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

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

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

Expression of CBX7-regulated genes in rat normal thyroid cells after suppression of cbx7 expression by RNAi.

<p><b>A</b>) PC Cl3 cells were transiently transfected with small interfering RNA (siRNA) against the rat cbx7 mRNA. After transfection we can observe an efficient knockdown of the cbx7 mRNA levels, as evaluated by qRT-PCR analysis. <b>B, C</b>) CBX7-regulated genes expression was evaluated by qRT-PCR in rat PC Cl3 cells after transfection with rat cbx7 siRNA. Expression was evaluated 48 hours after transfection. Values are expressed as Relative expression with respect to the PC Cl3 cells transfected with a non silencing control siRNA (scrambled), that were set equal to 1.</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

Validation of microarray data.

<p><b>A</b>) CBX7 expression was evaluated by qRT-PCR analysis in FRO (wt), and several FRO-EV (empty vector) and FRO-CBX7 cell clones. Values are expressed as Relative expression with respect to the FRO sample that was set equal to 1. <b>B, C</b>) The selected CBX7-regulated genes were analyzed by qRT-PCR analysis in FRO (wt), and several FRO-EV and FRO-CBX7 cell clones. FOS, FOSB and EGR1 gene expression was more abundant in FRO-CBX7 cell clones than in the FRO-EV cells, conversely, SPP1, SPINK1 and STEAP1 expression was less pronounced in FRO-CBX7 cells compared with the FRO-EV and FRO (wt) cells. Values are expressed as Relative expression with respect to the FRO sample that was set equal to 1. <b>D</b>) The expression of FOS, FOSB and EGR1 was evaluated in one FRO-CBX7 cell clone (FRO-CBX7-1), one FRO-EV (FRO-EV-1) and FRO wild-type cells. β-Actin was evaluated as loading control. Arrows indicate the bands corresponding to FOS and EGR1.</p

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 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

CCDC6 inhibits the phosphatase activity of PP4c.

<p>(<b>a</b>) PP4 complex immunopurified from HeLa cells transfected with CCDC6-specific shRNAs (shCCDC6) or with non-targeting control shRNAs (shCTRL), was incubated for 30 minutes at 30°C with pH2AX S139-enriched chromatin purified from irradiated cells. The phosphatase reactions were followed by western blot and probed with the indicated antibodies. (<b>b</b>) PP4c phosphatase was immunoprecipitated from shCCDC6 or shCTRL. 1, 0,7, 0,3 voulmes of total PP4c immunoprecipitated from 3 mg of total cell extract were mixed with 3 µg histones purified from cells exposed to 10 Gy IR and incubated in phosphatase buffer at 30°C for 30 minutes. Phosphatase reaction was terminated by the addition of 100 µl of Malachite Green solution and absorbance was measured at 630 nm. After the phosphatase assay, the actual amount of PP4c in each immunoprecipitate was determined by Western Blotting with the indicated antibody. PP4c activity is represented in arbitrary units (a.u.) calculated as the ratio between released free phosphate (absorbance at 630 nm) and PP4c densitometric signal at western blot. (<b>c</b>) Enzimatic activity of PP4c immunopurified from HeLa cells transfected with CCDC6-specific shRNA (shCCDC6) or with non-targeting control sh-RNAs (shCTRL) was assessed by Malachite Green phosphatase assay. 1, 0,7 and 0,3 volumes of total PP4c immunoprecipitated from 3 mg of total cell extract were incubated with 175 µM of RKpTIRR synthetic peptide for 30 minutes at 30°C. Phosphatase reaction was terminated by the addition of 100 µl of Malachite Green solution and absorbance was measured at 630 nm. After the phosphatase assay, the actual amount of PP4c in each immunoprecipitate was determined by Western Blotting with the indicated antibody. PP4c activity is represented in arbitrary units (a.u.) calculated as the ratio between released free phosphate (absorbance at 630 nm) and PP4c densitometric signal at western blot. (<b>d</b>) PP4 complex immunopurified from TPC-1 cells transfected with CCDC6 wt, CCDC6 (1–223) and (1–101) truncated mutants, was incubated for 30 minutes at 30°C with pH2AX S139-enriched chromatin, purified from irradiated cells. The phosphatase reactions were followed by immunoblotting and probed with the indicated antibodies. (<b>e</b>) (<b>f</b>) Enzimatic activity of PP4c immunopurified from TPC-1 cells transfected with epitope-tagged CCDC6 wt or empty vector, was determined as described in (<b>b</b>) and (<b>c</b>).</p