical treatment for melanoma at our institution between 1992 and 2010. We compared complications between three groups: wide local excision only (WLE), WLE and sentinel lymph node biopsy (SLNB), and WLE and completion lymph node dissection (CLND). Results One hundred twenty-five patients were identified: 37 patients received WLE only, 47 received WLE and SLNB, and 41 patients had WLE and CLND. Complication rates differed between the three groups: 19% in WLE, 11% in WLE+SLNB, and 39% in WLE+CLND ( P =.006). The risk of complications was significantly lower among patients having WLE+SLNB versus WLE+CLND (OR 0.19, 95% CI 0.06–0.57, P =.0032). Lymphedema was a common complication with a higher incidence in the CLND group compared to the SLNB group (19.5% vs. 2.1%, P =.01). Complications were more frequent in inguinal compared to axillary dissections (52.0% vs. 17.1%, P =.006). Conclusions In the surgical treatment of pediatric melanoma, the addition of a completion lymph node dissection significantly increases complication risk. Thus, it is critical to determine which patients truly benefit from this procedure.
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- 2013
15. UBE4B levels are correlated with clinical outcomes in neuroblastoma patients and with altered neuroblastoma cell proliferation and sensitivity to epidermal growth factor receptor inhibitors
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Brandon S. Brown, Shana L. Palla, Andrew J. Bean, Dennis P.M. Hughes, Yin Liu, Monica Gireud, Kristen Richards, Natalie Sirisaengtaksin, and Peter E. Zage
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Cancer Research ,Cetuximab ,biology ,Cell growth ,business.industry ,Cancer ,medicine.disease ,Oncology ,Growth factor receptor ,Neuroblastoma ,medicine ,Cancer research ,biology.protein ,Epidermal growth factor receptor ,business ,neoplasms ,Tyrosine kinase ,medicine.drug ,EGFR inhibitors - Abstract
BACKGROUND: The UBE4B gene, which is located on chromosome 1p36, encodes a ubiquitin ligase that interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a protein involved in epidermal growth factor receptor (EGFR) trafficking, suggesting a link between EGFR trafficking and neuroblastoma pathogenesis. The authors analyzed the roles of UBE4B in the outcomes of patients with neuroblastoma and in neuroblastoma tumor cell proliferation, EGFR trafficking, and response to EGFR inhibition. METHODS: The association between UBE4B expression and the survival of patients with neuroblastoma was examined using available microarray data sets. UBE4B and EGFR protein levels were measured in patient tumor samples, EGFR degradation rates were measured in neuroblastoma cell lines, and the effects of UBE4B on neuroblastoma tumor cell growth were analyzed. The effects of the EGFR inhibitor cetuximab were examined in neuroblastoma cells that expressed wild-type and mutant UBE4B. RESULTS: Low UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma. UBE4B overexpression reduced neuroblastoma tumor cell proliferation, and UBE4B expression was inversely related to EGFR expression in tumor samples. EGFR degradation rates correlated with cellular UBE4B levels. Enhanced expression of catalytically active UBE4B resulted in reduced sensitivity to EGFR inhibition. CONCLUSIONS: The current study demonstrates associations between UBE4B expression and the outcomes of patients with neuroblastoma and between UBE4B and EGFR expression in neuroblastoma tumor samples. Moreover, levels of UBE4B influence neuroblastoma tumor cell proliferation, EGFR degradation, and response to EGFR inhibition. These results suggest UBE4B-mediated growth factor receptor trafficking may contribute to the poor prognosis of patients who have neuroblastoma tumors with 1p36 deletions and that UBE4B expression may be a marker that can predict responses of neuroblastoma tumors to treatment. Cancer 2013. © 2012 American Cancer Society.
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- 2012
16. Driven to Death: Inhibition of Farnesylation Increases Ras Activity and Promotes Growth Arrest and Cell Death
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Dennis P.M. Hughes, Yanwen Yang, and Mandy Geryk-Hall
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Male ,MAPK/ERK pathway ,Cancer Research ,Adolescent ,Farnesyltransferase ,Drug Evaluation, Preclinical ,Antineoplastic Agents ,Bone Neoplasms ,Quinolones ,Article ,Geranylgeranylation ,Prenylation ,Cell Line, Tumor ,Anti-apoptotic Ras signalling cascade ,medicine ,Farnesyltranstransferase ,Humans ,Enzyme Inhibitors ,Cell Proliferation ,Osteosarcoma ,Cell Death ,biology ,Cell growth ,Kinase ,Cell Cycle ,U937 Cells ,Up-Regulation ,Cell biology ,Oncology ,ras Proteins ,biology.protein ,Tipifarnib ,medicine.drug - Abstract
To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition. Mol Cancer Ther; 9(5); 1111–9. ©2010 AACR.
- Published
- 2010
17. Signaling of ERBB receptor tyrosine kinases promotes neuroblastoma growth in vitro and in vivo
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Dennis P.M. Hughes, Nadine Van Roy, Patrick A. Zweidler-McKay, Jesus Trevino, Frank Speleman, Peter E. Zage, and Kristen Richards
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Cancer Research ,Morpholines ,Apoptosis ,Biology ,Article ,Receptor tyrosine kinase ,Erlotinib Hydrochloride ,Mice ,Neuroblastoma ,chemistry.chemical_compound ,ErbB ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Epidermal growth factor receptor ,Protein Kinase Inhibitors ,neoplasms ,Cell Proliferation ,Mice, Knockout ,medicine.disease ,Pediatric cancer ,ErbB Receptors ,Oncology ,chemistry ,Quinazolines ,Cancer research ,biology.protein ,Erlotinib ,Growth inhibition ,Neoplasm Transplantation ,Signal Transduction ,medicine.drug - Abstract
BACKGROUND: ERBB receptor tyrosine kinases can mediate proliferation, migration, adhesion, differentiation, and survival in many types of cells and play critical roles in many malignancies. Recent reports suggest a role for EGFR signaling in proliferation and survival of neuroblastoma, a common form of pediatric cancer that often has an extremely poor outcome. METHODS: The authors examined ERBB family expression in neuroblastoma cell lines and patient samples by flow cytometry, western blot, and quantitative real time polymerase chain reaction (Q-PCR). Response to ERBB inhibition was assessed in vitro by cell-cycle analysis and western blot and in vivo by serial tumor-size measurements. RESULTS: A panel of neuroblastoma cell lines and primary patient tumors expressed EGFR, HER-3, and HER-4, with HER-2 in some tumors. HER-4 mRNA was expressed predominantly in cleavable isoforms. Whereas EGFR inhibition with erlotinib and pan-ERBB inhibition with CI-1033 inhibited EGF-induced phosphorylation of EGFR, AKT, and ERK1/2, only CI-1033 induced growth inhibition and dose-dependent apoptosis in vitro. Both CI-1033 and erlotinib treatment of neuroblastoma xenograft tumors resulted in decreased tumor growth in vivo, although CI-1033 was more effective. In vivo expression of EGFR was observed predominantly in vascular endothelial cells. CONCLUSIONS: Pan-ERBB inhibition is required for ERBB-related neuroblastoma apoptosis in vitro, although EGFR contributes indirectly to tumor growth in vivo. Inhibition of EGFR in endothelial cells may be an important aspect of erlotinib's impact on neuroblastoma growth in vivo. Our results suggest that non-EGFR ERBB family members contribute directly to neuroblastoma growth and survival, and pan-ERBB inhibition represents a potential therapeutic target for treating neuroblastoma. Cancer 2010. © 2010 American Cancer Society.
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- 2010
18. Novel agents in development for pediatric sarcomas
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Dennis P.M. Hughes
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Cancer Research ,Pediatric practice ,business.industry ,Antineoplastic Agents ,Sarcoma ,medicine.disease ,Receptor, IGF Type 1 ,Metastasis ,ErbB Receptors ,Clinical trial ,Drug Delivery Systems ,src-Family Kinases ,Oncology ,Growth factor receptor ,Novel agents ,ErbB ,medicine ,Cancer research ,Humans ,Osteosarcoma ,Child ,business - Abstract
PURPOSE OF REVIEW The survival curves for many pediatric sarcomas have remained flat for the past 2 decades or more and novel therapeutics - those small molecule medicines that selectively inhibit specific signaling molecules - have been slow to enter into pediatric practice. The preclinical basis for their use is reviewed here. RECENT FINDINGS Preclinical and phase I studies showing efficacy of antiinsulin-like growth factor receptor 1 therapies for Ewing sarcoma have led to numerous ongoing clinical trials using these agents for Ewing and other sarcomas. Early studies of ERBB signaling as a target in sarcoma therapy have been tantalizing, but progress in this area has been controversial. In-vitro analysis of Src inhibitors suggested that these agents would prevent metastasis in osteosarcoma, whereas in-vivo analysis showed no effect on metastasis, underscoring the need for thorough preclinical investigations of promising new therapies to guide future clinical trials. Antiangiogenic and immunomodulatory therapies are gaining momentum in the pediatric arena and should be tested in combination with traditional cytotoxic agents for recurrent and high-risk primary pediatric sarcomas. SUMMARY Pediatric sarcomas have diverse biology and distinct signaling pathways, making detailed preclinical evaluation of small molecule inhibitors essential to guiding the design of further clinical investigations.
- Published
- 2009
19. Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity
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Irfan A. Asangani, Lothar R. Pilz, Erika Buchholz, Gordon B. Mills, Andrea Harms, Laura D. Nelson, Dessislava A. Nikolova, Christian Manegold, Heike Allgayer, Doris R. Siwak, and Dennis P.M. Hughes
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,Lung Neoplasms ,MAP Kinase Signaling System ,Proto-Oncogene Proteins c-jun ,Cetuximab ,Gene Expression ,Antineoplastic Agents ,Chick Embryo ,Antibodies, Monoclonal, Humanized ,Receptors, Urokinase Plasminogen Activator ,Metastasis ,Metastasis Suppression ,Carcinoma, Non-Small-Cell Lung ,Biomarkers, Tumor ,medicine ,Animals ,Humans ,Epidermal growth factor receptor ,Neoplasm Metastasis ,Promoter Regions, Genetic ,Lung cancer ,neoplasms ,Epidermal Growth Factor ,biology ,Cadherin ,business.industry ,Cell adhesion molecule ,JNK Mitogen-Activated Protein Kinases ,Antibodies, Monoclonal ,Cancer ,Cadherins ,medicine.disease ,digestive system diseases ,respiratory tract diseases ,ErbB Receptors ,Oncology ,Cancer research ,biology.protein ,business ,medicine.drug - Abstract
Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non–small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif −190/−171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab. [Cancer Res 2009;69(6):2461–70]
- Published
- 2009
20. Novel bone cancer drugs: investigational agents and control paradigms for primary bone sarcomas (Ewing's sarcoma and osteosarcoma)
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Pete Anderson, Kathleen Cornelius, Nicholas Anderson, Winston W. Huh, Lisa M. Kopp, Dennis P.M. Hughes, and Cynthia E. Herzog
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Oncology ,medicine.medical_specialty ,Drug Evaluation, Preclinical ,Antineoplastic Agents ,Sarcoma, Ewing ,Pharmacology ,Internal medicine ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Neoplasm Metastasis ,Osteosarcoma ,Temozolomide ,business.industry ,Bone cancer ,Ewing's sarcoma ,General Medicine ,medicine.disease ,Radiography ,Irinotecan ,Denosumab ,Topotecan ,Immunotherapy ,Sarcoma ,business ,medicine.drug - Abstract
New investigational agents and chemotherapy regimens including cyclophosphamide + topotecan, temozolomide + irinotecan, and anti-IGF-1R antibodies in Ewing's sarcoma (ES) and liposomal muramyltripeptide phosphatidylethanolamine (L-MTP-PE), aerosol therapy, and bone-specific agents in osteosarcoma (OS) may improve survival and/or quality of life on 'continuation' therapy.Review of investigational approaches and control paradigms for recurrent or metastatic primary bone tumors.Analyze temozolomide + irinotecan data and review in the context of other newer approaches including antiangiogenesis, anti-IGF-1R antibodies and bisphosphonates for ES. Review some current state-of-the-art approaches for OS including L-MTP-PE, anti-IGF-1R inhibition, aerosol therapies and bone specific agents.L-MTP-PE with chemotherapy in OS has been shown to improve survival; compassionate access is available for recurrence and/or metastases. Aerosol therapy (granulocyte-macrophage colony stimulating factor, cisplatin, gemcitabine) for lung metastases is a promising approach to reduce systemic toxicity. The bone-specific agents including denosumab (anti-receptor activator of NF-kappaB ligand antibody) and bisphosphonates may have benefit against giant cell tumor, ES and OS. Anti-IGF-1R antibody SCH717454 has preclinical activity in OS but best effectiveness will most likely be in combination with chemotherapy earlier in therapy. Both temozolomide + irinotecan and cyclophosphamide + topotecan combinations are very active in ES and are likely to be tested with anti-IGF-1R antibodies against ES.
- Published
- 2008
21. Recurrent, refractory, metastatic and/or unresectable pediatric sarcomas: treatment options for young people ‘off the roadmap’
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Dennis P.M. Hughes, David G. Kornguth, Anita Mahajan, Phil Phan, Winston W. Huh, Kathleen Cornelius, Pete Anderson, and Kamran Ahrar
- Subjects
medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,medicine.medical_treatment ,Ewing's sarcoma ,Cancer ,Interventional radiology ,Disease ,medicine.disease ,Pediatrics ,Surgery ,Family centered care ,Radiation therapy ,Pediatrics, Perinatology and Child Health ,medicine ,Sarcoma ,Intensive care medicine ,business ,Rhabdomyosarcoma - Abstract
Although sarcoma surgery is very important for cancer control, it is not always possible or practical to offer in some situations, including sarcoma recurrences, metastatic disease and/or unacceptable loss of function. We review some pragmatic approaches and examples of how to balance indications, risks and alternatives to control cancer in young people with sarcomas that are no longer using ‘front-line’ therapy. Radiotherapy combined with chemotherapy and outpatient ‘continuation’ chemotherapy regimens using drugs that cause less alopecia can improve function and quality of life. Some effective strategies to help cope when cure is not possible may include tumor ablation techniques performed in interventional radiology and percutaneous nerve blocks. Family centered care and effective problem solving of difficult issues can be greatly facilitated by consultation with a multidisciplinary team experienced in the management of very difficult cases. Treatment of young people with recurrent, relapsed and/or metastatic sarcoma still remains an art very much in the realm of compassion not protocol and persistent advocacy is required for the young person for whom cure may not be possible. A reduction of suffering and assistance in writing more chapters of a rich life narrative is the goal.
- Published
- 2008
22. Critical Role of Notch Signaling in Osteosarcoma Invasion and Metastasis
- Author
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Yanwen Yang, Pingyu Zhang, Patrick A. Zweidler-McKay, and Dennis P.M. Hughes
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,Blotting, Western ,Notch signaling pathway ,Gene Expression ,Biology ,Article ,Metastasis ,Mice ,Cell Line, Tumor ,Basic Helix-Loop-Helix Transcription Factors ,medicine ,Animals ,Humans ,Neoplasm Invasiveness ,HES1 ,Cell Proliferation ,Homeodomain Proteins ,Osteosarcoma ,Matrigel ,Receptors, Notch ,Reverse Transcriptase Polymerase Chain Reaction ,Neoplasms, Experimental ,medicine.disease ,Oncology ,Hes3 signaling axis ,Cancer research ,Transcription Factor HES-1 ,Cyclin-dependent kinase 8 ,Signal transduction ,Signal Transduction - Abstract
Purpose: Notch signaling is an important mediator of growth and survival in several cancer types, with Notch pathway genes functioning as oncogenes or tumor suppressors in different cancers. However, the role of Notch in osteosarcoma is unknown. Experimental Design: We assessed the expression of Notch pathway genes in human osteosarcoma cell lines and patient samples. We then used pharmacologic and retroviral manipulation of the Notch pathway and studied the effect on osteosarcoma cell proliferation, survival, anchorage-independent growth, invasion, and metastasis in vitro and in vivo. Results: Notch pathway genes, including Notch ligand DLL1, Notch1 and Notch2, and the Notch target gene HES1, were expressed in osteosarcoma cells, and expression of HES1 was associated with invasive and metastatic potential. Blockade of Notch pathway signaling with a small molecule inhibitor of γ secretase eliminated invasion in Matrigel without affecting cell proliferation, survival, or anchorage-independent growth. Manipulation of Notch and HES1 signaling showed a crucial role for HES1 in osteosarcoma invasiveness and metastasis in vivo. Conclusion: These studies identify a new invasion and metastasis-regulating pathway in osteosarcoma and define a novel function for the Notch pathway: regulation of metastasis. Because the Notch pathway can be inhibited pharmacologically, these findings point toward possible new treatments to reduce invasion and metastasis in osteosarcoma.
- Published
- 2008
23. Multimodality treatment of osteosarcoma: Radiation in a high-risk cohort
- Author
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Christopher E. Pelloski, Cynthia E. Herzog, Pete Anderson, Winston W. Huh, Anita Mahajan, Eric L. Chang, Shiao Y. Woo, Dennis P.M. Hughes, and David G. Kornguth
- Subjects
Adult ,Male ,Adolescent ,medicine.medical_treatment ,Bone Neoplasms ,Multimodality Therapy ,Medical Records ,Metastasis ,Cohort Studies ,Risk Factors ,Antineoplastic Combined Chemotherapy Protocols ,Humans ,Medicine ,Ifosfamide ,Child ,Survival rate ,Etoposide ,Retrospective Studies ,Osteosarcoma ,Chemotherapy ,business.industry ,Radiotherapy Dosage ,Hematology ,medicine.disease ,Combined Modality Therapy ,Survival Rate ,Radiation therapy ,Methotrexate ,Unresectable Osteosarcoma ,Oncology ,Doxorubicin ,Pediatrics, Perinatology and Child Health ,Female ,Cisplatin ,Neoplasm Recurrence, Local ,business ,Nuclear medicine ,Follow-Up Studies ,medicine.drug - Abstract
Purpose Chemotherapy during radiation and/or bone-seeking radioisotope therapy (153-samarium; 1 mCi/kg) during radiation may improve osteosarcoma cancer control. Patients and Methods We analyzed our preliminary radiation experience in high-risk, metastatic, and/or recurrent patients during a consecutive period of 20 months (May 2005–December 2006). Results Thirty-nine high-risk osteosarcoma patients had radiotherapy; 119 sites were irradiated. A median four sites were irradiated per patient (range 1–14). The median radiation dose and number of fractions of radiation was 30 Gy in 10 fractions (range 10–70 Gy in 4–35 fractions). Chemotherapy, most commonly ifosfamide or methotrexate, was used in 80% (100/119) radiotherapy courses. Of 38 painful sites, 29 had improvement (76%), 4 had no change (10%), and 5 had more pain (13%). Objective and potentially durable responses were documented using PET-CT and bone scans with persistent and sustained reduction of standard uptake values (SUVs; initial SUV of indication lesion 9.5 became
- Published
- 2008
24. Primary Tuberculosis of Bone Mimicking a Lytic Bone Tumor
- Author
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A. K. Raymond, Dennis P.M. Hughes, Christopher P. Cannon, Kimberly C. Smith, Scott D. Lemme, and Anne N. Normand
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Tuberculosis ,Biopsy, Fine-Needle ,Bone Neoplasms ,Lytic Bone Lesion ,Malignancy ,Tuberculosis, Osteoarticular ,Diagnosis, Differential ,Mycobacterium tuberculosis ,Lesion ,Biopsy ,medicine ,Humans ,Osteosarcoma ,biology ,medicine.diagnostic_test ,business.industry ,Osteomyelitis ,Hematology ,biology.organism_classification ,medicine.disease ,Magnetic Resonance Imaging ,Anti-Bacterial Agents ,Treatment Outcome ,Oncology ,Lytic cycle ,Child, Preschool ,Pediatrics, Perinatology and Child Health ,medicine.symptom ,business - Abstract
Causes of lytic bone lesions in children include benign, malignant, and infectious processes. Here, we present the case of a 3-year-old boy presenting with a lytic bone lesion and surrounding soft tissue mass sent for evaluation of possible malignancy versus osteomyelitis. Biopsy revealed granulomatous osteomyelitis, and subsequent purified protein derivative resulted in 20-mm induration. Lesion cultures eventually identified pan-sensitive Mycobacterium tuberculosis. We emphasize that tuberculosis can cause primary lytic bone lesions in children in the United States, even in the absence of pulmonary symptoms or known exposure, and advise clinicians to include mycobacterial cultures when analyzing biopsies of lytic bone lesions.
- Published
- 2007
25. Fate and function of anti-CD3/CD28-activated T cells following adoptive transfer: IL-2 promotes development of anti-tumor memory T cells in vivo
- Author
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Dennis P.M. Hughes, Thomas Braun, Michael S. Friedman, D. Baskar, F. A. Urban, and Kevin T. McDonagh
- Subjects
Cancer Research ,Time Factors ,CD3 Complex ,T-Lymphocytes ,Immunology ,Enzyme-Linked Immunosorbent Assay ,Mice, Transgenic ,CD8-Positive T-Lymphocytes ,Biology ,Lymphocyte Activation ,Immunotherapy, Adoptive ,Interferon-gamma ,Mice ,Mice, Congenic ,Interleukin 21 ,CD28 Antigens ,Cell Line, Tumor ,Animals ,Immunology and Allergy ,Cytotoxic T cell ,IL-2 receptor ,Antigen-presenting cell ,Genetics (clinical) ,Interleukin 3 ,Antigen Presentation ,Transplantation ,ZAP70 ,Neoplasms, Experimental ,Cell Biology ,Flow Cytometry ,Natural killer T cell ,Molecular biology ,Mice, Inbred C57BL ,Kinetics ,Oncology ,Leukemia, Myeloid ,Acute Disease ,Interleukin 12 ,Cancer research ,Interleukin-2 ,Lymph Nodes ,Spleen - Abstract
Adoptive immunotherapy with T cells activated through CD3 alone requires exogenous IL-2 for T-cell function and survival after transfer, but the in vivo cytokine requirement of T cells activated through CD3 and CD28 is unknown. We hypothesized that CD3/CD28-activated T cells, unlike those activated through CD3 alone, might develop into long-lived memory T cells, either with or without systemic IL-2.We used MHC class I-restricted TCR transgenic T cells from the OT-1 mouse, specific for the surrogate tumor Ag ovalbumin (OVA), to assess the trafficking kinetics, antigenic responsiveness and anti-tumor efficacy of dual-activated T cells in vivo as a function of IL-2 administration. At days 7, 14, and 28 after transfer, lymph node cells and splenocytes were examined for donor cell persistence and antigenic responsiveness by FACS and ELISA, respectively.In IL-2-treated mice, donor CD8+ T cells persisted and developed a memory phenotype, based on CD44 and Ly6c expression at day 28, while mice given no IL-2 had fewer donor cells at all time points. OVA-specific release of IFN-gamma was higher from lymphocytes of IL-2-treated mice compared with no-IL-2 mice (P0.02 at all time points). In mice challenged with an OVA-bearing subline of the AML leukemia model C1498, IL-2 did not confer added protection from tumor challenge at 1 or 2 weeks after adoptive transfer, but gave improved survival at 4 weeks post-transfer.We conclude that exogenous IL-2 is not required for anti-tumor activity of CD3/CD28-activated CD8+ cells early after adoptive transfer, but promotes T-cell persistence that confers disease protection at more remote times.
- Published
- 2005
26. Effect of concurrent metastatic disease on survival in children and adolescents undergoing lung resection for metastatic osteosarcoma
- Author
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Austen D. Slade, Dennis P.M. Hughes, Kevin P. Lally, Pamela A. Lally, Mary T. Austin, Andrea Hayes-Jordan, and Carla L. Warneke
- Subjects
Male ,medicine.medical_specialty ,Lung Neoplasms ,Adolescent ,medicine.medical_treatment ,Bone Neoplasms ,Disease ,Group A ,Group B ,Disease-Free Survival ,Young Adult ,medicine ,Humans ,Thoracotomy ,Child ,Pneumonectomy ,Retrospective Studies ,Osteosarcoma ,business.industry ,Neoplasms, Second Primary ,General Medicine ,medicine.disease ,Texas ,Surgery ,Log-rank test ,Survival Rate ,Child, Preschool ,Pediatrics, Perinatology and Child Health ,Metastatic osteosarcoma ,Female ,Lung resection ,business - Abstract
To evaluate the impact of treated extra-pulmonary metastatic disease on overall (OS) and event-free survival (EFS) for pediatric osteosarcoma patients undergoing pulmonary metastatectomy.We retrospectively reviewed pediatric patients who were treated for osteosarcoma at our institution from 2001 to 2011 and received pulmonary metastatectomy (n=76). We compared OS and EFS between patients with metastases limited to the lungs (Group A, n=58) to those with treated extra-pulmonary metastases (Group B, n=18) at the time of first pulmonary metastatectomy.The estimated median OS and EFS from first pulmonary metastatectomy were 2.0years (95% CI 1.5-2.8years) and 5.5months (95% CI 3.0-8.1months), respectively. Median OS was significantly greater for Group A (2.6years, 95% CI 1.9-3.8) compared to Group B (0.9years, 95% CI 0.6-1.5) (log rank p=0.0001). Median EFS was significantly greater for Group A (7.9months, 95% CI 5.0-10.7) compared to Group B (1.6months, 95% CI 0.8-2.7) (log rank p0.0001). Independent predictors of OS included extra-pulmonary metastatic disease at the time of first thoracotomy, bilateral pulmonary metastases, and4 nodules resected at first thoracotomy (all p0.001).Osteosarcoma patients with treated extra-pulmonary metastatic disease at the time of pulmonary metastatectomy have significantly worse survival compared to those with disease limited to the lungs.
- Published
- 2014
27. Health disparities are important determinants of outcome for children with solid tumor malignancies
- Author
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Kevin P. Lally, Linda S. Elting, Hoang Nguyen, Jan M. Eberth, Yuchia Chang, Mary T. Austin, Dennis P.M. Hughes, and Andras Heczey
- Subjects
Male ,Pediatrics ,medicine.medical_specialty ,Adolescent ,Health Status ,Ethnic group ,Outcome (game theory) ,Article ,Neoplasms ,parasitic diseases ,Ethnicity ,Medicine ,Humans ,Solid tumor ,Child ,Neoplasm Staging ,Retrospective Studies ,business.industry ,Racial Groups ,Infant, Newborn ,Infant ,Retrospective cohort study ,General Medicine ,Pediatric cancer ,Texas ,Health equity ,Socioeconomic Factors ,Disease Presentation ,Child, Preschool ,Pediatrics, Perinatology and Child Health ,Surgery ,Neoplasm staging ,Female ,business - Abstract
The purpose of this study was to identify health disparities in children with non-CNS solid tumor malignancies and examine their impact on disease presentation and outcome.We examined the records of all children (age≤18years) diagnosed with a non-CNS solid tumor malignancy and enrolled in the Texas Cancer Registry between 1995 and 2009 (n=4603). The primary outcome measures were disease stage and overall survival (OS). Covariates included gender, age, race/ethnicity, year of diagnosis, socioeconomic status (SES), and driving distance to the nearest pediatric cancer treatment facility. Statistical analyses included life table methods, logistic, and Cox regression. Statistical significance was defined as p0.05.Children with advanced-stage disease were more likely to be male,10years old, and Hispanic or non-Hispanic Blacks (all p0.05). Distance to treatment and SES did not impact stage of disease at presentation. However, Hispanic and non-Hispanic Blacks and patients in the lowest SES quartile had the worst 1- and 5-year survival (all p0.05). The adjusted OS differed by age, race, and stage, but not SES or distance to the nearest treatment facility.Race/ethnicity plays an important role in survival for children with non-CNS solid tumor malignancies. Future work should better define these differences to establish mechanisms to decrease their impact.
- Published
- 2014
28. Understanding the Role of Notch in Osteosarcoma
- Author
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Dennis P.M. Hughes, Madonna M. McManus, and Kurt R. Weiss
- Subjects
JAG1 ,Vasculogenesis ,Cancer stem cell ,Angiogenesis ,Wnt signaling pathway ,Cancer research ,Notch signaling pathway ,medicine ,Osteosarcoma ,Biology ,medicine.disease ,Gamma secretase - Abstract
The Notch pathway has been described as an oncogene in osteosarcoma, but the myriad functions of all the members of this complex signaling pathway, both in malignant cells and nonmalignant components of tumors, make it more difficult to define Notch as simply an oncogene or a tumor suppressor. The cell-autonomous behaviors caused by Notch pathway manipulation may vary between cell lines but can include changes in proliferation, migration, invasiveness, oxidative stress resistance, and expression of markers associated with stemness or tumor-initiating cells. Beyond these roles, Notch signaling also plays a vital role in regulating tumor angiogenesis and vasculogenesis, which are vital aspects of osteosarcoma growth and behavior in vivo. Further, osteosarcoma cells themselves express relatively low levels of Notch ligand, making it likely that nonmalignant cells, especially endothelial cells and pericytes, are the major source of Notch activation in osteosarcoma tumors in vivo and in patients. As a result, Notch pathway expression is not expected to be uniform across a tumor but likely to be highest in those areas immediately adjacent to blood vessels. Therapeutic targeting of the Notch pathway is likewise expected to be complicated. Most pharmacologic approaches thus far have focused on inhibition of gamma secretase, a protease of the presenilin complex. This enzyme, however, has numerous other target proteins that would be expected to affect osteosarcoma behavior, including CD44, the WNT/β-catenin pathway, and Her-4. In addition, Notch plays a vital role in tissue and organ homeostasis in numerous systems, and toxicities, especially GI intolerance, have limited the effectiveness of gamma secretase inhibitors. New approaches are in development, and the downstream targets of Notch pathway signaling also may turn out to be good targets for therapy. In summary, a full understanding of the complex functions of Notch in osteosarcoma is only now unfolding, and this deeper knowledge will help position the field to better utilize novel therapies as they are developed.
- Published
- 2014
29. Developmental Pathways Hijacked by Osteosarcoma
- Author
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Dennis P.M. Hughes, Jared R. Mortus, and Yi Zhang
- Subjects
musculoskeletal diseases ,Hippo signaling pathway ,Cellular differentiation ,Wnt signaling pathway ,Osteoblast ,Biology ,Fibroblast growth factor ,Bone morphogenetic protein ,medicine.disease ,Cell biology ,RUNX2 ,medicine.anatomical_structure ,medicine ,Osteosarcoma - Abstract
Cancer of any type often can be described by an arrest, alteration or disruption in the normal development of a tissue or organ, and understanding of the normal counterpart’s development can aid in understanding the malignant state. This is certainly true for osteosarcoma and the normal developmental pathways that guide osteoblast development that are changed in the genesis of osteogenic sarcoma. A carefully regulated crescendo–decrescendo expression of RUNX2 accompanies the transition from mesenchymal stem cell to immature osteoblast to mature osteoblast. This pivotal role is controlled by several pathways, including bone morphogenic protein (BMP), Wnt/β-catenin, fibroblast growth factor (FGF), and protein kinase C (PKC). The HIPPO pathway and its downstream target YAP help to regulate proliferation of immature osteoblasts and their maturation into non-proliferating mature osteoblasts. This pathway also helps regulate expression of the mature osteoblast protein osteocalcin. YAP also regulates expression of MT1-MMP, a membrane-bound matrix metalloprotease responsible for remodeling the extracellular matrix surrounding the osteoblasts. YAP, in turn, can be regulated by the ERBB family protein Her-4. Osteosarcoma may be thought of as a cell held at the immature osteoblast stage, retaining some of the characteristics of that developmental stage. Disruptions of several of these pathways have been described in osteosarcoma, including BMP, Wnt/b-catenin, RUNX2, HIPPO/YAP, and Her-4. Further, PKC can be activated by several receptor tyrosine kinases implicated in osteosarcoma, including the ERBB family (EGFR, Her-2 and Her-4 in osteosarcoma), IGF1R, FGF, and others. Understanding these functions may aid in the understanding the mechanisms underpinning clinical observations in osteosarcoma, including both the lytic and blastic phenotypes of tumors, the invasiveness of the disease, and the tendency for treated tumors to ossify rather than shrink. Through a better understanding of the relationship between normal osteoblast development and osteosarcoma, we may gain insights into novel therapeutic avenues and improved outcomes.
- Published
- 2014
30. Understanding the Biology of Bone Sarcoma from Early Initiating Events through Late Events in Metastasis and Disease Progression
- Author
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Limin Zhu, Madonna McManus, and Dennis P.M. Hughes
- Subjects
tumor dormancy ,Cancer Research ,Pathology ,medicine.medical_specialty ,Review Article ,Disease ,Bone Sarcoma ,lcsh:RC254-282 ,Metastasis ,Neovascularization ,cancer signaling ,medicine ,metastasis ,anoikis resistance ,Osteosarcoma ,intravasation ,business.industry ,Intravasation ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,medicine.disease ,Primary bone ,Oncology ,Cancer research ,Sarcoma ,neovascularization ,medicine.symptom ,business ,Ewing sarcoma - Abstract
The two most common primary bone malignancies, osteosarcoma and Ewing sarcoma, are both aggressive, highly metastatic cancers that most often strike teens, though both can be found in younger children and adults. Despite distinct origins and pathogenesis, both diseases share several mechanisms of progression and metastasis, including neovascularization, invasion, anoikis resistance, chemoresistance and evasion of the immune response. Some of these processes are well-studies in more common carcinoma models, and the observation from adult diseases may be readily applied to pediatric bone sarcomas. Neovascularization, which includes angiogenesis and vasculogenesis, is a clear example of a process that is likely to be similar between carcinomas and sarcomas, since the responding cells are the same in each case. Chemoresistance mechanisms also may be similar between other cancers and the bone sarcomas. Since osteosarcoma and Ewing sarcoma are mesenchymal in origin, the process of epithelial-to-mesenchymal transformation is largely absent in bone sarcomas, necessitating different approaches to study progression and metastasis in these diseases. One process that is less well-studied in bone sarcomas is dormancy, which allows micrometastatic disease to remain viable but not growing in distant sites – typically the lungs – for months or years before renewing growth to become overt metastatic disease. By understanding the basic biology of these processes, novel therapeutic strategies may be developed that could improve survival in children with osteosarcoma or Ewing sarcoma.
- Published
- 2013
31. Anti-tumor effects of the Notch pathway in gastrointestinal stromal tumors
- Author
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Yanwen Yang, A. G. Dumont, Dennis P.M. Hughes, Daniela Katz, Jonathan C. Trent, and David Reynoso
- Subjects
Adult ,Male ,Cancer Research ,Pathology ,medicine.medical_specialty ,Stromal cell ,medicine.drug_class ,Gastrointestinal Stromal Tumors ,Notch signaling pathway ,Apoptosis ,Original Manuscript ,PDGFRA ,Biology ,Hydroxamic Acids ,Cell Line, Tumor ,medicine ,Basic Helix-Loop-Helix Transcription Factors ,Humans ,RNA, Messenger ,HES1 ,Progenitor cell ,Aged ,Cell Proliferation ,Gastrointestinal Neoplasms ,Aged, 80 and over ,Homeodomain Proteins ,GiST ,Receptors, Notch ,Histone deacetylase inhibitor ,General Medicine ,Middle Aged ,digestive system diseases ,Repressor Proteins ,Proto-Oncogene Proteins c-kit ,Cancer research ,Transcription Factor HES-1 ,Female ,Histone deacetylase ,Signal Transduction - Abstract
Gastrointestinal stromal tumors (GISTs) are driven by gain-of-function mutations of KIT or PDGFRa. The introduction of imatinib has significantly extended survival for patients. However, most patients develop resistances. Notch signaling is a conserved developmental pathway known to play a critical role in the development of several cancers, functioning as a tumor promoter or a tumor suppressor. Given that the normal progenitor cell for GIST, the interstitial cell of Cajal, has characteristics similar to those of cells of neuroendocrine origin, we hypothesized that Notch pathway impacts the biology of GIST cells. In this study, we retrovirally and pharmacologically manipulated the Notch pathway in human GIST cells. We also performed a retrospective analysis of a cohort on 15 primary tumors to determine the role of Hes1, a major target gene of Notch, as a prognostic marker for GIST. Constitutively, active intracellular domain of Notch1 (ICN1) expression potently induced growth arrest and downregulated KIT expression in vitro. Additionally, treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid caused dose-dependent upregulation of Notch1 expression and a parallel decrease in viability in these cells. Retroviral silencing of downstream targets of Notch (dominant-negative Hes1) and pharmacological inhibition of Notch activation (γ-secretase inhibition) partially rescued GIST cells from suberoylanilide hydroxamic acid treatment. GIST patients with high Hes1 mRNA levels have a significantly longer relapse-free survival. These results identify a novel anti-tumor effect of Notch1 and cross talk between the Notch and KIT pathways. Thus, activation of this pathway by treatment with histone deacetylase inhibitors is an appealing potential therapeutic strategy for GISTs. Précis: This study is the first report of the tumor suppressor effects of Notch pathway in gastrointestinal stromal tumors via a negative feedback with the oncogene KIT and may lead the development of new therapeutic strategies for GISTs patients.
- Published
- 2012
32. Slow down to stay alive: HER4 protects against cellular stress and confers chemoresistance in neuroblastoma
- Author
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Wenyi Wang, Dennis P.M. Hughes, Nianxiang Zhang, Kirill Gorshkov, Yingqi Hua, and Yanwen Yang
- Subjects
Cancer Research ,animal structures ,Receptor, ErbB-4 ,Cell Survival ,Cyclin D ,Cell Culture Techniques ,Gene Expression ,Article ,Small hairpin RNA ,Neuroblastoma ,Cell Line, Tumor ,medicine ,Humans ,Anoikis ,RNA, Messenger ,RNA, Small Interfering ,Gene knockdown ,biology ,Cell growth ,Cell cycle ,medicine.disease ,Molecular biology ,ErbB Receptors ,Oncology ,Cell culture ,Drug Resistance, Neoplasm ,biology.protein ,Cancer research - Abstract
BACKGROUND: Neuroblastoma (NBL) is a common pediatric solid tumor, and outcomes for patients with advanced neuroblastoma remain poor despite extremely aggressive treatment. Chemotherapy resistance at relapse contributes heavily to treatment failure. The poor survival of patients with high-risk NBL prompted this investigation into novel treatment options with the objective of gaining a better understanding of resistance mechanisms. On the basis of previous work and on data from publicly available studies, the authors hypothesized that human epidermal growth factor receptor 4 (Her4) contributes to resistance. METHODS: Her4 expression was reduced with small-hairpin RNA (shRNA) to over express intracellular HER4, and the authors tested its impact on tumor cell survival under various culture conditions. The resulting changes in gene expression after HER4 knockdown were measured by using a messenger RNA (mRNA) array. RESULTS: HER4 expression was up-regulated in tumor spheres compared with the expression in monolayer culture. With HER4 knockdown, NBL cells became less resistant to anoikis and serum starvation. Moreover, HER4 knockdown increased the chemosensitivity of NBL cells to cisplatin, doxorubicin, etoposide, and activated ifosfamide. In mRNA array analysis, HER4 knockdown predominately altered genes related to cell cycle regulation. In NBL spheres compared with monolayers, cell proliferation was decreased, and cyclin D expression was reduced. HER4 knockdown reversed cyclin D suppression. Overexpressed intracellular HER4 slowed the cell cycle and induced chemoresistance. CONCLUSIONS: The current results indicated that HER4 protects NBL cells from multiple exogenous apoptotic stimuli, including anoikis, nutrient deficiency, and cytotoxic chemotherapy. The intracellular fragment of HER4 was sufficient to confer this phenotype. HER4 functions as a cell cycle suppressor, maintaining resistance to cellular stress. The current findings indicate that HER4 overexpression may be associated with refractory disease, and HER4 may be an important therapeutic target. Cancer 2012. © 2012 American Cancer Society.
- Published
- 2012
33. miR-20a encoded by the miR-17-92 cluster increases the metastatic potential of osteosarcoma cells by regulating Fas expression
- Author
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Dennis P.M. Hughes, Kazumasa Nishimoto, Zhichao Zhou, Eugenie S. Kleinerman, and Gangxiong Huang
- Subjects
Cancer Research ,Lung Neoplasms ,Mice, Nude ,Bone Neoplasms ,Biology ,Transfection ,Article ,Mice ,Cell Line, Tumor ,microRNA ,medicine ,Animals ,Humans ,fas Receptor ,Mice nude ,Osteosarcoma ,Lung ,respiratory system ,medicine.disease ,respiratory tract diseases ,Gene Expression Regulation, Neoplastic ,MicroRNAs ,medicine.anatomical_structure ,Oncology ,Cancer cell ,Immunology ,Cancer research ,Female ,RNA, Long Noncoding - Abstract
The ability of osteosarcoma cells to form lung metastases has been inversely correlated to cell surface Fas expression. Downregulation of Fas allows osteosarcoma cells to circumvent FasL-mediated apoptosis upon entrance into the FasL+ lung microenvironment. However, the mechanism of Fas regulation remains unclear. Here, we show that miRNA plays a role in the downregulation of Fas expression in osteosarcoma. Expression levels of several members of the miR-17–92 cluster including miR-20a and miR-19a were found to be higher in metastatic low-Fas–expressing LM7 cells than in the parental nonmetastatic high-Fas–expressing SAOS-2 cells. We also found an inverse correlation between Fas and miR-20a expression in all 8 cell lines derived from patient samples. Overexpression of miR-20a consistently resulted in the downregulation of Fas expression in SAOS-2 cells and thus in decreased sensitivity to FasL. Conversely, inhibiting miR-20a in LM7 cells increased Fas expression and their sensitivity to FasL. Mice injected with LM7 stably transfected with anti-miR-20a had fewer metastases than those with control plasmids. Taken together, our findings suggest that miR-20a, encoded by miR-17–92, downregulates Fas expression in osteosarcoma, thus contributing to the metastatic potential of osteosarcoma cells by altering the phenotype and allowing survival in the FasL+ lung microenvironment. Cancer Res; 72(4); 908–16. ©2011 AACR.
- Published
- 2011
34. Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness
- Author
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Dennis P.M. Hughes, Patrick A. Zweidler-McKay, Pingyu Zhang, Yanwen Yang, and Riitta Nolo
- Subjects
Cancer Research ,Chromatin Immunoprecipitation ,Blotting, Western ,INVASION ,Notch signaling pathway ,Bone Neoplasms ,Electrophoretic Mobility Shift Assay ,Biology ,Transfection ,Article ,Deltex1(DTX1) ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,Basic Helix-Loop-Helix Transcription Factors ,Humans ,Immunoprecipitation ,Neoplasm Invasiveness ,RNA, Messenger ,HES1 ,Luciferases ,Molecular Biology ,030304 developmental biology ,Homeodomain Proteins ,0303 health sciences ,Transcription Factor HES-1 ,Osteosarcoma ,Receptors, Notch ,Reverse Transcriptase Polymerase Chain Reaction ,Ubiquitination ,DNA-Binding Proteins ,NOTCH ,Notch proteins ,Hes3 signaling axis ,030220 oncology & carcinogenesis ,embryonic structures ,Cancer research ,Cyclin-dependent kinase 8 ,Signal transduction ,Carrier Proteins ,Chromatin immunoprecipitation ,Signal Transduction - Abstract
The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.
- Published
- 2010
35. Critical signaling pathways in bone sarcoma: candidates for therapeutic interventions
- Author
-
Mandy Geryk-Hall and Dennis P.M. Hughes
- Subjects
Akt/PKB signaling pathway ,business.industry ,Wnt signaling pathway ,Notch signaling pathway ,Antineoplastic Agents ,Bone Neoplasms ,Receptors, Cell Surface ,Sarcoma ,Bone Sarcoma ,medicine.disease ,Oncology ,ErbB ,Cancer research ,medicine ,Humans ,business ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Signal Transduction - Abstract
Bone sarcomas cause disproportionate morbidity and mortality and desperately need new therapies as there has been little improvement in outcomes in 20 years. Identification of critical signaling pathways, including type 1 insulin-like growth factor receptor (IGF-1R) for Ewing sarcoma and possibly osteosarcoma, and the ERBB and the Wnt signaling pathways for osteosarcoma, have emerged as receptors mediating vital signals for bone sarcoma. Akt, mammalian target of rapamycin (mTOR), phosphoinositide 3-kinases, mitogen-activated protein kinase kinase, extracellular signal-regulated kinases, and Ras pathway play key roles in at least some tumors, and inhibition of mTOR in particular will likely lead to improved survival, although clinical trials are still underway. The Notch pathway and ezrin are essential for osteosarcoma metastasis, and Fas down-regulation is necessary for survival of metastases in lungs. As little is known about chondrosarcoma signaling, more preclinical work is needed. By defining vital signaling pathways in bone sarcomas, small molecule inhibitors can be applied rationally, leading to longer survival and reducing morbidity and late effects from intensive chemotherapy.
- Published
- 2009
36. Strategies for the targeted delivery of therapeutics for osteosarcoma
- Author
-
Dennis P.M. Hughes
- Subjects
medicine.medical_specialty ,Limb salvage ,medicine.medical_treatment ,Pharmaceutical Science ,Antineoplastic Agents ,Bone Neoplasms ,Article ,Drug Delivery Systems ,medicine ,Animals ,Humans ,Bone formation ,Intrapleural chemotherapy ,Intensive care medicine ,Survival rate ,Chemotherapy ,Osteosarcoma ,business.industry ,Antibodies, Monoclonal ,medicine.disease ,Limb Salvage ,Surgery ,Survival Rate ,Sarcoma ,business ,Central venous catheter - Abstract
Background: Conventional therapy for osteosarcoma has reached a plateau of 60 – 70%, a 5-year survival rate that has changed little in two decades, highlighting the need for new approaches. Objective: To review the alternative means of delivering effective therapy for osteosarcoma that reach beyond the central venous catheter. Methods: Drawing on the author's own experiences providing care to high-risk osteosarcoma patients and reviewing the last two decades of literature describing sarcoma therapy, available information is summarized about potential osteosarcoma treatments that deliver therapy by a less conventional route. Results/conclusions: Intra-arterial chemotherapy has a limited impact on survival, but may help to achieve a better limb salvage. Intrapleural chemotherapy is important for managing malignant effusions. The development of inhalation therapies, treatments that target new bone formation such as bisphosphonates, chemically targeted radiation and antibody-based therapies all have potential...
- Published
- 2009
37. Expression levels and activation of a PXR variant are directly related to drug resistance in osteosarcoma cell lines
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Laurence H. Baker, Michelle L. Lizyness, Bruce Blumberg, James M. Rae, Dafydd G. Thomas, Edith J. Mensah-Osman, Thomas J. Giordano, Dennis P.M. Hughes, Jose M. Larios, Paul F. Hollenberg, and Michelle M. Tabb
- Subjects
Cancer Research ,Receptors, Steroid ,Blotting, Western ,Antineoplastic Agents ,Biology ,digestive system ,Article ,Western blot ,Cytochrome P-450 Enzyme System ,Annexin ,Cell Line, Tumor ,medicine ,Cytochrome P-450 CYP3A ,Humans ,Protein Isoforms ,Doxorubicin ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,RNA, Messenger ,RNA, Small Interfering ,Cytotoxicity ,Etoposide ,Pregnane X receptor ,Osteosarcoma ,medicine.diagnostic_test ,Reverse Transcriptase Polymerase Chain Reaction ,Pregnane X Receptor ,medicine.disease ,digestive system diseases ,Oncology ,Cell culture ,Drug Resistance, Neoplasm ,Immunology ,Cancer research ,Rifampin ,medicine.drug - Abstract
BACKGROUND. Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors. METHODS. Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining. RESULTS. Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole. CONCLUSION. The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance. Cancer 2007. © 2007 American Cancer Society.
- Published
- 2007
38. Ex vivo culture with interleukin (IL)-12 improves CD8(+) T-cell adoptive immunotherapy for murine leukemia independent of IL-18 or IFN-gamma but requires perforin
- Author
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Jennifer N. MacGregor, Dennis P.M. Hughes, Qiao Li, Thomas Braun, Alfred E. Chang, and Kevin T. McDonagh
- Subjects
Cytotoxicity, Immunologic ,Male ,Pore Forming Cytotoxic Proteins ,Cancer Research ,Adoptive cell transfer ,Epitopes, T-Lymphocyte ,Biology ,CD8-Positive T-Lymphocytes ,Lymphocyte Activation ,Immunotherapy, Adoptive ,Interleukin 21 ,Interferon-gamma ,Mice ,In vivo ,Cell Line, Tumor ,Cytotoxic T cell ,Animals ,Mice, Knockout ,Mice, Inbred BALB C ,Membrane Glycoproteins ,Perforin ,Interleukin-18 ,Drug Synergism ,Interleukin-12 ,Mice, Inbred C57BL ,Oncology ,Cell culture ,Leukemia, Myeloid ,Immunology ,Acute Disease ,Cancer research ,Interleukin 12 ,Female ,Ex vivo ,CD8 - Abstract
In animal models and clinical trials, adoptive transfer of activated, antigen-specific CD8+ T cells mediates tumor regression in a cell dose-dependent manner. The cytokine interleukin (IL)-12 promotes CD8+ T-cell cytotoxicity and, with IL-18, synergistically up-regulates IFN-γ release. We have shown that culturing CD8+ T cells ex vivo with IL-12 and IL-18 enhanced antitumor responses in vivo and in vitro using a model of C1498/ovalbumin, a murine acute myeloid leukemia cell line expressing the antigen ovalbumin. Activated ovalbumin-specific CD8+ T cells cultured with IL-12, IL-18, both, or neither were assayed for antigen-specific cytokine production and cytolytic activity and adoptively transferred to C57BL/6 mice with established tumors. Maximal IFN-γ release occurred after T-cell culture with IL-12 and IL-18. Tumor-specific in vitro cytotoxicity was enhanced by IL-12, unaffected by addition of IL-18, and abrogated in perforin-deficient T cells irrespective of cytokine exposure. T cells cultured with IL-12 more effectively eliminated tumors, and addition of IL-18 did not further augment responses. IFN-γ-deficient CD8+ T cells showed effective antitumor activity that was enhanced by IL-12 with or without IL-18. Perforin-deficient CD8+ T cells were poor mediators of antitumor activity, though, and showed no improvement after culture with IL-12 and/or IL-18. Thus, ex vivo culture with IL-12 was sufficient to augment antigen-specific in vitro cytotoxicity and antitumor activity in vivo in an IFN-γ-independent but perforin-dependent manner. Ex vivo culture with IL-12 may improve CD8+ T-cell immunotherapy of cancer in the absence of donor cell–derived IFN-γ via perforin-mediated cytolysis. (Cancer Res 2006; 66(9): 4913-21)
- Published
- 2006
39. Abstract A71: Her-4 mediates anoikis resistance, chemoresistance, and metastatic potential in osteosarcoma
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Rocio K. Rivera-Valentin, Yingqi Hua, Yanwen Yang, Nupur Lala, Dennis P.M. Hughes, and Yi Zhang
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Cisplatin ,Cancer Research ,Pathology ,medicine.medical_specialty ,business.industry ,Cancer ,Cell cycle ,medicine.disease ,Pediatric cancer ,Metastasis ,Oncology ,Cell culture ,Cancer research ,Medicine ,Osteosarcoma ,Anoikis ,business ,medicine.drug - Abstract
Osteosarcoma (OS) is the most common primary bone cancer in children, and despite more than 20 years of clinical trials, the 5-year survival for OS patients remains essentially unchanged (∼70% for non-metastatic patients and 30% for metastatic patients), with the vast majority deaths arising from metastasis to lungs or bones. We wanted to identify signaling pathways that mediate tumor survival and metastasis in OS, and we focused on Her-4 (ERBB4), which promoted survival on neuroblastoma cells in our previous work. Purpose: To evaluate the role of Her-4 in OS cell survival, metastasis and “stemness”. Methods: Her-4 expression was measured by flow cytometry, western blot and Q-PCR. Validated OS cell lines were manipulated with shRNA to knock-down Her-4 (compared to scrambled control). In vitro responses were measured by varying culture conditions (normal, high-density or anchorage-independent growth), serum starvation, hypoxia or chemotherapy (methotrexate, cisplatin, doxorubicin and 4-OH-ifosfamide) and measuring proliferation, cell cycle, and apoptosis (by both flow cytometry and PARP cleavage). A sarcosphere assay assessed expression of “stemness” markers, or tumor initiating cells (TICs), and Her-4 expression. The luciferase-labeled CCH-OS-O xenograft model was used to assess the impact of Her-4 knock-down on metastatic potential, with weekly luciferase measures (IVIS 100, Xenogen) assessing tumor burden. Immunohistochemistry (IHC) assessed Her-4 expression in archival material and murine xenografts. Results: Most OS cells express Her-4 which upregulated with increased culture density and with serum starvation. RT-PCR showed that the proportion of cleavable isoforms increased with increasing cell density (p < .05 with One-Way ANOVA analysis), while non-cleavable isoforms decreased (p < .05). Hypoxia increased the cleavage of Her-4 to the 80KD intracellular fragment without increasing Her-4 expression. IHC analysis of OS tissue microarrays showed that Her-4 expression is higher in metastatic lesions relative to primary tumors (p>0.01). Knock-down of Her-4 in OS cell lines reduced proliferation and increased apoptosis (by sub-diploid DNA and PARP cleavage) for cells grown in conventional culture, high density or spheres. Her-4 knock-down increased sensitivity to methotrexate (60% increase in apoptosis) but not other chemotherapies. To assess the role of Her-4 in metastasis, NOD/SCID/IL2Rg-/- mice were injected in the tibia with CCH-OD-O cells expressing luciferase and either Her-4 shRNA or scrambled control. With this model, a “primary” tumor arises in the tibia, with spontaneous metastasis to the lungs. Non-invasive imaging showed reduced growth of primary tumors in knock-down mice and complete elimination of tumor signal from lungs. Xenograft lungs showed a significant reduction in metastasis in knock-down mice. IHC for Her-4 showed that, while tibial tumors of knock-down mice still expressed no Her-4, the tumors present in these mice had re-expressed Her-4 despite the shRNA. Stress resistance, chemotherapy resistance and metastatic potential are TIC-associated behaviors, so we examined the association of Her-4 with other described TIC markers for OS. Sarcosphere culture induced expression of both Her-4 and stemness markers CD117 and Stro1 over a period of 2-7 days. Her-4 expression preceded upregulation of other markers by 1-2 days. Conclusions: Expression of Her-4 confers a survival benefit to OS cells under stressed conditions, including anoikis, serum starvation and methotrexate exposure. Her-4 is necessary for OS metastasis. Her-4 expression increases in sarcophere cultures and may be a mediator of stemness. Therapies targeting Her-4 in OS should be evaluated in further preclinical models. Citation Format: Rocio K. Rivera-Valentin, Yingqi Hua, Yi Zhang, Nupur Lala, Yanwen Yang, Dennis P.M. Hughes. Her-4 mediates anoikis resistance, chemoresistance, and metastatic potential in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A71.
- Published
- 2014
40. Abstract A53: Metadherin functions as a laminin receptor that is essential for metastasis and is associated with poor survival in osteosarcoma
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Dennis P.M. Hughes, Yanwen Yang, Dafydd G. Thomas, Pingyu Zhang, Wei-Lien Wang, Limin Zhu, and Adrianna S. Buford
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Cancer Research ,Tissue microarray ,business.industry ,Cancer ,MTDH ,medicine.disease ,Pediatric cancer ,Primary tumor ,Metastasis ,Oncology ,medicine ,Cancer research ,Osteosarcoma ,Cell adhesion ,business - Abstract
Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. In order to improve the clinical outcomes for patients with poor prognosis, it is urgent to find new therapeutic targets to block metastasis in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. To evaluate the expression of MTDH in primary and metastatic lesions of osteosarcoma, two tissue microarrays containing patient-derived primary and metastatic tumor specimens were examined by immunohistochemical staining with anti-MTDH antibody. We also examined MTDH in a cDNA array expression database made from pretreatment diagnostic biopsies of high-grade osteosarcoma patients to further assess the correlation between MTDH expression and clinical outcome. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. In addition, we developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction and to identify the extracellular binding partner for cell surface MTDH, a series of adhesion assays were performed, followed by bidirectional co-immunoprecipitation. We have demonstrated that MTDH is upregulated in human osteosarcoma cell lines and patient-derived specimens compared with normal human osteoblasts. Overexpression of MTDH is more profound in metastatic lesions compared to primary tumors and is correlated with poor clinical outcomes in osteosarcoma patients. MTDH knockdown and blockade of cell surface MTDH significantly reduced migration and invasion in osteosarcoma cells. In the in vivo experiments, downregulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Both in vitro and in vivo studies confirmed the critical role of MTDH in the invasive and metastatic capacity of osteosarcoma cells. More importantly, we have identified the significance of cell surface localization of MTDH in mediating osteosarcoma motility and invasiveness. We showed that MTDH exists as a type II membrane protein in osteosarcoma cells and its expression on cell surface is facilitating cell invasion by means of modulating cell adhesion to the ECM through interaction with laminin. In total, these observations establish MTDH as a promising target for therapeutic interventions in metastatic osteosarcoma. The novel connection between MTDH and extracellular laminin also establishes a new paradigm for the function of MTDH in mediating tumor cell metastasis. Citation Format: Limin Zhu, Pingyu Zhang, Yanwen Yang, Adrianna S. Buford, Wei-Lien Wang, Dafydd G. Thomas, Dennis P.M. Hughes. Metadherin functions as a laminin receptor that is essential for metastasis and is associated with poor survival in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A53.
- Published
- 2014
41. Abstract 1261: Targeting IL-11Rα inhibits osteosarcoma pulmonary metastasis in an orthotopic xenograft mouse model
- Author
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Valerae O. Lewis, Dennis P.M. Hughes, and Eswaran Devarajan
- Subjects
musculoskeletal diseases ,Cancer Research ,Pathology ,medicine.medical_specialty ,Gene knockdown ,business.industry ,Cancer ,medicine.disease ,Primary tumor ,Metastasis ,Small hairpin RNA ,Oncology ,Cancer research ,Medicine ,Osteosarcoma ,Immunohistochemistry ,Gastrointestinal cancer ,business - Abstract
Osteosarcoma is the most common primary tumor of bones. In the past three decades treatment paradigms and survival rates have not improved. While osteosarcoma clinical phenotype has been well characterized, the underlying tumor molecular biology remains unclear. We have previously demonstrated that IL-11Rα is overexpressed in primary and metastatic osteosarcoma. Recently investigators identified that IL-11 and IL-11Rα play a role in the development of metastasis in breast and gastrointestinal cancer. However the role of IL-11/IL-11Rα signaling in osteosarcoma is unknown. Using in vitro and in vivo osteosarcoma models we investigated the role of IL-11/IL-11Rα in osteosarcoma metastasis. The purpose of our study is to determine the effect of the knockdown of IL-11Rα in osteosarcoma. We stably knocked down IL-11Rα expression in a highly metastatic osteosarcoma cell line, using a lentiviral short hair-pin (shRNA) based approach. Knockdown of IL-11Rα led to inhibition of STAT-3(Tyr705) protein phosphorylation, cell growth, migration, invasion and colony formation in vitro. To determine the role of IL-11Rα in osteosarcoma tumorigeniesis and metastasis, we used an orthotopic metastatic model. In this model, mice injected with KRIB-IL-11Rα shRNA cells developed significantly less metastasis than control mice injected with KRIB control shRNA (P Citation Format: Valerae O. Lewis, Eswaran Devarajan, Dennis PM Hughes. Targeting IL-11Rα inhibits osteosarcoma pulmonary metastasis in an orthotopic xenograft mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2014-1261
- Published
- 2014
42. Abstract 3970: EGF has stimulatory and survival effects in osteosarcoma in vitro and pan-ERBB kinase inhibition causes cell growth inhibition in vitro and reduces lung metastases in vivo
- Author
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Diane Liu, Yanwen Yang, Wei-Lien Wang, Dennis P.M. Hughes, Tiffany N. Lynch, Laura D. Nelson, and Dafydd G. Thomas
- Subjects
Cancer Research ,Chemistry ,Cell growth ,Kinase ,Cell ,medicine.disease ,Molecular biology ,chemistry.chemical_compound ,medicine.anatomical_structure ,Oncology ,ErbB ,In vivo ,medicine ,Osteosarcoma ,Growth inhibition ,Protein kinase B - Abstract
Osteosarcoma is the most common primary bone tumor in children and adolescents, with recurring disease or lung metastases being the primary cause of death in patients. Expression of EGFR, Her-2, and Her-4 (ERBB1, 2 and 4) have been reported in human primary osteosarcoma, suggesting that they may be contributing to osteosarcoma pathogenesis, but the correlation of expression levels with patient outcomes has been controversial. We have shown that a pan-ERBB small molecule kinase inhibitor causes growth inhibition and apoptosis of osteosarcoma cell in vitro, suggesting a potential therapeutic benefit of using this drug in vivo. In the present study, we show that protein expression levels of EGFR, Her-2 and Her-4 are higher in osteosarcoma lung metastases than in primary tumors by TMA, and that these levels are significantly higher for Her-2 and Her-4. We created eight new osteosarcoma cell lines from primary patient tissues and found that all eight, in addition to six commonly used osteosarcoma cell lines, express detectable levels of EGFR and Her-2, and several express detectable Her-4. EGF ligand stimulation in vitro produces robust phosphorylation of EGFR, Her-2 and Her-4 in most cell lines and moderate activation in others. EGF stimulation also produces phosphorylation of Akt, MAPK and p70S6K in most cell lines and of STAT5 in six cell lines, suggesting that EGF has growth and survival stimulation effects. When adding a pan-ERBB kinase inhibitor, Pf-299804, to cells pre-stimulated with EGF, phosphorylation of EGFR, Her-2, Her-4, Akt, etc. are inhibited in a dose-dependent manner. Reverse phase protein array also shows an increase in pro-apoptotic proteins Bim and cleaved PARP with Pf-299804 treatment, confirmed by Western blots. Mouse xenograft experiments, where cells injected in the tibia spontaneously form lung metastases, showed a significant decrease in three categories of Her-4-positive lung metastases in drug-treated mice based on size: 1-4 cell oligomets (p=0.0055); 5 cell-200 micron micromets (p=0.0276); and greater than 200 micron macromets (p=0.0055). These data suggest that EGF produces mitogenic and survival stimulation in osteosarcoma cells and that pan ERBB-family kinase inhibition causes growth inhibition and apoptosis in vitro and reduction of lung metastases in a mouse model in vivo. This provides evidence that pan-ERBB kinase inhibition has therapeutic potential for osteosarcoma lung metastases. Citation Format: Laura D. Nelson, Tiffany N. Lynch, Yanwen Yang, Wei-Lien Wang, Diane Liu, Dafydd G. Thomas, Dennis P. M. Hughes. EGF has stimulatory and survival effects in osteosarcoma in vitro and pan-ERBB kinase inhibition causes cell growth inhibition in vitro and reduces lung metastases in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3970. doi:10.1158/1538-7445.AM2014-3970
- Published
- 2014
43. Abstract 2683: Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma
- Author
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Adrianna S. Buford, Pingyu Zhang, Limin Zhu, Yanwen Yang, Wei-Lien Wang, and Dennis P.M. Hughes
- Subjects
Cancer Research ,Matrigel ,Pathology ,medicine.medical_specialty ,Cell ,MTDH ,Biology ,medicine.disease ,medicine.disease_cause ,Metastasis ,Extracellular matrix ,medicine.anatomical_structure ,Oncology ,Cancer research ,medicine ,Osteosarcoma ,Cell adhesion ,Carcinogenesis - Abstract
Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines, and used immunohistochemistry to confirm expression in patient-derived material. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. We also developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction, a series of adhesion assays were performed to assess the adhesion ability of osteosarcoma cells with or without MTDH inhibition to major components of ECM. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed increased MTDH expression. MTDH knockdown significantly reduced migration and invasion in osteosarcoma cells. Blockade of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness. In the in vivo experiments, down-regulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Immunohistochemical analysis of tumors samples isolated from the mice showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether, these data indicated an important role for Metadherin in osteosarcoma metastasis. Further, knockdown of MTDH by shRNA and inhibition of cell surface MTDH by anti-MTDH antibodies both profoundly decreased cell adhesion to matrigel and specific ECM proteins, which suggested that MTDH, particularly the proportion localized at the cell surface, may be involved in the interaction between tumor cells and the non-cellular matrix proteins that constitute tissue stroma. The interaction will be confirmed by future experiments. By identifying the ECM protein binding partners for MTDH, we may establish a new paradigm for the function of MTDH in mediating tumor cell migration and invasion in osteosarcoma. Citation Format: Limin Zhu, Adrianna Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2013-2683
- Published
- 2013
44. Abstract B67: Metadherin mediates osteosarcoma invasion and metastasis
- Author
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Pingyu Zhang, Yanwen Yang, Adrianna S. Buford, Limin Zhu, Dennis P.M. Hughes, and Wei-Lien Wang
- Subjects
Cancer Research ,Matrigel ,Pathology ,medicine.medical_specialty ,business.industry ,Cell ,Cancer ,MTDH ,medicine.disease ,Primary tumor ,Metastasis ,medicine.anatomical_structure ,Oncology ,medicine ,Cancer research ,Osteosarcoma ,Immunohistochemistry ,business - Abstract
Osteosarcoma is the most common type of bone malignancy in children. It is a highly invasive mesenchymal tumor in which metastasis accounts for the vast majority of mortality and morbidity in patients. Understanding the mechanisms controlling metastasis in osteosarcoma and developing novel therapeutic options targeting metastasis are essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays a key role in mediating metastasis in a variety of human cancers. This study examined the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its oncogenic activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines. Patient-derived osteosarcoma samples were also examined for MTDH expression using immunohistochemistry. In parallel experiments we used MTDH-specific shRNA to down-regulate endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using migration assays and matrigel invasion assays. We also developed an osteosarcoma orthotopic xenograft model of NOD/SCID/IL2Rγ-deficient mice through intratibial injection of osteosarcoma cells with or without MTDH knockdown to study the relationship between MTDH expression and pulmonary metastatic potential of osteosarcoma cells. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed elevated MTDH expression as shown by western blot, qPCR and flow cytometry analyses. MTDH knockdown reduced migration and decreased invasion in matrigel. Inhibition of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness in vitro. In an orthotopic xenograft mouse model using CCHD cells, inhibition of MTDH delayed primary tumor growth and prohibited pulmonary metastasis. All mice in the control group developed lung metastases while fewer than 20% of the mice in the MTDH-knockdown group showed sign of metastasis at six weeks after inoculation. MTDH knockdown also reduced mean lung weight and mean number of pulmonary metastasis. Immunohistochemical analysis of tumor samples showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether these studies suggested that MTDH plays a critical role in promoting pulmonary metastasis of osteosarcoma and may serve as an attractive target for therapeutic intervention. Citation Format: Limin Zhu, Adrianna S. Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates osteosarcoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B67.
- Published
- 2013
45. Abstract C96: The effect of differential expression of HES1 on the metastatic dormancy of osteosarcoma
- Author
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Yanwen Yang, Madonna McManus, and Dennis P.M. Hughes
- Subjects
Cancer Research ,education.field_of_study ,Cell growth ,Population ,Notch signaling pathway ,Cell cycle ,Biology ,Molecular biology ,Oncology ,embryonic structures ,Cancer cell ,Cancer research ,HES1 ,education ,Notch 1 ,Transcription factor - Abstract
Osteosarcoma (OS) is the most common primary bone cancer in childhood. The majority of patients diagnosed with osteosarcoma will present with concurrent micro-metastatic disease. Interestingly, micro-metastatic lesions will typically remain dormant for anywhere from 6 months to 3 years after the surgical resection of the primary site. The Notch signaling pathway, a key component in normal bone development that is implicated as a key mediator in a number of various cancers, is initiated when membrane-bound ligands (Jagged 1 or 2, Delta-like 1, 3, or 4) on the surface of a cell interact with a membrane-bound Notch receptor (Notch 1, 2, 3, or 4) on another cell. This interaction induces the release of the intracellular domain of Notch (ICN). Once activated, ICN enters the nucleus where it forms a transcriptional complex with Mastermind-like 1 (MAM1) and promotes transcription of a number of CSL target genes including Hairy/Enhancer of Split 1 (HES1). The effects of Notch1 and HES1 on OS are still not well understood. To alter Notch pathway member expression, OS cells (HOS, CCHD, CCHO) were transduced with GFP labeled retroviral constructs containing ICN1, the constitutively active intracellular domain of the Notch1 receptor, HES1, a constitutively active form of the transcription factor and downstream target of Notch activation, dnMAM, a truncated form of MAM that inhibits ICN induced transcription by binding to ICN1 but not CSL, and dnHES1, a dominant negative form of HES1 that does not allow HES1 to bind to its downstream targets. Cell proliferation was characterized using colony formation and competitive proliferation assays. Colony formation assays measure the ability for a cancer cell to produce a colony after any given treatment, while the competitive proliferation assay monitors GFP positivity in a mixed population by flow cytometry over a period of up to 50 days to compare changes in positive and negative populations. PARP cleavage and caspase activity were monitored to determine if changes in proliferation were due to apoptotic death. Transduction of ICN1 and HES1 in HOS, CCHD, and CCHO incurs a proliferative disadvantage relative to non-transduced cells and cells transduced with dnMAM and dnHES1. HOS cells transduced with HES1 differentiate into two distinct populations: intermediate positive HES1 (HES1++) and high positive HES1 (HES1+++). HES1+++ expression increases caspase activity within 48 hours after transduction, while HES1++ did not incur an observed effect on the cell cycle throughout the 40 days of the experiment. Interestingly, however, when HES1++ cells are sorted from the GFP negative population, they fail to form colonies. This suggests that the GFP negative HOS population may provide growth stimulating signals to HES1++ HOS cells which are necessary for proliferation. This presents a potential mechanism that may explain clinical metastatic dormancy; as cells detach from the primary tumor, they are no longer exposed to the stimulatory effect of the tumor population, and are able to remain dormant and resistant to chemotherapeutics. Citation Format: Madonna McManus, Yanwen Yang, Dennis Hughes. The effect of differential expression of HES1 on the metastatic dormancy of osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C96.
- Published
- 2013
46. Erratum: Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness
- Author
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Patrick A. Zweidler-McKay, Dennis P.M. Hughes, Yanwen Yang, Pingyu Zhang, and Riitta Nolo
- Subjects
Genetics ,Cancer Research ,Oncogene ,Cancer research ,medicine ,Notch signaling pathway ,Reciprocal inhibition ,Osteosarcoma ,Biology ,HES1 ,medicine.disease ,Molecular Biology - Published
- 2012
47. Erratum
- Author
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Bruce Blumberg, Dennis P.M. Hughes, Michelle M. Tabb, Laurence H. Baker, Thomas J. Giordano, James M. Rae, Dafydd G. Thomas, Michelle L. Lizyness, Paul F. Hollenberg, and Jose M. Larios
- Subjects
Cancer Research ,Pregnane X receptor ,Oncology ,Cell culture ,business.industry ,Cancer research ,medicine ,Cancer ,Osteosarcoma ,Drug resistance ,Pharmacology ,medicine.disease ,business - Published
- 2012
48. Abstract 3239: The Notch pathway as a new target in gastrointestinal stromal tumors
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Jonathan C. Trent, Dennis P.M. Hughes, and A. G. Dumont
- Subjects
Cancer Research ,Stromal cell ,GiST ,medicine.drug_class ,Kinase ,Histone deacetylase inhibitor ,Notch signaling pathway ,Biology ,Oncology ,Immunology ,medicine ,Cancer research ,Progenitor cell ,HES1 ,Tyrosine kinase - Abstract
Small molecule kinase inhibitors have dramatically improved the survival of patients with Gastrointestinal Stromal Tumors (GIST), yet most patients develop resistance, urging us to understand better the molecular mechanisms underlying this disease and identify new therapeutic strategies. Notch signaling is a conserved developmental pathway known to play a critical role in the development of several cancers, functioning as a tumor promoter or a tumor suppressor. Given that the normal progenitor cell for GIST, the Interstitial Cell of Cajal, has characteristics similar to those of cells of neuroendocrine origin, we hypothesized that Notch pathway impacts the biology of GIST cells. In a previous study, we demonstrated that constitutively active intracellular domain of Notch1 (ICN-1) induced growth arrest in GIST cell lines. Here we report that ICN-1 downregulated KIT protein by a flow cytometric analysis in the three cell lines analyzed (GIST-T1, GIST882, GIST48IM). In parallel, inhibitions of Hes1, a major target gene of ICN-1, with a dominant negative construct, upregulated KIT protein expression in vitro (60% increase on western blot). This anti-tumor effect of Notch1 in GIST leads to a therapeutic opportunity as we showed in the present study that treatment with the histone deacetylase inhibitor, SAHA, caused a dose-dependent upregulation of Notch1 (6 fold increase of the mRNA with SAHA 2 µm/L on GIST-T1, P80 months for the high group; P=0.005). These results identify a novel anti-tumor effect of Notch1 via a crosstalk between the Notch1 and KIT pathways in GIST cells. This finding may lead to new therapeutic strategies for GIST patients, in particular after failure of tyrosine kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3239. doi:1538-7445.AM2012-3239
- Published
- 2012
49. Abstract 2876: Farnesyltransferase inhibitor induces p53-dependent cellular senescence in colon cancer cells
- Author
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Dennis P.M. Hughes, Jung Hyun Noh, Mandy Geryk-Hall, and Limin Zhu
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Cancer Research ,Cell cycle checkpoint ,Cell ,Farnesyltransferase inhibitor ,Biology ,Cell morphology ,medicine.anatomical_structure ,Oncology ,Prenylation ,Immunology ,Cancer research ,medicine ,Cytotoxic T cell ,Tipifarnib ,Viability assay ,medicine.drug - Abstract
Farnesyltransferase inhibitors (FTIs), small molecules that inhibit prenylation of multiple proteins such as Ras and Rho, have clinical promise for cancer. FTIs block malignant growth and promote cell death in a wide variety of tumor types in vitro and in vivo, but the mechanisms underlying the antitumor activity of FTIs are not completely understood. We have observed that colon cancer cell line OS187 responds to the FTI tipifarnib with a senescent phenotype, demonstrated as altered cell morphology, elevated senescence-associated β-galactosidase activity, upregulated protein and mRNA levels of senescence markers, and long-term cell cycle arrest. Our previous results also indicated that cells retaining intact p53 are more sensitive to tipifarnib than are cells with absent/inactive p53, suggesting a possible role for p53 in the senescent response to tipifarnib. To test this hypothesis, we used shRNA to reduce p53 expression in OS187 cells. Loss of p53 reversed the swollen and enlarged senescent-like cellular phenotype induced by tipifarnib. In addition, p53 knockdown cells showed less senescence-associated β-galactosidase activity than did scrambled control cells after 7-day treatment with tipifarnib. Cell viability assay suggested that p53 knockdown partially rescued the cytotoxic effects of tipifarnib at low concentrations. Western blot also demonstrated that cell cycle regulatory proteins such as p27, p21, and p15 were significantly upregulated in scrambled control cells while only a slight increase of these proteins were observed in p53 knockdown cells after tipifarnib treatment. Flow cytometry analysis showed that cells with decreased p53 level had a reduced percentage of cells in sub G1 phase when treated with a high concentration of tipifarnib, and fewer cells arresting in G2/M phase when treated with low concentration of tipifarnib compared to the scrambled control cells. This observation suggested that p53 may involve in different pathways depending on the strength of the stress signal imposed by different concentration of tipifarnib. In conclusion, these results confirm that p53 is important for inducing senescence when farnesylation is inhibited in OS187 cells. However, since p53 knockdown has very limited impact on the anti-proliferation effects of tipifarnib at high concentrations, other pathways are expected to play a role in mediating cell susceptibility to tipifarnib. Future experiments will focus on investigating the p53-independent pathways that are responsible for tipifarnib-induced senescence and apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2876. doi:1538-7445.AM2012-2876
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- 2012
50. Small Molecule Inhibitors of HER2 Inhibit Proliferative Signaling and Promote Apoptosis in Ph+ ALL
- Author
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Laura D. Nelson, Shulin Li, Janice M. Santiago-O'Farrill, Joya Chandra, Doris R. Siwak, Dennis P.M. Hughes, Mary E. Irwin, Claudia P. Miller, and Gordon B. Mills
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Cell signaling ,Canertinib ,Kinase ,Immunology ,Cell ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Receptor tyrosine kinase ,Cell biology ,chemistry.chemical_compound ,medicine.anatomical_structure ,Imatinib mesylate ,chemistry ,Ribosomal protein s6 ,medicine ,biology.protein ,Signal transduction ,skin and connective tissue diseases - Abstract
Abstract 1397 The ERBB family of receptor tyrosine kinases (EGFR, Her-2, Her-3 and Her-4) are receptor tyrosine kinases that, through mutation or aberrant expression, serve as oncogenes by promoting hallmark behaviors of cancer in many solid tumors. Previous work has suggested that HER2 is expressed in as much as 30% of B-ALL patients, and correlates with chemoresistance. We therefore hypothesized that HER2 signaling in Ph+ ALL may augment growth signaling and promote other malignant behaviors, such as resistance to cell death and independence from growth factors. Western blot and flow cytometric analyses of two human Ph+ ALL cell lines, Z119 and Z181, revealed cell surface expression of HER2, but not other family members. To determine the role of HER2 signaling in Ph+ ALL cell lines, the pan-HER family small molecule kinase inhibitor canertinib was used, and reverse phase protein array (RPPA) was conducted in Z119 and Z181 cell lines. Briefly, lysates from canertinib treated cells were spotted using a GeneTAC™ G3 arrayer onto nitrocellulose-coated FAST® slides. Incubation of the slides was performed with forty-three antibodies directed towards various cell signaling proteins followed by colorimetric detection and results were subsequently validated by western blotting. RPPA analyses revealed that treatment with canertinib effectively diminished HER2 phosphorylation in both cell lines. Additionally, we found decreased phosphorylation of the pro-survival molecules ribosomal protein S6, p70S6kinase, and c-Src, as well as increased expression of the pro-apoptotic molecules BIM and cleaved-PARP in both Ph+ ALL cell lines. Congruent with these findings, elevated activity of the executioner caspase 3 and increased DNA fragmentation, two distinct biochemical markers of apoptosis, were present after canertinib treatment in Z181 and Z119 cells, suggesting that inhibition of HER2 signaling results in programmed cell death of Ph+ ALL cell lines. This induction of apoptosis paralleled a decrease in overall proliferation of these cell lines, further implicating HER2 signaling in proliferation of Ph+ ALL. Next, we analyzed if clinically approved inhibitors of HER2 function could be utilized to produce the same biological consequence as canertinib in Ph+ ALL cell lines. Lapatinib (Tykerb) is a dual EGFR/HER2 small molecule kinase inhibitor approved by the FDA for the treatment of breast cancer. Consistent with our results utilizing canertinib, lapatinib was capable of inhibiting proliferation of both Z119 and Z181 cell lines. Interestingly, the FDA approved monoclonal antibody HER2 inhibitor trastuzumab (Herceptin) did not inhibit proliferation of these cell lines. Similarly, trimerized herceptin conjugates, which improve internalization of HER2 receptor, also had no effect on Ph+ ALL cell line proliferation. These results highlight an important distinction between the effects of the intracellular small molecule inhibitors of HER2 and monoclonal HER2 antibodies. In particular, extracellular engagement of the HER2 receptor by monoclonal antibodies may not be effective in targeting the HER2 signaling pathways required for proliferation and survival of Ph+ ALL. Taken together, our studies suggest that HER2 may play an important role in growth and survival signaling of Ph+ ALL cell lines and inhibition of HER2 with small molecule kinase inhibitors may improve treatment regimens. Thus, additional studies are warranted to determine the importance of HER2 in clinical specimens and the potential benefit of combining HER2 inhibitor therapy with imatinib treatment for Ph+ ALL. Disclosures: Mills: Glaxosmithkline: Research Funding; Pfizer: Research Funding.
- Published
- 2011
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