Dickkopf-1 (DKK1) drives growth and metastases in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is associated with a poor prognosis and remains an incurable fatal disease. Therefore, the identification of molecular markers involved in cancer progression is urgently needed to develop more-effective therapies. The present study investigated the role of the Wnt signaling modulator Dickkopf-1 (DKK1) in the growth and metastatic progression of mCRPC. DKK1 silencing through siRNA and deletion via CRISPR/Cas9 editing were performed in two different metastatic castration-resistant prostate cancer cell lines (PC3 and DU145). A xenograft tumor model was used to assess tumor growth and metastases. In in vitro experiments, both DKK1 silencing and deletion reduced cell growth and migration of both cell lines. DKK1 knockout clones (DKK1-KO) exhibited cell cycle arrest, tubulin reorganization, and modulation of tumor metastasis-associated genes. Furthermore, in DKK1-KO cells, E-cadherin re-expression and its membrane co-localization with ß-catenin were observed, contributing to reduced migration; Cadherin-11, known to increase during epithelial-mesenchymal transition, was down-regulated in DKK1-KO cells. In the xenograft mouse model, DKK1 deletion not only reduced tumor growth but also inhibited the formation of lung metastases. In conclusion, our findings support the key role of DKK1 in the growth and metastatic dissemination of mCRPC, both in vitro and in vivo.

Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients.

Chronic obstructive pulmonary disease (COPD) is due to structural changes and narrowing of small airways and parenchymal destruction (loss of the alveolar attachment as a result of pulmonary emphysema), which all lead to airflow limitation. Extracorporeal shock waves (ESW) increase cell proliferation and differentiation of connective tissue fibroblasts. To date no studies are available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects. We obtained primary bronchial fibroblasts from bronchial biopsies of 3 patients with mild/moderate COPD and 3 control smokers with normal lung function. 16HBE cells were also studied. Cells were treated with a piezoelectric shock wave generator at low energy (0.3 mJ/mm2, 500 pulses). After treatment, viability was evaluated and cells were recultured and followed up for 4, 24, 48, and 72 h. Cell growth (WST-1 test) was assessed, and proliferation markers were analyzed by qRT-PCR in cell lysates and by ELISA tests in cell supernatants and cell lysates. After ESW treatment, we observed a significant increase of cell proliferation in all cell types. C-Kit (CD117) mRNA was significantly increased in 16HBE cells at 4 h. Protein levels were significantly increased for c-Kit (CD117) at 4 h in 16HBE (p < 0.0001) and at 24 h in COPD-fibroblasts (p = 0.037); for PCNA at 4 h in 16HBE (p = 0.046); for Thy1 (CD90) at 24 and 72 h in CS-fibroblasts (p = 0.031 and p = 0.041); for TGFß1 at 72 h in CS-fibroblasts (p = 0.038); for procollagen-1 at 4 h in COPD-fibroblasts (p = 0.020); and for NF-κB-p65 at 4 and 24 h in 16HBE (p = 0.015 and p = 0.0002). In the peripheral lung tissue of a representative COPD patient, alveolar type II epithelial cells (TTF-1+) coexpressing c-Kit (CD117) and PCNA were occasionally observed. These data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in 16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects.

Modulating tumor reactive stroma by extracorporeal shock waves to control prostate cancer progression.

BACKGROUND: Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. METHODS: Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. RESULTS: ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. CONCLUSIONS: This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.

Benzene affects the response to octreotide treatment of growth hormone secreting pituitary adenoma cells.

Growth hormone (GH) secreting pituitary adenomas are the main cause of acromegaly. Somatostatin analogs are the gold standard of medical therapy; however, resistance represents a big drawback in acromegaly management. We recently demonstrated that benzene (BZ) modifies the aggressiveness of GH-secreting rat pituitary adenoma cells (GH3), increasing GH secretion and altering the synthesis of molecules involved in the somatostatin signaling pathway. Based on these pieces of evidence, this study aimed to evaluate the effects of BZ on octreotide (OCT) efficacy in GH-secreting adenoma cells. In GH3 cells, BZ counteracted the anti-proliferative action of OCT. GH gene expression, unmodified by OCT, remained high in BZ-treated cells as well as after treatment with the association of both. GH secretion, reduced by OCT, was increased after treatment with BZ alone or when the pollutant was used with OCT. The combination of BZ and OCT greatly reduced the gene expression of ZAC1 and SSTR2; and this reduction was also present at a protein level. BZ caused an increase in the protein level of the transcription factor STAT3 and in its phosphorylated form. In the presence of BZ, OCT lost the ability to reduce the phosphorylated protein levels. Finally, in primary cultures of human pituitary adenoma cells, BZ caused an increase in GH secretion. OCT decreased GH secretion, but the addition of BZ reversed the OCT effect. In conclusion, our results suggest that BZ may have an important role in the resistance of pituitary adenomas to the pharmacological treatment with somatostatin analogs.

FOXA1 and AR in invasive breast cancer: new findings on their co-expression and impact on prognosis in ER-positive patients.

BACKGROUND: The role of forkhead-box A1 (FOXA1) and Androgen receptor (AR) in breast cancer (BC) has been extensively studied. However, the prognostic role of their co-expression in Estrogen receptor positive (ER+) BC has not been investigated so far. The aim of the present study was thus to assess the co-expression (protein and mRNA) of FOXA1 and AR in BC patients, in order to evaluate their prognostic impact according to ER status. METHODS: Immunohistochemical expression of AR and FOXA1 was evaluated on 479 consecutive BC, with complete clinical-pathological and follow up data. Fresh-frozen tissues from 65 cases were available. The expression of AR and FOXA1 with ER was validated using mRNA analyses. Survival and Cox proportional hazard analyses were used to evaluate the relationship between FOXA1, AR and prognosis. RESULTS: Expression of ER, AR and FOXA1 was observed in 78, 60 and 85% of cases respectively. Most AR+ cases (97%) were also FOXA1+. The level of FOXA1 mRNA positively correlated with level of both AR mRNA (r = 0.8975; P < 0.001) and ER mRNA (r = 0.7326; P < 0.001). In ER+ BC, FOXA1 was associated with a good prognosis independently of AR expression in the three subgroups analyzed (FOXA1+/AR+; FOXA1+/AR-; FOXA1-/AR-). Multivariate analyses confirmed that FOXA1 may provide more information than AR in Disease-Free Interval (DFI) of ER+ BC patients. CONCLUSION: Our results suggest that in BC the expression of FOXA1 is directly related to the expression of AR. Despite that, FOXA1 is found as superior predicting marker of recurrences compared to AR in ER+ BC patients.

Fibulin-1 interacts with Sex Hormone Binding Globulin and is linked to less aggressive estrogen-dependent breast cancers.

AIMS: Interaction of Sex Hormone-Binding Globulin (SHBG) with estrogen-sensitive breast cancer cells has a protective role against estrogen exposure. No specific membrane receptor for SHBG had been identified by now, but a putative interaction of SHBG with extracellular matrix associated-proteins (e.g. fibulins) was suggested. In this study we investigated the expression of fibulins, their functional relationship with SHBG and involvement in behavior of estrogen-sensitive breast cancer. MAIN METHODS: Gene expression of fibulins was performed by Real time-PCR on two estrogen-sensitive breast cancer cell lines, MCF-7 and T47D. Fibulin-1 protein expression and localization were determined by Western blot and immunofluorescence. SHBG interaction with-fibulin-1 was assessed by GST-pull down assay. MCF-7 cell growth and gene expression, after fibulin-1 silencing by siRNA, were evaluated. Finally, the expression of fibulin-1 was correlated to clinical and pathological data of 21 breast cancer tissue samples. KEY FINDINGS: Fibulin-1 was expressed in both cell lines and it was increased by estradiol. SHBG interacted with fibulin-1C; proteins co-localized at MCF-7 cell membranes and SHBG localization at membranes disappeared after silencing fibulin-1. Fibulin-1 silencing, moreover, generated MCF-7 cells unresponsive to estradiol and SHBG and characterized by increased proliferation. Finally, in breast cancer tissue samples expressing fibulin-1 the proliferation index was significantly lower than in fibulin-1 negative samples. SIGNIFICANCE: Fibulin-1 interacts with SHBG, it is associated with a less aggressive behavior of breast cancer cells and correlates to a better prognosis of the tumor.

Extracorporeal shock waves trigger tenogenic differentiation of human adipose-derived stem cells.

PURPOSES: Incomplete tendon healing impairs the outcome of tendon ruptures and tendinopathies. Human Adipose-derived Stem Cells (hASCs) are promising for tissue engineering applications. Extracorporeal Shock Waves (ESW) are a leading choice for the treatment of several tendinopathies. In this study, we investigated the effects of ESW treatment and tenogenic medium on the differentiation of hASCs into tenoblast-like cells. MATERIALS AND METHODS: hASCs were treated with ESW generated by a piezoelectric device and tenogenic medium. Quantitative real-time PCR was used to check the mRNA expression levels of tenogenic transcription factors, extracellular matrix proteins, and integrins. Western blot and immunofluorescence were used to detect collagen 1 and fibronectin. Collagen fibers were evaluated by Masson staining. Calcium deposition was assessed by Alizarin Red staining. RESULTS: The combined treatment improved the expression of the tendon transcription factors scleraxis and eyes absent 2, and of the extracellular matrix proteins fibronectin, collagen I, and tenomodulin. Cells acquired elongated and spindle shaped fibroblastic morphology; Masson staining revealed the appearance of collagen fibers. Finally, the combined treatment induced the expression of alpha 2, alpha 6, and beta 1 integrin subunits, suggesting a possible role in mediating ESW effects. CONCLUSIONS: ESW in combination with tenogenic medium improved the differentiation of hASCs toward tenoblast-like cells, providing the basis for ESW and hASCs to be used in tendon tissue engineering.

Effects of environmental pollutants on signaling pathways in rat pituitary GH3 adenoma cells.

An increased rate of acromegaly was reported in industrialized areas, suggesting an involvement of environmental pollutants in the pathogenesis and behavior of GH-secreting pituitary adenomas. Based on these premises, the aim of the study was to evaluate the effects of some widely diffused pollutants (i.e. benzene, BZ; bis(2-ethylhexyl) phthalate, DEHP and polychlorinated biphenyls, PCB) on growth hormone secretion, the somatostatin and estrogenic pathways, viability and proliferation of rat GH-producing pituitary adenoma (GH3) cells. All the pollutants induced a statistically significant increase in GH secretion and interfered with cell signaling. They all modulated the expression of SSTR2 and ZAC1, involved in the somatostatin signaling, and the expression of the transcription factor FOXA1, involved in the estrogen receptor signaling. Moreover, all the pollutants increased the expression of the CYP1A1, suggesting AHR pathway activation. None of the pollutants impacted on cell proliferation or viability. Present data demonstrate that exposure to different pollutants, used at in vivo relevant concentrations, plays an important role in the behavior of GH3 pituitary adenoma cells, by increasing GH secretion and modulating several cellular signaling pathways. These observations support a possible influence of different pollutants in vivo on the GH-adenoma aggressiveness and biological behavior.

Combining doxorubicin-nanobubbles and shockwaves for anaplastic thyroid cancer treatment: preclinical study in a xenograft mouse model.

Anaplastic thyroid cancer is one of the most lethal diseases, and a curative therapy does not exist. Doxorubicin, the only drug approved for anaplastic thyroid cancer treatment, has a very low response rate and causes numerous side effects among which cardiotoxicity is the most prominent. Thus, doxorubicin delivery to the tumor site could be an import goal aimed to improve the drug efficacy and to reduce its systemic side effects. We recently reported that, in human anaplastic thyroid cancer cell lines, combining doxorubicin-loaded nanobubbles with extracorporeal shock waves, acoustic waves used in lithotripsy and orthopedics without side effects, increased the intracellular drug content and in vitro cytotoxicity. In the present study, we tested the efficacy of this treatment on a human anaplastic thyroid cancer xenograft mouse model. After 21 days, the combined treatment determined the greatest drug accumulation in tumors with consequent reduction of tumor volume and weight, and an extension of the tumor doubling time. Mechanistically, the treatment induced tumor apoptosis and decreased cell proliferation. Finally, although doxorubicin caused the increase of fibrosis markers and oxidative stress in animal hearts, loading doxorubicin into nanobubbles avoided these effects preventing heart damage. The improvement of doxorubicin anti-tumor effects together with the prevention of heart damage suggests that the combination of doxorubicin-loaded nanobubbles with extracorporeal shock waves might be a promising drug delivery system for anaplastic thyroid cancer treatment.

Thyroid Autoimmunity and Cancer.

Cancer and autoimmune diseases are often associated in the same individual. The functional link between the immune system and cancer development is only partially known. Even though the immune system can control the development of cancer through immune surveillance, cancer cell can escape it. It is debated whether autoimmune diseases have to be regarded as a cancer cause or its consequence. In particular, the association between autoimmune thyroiditis and thyroid cancer (TC; especially papillary carcinoma) is a fascinating model of this complex relationship. In this review, we present data reported in literature about autoimmune thyroiditis and papillary TC, and on the basis of available data, we try to clarify the present knowledge.

Extracorporeal shockwaves (ESWs) enhance the osteogenic medium-induced differentiation of adipose-derived stem cells into osteoblast-like cells.

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue engineering, given their potential to differentiate into osteoblast-like cells. Interactions among biochemical and mechanical signals result in bone formation and repair. In this process stem cells have a crucial role. Extracorporeal shockwaves (ESWs) are acoustic waves capable of enhancing bone regeneration, suggesting that ESWs may induce some signals for mesenchymal progenitor maturation. The aim of the present work is to investigate the effects of ESW treatment on the differentiation of hASCs into osteoblast-like cells and to better clarify the mechanisms involved. The hASCs were treated with ESWs and osteogenic medium, and the effects in terms of gene expression, alkaline phosphatase (ALP) activity and calcium deposition were then evaluated. Moreover, to investigate the mechanisms of ESW action, reactive oxygen species (ROS) production, extracellular-signal-regulated kinase (ERK) and small 'mothers against' decapentaplegic (Smad) phosphorylation, and bone morphogenetic protein 2 (BMP2) expression were assessed. The ESW treatment increased Runt-related transcription factor 2 (Runx2), ALP and BMP2 expression, as well as ALP activity and calcium deposits with respect to untreated cells. Moreover ESWs induced ROS formation, and both ERK and Smad phosphorylation. The present study shows the effects of ESWs on osteogenic differentiation in an in vitro model using hASCs and defines the mechanisms involved in this process. The observations suggest that the combination of autologous hASCs and ESW treatment may improve bone tissue repair in tissue engineering procedures. Copyright © 2014 John Wiley & Sons, Ltd.

Benzene and 2-ethyl-phthalate induce proliferation in normal rat pituitary cells.

PURPOSE: Endocrine disruptors are known to modulate a variety of endocrine functions and increase the risk for neoplasia. Epidemiological data reported increased prevalence of pituitary tumors in high industrial areas while genotyping studies showed that mutations in the aryl hydrocarbon receptor (AhR) interacting protein (AIP)-chaperone to the dioxin ligand AhR-gene are linked to predisposition to pituitary tumor development. Aim of the present study was to establish whether endocrine pollutants can induce cell proliferation in normal rat pituitary cells. METHODS: Pituitary primary cultures were incubated with 250, 650 and 1250 pM benzene or 2-ethyl-phthalate for up to 96 h and viability, energy content and cell proliferation assessed. Expression of pituitary tumor transforming gene (PTTG), cyclin D1 (Ccnd1), AhR and AIP was quantified by RT-qPCR. RESULTS: Incubation with benzene or 2-ethyl-phthalate increased viability and energy content in pituitary cells. The endocrine disruptors also increased cell proliferation as well as Ccnd1 and PTTG expression. Increased AhR and AIP expression was observed after incubation with the two pollutants. CONCLUSIONS: Our findings indicate that benzene and 2-ethyl-phthalate activate AhR/AIP expression and stimulate proliferation in normal rat pituitary cells. This study is the first demonstration that pollutants can induce normal pituitary cells to proliferate and provides a link between epidemiological and genomic findings in pituitary tumors.

Targeting Taxanes to Castration-Resistant Prostate Cancer Cells by Nanobubbles and Extracorporeal Shock Waves.

To target taxanes to castration-resistant prostate cancer cells, glycol-chitosan nanobubbles loaded with paclitaxel and docetaxel were constructed. The loaded nanobubbles were then combined with Extracorporeal Shock Waves, acoustic waves widely used in urology and orthopedics, with no side effects. Nanobubbles, with an average diameter of 353.3 ± 15.5 nm, entered two different castration-resistant prostate cancer cells (PC3 and DU145) as demonstrated by flow cytometry and immunofluorescence. The shock waves applied increased the amount of intracellular nanobubbles. Loading nanobubbles with paclitaxel and docetaxel and combining them with shock waves generated the highest cytotoxic effects, resulting in a paclitaxel GI50 reduction of about 55% and in a docetaxel GI50 reduction of about 45% respectively. Combined treatment also affected cell migration. Paclitaxel-loaded nanobubbles and shock waves reduced cell migration by more than 85% with respect to paclitaxel alone; whereas docetaxel-loaded nanobubbles and shock waves reduced cell migration by more than 82% with respect to docetaxel alone. The present data suggest that nanobubbles can act as a stable taxane reservoir in castration-resistant prostate cancer cells and shock waves can further increase drug release from nanobubbles leading to higher cytotoxic and anti-migration effect.

Valproic Acid, a Histone Deacetylase Inhibitor, in Combination with Paclitaxel for Anaplastic Thyroid Cancer: Results of a Multicenter Randomized Controlled Phase II/III Trial.

Anaplastic thyroid cancer (ATC) has a median survival less than 5 months and, to date, no effective therapy exists. Taxanes have recently been stated as the main drug treatment for ATC, and the histone deacetylase inhibitor valproic acid efficiently potentiates the effects of paclitaxel in vitro. Based on these data, this trial assessed the efficacy and safety of the combination of paclitaxel and valproic acid for the treatment of ATC. This was a randomized, controlled phase II/III trial, performed on 25 ATC patients across 5 centers in northwest Italy. The experimental arm received the combination of paclitaxel (80 mg/m2/weekly) and valproic acid (1,000 mg/day); the control arm received paclitaxel alone. Overall survival and disease progression, evaluated in terms of progression-free survival, were the primary outcomes. The secondary outcome was the pharmacokinetics of paclitaxel. The coadministration of valproic acid did not influence the pharmacokinetics of paclitaxel. Neither median survival nor median time to progression was statistically different in the two arms. Median survival of operated-on patients was significantly better than that of patients who were not operated on. The present trial demonstrates that the addition of valproic acid to paclitaxel has no effect on overall survival and disease progression of ATC patients. This trial is registered with EudraCT 2008-005221-11.

Doxorubicin-Loaded Nanobubbles Combined with Extracorporeal Shock Waves: Basis for a New Drug Delivery Tool in Anaplastic Thyroid Cancer.

BACKGROUND: No standard chemotherapy is available for anaplastic thyroid cancer (ATC). Drug-loaded nanobubbles (NBs) are a promising innovative anticancer drug formulation, and combining them with an externally applied trigger may further control drug release at the target region. Extracorporeal shock waves (ESWs) are acoustic waves widely used in urology and orthopedics, with no side effects. The aim of the present work was to combine ESWs and new doxorubicin-loaded glycol chitosan NBs in order to target doxorubicin and enhance its antitumor effect in ATC cell lines. METHODS: CAL-62 and 8305C cells were treated with empty NBs, fluorescent NBs, free doxorubicin, and doxorubicin-loaded NBs in the presence or in the absence of ESWs. NB entrance was evaluated by fluorescence microscopy and flow cytofluorimetry. Cell viability was assessed by Trypan Blue exclusion and WST-1 proliferation assays. Doxorubicin intracellular content was measured by high-performance liquid chromatography. RESULTS: Treatment with empty NBs and ESWs, even in combination, was safe, as cell viability and growth were not affected. Loading NBs with doxorubicin and combining them with ESWs generated the highest cytotoxic effect, resulting in drug GI50 reduction of about 40%. Mechanistically, ESWs triggered intracellular drug release from NBs, resulting in the highest nuclear drug content. CONCLUSIONS: Combined treatment with doxorubicin-loaded NBs and ESWs is a promising drug delivery tool for ATC treatment with the possibility of using lower doxorubicin doses and thus limiting its systemic side effects.

The pan-histone deacetylase inhibitor LBH589 (panobinostat) alters the invasive breast cancer cell phenotype.

Triple-negative breast cancer (TNBC) is a very aggressive type of tumour and its aggressiveness is linked to E-cadherin downregulation. In estrogen-sensitive breast cancer, high levels of E-cadherin fit with high levels of ERα and MTA3 (a component of the transcription Mi-2/NuRD complex with intrinsic DAC activity). In TNBC the E-cadherin downregulation could be due to epigenetic silencing of the CDH1 gene as well as to the lack of a fully functioning ERα-activated pathway. We report that the pan-histone deacetylase inhibitor LBH589, a potent anti-proliferative agent, induced E-cadherin expression on cell membranes of MDA-MB-231 cells (TNBC), determining a reduction of cell invasion and migration. Even though E-cadherin expression in breast cancer is also regulated by estradiol and the ERα/MTA3/Snail/Slug pathway, LBH589 is able to increase E-cadherin without affecting the estrogen pathway. In fact, no expression of ERα, PR and FoxA1 was observed in MDA-MB-231 cells before and after LBH589 treatment; furthermore, the drug caused an increase in Snail and Slug expression with a concomitant reduction of MTA3 levels. Taking into consideration its anti-proliferative and anti-invasive properties, we suggest the use of LBH589 in aggressive breast cancer refractory to hormonal therapy.

Histone deacetylase inhibition affects sodium iodide symporter expression and induces 131I cytotoxicity in anaplastic thyroid cancer cells.

BACKGROUND: Anaplastic thyroid cancers (ATCs) represent only 1%-2% of all thyroid tumors, but they account for up to 50% of the mortality. Treatment of differentiated thyroid carcinomas is well standardized and the use of radioiodine represents an essential step; in contrast, there is no standardized therapeutic approach for anaplastic tumors and their prognosis is poor. The resistance of ATC to radioiodine treatment is principally due to the absence of expression of the sodium iodide symporter (NIS), mainly due to epigenetic silencing. The acetylation status of histones is involved in the epigenetic control of gene expression and is usually disrupted in advanced thyroid cancer. Histone deacetylase inhibitors have been demonstrated as potent anticancer drugs with several different effects on cell viability and differentiation. METHODS: Stabilized ATC cell lines (BHT-101 and CAL-62) and primary cultures from patients who underwent thyroidectomy for ATC were treated with the histone deacetylase inhibitor LBH589. After treatment, we evaluated the expression and function of NIS. Gene expression was evaluated by real-time polymerase chain reaction (RT-PCR), NIS promoter activity was determined with a luciferase reporter assay, and protein expression was assessed through immunofluorescence. We tested the protein function by (125)I uptake and efflux experiments; finally the cytotoxic effect of (131)I was determined with a clonogenic assay. RESULTS: Our results demonstrate that treatment with LBH589 leads to NIS RNA expression as shown by RT-PCR and luciferase assay, and to protein expression as determined by immunofluorescence in vitro and by immunohistochemistry in xenograft tumors. Moreover, (125)I uptake and efflux experiments show the correct protein function and iodine retention, which translate into cytotoxicity effects, as demonstrated by a clonogenic assay with (131)I. CONCLUSIONS: This study supplies a new potential strategy for the treatment of ATC by modifying gene expression with the aim of inducing responsiveness towards radioiodine therapy.

Epigenetics modifications and therapeutic prospects in human thyroid cancer.

At present no successful treatment is available for advanced thyroid cancer, which comprises poorly differentiated, anaplastic, and metastatic or recurrent differentiated thyroid cancer not responding to radioiodine. In the last few years, biologically targeted therapies for advanced thyroid carcinomas have been proposed on the basis of the recognition of key oncogenic mutations. Although the results of several phase II trials look promising, none of the patients treated had a complete response, and only a minority of them had a partial response, suggesting that the treatment is, at best, effective in stabilizing patients with progressive disease. "Epigenetic" refers to the study of heritable changes in gene expression that occur without any alteration in the primary DNA sequence. The epigenetic processes establish and maintain the global and local chromatin states that determine gene expression. Epigenetic abnormalities are present in almost all cancers and, together with genetic changes, drive tumor progression. Various genes involved in the control of cell proliferation and invasion (p16INK4A, RASSF1A, PTEN, Rap1GAP, TIMP3, DAPK, RARß2, E-cadherin, and CITED1) as well as genes specific of thyroid differentiation (Na+/I- symport, TSH receptor, pendrin, SL5A8, and TTF-1) present aberrant methylation in thyroid cancer. This review deals with the most frequent epigenetic alterations in thyroid cancer and focuses on epigenetic therapy, whose goal is to target the chromatin in rapidly dividing tumor cells and potentially restore normal cell functions. Experimental data and clinical trials, especially using deacetylase inhibitors and demethylating agents, are discussed.

Histone deacetylase inhibition modulates E-cadherin expression and suppresses migration and invasion of anaplastic thyroid cancer cells.

CONTEXT: Anaplastic thyroid cancer cells are characterized by a mesenchymal phenotype, as revealed by spindle-shaped cells and absent or reduced levels of E-cadherin. Epigenetic silencing is considered one of the leading mechanisms of E-cadherin impairment, which causes the acquisition of the invasive and metastatic phenotype of anaplastic thyroid cancer. OBJECTIVES: In this study we investigated the effects of histone deacetylase inhibition on E-cadherin expression, cell motility, and invasion in anaplastic thyroid cancer cell cultures. DESIGN: Three stabilized cell lines and primary cultures of anaplastic thyroid cancer were treated with various histone deacetylase inhibitors. After treatment, we evaluated histone acetylation by Western blotting and E-cadherin expression by RT-real time PCR. The proper localization of E-cadherin/ß-catenin complex was assessed by immunofluorescence and Western blot. Transcription activity of ß-catenin was measured by luciferase reporter gene and cyclin D1 expression. The effect on cell motility and invasion was studied both in vitro using scratch-wound and transwell invasion assays and in anaplastic thyroid carcinomas tumor xenografts in mice in vivo. RESULTS: Histone deacetylase inhibition induced the E-cadherin expression and the proper membrane localization of the E-cadherin/ß-catenin complex, leading to reduced cancer cell migration and invasion. CONCLUSIONS: We here demonstrate an additional molecular mechanism for the anticancer effect of histone deacetylase inhibition. The antiinvasive effect in addition to the cytotoxic activity of histone deacetylase inhibitors opens up therapeutic perspectives for the anaplastic thyroid tumor that does not respond to conventional therapy.