Lab Anim Res.  2011 Dec;27(4):333-338. 10.5625/lar.2011.27.4.333.

Inhibitor of DNA-binding 4 contributes to the maintenance and expansion of cancer stem cells in 4T1 mouse mammary cancer cell line

• Park SJ
• Kim RJ
• Nam JS

• KMID: 1444966
• DOI: http://doi.org/10.5625/lar.2011.27.4.333

Abstract

The cancer stem cell (CSC) hypothesis proposes that CSCs are the root of cancer. CSC-targeted therapies may prevent cancer relapse and provide more effective treatment. The expression of aldehyde dehydrogenase 1, as assessed by the Aldefluor assay, has been recognized as a marker of CSCs in breast cancer. Inhibitors of DNA-binding proteins (IDs) have an important role in stem cell differentiation. In this study, we examined IDs necessary for the regulation of stem properties in Aldefluorpos 4T1 cells. When the expression profile of IDs in Aldefluorneg and Aldefluorpos 4T1 cells was compared, qRT-PCR analysis showed that ID4 expression was highly upregulated in Aldefluorpos 4T1 cells. In addition, knockdown of ID4 expression suppressed the properties of CSCs, including their sphere-forming ability and side population phenotype. The findings suggest that ID4 may be a therapeutic target for the treatment of advanced breast cancer.

Keyword

Breast cancer cell; cancer stem cell; inhibitor of DNA-binding 4

MeSH Terms

Aldehyde Dehydrogenase
Animals
Breast Neoplasms
Cell Line
DNA-Binding Proteins
Isoenzymes
Mice
Neoplastic Stem Cells
Phenotype
Recurrence
Retinal Dehydrogenase
Stem Cells
Aldehyde Dehydrogenase
DNA-Binding Proteins
Isoenzymes
Retinal Dehydrogenase

Figure

• 

  Figure 1 The Aldefluorpos cell population has high ID4 expression. (A) The dot plots show the percentage of Aldefluorneg (R1) and Aldefluorpos (R2) cells from 4T1 cells. (B) The expression profile of IDs in Aldefluorneg and Aldefluorpos 4T1 cells. qRT-PCR was performed using primers for IDs. Values are mean±SD. *P<0.05 is a statistically significant difference.

• 

  Figure 2 Knockdown of ID4 expression suppresses tumorsphere-forming ability. (A and B) qRT-PCR and immunoblot analysis for ID4 in control (Non, non-transfected cells), siCon (control siRNA) or siID (ID4 siRNA)-transfected 4T1 cells. (C) Effects of ID4 knockdown on tumorsphere formation in 4T1 cells. After siRNA transfection under adherent conditions, cells were detached and suspension cultured under serum free conditions for 7 days. Values are mean±SD. *P<0.05 is a statistically significant difference. Scale bar=100 µm.

• 

  Figure 3 ID4 knockdown affects SP fraction and ABCC3 expression. (A) Representative SP phenotype from 4T1 cells transfected with non-specific control siRNA (siCON) of ID4-targeted siRNA (siID4). The SP fraction, which actively pumps out the Hoechst dye, is identified as the poorly staining cell population (black triangle) that largely disappears when the ABC transporters are inhibited with verapamil. (B) qRT-PCR analysis of ABC transporters expression in 4T1 cells following transfection with siCON or siID4. Values are mean±SD. *P<0.05 is a statistically significant difference.

• 

  Figure 4 ID4 mRNA expression in human breast tumors as a function of recurrent status (extracted from Sorlie et al [16] and Finak et al [17]). In silico analysis of published clinical microarray data was performed using database and analysis tools (http://www.oncomine.org).

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