ABSTRACT
RAS-driven malignancies remain a major therapeutic challenge. The two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-o-tetradecanoylphorbol-13-acetate (TPA) model of mouse skin carcinogenesis has been used to study mechanisms of epithelial tumor development by oncogenic Hras. We used mice with an Hras(G12V) knock-in allele to elucidate the early events after Hras activation, and to evaluate the therapeutic effectiveness of farnesyltransferase inhibition (FTI). Treatment of Caggs-Cre/FR-Hras(G12V) mice with TPA alone was sufficient to trigger papilloma development with a shorter latency and an â¼10-fold greater tumor burden than DMBA/TPA-treated WT-controls. Hras(G12V) allele copy number was increased in all papillomas induced by TPA. DMBA/TPA treatment of Hras(G12V) knock-in mice induced an even greater incidence of papillomas, which either harbored Hras(G12V) amplification or developed an Hras(Q61L) mutation in the second allele. Laser-capture microdissection of normal skin, hyperplastic skin and papillomas showed that amplification occurred only at the papilloma stage. HRAS-mutant allelic imbalance was also observed in human cancer cell lines, consistent with a requirement for augmented oncogenic HRAS signaling for tumor development. The FTI SCH66336 blocks HRAS farnesylation and delocalizes it from the plasma membrane. NRAS and KRAS are not affected as they are alternatively prenylated. When tested in lines harboring HRAS, NRAS or KRAS mutations, SCH66336 delocalized, inhibited signaling and preferentially inhibited growth only of HRAS-mutant lines. Treatment with SCH66336 also induced near-complete regression of papillomas of TPA-treated Hras(G12V) knock-in mice. These data suggest that farnesyl transferase inhibitors should be reevaluated as targeted agents for human HRAS-driven cancers, such as those of bladder, thyroid and other epithelial lineages.
Subject(s)
Enzyme Inhibitors/pharmacology , Farnesyltranstransferase/antagonists & inhibitors , Genes, ras , Papilloma/genetics , Skin Neoplasms/genetics , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Cell Line, Tumor , Farnesyltranstransferase/metabolism , Gene Dosage , Gene Knock-In Techniques , Humans , Mice, Mutant Strains , Mutation , Papilloma/chemically induced , Papilloma/drug therapy , Piperidines/pharmacology , Pyridines/pharmacology , Skin Neoplasms/chemically induced , Skin Neoplasms/drug therapy , Tetradecanoylphorbol Acetate/toxicityABSTRACT
BACKGROUND: Radioiodine (RAI) remains the mainstay of therapy for RAI-avid (RAIA) distant metastatic thyroid carcinoma. We previously demonstrated that RAI-refractory distant metastatic thyroid cancers commonly harbor BRAF mutations. However, the molecular profile of RAIA metastatic thyroid cancer is unknown. Here we describe the mutational profile of thyroid tumors from follicular cell-derived cancer (FCDTC) patients presenting with RAIA distant metastases. In addition, we aimed to correlate clinical outcomes of RAI therapy with clinicopathological factors and tumor mutational status. METHODS: We retrospectively identified 43 patients with FCDTC who had RAI uptake in the lungs and/or bones on their initial ¹³¹I postablation scan. Primary tumors were genotyped for known mutations in thyroid cancer genes. Structural response to RAI was assessed 6-18 months after each administered RAI activity and at the end of follow-up. RESULTS: RAS, BRAF, RET/PTC, and PIK3CA mutations were found in 42, 23, 10, and 2% of tumors, respectively, and the remaining 23% were wild type. None of these patients achieved cure after repeat RAI therapies, and most patients (54%) experienced disease progression despite repeated RAI administration. There was an increased prevalence of RAS mutations in these RAIA tumors. RAS-mutant cancers were more likely to concentrate iodine on diagnostic whole body scans. Despite this, structural response to RAI was not influenced by tumor genotype. CONCLUSIONS: RAIA metastatic FCDTC are overrepresented with RAS mutations, whereas RAI refractory metastatic thyroid cancers are enriched with BRAF mutations. Despite a seemingly preserved ability to concentrate iodine, RAI therapy is ineffective in achieving cure in most patients with RAIA metastatic FCDTC, even in RAS-mutant disease. These poor outcomes may be improved based on recent evidence that pretreatment with MAPK kinase 1/2 inhibitors enhances responses to RAI, particularly in patients with RAS-mutant tumors.
Subject(s)
Adenocarcinoma, Follicular/secondary , Gene Expression Regulation, Neoplastic , Mutant Proteins/metabolism , Neoplasm Proteins/metabolism , Radiopharmaceuticals/therapeutic use , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/radiotherapy , Adenocarcinoma, Follicular/genetics , Adenocarcinoma, Follicular/metabolism , Adenocarcinoma, Follicular/therapy , Adolescent , Adult , Aged , Aged, 80 and over , Cohort Studies , Combined Modality Therapy , Female , Follow-Up Studies , Gene Expression Profiling , Genes, ras , Humans , Iodine Radioisotopes/therapeutic use , Male , Middle Aged , Neoplasm Proteins/genetics , Retrospective Studies , Survival Analysis , Thyroid Neoplasms/genetics , Thyroid Neoplasms/surgery , Thyroidectomy , Young AdultABSTRACT
CONTEXT: Elevated levels of TSH markedly enhance the effectiveness of radioiodine (RAI) therapy in metastatic thyroid cancer. OBJECTIVE: The objective of the study was to compare short-term overall survival in thyroid cancer patients with RAI-avid distant metastases prepared for RAI therapy with either traditional thyroid hormone withdrawal (THW) or recombinant human TSH (rhTSH) stimulation. DESIGN: This was a retrospective chart review. SETTING: The study was conducted at a tertiary care comprehensive cancer center. PATIENTS: Patients included 175 patients with RAI avid metastatic disease to lung and/or bone. INTERVENTIONS: In 58 patients, all RAI treatments (remnant ablation and therapy of metastatic disease) were done with rhTSH stimulation. In 35 patients, all RAI treatments were done after THW. In 82 patients, THW was used for initial RAI treatment(s) with subsequent administered activities given after rhTSH stimulation. MAIN OUTCOME MEASURE: Overall survival was measured. RESULTS: After a median follow-up of 5.5 yr, there were no significant differences in overall survival between patients prepared for RAI therapy with rhTSH alone, THW alone, or THW followed by rhTSH (Kaplan-Meier analysis, P = 0.80). In a multivariate analysis that included clinicopathological features and method of preparation (rhTSH or TWH), only age at diagnosis was an independent predictor of overall survival. CONCLUSIONS: Preparation for RAI therapy using either THW or rhTSH stimulation was associated with similar 5-yr overall survival rates in patients with RAI avid thyroid cancer metastases to lung or bone.
Subject(s)
Carcinoma/mortality , Carcinoma/therapy , Iodine Radioisotopes/therapeutic use , Recombinant Proteins/therapeutic use , Thyroid Neoplasms/mortality , Thyroid Neoplasms/therapy , Thyrotropin/therapeutic use , Adult , Aged , Aged, 80 and over , Carcinoma/secondary , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Neoplasm Staging , Retrospective Studies , Survival Rate , Thyroid Neoplasms/pathology , Treatment OutcomeABSTRACT
Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTCs) that are locally invasive and have well-defined foci of poorly differentiated thyroid carcinoma (PDTC). To investigate the PTC-PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of eight paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial-mesenchymal transition (EMT). This was confirmed by immunohistochemistry, as vimentin expression was increased and E-cadherin lost in PDTC compared with adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for transforming growth factor (TGF)ß in mediating this process. Accordingly, TGFß-induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFß-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required mitogen-activated protein kinase (MAPK) pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFß-induced EMT, through a MAPK-dependent process.
Subject(s)
Disease Progression , Epithelial-Mesenchymal Transition , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Thyroid Neoplasms/pathology , Transforming Growth Factor beta/metabolism , Animals , Carcinoma , Carcinoma, Papillary , Cattle , Enzyme Activation/drug effects , Epithelial-Mesenchymal Transition/drug effects , Gene Expression Profiling , Humans , MAP Kinase Signaling System/drug effects , Mice , Organ Specificity , Phosphorylation/drug effects , Proto-Oncogene Proteins B-raf/genetics , Smad2 Protein/metabolism , Thyroid Cancer, Papillary , Thyroid Gland/drug effects , Thyroid Gland/metabolism , Thyroid Gland/pathology , Thyroid Neoplasms/genetics , Thyroid Neoplasms/metabolism , Transcription, Genetic/drug effects , Transforming Growth Factor beta/pharmacologyABSTRACT
RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response.
Subject(s)
Carcinoma, Papillary/genetics , Immunologic Factors/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Recombination, Genetic , Thyroid Neoplasms/genetics , Animals , Carcinoma, Papillary/immunology , Cell Line, Tumor , Gene Expression Profiling , Proto-Oncogene Proteins c-ret , Rats , Thyroid Neoplasms/immunologyABSTRACT
BACKGROUND/AIM: Hepatic stellate cells (HSCs) express alpha-smooth muscle actin (alphaSMA) and acquire a profibrogenic phenotype upon activation by noxious stimuli. Insulin-like growth I (IGF-I) has been shown to stimulate HSCs proliferation in vitro, but it has been reported to reduce liver damage and fibrogenesis when given to cirrhotic rats. METHODS: The authors used transgenic mice (SMP8-IGF-I) expressing IGF-I under control of alphaSMA promoter to study the influence of IGF-I synthesised by activated HSCs on the recovery from liver injury. RESULTS: The transgene was expressed by HSCs from SMP8-IGF-I mice upon activation in culture and in the livers of these animals after CCl4 challenge. Twenty four hours after administration of CCl4 both transgenic and wild type mice showed similar extensive necrosis and increased levels of serum transaminases. However at 72 hours SMP8-IGF-I mice exhibited lower serum transaminases, reduced hepatic expression of alphaSMA, and improved liver morphology compared with wild type littermates. Remarkably, at this time all eight CCl4 treated wild type mice manifested histological signs of liver necrosis that was severe in six of them, while six out of eight transgenic animals had virtually no necrosis. In SMP8-IGF-I mice robust DNA synthesis occurred earlier than in wild type animals and this was associated with enhanced production of HGF and lower TGFbeta1 mRNA expression in the SMP8-IGF-I group. Moreover, Colalpha1(I) mRNA abundance at 72 hours was reduced in SMP8-IGF-I mice compared with wild type controls. CONCLUSIONS: Targeted overexpression of IGF-I by activated HSCs restricts their activation, attenuates fibrogenesis, and accelerates liver regeneration. These effects appear to be mediated in part by upregulation of HGF and downregulation of TGFbeta1. The data indicate that IGF-I can modulate the cytokine response to liver injury facilitating regeneration and reducing fibrosis.
Subject(s)
Adipocytes/metabolism , Insulin-Like Growth Factor I/physiology , Liver Cirrhosis, Experimental/physiopathology , Liver Regeneration , Actins , Animals , Carbon Tetrachloride , Cells, Cultured , Collagen/metabolism , Female , Fibronectins/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Mice , Mice, Transgenic , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1ABSTRACT
Treatment of patients with thyroid cancer is usually successful, and most patients are cured of the disease. However, we do not have effective therapies for patients with invasive or metastatic thyroid cancer if the disease is not surgically resectable and does not concentrate radio-iodine. Conventional external beam radiotherapy and chemotherapy are of marginal benefit. In other types of cancer, new therapies are being developed that take advantage of our knowledge of cancer pathogenesis to interfere with the activity of specific oncoproteins believed to be important in disease causation. Because these approaches are being considered for thyroid cancer, I will briefly describe in this review examples of recent breakthroughs in medical therapy of certain hematological malignancies and some solid tumors using drugs that work in this fashion, focusing in particular on compounds that block the enzymatic activity of specific tyrosine kinase oncoproteins. It should be noted, however, that cancers commonly harbor mutations or other disruptions of many genes, each of which could conceivably play a role in disease pathogenesis. This makes the choice of molecular target a difficult and critical decision if these approaches are to succeed. Here I will argue that priority should be given to blocking the function of oncoproteins activated early in tumor development. We have a fairly good understanding of the genetic changes involved in thyroid cancer initiation, and hence these cancers may prove to be particularly well suited for oncoprotein-specific therapies.
Subject(s)
Carcinoma, Medullary/pathology , Carcinoma, Papillary/pathology , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/pathology , Animals , Antineoplastic Agents/therapeutic use , Benzenesulfonates/therapeutic use , Carcinoma, Medullary/drug therapy , Carcinoma, Papillary/drug therapy , Humans , Mutation , Niacinamide/analogs & derivatives , Oncogenes , Phenylurea Compounds , Phosphotransferases/antagonists & inhibitors , Phosphotransferases/genetics , Proto-Oncogene Proteins B-raf/genetics , Pyridines/therapeutic use , SorafenibABSTRACT
Recent studies support a critical role for the paracrine IGF/IGF-binding protein system in the regulation of vascular smooth muscle cell growth. In this study we have explored the hypothesis that the abundance of individual IGF-binding proteins in smooth muscle is subject to regulation during postnatal life and in response to injury. IGF-binding protein-2 was the predominant binding protein secreted by neonatal rat vascular smooth muscle cells, whereas IGF-binding protein-4 was most prevalent in adult vascular smooth muscle cells coincident with increased IGF-binding protein-4 protease activity. After arterial injury, IGF-binding protein-4 mRNA increased, associated with greater IGF-binding protein-4 proteolytic activity, resulting in stable steady state levels of the IGF-binding protein-4 protein. Expression of pregnancy-associated plasma protein A mRNA, recently identified as an IGF-binding protein-4 protease, was expressed at higher levels in adult than neonatal vascular smooth muscle cell lines, but did not change significantly after arterial injury. The peak of immunoreactive pregnancy-associated plasma protein A from hydrophobic interaction chromatography fractions of smooth muscle cell-conditioned medium coincided, but did not fully overlap, with the fractions containing maximal IGF-binding protein-4 protease activity. In conclusion, our data point to a developmental switch from IGF-binding protein-2 to IGF-binding protein-4 in vascular smooth muscle cells postnatally. Moreover, IGF-binding protein-4 expression is coregulated with IGF-binding protein-4 protease activity, suggesting that biosynthesis and degradation of this binding protein are coordinated events important for regulating biological activity of IGF-I.
Subject(s)
Insulin-Like Growth Factor Binding Protein 4/physiology , Metalloendopeptidases/physiology , Muscle, Smooth, Vascular/physiology , Animals , Cells, Cultured , Gene Expression Regulation/physiology , Insulin-Like Growth Factor Binding Protein 2/physiology , Muscle, Smooth, Vascular/pathology , Pregnancy-Associated Plasma Protein-A , RNA, Messenger/physiology , RatsABSTRACT
The response of arterial smooth muscle cells to injury is governed by a complex series of events. Significant among them is the paracrine production of peptide growth factors. To determine the impact of local IGF-I gene expression on vascular injury, the left carotid arteries of SMP8-IGF-I mice (in which IGF-I is selectively overexpressed in smooth muscle cells by means of a smooth muscle alpha-actin promoter) and wild-type controls were injured mechanically with an epon resin probe. After 7 and 14 d, a progressive increase in medial area was seen in both SMP8-IGF-I and wild-type mice, but they were not significantly different from each other. However, by 14 d there was a more than 4-fold increase in neointimal area in transgenic vs. wild-type. The intima/media ratios were also strikingly increased at 14 d in the IGF-I-overexpressing animals. The mitotic index, determined in animals injected daily with bromodeoxyuridine for 3 d before death, was markedly elevated in both the media and neointima 7 d after injury in SMP8-IGF-I mice, but the effect had subsided by 14 d. Despite a higher rate of cell division, the relative increase in medial area was less in the SMP8-IGF-I mice than in wild-type mice at both 7 and 14 d, consistent with a stimulation of cell migration to the neointima. The experiments reported here provide compelling evidence that paracrine expression of IGF-I is a powerful stimulus for smooth muscle cell proliferation and migration in vivo.
Subject(s)
Carotid Artery Injuries/pathology , Carotid Artery Injuries/physiopathology , Insulin-Like Growth Factor I/pharmacology , Muscle, Smooth, Vascular/drug effects , Tunica Intima/growth & development , Actins/metabolism , Animals , Cell Division/drug effects , Cell Movement/drug effects , Insulin-Like Growth Factor I/genetics , Mice , Mice, Transgenic/genetics , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , RNA, Messenger/metabolism , Tunica Intima/drug effectsABSTRACT
Cancer is a disease of genes. Detection of genetic abnormalities associated with cancers of various cell types can now be used for genetic counseling, diagnosis or treatment selection. In the case of thyroid cancer, genetic testing for mutations of the RET oncogene has had a profound effect on the management of medullary thyroid carcinomas. There is also considerable information on the genetic changes associated with development and progression of cancers of thyroid follicular cells, although these have not yet proven to be of practical value for clinical diagnosis or to guide prognosis and therapy.
Subject(s)
Drosophila Proteins , Genetic Markers , Thyroid Neoplasms/genetics , Adenocarcinoma, Follicular/genetics , Carcinoma, Medullary/genetics , Carcinoma, Papillary/genetics , Humans , Multiple Endocrine Neoplasia Type 2a/genetics , Mutation , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ret , Receptor Protein-Tyrosine Kinases/geneticsSubject(s)
Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Thyroid Neoplasms/etiology , Thyroid Neoplasms/metabolism , Apoptosis , Blotting, Western , Cell Division , Humans , In Situ Nick-End Labeling , Microscopy, Fluorescence , Models, Biological , Protein Isoforms , Protein Kinase C-epsilon , Signal Transduction , Thyroid Gland/metabolism , Tumor Cells, CulturedABSTRACT
Insulin-like growth factor I (IGF-I) has been postulated to function as a vasodilator. We explored the vasoactive effects of chronic elevations of arterial IGF-I levels in SMP8-IGF-I mice, in which IGF-I is overexpressed in smooth muscle (SM) by means of a SM alpha-actin promoter. Denuded aortas from SMP8-IGF-I mice generated increased force in response to KCl or phenylephrine and had greater sensitivity to KCl depolarization. This is not due to desensitization of a SM NO pathway, as pretreatment with n-omega-nitro-L-arginine affected both wild-type and SMP8-IGF-I aortas to a similar degree. The increased contractility ex vivo is not associated with changes in heart rate or blood pressure. Total smooth muscle myosin heavy chain (SMHC) messenger RNA (mRNA) was greater in SMP8-IGF-I aortas, with preferential expression of SMHC-A. Reciprocal effects on contractility and SMHC mRNA were observed in SMP8-IGFBP-4 animals, in which IGF-binding protein-4 was overexpressed through the same promoter. Also, SM alpha-actin mRNA was increased in the aortas from SMP8-IGF-I mice. In summary, chronic arterial overexpression of IGF-I is associated with increased contractility. These effects differ from those seen after acute exposure to the growth factor and may relate to IGF-mediated changes in expression and relative isoform abundance of critical contractile proteins.