AXL Is a Novel Predictive Factor and Therapeutic Target for Radioactive Iodine Refractory Thyroid Cancer.

Papillary thyroid carcinomas (PTCs) have an excellent prognosis, but a fraction of them show aggressive behavior, becoming radioiodine (RAI)-resistant and/or metastatic. AXL (Anexelekto) is a tyrosine kinase receptor regulating viability, invasiveness and chemoresistance in various human cancers, including PTCs. Here, we analyze the role of AXL in PTC prognosis and as a marker of RAI refractoriness. Immunohistochemistry was used to assess AXL positivity in a cohort of human PTC samples. Normal and cancerous thyroid cell lines were used in vitro for signaling, survival and RAI uptake evaluations. 38.2% of human PTCs displayed high expression of AXL that positively correlated with RAI-refractoriness and disease persistence or recurrence, especially when combined with v-raf murine sarcoma viral oncogene homolog B(BRAF) V600E mutation. In human PTC samples, AXL expression correlated with V-akt murine thymoma viral oncogene homolog 1 (AKT1) and p65 nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation levels. Consistently, AXL stimulation with its ligand growth arrest-specific gene 6 (GAS6) increased AKT1- and p65 NF-kB-phosphorylation and promoted survival of thyroid cancer cell lines in culture. Enforced expression or activation of AXL in normal rat thyroid cells significantly reduced the expression of the sodium/iodide symporter (NIS) and the radioiodine uptake. These data indicate that AXL expression levels could be used as predictor of RAI refractoriness and as a possible novel therapeutic target of RAI resistant PTCs.

Recent advances in understanding immune phenotypes of thyroid carcinomas: prognostication and emerging therapies.

Tumors modulate the host immune cells within their microenvironment to avoid recognition and elimination by our immune system, a phenotype called cancer immune escape. Different mechanisms responsible for cancer immune escape that result either in decreased tumor immunogenicity or in increased tumor immunosuppressive activity have been identified. Recently, various immunotherapeutic approaches have been developed with the aim to revert tumor immune escape. The aims of this review are to explore the immunological aspects of thyroid cancer and to assess whether these features can be exploited in the prognosis and treatment of advanced forms of this disease. Therefore, we will describe the immune landscape and phenotypes of thyroid cancer, summarize studies investigating the expression of immunomodulatory molecules, and finally describe the preclinical and clinical trials investigating the utility of immunotherapies in the management of thyroid cancer. The aim of this review is to explore the immunological aspects of thyroid cancer and to assess whether these features can be exploited in the prognosis and treatment of advanced forms of this disease. Therefore, we will describe the immune-landscape and phenotypes of thyroid cancer, we will summarize studies investigating the expression of immunomodulatory molecules, and we will finally describe the preclinical and clinical trials investigating the utility of immunotherapies in the management of thyroid cancer.

Retraction for Fusco et al., "A mos Oncogene-Containing Retrovirus, Myeloproliferative Sarcoma Virus, Transforms Rat Thyroid Epithelial Cells and Irreversibly Blocks Their Differentiation Pattern".

[This retracts the article DOI: 10.1128/JVI.56.1.284-292.1985.].

Reduced expression of α-L-Fucosidase-1 (FUCA-1) predicts recurrence and shorter cancer specific survival in luminal B LN+ breast cancer patients.

BACKGROUND: The lysosomal enzyme α-L-Fucosidase-1 (FUCA-1) catalyzes the hydrolytic cleavage of terminal fucose residues. FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors as its inactivation perturbs the fucosylation of proteins involved in cell adhesion, migration and metastases. RESULTS: Negativity to FUCA-1 was significantly related to the development of later recurrences in breast cancer patients with lymph node involvement at diagnosis. Cancer specific survival of luminal B LN+ patients was influenced by FUCA-1 expression as luminal B LN+ patients with positive expression had a longer cancer specific survival. FUCA-1 mRNA expression was inversely related to cancer stage and lymph node involvement. WB and qPCR analysis of FUCA-1 expression in breast cancer-derived cell lines confirmed an inverse relationship with tumor aggressiveness. CONCLUSIONS: This study shows that, within LN+ breast cancer patients, FUCA-1 is able to identify a sub-set of non recurrent patients characterized by the positive expression of FUCA-1 and that, within luminal B LN+ patients, the expression of FUCA-1 predicts longer cancer specific survival. METHODS: We have analyzed FUCA-1 in 305 breast cancer patients by Immunohistochemistry (IHC), and by qPCR in breast cancer patients and in breast cancer cell lines.

FUCA1 is induced by wild-type p53 and expressed at different levels in thyroid cancers depending on p53 status.

Fucose residues of cell surface glycans, which play important roles in growth, invasion and metastasis, are added by fucosyltransferases (FUTs) and removed by α-L-fucosidases (FUCAs). By the differential display method, we isolated a 3' non-coding region of α-L-fucosidase-1 (FUCA1) (a gene coding for the lysosomal fucosidase-1 enzyme) as a wild-type p53-inducible gene: 18S and 20S FUCA1 mRNA species were induced in Saos-2 cells transfected with a temperature-sensitive p53 mutant at the permissive temperature. By microarray analyses of thyroid cancer biopsy samples, FUCA1 RNA expression levels were found to be lower in anaplastic thyroid cancer samples (ATCs), while they were higher in papillary thyroid cancer samples (PTCs) and in normal thyroid tissues. Since most ATCs were reported to carry the mutated form of p53, while PTCs carry mostly the wild-type form of p53, it is likely that FUCA1 expression levels are regulated, at least in part, by the p53 status in thyroid cancers. In order to better understand the role played by FUCA genes in thyroid tumorigenesis, we examined the clonogenic potential in vitro of thyroid cell lines transfected with either FUCA1 or FUCA2 (the latter gene coding for a secreted, non-lysosomal enzyme). We found that α-L-fucosidases did not suppress grossly cell growth. Contrary to what we observed with the expression of FUCA1, the FUT8 expression levels were found high in ATCs but lower in PTCs and normal thyroid tissues. Taken together, these results suggest the possibility that the higher fucose levels on cell surface glycans of aggressive ATCs, compared to those of less aggressive PTCs, may be at least in part responsible for the more aggressive and metastatic phenotype of ATCs compared to PTCs, as the expression levels of FUCA1 and FUT8 were inversely related in these two types of cancers.

Human a-L-fucosidase-1 attenuates the invasive properties of thyroid cancer.

Glycans containing α-L-fucose participate in diverse interactions between cells and extracellular matrix. High glycan expression on cell surface is often associated with neoplastic progression. The lysosomal exoenzyme, α-L-fucosidase-1 (FUCA-1) removes fucose residues from glycans. The FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors. However, the role of FUCA-1 in tumor progression remains unclear. It is speculated that its inactivation perturbs glycosylation of proteins involved in cell adhesion and promotes cancer. FUCA-1 expression of various thyroid normal and cancer tissues assayed by immunohistochemical (IHC) staining was high in normal thyroids and papillary thyroid carcinomas (PTC), whereas it progressively decreased in poorly differentiated, metastatic and anaplastic thyroid carcinomas (ATC). FUCA-1 mRNA expression from tissue samples and cell lines and protein expression levels and enzyme activity in thyroid cancer cell lines paralleled those of IHC staining. Furthermore, ATC-derived 8505C cells adhesion to human E-selectin and HUVEC cells was inhibited by bovine α-L-fucosidase or Lewis antigens, thus pointing to an essential role of fucose residues in the adhesive phenotype of this cancer cell line. Finally, 8505C cells transfected with a FUCA-1 containing plasmid displayed a less invasive phenotype versus the parental 8505C. These results demonstrate that FUCA-1 is down-regulated in ATC compared to PTC and normal thyroid tissues and cell lines. As shown for other human cancers, the down-regulation of FUCA-1 correlates with increased aggressiveness of the cancer type. This is the first report indicating that the down-regulation of FUCA-1 is related to the increased aggressiveness of thyroid cancer.

Differential diagnosis of thyroid nodules using fine-needle aspiration cytology and oncogene mutation screening: are we ready?

Thyroid nodules are a very common clinical finding, and although the majority of them are benign, thyroid carcinoma accounts for about 5-15% of nodules. Fine-needle aspiration cytology (FNAC) is actually used for the differential diagnosis of these lesions. Although in most cases this examination clearly distinguishes benign from malignant lesions, some fine-needle aspiration (FNA) samples fall into undetermined thyroid cytology categories, which according to the most recent classification of thyroid FNAC consist of 'suspicious for malignancy', 'suspicious for follicular or Hurtle cell neoplasm', and 'follicular lesion of undetermined significance/atypia of undetermined significance'. Moreover, some samples are insufficient for diagnosis. Taken together, these categories account for almost 20-30% of nodules. Owing to the high risk of papillary thyroid carcinoma, patients with lesions that are 'suspicious for malignancy' are currently subjected to lobectomy or total thyroidectomy. On the other hand, patients with 'atypia of undetermined significance' undergo repeated FNAs, and patients with 'suspicious for follicular or Hurtle cell neoplasm' are subjected to diagnostic lobectomy and subsequently, in the case of histological diagnosis of carcinoma, total thyroidectomy. Recent studies clearly indicate that molecular analysis of thyroid nodules can significantly improve the diagnostic power of cytology and drive the appropriate clinical management of these patients.

RET is a heat shock protein 90 (HSP90) client protein and is knocked down upon HSP90 pharmacological block.

CONTEXT: Mutations of the RET receptor tyrosine kinase are associated to multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinoma (MTC). The heat shock protein (HSP) 90 chaperone is required for folding and stability of several kinases. HSP90 is specifically inhibited by 17-allyl-amino-17-demethoxygeldanamycin (17-AAG). OBJECTIVE: Our aim was to investigate whether RET protein half-life depends on HSP90 and to dissect the molecular pathway responsible for the degradation of RET upon HSP90 inhibition by 17-AAG. DESIGN: 17-AAG effects were studied in RAT1 fibroblasts exogenously expressing MEN2-associated RET mutants and human MTC-derived cell lines. RESULTS: 17-AAG induced a 26S proteasome-dependent degradation of wild-type RET and MEN2-associated RET mutants. The compound hampered HSP90/RET interaction and stabilized RET binding to HSP70, leading to the recruitment of the HSP70-associated E3 ligase C-terminus of Hsc70-interacting protein. In turn, C-terminus of Hsc70-interacting protein polyubiquitinated RET, promoting its proteasomal degradation. 17-AAG blocked RET downstream effectors and RET-dependent transcriptional activation of gene promoters. In human MTC cells carrying oncogenic RET mutants, HSP90 inhibition induced receptor degradation and signaling hindrance leading to cell cycle arrest. CONCLUSION: RET and MEN2-associated RET mutants rely on HSP90 for protein stability, and HSP90 blockade by 17-AAG promotes RET degradation.

Cytostatic activity of adenosine triphosphate-competitive kinase inhibitors in BRAF mutant thyroid carcinoma cells.

CONTEXT: The V600E mutation accounts for the vast majority of thyroid carcinoma-associated BRAF mutations. OBJECTIVE: The aim was to study the effects of the two BRAF V600E ATP-competitive kinase inhibitors, PLX4032 and PLX4720, in thyroid carcinoma cell lines. EXPERIMENTAL DESIGN: We examined the activity of PLX4032 and PLX4720 in thyroid carcinoma cell lines harboring BRAF V600E (8505C, BCPAP, SW1736, BHT101), NRAS Q61R (HTH7), KRAS G12R (CAL62), HRAS G13R (C643), or RET/PTC1 (TPC-1) oncogenes. Normal thyrocytes (PC Cl 3) were used as control. RESULTS: Both compounds inhibited the proliferation of BRAF mutant cell lines, but not normal thyrocytes, with a half maximal effective concentration (EC(50)) ranging from 78-113 nm for PLX4720 and from 29-97 nm for PLX4032. Doses equal to or higher than 500 nm were required to achieve a similar effect in BRAF wild-type cancer cells. Phosphorylation of ERK 1/2 and MAPK kinase (MEK) 1/2 decreased upon PLX4032 and PLX4720 treatment in BRAF mutant thyroid carcinoma cells but not in normal thyroid cells or in cell lines harboring mutations of RAS or RET/PTC1 rearrangements. PLX4032 and PLX4720 treatment induced a G(1) block and altered expression of genes involved in the control of G(1)-S cell-cycle transition. 8505C cell tumor xenografts were smaller in nude mice treated with PLX4032 than in control mice. This inhibition was associated with reduction of phospho-ERK and phospho-MEK levels. CONCLUSIONS: This study provides additional evidence of the promising nature of mutant BRAF as a molecular target for thyroid carcinoma cells.

Effects of VEGF on phenotypic severity in children with hereditary hemorrhagic telangiectasia.

The purpose of this study was to estimate vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta1 serum levels in children with hereditary hemorrhagic telangiectasia (HHT) type 1 and type 2 and to correlate them to the presence of arteriovenous malformations (AVMs). High VEGF levels were initially found in an infant who had been hospitalized with intestinal bleeding and suspected HHT. This case led to the evaluation of VEGF and TGF-beta1 by standard enzyme-linked immunosorbent assay in 13 children with HHT and familiarity. Patients were divided into 2 groups on the basis of the presence/absence of pulmonary AVMs. No significant difference was found for VEGF and TGF-beta1 levels in HHT patients versus controls. Among HHT patients, serum levels of VEGF in those without AVM were significantly lower than those with AVM and normal controls. No difference for TGF-beta1 levels was found in these patient subgroups. Low VEGF levels may represent a protection factor against the onset of pulmonary AVMs in HHT children. However, neither VEGF nor TGF-beta1 can be used as biochemical markers for an early diagnosis in HHT. The diagnosis of HHT still requires clinical criteria, which permitted to confirm the presence of the disease in the infant with intestinal bleeding.

Pathophysiology of ageing, longevity and age related diseases.

On April 18, 2007 an international meeting on Pathophysiology of Ageing, Longevity and Age-Related Diseases was held in Palermo, Italy. Several interesting topics on Cancer, Immunosenescence, Age-related inflammatory diseases and longevity were discussed. In this report we summarize the most important issues. However, ageing must be considered an unavoidable end point of the life history of each individual, nevertheless the increasing knowledge on ageing mechanisms, allows envisaging many different strategies to cope with, and delay it. So, a better understanding of pathophysiology of ageing and age-related disease is essential for giving everybody a reasonable chance for living a long and enjoyable final part of the life.

45th annual meeting of the Italian Cancer Society. Bergamo, 9-12 November 2003.

The 45th Annual Meeting of the Italian Cancer Society (SIC), held at the Centro Congressi Giovanni XXIII in Bergamo, Italy on 9-12 November, 2003, attracted almost 400 participants. The Scientific Committee chaired by R Giavazzi (Mario Negri Institute, Bergamo) and the board of the Italian Cancer Society produced a packed and varied program. Plenary sessions with keynote speakers (24 lectures) were combined with oral proffered papers (20 selected presentations), providing a platform from laboratory science to clinical interventions. Two hundred and sixty-one abstracts were submitted to the conference and presented in poster and poster discussion sessions, providing a stimulating ground for discussion and a network of interactive collaboration among young investigators. The venue of Bergamo offered exceptional hospitality with social events in the beautiful old town and at the Modern Art Museum. We hope that the following report gives an idea of the event and will encourage the reader to attend the 46th meeting of the SIC, 24-26 October, 2004 in Pisa.

Minireview: RET: normal and abnormal functions.

The RET gene encodes a single-pass transmembrane receptor tyrosine kinase. RET is the oncogene that causes papillary thyroid carcinoma and medullary thyroid carcinoma. The latter may arise as a component of multiple endocrine neoplasia type 2 syndromes; germline mutations in RET are responsible for multiple endocrine neoplasia type 2 inheritance. In this report we review data on the mechanisms leading to RET oncogenic conversion and on RET targeting as a strategy in thyroid cancer treatment.

Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors.

We have recently demonstrated that the pyrazolopyrimidines PP1 and PP2 and the 4-anilinoquinazoline ZD6474 display a strong inhibitory activity (IC(50)< or =100 nM) towards constitutively active oncogenic RET kinases. Here, we show that most oncogenic MEN2-associated RET kinase mutants are highly susceptible to PP1, PP2 and ZD6474 inhibition. In contrast, MEN2-associated swap of bulky hydrophobic leucine or methionine residues for valine 804 in the RET kinase domain causes resistance to the three compounds. Substitution of valine 804 with the small amino- acid glycine renders the RET kinase even more susceptible to inhibition (ZD6474 IC(50): 20 nM) than the wild-type kinase. Our data identify valine 804 of RET as a structural determinant mediating resistance to pyrazolopyrimidines and 4-anilinoquinazolines.

H4(D10S170), a gene frequently rearranged with RET in papillary thyroid carcinomas: functional characterization.

Human thyroid papillary carcinomas are characterized by rearrangements of the RET protooncogene with a number of heterologous genes, which generate the RET/papillary thyroid carcinoma (PTC) oncogenes. One of the most frequent variants of these recombination events is the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of a gene named H4(D10S170). We have characterized the H4(D10S170) gene product, showing that it is a ubiquitously expressed 55 KDa nuclear and cytosolic protein that is phosphorylated following serum stimulation. This phosphorylation was found to depend on mitogen-activated protein kinase (MAPK) Erk1/2 activity and to be associated to the relocation of H4(D10S170) from the nucleus to the cytosol. Overexpression of the H4(D10S170) gene was able to induce apoptosis of thyroid follicular epithelial cells; conversely a carboxy-terminal truncated H4(D10S170) mutant H4(1-101), corresponding to the portion included in the RET/PTC1 oncoprotein, behaved as dominant negative on the proapoptotic function and nuclear localization of H4(D10S170). Furthermore, conditional expression of the H4(D10S170)-dominant negative truncated mutant protected cells from stress-induced apoptosis. The substitution of serine 244 with alanine abrogated the apoptotic function of H4(D10S170). These data suggest that loss of the H4(D10S170) gene function might have a role in thyroid carcinogenesis by impairing apoptosis.

ONYX-015 enhances radiation-induced death of human anaplastic thyroid carcinoma cells.

ONYX-015 is a genetically modified adenovirus with a deletion of the E1B early gene and therefore is designed to replicate preferentially in p53-mutated cells causing their death. We previously demonstrated that the ONYX-015 virus kills anaplastic thyroid carcinoma (ATC) cells and enhances the antineoplastic effects of doxorubicin and paclitaxel. Here we report that ONYX-015 increased the cytopathic effect of radiotherapy in three ATC cell lines. In fact, ONYX-015 and radiation induced a significant cytopathic effect on ATC cells. DNA fragmentation analysis showed that ATC ONYX-015-treated cells were very sensitive to radiation-induced apoptosis. In addition, low doses of ONYX-015 associated with a single radiation dose of 10 Gy delayed the growth of a xenograft tumor induced by ARO cells in athymic mice. Our results suggest that the combination of ONYX-015 and radiotherapy should be considered for experimental trials in patients with anaplastic thyroid carcinoma.