Giuliana Salvatore

The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells

In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle-dependent element (CDE)/cell cycle gene homology region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the negative control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest associated with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential molecular target for ATC treatment.

Potent Mitogenicity of the RET/PTC3 Oncogene Correlates with Its Prevalence in Tall-Cell Variant of Papillary Thyroid Carcinoma

The tall-cell variant (TCV) of papillary thyroid carcinoma (PTC), characterized by tall cells bearing an oxyphilic cytoplasm, is more clinically aggressive than conventional PTC. RET tyrosine kinase rearrangements, which represent the most frequent genetic alteration in PTC, lead to the recombination of RET with heterologous genes to generate chimeric RET/PTC oncogenes. RET/PTC1 and RET/PTC3 are the most prevalent variants. We have found RET rearrangements in 35.8% of TCV (14 of 39 cases). Whereas the prevalences of RET/PTC1 and RET/PTC3 were almost equal in classic and follicular PTC, all of the TCV-positive cases expressed the RET/PTC3 rearrangement. These findings prompted us to compare RET/PTC3 and RET/PTC1 in an in vitro thyroid model system. We have expressed the two oncogenes in PC Cl 3 rat thyroid epithelial cells and found that RET/PTC3 is endowed with a strikingly more potent mitogenic effect than RET/PTC1. Mechanistically, this difference correlated with an increased signaling activity of RET/PTC3. In conclusion, we postulate that the correlation between the RET/PTC rearrangement type and the aggressiveness of human PTC is related to the efficiency with which the oncogene subtype delivers mitogenic signals to thyroid cells.

Biological Role and Potential Therapeutic Targeting of the Chemokine Receptor CXCR4 in Undifferentiated Thyroid Cancer

Anaplastic thyroid carcinoma (ATC) is a rare thyroid cancer type with an extremely poor prognosis. Despite appropriate treatment, which includes surgery, radiotherapy, and chemotherapy, this cancer is invariably fatal. CXCR4 is the receptor for the stromal cell-derived factor-1 (SDF-1)/CXCL12 chemokine and it is expressed in a variety of solid tumors, including papillary thyroid carcinoma. Here, we show that ATC cell lines overexpress CXCR4, both at the level of mRNA and protein. Furthermore, we found that CXCR4 was overexpressed in ATC clinical samples, with respect to normal thyroid tissues by real-time PCR and immunohistochemistry. Treatment of ATC cells with SDF-1 induced proliferation and increase in phosphorylation of extracellular signal-regulated kinases and protein kinase B/AKT. These effects were blocked by the specific CXCR4 antagonist AMD3100 and by CXCR4 RNA interference. Moreover, AMD3100 effectively reduced tumor growth in nude mice inoculated with different ATC cells. Thus, we suggest that CXCR4 targeting is a novel potential strategy in the treatment of human ATC.

Identification of Polo-like Kinase 1 as a Potential Therapeutic Target in Anaplastic Thyroid Carcinoma

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and chemoresistant cancers. The serine/threonine kinase Polo-like kinase 1 (PLK1), a key regulator of multiple steps during mitotic progression, is highly expressed in ATC. Here, we used the BI 2536 PLK1 inhibitor on ATC and nontransformed thyroid follicular cell lines. Our data show that ATC cells are addicted to high levels of PLK1 activity for proliferation, survival, anchorage-independent growth, and tumorigenicity. On treatment with nanomolar doses of BI 2536, ATC cells progressed normally through S phase but died thereafter, directly from mitotic arrest. Immunofluorescence microscopy, immunoblot, and flow cytometry analysis showed that, on PLK1 blockade, ATC cells arrested in prometaphase with a 4N DNA content. Treated ATC cells accumulated phosphohistone H3 and displayed characteristic mitotic (Polo) spindle aberrations. Nontransformed thyroid cells were 3.2- to 18.4-fold less susceptible to BI 2536-induced cell cycle effects compared with ATC cells. These findings identify PLK1 as a promising target for the molecular therapy of ATC.

TWIST1 plays a pleiotropic role in determining the anaplastic thyroid cancer phenotype.

CONTEXT Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors; it is characterized by chemoresistance, local invasion, and distant metastases. ATC is invariably fatal. OBJECTIVE The aim was to study the role of TWIST1, a basic helix-loop-helix transcription factor, in ATC. DESIGN Expression of TWIST1 was studied by immunohistochemistry and real-time PCR in normal thyroids and well-differentiated, poorly differentiated, and ATC. The function of TWIST1 was studied by RNA interference in ATC cells and by ectopic expression in well-differentiated thyroid carcinoma cells. RESULTS ATCs up-regulate TWIST1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Knockdown of TWIST1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of TWIST1 in thyroid cells induced resistance to apoptosis and increased cell migration and invasion. CONCLUSIONS TWIST1 plays a key role in determining malignant features of the anaplastic phenotype in vitro.

FOXM1 is a molecular determinant of the mitogenic and invasive phenotype of anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) is a very aggressive thyroid cancer. forkhead box protein M1 (FOXM1) is a member of the forkhead box family of transcription factors involved in control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Here, we show that FOXM1 is significantly increased in ATCs compared with normal thyroid, well-differentiated thyroid carcinomas (papillary and/or follicular), and poorly differentiated thyroid carcinomas (P=0.000002). Upregulation of FOXM1 levels in ATC cells was mechanistically linked to loss-of-function of p53 and to the hyperactivation of the phosphatidylinositol-3-kinase/AKT/FOXO3a pathway. Knockdown of FOXM1 by RNA interference inhibited cell proliferation by arresting cells in G2/M and reduced cell invasion and motility. This phenotype was associated with decreased expression of FOXM1 target genes, like cyclin B1 (CCNB1), polo-like kinase 1 (PLK1), Aurora B (AURKB), S-phase kinase-associated protein 2 (SKP2), and plasminogen activator, urokinase: uPA (PLAU). Pharmacological inhibition of FOXM1 in an orthotopic mouse model of ATC reduced tumor burden and metastasization. All together, these findings suggest that FOXM1 represents an important player in thyroid cancer progression to the anaplastic phenotype and a potential therapeutic target for this fatal cancer.

Glial cell line-derived neurotrophic factor induces proliferative inhibition of NT2/D1 cells through RET-mediated up-regulation of the cyclin-dependent kinase inhibitor p27 kip 1

Growth factors of the glial cell line-derived neurotrophic factor (GDNF) family control the differentiation of neuronal cells of the central and peripheral nervous systems. Intracellular signalling of these growth factors is, at least in part, mediated by activation of the RET receptor tyrosine kinase. Here, we demonstrate that GDNF triggering inhibits the proliferation of the embryonal carcinoma cell line NT2/D1. This anti-proliferative effect is accompanied by down-regulation of the SSEA-3 antigen, a marker typical of undifferentiated NT2/D1 cells. We show that these effects are mediated by activation of RET signalling. The block of RET by a kinase-deficient dominant negative mutant impairs GDNF-dependent growth inhibition, whereas the adoptive expression of a constitutively active RET, the RET-MEN2A oncogene, promotes effects similar to those exerted by GDNF. We show that RET signalling increases the expression of the cyclin-dependent kinase inhibitor p27kip1 in NT2/D1 cells. Both DNA synthesis inhibition and SSEA-3 down-regulation are prevented if p27kip1 expression is blocked by an antisense construct, which demonstrates that RET-triggered effects are mediated by p27kip1.

Extracellular superoxide dismutase is a thyroid differentiation marker down-regulated in cancer

Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H(2)O(2) synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H(2)O(2)-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C-Ca(2+) and cAMP-protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.

Morphological Ultrasound Microimaging of Thyroid in Living Mice

The objective of the study was to explore high-frequency ultrasound (HFUS) for noninvasive microimaging of thyroid in living mice. Thyroid examination was performed by HFUS in 10 normal C57BL/6 mice, eight mice treated by propylthiouracil, and 22 Tg-TRK-T1 transgenic mice. The dimension of the gland and the presence of nodules were evaluated. Nodules were classified as malignant (hypoechogenicity, poorly defined margins, internal microcalcification, irregular shapes, and extra glandular extension) or not, and the findings were compared with histological data. Thyroid images were successfully obtained in all the animals analyzed. Normal thyroid reached a volume of 4.92 microl (range 2.11-4.92 microl). Mice with propylthiouracil-induced goiter showed diffuse thyroid enlargement (median volume 6.67 microl, range 4.09-8.82 microl). In 19 of 22 Tg-TRK-T1 mice (86%), HFUS identified a nodular process (the smallest detected nodule had a diameter of 0.46 mm). Eleven nodules were classified as malignant and eight as benign. Compared with histological analysis, HFUS showed a sensitivity of 100% in the detection of thyroid nodules and a specificity of 60% (two of the nodules identified by HFUS were not confirmed at the histology). The specificity and sensitivity of HFUS in predicting the malignancy of the thyroid nodules were 83 and 91%, respectively. Thus, HFUS is an accurate imaging modality that can potentially replace more invasive techniques, and, therefore, it represents a significant advancement in phenotypic assessment of mouse models of thyroid cancer.