ABSTRACT
Gain-of-function point mutations in the receptor tyrosine kinase RET, a driver oncogene in medullary thyroid carcinoma (MTC), prevent apoptosis through inhibition of ATF4, a critical transcriptional regulator of endoplasmic reticulum stress. However, the critical regulatory mechanisms driving RET-dependent oncogenesis remain elusive, and there is a clinical need to identify a transcriptional RET inhibitor. Here, we found that RET depletion decreased IGFBP2 and VEGFR2 mRNA and protein expression in MTC cells. IGFBP2 knockdown decreased cell survival and migration of MTC cells. In patients, IGFBP2 expression increased in metastatic MTC, and high IGFBP2 associated with poor overall survival. VEGFR2 protein levels were positively associated with RET expression in primary tumors, and VEGF-mediated increased cell viability was RET dependent. The small-molecule ONC201 treatment of MTC cells caused apoptotic cell death, decreased transcription of RET, VEGFR2, IGFBP2, increased mRNA levels of ATF4, and ATF4 target genes including DDIT3, BBC3, DUSP8, MKNK2, KLF9, LZTFL1, and SESN2 Moreover, IGFBP2 depletion increased ONC201-induced cell death. ONC201 inhibited tumor growth at a well-tolerated dose of 120 mg/kg/week administered by oral gavage and decreased MTC xenograft cell proliferation and angiogenesis. The protein levels of RET, IGFBP2, and VEGFR2 were decreased in ONC201-treated xenografts. Our study uncovered a novel ONC201 mechanism of action through regulation of RET and its targets, VEGFR2 and IGFBP2; this mechanism could be translated into the clinic and represent a promising strategy for the treatment of all patients with MTC, including those with TKI-refractory disease and other cancer with RET abnormalities.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Neuroendocrine/drug therapy , Imidazoles/therapeutic use , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-ret/antagonists & inhibitors , Pyridines/therapeutic use , Pyrimidines/therapeutic use , Thyroid Neoplasms/drug therapy , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Humans , Imidazoles/pharmacology , Male , Mice , Mice, Inbred NOD , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Pyrimidines/pharmacology , Retrospective StudiesABSTRACT
The brain is often reported as the first site of recurrence among breast cancer patients overexpressing human epidermal growth factor receptor 2 (HER2). Although most HER2+tumors metastasize to the subcortical region of the brain, a subset develops in the cortical region. We hypothesize that factors in cerebrospinal fluid (CSF) play a critical role in the adaptation, proliferation, and establishment of cortical metastases. We established novel cell lines using patient biopsies to model breast cancer cortical and subcortical metastases. We assessed the localization and growth of these cells in vivo and proliferation and apoptosis in vitro under various conditions. Proteomic analysis of human CSF identified astrocyte-derived factors that support the proliferation of cortical metastases, and we used neutralizing antibodies to test the effects of inhibiting these factors both in vivo and in vitro. The cortical breast cancer brain metastatic cells exhibited greater proliferation than subcortical breast cancer brain metastatic cells in CSF containing several growth factors that nourish both the CNS and tumor cells. Specifically, the astrocytic paracrine factors IGFBP2 and CHI3LI promoted the proliferation of cortical metastatic cells and the formation of metastatic lesions. Disruption of these factors suppressed astrocyte-tumor cell interactions in vitro and the growth of cortical tumors in vivo. Our findings suggest that inhibition of IGFBP2 and CHI3LI signaling, in addition to existing treatment modalities, may be an effective therapeutic strategy targeting breast cancer cortical metastasis.
Subject(s)
Astrocytes/pathology , Brain Neoplasms/secondary , Breast Neoplasms/pathology , Cerebrospinal Fluid/cytology , Chitinase-3-Like Protein 1/metabolism , Insulin-Like Growth Factor Binding Protein 2/metabolism , Animals , Apoptosis/drug effects , Brain Neoplasms/cerebrospinal fluid , Brain Neoplasms/drug therapy , Breast Neoplasms/cerebrospinal fluid , Cell Proliferation/drug effects , Cerebral Cortex/pathology , Chitinase-3-Like Protein 1/antagonists & inhibitors , Coculture Techniques , Female , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Mice , Paracrine Communication , Primary Cell Culture , Proteomics , Receptor, ErbB-2/metabolism , Signal Transduction/drug effects , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Adeno-associated virus 2 (AAV2) is prepotent in the biological treatment of breast tumor because of its low pathogenicity and immunogenicity. Our previous study demonstrated that insulinlike growth factorbinding protein 2 (IGFBP2) was highly expressed in patients with breast metastasis. In the present study, the effects of recombinant AAV2 on the growth and metastasis of breast cancer cells were determined in vitro, and in vivo. rAAV2-ZsGreen-shRNA-scramble and rAAV2ZsGreenshRNAhIGFBP2 were used to transfect MDAMB468, and MCF10A cells respectively, and observed that these virus could not penetrate the normal human breast epithelia MCF10A cell line. To investigate the effect of the recombinant virus on chemotherapeutics, paclitaxel was added to MDAMB468 cells and it was demonstrated that rAAV2ZsGreenshRNAhIGFBP-2-infected MDA-MB-468 cells were highly chemosensitive to paclitaxel compared with rAAV2ZsGreenshRNAscrambleinjected cells. In addition, it was demonstrated that the invasive ability of rAAV2ZsGreenshRNAhIGFBP2infected MDA-MB-468 cells was highly impaired compared with the rAAV2ZsGreenshRNAscramble group. In the nude mice xenografts, the rAAV2ZsGreenshRNAhIGFBP2 injection inhibited tumor growth and Ki67 expression was significantly downregulated compared with the scramble group. Following IGFBP2 knockdown using rAAV2-ZsGreen-shRNA-hIGFBP2, matrix metalloproteinase2 expression was significantly reduced in tumor tissues compared with that in rAAV2ZsGreenshRNAscramble treated tumor tissues. These findings have provided a direction for the application of novel AAV2based therapeutics for treating aggressive triplenegative breast cancer types.
Subject(s)
Insulin-Like Growth Factor Binding Protein 2/metabolism , RNA, Small Interfering/metabolism , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Dependovirus/genetics , Down-Regulation , Female , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/genetics , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , RNA Interference , RNA, Small Interfering/therapeutic use , Transplantation, HeterologousABSTRACT
OBJECTIVE: Gliomas are accompanied with high mortality owning to their invasive peculiarity and vulnerability to drug resistance. miR-21 is a vital oncogenic miRNA that regulates drug resistance of tumor cells. This study aims to elucidate the function of miR-21 in human glioma cells resistant to carmustine (BCNU) and to demonstrate the underlying molecular mechanism. MATERIALS AND METHODS: BCNU-sensitive cells (SWOZ2 cells) were transfected with miR-21 agomir and negative control, and BCNU-resistance cells (SWOZ2-BCNU cells) were transfected with miR-21 antagomir and negative control. The Real-time fluorescence quantitative PCR was used to detect and compare the levels of miR-21expression between SWOZ2-BCNU and SWOZ2 cells. The drug sensitivity of these cells to BCNU was determined by Cell Counting Kit-8 (CCK-8) assay. The protein expression of Spry2 was detected by Western blotting. RESULTS: The expression level of miR-21 was remarkably higher in SWOZ2-BCNU cells than that in SWOZ2 cells. The half-maximal inhibitory concentration (IC50) of BCNU was obviously higher for SWOZ2-BCNU cells than that for SWOZ2 cells. Besides, we found that aberrant expression of miR-21 in SWOZ2-BCNU cells is responsible for glioma BCNU-resistance. Consistently, Spry2 protein levels were significantly reduced in SWOZ2-BCNU as well as in miR-21 agomir-transfected cells, inversely correlated to miR-21 expression. The results of si-Spry2 co-transfection suggested that the effect of miR-21 on glioma BCNU-resistance is mediated through Spry2. CONCLUSIONS: miR-21 enhances the resistance of human glioma cells to BCNU by decreasing the expression of Spry2 protein. Thus, Spry2 may be a novel therapeutic target for treating glioma BCNU-resistance.
Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Insulin-Like Growth Factor Binding Protein 2/metabolism , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Transcription Factor RelA/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/mortality , Cadherins/metabolism , Cell Line, Tumor , CpG Islands , Disease-Free Survival , Glioblastoma/genetics , Glioblastoma/mortality , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Kaplan-Meier Estimate , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Protein Interaction Maps , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction , Snail Family Transcription Factors/metabolism , Vimentin/metabolismABSTRACT
MicroRNAs are small noncoding RNAs that post-transcriptionally control the expression of genes involved in glioblastoma multiforme (GBM) development. Although miR-302b functions as a tumor suppressor, its role in GBM is still unclear. Therefore, this study comprehensively explored the roles of miR-302b-mediated gene networks in GBM cell death. We found that miR-302b levels were significantly higher in primary astrocytes than in GBM cell lines. miR-302b overexpression dose dependently reduced U87-MG cell viability and induced apoptosis through caspase-3 activation and poly(ADP ribose) polymerase degradation. A transcriptome microarray revealed 150 downregulated genes and 380 upregulated genes in miR-302b-overexpressing cells. Nuclear factor IA (NFIA), higher levels of which were significantly related to poor survival, was identified as a direct target gene of miR-302b and was involved in miR-302b-induced glioma cell death. Higher NFIA levels were observed in GBM cell lines and human tumor sections compared with astrocytes and non-tumor tissues, respectively. NFIA knockdown significantly enhanced apoptosis. We found high levels of insulin-like growth factor-binding protein 2 (IGFBP2), another miR-302b-downregulated gene, in patients with poor survival. We verified that NFIA binds to the IGFBP2 promoter and transcriptionally enhances IGFBP2 expression levels. We identified that NFIA-mediated IGFBP2 signaling pathways are involved in miR-302b-induced glioma cell death. The identification of a regulatory loop whereby miR-302b inhibits NFIA, leading to a decrease in expression of IGFBP-2, may provide novel directions for developing therapies to target glioblastoma tumorigenesis.
Subject(s)
Glioma/genetics , Glioma/metabolism , Insulin-Like Growth Factor Binding Protein 2/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , NFI Transcription Factors/antagonists & inhibitors , NFI Transcription Factors/genetics , Apoptosis/genetics , Apoptosis/physiology , Astrocytes/cytology , Astrocytes/metabolism , Cell Line, Tumor , Cell Survival/genetics , Down-Regulation , Gene Knockdown Techniques , Gene Regulatory Networks , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Glioma/pathology , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/metabolism , NFI Transcription Factors/metabolism , Promoter Regions, Genetic , Signal Transduction , TranscriptomeABSTRACT
IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3-6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Autophagy , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor I/metabolism , Osteoblasts/cytology , Osteogenesis , Protein Processing, Post-Translational , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/chemistry , AMP-Activated Protein Kinases/genetics , Amino Acid Substitution , Animals , Autophagy/drug effects , Cell Line , Cells, Cultured , Enzyme Activation/drug effects , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutant Proteins , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteogenesis/drug effects , Phagosomes/drug effects , Phagosomes/enzymology , Phagosomes/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , RNA InterferenceABSTRACT
Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface α5ß1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 × 10(8)) and Tomlinson J (Library size 1.37 × 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.
Subject(s)
Brain Neoplasms/prevention & control , Cell Adhesion , Cell Movement , Glioma/prevention & control , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Single-Chain Antibodies/pharmacology , Apoptosis , Blotting, Western , Brain Neoplasms/immunology , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Neoplastic , Glioma/immunology , Glioma/metabolism , Glioma/pathology , Humans , Insulin-Like Growth Factor Binding Protein 2/immunology , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Neoplasm Invasiveness , Peptide Library , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Single-Chain Antibodies/immunology , Surface Plasmon Resonance , Tumor Cells, CulturedABSTRACT
A multiantigen multipeptide vaccine, targeting proteins expressed in preinvasive breast lesions, can stimulate type I CD4(+) T cells which have been shown to be deficient in both patients with breast cancer and mice that develop mammary tumors. Transgenic mice (TgMMTV-neu) were immunized with a multiantigen peptide vaccine specific for neu, insulin-like growth factor-binding protein 2 and insulin-like growth factor receptor-I at a time when some of the animals already had preinvasive lesions (18 weeks of age). Although immunization with each individual antigen was partially effective in inhibiting tumor growth, immunization with the multiantigen vaccine was highly effective, blocking development of palpable lesions in 65% of mice and slowing tumor growth in the infrequent palpable tumors, which did arise. Protection was mediated by CD4(+) T cells, and the few slow-growing tumors that did develop demonstrated a significant increase in intratumoral CD8(+) T cells as compared with controls (P = 0.0007). We also combined the vaccine with agents that were, by themselves, partially effective inhibitors of tumor progression in this model; lapatinib and the RXR agonist bexarotene. Although the combination of lapatinib and vaccination performed similarly to vaccination alone (P = 0.735), bexarotene and vaccination significantly enhanced disease-free survival (P < 0.0001), and approximately 90% of the mice showed no pathologic evidence of carcinomas at one year. The vaccine also demonstrated significant clinical efficacy in an additional transgenic model of breast cancer (TgC3(I)-Tag). Chemoimmunoprevention combinations may be an effective approach to breast cancer prevention even when the vaccine is administered in the presence of subclinical disease.
Subject(s)
Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Mammary Neoplasms, Animal/prevention & control , Precancerous Conditions/prevention & control , Receptor, ErbB-2/antagonists & inhibitors , Receptor, IGF Type 1/antagonists & inhibitors , Vaccines, Subunit/therapeutic use , Adoptive Transfer , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bexarotene , Female , Insulin-Like Growth Factor Binding Protein 2/immunology , Lapatinib , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Mammary Neoplasms, Animal/immunology , Mammary Neoplasms, Animal/pathology , Mice , Mice, Transgenic , Neoplasm Invasiveness , Precancerous Conditions/immunology , Precancerous Conditions/pathology , Quinazolines/administration & dosage , Receptor, ErbB-2/immunology , Receptor, IGF Type 1/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Tetrahydronaphthalenes/administration & dosage , Tumor Cells, CulturedABSTRACT
In breast tumors IGF binding protein-2 (IGFBP-2) is elevated, and the presence of IGFBP-2 has been shown to correlate with malignancy. However, how IGFBP-2 contributes to the malignant state is still unclear. Silencing IGFBP-2 blocked cell proliferation and in MCF-7 cells increased cell death, indicating that IGFBP-2 was acting in both a mitogenic and a survival capacity. Exogenous IGFBP-2 acting via integrin receptors to reduce phosphatase and tensin homolog deleted from chromosome 10 (PTEN) levels protected these cells against death induced by various chemotherapeutic agents. This was dependent on a functional estrogen receptor (ER)-α because silencing ER-α blocked the ability of IGFBP-2 to confer cell survival. Loss of IGFBP-2 increased levels of PTEN and improved chemosensitivity of the cells, confirming its role as a survival factor. Silencing IGFBP-2 had no effect on the response to IGF-II, but responses to estrogen and tamoxifen were no longer observed due to loss of ER-α, which could be prevented by the inhibition of PTEN. Conversely, exogenous IGFBP-2 increased ER-α mRNA and protein in both normal and cancer cells via its interaction with integrin receptors. These actions of IGFBP-2 on ER-α involved the IGF-I receptor and activation of phosphatidylinositol 3-kinase in the cancer cells but were independent of this in normal breast cells. The production of IGFBP-2 by breast cancer cells enhances their proliferative potential, increases their survival, and protects them against chemotherapy-induced death. IGFBP-2 not only modulates IGFs and directly regulates PTEN but also has a role in maintaining ER-α expression.
Subject(s)
Estrogen Receptor alpha/genetics , Insulin-Like Growth Factor Binding Protein 2/physiology , Mammary Glands, Human/physiology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Drug Evaluation, Preclinical , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation/drug effects , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor Binding Protein 2/metabolism , Mammary Glands, Human/drug effects , Mammary Glands, Human/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , RNA, Small Interfering/pharmacologyABSTRACT
To investigate the mechanisms by which tretinoin and N-acetyl-L-cysteine (NAC) reverse the growth inhibition of alveolar epithelial cells induced by cigarette smoke extract (CSE), MTT assay was used to evaluate cell viability. It was observed that both tretinoin and NAC could restore the viability of CSE-inhibited A549 cells. By incubation with fluorescent indicator H2DCFDA, it was documented that CSE-stimulated accumulation of intracellular reactive oxygen species (ROS) was obviously decreased by tretinoin or NAC. Furthermore, using semi-quantitative and real-time quantitative RT-PCR as well as western blot methods, high expression of insulin-like growth factor binding protein-2 (IGFBP-2) in A549 cells treated with CSE was found at both transcriptional and protein levels, and concomitant with the restoration of cell growth after treatment with tretinoin or NAC, down regulation of IGFBP-2 was observed. From the present study, it is concluded that both RA and NAC can antagonize CSE-induced growth arrest of alveolar epithelial cells and that down regulation of IGFBP-2 may play an important role in the process.
Subject(s)
Acetylcysteine/pharmacology , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Free Radical Scavengers/pharmacology , Nicotiana/chemistry , Pulmonary Alveoli/cytology , Smoke/adverse effects , Tretinoin/pharmacology , Blotting, Western , Cell Survival/drug effects , Down-Regulation/drug effects , Epithelial Cells/drug effects , Humans , Indicators and Reagents , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/biosynthesis , Pulmonary Alveoli/drug effects , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: Primary pigmented nodular adrenocortical disease (PPNAD) can occur as an isolated trait or part of Carney complex, a familial lentiginosis-multiple endocrine neoplasia syndrome frequently caused by mutations in PRKAR1A, which encodes the 1alpha regulatory subunit of protein kinase A (PKA). Because alterations in the insulin-like growth factor (IGF) axis, particularly IGF-II and IGF binding protein (IGFBP)-2 overexpression, have been implicated in sporadic adrenocortical tumors, we sought to examine the IGF axis in PPNAD. DESIGN: RNA samples and paraffin-embedded sections were procured from adrenalectomy specimens of patients with PPNAD. Changes in expression of IGF axis components were evaluated by real-time quantitative RT-PCR and immunohistochemistry. NCI-H295R cells were used to study PKA and IGF axis signaling in adrenocortical cells in vitro. RESULTS: IGFBP-2 mRNA level distinguished between the two genetic subtypes of this disease; increased IGFBP-2 expression in PRKAR1A mutation-positive PPNAD tissues was also confirmed by immunohistochemistry. Moreover, PKA inhibitors increased IGFBP-2 expression in NCI-H295R adrenocortical cells, and anti-IGFBP-2 antibody reduced their proliferation. CONCLUSIONS: IGFBP-2 expression is increased in PPNAD caused by PRKAR1A mutations, and in adrenocortical cancer cells. This is the first evidence for PKA-dependent regulation of IGFBP-2 expression in adrenocortical cells.
Subject(s)
Adrenal Cortex Neoplasms/enzymology , Cyclic AMP-Dependent Protein Kinases/physiology , Insulin-Like Growth Factor Binding Protein 2/metabolism , Multiple Endocrine Neoplasia/enzymology , Adrenal Cortex Neoplasms/genetics , Cell Line , Cell Proliferation/drug effects , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/genetics , Humans , Immunohistochemistry , Insulin-Like Growth Factor Binding Protein 2/analysis , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor II/metabolism , Multiple Endocrine Neoplasia/genetics , Mutation , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/analysis , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The insulin-like growth factor binding proteins (IGFBPs) represent a unique class of IGF antagonists regulating the bioavailability of the IGFs extracellularly. Accordingly, they represent an important class of proteins for cancer therapeutics and chemoprevention. IGF-F1-1 is a cyclic hexadecapeptide identified by high throughput phage display that binds to the IGFBP-binding domain on IGF-1. It acts as an IGFBP-mimetic, capable of inhibiting IGF-1 binding to the IGFBPs. To further examine the utility of IGF-F1-1 as an IGF-1 antagonist we tested its ability to inhibit IGFBP-2 and IGFBP-3 binding to IGF-1, (125)I-IGF-1 binding to IGF-1Rs and to block IGF-1 induced Akt activation, cell cycle changes and [(3)H]thymidine incorporation in MCF-7 cells. These biological activities were inhibited by treatment with IGFBP-2, wortmannin or the IGF-1R tyrosine kinase inhibitor, NVP-AEW541, but not by IGF-F1-1. Our findings confirm previous studies indicating that IGF-F1-1 is a weak antagonist of IGF-1 binding to the IGFBPs and the IGF-1R and suggest that it does not effectively inhibit downstream events stimulated by IGF-1. We further demonstrated that IGF-F1-1 treatment of MCF-7 cells results in the paradoxical activation of Akt, S-phase transition and [(3)H]thymidine incorporation. These results suggest that IGF-F1-1 is a weak agonist, exhibiting mitogenic actions. IGF-F1-1 may act in conjunction with IGF-1 at the IGF-1R or independently of IGF-1 at the IGF-1R or another receptor.
Subject(s)
Insulin-Like Growth Factor I/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Receptor, IGF Type 1/metabolism , Signal Transduction , Androstadienes/pharmacology , Cell Cycle/physiology , Cell Line, Tumor/drug effects , DNA Replication , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor Binding Protein 2/pharmacology , Insulin-Like Growth Factor Binding Protein 3/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 3/metabolism , Insulin-Like Growth Factor I/metabolism , Peptide Library , Phosphorylation , Protein Binding/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/pharmacology , Pyrroles/pharmacology , WortmanninABSTRACT
During perinatal development, the regulation of IGF system appears to be growth hormone (GH) independent. By using highly purified primary fetal hepatocytes, we investigated the role of prolactin (PRL) in the regulation of IGF system and hepatocyte proliferation. We also analyzed the consequence of a maternal low-protein (LP) diet on the regulation of IGF, IGF-binding protein (IGFBP), and hepatocyte proliferation by prolactin. Pregnant Wistar rats were fed a control (C) diet (20% protein) or isocaloric (LP; 8%) diet throughout gestation. On day 21.5, fetal hepatocytes were cultured for 4 days and incubated with rat prolactin. In the C hepatocytes, PRL at 100 ng/ml decreased the abundance of IGFBP-1 and IGFBP-2 by 50 (P < 0.05) and 60% (P < 0.01), respectively. It also reduced by 70% the level of IGF-II mRNA (P < 0.01). By contrast, PRL failed to modulate IGFBP-1 and IGFBP-2 production by LP hepatocytes, and this was associated with reduced abundance of the short form of PRL receptor (P < 0.05). PRL had no effect on either the proliferation or the IGF-I production by C and LP hepatocytes, although it reduced the expression of IGF-II. These results suggest that prolactin influences hepatocyte proliferation in vitro by inhibiting IGFBP-1, IGFBP-2, and IGF-II levels, which may coincide with the decline of IGF-II observed in rodents during late gestation in vivo. On the other hand, maternal LP diet induces a resistance of fetal hepatocytes to PRL.
Subject(s)
Insulin-Like Growth Factor Binding Protein 1/metabolism , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor II/metabolism , Insulin-Like Growth Factor I/metabolism , Liver/metabolism , Malnutrition/metabolism , Prolactin/pharmacology , Animals , Blotting, Western , Cell Growth Processes/physiology , Culture Media, Conditioned/metabolism , Dietary Proteins/metabolism , Female , Fetal Development/physiology , Hepatocytes/cytology , Hepatocytes/metabolism , Insulin-Like Growth Factor Binding Protein 1/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor I/antagonists & inhibitors , Insulin-Like Growth Factor II/antagonists & inhibitors , Insulin-Like Growth Factor II/genetics , Liver/cytology , Liver/embryology , Male , Pregnancy , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Random Allocation , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Insulin-like growth factor binding protein-2 (IGFBP-2), the second most abundant IGFBP in the circulation, is dramatically increased in the serum and ovarian cyst fluid of women with epithelial ovarian cancer. The specific role of IGFBP-2 in ovarian carcinogenesis remains elusive. Using NIH-OVCAR3 human epithelial ovarian cancer cells, we have evaluated the effects of IGFBP-2 and its antibody on cell proliferation, activation of mitogen-activated protein kinase (MAP kinase) pathways and on cytokine expression. Treatment of the cells with IGFBP-2 stimulates cell growth significantly (p<.05) and potentiates the activation of (1) the extracellular signal regulated kinase (ERK1/2) signaling pathway, which transduces cell-specific growth and differentiation signals; (2) the stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) pathway, which is activated by environmental stresses, inflammatory cytokines, growth factors and G-protein coupled receptor (GPCR) agonists; and (3) the p38 MAP kinase pathway, which mediates inflammatory and stress responses. Suppression of IGFBP-2, with its neutralizing antibody, significantly (p<.05) retards cell growth, blocks the activation of all three cascades of the MAPK pathways and downregulates the expression of a number of potential cancer-promoting cytokines. These novel findings may have important clinical implications for developing innovative strategies for the treatment and management of ovarian cancer.
Subject(s)
Adenocarcinoma/enzymology , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/pharmacology , MAP Kinase Signaling System/drug effects , Ovarian Neoplasms/enzymology , Antibodies/immunology , Antibodies/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cytokines/metabolism , Enzyme Activation/drug effects , Female , Humans , Insulin-Like Growth Factor Binding Protein 2/immunology , Phosphorylation/drug effectsABSTRACT
BACKGROUND: Insulin-like growth factor binding protein-2 (IGFBP-2) is expressed by all human prostate cancer cell lines and dramatically increases in the serum of prostate cancer patients. However, the role of IGFBP-2 in prostatic tumorigenesis is not known. The aim of the present study was to investigate the effects of IGFBP-2 on the proliferation of DU145 human prostate cancer cells in culture. METHODS: Using cell proliferation assays, we examined the effects of exogenously administered and endogenously modulated levels of IGFBP-2 on the proliferation of DU145 cells. RESULT: Cell growth was stimulated by exogenously administered IGFBP-2, but significantly retarded (P < 0.05) by its neutralizing antibody. Overexpression of IGFBP-2 by transfection also stimulated cell growth, which was significantly (P < 0.05) inhibited in transfectants expressing antisense mRNA to IGFBP-2. Furthermore, the proliferation of IGFBP-2 overexpressing cells was significantly dampened by exogenously administered IGFBP-2 antibody. CONCLUSIONS: IGFBP-2 is an autocrine growth factor for DU145 human prostate cancer cells and cell proliferation can be significantly retarded by neutralizing or inhibiting its synthesis. These findings provide a strong rationale for targeting IGFBP-2 in the testing of novel strategies to treat prostate cancer.
Subject(s)
Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Cell Division/drug effects , Humans , Insulin-Like Growth Factor Binding Protein 2/administration & dosage , Insulin-Like Growth Factor Binding Protein 2/physiology , Male , Prostatic Neoplasms/etiology , Tumor Cells, CulturedABSTRACT
Our previous studies have shown that insulin-like growth factor binding protein 2 (IGFBP-2) is frequently overexpressed in the highly invasive glioblastoma multiforme (GBM). By using a yeast two-hybrid system, we identified a gene, invasion inhibitory protein 45 (IIp45), whose protein product bound to IGFBP-2 through the thyroglobulin-RGD region of the C terminus of IGFBP-2. The IIp45 gene is located on chromosome 1p36 and has nine exons. The IIp45 protein has three SEG (segment of low compositional complexity) domains and an integrin-binding RGD motif. The IIp45 protein was not expressed in some GBMs. Functional studies showed that IIp45 inhibited GBM cell invasion both in vitro and in xenograft model. Gene expression profiling studies showed that IIp45 consistently inhibited the expression of cell invasion-associated genes, such as the transcriptional NFkappaB, and its downstream target gene, intercellular adhesion molecule 1. Thus, we report here the isolation and characterization of a gene, IIp45, whose protein product binds to IGFBP-2 and inhibits glioma cell invasion.