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Down-regulation of HtrA1 activates the epithelial-mesenchymal transition and ATM DNA damage response pathways.

Wang N, Eckert KA, Zomorrodi AR, Xin P, Pan W, Shearer DA, Weisz J, Maranus CD, Clawson GA - PLoS ONE (2012)

Bottom Line: Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers.Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation.Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

View Article: PubMed Central - PubMed

Affiliation: Gittlen Cancer Research Institute & Department of Pathology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, United States of America.

ABSTRACT
Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

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HtrA1 Gene Expression in hBC cell lines.RNA was isolated from the various human breast epithelial cell lines and expression levels of HtrA1 mRNA were determined using QPCR (Lower Panel) and Northern blot analyses (Upper Panel) as described. Results are representative of multiple independent analyses. Expression levels were 20–25X higher in the non-tumorigenic MCF10A and 12A cell lines, with very low expression levels in most of the hBC cell lines (MDA-MB-231 was the exception; see text). β-actin transcript were used as to assess loading on Northern blots.
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pone-0039446-g002: HtrA1 Gene Expression in hBC cell lines.RNA was isolated from the various human breast epithelial cell lines and expression levels of HtrA1 mRNA were determined using QPCR (Lower Panel) and Northern blot analyses (Upper Panel) as described. Results are representative of multiple independent analyses. Expression levels were 20–25X higher in the non-tumorigenic MCF10A and 12A cell lines, with very low expression levels in most of the hBC cell lines (MDA-MB-231 was the exception; see text). β-actin transcript were used as to assess loading on Northern blots.

Mentions: We next examined HtrA1 transcript levels in 7 breast epithelial cell lines, including 5 human breast cancer (hBC) cell lines (MCF7, MDA-MB-231, MDA-MB-468, NM2C5, and M4A4), and 2 immortalized non-tumorigenic cell lines (MCF10A and MCF12A), by QPCR. HtrA1 gene expression was dramatically decreased in hBC cells compared to their non-tumorigenic counterparts (Figure 2). The differences were generally greater than 20X (P<0.005), except for the MDA-MB-231 cell line, which showed HtrA1 mRNA levels which were ∼50% of those found in MCF10A cells. We further confirmed the expression differences by Northern blot analysis (Figure 2), which showed a single transcript. Immunoblot analyses demonstrated that HtrA1 protein expression was high in the non-tumorigenic MCF12A and MCF10A cell lines, but undetectable in all of the hBC cell lines tested (Figure 3). Results from the MDA-MD-231 cell line indicate a translational block, since HtrA1 mRNA is relatively high but the protein is absent.


Down-regulation of HtrA1 activates the epithelial-mesenchymal transition and ATM DNA damage response pathways.

Wang N, Eckert KA, Zomorrodi AR, Xin P, Pan W, Shearer DA, Weisz J, Maranus CD, Clawson GA - PLoS ONE (2012)

HtrA1 Gene Expression in hBC cell lines.RNA was isolated from the various human breast epithelial cell lines and expression levels of HtrA1 mRNA were determined using QPCR (Lower Panel) and Northern blot analyses (Upper Panel) as described. Results are representative of multiple independent analyses. Expression levels were 20–25X higher in the non-tumorigenic MCF10A and 12A cell lines, with very low expression levels in most of the hBC cell lines (MDA-MB-231 was the exception; see text). β-actin transcript were used as to assess loading on Northern blots.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383700&req=5

pone-0039446-g002: HtrA1 Gene Expression in hBC cell lines.RNA was isolated from the various human breast epithelial cell lines and expression levels of HtrA1 mRNA were determined using QPCR (Lower Panel) and Northern blot analyses (Upper Panel) as described. Results are representative of multiple independent analyses. Expression levels were 20–25X higher in the non-tumorigenic MCF10A and 12A cell lines, with very low expression levels in most of the hBC cell lines (MDA-MB-231 was the exception; see text). β-actin transcript were used as to assess loading on Northern blots.
Mentions: We next examined HtrA1 transcript levels in 7 breast epithelial cell lines, including 5 human breast cancer (hBC) cell lines (MCF7, MDA-MB-231, MDA-MB-468, NM2C5, and M4A4), and 2 immortalized non-tumorigenic cell lines (MCF10A and MCF12A), by QPCR. HtrA1 gene expression was dramatically decreased in hBC cells compared to their non-tumorigenic counterparts (Figure 2). The differences were generally greater than 20X (P<0.005), except for the MDA-MB-231 cell line, which showed HtrA1 mRNA levels which were ∼50% of those found in MCF10A cells. We further confirmed the expression differences by Northern blot analysis (Figure 2), which showed a single transcript. Immunoblot analyses demonstrated that HtrA1 protein expression was high in the non-tumorigenic MCF12A and MCF10A cell lines, but undetectable in all of the hBC cell lines tested (Figure 3). Results from the MDA-MD-231 cell line indicate a translational block, since HtrA1 mRNA is relatively high but the protein is absent.

Bottom Line: Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers.Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation.Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

View Article: PubMed Central - PubMed

Affiliation: Gittlen Cancer Research Institute & Department of Pathology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, United States of America.

ABSTRACT
Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

Show MeSH
Related in: MedlinePlus