Kirk Jensen

Highly Prevalent Genetic Alterations in Receptor Tyrosine Kinases and Phosphatidylinositol 3-Kinase/Akt and Mitogen-Activated Protein Kinase Pathways in Anaplastic and Follicular Thyroid Cancers

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, PDGFRalpha and -beta, 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.

GABA receptor expression in benign and malignant thyroid tumors.

Neurotransmitter systems have recently been shown to be involved in multiple malignancies including breast, colon and prostate cancers. The role of neurotransmitters and neurotrophic factors has not yet been examined in thyroid cancer. To determine the possible involvement of neurotransmitter systems in thyroid carcinogenesis we characterized the patterns of gamma-aminobutyric acid (GABA) receptor expression in normal thyroid and thyroid tumors. We examined the expression patterns of the GABAergic system in 70 human thyroid tumor samples (13 follicular adenomas, 14 follicular carcinomas, 43 papillary carcinomas) and adjacent normal thyroid by immunohistochemistry. GABAergic system mRNA expression in thyroid cancer cell lines derived from primary (FTC133) and metastatic tumors (FTC236 and FTC238) was examined by real time PCR. Overall, GABA receptor expression is increased in tumors compared to normal thyroid tissue. Expression of GABAA receptor β2 was detected in the vasculature of normal thyroid and thyroid tumors but not in thyroid cancer cells. GABAA α2 was detected in metastatic-derived but not in primary-tumor derived cell lines. Expression levels of GABAB R2 and GABA receptor associated protein (GABARAP) are increased in adenomas and thyroid cancer suggesting their role in early stages of thyroid tumorigenesis. This study represents the first demonstration of GABA receptor expression in human thyroid tissue and suggests that the GABAergic system is involved in thyroid carcinogenesis.

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 NFκB 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. Copyright

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.

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

The BRAFV600E mutation is specific for thyroid papillary cancer (PTC) and correlates with PTCs invasiveness. This study investigated whether detection of BRAFV600E mutation can be performed on routinely stained FNABs. We also examined if establishment of the BRAFV600E 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. BRAFV600E 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, BRAFV600E 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%) BRAFV600E positive and in 24/57 (42.1%) wild-type BRAF cases (p = 0.001). Metastases were detected in 37/47 (78.7%) BRAFV600E 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 BRAFV600E mutation. Detection of BRAFV600E 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, BRAFV600E 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.

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.

Transient Hypothyroidism in Premature Infants After Short-term Topical Iodine Exposure: An Avoidable Risk?

Studies in preterm infants have shown that prolonged treatment with topical iodine (multiple doses, often over multiple days) can transiently suppress thyroid function. However, it is uncertain if topical iodine exposure for very short periods of time can cause significant changes in thyroid function. We report two cases of transient hypothyroidism in preterm infants after short-term exposure to topical iodine during surgical preparation, and review their clinical and laboratory findings before and after iodine exposure. We conclude that premature infants are at risk of developing transient hypothyroidism in response to a single, short-term exposure to topical iodine, even in iodine-sufficient geographical areas. We advise monitoring of thyroid function in these infants after iodine exposure, as treatment with levothyroxine may be needed for a limited duration to prevent the sequelae of untreated hypothyroidism. Consideration of using alternative cleansing agents is also advised.

Targeted therapies in thyroid cancer: an extensive review of the literature

ABSTRACT Introduction: Patients with progressive, metastatic, RAI-refractory differentiated thyroid cancer (DTC), as well as patients with advanced medullary (MTC) and anaplastic thyroid cancer represent a cohort for which therapeutic options are limited. The recent discoveries in the molecular mechanisms implicated in TC have provided insight of the pathogenesis and progression of disease. In that respect, targeted therapies have emerged as a promising alternative for the treatment of those patients. Areas covered: Tyrosine Kinase Inhibitors (TKIs) have been studied extensively in TC: sorafenib and lenvatinib have been approved by the FDA for the treatment of metastatic, RAI-refractory DTC, while vandetanib and cabozantinib are FDA approved for use in advanced MTC. Moreover, several additional TKIs, multi-targeted or specific, are currently under investigation in TC. The current manuscript provides an extensive review of the literature regarding targeted therapies in TC including the rationale behind their use, the clinical trials and an expert opinion on their use. Literature in English appearing at PubMed was thoroughly reviewed, especially manuscripts of the last 5 years. Expert commentary: Patients with advanced, progressive, metastatic TC should be evaluated for enrollment in a clinical trial or should be placed on treatment with one of the FDA- and EMA- approved agents.

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.