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Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Meng Y, Gu C, Wu Z, Zhao Y, Si Y, Fu X, Han W - BMC Cancer (2009)

Bottom Line: The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells.Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2.E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing, 100853, PR China. mengyg6512@vipsina.com

ABSTRACT

Background: Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor alpha (ERalpha)-positive MCF-7 and SKOV-3 cancer cells.

Methods: MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.

Results: Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.

Conclusion: Overexpression of Id2 in ERalpha-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

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(A) Effect of overexpression of Id2 or its mutants on E-cadherin protein expression. Total proteins were extracted from the indicated cells for analysis of E-cadherin protein by western blot. (B) Effect of overexpression of Id2 or its mutants on the transactivation of E-cadherin gene promoter. The indicated cells were transiently cotransfected with the indicated plasmids for luciferase assay as described in "Methods". Data are expressed as mean ± SEM for at least three separate determinations.
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Figure 6: (A) Effect of overexpression of Id2 or its mutants on E-cadherin protein expression. Total proteins were extracted from the indicated cells for analysis of E-cadherin protein by western blot. (B) Effect of overexpression of Id2 or its mutants on the transactivation of E-cadherin gene promoter. The indicated cells were transiently cotransfected with the indicated plasmids for luciferase assay as described in "Methods". Data are expressed as mean ± SEM for at least three separate determinations.

Mentions: E-cadherin (encoded by the CAD1 gene in humans) is a cell-surface glycoprotein involved in calcium-dependent cell-cell adhesion [26]. The increased invasion and migratory capacity of epithelial tumor cells are always associated with reduced level of E-cadherin [27-29]. To ascertain whether Id2-induced increase of in vitro invasiveness and migration of MCF-7 and SKOV-3 cells is accompanied by changes in E-cadherin, we analyzed the protein expression of E-cadherin by western blot assay. Transfection of the wild-type Id2 did not significantly change the expression of E-cadherin protein in either cell line (Figure 6A); however, transfection with Id2-DBM or Id2-DBM-δHLH caused a 2- to 5-fold decrease of E-cadherin protein expression as compared with the mock transfection in both cell lines. Next, to determine whether the Id2 mutants are repressors of transcriptional activity of E-cadherin gene, the promoter construct of E-cadherin, Id2 or either of its mutants were cotransfected into MCF-7 and SKOV-3 cells. Since the proximal E-cadherin promoter was shown to mainly confer the transcriptional inactivation of E-cadherin in some cancer cell lines [30-33], we used the most proximal E-cadherin human promoter construct (EcadK1-Luc, -108 to +125), which contained three E-box elements. E-cadK1-Luc activities were efficiently repressed by Id2-DBM and Id2-DBM-δHLH (2- to 2.5- fold) but not by the wild-type Id2 (Figure 6B). Thus, excessive accumulation of Id2 by abolishing its degradation in MCF-7 and SKOV-3 cells represses the expression of E-cadherin gene by inhibiting its transactivation. As well, this repression is not dependent on the HLH domain of Id2.


Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Meng Y, Gu C, Wu Z, Zhao Y, Si Y, Fu X, Han W - BMC Cancer (2009)

(A) Effect of overexpression of Id2 or its mutants on E-cadherin protein expression. Total proteins were extracted from the indicated cells for analysis of E-cadherin protein by western blot. (B) Effect of overexpression of Id2 or its mutants on the transactivation of E-cadherin gene promoter. The indicated cells were transiently cotransfected with the indicated plasmids for luciferase assay as described in "Methods". Data are expressed as mean ± SEM for at least three separate determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654660&req=5

Figure 6: (A) Effect of overexpression of Id2 or its mutants on E-cadherin protein expression. Total proteins were extracted from the indicated cells for analysis of E-cadherin protein by western blot. (B) Effect of overexpression of Id2 or its mutants on the transactivation of E-cadherin gene promoter. The indicated cells were transiently cotransfected with the indicated plasmids for luciferase assay as described in "Methods". Data are expressed as mean ± SEM for at least three separate determinations.
Mentions: E-cadherin (encoded by the CAD1 gene in humans) is a cell-surface glycoprotein involved in calcium-dependent cell-cell adhesion [26]. The increased invasion and migratory capacity of epithelial tumor cells are always associated with reduced level of E-cadherin [27-29]. To ascertain whether Id2-induced increase of in vitro invasiveness and migration of MCF-7 and SKOV-3 cells is accompanied by changes in E-cadherin, we analyzed the protein expression of E-cadherin by western blot assay. Transfection of the wild-type Id2 did not significantly change the expression of E-cadherin protein in either cell line (Figure 6A); however, transfection with Id2-DBM or Id2-DBM-δHLH caused a 2- to 5-fold decrease of E-cadherin protein expression as compared with the mock transfection in both cell lines. Next, to determine whether the Id2 mutants are repressors of transcriptional activity of E-cadherin gene, the promoter construct of E-cadherin, Id2 or either of its mutants were cotransfected into MCF-7 and SKOV-3 cells. Since the proximal E-cadherin promoter was shown to mainly confer the transcriptional inactivation of E-cadherin in some cancer cell lines [30-33], we used the most proximal E-cadherin human promoter construct (EcadK1-Luc, -108 to +125), which contained three E-box elements. E-cadK1-Luc activities were efficiently repressed by Id2-DBM and Id2-DBM-δHLH (2- to 2.5- fold) but not by the wild-type Id2 (Figure 6B). Thus, excessive accumulation of Id2 by abolishing its degradation in MCF-7 and SKOV-3 cells represses the expression of E-cadherin gene by inhibiting its transactivation. As well, this repression is not dependent on the HLH domain of Id2.

Bottom Line: The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells.Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2.E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing, 100853, PR China. mengyg6512@vipsina.com

ABSTRACT

Background: Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor alpha (ERalpha)-positive MCF-7 and SKOV-3 cancer cells.

Methods: MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.

Results: Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.

Conclusion: Overexpression of Id2 in ERalpha-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

Show MeSH
Related in: MedlinePlus