Limits...
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.

Show MeSH

Related in: MedlinePlus

Migration and Invasion Assay.The MCF10A-derived cell lines were tested for migration and invasive capability in a transwell assay, using uncoated (migration) or basement membrane-extract coated (invasion) wells. Results are from 3 independent experiments. Panel A: Migration. The vector control cell line MCF10A/HPVsh did not differ in migration from the parental MCF10A cell line. The MCF10A/HtrA1 cell line over-expressing HtrA1 showed significantly decreased (p<0.01) migration vs. the control cell lines. However, MCF10A/siRNA4 cell line showed significantly increased migration (p<0.01), whereas the MCF10A/siRNA1 showed an increase of borderline significance. Panel B: Invasion. The MCF10A/HPVsh and MCF10A/HtrA1 cell lines did not differ in invasion capability from the parental MCF10A cell line. However, both MCF10A/siRNA1 and 4 cell lines showed significant increases (p<0.01) in invasive capability.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3383700&req=5

pone-0039446-g007: Migration and Invasion Assay.The MCF10A-derived cell lines were tested for migration and invasive capability in a transwell assay, using uncoated (migration) or basement membrane-extract coated (invasion) wells. Results are from 3 independent experiments. Panel A: Migration. The vector control cell line MCF10A/HPVsh did not differ in migration from the parental MCF10A cell line. The MCF10A/HtrA1 cell line over-expressing HtrA1 showed significantly decreased (p<0.01) migration vs. the control cell lines. However, MCF10A/siRNA4 cell line showed significantly increased migration (p<0.01), whereas the MCF10A/siRNA1 showed an increase of borderline significance. Panel B: Invasion. The MCF10A/HPVsh and MCF10A/HtrA1 cell lines did not differ in invasion capability from the parental MCF10A cell line. However, both MCF10A/siRNA1 and 4 cell lines showed significant increases (p<0.01) in invasive capability.

Mentions: We next examined the migration and invasion capabilities of the various cell lines in a transwell assay. We found that one of the two MCF10A/siRNA cell lines tested showed significantly increased migration ability (p-value <0.01), while the MCF10A/HtrA1 over-expressing cell line showed a significant decrease in migration (p-value <0.01) (Figure 7A). Both the MCF10A/siRNA1 and/siRNA4 cell lines showed significantly increased invasion ability compared to the control cell line (p-value <0.01; see Figure 7B), while the vector control and over-expressing MCF10A/HtrA1 cell lines showed no change in invasion capability.


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)

Migration and Invasion Assay.The MCF10A-derived cell lines were tested for migration and invasive capability in a transwell assay, using uncoated (migration) or basement membrane-extract coated (invasion) wells. Results are from 3 independent experiments. Panel A: Migration. The vector control cell line MCF10A/HPVsh did not differ in migration from the parental MCF10A cell line. The MCF10A/HtrA1 cell line over-expressing HtrA1 showed significantly decreased (p<0.01) migration vs. the control cell lines. However, MCF10A/siRNA4 cell line showed significantly increased migration (p<0.01), whereas the MCF10A/siRNA1 showed an increase of borderline significance. Panel B: Invasion. The MCF10A/HPVsh and MCF10A/HtrA1 cell lines did not differ in invasion capability from the parental MCF10A cell line. However, both MCF10A/siRNA1 and 4 cell lines showed significant increases (p<0.01) in invasive capability.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039446-g007: Migration and Invasion Assay.The MCF10A-derived cell lines were tested for migration and invasive capability in a transwell assay, using uncoated (migration) or basement membrane-extract coated (invasion) wells. Results are from 3 independent experiments. Panel A: Migration. The vector control cell line MCF10A/HPVsh did not differ in migration from the parental MCF10A cell line. The MCF10A/HtrA1 cell line over-expressing HtrA1 showed significantly decreased (p<0.01) migration vs. the control cell lines. However, MCF10A/siRNA4 cell line showed significantly increased migration (p<0.01), whereas the MCF10A/siRNA1 showed an increase of borderline significance. Panel B: Invasion. The MCF10A/HPVsh and MCF10A/HtrA1 cell lines did not differ in invasion capability from the parental MCF10A cell line. However, both MCF10A/siRNA1 and 4 cell lines showed significant increases (p<0.01) in invasive capability.
Mentions: We next examined the migration and invasion capabilities of the various cell lines in a transwell assay. We found that one of the two MCF10A/siRNA cell lines tested showed significantly increased migration ability (p-value <0.01), while the MCF10A/HtrA1 over-expressing cell line showed a significant decrease in migration (p-value <0.01) (Figure 7A). Both the MCF10A/siRNA1 and/siRNA4 cell lines showed significantly increased invasion ability compared to the control cell line (p-value <0.01; see Figure 7B), while the vector control and over-expressing MCF10A/HtrA1 cell lines showed no change in invasion capability.

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