Veronica Vella

BRAF(V600E) mutation and the biology of papillary thyroid cancer

BRAF((V600E)) mutation is the most frequent genetic alteration in papillary thyroid carcinomas (PTCs) that are 80-90% of all thyroid cancers. We evaluated the relationship between BRAF((V600E)) and tumor, host, and environmental factors in PTCs from all geographical areas of Sicily. By PCR, BRAF((V600E)) was investigated in a series of 323 PTCs diagnosed in 2002-2005. The correlation between clinicopathological tumor, host, and environmental characteristics and the presence of BRAF((V600E)) were evaluated by both univariate and multivariate analyses. BRAF((V600E)) was found in 38.6% PTCs, with a 52% frequency in the classical PTCs and 26.4% in the tall cell variant. Univariate analysis indicated that BRAF((V600E)) was associated with greater tumor size (P=0.0048), extra-thyroid invasion (P<0.0001), and cervical lymph nodal metastases (P=0.0001). Multivariate logistic regression analysis confirmed that BRAF((V600E)) was an independent predictor of extra-thyroid invasion (P=0.0001) and cervical lymph nodal metastasis (P=0.0005). The association between BRAF((V600E)) and extra-thyroid invasion was also found in micro-PTCs (P=0.006). In 60 classical PTCs, BRAF((V600E)) was positively correlated with matrix metalloproteinase-9 expression (P=0.0047), suggesting a possible mechanism for BRAF((V600E)) effect on PTC invasiveness. No association was found between BRAF((V600E)) and patient age, gender, or iodine intake. In contrast, a strong association was found with residency in Eastern Sicily (P<0.0001 compared with Western Sicily). These results indicate that BRAF((V600E)) mutation is a marker of aggressive disease in both micro- and macro-PTCs. Moreover, for the first time, a possible link between BRAF((V600E)) mutation and environmental carcinogens is suggested.

Insulin/IGF-I hybrid receptors play a major role in IGF-I signaling in thyroid cancer.

The insulin-like growth factor-I (IGF-I) plays an important role in determining the biological behavior of a variety of malignancies. We measured IGF-I, its receptor and related receptors in thyroid cancer. IGF-I was present both in normal thyroid tissue and in thyroid cancer tissue and it was produced by stromal cells but not by thyrocytes. Values were significantly higher in malignant than in normal tissue. IGF-I receptors (IGF-I-Rs) and the homologous insulin receptors (IRs) were found overexpressed in both thyroid cancer cell lines (n = 4) and specimens (n = 17) as compared to normal values. In addition, high levels of hybrid IGF-I/insulin receptors (IR/IGF-I-Rs) were present in both thyroid cancer specimens and cell lines. IR/IGF-I-R hybrids were the most represented type of receptor in 14/17 specimens and exceeded the IGF-I-R content in all cases. Hybrid content correlated with the IR and IGF-I-R content, suggesting that in thyroid tissue hybrid formation occurs by random assembly of IR and IGF-I-R half receptors. Hybrid receptor autophosphorylation was stimulated by IGF-I with high affinity. In cells with a high IR/IGF-I-Rs content, blocking antibodies specific to these receptors substantially inhibited IGF-I induced cell growth. These data indicate that the IGF-I system is overactivated in thyroid cancer and that IR/IGF-I-R hybrid receptors play an important role in IGF-I mitogenic signaling in these tumors.

Insulin Receptor Isoforms and Insulin-Like Growth Factor Receptor in Human Follicular Cell Precursors from Papillary Thyroid Cancer and Normal Thyroid

CONTEXT Factors involved in the biology of normal and cancer stem/precursor cells from the thyroid are unknown. Thyroid cancer cells are responsive to insulin and IGF-I and IGF-II and often overexpress the insulin receptor (IR) and the IGF-I receptor (IGF-IR). OBJECTIVE We investigated the role of IR isoforms (IR-A and IR-B), IGF-IR, and their ligands in thyroid follicular cell precursors both normal and malignant. DESIGN We established cultures of follicular cell precursors as thyrospheres from three papillary thyroid cancers and the corresponding nonaffected tissues. The expression of IR, IGF-IR, and their ligands was evaluated by quantitative RT-PCR and, in one case, also by Western blot. The effects of insulin and IGFs on thyrosphere growth and self-renewal were evaluated. RESULTS Thyrospheres were characterized by the expression of stem cell markers and low/absent thyroid specific markers. Thyrospheres from normal tissue, but not from cancer tissue, could be induced to differentiate. Both IR isoforms, IGF-IR, IGF-I and IGF-II, were expressed at high levels in thyrospheres and markedly decreased in differentiating cells. IR-A was the predominant isoform in thyrospheres, especially from cancer, while IR-B was predominant in differentiating cells. Cancer thyrosphere growth was stimulated by insulin and IGFs. CONCLUSIONS Our data suggest that IR isoforms and IGF-IR play a role in the biology of follicular thyroid precursors. Cell differentiation is associated with marked changes in the expression of these receptors and cognate ligands. These data may provide insight for future differentiation therapies in thyroid cancer.

Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to Hepatocyte Growth Factor

The Hepatocyte Growth Factor (HGF) and its receptor Met are physiological regulators of cell migration. HGF and Met have also been implicated in tumor progression and metastasis. We show here that the tyrosine kinase inhibitor STI571 has a stimulatory effect on HGF-induced migration and branching morphogenesis in thyroid cancer but not in primary or immortalized thyroid epithelial cells. These stimulatory effects of STI571 are observed at a concentration that is clinically relevant. The STI571-enhanced motile response can be correlated with an increase in the Met receptor tyrosine phosphorylation as well as ERK and Akt activation by HGF. Interestingly, one of the targets of STI571, namely the c-Abl tyrosine kinase, is activated by HGF and is recruited at the migrating edge of thyroid cancer cells. These data suggests that c-Abl and/or STI571-inhibited tyrosine kinases can negatively regulate the Met receptor to restrain the motile response in thyroid cancer cells.

Reactivation of p53 mutants by p53 reactivation and induction of massive apoptosis in thyroid cancer cells

Most undifferentiated thyroid carcinomas express p53 mutants and thereafter, are very resistant to chemotherapy. p53 reactivation and induction of massive apoptosis (Prima‐1) is a compound restoring the tumor‐suppressor activity of p53 mutants. We tested the effect of Prima‐1 in thyroid cancer cells harboring p53 mutations. Increasing doses of Prima‐1 reduced viability of thyroid cancer cells at a variable extent (range 20–80%). Prima‐1 up‐regulated p53 target genes (p21WAF1, BCL2‐associated X protein (Bax), and murine double minute 2 (MDM2)), in BC‐PAP and Hth‐74 cells (expressing D259Y/K286E and K286E p53 mutants) but had no effect in SW1736 (p53 null) and TPC‐1 (expressing wild‐type p53) thyroid cancer cells. Prima‐1 also increased the cytotoxic effects of either doxorubicin or cisplatin in thyroid cancer cells, including the chemo‐resistant 8305C, Hth‐74 and BC‐PAP cells. Moreover, real‐time PCR and Western blot indicated that Prima‐1 increases the mRNA of thyroid‐specific differentiation markers in thyroid cancer cells. Fluorescence‐activated cell sorting analysis revealed that Prima‐1 effect on thyroid cancer cells occurs via the enhancement of both cell cycle arrest and apoptosis. Small interfering RNA experiments indicated that Prima‐1 effect is mediated by p53 mutants but not by the p53 paralog p73. Moreover, in C‐643 thyroid cancer cells, forced to ectopically express wild‐type p53, Prima‐1 prevented the dominant negative effect of double K248Q/K286E p53 mutant. Finally, co‐IP experiments indicated that in Hth‐74 cells Prima‐1 prevents the ability of p53 mutants to sequestrate the p53 paralog TAp73. These in vitro studies imply that p53 mutant reactivation by small compounds may become a novel anticancer therapy in undifferentiated thyroid carcinomas.

Immunostaining for Met/HGF receptor may be useful to identify malignancies in thyroid lesions classified suspicious at fine-needle aspiration biopsy.

The receptor for hepatocyte growth factor (Met) is not expressed in the normal thyroid but it is overexpressed in most thyroid carcinomas. We evaluated whether Met immunostaining of cytological smears from fine-needle aspiration biopsy (FNAB) may be useful for the preoperative diagnosis of thyroid cancer. Notably, routine cytological examination often fails to distinguish well-differentiated follicular carcinomas and a proportion of papillary carcinomas (low-grade papillary carcinomas and papillary carcinomas follicular variant [FVPTC]) from benign lesions: all these lesions are usually classified as suspicious. We examined 80 thyroid lesions diagnosed as suspicious at cytology that had subsequently undergone surgery. The histologic diagnosis had been: papillary carcinomas (n = 14), FVPTC (n = 11), follicular carcinomas (n = 25), atypical follicular adenomas (n = 5), follicular adenomas (n = 20), and nodular goiters (n = 5). We also studied typical papillary carcinomas (n = 30) and nodular goiters (n = 10), all correctly diagnosed at cytology. In lesions classified suspicious at routine cytology, Met immunostaining was positive in 12 of 14 (85.7%) papillary carcinomas, 8 of 11 (72.7%) FVPTC, 7 of 25 (28%) follicular carcinomas, and 5 of 5 atypical adenomas. In contrast, none of the 25 lesions cytologically suspicious but benign at histology were positive. These data suggest that Met immunostaining of suspicious cytological smears are useful for identifying malignant lesions, especially those with a papillary histotype.

Insulin Receptor Isoforms in Physiology and Disease: An Updated View

The insulin receptor (IR) gene undergoes differential splicing that generates two IR isoforms, IR-A and IR-B. The physiological roles of IR isoforms are incompletely understood and appear to be determined by their different binding affinities for insulin-like growth factors (IGFs), particularly for IGF-2. Predominant roles of IR-A in prenatal growth and development and of IR-B in metabolic regulation are well established. However, emerging evidence indicates that the differential expression of IR isoforms may also help explain the diversification of insulin and IGF signaling and actions in various organs and tissues by involving not only different ligand-binding affinities but also different membrane partitioning and trafficking and possibly different abilities to interact with a variety of molecular partners. Of note, dysregulation of the IR-A/IR-B ratio is associated with insulin resistance, aging, and increased proliferative activity of normal and neoplastic tissues and appears to sustain detrimental effects. This review discusses novel information that has generated remarkable progress in our understanding of the physiology of IR isoforms and their role in disease. We also focus on novel IR ligands and modulators that should now be considered as an important strategy for better and safer treatment of diabetes and cancer and possibly other IR-related diseases.

Levels of histone acetylation in thyroid tumors

Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells.

Type 3 Deiodinase: Role in Cancer Growth, Stemness, and Metabolism

Deiodinases are selenoenzymes that catalyze thyroid hormones (THs) activation (type 1 and type 2, D1 and D2, respectively) or inactivation (type 3, D3). THs are essential for proper body development and cellular differentiation. Their intra- and extra-cellular concentrations are tightly regulated by deiodinases with a pre-receptorial control thus generating active or inactive form of THs. Changes in deiodinases expression are anatomically and temporally regulated and influence the downstream TH signaling. D3 overexpression is a feature of proliferative tissues such as embryo or cancer tissues. The enhanced TH degradation by D3 induces a local hypothyroidism, thus inhibiting THs transcriptional activity. Of note, overexpression of D3 is a feature of several highly proliferative cancers. In this paper, we review recent advances in the role of D3 in cancer growth, stemness, and metabolic phenotype. In particular, we focus on the main signaling pathways that result in the overexpression of D3 in cancer cells and are known to be relevant to cancer development, progression, and recurrence. We also discuss the potential role of D3 in cancer stem cells metabolic phenotype, an emerging topic in cancer research.

ΔNp73α inhibits PTEN expression in thyroid cancer cells

ΔNp73 is a N‐terminally truncated p53 family member with a dominant negative function, which is upregulated in cancer. PTEN is a lipid phosphatase, which is involved in the attenuation of tyrosine kinase signaling. PTEN expression is increased by p53, and its function is blunted in several malignancies. Because in most of the thyroid carcinomas, ΔNp73α is upregulated, whereas PTEN expression down regulated, we investigated whether ΔNp73α may influence PTEN expression in this cell model. We found that ΔNp73α overexpression in thyroid cancer cells reduces PTEN expression, whereas ΔNp73α down‐regulation by siRNA increases PTEN expression. Real‐time PCR indicated that overexpression of ΔNp73α is able to reduce PTEN mRNA levels. Moreover, chromatin immunoprecipitation (ChIP) and luciferase assays indicated that ΔNp73α binds to −1031‐779 region of the PTEN promoter, which is a different site than that for p53, thereby inhibiting promoter activity. Interestingly, also the transcriptionally active p73 isoforms (TAp73α and TAp73β) bound to this DNA sequence and, at variance with ΔNp73α, stimulated PTEN promoter activity to an extent similar to that of p53. In accordance with its effect on PTEN protein levels, ΔNp73α increased phospho‐Akt protein content and, as a consequence, Mdm2‐mediated p53 degradation. This effect of ΔNp73α resulted in increased thyroid cancer cell proliferation and reduced apoptosis and was reverted by the PI3‐kinase inhibitor LY294002, indicating the role of Akt pathway in this effect. Taken together, these results indicate a novel p73 regulated mechanism for PTEN expression in thyroid cancer cells, and that, also through this mechanism, ΔNp73α exerts its protumorigenic effect.