Clinical and molecular characterization of thyroid cancer when seen as a second malignant neoplasm.

BACKGROUND: Second malignant neoplasms (SMN) are among the most serious long-term adverse health conditions in cancer survivors. The aim of this study was to characterize clinical findings of patients who developed thyroid cancers as SMN, and to examine genomic alterations in thyroid cancer tissue. METHODS: Retrospective analysis of medical records from patients seen for management of thyroid cancer over 10-year period was performed. Clinical and pathologic data were retrieved from their medical charts. Tumor DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and subjected to next-generation sequencing (NGS) using Ion Torrent Oncomine Focus Assay. Microfluidic digital polymerase chain reactions (PCRs) were performed using QIAcuity Digital PCR System to identify BRAF V600E mutations and RET/PTC fusions. RESULTS: Sixteen of 620 patients operated for thyroid cancer had history of previously diagnosed malignancy. Eight patients were male and eight patients were female, with a median age at diagnosis of 58.5 years (range, 4-78). Four patients had history of pediatric malignancy (PedCa), and 12 patients had a history of prior malignancy as an adult (AdCa). The latency periods for development of SMN in PedCa and AdCa patients were 10.8 (±5.2) years and 9.5 (±5.2) years, respectively. Histopathology revealed papillary thyroid cancers in 15 cases, and follicular thyroid cancer in 1 case. All tumors were classified as T1 or T2, and there were no patients presenting with metastases at the time of surgery. Genomic alterations were detected in 13/16 (81.2%) tumors including eight gene mutations (BRAF V600E (N = 4), RAS (N = 2), PI3CA (N = 2) and five gene fusions (RET/PTC1 (N = 4) and STRN/ALK (N = 1). In patients with PedCa and AdCa, mutations were detected in 1/4 (25%) and 7/12 (58.3%), respectively, p = 0.56; and fusions were detected in 3/4 (75%) and 2/12 (16.6%), respectively, p = 0.06. In patients with and without history of therapeutic irradiation, mutations were detected with the same frequencies (5/10 (50%), and 3/6 (50%), respectively, p = 1.0). Gene fusions were detected in patients with and without history of irradiation in 5/10 (55.5%) and 0/6 (0%), respectively, p = 0.09. CONCLUSIONS: Monitoring of cancer survivors for thyroid disorders allowed diagnosis of second thyroid cancers at early stages. Second thyroid cancers harbor genomic alterations that are typical for sporadic as well as for radio-induced thyroid cancers.

Cytochrome C Oxidase Subunit 4 (COX4): A Potential Therapeutic Target for the Treatment of Medullary Thyroid Cancer.

The nuclear-encoded subunit 4 of cytochrome c oxidase (COX4) plays a role in regulation of oxidative phosphorylation and contributes to cancer progression. We sought to determine the role of COX4 in differentiated (DTC) and medullary (MTC) thyroid cancers. We examined the expression of COX4 in human thyroid tumors by immunostaining and used shRNA-mediated knockdown of COX4 to evaluate its functional contributions in thyroid cancer cell lines. In human thyroid tissue, the expression of COX4 was higher in cancers than in either normal thyroid (p = 0.0001) or adenomas (p = 0.001). The level of COX4 expression correlated with tumor size (p = 0.04) and lymph-node metastases (p = 0.024) in patients with MTCs. COX4 silencing had no effects on cell signaling activation and mitochondrial respiration in DTC cell lines (FTC133 and BCPAP). In MTC-derived TT cells, COX4 silencing inhibited p70S6K/pS6 and p-ERK signaling, and was associated with decreased oxygen consumption and ATP production. Treatment with potassium cyanide had minimal effects on FTC133 and BCPAP, but inhibited mitochondrial respiration and induced apoptosis in MTC-derived TT cells. Our data demonstrated that metastatic MTCs are characterized by increased expression of COX4, and MTC-derived TT cells are vulnerable to COX4 silencing. These data suggest that COX4 can be considered as a novel molecular target for the treatment of MTC.

Detection of BRAFV600E in Liquid Biopsy from Patients with Papillary Thyroid Cancer Is Associated with Tumor Aggressiveness and Response to Therapy.

The detection of rare mutational targets in plasma (liquid biopsy) has emerged as a promising tool for the assessment of patients with cancer. We determined the presence of cell-free DNA containing the BRAFV600E mutations (cfBRAFV600E) in plasma samples from 57 patients with papillary thyroid cancer (PTC) with somatic BRAFV600E mutation-positive primary tumors using microfluidic digital PCR, and co-amplification at lower denaturation temperature (COLD) PCR. Mutant cfBRAFV600E alleles were detected in 24/57 (42.1%) of the examined patients. The presence of cfBRAFV600E was significantly associated with tumor size (p = 0.03), multifocal patterns of growth (p = 0.03), the presence of extrathyroidal gross extension (p = 0.02) and the presence of pulmonary micrometastases (p = 0.04). In patients with low-, intermediate- and high-risk PTCs, cfBRAFV600E was detected in 4/19 (21.0%), 8/22 (36.3%) and 12/16 (75.0%) of cases, respectively. Patients with detectable cfBRAFV600E were characterized by a 4.68 times higher likelihood of non-excellent response to therapy, as compared to patients without detectable cfBRAFV600E (OR (odds ratios), 4.68; 95% CI (confidence intervals)) 1.26-17.32; p = 0.02). In summary, the combination of digital polymerase chain reaction (dPCR) with COLD-PCR enables the detection of BRAFV600E in the liquid biopsy from patients with PTCs and could prove useful for the identification of patients with PTC at an increased risk for a structurally or biochemically incomplete or indeterminate response to treatment.

Microfluidic Droplet Digital PCR Is a Powerful Tool for Detection of BRAF and TERT Mutations in Papillary Thyroid Carcinomas.

We examined the utility of microfluidic digital PCR (dPCR) for detection of BRAF and TERT mutations in thyroid tumors. DNA extracted from 100 thyroid tumors (10 follicular adenomas, 10 follicular cancers, 5 medullary cancers, and 75 papillary thyroid cancer (PTC) were used for detection of BRAF and TERT mutations. Digital PCRs were performed using rare mutation SNP genotyping assays on QuantStudio 3D platform. In PTCs, BRAFV600E was detected by dPCR and Sanger sequencing in 42/75 (56%) and in 37/75 (49%), respectively. BRAFV600E was not detected in other tumors. The ratio of mutant/total BRAF alleles varied from 4.7% to 47.5%. These ratios were higher in classical PTCs (27.1%) as compared to follicular variant PTCs (9.4%) p = 0.001. In PTCs with and without metastases, the ratios of mutant/total BRAF alleles were 27.6% and 18.4%, respectively, (p = 0.03). In metastatic lesions percentages of mutant/total BRAF alleles were similar to those detected in primary tumors. TERTC228T and TERTC250T were found in two and one cases, respectively, and these tumors concomitantly harbored BRAFV600E. These tumors exhibited gross extra-thyroidal extension, metastases to lymph nodes, and pulmonary metastases (one case). Our results showed that dPCR allows quantitative assessment of druggable targets in PTCs and could be helpful in a molecular-based stratification of prognosis in patients with thyroid cancer.

Limited Utility of Circulating Cell-Free DNA Integrity as a Diagnostic Tool for Differentiating Between Malignant and Benign Thyroid Nodules With Indeterminate Cytology (Bethesda Category III).

Background: Analysis of plasma circulating cell-free DNA integrity (cfDI) has emerged as a promising tool in the diagnosis of malignant vs. benign tumors. There is limited data on the role of cfDI in thyroid cancer. The goal of this study was to analyze cfDI as a biomarker of malignancy in patients with cytologically indeterminate thyroid nodules. Methods: The cfDI was measured in the plasma of patients with cytologically indeterminate thyroid nodules. All patients underwent plasma collection within 24-72 h before surgical treatment for thyroid nodules. Additionally, samples were collected from seven patients via the vein draining the thyroid and peripheral vein during surgery. Quantitative real-time PCR was performed on the isolated cell-free DNA using two different primer sets (115 and 247 bp) to amplify consensus ALU sequences. The cfDI was calculated as the ratio of ALU247 to ALU115. Results: All data are given as median [25th-75th percentile]. The study group consisted of 67 patients with 100 nodules, 80.6% (54/67) women, aged 43 [33-60] years. There was no difference in cfDI between 29 patients with benign nodules (0.49 [0.41-0.59]) and 38 patients with malignant lesions (0.45 [0.36-0.57], p = 0.19). There was no difference in cfDI in the vein draining the thyroid (0.47 [0.24-1.05]) and peripheral vein (0.48 [0.36-0.56], p = 0.44). In comparison to thyroid cancer patients, patients with benign nodules were characterized by significantly higher concentrations of ALU115 (1,064 [529-2,960] vs. 411 [27-1,049] ng/ml; p = 0.002) and ALU247 (548 [276-1,894] vs. 170 [17-540] ng/ml; p = 0.0005), most likely because benign tumors were larger (3, [1.8-4.1 cm]) than malignant lesions (0.7 [0.23-1.45], p < 0.0001). Women had significantly lower cfDI (0.45 [0.27-0.54]) than men (0.56 [0.44-0.8], p = 0.011). Conclusion: The cfDI measured in the vein draining the thyroid is similar to the cfDI measured in the antecubital vein, validating cfDI measurements by peripheral liquid biopsy. Analysis of cfDI needs to be stratified by patients gender. In contrast to its diagnostic utility in aggressive cancers, cfDI has limited utility as a biomarker of malignancy in cytologically indeterminate thyroid nodules.

Mitotane induces mitochondrial membrane depolarization and apoptosis in thyroid cancer cells.

Mitotane is used for the treatment of adrenocortical cancer and elicits its anticancer effects via inhibition of mitochondrial respiration. Targeting mitochondria­dependent metabolism has emerged as a promising strategy for thyroid cancer (TC) treatment. We hypothesized that mitotane targets mitochondria and induces apoptosis in TC cells. Cell lines representative of the major histological variants of TC were chosen: Follicular (FTC­133), poorly differentiated (BCPAP), anaplastic (SW1736 and C643) and medullary (TT) TC cells, and were treated with mitotane (0­100 µM). Mitochondrial membrane potential, cell viability and apoptosis were examined by JC­1 staining and by western blot analysis using an antibody against caspase­3. The expression of mitochondrial molecules and DNA damage markers and the activation of endoplasmic reticulum (ER) stress were determined by western blotting. The expression of mitochondrial ATP synthase subunit ß (ATP5B) was examined by immunostaining in 100 human TC tissue samples. Treatment with mitotane (50 µM for 24 h) decreased the viability of FTC­133, BCPAP, SW1736, C643 and TT cells by 12, 59, 54, 31 and 66%, respectively. Morphological evidence of ER stress and overexpression of ER markers was observed in TC cells following exposure to mitotane. The treatment led to increased expression of histone γH2AX, indicating DNA damage, and to caspase­3 cleavage. Consistent with the results of the cell viability assays, the overexpression of pro­apoptotic genes following treatment with mitotane was more prominent in TC cells harboring mutations in the serine/threonine­protein kinase B­raf gene and proto­oncogene tyrosine­protein kinase receptor Ret. Treatment with mitotane was associated with loss of mitochondrial membrane potential and decreased expression of ATP5B, particularly in the medullary TC (MTC)­derived TT cells. Immunohistochemical analysis of mitochondrial ATP5B in human TC specimens demonstrated its overexpression in cancer compared with normal thyroid tissue. The level of ATP5B expression was higher in MTC compared with the follicular, papillary or anaplastic types of TC. Mitotane elicited pleiotropic effects on TC cells, including induction of ER stress, inhibition of mitochondrial membrane potential and induction of apoptosis. The results of the present study suggest that mitotane could be considered as a novel agent for the treatment of aggressive types of TC.

Amifostine does not protect thyroid cancer cells in DNA damaging in vitro models.

BACKGROUND: Amifostine is a potent scavenger of reactive oxygen species that is used for the salivary gland protection during therapy with radioactive iodine for thyroid cancer. There are no data on the potential effect of amifostine on thyroid cancer cells. METHODS: We investigated the effects of the active form of amifostine (WR-1065) on the response of thyroid cancer cells to treatment with DNA-damaging agents. WR-1065 was examined in human thyroid cancer cell lines (FTC133, TPC1, BCPAP and C643) and embryonic fibroblast cells NIH3T3. DNA damage was induced by exposure to H2O2 (0.1 mM), by treatment with the radiomimetic neocarzinostatin (NCS 250 ng/mL) and by γ-radiation (6 Gy). DNA damage, cell viability and apoptosis were examined. RESULTS: We demonstrated the selective action of WR-1065 (0.1 mM), which prevented oxidative stress-induced DNA damage in fibroblasts, but did not protect thyroid cancer cells from DNA damage and apoptosis documented by caspase-3 and PARP cleavage after exposure to H2O2, NCS and γ-radiation. Prolonged exposure to WR-1065 (0.1 mM for 24 h) was toxic for thyroid cancer cells; this treatment decreased the number of viable cells by 8% in C643 cells, 47% in TPC cells, 92% in BCPAP cells and 82% in FTC 133 cells. The cytotoxic effects of WR-1065 were not associated with induction of apoptosis. CONCLUSIONS: Our data show that amifostine has no protective effect on thyroid cancer cells against DNA-damaging agents in vitro and suggest that amifostine will not attenuate the efficacy of radioiodine treatment in patients with thyroid cancer.

Nelfinavir inhibits proliferation and induces DNA damage in thyroid cancer cells.

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 determined in vitro using 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. Our in vitro data suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.

Metformin Attenuates 131I-Induced Decrease in Peripheral Blood Cells in Patients with Differentiated Thyroid Cancer.

BACKGROUND: 131I treatment (tx) of differentiated thyroid cancer (DTC) is associated with hematopoietic toxicity. It was hypothesized that metformin could have radioprotective effects on bone-marrow function. The objective was to determine whether metformin prevents 131I-induced changes in complete blood counts (CBC) in patients with DTC. METHODS: A retrospective analysis was performed of CBC values in DTC patients who were (40 patients: metformin group) or were not taking metformin (39 patients: control group) at the time of administration of 131I. Repeated measures analysis of variance was used for the analysis of the differences in the averages of CBC that were documented at baseline and at 1, 6, and 12 months post 131I tx. RESULTS: The groups were comparable in terms of age, sex, stage of DTC, 131I dose administered, and baseline CBC values. In the control group, the decrease in white blood cells (WBC) was 35.8% (p < 0.0001) at one month, 21.8% (p < 0.0001) at six months, and 19.4% (p < 0.0001) at 12 months. In the metformin group, the decrease in WBC was 17.1% (p < 0.0001) at one month, and 8.6% at six months (p = 0.01), while at 12 months WBC had returned to baseline values (p = 0.9). Differences between the two groups were highly statistically significant at all time points (p < 0.0001, p = 0.0027, and p < 0.0001, respectively). Lymphocytes were more sensitive to 131I, but metformin's radioprotective properties were more prominent in neutrophils. At 12 months, the decrease in platelets in the control group was 15.5% (p < 0.0001) versus 5.6% (p = 0.056) in the metformin group, while at one and six months the reductions in the two groups were comparable. No statistically significant differences were observed between the two groups in the change from baseline values for hemoglobin. CONCLUSIONS: Metformin attenuated the 131I-induced decrease in CBC parameters, and its radioprotective properties were more prominent in WBC. Patients who were taking metformin during 131I tx also experienced a faster recovery in their blood counts, when compared to the control group. Further study is warranted in order to examine if the radioprotective properties of metformin observed in the current study for 131I tx can also apply to other forms of therapeutic chemo- and radiotherapy.

Glucose-deprivation increases thyroid cancer cells sensitivity to metformin.

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 down-regulates RET signaling and induces apoptosis in medullary thyroid cancer cells.

CONTEXT: Mutations of RET tyrosine kinase are associated with the development of medullary thyroid cancer (MTC). The heat shock protein (HSP) 90 chaperone is required for folding and stability of RET mutants. HSP90 is a molecular target for the HIV protease inhibitor nelfinavir (NFV). OBJECTIVE: We hypothesized that treatment with NFV may lead to the inhibition of RET signaling and induction of apoptosis in MTC cells. DESIGN: Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to clinically achievable concentrations of NFV. JC-1 staining and caspase-3 cleavage assays were performed to measure mitochondrial membrane potential and apoptosis. Activation of RET signaling was examined by Western blot. Autophagy was monitored by the detection of the light-chain 3BII. Expression of HSP90 and LC3B were examined in 36 human MTCs. RESULTS: At a therapeutic serum concentration (10 µM), NFV inhibited the viability of TT and MZ-CRC-1 cells by 55% and 10%, respectively. In a dose-dependent manner, NFV inhibited cyclin D1 and caused caspase-3 cleavage. NFV decreased the level of RET protein and blocked the activation of RET downstream targets (phosphorylated ERK, phosphorylated AKT, and p70S6K/pS6). NFV induced metabolic stress, activated AMP-activated protein kinase and increased autophagic flux. Pharmacological inhibition of autophagy (chloroquine) augmented NFV-inducible cytotoxicity, suggesting that autophagy was protective in NFV-treated cells. NFV led to mitochondrial membrane depolarization and induced both oxidative stress and DNA damage. An antioxidant (n-acetylcysteine) attenuated DNA damage and prevented NFV-inducible apoptosis. HSP90 overexpression was found in 17 of 36 human MTCs and correlated with metastases and RET mutations. LC3B was detected in 20 of 36 human MTCs. CONCLUSIONS: NFV has a wide spectrum of activity against MTC cells, and its cytotoxicity can be augmented by inhibiting autophagy. Expression of NFV molecular targets in metastatic MTC suggests that NFV has a potential to become a thyroid cancer therapeutic agent.

Treatment with metformin is associated with higher remission rate in diabetic patients with thyroid cancer.

CONTEXT: Clinical trials demonstrated that metformin increases the efficiency of systemic therapy in cancer patients. OBJECTIVE: We examined whether the efficacy of conventional treatment of differentiated thyroid cancer (DTC) is affected by therapy with metformin in diabetic patients. DESIGN: We compared the rate of complete response (CR) between diabetics who were treated with metformin (group MF+; n = 34) or not treated (group MF-; n = 21) and control nondiabetic patients (group C; n = 185). We also examined the effects of metformin on DTC cells in vitro. RESULTS: The groups were comparable in terms of age, sex, body mass index, diabetes management, frequencies of multifocal tumor growth, extrathyroidal extension, and locoregional and distant metastases. Tumor size was significantly smaller in the MF+ group compared with the MF- and C groups (1.37 ± 0.97 vs 2.44 ± 1.49 vs 2.39 ± 1.73 cm, respectively; P = .026). A multivariate model revealed that extrathyroidal extension (P = .018), distant metastases (P < .0001), and lack of treatment with metformin of diabetics (P < .0001) decreased the likelihood of CR. A Cox hazards model revealed that age (P = .025), locoregional metastases (P = .022), distant metastases (P = .003), and lack of treatment with metformin of patients with diabetes (P = .014) are associated with increased risk for shortened progression-free survival. In vitro data revealed that metformin inhibited cancer cell growth, activated cAMP-inducible protein kinase (5'-AMP-activated protein kinase [AMPK]), and down-regulated p70S6K/pS6. Metformin potentiated H2O2-inducible activation of AMPK but attenuated pERK and p70S6K. Tumors from MF+ patients demonstrated a lower level of phospho-p70S6K compared with the MF- group. CONCLUSIONS: Tumor size is smaller in patients treated with metformin, suggesting inhibition of tumor growth by the drug. Among diabetics, the absence of metformin therapy is an independent factor for decreased likelihood of CR and increased risk of shorter progression-free survival. In vitro data suggest that p70S6K/pS6 is likely a molecular target of metformin in DTC cells.

The expression of translocator protein in human thyroid cancer and its role in the response of thyroid cancer cells to oxidative stress.

The translocator protein (TSPO), formerly known as a peripheral benzodiazepine receptor, exerts pro-apoptotic function via regulation of mitochondrial membrane potential. We examined TSPO expression in human thyroid tumors (25 follicular adenomas (FA), 15 follicular cancers (FC), and 70 papillary cancers (PC)). The role of TSPO in the regulation of cell growth, migration, and apoptosis was examined in thyroid cancer cell lines after TSPO knockdown with siRNA and after treatment with TSPO antagonist (PK11195). Compared with normal thyroid, the level of TSPO expression was increased in FA, FC, and PC in 24, 26.6, and 55.7% of cases respectively. Thyroid cancer cell lines demonstrated variable levels of TSPO expression, without specific association with thyroid oncogene mutations. Treatment with inhibitors of PI3K/AKT or MEK/ERK signaling was not associated with changes in TSPO expression. Treatment with histone deacetylase inhibitor (valproic acid) increased TSPO expression in TSPO-deficient cell lines (FTC236 cells). TSPO gene silencing or treatment with PK11195 did not affect thyroid cancer cell growth and migration but prevented depolarization of mitochondrial membranes induced by oxidative stress. Induction of TSPO expression by valproic acid was associated with increased sensitivity of FTC236 to oxidative stress-inducible apoptosis. Overall, we showed that TSPO expression is frequently increased in PC. In vitro data suggested the role of epigenetic mechanism(s) in the regulation of TSPO in thyroid cells. Implication of TSPO in the thyroid cancer cell response to oxidative stress suggested its potential role in the regulation of thyroid cancer cell response to treatment with radioiodine and warrants further investigation.

Metformin inhibits growth and decreases resistance to anoikis in medullary thyroid cancer cells.

Medullary thyroid cancer (MTC) is associated with activation of mammalian target of rapamycin (mTOR) signaling pathways. Recent studies showed that the antidiabetic agent metformin decreases proliferation of cancer cells through 5'-AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR. In the current study, we assessed the effect of metformin on MTC cells. For this purpose, we determined growth, viability, migration, and resistance to anoikis assays using two MTC-derived cell lines (TT and MZ-CRC-1). Expressions of molecular targets of metformin were examined in MTC cell lines and in 14 human MTC tissue samples. We found that metformin inhibited growth and decreased expression of cyclin D1 in MTC cells. Treatment with metformin was associated with inhibition of mTOR/p70S6K/pS6 signaling and downregulation of pERK in both TT and MZ-CRC-1 cells. Metformin had no significant effects on pAKT in the cell lines examined. Metformin-inducible AMPK activation was noted only in TT cells. Treatment with AMPK inhibitor (compound C) or AMPK silencing did not prevent growth inhibitory effects of metformin in TT cells. Metformin had no effect on MTC cell migration but reduced the ability of cells to form multicellular spheroids in nonadherent conditions. Immunostaining of human MTC showed over-expression of cyclin D1 in all tumors compared with corresponding normal tissue. Activation of mTOR/p70S6K was detected in 8/14 (57.1%) examined tumors. Together, these findings indicate that growth inhibitory effects in MTC cells are associated with downregulation of both mTOR/6SK and pERK signaling pathways. Expression of metformin's molecular targets in human MTC cells suggests its potential utility for the treatment of MTC in patients.

BRAF(V600E) mutation analysis from May-Grünwald Giemsa-stained cytological samples as an adjunct in identification of high-risk papillary thyroid carcinoma.

The BRAF(V600E) mutation is specific for thyroid papillary cancer (PTC) and correlates with PTCs invasiveness. This study investigated whether detection of BRAF(V600E) mutation can be performed on routinely stained FNABs. We also examined if establishment of the BRAF(V600E) mutation could help in identification of patients at higher risk for metastatic disease. DNA was isolated from 134 FNABs samples (20 follicular neoplasm, ten suspicious for malignancy, and 104 malignant) using Pinpoint Slide DNA Isolation System. BRAF(V600E) mutation was detected by PCR followed by sequencing. DNA was successfully extracted from all examined FNABs samples. In follicular neoplasm, suspicious for malignancy and malignant FNABs, BRAF(V600E) mutation was found in 0/20 (0%), 2/10 (20%), and 47/104 (45.2%) of cases, respectively. Extra-thyroidal extension was detected in 35/47 (74.4%) BRAF(V600E) positive and in 24/57 (42.1%) wild-type BRAF cases (p = 0.001). Metastases were detected in 37/47 (78.7%) BRAF(V600E) positive and in 28/57 (49.1%) wild-type BRAF cases (p = 0.002). Our results showed that stained FNAB specimens can be used for DNA extraction and assessment of BRAF(V600E) mutation. Detection of BRAF(V600E) mutation had limited value in diagnoses of malignancy in follicular neoplasms but can ascertain malignancy in subset of suspicious for malignancy FNABs. In malignant FNABs, BRAF(V600E) mutation was significantly associated with presence of extra-thyroidal extension and metastases after surgery.

Inhibition of gap junction transfer sensitizes thyroid cancer cells to anoikis.

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.

Human herpes simplex viruses in benign and malignant thyroid tumours.

To test the hypothesis that herpes viruses may have a role in thyroid neoplasia, we analysed thyroid tissues from patients with benign (44) and malignant (65) lesions for HSV1 and HSV2 DNA. Confirmatory studies included direct sequencing, analysis of viral gene expression, and activation of viral-inducible signalling pathways. Expression of viral entry receptor nectin-1 was examined in human samples and in cancer cell lines. In vitro experiments were performed to explore the molecular mechanisms underlying thyroid cancer cell susceptibility to HSV. HSV DNA was detected in 43/109 (39.4%) examined samples. HSV capsid protein expression correlated with HSV DNA status. HSV-positive tumours were characterized by activation of virus-inducible signalling such as interferon-beta expression and nuclear NFkappaB expression. Lymphocyte infiltration and oncocytic cellular features were common in HSV-positive tumours. HSV1 was detected with the same frequency in benign and malignant thyroid tumours. HSV2 was significantly associated with papillary thyroid cancer and the presence of lymph node metastases. The expression of HSV entry receptor nectin-1 was increased in thyroid tumours compared to normal thyroid tissue and further increased in papillary thyroid cancer. Nectin-1 expression was detected in all examined thyroid cancer cell lines. Nectin-1 expression in cancer cells correlated with their susceptibility to HSV. Inhibition of PI3K/AKT or MAPK/ERK signalling did not affect the level of nectin-1 expression but decreased thyroid cancer cell susceptibility to HSV. These findings showed that HSV is frequently detected in thyroid cancer. During tumour progression, thyroid cells acquire increased susceptibility to HSV due to increased expression of viral entry mediator nectin-1 and activation of mitogenic signalling in cancer cells.

Dynamic changes in E-cadherin gene promoter methylation during metastatic progression in papillary thyroid cancer.

Thyroid cancer cells undergo epithelial to mesenchymal transition (EMT) in the invasive front of tumor. To determine whether the mesenchymal feature of invasive cancer cells is maintained in metastatic sites, we examined E-cadherin methylation and E-cadherin expression in 66 papillary thyroid cancer (PTC) samples and in 34 corresponding lymph node metastases (LNM). Relationships between E-cadherin and cell motility were evaluated using thyroid cancer cell lines. Hypermethylation of the E-cadherin gene promoter was detected in 39.3% of the PTCs, and loss of E-cadherin expression correlated with lymphocytic infiltration, extrathyroidal invasion and the presence of metastases. Comparing primary PTCs to the corresponding LNM, E-cadherin methylation status was identical in 60% of the cases. The switch in E-cadherin promoter status from unmethylated in PTCs to hypermethylated in LNM was detected in 5%; and from hypermethylatated in PTCs to unmethylated in LNM in 35%. Loss of epigenetic silencing in LNM was associated with a gain of E-cadherin expression. Hypermethylation of the E-cadherin gene promoter was detected in thyroid cancer cell lines with mesenchymal-like morphology. Loss of E-cadherin expression in these cells correlated with high migratory ability. Inhibition of RAS/ERK or PI3K/AKT signaling decreased the migratory ability of these cells but did not induce E-cadherin expression. In the cells with epithelial-like morphology, treatments with phorbol-ester or tumor necrosis factor (TNF)-α resulted in translocation of membranous E-cadherin to the cytoplasm and induction of migration. E-cadherin promoter methylation status and E-cadherin expression were not affected by TNF. Demethylating agents induced apoptosis in the mesenchymal-like cells but had no effect on E-cadherin expression or on migratory ability. Together, dynamic changes in E-cadherin methylation occur during metastatic progression in thyroid cancer. Epigenetic mechanisms and TNF-inducible signaling independently contribute to the regulation of E-cadherin expression and localization. These mechanisms may play a role in the induction of EMT in primary tumors and in the conversion from the mesenchymal to the epithelial phenotype in metastases.

Capecitabine therapy for refractory metastatic thyroid carcinoma: a case series.

OBJECTIVE: There are few effective therapies for metastatic medullary (MTC) or radioiodine-resistant follicular thyroid carcinomas (FTC). We report a single institution's experience with capecitabine, a thymidylate synthase (TS) inhibitor, in the treatment of MTC and FTC. DESIGN: We retrospectively analyzed five cases of metastatic thyroid carcinoma, three MTCs and two radioiodine-resistant FTCs, treated with capecitabine alone or in combination with other chemotherapeutics. Patients were selected for treatment based on low tumor TS immunohistochemical staining (< or =5%). Staining for thymidylate phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) was also performed. Therapeutic response was assessed by imaging studies and serum tumor markers: calcitonin and carcinoembryonic antigen (MTC), and thyroglobulin (FTC). MAIN OUTCOME: Two of three patients with MTC had stable disease or disease regression on capecitabine. One of these patients had a 90% reduction in calcitonin and stabilization by imaging that lasted 4 years. Both patients with FTC initially had stable disease on capecitabine. One patient, who was treated with capecitabine in combination first with doxorubicin and then etoposide, had an initial decrease in tumor burden, followed by stable disease for 2.8 years. The second patient had stable disease, but capecitabine was discontinued after 11 months because of hand/foot syndrome. CONCLUSIONS: This series demonstrates promising results for the use of capecitabine in treatment of MTC and radioiodine-resistant FTC, for which there is a limited repertoire of therapeutic agents. Larger studies are needed to confirm these findings and to establish the role of fluoropyramidine metabolism markers in predicting response.

Aplidin reduces growth of anaplastic thyroid cancer xenografts and the expression of several angiogenic genes.

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the most aggressive and highly lethal human cancers. Median survival after diagnosis is 4-6 months despite available radiotherapy and chemotherapy. Additional treatments are needed for ATC. Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulus, which is expressed by ATC. Previously, anti-VEGF antibody was used to block VEGF-dependent angiogenesis in ATC xenografts. This treatment induced partial (56%) but not complete tumor regression. Aplidin (APLD) is a marine derived antitumor agent currently in phase II clinical studies. Multiple activities of this compound have been described which likely contribute to its antiproliferative effect. Notably, APLD has been shown to have antiangiogenic properties which include: inhibition of VEGF secretion, reduction in the synthesis of the VEGF receptor (FLT-1), and blockade of matrix metalloproteinase production by endothelial cells. We hypothesized that Aplidin, with its broad spectrum of action and antiangiogenic properties, would be a potentially effective drug against ATC. METHODS: Thirty BALB/c nu/nu mice were injected with ATC cells (ARO-81, 1 x 10(6)) and allowed to implant for 3 weeks. Animals were randomized to receive daily intraperitoneal injections of vehicle, low dose (0.5 mg/kg/day), or high dose (1.0 mg/kg/day) APLD. After 3 days, the animals were killed and the tumors were removed, weighed, and divided for RNA and protein analyses. RESULTS: APLD significantly reduced ATC xenograft growth (low dose, 20% reduction, P = 0.01; high dose, 40% reduction, P < 0.001). This was associated with increased levels of apoptosis related proteins polyadenosylribose polymerase 85 (PARP-85, 75% increase, P = 0.024) and caspase 8 (greater than fivefold increase, P = 0.03). APLD treatment was further associated with lost or reduced expression of several genes that support angiogenesis to include: VEGF, hypoxia inducible factor 1(HIF-1), transforming growth factor-beta (TGFbeta), TGFbeta receptor 2 (TGFbetaR2), melanoma growth stimulating factor 1 (GRO1), cadherin, and vasostatin. CONCLUSIONS: This data supports the hypothesis that APLD may be an effective adjunctive therapy against ATC. The demonstrated molecular impact against angiogenic related genes specifically supports future strategies combining APLD with VEGF interacting agents.