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Supplementary Figures S1-S2; Supplementary Table S1.

Purpose: This study was designed to explore the role of IQGAP1 in the invasiveness of thyroid cancer and its potential as a novel prognostic marker and therapeutic target in this cancer.Experimental Design: We examined IQGAP1 copy gain and its relationship with clinicopathologic outcomes of thyroid cancer and investigated its role in cell invasion and molecules involved in the process.Results: We found IQGAP1 copy number (CN) gain ≥3 in 1 of 30 (3%), 24 of 74 (32%), 44 of 107 (41%), 8 of 16 (50%), and 27 of 41 (66%) of benign thyroid tumor, follicular variant papillary thyroid cancer (FVPTC), follicular thyroid cancer (FTC), tall cell papillary thyroid cancer (PTC), and anaplastic thyroid cancer, respectively, in the increasing order of invasiveness of these tumors. A similar tumor distribution trend of CN ≥4 was also seen. IQGAP1 copy gain was positively correlated with IQGAP1 protein expression. It was significantly associated with extrathyroidal and vascular invasion of FVPTC and FTC and, remarkably, a 50%–60% rate of multifocality and recurrence of BRAF mutation–positive PTC (P = 0.01 and 0.02, respectively). The siRNA knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion and colony formation. Coimmunoprecipitation assay showed direct interaction of IQGAP1 with E-cadherin, a known invasion-suppressing molecule, which was upregulated when IQGAP1 was knocked down. This provided a mechanism for the invasive role of IQGAP1 in thyroid cancer. In contrast, IQGAP3 lacked all these functions.Conclusions: IQGAP1, through genetic copy gain, plays an important role in the invasiveness of thyroid cancer and may represent a novel prognostic marker and therapeutic target for this cancer. Clin Cancer Res; 16(24); 6009–18. ©2010 AACR.

Management of metastatic radioiodine refractory differentiated thyroid cancer (DTC) can be a therapeutic challenge. Generally, little is known about the paired molecular profile of the primary tumor and the metastases and whether they harbor the same genetic abnormalities. The present study compared the molecular profile of paired tumor specimens (primary tumor/metastatic sites) from patients with radioiodine refractory DTC in order to gain insight into a possible basis for resistance to radioiodine. Twelve patients with radioiodine refractory metastases were studied; median age at diagnosis of 61 years (range, 25–82). Nine patients had papillary TC (PTC), one had follicular TC (FTC), and two had Hürthle cell TC (HTC). Distant metastases were present in the lungs (n = 10), bones (n = 4), and liver (n = 1). The molecular profiling of paired tumors was performed with a panel of 592 genes for Next Generation Sequencing, RNA-sequencing, and immunohistochemistry. Digital microfluidic PCR was used to investigateTERTpromoter mutations. The genetic landscape of all paired sites comprisedBRAF,NRAS,HRAS,TP53,ATM,MUTYH,POLE, andNTRKgenes, includingBRAFandNTRKfusions.BRAFV600E was the most common point mutation in the paired specimens (5/12).TERTpromoter mutation C228T was detected in one case. PD-L1 expression at metastatic sites was highly positive (95%) for one patient with HTC. All specimens were stable for microsatellite instability testing, and the tumor mutation burden was low to intermediate. Therefore, the molecular profile of DTC primary and metastatic lesions can show heterogeneity, which may help explain some altered responses to therapeutic intervention.

Management of metastatic radioiodine refractory differentiated thyroid cancer (DTC) can be a therapeutic challenge. Generally, little is known about the paired molecular profile of the primary tumor and the metastases and whether they harbor the same genetic abnormalities. The present study compared the molecular profile of paired tumor specimens (primary tumor/metastatic sites) from patients with radioiodine refractory DTC in order to gain insight into a possible basis for resistance to radioiodine. Twelve patients with radioiodine refractory metastases were studied; median age at diagnosis of 61 years (range, 25–82). Nine patients had papillary TC (PTC), one had follicular TC (FTC), and two had Hürthle cell TC (HTC). Distant metastases were present in the lungs (n = 10), bones (n = 4), and liver (n = 1). The molecular profiling of paired tumors was performed with a panel of 592 genes for Next Generation Sequencing, RNA-sequencing, and immunohistochemistry. Digital microfluidic PCR was used to investigate TERT promoter mutations. The genetic landscape of all paired sites comprised BRAF, NRAS, HRAS, TP53, ATM, MUTYH, POLE, and NTRK genes, including BRAF and NTRK fusions. BRAF V600E was the most common point mutation in the paired specimens (5/12). TERT promoter mutation C228T was detected in one case. PD-L1 expression at metastatic sites was highly positive (95%) for one patient with HTC. All specimens were stable for microsatellite instability testing, and the tumor mutation burden was low to intermediate. Therefore, the molecular profile of DTC primary and metastatic lesions can show heterogeneity, which may help explain some altered responses to therapeutic intervention.

Metformin inhibits thyroid cancer cell growth. We sought to determine if variable glucose concentrations in medium alter the anti-cancer efficacy of metformin. Thyroid cancer cells (FTC133 and BCPAP) were cultured in high-glucose (20 mM) and low-glucose (5 mM) medium before treatment with metformin. Cell viability and apoptosis assays were performed. Expression of glycolytic genes was examined by real-time PCR, western blot, and immunostaining. Metformin inhibited cellular proliferation in high-glucose medium and induced cell death in low-glucose medium. In low-, but not in high-glucose medium, metformin induced endoplasmic reticulum stress, autophagy, and oncosis. At micromolar concentrations, metformin induced phosphorylation of AMP-activated protein kinase and blocked p-pS6 in low-glucose medium. Metformin increased the rate of glucose consumption from the medium and prompted medium acidification. Medium supplementation with glucose reversed metformin-inducible morphological changes. Treatment with an inhibitor of glycolysis (2-deoxy-d-glucose (2-DG)) increased thyroid cancer cell sensitivity to metformin. The combination of 2-DG with metformin led to cell death. Thyroid cancer cell lines were characterized by over-expression of glycolytic genes, and metformin decreased the protein level of pyruvate kinase muscle 2 (PKM2). PKM2 expression was detected in recurrent thyroid cancer tissue samples. In conclusion, we have demonstrated that the glucose concentration in the cellular milieu is a factor modulating metformin's anti-cancer activity. These data suggest that the combination of metformin with inhibitors of glycolysis could represent a new strategy for the treatment of thyroid cancer.

The HIV protease inhibitor Nelfinavir (NFV) inhibits PI3K/AKT and MAPK/ERK signaling pathways, emerging targets in thyroid cancers. We examined the effects of NFV on cancer cells that derived from follicular (FTC), papillary (PTC) and anaplastic (ATC) thyroid cancers. NFV (1–20 µM) was tested in FTC133, BCPAP and SW1736 cell lines. The effects of NFV on cell proliferation were determinedin vitrousing real-time microscopy and by flow cytometry. DNA damage, apoptotic cell death and expression of molecular markers of epithelial–mesenchymal transition (EMT) were determined by Western blot and real-time PCR. Real-time imaging demonstrated that NFV (10 µM) increased the time required for the cell passage through the phases of cell cycle and induced DNA fragmentation. Growth inhibitory effects of NFV were associated with the accumulation of cells in G0/G1 phase, downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). NFV also induced the expression of γH2AX and p53BP1 indicating DNA damage. Treatment with NFV (20 µM) resulted in caspase-3 cleavage in all examined cells. NFV (20 µM) decreased the levels of total and p-AKT in PTEN-deficient FTC133 cells. NFV had no significant effects on total ERK and p-ERK in BRAF-positive BCPAP and SW1736 cells. NFV had no effects on the expression of EMT markers (Twist, Vimentin, E- and N-Cadherin), but inhibited the migration and decreased the abilities of thyroid cancer cells to survive in non-adherent conditions. We conclude that NFV inhibits proliferation and induces DNA damage in thyroid cancer cell lines. Ourin vitrodata suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.

et al., [Context]: Individualized management, incorporating papillary thyroid cancer (PTC) variant-specific risk, is conceivably a useful treatment strategy for PTC, which awaits comprehensive data demonstrating differential risks of PTC variants to support. [Objective]: This study sought to establish the differential clinicopathological risk of major PTC variants: conventional PTC (CPTC), follicular-variant PTC (FVPTC), and tall-cell PTC (TCPTC). [Methods]: This was a retrospective study of clinicopathological outcomes of 6282 PTC patients (4799 females and 1483 males) from 26 centers and The Cancer Genome Atlas in 14 countries with a median age of 44 years (interquartile range, 33-56 y) and median follow-up time of 37 months (interquartile range, 15-82 mo)., [Results]: The cohort consisted of 4702 (74.8%) patients with CPTC, 1126 (17.9%) with FVPTC, and 239 (3.8%) with TCPTC. The prevalence of high-risk parameters was significantly different among the three variants, including extrathyroidal invasion, lymph node metastasis, stages III/IV, disease recurrence, mortality, and the use (need) of radioiodine treatment (all P < .001), being highest in TCPTC, lowest in FVPTC, and intermediate in CPTC, following an order of TCPTC > CPTC ≫ FVPTC. Recurrence and mortality in TCPTC, CPTC, and FVPTC were 27.3 and 6.7%, 16.1 and 2.5%, and 9.1 and 0.6%, corresponding to events per 1000 person-years (95% confidence interval [CI]) of 92.47 (64.66-132.26) and 24.61 (12.31-49.21), 34.46 (30.71-38.66), and 5.87 (4.37-7.88), and 24.73 (18.34-33.35) and 1.68 (0.54-5.21), respectively. Mortality hazard ratios of CPTC and TCPTC over FVPTC were 3.44 (95% CI, 1.07-11.11) and 14.96 (95% CI, 3.93-56.89), respectively. Kaplan-Meier survival analyses showed the best prognosis in FVPTC, worst in TCPTC, and intermediate in CPTC in disease recurrence-free probability and disease-specific patient survival. This was particularly the case in patients at least 45 years old. [Conclusion]: This large multicenter study demonstrates differential prognostic risks of the three major PTC variants and establishes a unique risk order of TCPTC > CPTC ≫ FVPTC, providing important clinical implications for specific variant-based management of PTC., This work was supported by the US National Institutes of Health (NIH) Grants No. RO1CA113507 and R01CA189224 (to M.X.). In addition, the studies at individual centers were supported as follows: National Science Centre Poland Grants No. N403 194340 and N N401 612440 to A.C. and B.J., respectively, and Milestone Grant No. 267398 to both (Poland); Grants from Queensland Government Smart State Fellowship and Griffith Health Institute to A.K.L. (Australia); Grants No. RD12/0036/0030 FIS-ISCIII, S2011/BMD-2328 TIRONET, and SAF2013-44709-R to P.So. (Spain); grants from Fondazione Cassa di Risparmio di Perugia and Associazione Italiana per la Ricerca sul Cancro (IG 9338) (Italy) and the Beadle Family Foundation (San Antonio, TX) to E.P.; Grant IGA MH CR NT 13901-4 to V.S. and B.B. (the Czech Republic); grants from the New South Wales Cancer Institute to C.J.O. and from Cancer Council of New South Wales to R.C.-B. (Australia); Italian Government-Ministero della Salute Grant No. RF-2011-02350857 to G.T. (Italy); Grant NIH/NIA 5R03AG042334-02 to L.Y. (United States); Grants from the Ministerodella Istruzione Universitaria e Ricerca Scientifica, the AssociazioneItaliana per la Ricerca sul Cancro, the Istituto Toscano Tumori, and the Ministero della Salute to D.V. and R.E (Italy); and Grant No. CB-2011-03-02 from the Korean Foundation for Cancer Research to Y.K.S. and T.Y.K. (South Korea); Research Grants 2012/02902-9 and 2013/03867-5 from The São Paulo State Research Foundation (FAPESP) to J.M.C. (G.O. is a FAPESP scholar and J.M.C. is a Brazilian Research Council investigator (Brazil); AIRC Grant No. IG 10316 to F.B. (Italy); Grant No. SHDC 12014229 from Shanghai Hospital Development Center to H.X. (China); Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional, and through the Fundo Europeu de Desenvolvimento Regional to M.S.-S. and P.So.

Resistance to anoikis (matrix deprivation-induced apoptosis) is a critical component of the metastatic cascade. Molecular mechanisms underlying resistance to anoikis have not been reported in thyroid cancer cells. For an in vitro model of anoikis, we cultured follicular, papillary, and anaplastic thyroid cancer cell lines on poly-HEMA-treated low-adherent plates. We also performed immunohistochemical analysis of human cancer cells that had infiltrated blood and/or lymphatic vessels. Matrix deprivation was associated with establishment of contacts between floating thyroid cancer cells and formation of multi-cellular spheroids. This process was associated with activation of gap junctional transfer. Increased expression of the gap junction molecule Connexin43 was found in papillary and anaplastic cancer cells forming spheroids. All non-adherent cancer cells showed a lower proliferation rate compared with adherent cells but were more resistant to serum deprivation. AKT was constitutively activated in cancer cells forming spheroids. Inhibition of gap junctional transfer through Connexin43 silencing, or by treatment with the gap junction disruptor carbenoxolone, resulted in loss of pAKT and induction of apoptosis in a cell-type-specific manner. In human thyroid tissue, cancer cells that had infiltrated blood vessels showed morphological similarity to cancer cells forming spheroids in vitro. Intra-vascular cancer cells demonstrated prominent AKT activation in papillary and follicular cancers. Increased Connexin43 immunoreactivity was observed only in intra-vascular papillary cancer cells. Our data demonstrate that establishment of inter-cellular communication contributes to thyroid cancer cell resistance to anoikis. These findings suggest that disruption of gap junctional transfer could represent a potential therapeutic strategy for prevention of metastases.

Purpose Biliary cancers (BCs) carry a poor prognosis, but targeting the RAS/RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) pathway is of significance. Selumetinib is an inhibitor of MEK1/2, so this trial was designed to determine the safety and efficacy of selumetinib in BC. Patients and Methods This was a multi-institutional phase II study of selumetinib at 100 mg given orally twice per day to patients with advanced BC. The primary end point was response rate. All patients were required to provide tissue before enrolling. The levels of phosphorylated ERK (pERK) and AKT (pAKT) were assessed by immunohistochemistry. Tumors were genotyped for the presence of BRAF- and/or RAS-activating mutations. Results Twenty-eight eligible patients with a median age of 55.6 years were enrolled. Thirty-nine percent of patients had received one prior systemic therapy. Three patients (12%) had a confirmed objective response. Another 17 patients (68%) experienced stable disease (SD), 14 of whom (56%) experienced prolonged SD (> 16 weeks). Patients gained an average nonfluid weight of 8.6 pounds. Median progression-free survival was 3.7 months (95% CI, 3.5 to 4.9) and median overall survival was 9.8 months (95% CI, 5.97 to not available). Toxicities were mild, with rash (90%) and xerostomia (54%) being most frequent. Only one patient experienced grade 4 toxicity (fatigue). All patients had tissue available for analysis. No BRAF V600E mutations were found. Two patients with short-lived SD had KRAS mutations. Absence of pERK staining was associated with lack of response. Conclusion Selumetinib displays interesting activity and acceptable tolerability in patients with metastatic BC. Our results warrant further evaluation of selumetinib in patients with metastatic BC.

Purpose: This study was designed to explore the role of IQGAP1 in the invasiveness of thyroid cancer and its potential as a novel prognostic marker and therapeutic target in this cancer. Experimental Design: We examined IQGAP1 copy gain and its relationship with clinicopathologic outcomes of thyroid cancer and investigated its role in cell invasion and molecules involved in the process. Results: We found IQGAP1 copy number (CN) gain ≥3 in 1 of 30 (3%), 24 of 74 (32%), 44 of 107 (41%), 8 of 16 (50%), and 27 of 41 (66%) of benign thyroid tumor, follicular variant papillary thyroid cancer (FVPTC), follicular thyroid cancer (FTC), tall cell papillary thyroid cancer (PTC), and anaplastic thyroid cancer, respectively, in the increasing order of invasiveness of these tumors. A similar tumor distribution trend of CN ≥4 was also seen. IQGAP1 copy gain was positively correlated with IQGAP1 protein expression. It was significantly associated with extrathyroidal and vascular invasion of FVPTC and FTC and, remarkably, a 50%–60% rate of multifocality and recurrence of BRAF mutation–positive PTC (P = 0.01 and 0.02, respectively). The siRNA knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion and colony formation. Coimmunoprecipitation assay showed direct interaction of IQGAP1 with E-cadherin, a known invasion-suppressing molecule, which was upregulated when IQGAP1 was knocked down. This provided a mechanism for the invasive role of IQGAP1 in thyroid cancer. In contrast, IQGAP3 lacked all these functions. Conclusions: IQGAP1, through genetic copy gain, plays an important role in the invasiveness of thyroid cancer and may represent a novel prognostic marker and therapeutic target for this cancer. Clin Cancer Res; 16(24); 6009–18. ©2010 AACR.

Context: Genetic alterations in receptor tyrosine kinases (RTKs) and phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK pathways have not been fully defined in anaplastic and follicular thyroid cancers [anaplastic thyroid cancer (ATC), follicular thyroid cancer (FTC)]. Objective: The objective of the study was to explore a wide-range genetic basis for the involvement of these pathways in ATC. Design: We examined mutations and copy number gains of a large panel of genes in these pathways and corresponding phosphorylation of ERK (p-ERK) and Akt. Results: We found frequent copy gains of RTK genes, including EGFR, PDGFRα and -β, VEGFR1 and 2, KIT, and MET and in PIK3Ca, PIK3Cb, and PDK1 genes in the PI3K/Akt pathway. Mutations of Ras, PIK3Ca, PTEN, and BRAF genes and RET/PTC rearrangements were common, whereas mutations in PDK1, Akt1, Akt2, and RTK genes were uncommon in ATC. Overall, 46 of 48 ATC (95.8%) harbored at least one genetic alteration, and coexistence of two or more was seen in 37 of 48 ATC (77.1%). These genetic alterations were somewhat less common in FTC. Genetic alterations that could activate both the PI3K/Akt and MAPK pathways were found in 39 of 48 ATC (81.3%). RTK gene copy gains were preferentially associated with p-Akt, suggesting their dominant role in activating the PI3K/Akt pathway. The phosphorylation of Akt was far more common than p-ERK in FTC, and both were relatively common and often coexisted in ATC. Conclusions: Genetic alterations in the RTKs and PI3K/Akt and MAPK pathways are extremely prevalent in ATC and FTC, providing a strong genetic basis for an extensive role of these signaling pathways and the development of therapies targeting these pathways for ATC and FTC, particularly the former.

Papillary thyroid carcinomas (PTCs) that invade into local structures are associated with a poor prognosis, but the mechanisms for PTC invasion are incompletely defined, limiting the development of new therapies. To characterize biological processes involved in PTC invasion, we analyzed the gene expression profiles of microscopically dissected intratumoral samples from central and invasive regions of seven widely invasive PTCs and normal thyroid tissue by oligonucleotide microarray and performed confirmatory expression and functional studies. In comparison with the central regions of primary PTCs, the invasive fronts overexpressed TGF β, NFκB and integrin pathway members, and regulators of small G proteins and CDC42. Moreover, reduced levels of mRNAs encoding proteins involved in cell–cell adhesion and communication were identified, consistent with epithelial-to-mesenchymal transition (EMT). To confirm that aggressive PTCs were characterized by EMT, 34 additional PTCs were examined for expression of vimentin, a hallmark of EMT. Overexpression of vimentin was associated with PTC invasion and nodal metastasis. Functional, in vitro studies demonstrated that vimentin was required both for the development and maintenance of a mesenchymal morphology and invasiveness in thyroid cancer cells. We conclude that EMT is common in PTC invasion and that vimentin regulates thyroid cancer EMT in vitro .

Although the follicular variant of papillary thyroid carcinoma (FVPTC) has been classified as a papillary cancer based on nuclear features, its follicular growth pattern and potential for hematogenous spread are more characteristic of follicular carcinoma. To gain insight into the biologic nature of FVPTC, we compared genetic alterations characteristic of papillary and follicular thyroid carcinomas in 24 FVPTCs and 26 classic PTC (CPTCs). In FVPTCs, we observed ras mutation in 6 of 24 cases (25%), BRAF mutation in 1 of 13 cases (7.6%), and ret rearrangement in 5 of 12 cases (41.7%). In CPTCs, we found ras mutation in no case, BRAF mutation in 3 of 10 cases (30%), and ret rearrangement in 5 of 11 cases (45%). One FVPTC exhibited simultaneous ras mutation and ret/PTC1 rearrangement, and one CPTC harbored simultaneous BRAF mutation and ret/PTC3 rearrangement. Based on these findings, we concluded that ras mutation correlates with follicular differentiation of thyroid tumors whereas ret activation is associated with papillary nuclei but not with papillary architecture. ret activation is not exclusive of ras or BRAF mutation, whereas ras and BRAF mutations are mutually exclusive. The implications of these results for follicular and papillary carcinogenesis are discussed.

The role of the T1799A BRAF mutation in lymph node metastasis of papillary thyroid cancer (PTC) is not clear.Our objective was to explore the relationship between BRAF mutation and lymph node metastasis of PTC by examining the mutation in both the primary tumors and their paired lymph node metastases.We isolated genomic DNA from primary thyroid tumors and paired lymph node metastases and performed direct sequencing of exon 15 of the BRAF gene mutation that carries the T1799A mutation.In a series of 33 cases, 21 harbored the T1799A mutation in the primary tumors, and 17 (81%) of them harbored the same mutation also in the paired lymph node metastases. Twelve cases did not harbor the T1799A mutation in the primary tumors, among which nine cases also did not harbor BRAF mutation in the lymph node-metastasized tumors, whereas the other three did harbor the T1799A mutation in lymph node-metastasized tumor tissues. A novel tandem TG1799-1800AA mutation within one allele was found in a lymph node-metastasized tumor but not in the primary tumor. This mutation results in the change of codon 600 (GTG) of the gene to GAA with the consequent amino acid change (V600E) in the B-type Raf (BRAF) protein, same as that caused by the T1799A mutation alone.The high prevalence of BRAF mutation in lymph node-metastasized PTC tissues from BRAF mutation-positive primary tumors and the possible de novo formation of BRAF mutation in lymph node-metastasized PTC were consistent with a role of BRAF mutation in facilitating the metastasis and progression of PTC in lymph nodes.

Thyroid follicular adenomas (FA) are encapsulated tumors lacking vascular, capsular or lymphatic invasion and the typical nuclear features of papillary carcinoma (PC). However, some FA demonstrate nuclear atypia reminiscent of either follicular carcinomas (FC) or follicular variant of papillary carcinomas (FVPC), suggesting they may represent precursors of malignant transformation. We hypothesized that an objective evaluation of nuclear chromatin patterns could be used to define atypical follicular tumors (AFT) that are likely to be premalignant. To test this hypothesis, we used a computer-aided image analysis system to define the chromatin pattern of nuclei from thyroid tumors. To validate the system, we analyzed 3000 nuclei from 10 FA, 10 FC, and 10 FVPC samples and accurately distinguished between these classes of tumors. Then, we analyzed nine AFT and, in parallel, we analyzed the tumors for activating mutations of N2-RAS and over-expression of RET. The predominant chromatin pattern of AFT was of FA type in two cases, FC type in two cases, and PC type in three cases. One case contained similar numbers of FC and PC nuclei and one was comprised of a mixture of the three nuclear types. Neither RAS mutation nor RET overexpression were detected in FA. N2-RAS mutations were found in 33% of AFT, 20% of FC and 20% of FVPC without correlation with chromatin pattern. Over-expression of RET was detected in 45% of AFT, 20% of FC and 50% of FVPC and was correlated with PC nuclei. These results show that AFT are a heterogenous group of tumors, containing genuine benign tumors and tumors that share morphological and molecular features with follicular and papillary carcinomas that might be precursors of both types of thyroid carcinomas.

The RET/PTC3 oncogene is a genetically rearranged and constitutively activated tyrosine kinase receptor that is common in papillary thyroid cancer. Because RET/PTC3 is chronically overexpressed in these thyroid cancer cells, and RET/PTC3-expressing tumors are associated with overactivity of tyrosine kinase signaling pathways and a more aggressive clinical course, we questioned whether chronic RET/PTC3 expression enhances cellular responses to thyroid mitogens in vitro. We stably transfected FRTL-5 cells with the RET/PTC3 gene; transfected and control cell lines were cultured without insulin, TSH, or serum. Thymidine incorporation into DNA was enhanced in the RET/PTC3 cells, but transformation was not observed. RET/PTC3 cells demonstrated higher basal and insulin-stimulated levels of activated Akt, both of which were reduced by LY294002, a PI3 kinase inhibitor, but not PD98059, a MEK inhibitor. By contrast, mitogen activated protein kinase (MAP kinase) was only minimally activated in RET/PTC3 cells before and after stimulation. Consistent with preferential activation of PI3 kinase, increased levels of total and phosphorylated IRS2 protein, relative activation of PDK-1, and enhanced IRS2-p85 interactions were identified in RET/PTC3-expressing cells. RET/PTC3 cells were also sensitized to insulin-induced thymidine incorporation; this effect was blocked by PI3 kinase (LY294002) rather than MEK 1/2 (PD98059) inhibitors. In summary, we have demonstrated that RET/PTC3 expression enhances basal and insulin-stimulated DNA synthesis through PI3 kinase, cooperatively activates Akt with insulin via PI3 kinase, and preferentially activates the Akt rather than MAP kinase pathway in FRTL-5 cells.

Context. There is limited data comparing results of fine needle aspiration biopsies (FNABs) to histological diagnosis in children. Design. FNABs were performed in 707 children and cytological results were compared to histology in 165 cases. The usefulness of immunostaining with anti-TPO monoclonal antibodies (MoAb47) on FNAB samples was examined in 54 operated patients. Results. Among unsatisfactory, benign, suspicious, and malignant FNAB, the histological diagnoses were benign in 12/12 (100%), 69/70 (98.5%), 40/50 (80.0%), and 0/33 (0%), respectively. After surgery, malignancy was established in 44/165 (26.6%) cases. The sensitivity, specificity, and positive and negative predictive values were 95.4%, 55.8%, 61.7%, and 95% with standard FNAB; and 100%, 75%, 73.3, and 100% with MoAb47. Among suspicious FNAB, positive MoAb47 staining was a reliable marker for exclusion of malignancy. Conclusion. Benign and malignant FNAB accurately predict histological diagnosis. In suspicious FNAB, MoAb47 immunostaining may be a useful adjunct to standard cytology.

Purpose: To investigate the overall occurrence and relationship of genetic alterations in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in thyroid tumors and explore the scope of this pathway as a therapeutic target for thyroid cancer. Experimental Design: We examined collectively the major genetic alterations and their relationship in this pathway, including PIK3CA copy number gain and mutation, Ras mutation, and PTEN mutation, in a large series of primary thyroid tumors. Results: Occurrence of any of these genetic alterations was found in 25 of 81 (31%) benign thyroid adenoma (BTA), 47 of 86 (55%) follicular thyroid cancer (FTC), 21 of 86 (24%) papillary thyroid cancer (PTC), and 29 of 50 (58%) anaplastic thyroid cancer (ATC), with FTC and ATC most frequently harboring these genetic alterations. PIK3CA copy gain was associated with increased PIK3CA protein expression. A mutual exclusivity among these genetic alterations was seen in BTA, FTC, and PTC, suggesting an independent role of each of them through the PI3K/Akt pathway in the tumorigenesis of the differentiated thyroid tumors. However, coexistence of these genetic alterations was increasingly seen with progression from differentiated tumor to undifferentiated ATC. Their coexistence with BRAF mutation was also frequent in PTC and ATC. Conclusions: The data provide strong genetic implication that aberrant activation of PI3K/Akt pathway plays an extensive role in thyroid tumorigenesis, particularly in FTC and ATC, and promotes progression of BTA to FTC and to ATC as the genetic alterations of this pathway accumulate. Progression of PTC to ATC may be facilitated by coexistence of PI3K/Akt pathway–related genetic alterations and BRAF mutation. The PI3K/Akt pathway may thus be a major therapeutic target in thyroid cancers.

The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation-induced gene silencing in PTC-derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase-3 (TIMP3), SLC5A8, death-associated protein kinase (DAPK) and retinoic acid receptor b2 (RARb2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall-cell PTC subtypes than in less aggressive follicularvariant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARb2 ,i n association with BRAF mutation, may be an important step in PTC tumorigenesis and progression. ' 2006 Wiley-Liss, Inc.

The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregulation in tumors is a common finding. The molecular basis of follicular thyroid cancer metastasis is not well understood but may also be influenced by AKT activation. We previously created a knockin mutant mouse that expresses a mutant thyroid hormone receptor-beta gene (TRbetaPV mouse) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. In this study, we investigated whether our mouse model exhibits similar AKT activation as human follicular thyroid cancer. Western blot analysis on thyroids from both wild-type and TRbeta(PV/PV) mice revealed elevation of activated AKT in TRbeta(PV/PV) mice. Immunohistochemistry and confocal microscopy reveal activated AKT in both the thyroid and metastatic lesions of TRbeta(PV/PV) mice. Whereas all three AKT isoforms were overexpressed in primary tumors from TRbeta(PV/PV) mice in the cytoplasm of thyroid cancer cells, only AKT1 was also found in the nucleus, matching the localization of activated AKT in a pattern similar to human follicular thyroid cancer. In the metastases, all AKT isoforms correlated with phosphorylated AKT nuclear localization. We created primary thyroid cell lines derived from TRbeta(PV/PV) mice and found reduction of phosphorylated AKT levels or AKT downstream targets diminishes cell motility. Activated AKT is common to both human and mouse follicular thyroid cancer and is correlated with increased cell motility in vitro and metastasis in vivo. Thus, TRbeta(PV/PV) mice could be used to further dissect the detailed pathways underlying the progression and metastasis of follicular thyroid carcinoma.

Tumor metastasis is a critical determinant of death from cancer. Metastin, a product of the KiSS-1 gene, is an endogenously expressed metastasis suppressor that is the ligand for G protein-coupled receptor 54 (GPR54), a Gq/11-coupled receptor. In the present study, our goal was to define the basis of GPR54 action using thyroid cancer cells as a model.We used GPR54-null thyroid cancer cells to create a stable GPR54 overexpression model. Cell growth and cell migration of the GPR54-expressing lines were inhibited by recombinant metastin, and metastin stimulated the protein kinase C, ERK, and phosphatidylinositol-3-kinase pathways. To identify metastin-regulated genes, we performed microarray analyses using RNA isolated from GPR54 stable transfectants before and after 1 and 24 h of metastin stimulation. Consistent increases in expression of the gene encoding myocyte-enriched calcineurin interacting protein 1 (MCIP-1), an inhibitor of calcineurin, were identified and confirmed using real-time RT-PCR and Western blot. Functionally, metastin treatment of GPR54-expressing cells initially increased calcineurin activity, followed by a prolonged reduction in calcineurin activity for 24 and 48 h, consistent with the pattern of MCIP-1 expression. In addition, treatment with cyclosporin A, a calcineurin inhibitor, blocked cell migration. Lymph node metastasis in papillary thyroid cancers demonstrated loss of MCIP-1 expression in comparison with primary tumors.These data suggest a role for MCIP-1 and calcineurin inhibition in GPR54-mediated metastasis suppression in human cancers.

Nuclear Akt1 expression and Akt activation are common in cancer invasion. However, the mechanisms for this association and its causal role in invasion are uncertain. In an effort to identify potential mechanisms for regulating Akt subcellular localization, we analyzed the Akt gene sequences and identified a highly conserved leucine-rich potential nuclear export sequence (NES). Initial experiments demonstrated that leptomycin B induced nuclear Akt1 localization. Transient expression experiments demonstrated that, in comparison to wild-type Akt1, NES-mutated (AKT/NES) Akt1 has reduced interactions with CRM-1 and persistent nuclear localization. Subsequent stable transfection experiments in Akt1{sup -/-} fibroblasts confirmed that expression of AKT/NES resulted in persistent nuclear localization and activation1. Finally, stable expression of AKT/NES in Akt1{sup -/-} fibroblasts was sufficient to enhance cell migration in vitro. Thus, Akt1 contains a functional NES and mutation of the NES results in nuclear-predominant Akt1 activation that is sufficient to induce migration.

Context: Use of BRAF mutation in papillary thyroid cancer (PTC) has the potential to improve risk stratification of this cancer. Objective: The objective of the study was to investigate the prognostic value of BRAF mutation in patients with PTC. Design, Setting, and Subjects: In a multicenter study of 219 PTC patients, data on their clinicopathological characteristics and clinical courses between 1990 and 2004 were retrospectively collected, and their tumor BRAF mutation status was determined. Associations of BRAF mutation with initial tumor characteristics and subsequent recurrence were analyzed. Main Outcome Measure: Relationships between the BRAF mutation status and clinicopathological outcomes, including recurrence, were measured. Results: We found a significant association between BRAF mutation and extrathyroidal invasion (P < 0.001), lymph node metastasis (P < 0.001), and advanced tumor stage III/IV (P = 0.007) at initial surgery. This association remained significant on multivariate analysis, adjusting for conventional clinicopathological predictors of recurrence excluding the histological PTC subtype, but was lost when the tumor subtype was included in the model. BRAF mutation was also significantly associated with tumor recurrence, 25 vs. 9% with and without mutation, respectively (P = 0.004), during a median of 15 (interquartile range, 3–29) months of follow-up. This association remained significant on multivariate analysis adjusting for conventional clinicopathological predictors of recurrence, even including the PTC subtype (odds ratio, 4.0; 95% confidence interval, 1.1–14.1; P = 0.03). BRAF mutation was even an independent predictor of recurrence in patients with stage I/II disease, 22 vs. 5% with and without BRAF mutation, respectively (P = 0.002). BRAF mutation was also more frequently associated with absence of tumor I-131 avidity and treatment failure of recurrent disease. Conclusions: In patients with PTC, BRAF mutation is associated with poorer clinicopathological outcomes and independently predicts recurrence. Therefore, BRAF mutation may be a useful molecular marker to assist in risk stratification for patients with PTC.

A high prevalence of activating mutation of the B type Raf kinase (BRAF) gene was recently reported in papillary thyroid cancer (PTC). However, the frequency of this mutation in several other types of thyroid neoplasms was not thoroughly investigated. In the present study, in addition to PTC, we evaluated various thyroid tumor types for the most common BRAF T1796A mutation by direct genomic DNA sequencing. We found a high and similar frequency (45%) of the BRAF T1796A mutation in two geographically distinct PTC patient populations: one composed of sporadic cases from North America, and the other from Kiev, Ukraine, that included individuals who were exposed to the Chernobyl nuclear accident. In contrast, we found BRAF mutation in only 20% of anaplastic thyroid cancers and no mutation in medullary thyroid cancers and benign thyroid hyperplasia. We also confirmed previous reports that the BRAF T1796A mutation did not occur in benign thyroid adenomas and follicular thyroid cancers. Specific analysis of the Ukraine patients with confirmed history of radiation exposure failed to show a higher incidence of BRAF mutation. Our results suggest that frequent occurrence of BRAF mutation is inherently associated with PTC, irrespective of geographic origin, and is apparently not a radiation-susceptible mutation. The lack or low prevalence of BRAF mutation in other thyroid neoplasms is consistent with the notion that other previously defined genetic alterations on the same signaling pathway are sufficient to cause tumorigenesis in most thyroid neoplasms.

Introduction: Akt activation is involved in the pathogenesis of inherited thyroid cancer in Cowden's syndrome and in sporadic thyroid cancers. In cell culture, Akt regulates thyroid cell growth and survival; but recent data suggest that Akt also regulates cell motility in non-thyroid cell lines. We therefore sought to evaluate the role of Akt in thyroid cancer progression. Methods: We evaluated 46 thyroid cancer, 20 thyroid follicular adenoma, and adjacent normal tissues samples by immunohistochemistry for activated Akt (pAkt), Akt 1, 2, and 3, and p27 expression. Immunoblots were performed in 14 samples. Results: Akt activation was identified in 10/10 follicular cancers, 26/26 papillary cancers, and 2/10 follicular variant of papillary cancers, but in only 4/66 normal tissue samples and 2/10 typical benign follicular adenomas. Immunoactive pAkt was greatest in regions of capsular invasion; and was localised to the nucleus in follicular cancers and the cytoplasm in papillary cancers, except for invasive regions of papillary cancers where it localised to both compartments. Immunoactive Akt 1, but not Akt 2 or Akt 3, correlated with pAkt localisation, and nuclear pAkt was associated with cytoplasmic expression of p27. In vitro studies using human thyroid cancer cells demonstrated that nuclear translocation of Akt 1 and pAkt were associated with cytoplasmic p27 and cell invasion and migration. Cell migration and the localisation of Akt 1, pAkt, and p27 were inhibited by PI3 kinase, but not MEK inhibition. Discussion: These data suggest an important role for nuclear activation of Akt 1 in thyroid cancer progression.