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Characterization of a tissue-specific CDP/Cux isoform, p75, activated in breast tumor cells
Brigitte Goulet1, Peter Watson, Madeleine Poirier
1Molecular Oncology Group, McGill University Health Center, Montreal, Quebec, QC H3A 1A1, Canada.
Abstract:
Two isoforms of the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor have been characterized thus far. The full length protein, p200, which contains four DNA binding domains, transiently binds to DNA and carries the CCAAT-displacement activity. The p110 isoform is generated by proteolytic processing at the G1-S transition and is capable of stable interaction with DNA. Here we demonstrate the existence of a shorter CDP/Cux isoform, p75, which contains only two DNA binding domains, Cut repeat 3 and the Cut homeodomain, and binds more stably to DNA. CDP/Cux p75 was able to repress a reporter carrying the promoter for the cyclin-dependent kinase inhibitor p21 gene and to activate a DNA polymerase alpha gene reporter. Expression of CDP/Cux p75 involved a novel mechanism: transcription initiation within intron 20. The intron 20-initiated mRNA (I20-mRNA) was expressed at higher level in the thymus and in CD4+/CD8+ and CD4+ T cells. I20-mRNA was expressed only weakly or not at all in normal human mammary epithelial cells and normal breast tissues but was detected in many breast tumor cells lines and breast tumors. In invasive tumors a significant association was established between higher I20-mRNA expression and a diffuse infiltrative growth pattern (n = 41, P = 0.0137). In agreement with these findings, T47D breast cancer cells stably expressing p75 could not form tubule structures in collagen but rather developed as solid undifferentiated aggregates of cells. Taken together, these results suggest that aberrant expression of the CDP/Cux p75 isoform in mammary epithelial cells may be associated with the process of tumorigenesis in breast cancer.
Insights
A new, shorter CCAAT-displacement protein/cut homeobox (CDP/Cux) isoform, p75, binds DNA stably. Aberrant expression of this CDP/Cux p75 in breast cells correlates with tumor growth and invasiveness.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Two CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor isoforms, p200 and p110, are known.
- CDP/Cux p200 has transient DNA binding and CCAAT-displacement activity.
- CDP/Cux p110 is proteolytically processed and stably binds DNA.
Purpose of the Study:
- To identify and characterize novel CDP/Cux isoforms.
- To investigate the function and expression of a newly identified CDP/Cux p75 isoform.
- To explore the association of CDP/Cux p75 with breast cancer development and progression.
Main Methods:
- Identification and characterization of the CDP/Cux p75 isoform.
- Reporter gene assays to assess transcriptional activity of CDP/Cux p75.
- Analysis of I20-mRNA expression in various cell types and tissues using RT-PCR.
- Correlation analysis between I20-mRNA levels and clinicopathological features in breast tumors.
- In vitro cell culture experiments to evaluate the effect of p75 expression on cell morphology and growth.
Main Results:
- A novel, shorter CDP/Cux isoform, p75, was identified, containing two DNA binding domains and exhibiting stable DNA binding.
- CDP/Cux p75 repressed the p21 gene promoter and activated the DNA polymerase alpha gene promoter.
- A novel transcription mechanism involving initiation within intron 20 generated I20-mRNA, which was highly expressed in thymus, T cells, and breast tumors.
- Higher I20-mRNA expression in invasive breast tumors was significantly associated with a diffuse infiltrative growth pattern.
- Breast cancer cells expressing p75 failed to form tubule structures, instead forming undifferentiated aggregates.
Conclusions:
- CDP/Cux p75 represents a distinct functional isoform with altered DNA binding properties.
- Aberrant expression of CDP/Cux p75, driven by intron 20-initiated transcription, is linked to breast tumorigenesis.
- CDP/Cux p75 may play a role in promoting invasive growth and dedifferentiation in breast cancer.