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1.
J Biol Chem ; 289(38): 26533-26541, 2014 Sep 19.
Article in English | MEDLINE | ID: mdl-25104357

ABSTRACT

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.


Subject(s)
Protein Processing, Post-Translational , Pyruvate Dehydrogenase (Lipoamide)/metabolism , 3T3 Cells , Amino Acid Substitution , Animals , Carbohydrate Metabolism , Catalytic Domain , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation , Epidermal Growth Factor/physiology , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Oxidative Phosphorylation , Phosphorylation , Protein Binding , Pyruvate Dehydrogenase (Lipoamide)/chemistry , Pyruvate Dehydrogenase (Lipoamide)/genetics , Pyruvic Acid/chemistry , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Tumor Burden , Tyrosine/metabolism
2.
J Biol Chem ; 289(31): 21413-22, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24962578

ABSTRACT

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.


Subject(s)
Cell Division , Neoplasms/pathology , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/antagonists & inhibitors , Tyrosine/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line, Tumor , DNA Primers , Humans , Lactic Acid/metabolism , Molecular Sequence Data , Neoplasms/enzymology , Oxygen Consumption , Phosphorylation , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/chemistry , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/genetics , Receptor, Fibroblast Growth Factor, Type 1/physiology , Sequence Homology, Amino Acid
3.
Mol Cell ; 53(4): 534-48, 2014 Feb 20.
Article in English | MEDLINE | ID: mdl-24486017

ABSTRACT

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here, we report that lysine acetylation of PDHA1 and PDP1 is common in epidermal growth factor (EGF)-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1, and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important in promoting glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct posttranslational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.


Subject(s)
Acetyl-CoA C-Acetyltransferase/metabolism , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/metabolism , Pyruvate Dehydrogenase (Lipoamide)/metabolism , Sirtuin 3/metabolism , Tyrosine/chemistry , Animals , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Glycolysis , Humans , Lysine/chemistry , Male , Mice , Mice, Nude , Mitochondria/metabolism , Neoplasm Transplantation , Neoplasms/metabolism , Phosphorylation
4.
PLoS One ; 8(1): e54499, 2013.
Article in English | MEDLINE | ID: mdl-23349909

ABSTRACT

Considerable evidence has demonstrated that transforming growth factor ß (TGF-ß) plays a key role in hepatic fibrosis, the final common pathway for a variety of chronic liver diseases leading to liver insufficiency. Although a few studies have reported that blocking TGF-ß with soluble receptors or siRNA can prevent the progression of hepatic fibrosis, as yet no evidence has been provided that TGF-ß antagonism can improve pre-existing hepatic fibrosis. The aim of this study was to examine the effects of a murine neutralizing TGF-ß monoclonal antibody (1D11), in a rat model of thioacetamide (TAA)-induced hepatic fibrosis. TAA administration for 8 weeks induced extensive hepatic fibrosis, whereupon 1D11 dosing was initiated and maintained for 8 additional weeks. Comparing the extent of fibrosis at two time points, pre- and post-1D11 dosing, we observed a profound regression of tissue injury and fibrosis upon treatment, as reflected by a reduction of collagen deposition to a level significantly less than that observed before 1D11 dosing. Hepatic TGF-ß1 mRNA, tissue hydroxyproline, and plasminogen activator inhibitor 1 (PAI-1) levels were significantly elevated at the end of the 8 week TAA treatment. Vehicle and antibody control groups demonstrated progressive injury through 16 weeks, whereas those animals treated for 8 weeks with 1D11 showed striking improvement in histologic and molecular endpoints. During the course of tissue injury, TAA also induced cholangiocarcinomas. At the end of study, the number and area of cholangiocarcinomas were significantly diminished in rats receiving 1D11 as compared to control groups, presumably by the marked reduction of supporting fibrosis/stroma. The present study demonstrates that 1D11 can reverse pre-existing hepatic fibrosis induced by extended dosing of TAA. The regression of fibrosis was accompanied by a marked reduction in concomitantly developed cholangiocarcinomas. These data provide evidence that therapeutic dosing of a TGF-ß antagonist can diminish and potentially reverse hepatic fibrosis and also reduce the number and size of attendant cholangiocarcinomas.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/administration & dosage , Cholangiocarcinoma/metabolism , Liver Cirrhosis/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cholangiocarcinoma/drug therapy , Cholangiocarcinoma/immunology , Humans , Liver/metabolism , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Liver Cirrhosis/immunology , Male , Mice , Molecular Targeted Therapy , Plasminogen Activator Inhibitor 1/metabolism , Rats , Signal Transduction , Thioacetamide/toxicity , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/immunology
5.
Cancer Res ; 72(18): 4840-5, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22787119

ABSTRACT

The TGF-ß pathway is under active consideration as a cancer drug target based on its capacity to promote cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-ß inhibitors remains uncertain as genetic studies show a tumor suppressor function of TGF-ß in pancreatic cancer and other epithelial malignancies. Here, we used genetically engineered mouse models to investigate the therapeutic impact of global TGF-ß inhibition in pancreatic cancer in relation to tumor stage, genetic profile, and concurrent chemotherapy. We found that αvß6 integrin acted as a key upstream activator of TGF-ß in evolving pancreatic cancers. In addition, TGF-ß or αvß6 blockade increased tumor cell proliferation and accelerated both early and later disease stages. These effects were dependent on the presence of Smad4, a central mediator of TGF-ß signaling. Therefore, our findings indicate that αvß6 and TGF-ß act in a common tumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression.


Subject(s)
Antigens, Neoplasm/metabolism , Integrins/metabolism , Pancreatic Neoplasms/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta/metabolism , Animals , Disease Models, Animal , Disease Progression , Immunohistochemistry , Mice
6.
Cancer Res ; 72(16): 4119-29, 2012 Aug 15.
Article in English | MEDLINE | ID: mdl-22693253

ABSTRACT

The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma-initiating cells (GIC). Other studies indicate that although GIC are sensitive, the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-ß (TGF-ß), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased by approximately 25% when treated with LY364947, a small-molecule inhibitor of TGF-ß type I receptor kinase, before irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGF-ß neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on the primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 treatment before IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGF-ß per cell compared with conventional culture, suggesting that TGF-ß production by GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGF-ß inhibitors with radiation to improve therapeutic response in patients with GBM.


Subject(s)
Brain Neoplasms/radiotherapy , Glioblastoma/radiotherapy , Pyrazoles/pharmacology , Pyrroles/pharmacology , Radiation-Sensitizing Agents/pharmacology , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Antibodies, Neutralizing/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Combined Modality Therapy , DNA Damage , DNA, Neoplasm/radiation effects , Female , Glioblastoma/drug therapy , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Mice , Mice, Inbred C57BL , Mink , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/radiation effects , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , Neural Stem Cells/radiation effects , Radiation Tolerance , Signal Transduction , Transforming Growth Factor beta/metabolism , Tumor Microenvironment
7.
Int J Cancer ; 131(11): 2584-95, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-22487809

ABSTRACT

One great challenge in our understanding of TGF-ß cancer biology and the successful application of TGF-ß-targeted therapy is that TGF-ß works as both a tumor suppressor and a tumor promoter. The underlying mechanisms for its functional change remain to be elucidated. Using 4T1 mammary tumor model that shares many characteristics with human breast cancer, particularly its ability to spontaneously metastasize to the lungs, we demonstrate that Gr-1+CD11b+ cells or myeloid derived suppressor cells are important mediators in TGF-ß regulation of mammary tumor progression. Depletion of Gr-1+CD11b+ cells diminished the antitumor effect of TGF-ß neutralization. Two mechanisms were involved: first, treatment with TGF-ß neutralization antibody (1D11) significantly decreased the number of Gr-1+CD11b+ cells in tumor tissues and premetastatic lung. This is mediated through increased Gr-1+CD11b+ cell apoptosis. In addition, 1D11 treatment significantly decreased the expression of Th2 cytokines and Arginase 1. Interestingly, the number and property of Gr-1+CD11b+ cells in peripheral blood/draining lymph nodes correlated with tumor size and metastases in response to 1D11 treatment. Our data suggest that the efficacy of TGF-ß neutralization depends on the presence of Gr-1+CD11b+ cells, and these cells could be good biomarkers for TGF-ß-targeted therapy.


Subject(s)
CD11b Antigen/immunology , Mammary Neoplasms, Experimental/immunology , Receptors, Chemokine/immunology , Transforming Growth Factor beta/immunology , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Apoptosis/genetics , Apoptosis/immunology , Arginase/genetics , Arginase/immunology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , CD11b Antigen/biosynthesis , CD11b Antigen/genetics , Cell Line, Tumor , Cytokines/genetics , Cytokines/immunology , Disease Progression , Female , Lung/immunology , Lung/pathology , Lymph Nodes/immunology , Lymph Nodes/pathology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Myeloid Cells/immunology , Myeloid Cells/pathology , Receptors, Chemokine/biosynthesis , Receptors, Chemokine/genetics , Th2 Cells/immunology , Transforming Growth Factor beta/genetics , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
8.
PLoS One ; 6(11): e27090, 2011.
Article in English | MEDLINE | ID: mdl-22096521

ABSTRACT

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFß. Because TGFß favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFß antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFß antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFß antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFß treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFß antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFß antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFß antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFß treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.


Subject(s)
Antibodies/therapeutic use , Bone Neoplasms/prevention & control , Bone and Bones/drug effects , Bone and Bones/metabolism , Breast Neoplasms/complications , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Antibodies/pharmacology , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Bone and Bones/cytology , Cell Differentiation/drug effects , Cell Line, Tumor , Collagen/metabolism , Female , Humans , Mice , Mice, Nude , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects , Osteogenesis/drug effects , Real-Time Polymerase Chain Reaction
9.
Clin Cancer Res ; 17(21): 6754-65, 2011 Nov 01.
Article in English | MEDLINE | ID: mdl-22028490

ABSTRACT

PURPOSE: To determine whether inhibition of TGFß signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: TGFß-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFß type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFß signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFß neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. RESULTS: Human breast cancer cell lines pretreated with TGFß small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFß growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFß type I receptor kinase inhibitor. Moreover, TGFß neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. CONCLUSION: These results show that TGFß inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/radiotherapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrroles/pharmacology , Radiation-Sensitizing Agents/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta1/antagonists & inhibitors , Amino Acids/pharmacology , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Growth Processes/drug effects , Cell Growth Processes/radiation effects , Cell Line, Tumor , Combined Modality Therapy , Female , Humans , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mammary Neoplasms, Experimental/radiotherapy , Mice , Mice, Inbred BALB C , Mice, SCID , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1/metabolism , Xanthenes/pharmacology , Xenograft Model Antitumor Assays
10.
PLoS One ; 6(9): e25168, 2011.
Article in English | MEDLINE | ID: mdl-21980391

ABSTRACT

BACKGROUND: DNA methyltransferase (DNMT) is one of the major factors mediating the methylation of cancer related genes such as TGF-ß receptors (TßRs). This in turn may result in a loss of sensitivity to physiologic levels of TGF-ß in aggressive prostate cancer (CaP). The specific mechanisms of DNMT's role in CaP remain undetermined. In this study, we describe the mechanism of TGF-ß-mediated DNMT in CaP and its association with clinical outcomes following radical prostatectomy. METHODOLOGY/PRINCIPAL FINDINGS: We used human CaP cell lines with varying degrees of invasive capability to describe how TGF-ß mediates the expression of DNMT in CaP, and its effects on methylation status of TGF-ß receptors and the invasive capability of CaP in vitro and in vivo. Furthermore, we determined the association between DNMT expression and clinical outcome after radical prostatectomy. We found that more aggressive CaP cells had significantly higher TGF-ß levels, increased expression of DNMT, but reduced TßRs when compared to benign prostate cells and less aggressive prostate cancer cells. Blockade of TGF-ß signaling or ERK activation (p-ERK) was associated with a dramatic decrease in the expression of DNMT, which results in a coincident increase in the expression of TßRs. Blockade of either TGF-ß signaling or DNMT dramatically decreased the invasive capabilities of CaP. Inhibition of TGF-ß in an TRAMP-C2 CaP model in C57BL/6 mice using 1D11 was associated with downregulation of DNMTs and p-ERK and impairment in tumor growth. Finally, independent of Gleason grade, increased DNMT1 expression was associated with biochemical recurrence following surgical treatment for prostate cancer. CONCLUSIONS AND SIGNIFICANCE: Our findings demonstrate that CaP derived TGF-ß may induce the expression of DNMTs in CaP which is associated with methylation of its receptors and the aggressive potential of CaP. In addition, DNMTs is an independent predictor for disease recurrence after prostatectomy, and may have clinical implications for CaP prognostication and therapy.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/physiopathology , Transforming Growth Factor beta/metabolism , Animals , Blotting, Western , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferases/genetics , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Humans , Male , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Prostatectomy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/surgery , Receptors, Transforming Growth Factor beta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacology
11.
Cell Metab ; 14(1): 67-79, 2011 Jul 06.
Article in English | MEDLINE | ID: mdl-21723505

ABSTRACT

Imbalances in glucose and energy homeostasis are at the core of the worldwide epidemic of obesity and diabetes. Here, we illustrate an important role of the TGF-ß/Smad3 signaling pathway in regulating glucose and energy homeostasis. Smad3-deficient mice are protected from diet-induced obesity and diabetes. Interestingly, the metabolic protection is accompanied by Smad3(-)(/-) white adipose tissue acquiring the bioenergetic and gene expression profile of brown fat/skeletal muscle. Smad3(-/-) adipocytes demonstrate a marked increase in mitochondrial biogenesis, with a corresponding increase in basal respiration, and Smad3 acts as a repressor of PGC-1α expression. We observe significant correlation between TGF-ß1 levels and adiposity in rodents and humans. Further, systemic blockade of TGF-ß signaling protects mice from obesity, diabetes, and hepatic steatosis. Together, these results demonstrate that TGF-ß signaling regulates glucose tolerance and energy homeostasis and suggest that modulation of TGF-ß activity might be an effective treatment strategy for obesity and diabetes.


Subject(s)
Diabetes Mellitus/prevention & control , Obesity/prevention & control , Signal Transduction , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Adipose Tissue, Brown/metabolism , Adipose Tissue, Brown/physiology , Adipose Tissue, White/metabolism , Adipose Tissue, White/physiology , Animals , Antibodies/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Diabetes Mellitus/metabolism , Energy Metabolism , Glucose Tolerance Test , Mice , Mice, Knockout , Mice, Obese , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/physiology , Obesity/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Smad3 Protein/deficiency , Smad3 Protein/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/immunology
12.
Curr Pharm Biotechnol ; 12(12): 2176-89, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21619535

ABSTRACT

The role of TGF-ß in tumor development and progression is complex. Genetic mutations that disrupt the antiproliferative signaling effects of TGF-ß play a key role in the process of malignant transformation for many types of tumors. Paradoxically, this loss of sensitivity to TGF-ß's inhibitory actions often leads to TGF-ß overexpression by the tumor cells or by normal cells that are recruited to the tumor microenvironment. Elevated concentrations of TGF-ß in the tumor microenvironment have been shown to facilitate tumor growth and metastasis. Numerous published studies have provided evidence that inhibition of TGF-ß using antibodies, soluble receptors and small molecule inhibitors of TGF-ß signal transduction can have beneficial effects in murine models of cancer. Given the pleiotropic nature of TGF-ß and its homeostatic role in numerous biological processes, serious concerns have been expressed regarding the safety of administering TGF-ß antagonists to human patients. Interestingly, the results of numerous animal toxicology studies of TGF-ß antibodies in normal rodents and primates have shown that administration of neutralizing anti-TGF-ß antibodies is well tolerated and any adverse effects were reversible or self-limiting. Likewise, administration of a human anti-TGF-ß antibody (fresolimumab) in three separate human phase 1 clinical trials has also been shown to be well tolerated.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Neoplasms/drug therapy , Transforming Growth Factor beta/immunology , Animals , Antibodies, Monoclonal/pharmacology , Disease Models, Animal , Humans
13.
Int J Oncol ; 38(1): 51-9, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21109925

ABSTRACT

Even with current standard-of-care therapies, the prognosis for patients with malignant gliomas is very poor and several new treatment modalities for glioblastomas are currently under investigation. Given the role of TGF-ß in gliomas, anti-TGF-ß strategies against gliomas are currently being investigated. Biodistribution of intravenously injected AF680-labeled 1D11, a pan-neutralizing TGF-ß antibody, was monitored in mice bearing either subcutaneous or orthotopic gliomas using in vivo imaging and fluorescence microscopy. AF680-labeled 1D11 entered both the subcutaneous and intracranial tumors and the antibody was detected within the tumor tissue for several days whereas only low fluorescence was found in organs. The effects of 1D11 on subcutaneous versus orthotopic U87MG and GL261 gliomas in immunocompetent C57BL/6J versus immunodeficient CD1-Foxn1nu mice were observed by direct tumor size measurement, H&E staining and immunohistochemistry. Treatment of immunocompetent mice bearing subcutaneous GL261 tumors with 1D11 resulted in complete remission. In immune deficient mice, the growth of subcutaneous GL261 tumors was increased following treatment with 1D11. Intracranially implanted gliomas in C57Bl/6J mice showed no size reduction after 1D11 treatment but there was reduced invasion of the glioma cells into the adjacent normal brain. Together these data demonstrate that TGF-ß plays different roles in combating the tumor depending on subcutaneous versus orthotopic implantation site.


Subject(s)
Antibodies, Monoclonal/pharmacology , Brain Neoplasms/drug therapy , Glioma/drug therapy , Transforming Growth Factor beta/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Antibody Specificity , Brain Neoplasms/immunology , Brain Neoplasms/pathology , Cell Line, Tumor , Glioma/immunology , Glioma/pathology , Humans , Immunocompetence , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Nude , Microscopy, Fluorescence , Neoplasm Invasiveness , Spectroscopy, Near-Infrared , Tissue Distribution , Xenograft Model Antitumor Assays
14.
J Bone Miner Res ; 25(11): 2419-26, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20499365

ABSTRACT

Transforming growth factor ß (TGF-ß) is an abundant bone matrix protein that influences osteoblast and osteoclast interactions to control bone remodeling. As such, TGF-ß represents an obvious pharmacologic target with the potential to regulate both bone formation and resorption to improve bone volume and strength. To investigate the skeletal effect of TGF-ß inhibition in vivo, we used an antibody (1D11) specifically directed at all three isoforms of TGF-ß. Normal mice were treated with 1D11 or control antibody (4 weeks), and cortical and trabecular bone was assessed by micro-computed tomographic (µCT) scanning. Bone volume and cellular distribution were determined by histomorphometric analysis of vertebrae and long bones. Also, whole-bone strength was assessed biomechanically by three-point bend testing, and tissue-level modulus and composition were analyzed by nanoindentation and Raman microspectroscopy, respectively. TGF-ß blockade by 1D11 increased bone mineral density (BMD), trabecular thickness, and bone volume by up to 54%, accompanied by elevated osteoblast numbers and decreased osteoclasts. Biomechanical properties of bone also were enhanced significantly by 1D11 treatment, with increased bending strength and tissue-level modulus. In addition, Raman microspectroscopy demonstrated that 1D11-mediated TGF-ß inhibition in the bone environment led to an 11% increase in the mineral-to-collagen ratio of trabecular bone. Together these studies demonstrate that neutralizing TGF-ß with 1D11 increases osteoblast numbers while simultaneously decreasing active osteoclasts in the marrow, resulting in a profound increase in bone volume and quality, similar to that seen in parathyroid hormone (PTH)-treated rodent studies.


Subject(s)
Antibodies/pharmacology , Bone and Bones/drug effects , Bone and Bones/pathology , Signal Transduction/drug effects , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolism , Animals , Biomechanical Phenomena/drug effects , Male , Mice , Mice, Inbred C57BL , Organ Size/drug effects
15.
Mol Cancer ; 9: 122, 2010 May 26.
Article in English | MEDLINE | ID: mdl-20504320

ABSTRACT

BACKGROUND: Transforming Growth Factor beta (TGF-beta) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-beta antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-beta pathway antagonists (1D11, a mouse monoclonal pan-TGF-beta neutralizing antibody and LY2109761, a chemical inhibitor of TGF-beta type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR). RESULTS: Both 1D11 and LY2109761 effectively blocked TGF-beta-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones in vitro. Moreover, both antagonists inhibited TGF-beta stimulated in vitro migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-beta. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones in vivo, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-beta antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer. CONCLUSIONS: In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Pyrazoles/pharmacology , Pyrroles/pharmacology , Signal Transduction/drug effects , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Female , Humans , Mice , Mice, Nude , Neoplasm Metastasis/drug therapy , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Xenograft Model Antitumor Assays
16.
Cell Stem Cell ; 6(2): 130-40, 2010 Feb 05.
Article in English | MEDLINE | ID: mdl-20144786

ABSTRACT

Melanocyte stem cells in the bulge area of hair follicles are responsible for hair pigmentation, and defects in them cause hair graying. Here we describe the process of melanocyte stem cell entry into the quiescent state and show that niche-derived transforming growth factor beta (TGF-beta) signaling plays important roles in this process. In vitro, TGF-beta not only induces reversible cell cycle arrest, but also promotes melanocyte immaturity by downregulating MITF, the master transcriptional regulator of melanocyte differentiation, and its downstream melanogenic genes. In vivo, TGF-beta signaling is activated in melanocyte stem cells when they reenter the quiescent noncycling state during the hair cycle and this process requires Bcl2 for cell survival. Furthermore, targeted TGF-beta type II receptor (TGFbRII) deficiency in the melanocyte lineage causes incomplete maintenance of melanocyte stem cell immaturity and results in mild hair graying. These data demonstrate that the TGF-beta signaling pathway is one of the key niche factors that regulate melanocyte stem cell immaturity and quiescence.


Subject(s)
Melanocytes/metabolism , Signal Transduction , Stem Cells/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta2/metabolism , Transforming Growth Factor beta3/metabolism , Animals , Cell Cycle , Cell Differentiation , Cell Lineage , Cells, Cultured , Humans , Melanocytes/cytology , Melanocytes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/deficiency , Receptors, Transforming Growth Factor beta/immunology , Receptors, Transforming Growth Factor beta/metabolism , Stem Cells/cytology , Stem Cells/immunology , Transforming Growth Factor beta1/immunology , Transforming Growth Factor beta2/immunology , Transforming Growth Factor beta3/immunology
17.
Int J Cancer ; 126(7): 1666-74, 2010 Apr 01.
Article in English | MEDLINE | ID: mdl-19830696

ABSTRACT

Though TGF-beta inhibition enhances antitumor immunity mediated by CD8(+) T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF-beta blockade, we tested the effect of anti-TGF-beta combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti-TGF-beta antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-beta antibodies compared to vaccinated mice without anti-TGF-beta, suggesting that anti-TGF-beta synergistically enhanced irradiated tumor vaccine efficacy. CD8(+) T-cell depletion completely abrogated the vaccine efficacy, and so protection required CD8(+) T cells. Depletion of CD25(+) T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-beta did not change the number of CD25(+) T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-beta production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-beta enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-beta levels found in patients with cancer and that the effect is not dependent on TGF-beta solely from CD4(+)CD25(+) T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/therapeutic use , Colonic Neoplasms/prevention & control , T-Lymphocytes, Regulatory/immunology , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Cancer Vaccines/immunology , Cell Line, Tumor , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Female , Flow Cytometry , Interleukin-13/physiology , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-4/physiology , Mice , Mice, Inbred BALB C , Mice, Knockout , Natural Killer T-Cells/metabolism , Receptors, Interleukin-4/metabolism , STAT6 Transcription Factor/metabolism , Signal Transduction , Survival Rate , Vaccination
18.
Clin Cancer Res ; 15(21): 6560-9, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19861451

ABSTRACT

PURPOSE: Transforming growth factor-beta (TGF-beta) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4(+) and CD8(+)T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti-TGF-beta (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy. EXPERIMENTAL DESIGN: The effect of combination treatment with a peptide vaccine and anti-TGF-beta was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment. RESULTS: Anti-TGF-beta significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti-TGF-beta alone had no impact. The number of tumor antigen-specific CTL with high functional avidity as measured by IFN-gamma production and lytic activity was significantly increased in vaccinated mice by TGF-beta neutralization. Although TGF-beta is known to play a critical role in CD4(+)Foxp3(+) Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti-TGF-beta did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-beta neutralization had no effect on interleukin-17-producing T cells, which are induced by TGF-beta and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-beta, was not sufficient to eliminate all sources of suppressive TGF-beta. Finally, the synergistic protection induced by anti-TGF-beta vaccine augmentation was mediated by CD8(+) T cells since anti-CD8 treatment completely abrogated the effect. CONCLUSIONS: These results suggest that TGF-beta blockade may be useful for enhancing cancer vaccine efficacy.


Subject(s)
Antibodies, Monoclonal/therapeutic use , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/therapeutic use , T-Lymphocytes, Cytotoxic/immunology , Transforming Growth Factor beta/immunology , Vaccines, Subunit/therapeutic use , Animals , Cell Line, Tumor , Immunosuppression Therapy , Mice , Mice, Inbred C57BL
19.
Clin Cancer Res ; 15(21): 6551-9, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19861464

ABSTRACT

PURPOSE: A variety of cancers, including malignant gliomas, overexpress transforming growth factor-beta (TGF-beta), which helps tumors evade effective immune surveillance through a variety of mechanisms, including inhibition of CD8(+) CTLs and enhancing the generation of regulatory T (T(reg)) cells. We hypothesized that inhibition of TGF-beta would improve the efficacy of vaccines targeting glioma-associated antigen (GAA)-derived CTL epitopes by reversal of immunosuppression. EXPERIMENTAL DESIGN: Mice bearing orthotopic GL261 gliomas were treated systemically with a TGF-beta-neutralizing monoclonal antibody, 1D11, with or without s.c. vaccinations of synthetic peptides for GAA-derived CTL epitopes, GARC-1 (77-85) and EphA2 (671-679), emulsified in incomplete Freund's adjuvant. RESULTS: Mice receiving the combination regimen exhibited significantly prolonged survival compared with mice receiving either 1D11 alone, GAA vaccines alone, or mock treatments alone. TGF-beta neutralization enhanced the systemic induction of antigen-specific CTLs in glioma-bearing mice. Flow cytometric analyses of brain-infiltrating lymphocytes revealed that 1D11 treatment suppressed phosphorylation of Smad2, increased GAA-reactive/IFN-gamma-producing CD8(+) T cells, and reduced CD4(+)/FoxP3(+) T(reg) cells in the glioma microenvironment. Neutralization of TGF-beta also upregulated plasma levels of interleukin-12, macrophage inflammatory protein-1 alpha, and IFN-inducible protein-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment upregulated plasma interleukin-15 levels and promoted the persistence of GAA-reactive CD8(+) T cells in glioma-bearing mice. CONCLUSIONS: These data suggest that systemic inhibition of TGF-beta by 1D11 can reverse the suppressive immunologic environment of orthotopic tumor-bearing mice both systemically and locally, thereby enhancing the therapeutic efficacy of GAA vaccines.


Subject(s)
Antibodies, Monoclonal/pharmacology , Brain Neoplasms/immunology , Brain Neoplasms/therapy , Glioma/immunology , Glioma/therapy , Transforming Growth Factor beta/immunology , Vaccines, Subunit/therapeutic use , Animals , Antibodies, Neutralizing/immunology , Antigens, Neoplasm , Cancer Vaccines , Cell Line, Tumor , Epitopes, T-Lymphocyte , Male , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Regulatory/immunology
20.
Am J Pathol ; 174(6): 2137-49, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19406991

ABSTRACT

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor; its overexpression has been implicated in many diseases, making it a desirable target for therapeutic neutralization. In initial safety studies, mice were chronically treated (three times per week) with high doses (50 mg/kg) of a murine, pan-neutralizing, anti-TGF-beta antibody. Nine weeks after the initiation of treatment, a subset of mice exhibited weight loss that was concurrent with decreased food intake. Histopathology revealed a unique, nonneoplastic cystic epithelial hyperplasia and tongue inflammation, as well as dental dysplasia and epithelial hyperplasia and inflammation of both the gingiva and esophagus. In an effort to determine the cause of this site-specific pathology, we examined TGF-beta expression in these tissues and saliva under normal conditions. By immunostaining, we found higher expression levels of active TGF-beta1 and TGF-beta3 in normal tongue and esophageal submucosa compared with gut mucosal tissues, as well as detectable TGF-beta1 in normal saliva by Western blot analysis. Interestingly, mast cells within the tongue, esophagus, and skin co-localized predominantly with the TGF-beta1 expressed in these tissues. Our findings demonstrate a novel and restricted pathology in oral and esophageal tissues of mice chronically treated with anti-TGF-beta that is associated with basal TGF-beta expression in saliva and by mast cells within these tissues. These studies illustrate a previously unappreciated biological role of TGF-beta in maintaining homeostasis within both oral and esophageal tissues.


Subject(s)
Esophagus/metabolism , Homeostasis/physiology , Mast Cells/metabolism , Mouth/metabolism , Transforming Growth Factor beta/metabolism , Animals , Blotting, Western , Esophagus/immunology , Esophagus/pathology , Female , Image Processing, Computer-Assisted , Immunohistochemistry , Mice , Mice, Inbred BALB C , Mice, Knockout , Mouth/immunology , Mouth/pathology , Saliva/chemistry , Saliva/immunology
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