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Epidermal growth factor receptor 1 (EGFR1) signaling is involved in human cancer cell progression and is responsible for aggressive biological behavior and poor clinical outcome in several human malignancies. Activation of the EGFR1 pathway has been proposed, among others, as being involved in the progression of thyroid cancer toward a thyroid-stimulating hormone (TSH)-independent phenotype. We have previously observed that undifferentiated thyroid carcinoma cells are hyper-sensitive to EGF signaling of downstream intracellular pathways, and this correlated both with the loss of TSH-dependency and increase in EGF-dependent proliferation and migration. Thus, we hypothesized that the upregulation of EGFR1 protein expression may be enhanced in parallel with transition toward a poorly differentiated phenotype in human thyroid carcinomas.The expression of EGFR1 was evaluated, by immunohistochemistry, in a series of 49 human thyroid carcinomas at different degrees of tumor differentiation.The expression of EGFR1 protein was significantly upregulated in poorly differentiated and anaplastic thyroid carcinomas, whereas it was absent or faint in normal thyroid gland tissue and in differentiated thyroid papillary carcinomas. Of note, selected thyroid tumors characterized by a mixed population of differentiated and undifferentiated tumor cells, likely progressing from well to poorly differentiated and anaplastic phenotypes, exhibited EGFR1-negative differentiated fields together with EGFR1-positive poorly differentiated and anaplastic areas.Upregulation of EGFR1 expression may be a molecular marker of dedifferentiation in thyroid epithelial carcinomas, likely being responsible for the activation of EGF signaling observed in tumor cells and favoring progression toward an angiogenic, poorly differentiated, TSH-independent phenotype.

BACKGROUND Prostate carcinomas are androgen-dependent neoplasms which progress toward a hormone-independent phenotype during hormone-deprivation therapy. We evaluated nevirapine, a reverse transcriptase inhibitor, as a new treatment in hormone-refractory prostate carcinoma cells with the aim of restoring the androgen-dependency of tumor cells, the rationale being that endogenous reverse transcriptase is up-regulated in transformed cells and reverse transcriptase inhibitors exert a differentiating activity in human tumors. METHODS AND RESULTS Nevirapine induced extensive reprogramming of gene expression in vitro with up-regulation of genes that might be silenced during prostate tumor progression (i.e., K18, PSA and androgen receptor) and down-regulation of genes involved in the progression toward an androgen-independent phenotype (i.e., K5, EGFR1, EGF and VEGF-A). Furthermore, nevirapine down-regulated the growth of prostate carcinoma xenografts in athymic mice and induced a differentiated phenotype in vivo with increased K18 expression. Interestingly, the drug restored androgen signaling by enhancing the ability of tumor cells to respond to dihydrotestosterone stimulation and to the antiproliferative activity of the androgen receptor blocker bicalutamide. Finally, nevirapine pretreatment increased the susceptibility of tumor cells to docetaxel, by enhancing their ability to undergo apoptosis. CONCLUSIONS These data suggest that nevirapine may be clinically tested in human hormone-refractory prostate carcinoma to restore the susceptibility to androgen deprivation therapy or to docetaxel. Prostate 69: 744–754, 2009. © 2009 Wiley-Liss, Inc.

Background: Nevirapine (NVP) is a nonnucleoside reverse transcriptase inhibitor used in HIV patients and recently evaluated as a differentiating and antiproliferative agent in human malignancies. However, while NVP is a safe treatment in immunocompromised patients, NVP-containing regimens have been associated with severe immune-mediated toxicities in non-HIV individuals. Methods and Results: We describe the toxicity profile of single-agent NVP in 6 non-HIV cancer patients treated for a median period of 7.3 months (range 1–24), reporting only a reversible grade III increase in glutamyl transpeptidase and glutamic pyruvic transaminase serum values. Interestingly, NVP treatment correlates with either a decrease in CD8+ T cell counts or a parallel increase in CD4/CD8 ratio, antithyroglobulin and antithyroid peroxidase autoantibody titers. Conclusions: These results, although obtained in a small cohort of patients, suggest that the toxicity profile of single-agent NVP may be worth testing in a phase I/II study in non-HIV cancer patients and that NVP toxicity may depend on its capacity to trigger autoimmune responses in susceptible individuals.

Two classes of repeated genomic elements, retrotransposons and endogenous retroviruses, encode for endogenous nontelomeric reverse transcriptase (RT), a gene that is down-regulated in differentiated cells but is highly expressed in embryonic and transformed tissues. Two nonnucleosidic RT inhibitors, efavirenz and nevirapine, currently used in HIV treatment, reversibly down-regulate tumor growth and induce differentiation in several human tumor cell models.Aggressive biological behavior and loss of specific thyroid cell functions, such as thyroglobulin, thyroid peroxidase, TSH receptor, Na/I symporter expression, and iodine uptake are features of anaplastic thyroid cancer. Thus, we evaluated the use of RT inhibitors as a potentially differentiating and molecular-targeted treatment of this neoplasm.Our findings showed that nevirapine and efavirenz reversibly inhibit cell proliferation without triggering cell death in the undifferentiated thyroid carcinoma ARO and FRO cells, which exhibited high levels of endogenous RT activity. Inhibition of cell growth was correlated with accumulation of cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the S phase. Moreover, treated cells demonstrated a differentiated phenotype and a significant reprogramming of gene expression characterized by up-regulation of the TSH receptor, thyroglobulin, thyroid peroxidase, and Na/I symporter genes. Interestingly, RT inhibition reestablished the ability to uptake iodine in response to TSH either in vitro or in vivo and reversibly down-regulated tumor growth in mice xenografts of ARO cells.These findings support the need for clinical trials to clarify whether RT inhibitors may restore the sensitivity to radiometabolic therapy in anaplastic thyroid tumors.

The therapeutic role of EGFR inhibitors in thyroid malignancies is still controversial even though the full activation of EGF signaling has recently been proposed as involved in the dedifferentiation of human thyroid cancers.Agents which target EGFR signaling (erlotinib, cetuximab and panitumumab) were evaluated at preclinical level in a panel of thyroid tumor cell lines.Erlotinib induced a dose-dependent inhibition of cell proliferation together with inhibition of EGF-induced AKT and ERK1/2 signaling only in poorly-differentiated thyroid carcinoma FRO cells. By contrast, anti-EGFR monoclonal antibodies were inactive. Of note, erlotinib enhanced the proapoptotic activity of doxorubicin and paclitaxel and exhibited synergy with paclitaxel in poorly-differentiated thyroid carcinoma cells.EGFR signaling may represent a molecular target only in poorly-differentiated thyroid carcinoma cells, and agents that inhibit EGFR tyrosine kinase may be more effective than monoclonal antibodies which target the extracellular domain of the receptor.

Our recent findings have shown that the reverse transcriptase (RT) inhibitors, nevirapine and efavirenz, used for 10 years in human immunodeficiency virus (HIV) disease, act as cytostatic and differentiating agents by modulating gene expression in several human tumor cell models. In dedifferentiated thyroid cancer, they reestablish thyroid-stimulating hormone (TSH) signaling, Na/I symporter (NIS), thyroglobulin peroxidase (TPO) expression, and even radioiodine uptake (RIU). In this paper, we describe the case of a 76-year-old woman who was affected by thyroid papillary carcinoma and who underwent a total thyroidectomy and a debulking of the right laterocervical region for lymph-node metastases, vessel infiltration, and neoplastic thrombosis of the internal jugular vein, followed by 3 radioiodine treatments. At restaging, a computed tomography scan revealed that distant metastases were mostly not taking up the radioiodine at the 131I whole-body scan (WBS). An analysis of tumor cells obtained by fine-needle aspiration biopsy of a right laterocervical lymph-node revealed cell anisokaryosis, nuclear pleomorphism, and scanty colloid, as well as the undetectable expression of thyroglobulin and NIS proteins. After starting a nevirapine treatment (NT), higher thyroglobulin levels were observed and some metastases exhibited a significant increase in radioiodine uptake, which led us to again treat the patient with 131I. Five (5) months later, the 131I-WBS revealed the disappearance of RIU in some metastases and its significant reduction in other lesions, with a parallel drop in serum thyroglobulin. No new metastatic lesion was revealed by rh-TSH-stimulated 131IWBS and 18F-flourodeoxyglucose positron emission tomography scan. Cells obtained from the right laterocervical lymph-node 2 months after NT exhibited a reduced nuclear pleomorphism, an increase in colloid production, and a significant upregulation of thyroglobulin and NIS protein expression. This first in vivo molecular and morphologic evidence of cell differentiation in human cancer and low toxicity of nevirapine strongly encourage its use in dedifferentiated thyroid cancer treatment.

In rat liver, in addition to their intrinsic transferase activity, alpha-class GSTs have Se-independent glutathione peroxidase activity toward fatty acid hydroperoxides, cumene hydroperoxide and phospholipids hydroperoxides but not toward H 2 O 2. We have previously shown that hepatic GST activity by these isoenzymes is significantly increased 24 h after cadmium or manganese administration ( Casalino et al., 2004 ). Here it is reported that Se-independent glutathione peroxidase activity by alpha-class GSTs is also stimulated in the liver of intoxicated rats. The stimulation is associated with a higher level of alpha-class GST proteins, whose induction is blocked by actinomycin D co-administration. The observed Se-independent glutathione peroxidase activity is due to alpha-class GST isoenzymes, as indicated by the studies with diethyldithiocarbamate which, at any concentration, equally inhibits both GST and Se-independent glutathione peroxidase and is an uncompetitive inhibitor of both enzymes. As for liver Se-GSPx, it is not at all affected under these toxic conditions. For comparison, we have evaluated the status of another important antioxidant enzyme, NAD(P)H:quinone reductase, 24 h after cadmium or manganese administration. NQO1 too results strongly stimulated in the liver of the intoxicated rats. In these animals, a higher expression of Nrf2 protein is observed, actively translocated from the cytoplasm to the nucleus. The results with the transcription inhibitor, actinomycin D, and the effects on Nrf2 protein are the first clear indication that acute manganese intoxication, similarly to that of cadmium and other heavy metals, increases both the hepatic level of Nrf2 and its transfer from the cytoplasm to the nucleus where it actively regulates the induction of phase II enzymes.

On Feb 13, 2003, a 76-year-old woman was referred with a follicular variant of a thyroid papillary carcinoma and underwent a total thyroidectomy and debulking of the right laterocervical region, which showed several metastases in the right laterocervical lymph nodes, vessel infi ltration, and a neoplastic thrombosis of the internal jugular vein (pathological stage T4bN1aMx). During the next 2 years, apart from the persistence of the disease in the upper mediastinum and right laterocervical lymph nodes, the rapid and progressive appearance of multiple lung and bone metastases was noted during whole-body scans with recombinant thyroid-stimulating hormone (TSH)-stimulated iodine-131. She therefore underwent another surgical debulking of the right laterocervical region and upper mediastinum, and excision of the right internal jugular vein with the neoplastic thrombus on July 29, 2003, with external-beam radiotherapy (50 Gy in 28 fractions) of the laterocervical region and upper mediastinum in November, 2003, and three applications of recombinant TSH-stimulated I metabolic treatment (4834 MBq on June 6, 2003; 7400 MBq on Feb 23, 2004; and 5374 MBq on Nov 15, 2004). However, these treatments had little eff ect on disease progression, as shown by persistence of the disease in the upper mediastinum and right laterocervical lymph nodes in the fi rst I whole-body scan after treatment, followed rapidly by multiple bone and lung metastases seen in the second and third scans after treatment. On May 27, 2005, a whole-body scan with CT showed a region of contrast enhancement in the left retromandibular region, with multiple metastases in the right upper mediastinum (the region of the previous surgical excision of the neoplastic thrombus), both lungs, a dorsal vertebra, the right ilium, and the left femur. However, only the right upper mediastinal and retromandibular lesions showed any signs of radioiodine uptake, albeit low, during the I whole-body scan done before the CT scan (fi gure 1). These features were interpreted as probable signs of rapid and progressive dediff erentiation of the initial thyroid papillary carcinoma, which was confi rmed by the presence of cell anisokaryosis, nuclear pleiomorphism, and scanty colloid, and undetectable expression of thyroglobulin and sodium iodine symporter proteins in tumour cells (fi gure 2), which were obtained by an echo-guided fi ne-needle aspiration biopsy of a right laterocervical lymph node. Antibodies against thyroglobulin and the Na/I symporter were initially tested in primary cultures of human thyroid cells (positive control) and in anaplastic thyroid carcinoma ARO cells (negative control), and showed positive cytoplasmic staining in normal thyrocytes and a low signal in anaplastic thyroid tumour cells (fi gure 3). At that time, serum thyroblobulin was 795 μg/L, with a low serum thyroid peroxidase antibody titre. After consent from the ethics committee of the Riuniti Hospital (Foggia, Italy) was obtained, the patient started treatment with nevirapine (200 mg once a day for 14 days, then 200 mg twice a day for 7 months). A progressive increase in serum thyroglobulin was noted, which reached a peak concentration of 3925 μg/L 3 months after nevirapine treatment started. Moreover, 2 months after starting nevirapine, the retromandibular metastasis, which was metabolically silent before treatment with nevirapine, showed radioiodine uptake of 2·1 times higher than that seen before treatment, whereas the right upper mediastinal lesion showed 54% upregulation of radioiodine uptake (fi gure 1, table). The patient received a fourth dose of I metabolic treatment (9250 MBq on Aug 22, 2005), and 5 months later, the I whole-body scan Lancet Oncol 2006; 7: 877–79

Poorly differentiated and anaplastic thyroid cancers are aggressive malignancies unresponsive to standard treatments. The mechanisms responsible for the progression of thyroid tumors toward a thyroid-stimulating hormone (TSH)-independent phenotype are still under discussion, and a better understanding of them may provide novel molecular targets for the treatment of this disease. We evaluated the hypothesis that epithelial growth factor (EGF) signaling may play a role in favoring the loss of TSH dependency in human differentiated thyroid tumor cells.The sensitivity to EGF stimulation was evaluated in follicular thyroid carcinoma WRO cells that retain some features of thyroid cell differentiation and in undifferentiated TSH-independent thyroid carcinoma FRO cells.It was observed that, while both cell lines are characterized by a similar EGF-dependent activation of the RAS/MAPK signaling pathway, only FRO cells exhibited a significant induction of phosphoAKT, cell proliferation, and migration as well as the up-regulation of vascular endothelial growth factor-A expression in response to EGF. On the other hand, the inhibition of epidermal growth factor receptor 1 signaling by its tyrosine kinase inhibitor, erlotinib, caused a selective down-regulation of FRO cell proliferation and induced a phenotype more sensitive to the proapoptotic activity of anthracyclins and taxoids. By contrast, the protracted stimulation of TSH-dependent WRO cells with EGF induced the loss of TSH dependency and the rearrangement of F-actin cytoskeleton.These results suggest that the acquired sensitivity to EGF in these thyroid tumor cells may be responsible for the loss of differentiation in the transition toward a TSH-independent, invasive, and chemoresistant phenotype.