The CBX7 protein, whose expression is decreased in human carcinomas, positively regulates E-cadherin expression by interacting with the HDAC2 protein.

CBX7, chromobox homolog 7, is a chromobox family protein encoding a novel Polycomb protein, component of the Polycomb repressive complex 1 (PRC1). The Polycomb group (PcG) proteins are epigenetic transcriptional repressors involved in the control of cellular proliferation and oncogenesis. CBX7 protein levels show a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, its expression decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins among which we selected Histone Deacetylase 2 (HDAC2) that is well known to play a key role in neoplastic cell transformation and to downregulate E-cadherin expression, whose loss is a critical event in the Epithelial-Mesenchymal Transition, and therefore emerging as one of the caretakers of the epithelial phenotype. We confirmed by co-immunoprecipitation that CBX7 physically interacts with the HDAC2 and demostrated that is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter, and that CBX7 is able to upregulate E-cadherin expression. Consistent with these data we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we demonstrated that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression, by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter, would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype in thyroid cancer patients. Thus, several important interacting proteins of CBX7 and the pathways in which they are involved strongly suggest that CBX7 can be considerated a very important regulator of thyroid malignant transformation

Altered Chromogranin A Circulating Levels in Meniere’s Disease

Objectives. Meniere’s disease (MD) is an inner ear disorder characterized by episodic vertigo, ear fullness, and hearing loss; usually vertigo attacks cluster in specific period. We studied in MD patients the circulating levels of chromogranin A (CgA) and vasostatin-1 (VS-1), secreted by the neuroendocrine system and involved in the regulation of the endothelial barrier function. Methods. Serum levels were assessed in 37 MD patients and 36 controls. The ratio between VS-1 and CgA was calculated. Results. CgA was increased in patients compared to controls (1.46 versus 0.67 nM, p=0.01) while no difference was detected for VS-1 (0.41 versus 0.39, resp.). CgA levels in patients positively correlated with the frequency of vertigo spells in the previous four weeks (p=0.008) and negatively with the time in days from the last vertigo attack (p=0.018). Furthermore, the VS-1/CgA ratio negatively correlated with the frequency of vertigo spells (p=0.029) and positively correlated with the time from the last attack (p=0.003). Conclusion. The results indicate that variations of CgA levels, but not of VS-1, occur in the blood of patients with active MD, depending on the frequency of vertigo spells and the time from the last crisis

CBX7 Modulates the Expression of Genes Critical for Cancer Progression

<div><p>Background</p><p>We have previously shown that the expression of CBX7 is drastically decreased in several human carcinomas and that its expression progressively decreases with the appearance of a highly malignant phenotype. The aim of our study has been to investigate the mechanism by which the loss of CBX7 expression may contribute to the emergence of a more malignant phenotype.</p><p>Methods</p><p>We analyzed the gene expression profile of a thyroid carcinoma cell line after the restoration of CBX7 and, then, analyzed the transcriptional regulation of identified genes. Finally, we evaluated the expression of CBX7 and regulated genes in a panel of thyroid and lung carcinomas.</p><p>Results</p><p>We found that CBX7 negatively or positively regulates the expression of several genes (such as SPP1, SPINK1, STEAP1, and FOS, FOSB, EGR1, respectively) associated to cancer progression, by interacting with their promoter regions and modulating their transcriptional activity. Quantitative RT-PCR analyses in human thyroid and lung carcinoma tissues revealed a negative correlation between CBX7 and its down-regulated genes, while a positive correlation was observed with up-regulated genes.</p><p>Conclusion</p><p>In conclusion, the loss of CBX7 expression might play a critical role in advanced stages of carcinogenesis by deregulating the expression of specific effector genes.</p></div

Identification of a New Pathway for Tumor Progression: MicroRNA-181b Up-Regulation and CBX7 Down-Regulation by HMGA1 Protein

High mobility group A (HMGA) overexpression plays a critical role in neoplastic transformation. To investigate whether HMGA acts by regulating the expression of microRNAs, we analyzed the microRNA expression profile of human breast adenocarcinoma cells (MCF7) transfected with the HMGA1 gene, which results in a highly malignant phenotype. Among the microRNAs induced by HMGA1, we focused on miR-181b, which was overexpressed in several malignant neoplasias including breast carcinomas. We show that miR-181b regulates CBX7 protein levels, which are down-regulated in cancer, and promotes cell cycle progression. We also demonstrate that CBX7, being negatively regulated by HMGA, is able to negatively regulate miR-181b expression. Finally, there was a direct correlation between HMGA1 and miR-181b expression and an inverse correlation between HMGA1 and CBX7 expression in human breast carcinomas. These data indicate the presence of a novel pathway involving HMGA1, miR-181b, and CBX7, which leads to breast cancer progression

CBX7 binds to the promoters of the CBX7-regulated genes.

<p><b>A</b>) FRO-EV-1 and FRO-CBX7-1 cells were subjected to a ChIP assay using antibodies against CBX7. As negative controls, unrelated IgG antibodies were used. The associated DNA was amplified by qPCR using primers specific for the corresponding gene promoter and, as a control of ChIP specificity, primers recognizing the human GAPDH gene promoter. <b>B</b>) MEFs obtained from cbx7^+/+ and cbx7^-/- were analyzed for the binding of cbx7 protein to the promoters of its regulated genes. As negative controls, unrelated IgG antibodies were used and, as a control of ChIP specificity, primers recognizing the mouse Gapdh gene promoter were used. Data are reported as percent input and were calculated by using the following formula: 2^ΔCt×3, where ΔCt is the difference between Ct_input and Ct_IP.</p

CBX7 modulates the activity of the CBX7-regulate gene promoters.

<p>HEK 293 cells were transiently co-transfected with increasing amounts of CBX7 expression vector and a constant amount of vector containing the luciferase gene under the control of the promoters of CBX7-regulated genes. Increasing amounts of CBX7 enforced the expression of the reporter gene under the control of the FOS, FOSB and EGR1 promoter regions (<b>A</b>), whereas repressed the activity of the reporter gene under the control of the SPP1, SPINK1 and STEAP1 promoters (<b>B</b>).The relative activity of firefly luciferase expression was standardized to a transfection control, using β-galactosidase. The scale bars represent the mean ± SD (n = 3).</p

CBX7 and CBX7-regulated gene expression levels are correlated in human carcinoma samples.

<p>We analyzed the expression of CBX7, FOS, FOSB, EGR1, SPP1, SPINK1 and STEAP1 by qRT-PCR in papillary thyroid carcinomas (PTC) (<b>A</b>) and human lung carcinoma samples (<b>B</b>). The expression of FOS, FOSB and EGR1 is down-regulated, as it occurs for CBX7, in the neoplastic tissues with respect to the normal counterparts. Conversely, the expression of SPP1, SPINK1 and STEAP1 was up-regulated in the neoplastic samples with respect to the normal tissues, showing an opposite tendency if compared with that of CBX7. Results are expressed as Fold Change (for PTC) or Relative Expression (for lung carcinomas) with respect to a pool of normal samples which were set equal to 1. The range of variability of CBX7 and CBX7-regulated gene expression in normal thyroid and lung tissues was less than 10%.</p

Genes differentially expressed between FRO-CBX7-1 and FRO-EV-1 with a fold change ≤−2,0.

<p>Genes differentially expressed between FRO-CBX7-1 and FRO-EV-1 with a fold change ≤−2,0.</p

Gene expression in cbx7 knockout MEFs.

<p>Quantitative RT-PCR analysis was performed to analyze the expression levels of CBX7-regulated genes in cbx7^+/+ and cbx7^-/- MEFs. Values are expressed as Relative expression with respect to the cbx7^+/+, that was set equal to 1. <b>A</b>) Fos, fosb and egr1 were less expressed in cbx7^-/- MEFs compared to the cbx7^+/+ MEFs. <b>B</b>) On the contrary, spp1, spink1 and steap1 genes were more expressed in MEFs cbx7^-/- compared to the MEFs cbx7^+/+. <b>C</b>) Cbx7^-/- MEFs were transiently transfected with a mammalian vector expressing mouse cbx7 (myc-His-tagged) mRNA (cbx7^-/—R). Restoration of cbx7 expression is able to revert the phenotype of the cbx7^-/- MEFs (<b>A, B</b>). Values are expressed as Relative expression with respect to the cbx7^+/+, that was set equal to 1. <b>D</b>) The expression of fos and egr1 proteins was evaluated by western blot in MEFs obtained from cbx7^-/- mice in comparison to cbx7^+/+ MEFs. β-Actin expression was evaluated to normalize protein loading.</p