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The tumor suppressor gene KCTD11REN is regulated by Sp1 and methylation and its expression is reduced in tumors.

Mancarelli MM, Zazzeroni F, Ciccocioppo L, Capece D, Po A, Murgo S, Di Camillo R, Rinaldi C, Ferretti E, Gulino A, Alesse E - Mol. Cancer (2010)

Bottom Line: A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11).We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events.Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, University of L'Aquila, L'Aquila 67100, Italy.

ABSTRACT
A hallmark of several human cancers is loss of heterozygosity (LOH) of chromosome 17p13. The same chromosomal region is also frequently hypermethylated in cancer. Although loss of 17p13 has been often associated with p53 genetic alteration or Hypermethylated in Cancer 1 (HIC1) gene hypermethylation, other tumor suppressor genes (TSGs) located in this region have critical roles in tumorigenesis. A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11). We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events. Using a panel of human 177 tumor samples and their normal matching samples representing 18 different types of cancer, we show here that the down-regulation of KCTD11 protein level is a specific and a diffusely common event in tumorigenesis. Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression. Our findings identify KCTD11 as a widely down-regulated gene in human cancers, and provide a basis to understand how its expression might be deregulated in tumor cells.

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KCTD11 promoter is strongly activated by Sp1 TF. (A) D. Mel-2 cells (Invitrogen) were cultured in Drosophila Serum-Free Medium (Gibco Life Technologies) plus18 mM L-glutamine (Sigma) at room temperature. KCTD11-Luc reporter constructs (1 μg) and pPac-LacZ (200 ng) were co-transfected along with 1 μg of pPac empty vector, pPac-Sp1 or pPac-Sp2 (left panel) or pPac-Sp3 and pPac-Sp1+pPac-Sp3 (right panel) using Cellfectin (Invitrogen). After 48 h, the cells were harvested and the luciferase activity was assayed using the Single Luciferase Assay System (Promega), normalized to β-galactosidase activity using β-Galactosidase Enzyme Assay System (Promega). Each experiment has been done in triplicates. Values are the means ± S.D. (B) EMSAs showing the binding of Sp1 to KCTD11 promoter. Whole extracts were prepared from 293T HEK cells transfected with pCDNA-Sp1 (12 μg), using buffer C [16]. Cell extracts were incubated in vitro with 32P-labeled KCTD11-Sp1 probes (Sp1-A-F) or 32P-labeled canonical Sp1 probe [additional file 2]. Binding specificity was evaluated by competition with an excess (100x) of the cold probe or with a non-specific probe. For the supershift assays, the proteins were pre-incubated with two different anti-Sp1 antibodies (Santa-Cruz) at 4°C for 30 min. (C) ARO (thyroid cancer) and HCT15 (colon cancer) cell line were cultured, respectively, in RPMI-1640 and DMEM supplemented with 10% FCS. Chromatin Immunoprecipitations (ChIP) were performed by using the following antibodies: anti-Sp1 (1C6) X (sc-420; Santa Cruz Biotech), anti-Sp3 (F-7) X (sc-28305; Santa Cruz Biotech), anti-Acetyl-Histone3 (Cell Signaling). Eluted DNA has been analyzed with real-time q-PCR, normalized to GAPDH (left panels). Bars represent the mean of 3 independent experiments ± SD (*, p < 0.05, HCT15 versus ARO). Total protein levels of Sp1 and Sp3 were analyzed by Western blot (right panel).
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Figure 3: KCTD11 promoter is strongly activated by Sp1 TF. (A) D. Mel-2 cells (Invitrogen) were cultured in Drosophila Serum-Free Medium (Gibco Life Technologies) plus18 mM L-glutamine (Sigma) at room temperature. KCTD11-Luc reporter constructs (1 μg) and pPac-LacZ (200 ng) were co-transfected along with 1 μg of pPac empty vector, pPac-Sp1 or pPac-Sp2 (left panel) or pPac-Sp3 and pPac-Sp1+pPac-Sp3 (right panel) using Cellfectin (Invitrogen). After 48 h, the cells were harvested and the luciferase activity was assayed using the Single Luciferase Assay System (Promega), normalized to β-galactosidase activity using β-Galactosidase Enzyme Assay System (Promega). Each experiment has been done in triplicates. Values are the means ± S.D. (B) EMSAs showing the binding of Sp1 to KCTD11 promoter. Whole extracts were prepared from 293T HEK cells transfected with pCDNA-Sp1 (12 μg), using buffer C [16]. Cell extracts were incubated in vitro with 32P-labeled KCTD11-Sp1 probes (Sp1-A-F) or 32P-labeled canonical Sp1 probe [additional file 2]. Binding specificity was evaluated by competition with an excess (100x) of the cold probe or with a non-specific probe. For the supershift assays, the proteins were pre-incubated with two different anti-Sp1 antibodies (Santa-Cruz) at 4°C for 30 min. (C) ARO (thyroid cancer) and HCT15 (colon cancer) cell line were cultured, respectively, in RPMI-1640 and DMEM supplemented with 10% FCS. Chromatin Immunoprecipitations (ChIP) were performed by using the following antibodies: anti-Sp1 (1C6) X (sc-420; Santa Cruz Biotech), anti-Sp3 (F-7) X (sc-28305; Santa Cruz Biotech), anti-Acetyl-Histone3 (Cell Signaling). Eluted DNA has been analyzed with real-time q-PCR, normalized to GAPDH (left panels). Bars represent the mean of 3 independent experiments ± SD (*, p < 0.05, HCT15 versus ARO). Total protein levels of Sp1 and Sp3 were analyzed by Western blot (right panel).

Mentions: Because of Sp1 protein is constitutively expressed TF [9], to assess specifically the role of Sp1 in the regulation of KCTD11 promoter, we co-transfected the KCTD11 promoter constructs (Fig. 2B) along with Sp1, Sp2 or Sp3 expression vectors into Drosophila Mel-2 cells (Fig. 3A), which provide a background for Sp TFs activity [9]. In this cellular system, none of KCTD11 promoter constructs showed basal activity when co-transfected along with an empty vector, whereas Sp1 co-transfection strongly activated the promoter (Fig. 3A). Moreover, the functional significance of each Sp1 site in the KCTD11 promoter was evaluated. Sp1-A and Sp1-C resulted to drive KCTD11 promoter activity (Fig. 3A, compare -398/+99 + pPac-Sp1 vs -522/+99 + pPac-Sp1 and - 268/+99 + pPac-Sp1 vs -338/+99 + pPac-Sp1), whereas Sp1-B and Sp1-D did not (Fig. 3A, compare -338/+99 + pPac-Sp1 vs -398/+99 + pPac-Sp1 and -156/+99 + pPac-Sp1 vs -268/+99 + pPac-Sp1). As a control, deletion of all Sp-1 sites completely abrogated KCTD11 promoter activation by Sp1 (Fig. 3A, -37/+99 + pPac-Sp1 column).


The tumor suppressor gene KCTD11REN is regulated by Sp1 and methylation and its expression is reduced in tumors.

Mancarelli MM, Zazzeroni F, Ciccocioppo L, Capece D, Po A, Murgo S, Di Camillo R, Rinaldi C, Ferretti E, Gulino A, Alesse E - Mol. Cancer (2010)

KCTD11 promoter is strongly activated by Sp1 TF. (A) D. Mel-2 cells (Invitrogen) were cultured in Drosophila Serum-Free Medium (Gibco Life Technologies) plus18 mM L-glutamine (Sigma) at room temperature. KCTD11-Luc reporter constructs (1 μg) and pPac-LacZ (200 ng) were co-transfected along with 1 μg of pPac empty vector, pPac-Sp1 or pPac-Sp2 (left panel) or pPac-Sp3 and pPac-Sp1+pPac-Sp3 (right panel) using Cellfectin (Invitrogen). After 48 h, the cells were harvested and the luciferase activity was assayed using the Single Luciferase Assay System (Promega), normalized to β-galactosidase activity using β-Galactosidase Enzyme Assay System (Promega). Each experiment has been done in triplicates. Values are the means ± S.D. (B) EMSAs showing the binding of Sp1 to KCTD11 promoter. Whole extracts were prepared from 293T HEK cells transfected with pCDNA-Sp1 (12 μg), using buffer C [16]. Cell extracts were incubated in vitro with 32P-labeled KCTD11-Sp1 probes (Sp1-A-F) or 32P-labeled canonical Sp1 probe [additional file 2]. Binding specificity was evaluated by competition with an excess (100x) of the cold probe or with a non-specific probe. For the supershift assays, the proteins were pre-incubated with two different anti-Sp1 antibodies (Santa-Cruz) at 4°C for 30 min. (C) ARO (thyroid cancer) and HCT15 (colon cancer) cell line were cultured, respectively, in RPMI-1640 and DMEM supplemented with 10% FCS. Chromatin Immunoprecipitations (ChIP) were performed by using the following antibodies: anti-Sp1 (1C6) X (sc-420; Santa Cruz Biotech), anti-Sp3 (F-7) X (sc-28305; Santa Cruz Biotech), anti-Acetyl-Histone3 (Cell Signaling). Eluted DNA has been analyzed with real-time q-PCR, normalized to GAPDH (left panels). Bars represent the mean of 3 independent experiments ± SD (*, p < 0.05, HCT15 versus ARO). Total protein levels of Sp1 and Sp3 were analyzed by Western blot (right panel).
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Figure 3: KCTD11 promoter is strongly activated by Sp1 TF. (A) D. Mel-2 cells (Invitrogen) were cultured in Drosophila Serum-Free Medium (Gibco Life Technologies) plus18 mM L-glutamine (Sigma) at room temperature. KCTD11-Luc reporter constructs (1 μg) and pPac-LacZ (200 ng) were co-transfected along with 1 μg of pPac empty vector, pPac-Sp1 or pPac-Sp2 (left panel) or pPac-Sp3 and pPac-Sp1+pPac-Sp3 (right panel) using Cellfectin (Invitrogen). After 48 h, the cells were harvested and the luciferase activity was assayed using the Single Luciferase Assay System (Promega), normalized to β-galactosidase activity using β-Galactosidase Enzyme Assay System (Promega). Each experiment has been done in triplicates. Values are the means ± S.D. (B) EMSAs showing the binding of Sp1 to KCTD11 promoter. Whole extracts were prepared from 293T HEK cells transfected with pCDNA-Sp1 (12 μg), using buffer C [16]. Cell extracts were incubated in vitro with 32P-labeled KCTD11-Sp1 probes (Sp1-A-F) or 32P-labeled canonical Sp1 probe [additional file 2]. Binding specificity was evaluated by competition with an excess (100x) of the cold probe or with a non-specific probe. For the supershift assays, the proteins were pre-incubated with two different anti-Sp1 antibodies (Santa-Cruz) at 4°C for 30 min. (C) ARO (thyroid cancer) and HCT15 (colon cancer) cell line were cultured, respectively, in RPMI-1640 and DMEM supplemented with 10% FCS. Chromatin Immunoprecipitations (ChIP) were performed by using the following antibodies: anti-Sp1 (1C6) X (sc-420; Santa Cruz Biotech), anti-Sp3 (F-7) X (sc-28305; Santa Cruz Biotech), anti-Acetyl-Histone3 (Cell Signaling). Eluted DNA has been analyzed with real-time q-PCR, normalized to GAPDH (left panels). Bars represent the mean of 3 independent experiments ± SD (*, p < 0.05, HCT15 versus ARO). Total protein levels of Sp1 and Sp3 were analyzed by Western blot (right panel).
Mentions: Because of Sp1 protein is constitutively expressed TF [9], to assess specifically the role of Sp1 in the regulation of KCTD11 promoter, we co-transfected the KCTD11 promoter constructs (Fig. 2B) along with Sp1, Sp2 or Sp3 expression vectors into Drosophila Mel-2 cells (Fig. 3A), which provide a background for Sp TFs activity [9]. In this cellular system, none of KCTD11 promoter constructs showed basal activity when co-transfected along with an empty vector, whereas Sp1 co-transfection strongly activated the promoter (Fig. 3A). Moreover, the functional significance of each Sp1 site in the KCTD11 promoter was evaluated. Sp1-A and Sp1-C resulted to drive KCTD11 promoter activity (Fig. 3A, compare -398/+99 + pPac-Sp1 vs -522/+99 + pPac-Sp1 and - 268/+99 + pPac-Sp1 vs -338/+99 + pPac-Sp1), whereas Sp1-B and Sp1-D did not (Fig. 3A, compare -338/+99 + pPac-Sp1 vs -398/+99 + pPac-Sp1 and -156/+99 + pPac-Sp1 vs -268/+99 + pPac-Sp1). As a control, deletion of all Sp-1 sites completely abrogated KCTD11 promoter activation by Sp1 (Fig. 3A, -37/+99 + pPac-Sp1 column).

Bottom Line: A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11).We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events.Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, University of L'Aquila, L'Aquila 67100, Italy.

ABSTRACT
A hallmark of several human cancers is loss of heterozygosity (LOH) of chromosome 17p13. The same chromosomal region is also frequently hypermethylated in cancer. Although loss of 17p13 has been often associated with p53 genetic alteration or Hypermethylated in Cancer 1 (HIC1) gene hypermethylation, other tumor suppressor genes (TSGs) located in this region have critical roles in tumorigenesis. A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11). We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events. Using a panel of human 177 tumor samples and their normal matching samples representing 18 different types of cancer, we show here that the down-regulation of KCTD11 protein level is a specific and a diffusely common event in tumorigenesis. Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression. Our findings identify KCTD11 as a widely down-regulated gene in human cancers, and provide a basis to understand how its expression might be deregulated in tumor cells.

Show MeSH
Related in: MedlinePlus