Transformation by Hras(G12V) is consistently associated with mutant allele copy gains and is reversed by farnesyl transferase inhibition.

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 ∼10-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.

Clinical outcomes and molecular profile of differentiated thyroid cancers with radioiodine-avid distant metastases.

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.

Five-year survival is similar in thyroid cancer patients with distant metastases prepared for radioactive iodine therapy with either thyroid hormone withdrawal or recombinant human TSH.

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.

Progression of BRAF-induced thyroid cancer is associated with epithelial-mesenchymal transition requiring concomitant MAP kinase and TGFß signaling.

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.

RET/PTC-induced gene expression in thyroid PCCL3 cells reveals early activation of genes involved in regulation of the immune response.

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.

Expression of insulin-like growth factor I by activated hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury.

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.

How thyroid tumors start and why it matters: kinase mutants as targets for solid cancer pharmacotherapy.

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.

IGF-binding protein-4 expression and IGF-binding protein-4 protease activity are regulated coordinately in smooth muscle during postnatal development and after vascular injury.

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.

Genetic markers in thyroid neoplasia.

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.

Targeted overexpression of IGF-I in smooth muscle cells of transgenic mice enhances neointimal formation through increased proliferation and cell migration after intraarterial injury.

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.

Smooth muscle-targeted overexpression of insulin-like growth factor I results in enhanced vascular contractility.

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.

Conditional apoptosis induced by oncogenic ras in thyroid cells.

Mutations of ras are tumor-initiating events for many cell types, including thyrocytes. To explore early consequences after oncogenic Ras activation, we developed a doxycycline-inducible expression system in rat thyroid PCCL3 cells. Beginning 3-4 days after H-Ras(v12) expression, cells underwent apoptosis. The H-Ras(v12) effects on apoptosis were decreased by a mitogen-activated protein kinase kinase (MEK1) inhibitor and recapitulated by doxycycline-inducible expression of an activated MEK1 mutant (MEK1(S217E/S221E)). As reported elsewhere, acute expression of H-Ras(v12) also induces mitotic defects in PCCL3 cells through ERK (extracellular ligand-regulated kinase) activation, suggesting that apoptosis may be secondary to DNA damage. However, acute activation of SAPK/JNK (stress-activated protein kinase/Jun N-terminal kinase) through acute expression of Rac1(v12) also triggered apoptosis, without inducing large-scale genomic abnormalities. H-Ras(v12)-induced apoptosis was dependent on concomitant activation of cAMP by either TSH or forskolin, in a protein kinase A-independent manner. Thus, coactivation of cAMP-dependent pathways and ERK or JNK (either through H-Ras(v12), Rac1(v12), or MEK1(S217E/S221E)) is inconsistent with cell survival. The fate of thyrocytes within the first cell cycles after expression of oncogenic Ras is dependent on ambient TSH levels. If both cAMP and Ras signaling are simultaneously activated, most cells will die. Those that survive will eventually lose TSH responsiveness and/or inactivate the apoptotic cascade through secondary events, thus enabling clonal expansion.

Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells.

Rearrangements involving the RET gene are common in radiation-associated papillary thyroid cancer (PTC). The RET/PTC1 type of rearrangement is an inversion of chromosome 10 mediated by illegitimate recombination between the RET and the H4 genes, which are 30 megabases apart. Here we ask whether despite the great linear distance between them, RET and H4 recombination might be promoted by their proximity in the nucleus. We used two-color fluorescence in situ hybridization and three-dimensional microscopy to map the positions of the RET and H4 loci within interphase nuclei. At least one pair of RET and H4 was juxtaposed in 35% of normal human thyroid cells and in 21% of peripheral blood lymphocytes, but only in 6% of normal mammary epithelial cells. Spatial contiguity of RET and H4 may provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double-strand break in each gene at the same site in the nucleus.

The highly malignant phenotype of anaplastic thyroid carcinoma cell lines is recessive.

OBJECTIVE: The aim of our studies was to determine whether the phenotype of the anaplastic thyroid carcinomas is dominant or recessive. In fact, it is hypothesized, on the basis of epidemiological and pathological data, that undifferentiated thyroid carcinomas are derived from differentiated tumours through a mechanism of tumour progression. DESIGN: Cell hybrids have been generated by cell fusion of anaplastic thyroid carcinoma cell lines, which show a highly malignant phenotype, to cell lines deriving from differentiated thyroid carcinoma, which show a non-tumorigenic or a poorly tumorigenic phenotype. All of the parental cell lines showed impaired p53 gene function. RESULTS: The cell hybrids contained alleles from the parental cell lines. All of the cell hybrids showed a lower growth rate compared with the parental undifferentiated carcinoma cell lines and were unable to grow in soft agar and to induce tumours after injection into athymic mice. CONCLUSION: Taken together, these findings suggest that the highly malignant phenotype of the anaplastic thyroid carcinoma is achieved by the impairment of gene functions that negatively regulate cell growth, rather than by the activation of dominant oncogenes.

The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway.

Activating mutations of RAS are thought to be early events in the evolution of thyroid follicular neoplasms. We used a doxycycline-inducible expression system to explore the acute effects of H-RAS12 on genomic stability in thyroid PCCL3 cells. At 2-3 days (first or second cell cycle) there was a significant increase in the frequency of micronucleation. Treatment of cells with YVAD-CHO inhibited RAS-induced apoptosis, but had no effect on micronucleation. The effects of H-RAS(V12) were mediated by activation of MAPK, as treatment with PD98059 at concentrations verified to selectively inhibit MEK1 reduced the frequency of prevalence of cells with micronuclei. In addition, doxycycline-inducible expression of a constitutively active MEK1, but not of a mutant RAC1, mimicked the effects of H-RAS(V12). The effects of H-RAS(V12) on genome destabilization were apparent even though the sequence of p53 in PCCL3 cells was confirmed to be wild-type. Acute activation of H-RAS(V12) evoked a proportional increase in both CREST negative and CREST positive micronuclei, indicating that both clastogenic and aneugenic effects were involved. H-RAS(V12) and activated MEK1 also induced centrosome amplification, and chromosome misalignment. Evidence that acute expression of constitutively activated RAS destabilizes the genome of PCCL3 cells is consistent with a mode of tumor initiation in which this oncogene promotes phenotypic progression by predisposing to large scale genomic abnormalities.