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We developed mice with germline endogenous expression of oncogenic Hras to study effects on development and mechanisms of tumor initiation. They had high perinatal mortality, abnormal cranial dimensions, defective dental ameloblasts, and nasal septal deviation, consistent with some of the features of human Costello syndrome. These mice developed papillomas and angiosarcomas, which were associated with Hras(G12V) allelic imbalance and augmented Hras signaling. Endogenous expression of Hras(G12V) was also associated with a higher mutation rate in vivo. Tumor initiation by Hras(G12V) likely requires augmentation of signal output, which in papillomas and angiosarcomas is achieved via increased Hras-gene copy number, which may be favored by a higher mutation frequency in cells expressing the oncoprotein.

Constitutive activation of MAPK in cancer occurs through activating mutations or overexpression of upstream effectors in the pathway, primarily of genes encoding receptor tyrosine kinases, RAS and BRAF. Arguably, the evidence for MAPK activation is most compelling in thyroid cancers and in melanomas. In this review we discuss the mechanisms of tumor development by oncogenic BRAF in these two cancer cell lineages, since this kinase signals preferentially through this pathway. We describe recent information on the mediators of BRAF-induced tumor initiation and escape from senescence. In addition, we review the biochemical events implicated in cellular growth triggered by oncogenic BRAF and the determinants of oncogene addiction. The biology of thyroid cancers induced by oncogenic BRAF is quite distinct, both in humans and in mice. There is great interest in using these insights to design rational new therapies, for which it will become crucial to understand the determinants of sensitivity and resistance to compounds designed to block the pathway. In thyroid cancer, this interest is further heightened by new information on the role of activated BRAF and MAPK pathway activation in disrupting iodine transport and thyroid hormonogenesis.

Thyroid cancers are infiltrated with tumor-associated macrophages (TAMs), yet their role in cancer progression is not known. The objectives of this study were to characterize the density of TAMs in well-differentiated (WDTC), poorly differentiated (PDTC), and anaplastic thyroid cancers (ATC) and to correlate TAM density with clinicopathologic parameters. Immunohistochemistry was performed on tissue microarray sections from WDTC (n=33), PDTC (n=37), and ATC (n=20) using macrophage-specific markers. Electronic medical records were used to gather clinical and pathologic data. Follow-up information of PDTC patients was available for 0-12 years. In total, 9 out of 33 WDTC (27%), 20 out of 37 PDTC (54%), and 19 out of 20 ATC (95%) had an increased density of CD68(+) TAMs (> or = 10 per 0.28 mm(2); WDTC versus PDTC, P=0.03; WDTC versus ATC, P<0.0001; PDTC versus ATC, P<0.002). Increased TAMs in PDTC was associated with capsular invasion (P=0.034), extrathyroidal extension (P=0.009), and decreased cancer-related survival (P=0.009) compared with PDTC with a low density of TAMs. In conclusion, the density of TAMs is increased in advanced thyroid cancers. The presence of a high density of TAMs in PDTC correlates with invasion and decreased cancer-related survival. These results suggest that TAMs may facilitate tumor progression. As novel therapies directed against thyroid tumor cell-specific targets are being tested, the potential role of TAMs as potential modulators of the thyroid cancer behavior will need to be considered.

Context: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated., Objective: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis., Results: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines., Conclusions: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

Context: Mutually exclusive mutations of RET, RAS, or BRAF are present in about 70% of papillary thyroid carcinomas, whereas only the latter two are seen in poorly differentiated and anaplastic cancers. Although the signal output common to these oncoproteins is ERK, a recent report showed that only BRAF mutations consistently predicted responsiveness to MAPK kinase (MEK) inhibitors., Objectives: Here we investigated whether sensitivity to MEK inhibition was determined by oncogene status in 13 human thyroid cancer cell lines: four with BRAF mutations, four RAS, one RET/PTC1, and four wild type., Results: Growth of BRAF (+) cells was inhibited by the MEK antagonist PD0325901 with an IC(50) of less than 5 nm. By contrast, RAS, RET/PTC1, or wild-type cells had IC(50) of 4 nm to greater than 1000 nm. Sensitivity was not predicted by coexisting mutations in PIK3CA or by PTEN status. Similar effects were obtained with the MEK inhibitor AZD6244. PD0325901 induced a sustained G1/S arrest in BRAF (+) but not BRAF (-) lines. PD0325901 was equipotent at inhibiting pERK1/2 after 2 h, regardless of genetic background, but pERK rebounded at 24 h in most lines. MEK inhibitor resistance was associated with partial refractoriness of pERK to further inhibition by the compounds. AZD6244 was more potent at inhibiting growth of NPA (BRAF +) than Cal62 (KRAS +) xenografts., Conclusion: Thyroid cancers with BRAF mutation are preferentially sensitive to MEK inhibitors, whereas tumors with other MEK-ERK effector pathway gene mutations have variable responses, either because they are only partially dependent on ERK and/or because feedback responses elicit partial refractoriness to MEK inhibition.

RET/PTC rearrangements are one of the genetic hallmarks of papillary thyroid carcinomas. RET/PTC oncoproteins lack extracellular or transmembrane domains, and activation takes place through constitutive dimerization mediated through coiled-coil motifs in the NH(2) terminus of the chimeric protein. Based on the observation that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 decreased RET/PTC kinase autophosphorylation and activation of downstream effectors in thyroid cells, despite lacking activity on the purified RET kinase, we proceeded to examine possible functional interactions between RET/PTC and EGFR. Conditional activation of RET/PTC oncoproteins in thyroid PCCL3 cells markedly induced expression and phosphorylation of EGFR, which was mediated in part through mitogen-activated protein kinase signaling. RET and EGFR were found to coimmunoprecipitate. The ability of RET to form a complex with EGFR was not dependent on recruitment of Shc or on their respective kinase activities. Ligand-induced activation of EGFR resulted in phosphorylation of a kinase-dead RET, an effect that was entirely blocked by PKI166. These effects were biologically relevant, as the EGFR kinase inhibitors PKI166, gefitinib, and AEE788 inhibited cell growth induced by various constitutively active mutants of RET in thyroid cancer cells as well as NIH3T3 cells. These data indicate that EGFR contributes to RET kinase activation, signaling, and growth stimulation and may therefore be an attractive therapeutic target in RET-induced neoplasms.

The RET kinase has emerged as a promising target for the therapy of medullary thyroid cancers (MTC) and of a subset of papillary thyroid cancers. NVP-AST487, a N,N'-diphenyl urea with an IC(50) of 0.88 mumol/L on RET kinase, inhibited RET autophosphorylation and activation of downstream effectors, and potently inhibited the growth of human thyroid cancer cell lines with activating mutations of RET but not of lines without RET mutations. NVP-AST487 induced a dose-dependent growth inhibition of xenografts of NIH3T3 cells expressing oncogenic RET, and of the MTC cell line TT in nude mice. MTCs secrete calcitonin, a useful indicator of tumor burden. Human plasma calcitonin levels derived from the TT cell xenografts were inhibited shortly after treatment, when tumor volume was still unchanged, indicating that the effects of RET kinase inhibition on calcitonin secretion were temporally dissociated from its tumor-inhibitory properties. Accordingly, NVP-AST487 inhibited calcitonin gene expression in vitro in TT cells, in part, through decreased gene transcription. These data point to a previously unknown physiologic role of RET signaling on calcitonin gene expression. Indeed, the RET ligands persephin and GDNF robustly stimulated calcitonin mRNA, which was blocked by pretreatment with NVP-AST487. Antagonists of RET kinase activity in patients with MTC may result in effects on plasma calcitonin that are either disproportionate or dissociated from the effects on tumor burden, because RET kinase mediates a physiologic pathway controlling calcitonin secretion. The role of traditional tumor biomarkers may need to be reassessed as targeted therapies designed against oncoproteins with key roles in pathogenesis are implemented.

Papillary thyroid cancers (PTC) are associated with nonoverlapping mutations of genes coding for mitogen-activated protein kinase signaling effectors (i.e., the TK receptors RET or NTRK and the signaling proteins RAS and BRAF). We examined the pattern of gene expression after activation of these oncoproteins in thyroid PCCL3 cells, with the goal of identifying pathways or gene subsets that may account for the phenotypic differences observed in human cancers. We hybridized cDNA from cells treated with or without doxycycline to induce expression of BRAF(V600E), RET/PTC3, or RET/PTC3 with small interfering RNA-mediated knockdown of BRAF, respectively, to slides arrayed with a rat 70-mer oligonucleotide library consisting of 27,342 oligos. Among the RET/PTC3-induced genes, 2,552 did not require BRAF as they were similarly regulated by RET/PTC3 with or without BRAF knockdown and not by expression of BRAF(V600E). Immune response and IFN-related genes were highly represented in this group. About 24% of RET/PTC3-regulated genes were BRAF dependent, as they were similarly modified by RET/PTC3 and BRAF(V600E) but not in cells expressing RET/PTC3 with knockdown of BRAF. A gene cluster coding for components of the mitochondrial electron transport chain pathway was down-regulated in this group, potentially altering regulation of cell viability. Metalloproteinases were also preferentially induced by BRAF, particularly matrix metalloproteinase 3 (MMP3), MMP9, and MMP13. Accordingly, conditional expression of BRAF was associated with markedly increased invasion into Matrigel compared with cells expressing RET/PTC3. The preferential induction of MMPs by BRAF could explain in part the more invasive behavior of thyroid cancers with BRAF mutations.

Purpose: Papillary thyroid carcinomas are associated with nonoverlapping activating mutations of RET, NTRK, RAS and BRAF, which altogether are present in approximately 70% of cases. We postulated that compounds that inhibit a distal effector in the mitogen-activated protein kinase (MAPK) pathway would inhibit growth and tumorigenicity of human thyroid cancer cell lines with mutations of RET or BRAF., Experimental Design and Results: We first examined the effects of AAL-881 and LBT-613, two inhibitors of RAF kinase activity, on RAF-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK activation in thyroid PCCL3 cells after conditional induction of expression of H-RAS(G12V) or BRAF(V600E). Both compounds blocked RAS and RAF-dependent MEK and ERK phosphorylation. They also potently blocked MEK phosphorylation in human thyroid cancer cell lines with either RET/PTC1 (TPC1) or BRAF(V600E) (NPA, ARO, and FRO) mutations. Inhibition of ERK phosphorylation was transient in TPC1 and ARO cells, with recovery of ERK phosphorylation associated with concomitant down-regulation of the MAPK phosphatases MKP-3 and DUSP5. Both compounds inhibited growth of all cell lines, with LBT-613 being approximately 10-fold more potent than AAL-881. TPC1 cells were more sensitive to growth inhibition (IC50 0.1-0.25 and approximately 0.05 micromol/L for AAL-881 and LBT-613, respectively) than BRAF + lines (IC50 2.5-5 and 0.1-0.5 micromol/L, respectively). Growth inhibition was associated with G1 arrest, and induction of cell death. Growth of ARO and NPA tumor xenografts was inhibited by LBT-613 or AAL-881. MEK and ERK phosphorylation was inhibited by both compounds in ARO but not in NPA cell xenografts., Conclusions: Compounds that inhibit kinase activity are effective growth inhibitors for poorly differentiated thyroid cancer cell lines with either RET or RAF mutations, and hold promise for treatment of most forms of papillary thyroid carcinoma.

Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRAS(V12) increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRAS(V12) or HRAS(V12) effector mutants were presynchronized at the G(1)/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS(V12) accelerated the G(2)/M phase by approximately 4 h and promoted bypass of the G(2) DNA damage and mitotic spindle checkpoints. Accelerated passage through G(2)/M and bypass of the G(2) DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G(2) DNA damage checkpoint, because cells arrested appropriately in G(2) despite conditional expression of HRAS(V12,S35) or BRAF(V600E). By contrast to the MAPK requirement for radiation-induced G(2) arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G(2) progression and regulation of the G(2) DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G(2)/M.

In human papillary thyroid cancers (PTCs), mutations of RET/PTC, NTRK, RAS, or BRAF are found in about two thirds of cases with practically no overlap, providing genetic evidence that constitutive signaling along RET-RAS-BRAF-MAPK is key to their development. The requirement for BRAF in RET/PTC-mediated MAPK activation and gene expression has not been tested functionally. There are three RAF isoforms: ARAF, BRAF, and CRAF. Compared with the others, ARAF is a much weaker stimulator of MAPK. To determine the key RAF isoform mediating RET/PTC-induced ERK phosphorylation, we stably transfected doxycycline-inducible RET/PTC3-expressing thyroid PCCL3 cells with small interfering RNA vectors to induce selective knockdown of BRAF or CRAF. Conditional RET/PTC3 expression induced comparable ERK phosphorylation in CRAF knockdown and control cells but negligible ERK phosphorylation in BRAF knockdown cells. Selective knockdown of BRAF prevented RET/PTC-dependent down-regulation of the sodium iodide symporter, a gene that confers key biological effects of RET/PTC in PTCs. Moreover, microarray analysis revealed numerous RET/PTC-regulated genes showing requirement of BRAF for appropriate expression. These data indicate that BRAF is required for RET/PTC-induced MAPK activation in thyroid cells and support the notion that BRAF inactivation may be an attractive target for PTCs.

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.

The BRAFT1799A mutation is the most common genetic alteration in papillary thyroid carcinomas (PTC). It is also found in a subset of papillary microcarcinomas, consistent with a role in tumor initiation. PTCs with BRAFT1799A are often invasive and present at a more advanced stage. BRAFT1799A is found with high prevalence in tall-cell variant PTCs and in poorly differentiated and undifferentiated carcinomas arising from PTCs. To explore the role of BRAFV600E in thyroid cancer pathogenesis, we targeted its expression to thyroid cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Two Tg-BRAFV600E lines (Tg-BRAF2 and Tg-BRAF3) were propagated for detailed analysis. Tg-BRAF2 and Tg-BRAF3 mice had increased thyroid-stimulating hormone levels (>7- and approximately 2-fold, respectively). This likely resulted from decreased expression of thyroid peroxidase, sodium iodine symporter, and thyroglobulin. All lines seemed to successfully compensate for thyroid dysfunction, as serum thyroxine/triiodothyronine and somatic growth were normal. Thyroid glands of transgenic mice were markedly enlarged by 5 weeks of age. In Tg-BRAF2 mice, PTCs were present at 12 and 22 weeks in 14 of 15 and 13 of 14 animals, respectively, with 83% exhibiting tall-cell features, 83% areas of invasion, and 48% foci of poorly differentiated carcinoma. Tg-BRAF3 mice also developed PTCs, albeit with lower prevalence (3 of 12 and 4 of 9 at 12 and 22 weeks, respectively). Tg-BRAF2 mice had a 30% decrease in survival at 5 months. In summary, thyroid-specific expression of BRAFV600E induces goiter and invasive PTC, which transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAF-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis.

Activating point mutations of the BRAF gene have been recently described in a variety of human tumors. In a study published in the Journal of Clinical Investigation, we reported a novel mechanism of activation of this gene via paracentric inversion of chromosome 7q. The fusion protein, AKAP9-BRAF, contains the intact kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It exhibited constitutive activation of BRAF kinase and was transforming for NIH3T3 cells. This finding represents the first demonstration of RAF activation by chromosomal rearrangement in human tumors. AKAP9-BRAF was more common in radiation-induced thyroid tumors, whereas point mutations of BRAF predominated in sporadic tumors of the same type, demonstrating the association between environmental factors and specific mechanisms of BRAF activation.

The activating mutation BRAF(T1796A) is the most prevalent genetic alteration in papillary thyroid carcinomas (PTC). It is associated with advanced PTCs, suggesting that this oncoprotein confers thyroid cancers with more aggressive properties. BRAF(T1796A) is also observed in thyroid micropapillary carcinomas and may thus be an early event in tumor development. To explore its biological consequences, we established doxycycline-inducible BRAF(V600E)-expressing clonal lines derived from well-differentiated rat thyroid PCCL3 cells. Expression of BRAF(V600E) did not induce growth in the absence of thyrotropin despite increasing DNA synthesis, which is likely explained because of a concomitant increase in apoptosis. Thyrotropin-dependent cell growth and DNA synthesis were reduced by BRAF(V600E) because of decreased thyrotropin responsiveness associated with inhibition of thyrotropin receptor gene expression. These results are similar to those obtained following conditional expression of RET/PTC. However, in contrast to RET/PTC, BRAF activation did not impair key activation steps distal to the thyrotropin receptor, such as forskolin-induced adenylyl cyclase activity or cyclic AMP-induced DNA synthesis. We reported previously that acute RET/PTC expression in PCCL3 cells did not induce genomic instability. By contrast, induction of BRAF(V600E) expression increased the frequency of micronuclei by both clastogenic and aneugenic events. These data indicate that BRAF(V600E) expression confers thyroid cells with little growth advantage because of concomitant activation of DNA synthesis and apoptosis. However, in contrast to RET/PTC, BRAF(V600E) may facilitate the acquisition of secondary genetic events through induction of genomic instability, which may account for its aggressive properties.

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.

Point mutations of the BRAF gene have been recently described with high prevalence in papillary thyroid carcinomas. However, this molecular alteration has not been studied in radiation-induced thyroid tumors. We analyzed the prevalence of BRAF point mutations and RET/PTC rearrangements in 55 post-Chernobyl papillary carcinomas, compared with 82 sporadic papillary carcinomas. Radiation-induced tumors demonstrated a low prevalence (4%) of BRAF point mutations and high prevalence (58%) of RET/PTC rearrangements. Sporadic papillary carcinomas revealed a clearly distinct pattern, with 37% of tumors harboring BRAF mutations and 20% RET/PTC rearrangements. These results demonstrate a significant difference in the molecular genetic profile of sporadic and radiation-induced thyroid tumors.

RET/PTC rearrangements are believed to be tumor-initiating events in papillary thyroid carcinomas. We identified microsomal prostaglandin E2 synthase-1 (mPGES-1) as a RET/PTC-inducible gene through subtraction hybridization cloning and expression profiling with custom microarrays. The inducible prostaglandin E2 (PGE2) biosynthetic enzymes cyclooxygenase-2 (COX-2) and mPGES-1 are up-regulated in many cancers. COX-2 is overexpressed in thyroid malignancies compared with benign nodules and normal thyroid tissues. Eicosanoids may promote tumorigenesis through effects on tumor cell growth, immune surveillance, and angiogenesis. Conditional RET/PTC1 or RET/PTC3 expression in PCCL3 thyroid cells markedly induced mPGES-1 and COX-2. PGE2 was the principal prostanoid and up-regulated (by approximately 60-fold), whereas hydroxyeicosatetraenoic acid metabolites were decreased, consistent with shunting of prostanoid biosynthesis toward PGE2 by coactivation of the two enzymes. RET/PTC activated mPGES-1 gene transcription. Based on experiments with kinase inhibitors, with PCCL3 cell lines with doxycycline-inducible expression of RET/PTC mutants with substitutions of critical tyrosine residues in the kinase domain, and lines with inducible expression of activated mutants of H-RAS and MEK1, RET/PTC was found to regulate mPGES-1 through Shc-RAS-MEK-ERK. These data show a direct relationship between activation of a tyrosine kinase receptor oncogene and regulation of PGE2 biosynthesis. As enzymes involved in prostanoid biosynthesis can be targeted with pharmacological inhibitors, these findings may have therapeutic implications.

Most papillary thyroid carcinomas (PTC) have an isozyme-specific reduction of protein kinase C (PKC)epsilon, which occurs through a post-transcriptional mechanism. Here, we test whether the oncoprotein RET/PTC could be responsible for this effect, since RET/PTC rearrangements are quite prevalent in PTC and RET/PTC activates PLCgamma, an upstream modulator of PKCs. At 3 h after induction of RET/PTC1 or RET/PTC3 expression, there was evidence of PKCepsilon activation. Activation was restricted to PKCepsilon, as acute expression of RET/PTC did not change the subcellular distribution of other PKC isozymes expressed in PCCL3 cells. Prolonged RET/PTC expression (2-6 days) produced an isozyme-specific change in PKCepsilon subcellular localization and a decrease in total PKCepsilon levels. The expression of RET/PTC3(Y541F), which does not interact with PLCgamma, but signals normally through other RET effectors, had no effect on PKCepsilon distribution at any of the time points examined. However, downregulation of total PKCepsilon levels was only partially prevented by expression of RET/PTC(Y541F). Cells with decreased PKCepsilon following prolonged expression of RET/PTC were relatively resistant to doxorubicin-induced apoptosis. Based on our previous observation that PCCL3 cells expressing a dominant-negative PKCepsilon are also markedly resistant to apoptosis, we propose that selective downregulation of PKCepsilon following prolonged RET/PTC activation promotes cell survival and clonal expansion.

Constitutive activation of the RET proto-oncogene in papillary thyroid carcinomas results from rearrangements linking the promoter(s) and N-terminal domains of unrelated genes to the C-terminus of RET tyrosine kinase (RET/PTC). RET/PTC expression has been demonstrated to inhibit transcription of thyroid-specific genes. To study the signal transduction pathways responsible for this, we generated PCCL3 thyroid cells with doxycycline-inducible expression of RET/PTC3, RET/PTC3(Y541F), or PTC2/PDZ. Acute expression of RET/PTC(Y541F) appropriately interacted with Shc, an intermediate in the activation of the Ras pathway, but failed to activate PLCgamma. By contrast, PTC2/PDZ failed to bind Shc, but interacted normally with PLCgamma. Acute expression of RET/PTC3 or RET/PTC3(Y541F), but not PTC2/PDZ, inhibited TSH-induced Tg and NIS expression, suggesting that activation of Shc-Ras, but not PLCgamma, is required for RET/PTC-induced dedifferentiation. Accordingly, acute expression of H-Ras(V12) or of a constitutively active MEK1 also blocked TSH-induced expression of Tg and NIS. Moreover, MEK inhibitors restored Tg and NIS levels. In conclusion, activation of the Ras/Raf/MEK/MAPK pathway through Shc mediates RET/PTC-induced thyroid cell dedifferentiation. This suggests that inhibition of this pathway may promote redifferentiation in poorly differentiated thyroid carcinomas with constitutive activation of either Ras or RET/PTC.

Chromosomal rearrangements linking the promoter(s) and N-terminal domain of unrelated gene(s) to the C terminus of RET result in constitutively activated chimeric forms of the receptor in thyroid cells (RET/PTC). RET/PTC rearrangements are thought to be tumor-initiating events; however, the early biological consequences of RET/PTC activation are unknown. To explore this, we generated clonal lines derived from well-differentiated rat thyroid PCCL3 cells with doxycycline-inducible expression of either RET/PTC1 or RET/PTC3. As previously shown in other cell types, RET/PTC1 and RET/PTC3 oligomerized and displayed constitutive tyrosine kinase activity. Neither RET/PTC1 nor RET/PTC3 conferred cells with the ability to grow in the absence of TSH, likely because of concomitant stimulation of both DNA synthesis and apoptosis, resulting in no net growth in the cell population. Effects of RET/PTC on DNA synthesis and apoptosis did not require direct interaction of the oncoprotein with either Shc or phospholipase Cgamma. Acute expression of the oncoprotein decreased TSH-mediated growth stimulation due to interference of TSH signaling by RET/PTC at multiple levels. Taken together, these data indicate that RET/PTC is a weak tumor-initiating event and that TSH action is disrupted by this oncoprotein at several points, and also predict that secondary genetic or epigenetic changes are required for clonal expansion.

Thyroid papillary cancers (PTCs) are associated with activating mutations of genes coding for RET or TRK tyrosine kinase receptors, as well as of RAS genes. Activating mutations of BRAF were reported recently in most melanomas and a small proportion of colorectal tumors. Here we show that a somatic mutation of BRAF, V599E, is the most common genetic change in PTCs (28 of 78; 35.8%). BRAF(V599E) mutations were unique to PTCs, and not found in any of the other types of differentiated follicular neoplasms arising from the same cell type (0 of 46). Moreover, there was no overlap between PTC with RET/PTC, BRAF, or RAS mutations, which altogether were present in 66% of cases. The lack of concordance for these mutations was highly unlikely to be a chance occurrence. Because these signaling proteins function along the same pathway in thyroid cells, this represents a unique paradigm of human tumorigenesis through mutation of three signaling effectors lying in tandem.

PKC isozymes are the major binding proteins for tumor-promoting phorbol esters, and PKC activity is abnormal in a number of different human cancers. Less is known about putative structural and functional changes of specific PKC isozymes in human neoplasms. A single-point mutation of PKCalpha at position 881 of the coding sequence has been observed in human pituitary adenomas and up to 50% of thyroid follicular neoplasms, and a rearrangement of PKCepsilon was reported in a thyroid follicular carcinoma cell line, suggesting that these signaling proteins may play a role in thyroid tumorigenesis. To explore this possibility, we examined thyroid neoplasms for mutations and changes in expression levels of PKCepsilon or alpha. None of the 57 follicular adenomas, 26 papillary carcinomas (PCs), 7 follicular carcinomas, or the anaplastic carcinoma harbored the PKCalpha 881A>G mutation. Moreover, none of 15 PCs, 10 follicular adenomas, or 6 follicular carcinomas showed evidence of mutations of PKCepsilon. However, 8 of 11 PCs had major isozyme-specific reductions of the PKCepsilon protein, which occurred through either translational or posttranslational mechanisms. These data indicate that post-transcriptional changes in PKCepsilon are highly prevalent in thyroid tumors and may play a significant role in their development.

Objective: Thyroid follicular adenomas (FA) and carcinomas (FC) have a high prevalence of aneuploidy. We examined the contribution of mitotic checkpoint dysfunction and mutations of BUB1 or BUBR1, components of the spindle assembly checkpoint pathway, to chromosomal instability in thyroid cancer., Design: The integrity of the mitotic checkpoint was studied in 8 aneuploid thyroid tumour cell lines. All cell lines as well as 9 FA, 9 FC, and 1 aneuploid papillary carcinoma were screened for mutations of BUB1 by SSCP and direct sequencing. Cell lines were also examined for mutations of BUBR1., Results: Neither FRO, NPA nor WRO cells arrested in mitosis after treatment with nocodazole, whereas other aneuploid cell lines paused appropriately following microtubule disruption. One FC had a 2-bp somatic deletion (G2480/A2481) of BUB1 leading to a frameshift, and one FC had a silent polymorphism at nucleotide 1049 (TGT-TGC). There was a silent polymorphism of BUBR1 (G1271A) in one sample., Conclusion: Some, but not all thyroid cancer cell lines with aneuploidy have an abnormal mitotic checkpoint, indicating that chromosomal instability may arise through alternative cell cycle defects. Moreover, mutations of BUB1 or BUBR1 are infrequent in follicular neoplasms, and do not account for aneuploidy in thyroid cancer.

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.

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.

The protein kinase C (PKC) family has been implicated in the regulation of apoptosis. However, the contribution of individual PKC isozymes to this process is not well understood. We reported amplification of the chromosome 2p21 locus in 28% of thyroid neoplasms, and in the WRO thyroid carcinoma cell line. By positional cloning we identified a rearrangement and amplification of the PKCepsilon gene, that maps to 2p21, in WRO cells. This resulted in the overexpression of a chimeric/truncated PKCepsilon (Tr-PKCepsilon) mRNA, coding for N-terminal amino acids 1-116 of the isozyme fused to an unrelated sequence. Expression of the Tr-PKCepsilon protein in PCCL3 cells inhibited activation-induced translocation of endogenous PKCepsilon, but its kinase activity was unaffected, consistent with a dominant negative effect of the mutant protein on activation-induced translocation of wild-type PKCepsilon and/or displacement of the isozyme to an aberrant subcellular location. Cell lines expressing Tr-PKCepsilon grew to a higher saturation density than controls. Moreover, cells expressing Tr-PKCepsilon were resistant to apoptosis, which was associated with higher Bcl-2 levels, a marked impairment in p53 stabilization, and dampened expression of Bax. These findings point to a role for PKCepsilon in apoptosis-signaling pathways in thyroid cells, and indicate that a naturally occurring PKCepsilon mutant that functions as a dominant negative can block cell death triggered by a variety of stimuli.

Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. We now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. We used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3-6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKCepsilon), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKCepsilon as a previously unmapped candidate gene involved in thyroid tumorigenesis.

The mRNAs of transiently expressed proteins such as cytokines and proto-oncogenes are commonly subject to rapid transcriptional activation and degradation. Transcript turnover is determined in part by association of certain proteins with consensus AU-rich motifs (AUUUA) in the 3'-untranslated region of the transcripts. Here we report a modification of differential RNA display (DRD) to detect differentially expressed rapid turnover mRNAs containing AU-rich motifs from thyroid cancer tissues and cell lines. RNA of normal and thyroid cancer tissues was differentially displayed using a 3'anchor primer to the poly(A) tail and an arbitrary 5'primer incorporating an AUUUA sequence. The appropriateness of the strategy was established by its ability to display known early response genes, such as c- fos, using partially degenerate primers. To test whether the novel cDNAs isolated coded for transcripts subject to rapid turnover, they were used as probes for Northern blots of RNA from clonal human thyroid carcinoma cell lines treated for varying periods with either cycloheximide or actinomycin D. A number of novel differentially expressed cDNA fragments were isolated from human papillary thyroid carcinoma tissues, among them a cDNA with zinc finger motifs and homology to other zinc finger proteins. Using this fragment to probe a cDNA library, a full-length cDNA (ZnF20) was isolated that was 4333 bp in length and contained an open reading frame of 1029 amino acids. The ZnF20 cDNA hybridized to multiple transcripts in a thyroid cancer cell line (8.0, 4.5 and 2 kb) that increased after cycloheximide treatment and decayed <2 h after addition of actinomycin D. The ZnF20 mRNA was overexpressed in three of six thyroid papillary carcinomas as compared with paired normal thyroid tissue controls. The data presented here support the use of a targeted DRD approach for the isolation of rapid turnover mRNAs, many of which may be interesting candidate oncogenes.

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using beta-galactosidase-XPG fusion constructs (beta-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized beta-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic beta-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus.

We report here evidence for the mechanism of nuclear localization of XPG nuclease in human cells. Several candidate nuclear localization signal (NLS) peptides have been proposed for XPG protein. We have identified XPG peptides containing functional NLS and a potential nuclear retention signal (NRS) using in situ immunofluorescene localization of transiently expressed beta-galactosidase fusion proteins. Two XPG regions with putative NLS [amino acid (AA) coordinates: NLS-B (AA 1057-1074) and NLS-C (AA 1171-1185)] were each shown to independently localize the beta-gal extensively (> 80%) to the nucleus of HeLa cells. The C-terminus peptide containing NLS-C, an NLS conserved evolutionarily between yeasts and humans, also directed sub-localization of beta-galactosidase to intranuclear foci reminiscent of native XPG protein, as well as to peri-nucleolar regions. Peptides in the putative XPG 'NLS domain' (AA approximately 1051-1185) apparently function in concert for nuclear localization and also for retention of XPG in nuclear matrix-associated foci. Evidence presented elsewhere (Park et al., 1995) indicates that the peptide containing NLS-C (AA 1146-1185) also regulates the dynamic localization of XPG in the nucleus following UV-irradiation.