Nadia Passon

Expression of Dicer and Drosha in triple-negative breast cancer

Aims Dicer and Drosha are components of the miRNA-producing machinery and their altered expression may play a role in cancer progression. The main purpose of this study was a detailed investigation of Dicer and Drosha expression and localisation in triple-negative breast cancers. Methods Thirty-one triple-negative breast cancers and several breast cancer cell lines were investigated. Expression of Dicer and Drosha was evaluated at the mRNA level by quantitative reverse transcription PCR and at the protein level by immunohistochemistry or western blot. Results Compared with normal breast tissues, a wide variation of Dicer and Drosha mRNA levels was detected in triple-negative breast cancers. As a group, Drosha mRNA levels in triple-negative breast cancers were significantly higher than those in normal breast tissues. Immunohistochemical data confirmed higher expression of Drosha protein in triple-negative breast cancers. In normal breast tissues Dicer was detectable predominantly in the cytoplasm of basal/myoepithelial cells only. In contrast, in the majority of triple-negative breast cancers, intense Dicer staining was detectable also in the nuclear compartment. Detection of Dicer and Drosha mRNA and protein levels in breast cancer cell lines confirmed the nuclear localisation of Dicer, suggesting, in addition, that the steady-state protein levels could be controlled by post-mRNA regulatory events. Conclusions These findings indicate that Dicer and Drosha expression is deregulated in triple-negative breast cancers.

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.

Nucleophosmin Delocalization in Thyroid Tumour Cells

Nucleophosmin (NPM) is a multifunctional nucleolar protein that, depending on the context, can act as oncogene or tumour suppressor. Mutations of the NPM1 gene induce delocalization of NPM in acute myeloid leukaemia. Differently, in solid tumours, only NPM overexpression, but not delocalization, has been so far reported. Here, NPM localization in thyroid tumours was investigated. By using immunohistochemistry, we show increase of NPM cytoplasmic localization in follicular adenomas and papillary carcinomas compared to normal thyroid tissue (p = 0.0125 and <0.0001, respectively). NPM1 mutations commonly found in human leukaemia are not present in thyroid tumours. Immunofluorescence in cultured cell lines was utilized to discriminate between nucleolar and nuclear localization. We show that in thyroid cancer cell lines NPM localizes both in the nucleolus and in nucleus, while in non-tumorigenic thyroid cell lines localizes only in nucleolus. Either presence of the histone deacetylase inhibitor trichostatin A or absence of thyroid-stimulating hormone induces NPM nuclear localization in non-tumorigenic thyroid cell lines.

The PARP inhibitor PJ34 modifies proliferation, NIS expression and epigenetic marks in thyroid cancer cell lines.

Since PARP-1 is supposed to be part of a multimeric repressor of sodium iodide symporter (NIS) expression, in this study the effect of the PARP inhibitor PJ34 on several properties of thyroid cancer cell lines was investigated. In TPC1, BCPAP, FRO, WRO cell lines PJ34 induced a strong increase in NIS mRNA levels. In BCPAP and TPC1 cells also significant increase of radio-iodine uptake was induced. Accordingly, in transfection experiments performed in TPC1 cells, treatment with PJ34 increased NIS promoter activity without affecting PARP-1 binding to the promoter sequence. We also investigated the epigenetic status of NIS promoter after PJ34 treatment in TPC1 cell line: in addition to an increase of histone modification activation marks (H3K9K14ac, H3K4me3), surprisingly we observed also an increase of H3K27me3, a classical repressive mark. Our data demonstrate that in various thyroid cancer cell lines PARP inhibition increases NIS gene expression through a particular modulation of transcriptional regulatory mechanisms. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of thyroid cancer.

A Simple Multiplex Real-Time PCR Methodology for the SMN1 Gene Copy Number Quantification

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused, in about 95% of SMA cases, by homozygous deletion of the survival motor neuron 1 (SMN1) gene or its conversion to the highly homologous SMN2 gene. The molecular diagnosis of SMA is usually carried out by a PCR-Restriction fragment length polymorphism (RFLP) approach. However, this approach is not useful for identification of healthy deletion carriers. TaqMan technology is one of the most reliable and widely adopted techniques for the SMN1 copy number evaluation. However, several limitations of this technique have been described. Particularly, DNA extraction methods and accurate template quantification have been shown to be critical for reliable results. In this work, we set up a reliable, highly reproducible, and easy-to-perform TaqMan technology-based protocol to obtain the SMN1 gene copy number assessment. We demonstrate that PCR amplification of both target gene and reference gene in the same reaction mix, instead of separated mixes, greatly reduces reported criticisms of simplex TaqMan technology. The multiplex real-time PCR we describe allows interlaboratory samples and data exchange, without the need to equalize the DNA isolation technique. Further, the protocol described below requires fewer replica tests than the simplex methodology does, leading to reduced overall cost for the diagnostic assay.

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

In thyroid cancer cell lines expression of periostin gene is controlled by p73 and is not related to epigenetic marks of active transcription

BackgroundPeriostin expression is a feature of the epithelial-mesenchymal transition, which occurs during cancer progression. Previous reports indicate that periostin expression is related to tumour aggressiveness.MethodsIn order to identify mechanisms regulating periostin expression in thyroid cancer, a panel of continuous thyroid cancer cell lines was investigated. Levels of posttranslational modifications of the H3 histone were investigated by chromatin immunoprecipitation. Moreover, treatment of cell lines with deacetylase inhibitors and transfection experiments were performed.ResultsOur insights show that levels of H3 histone acetylated at lysines 9 and 14 (which are epigenetic marks of active transcription) are not related to periostin mRNA levels. Moreover, treatment of WRO and FRO thyroid cancer cell lines with the deacetylase inhibitor tricostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) increases levels of acetylated H3 histone to periostin promoter however, unpredictably, reduces periostin mRNA levels. Interestingly, treatment of WRO cells with either TSA or SAHA increases levels of the H3 histone trimethylated at lysine 4, which is a different epigenetic mark of active transcription. Instead, data obtained by cell transfection indicate that ΔNp73, a member of p53 family selectively expressed in thyroid carcinomas, plays a role in activating periostin gene expression.ConclusionsLevels of epigenetic marks of active transcription do not contribute to regulation of periostin gene expression. The ΔNp73 effects suggest a novel molecular mechanism involved in thyroid cancer progression.

Down-regulation of SM22/transgelin gene expression during H9c2 cells differentiation

The embryonic rat ventricle H9c2 cells maintain a proliferative state (P condition) in the presence of 10% FCS. However, by reducing serum concentration and in the presence of retinol acetate, proliferation is stopped, myogenic transdifferentiation is inhibited while cardiac differentiation is preserved (D condition). Two-dimensional gel electrophoresis and mass spectrometry analysis was used to define the modifications of the nuclear proteome occurring during the P-to-D transition. Among the proteins observed as modified, a reduced expression of the SM22/transgelin protein was associated with the D state. Also SM22 mRNA levels were reduced during P-to-D transition. Cell transfection experiments indicated that this decrease was partially due to a reduction of the SM22 promoter activity. GATA-4 had a repressive effect on SM22 promoter activity. Thus, since GATA-4 is known as a target of retinoids and may act as a transcriptional repressor, a mechanism to explain the SM22 reduction during the P-to-D transition is tentatively proposed. Immunohistochemical studies on heart cells confirmed the nuclear localization of SM22. Moreover, a differential expression of this protein in different districts of the human heart embryo was detected. Therefore, these data suggest that SM22 expression is regulated during heart development.

Quick MLPA test for quantification of SMN1 and SMN2 copy numbers.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused in about 95% of SMA patients by homozygous deletion of the survival motor neuron 1 (SMN1) gene or its conversion to the highly homologous SMN2 gene. In the majority of cases, disease severity correlates inversely with increased SMN2 copy number. Because of the comparatively high incidence of healthy carriers and severity of the disease, detection of sequence alterations and quantification of SMN1 and SMN2 copy numbers are essential for exact diagnosis and genetic counselling. Several assays have been developed for this purpose. Multiplex ligation-dependent probe amplification (MLPA) is a versatile technique for relative quantification of different nucleic acid sequences in a single reaction. Here, we establish a quick MLPA-based assay for the detection of SMN1 and SMN2 copy numbers with high specificity and low complexity.

Cooperative effects of SAHA and VPA on NIS gene expression and proliferation of thyroid cancer cells

Histone deacetylase inhibitors (HDACi) have shown both anti-proliferative and redifferentiating effects in thyroid cancer cells. Also, they induce the expression of the sodium–iodide symporter gene (NIS (SLC5A5)), a crucial step for radioiodine treatment of thyroid malignancies. Here we investigated the effects of suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) on BCPAP and FRO thyroid cancer cells, extending our analysis on the epigenetic mechanisms underlying the NIS gene expression stimulation. In both cell lines we found a cooperative effect of the two compounds on either cell viability and NIS gene expression, resulting in acquired/increased ability to uptake the radioiodine. Such effect was specific since it was not observed for expression of other genes or when SAHA was used in combination with trichostatin A. By using chromatin immunoprecipitation, we investigated epigenetic mechanisms underlying SAHA and VPA effects. Cooperation among the two HDACi occurred on H3 histone trimethylation at lysine 4 (H3K4me3) and not on histone acetylation. However, effects on H3K4me3 were detected only at the level of NIS Proximal Basal Promoter (NIS-PBP) in FRO cells and only at the level of NIS Upstream Enhancer (NIS-NUE) in BCPAP cells. Our data indicate that epigenetic changes are involved in the synergistic effects of VPA and SAHA on NIS gene expression and that the cellular context modifies effects of HDACi in terms of H3K4me3 target sequence. Investigation of cooperation among different HDACi may provide clues for better defining their mechanism of action in view of their use in thyroid cancer treatment.