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Metastasis-associated protein 1 is an upstream regulator of DNMT3a and stimulator of insulin-growth factor binding protein-3 in breast cancer

View Article: PubMed Central - PubMed

ABSTRACT

Despite a recognized role of DNA methyltransferase 3a (DNMT3a) in human cancer, the nature of its upstream regulator(s) and relationship with the master chromatin remodeling factor MTA1, continues to be poorly understood. Here, we found an inverse relationship between the levels of MTA1 and DNMT3a in human cancer and that high levels of MTA1 in combination of low DNMT3a status correlates well with poor survival of breast cancer patients. We discovered that MTA1 represses DNMT3a expression via HDAC1/YY1 transcription factor complex. Because IGFBP3 is an established target of DNMT3a, we investigated the effect of MTA1 upon IGFBP3 expression, and found a coactivator role of MTA1/c-Jun/Pol II coactivator complex upon the IGFBP3 transcription. In addition, MTA1 overexpression correlates well with low levels of DNMT3a which, in turn also correlates with a high IGFBP3 status in breast cancer patients and predicts a poor clinical outcome for breast cancer patients. These findings suggest that MTA1 could regulate the expression of IGFBP3 in both DNMT3a-dependent and -independent manner. Together findings presented here recognize an inherent role of MTA1 as a modifier of DNMT3a and IGFBP3 expression, and consequently, the role of MTA1-DNMT3a-IGFBP3 axis in breast cancer progression.

No MeSH data available.


MTA1 modulation of DNMT3a target genes.(A) Western blot showing secreted IGFBP-3 in the conditioned medium from MCF-7 and MCF-7/T7-MTA1 cells; ponceau staining was used to show the equal loading of protein. (B) Western blot showing the IGFBP3 levels from MCF-7 and MCF-7/T7-MTA1 cells. β-actin is shown as the loading control. (C) qPCR analysis of IGFBP-3 in MTA1 overexpressed MCF-7 cells as compared with the vector control. Results were represented in fold change normalized to mRNA levels of 18 S rRNA. (D) qPCR analysis of IGFBP-3 in MTA1+/+ and MEF MTA1−/− MEFs. (E) qPCR showing the levels of DNMT3a and HMMR mRNAs in MCF-7 and in MCF-7 cells overexpressing DNMT3a-YFP or pcDNA3.1 as a vector control. (F) qPCR analysis of HMMR mRNAs in wild-type and MEF MTA1−/− MEFs. Results are presented as fold change after normalizing with β-actin. (G) qPCR analysis of MTA1 and HMMR mRNAs in MCF-7 and in MTA1 overexpressing MCF-7 cells. Results are presented as fold change after normalizing with β-actin.
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f5: MTA1 modulation of DNMT3a target genes.(A) Western blot showing secreted IGFBP-3 in the conditioned medium from MCF-7 and MCF-7/T7-MTA1 cells; ponceau staining was used to show the equal loading of protein. (B) Western blot showing the IGFBP3 levels from MCF-7 and MCF-7/T7-MTA1 cells. β-actin is shown as the loading control. (C) qPCR analysis of IGFBP-3 in MTA1 overexpressed MCF-7 cells as compared with the vector control. Results were represented in fold change normalized to mRNA levels of 18 S rRNA. (D) qPCR analysis of IGFBP-3 in MTA1+/+ and MEF MTA1−/− MEFs. (E) qPCR showing the levels of DNMT3a and HMMR mRNAs in MCF-7 and in MCF-7 cells overexpressing DNMT3a-YFP or pcDNA3.1 as a vector control. (F) qPCR analysis of HMMR mRNAs in wild-type and MEF MTA1−/− MEFs. Results are presented as fold change after normalizing with β-actin. (G) qPCR analysis of MTA1 and HMMR mRNAs in MCF-7 and in MTA1 overexpressing MCF-7 cells. Results are presented as fold change after normalizing with β-actin.

Mentions: To understand the potential implication of MTA1 regulation of DNMT3a expression on downstream targets, we selected IGFBP3, a previously characterized repressed target gene by DNMT3a15. The IGFBP3, a secreted factor which could act both as an oncogene or a tumor suppressor in a context dependent manner21. We first analyzed the level of secreted IGFBP3 and cellular IGFBP3 in MCF-7 and MTA1 overexpressing breast cancer cells. We noticed an increased amount of secreted IGFBP3 in the concentrated conditioned medium from MTA1-overexpressing cells as compared to control cells (Fig. 5A). In addition, there was a modest increased expression in the levels of cellular IGFBP3 in MCF-7 overexpressing cells (Fig. 5B). Further, MTA1-overexpressed MCF-7 cells showed increased levels of IGFBP3 mRNA (Fig. 5C) when compared to the control cells. We also observed a significant decrease in the levels of IGFBP-3 mRNA in MTA1-KO-MEF as compared to wild-type MEF (Fig. 5D).


Metastasis-associated protein 1 is an upstream regulator of DNMT3a and stimulator of insulin-growth factor binding protein-3 in breast cancer
MTA1 modulation of DNMT3a target genes.(A) Western blot showing secreted IGFBP-3 in the conditioned medium from MCF-7 and MCF-7/T7-MTA1 cells; ponceau staining was used to show the equal loading of protein. (B) Western blot showing the IGFBP3 levels from MCF-7 and MCF-7/T7-MTA1 cells. β-actin is shown as the loading control. (C) qPCR analysis of IGFBP-3 in MTA1 overexpressed MCF-7 cells as compared with the vector control. Results were represented in fold change normalized to mRNA levels of 18 S rRNA. (D) qPCR analysis of IGFBP-3 in MTA1+/+ and MEF MTA1−/− MEFs. (E) qPCR showing the levels of DNMT3a and HMMR mRNAs in MCF-7 and in MCF-7 cells overexpressing DNMT3a-YFP or pcDNA3.1 as a vector control. (F) qPCR analysis of HMMR mRNAs in wild-type and MEF MTA1−/− MEFs. Results are presented as fold change after normalizing with β-actin. (G) qPCR analysis of MTA1 and HMMR mRNAs in MCF-7 and in MTA1 overexpressing MCF-7 cells. Results are presented as fold change after normalizing with β-actin.
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Related In: Results  -  Collection

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f5: MTA1 modulation of DNMT3a target genes.(A) Western blot showing secreted IGFBP-3 in the conditioned medium from MCF-7 and MCF-7/T7-MTA1 cells; ponceau staining was used to show the equal loading of protein. (B) Western blot showing the IGFBP3 levels from MCF-7 and MCF-7/T7-MTA1 cells. β-actin is shown as the loading control. (C) qPCR analysis of IGFBP-3 in MTA1 overexpressed MCF-7 cells as compared with the vector control. Results were represented in fold change normalized to mRNA levels of 18 S rRNA. (D) qPCR analysis of IGFBP-3 in MTA1+/+ and MEF MTA1−/− MEFs. (E) qPCR showing the levels of DNMT3a and HMMR mRNAs in MCF-7 and in MCF-7 cells overexpressing DNMT3a-YFP or pcDNA3.1 as a vector control. (F) qPCR analysis of HMMR mRNAs in wild-type and MEF MTA1−/− MEFs. Results are presented as fold change after normalizing with β-actin. (G) qPCR analysis of MTA1 and HMMR mRNAs in MCF-7 and in MTA1 overexpressing MCF-7 cells. Results are presented as fold change after normalizing with β-actin.
Mentions: To understand the potential implication of MTA1 regulation of DNMT3a expression on downstream targets, we selected IGFBP3, a previously characterized repressed target gene by DNMT3a15. The IGFBP3, a secreted factor which could act both as an oncogene or a tumor suppressor in a context dependent manner21. We first analyzed the level of secreted IGFBP3 and cellular IGFBP3 in MCF-7 and MTA1 overexpressing breast cancer cells. We noticed an increased amount of secreted IGFBP3 in the concentrated conditioned medium from MTA1-overexpressing cells as compared to control cells (Fig. 5A). In addition, there was a modest increased expression in the levels of cellular IGFBP3 in MCF-7 overexpressing cells (Fig. 5B). Further, MTA1-overexpressed MCF-7 cells showed increased levels of IGFBP3 mRNA (Fig. 5C) when compared to the control cells. We also observed a significant decrease in the levels of IGFBP-3 mRNA in MTA1-KO-MEF as compared to wild-type MEF (Fig. 5D).

View Article: PubMed Central - PubMed

ABSTRACT

Despite a recognized role of DNA methyltransferase 3a (DNMT3a) in human cancer, the nature of its upstream regulator(s) and relationship with the master chromatin remodeling factor MTA1, continues to be poorly understood. Here, we found an inverse relationship between the levels of MTA1 and DNMT3a in human cancer and that high levels of MTA1 in combination of low DNMT3a status correlates well with poor survival of breast cancer patients. We discovered that MTA1 represses DNMT3a expression via HDAC1/YY1 transcription factor complex. Because IGFBP3 is an established target of DNMT3a, we investigated the effect of MTA1 upon IGFBP3 expression, and found a coactivator role of MTA1/c-Jun/Pol II coactivator complex upon the IGFBP3 transcription. In addition, MTA1 overexpression correlates well with low levels of DNMT3a which, in turn also correlates with a high IGFBP3 status in breast cancer patients and predicts a poor clinical outcome for breast cancer patients. These findings suggest that MTA1 could regulate the expression of IGFBP3 in both DNMT3a-dependent and -independent manner. Together findings presented here recognize an inherent role of MTA1 as a modifier of DNMT3a and IGFBP3 expression, and consequently, the role of MTA1-DNMT3a-IGFBP3 axis in breast cancer progression.

No MeSH data available.