Francesca Marano

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/β-catenin complex was assessed by immunofluorescence and Western blot. Transcription activity of β-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/β-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.

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.

The pan-DAC inhibitor LBH589 is a multi-functional agent in breast cancer cells: cytotoxic drug and inducer of sodium-iodide symporter (NIS)

New drugs with anti-tumor activity, also able to modify the expression of selected molecules, are under evaluation in breast cancer which is becoming resistant to conventional treatment, or in metastatic disease. The sodium-iodide symporter (NIS), which mediates iodide uptake into thyroid cells, and is the molecular basis of radioiodine imaging and therapy in thyroid cancer, is also expressed in a large portion of breast tumors. Since NIS expression in breast cancer is not sufficient for a significant iodide uptake, drugs able to induce its expression and correct function are under evaluation. In the present study, we report for the first time that the pan-deacetylase (DAC) inhibitor LBH589 (panobinostat) significantly induced NIS, both as mRNA and as protein, through the increase of NIS promoter activity, with the final consequence of obtaining a significant up-take of iodide in MCF7, T47D, and MDA-MB231 breast cancer cells. Moreover, we observed that LBH589 causes a significant reduction in cell viability of estrogen-sensitive and -insensitive breast cancer cells within nanomolar range. The anti-tumor effect of LBH589 is sustained by apoptosis induction and cell cycle arrest in G2/M. In conclusion, our data suggest that LBH589 might be a powerful tool in the management of breast cancer due to its multiple effects and support a potential application of LBH589 in the diagnosis and treatment of this disease.

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

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.

Extracorporeal shock waves modulate myofibroblast differentiation of adipose‐derived stem cells

Mesenchymal stem cells are precursors of myofibroblasts, cells deeply involved in promoting tissue repair and regeneration. However, since myofibroblast persistence is associated with the development of tissue fibrosis, the use of tools that can modulate stem cell differentiation toward myofibroblasts is central. Extracorporeal shock waves are transient short‐term acoustic pulses first employed to treat urinary stones. They are a leading choice in the treatment of several orthopedic diseases and, notably, they have been reported as an effective treatment for patients with fibrotic sequels from burn scars. Based on these considerations, the aim of this study is to define the role of shock waves in modulating the differentiation of human adipose‐derived stem cells toward myofibroblasts. Shock waves inhibit the development of a myofibroblast phenotype; they down‐regulate the expression of the myofibroblast marker alpha smooth muscle actin and the extracellular matrix protein type I collagen. Functionally, stem cells acquire a more fibroblast‐like profile characterized by a low contractility and a high migratory ability. Shock wave treatment reduces the expression of integrin alpha 11, a major collagen receptor in fibroblastic cells, involved in myofibroblast differentiation. Mechanistically, the resistance of integrin alpha 11‐overexpressing cells to shock waves in terms of alpha smooth muscle actin expression and cell migration and contraction suggests also a role of this integrin in the translation of shock wave signal into stem cell responses. In conclusion, this in vitro study shows that stem cell differentiation toward myofibroblasts can be controlled by shock waves and, consequently, sustains their use as a therapeutic approach in reducing the risk of skin and tissue fibrosis.

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.

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.

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

ABSTRACT 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.