Down-Regulation of HtrA1 Activates the Epithelial-Mesenchymal Transition and ATM DNA Damage Response Pathways

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

HtrA1 Protein Expression in hBC cell lines.

<p>Cells (as indicated) were extracted into nuclear (N) and cytoplasmic (C) fractions as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039446#s4" target="_blank">Materials & Methods</a>), and proteins were analyzed by Immunoblot analyses using the polyclonal antibody against human HtrA1. β-actin was used as a loading control, and DEK was used to assess the nuclear/cytoplasmic fractionation (DEK is exclusively nuclear). The larger Mr bands seen in the MCF12A and MCF10A/Flp cell lines are consistent with dimers, trimers, etc., although this was not confirmed. MCF10A/Flp is the parental Flp-in cell line which was used to produce the various MCF10A/siRNA or Htra1 cell lines. Results are from a representative experiment.</p

Examination of MCF10A-derived cell lines for p-ATM and formation of γH2AX foci. Upper Panel.

<p>Immunoblot analyses for phosphorylated ATM were performed as described, specifically examining the Ser1981 site. The Upper Panel shows phosphorylated ATM (with β-actin as a loading control). “+” indicates H₂O₂ treatment with 100 µM H₂O₂ for 2 h to induce oxidative stress, vs. “-“ for no treatment. Numbers indicate cell lines as follows: 1 = MCF10A, 2 = MCF10A/HtrA1, 3 = MCF10A/HPVsh, 4 = MCF10A/siRNA1, and 5 = MCF10A/siRNA4. Phosphorylated ATM (pATM, at Ser1981) is observed in the absence of any treatment in both MCF10A/siRNA cell lines examined, whereas no pATM is observed in the MCF10A and MCF10A/HPVsh control cell lines, or the over-expressing MCF10A/HtrA1 cell line. <b>Lower Panels.</b> Cells were either untreated or treated with 200 µM H₂O₂ for 1 h, and then IHC analysis was performed, with staining for γH2AX foci using an antibody specific for phosphorylated Ser139 as described. Over-expression of HtrA1 effectively blocked formation of γH2AX foci following acute treatment with H₂O₂, whereas foci formation was increased in MCF10A/siRNA1 (generally by ∼50%) and/siRNA4 cells, dramatically so in the latter. Etoposide treatment was used as a positive control with MCF10A cells. Foci formation is unchanged in the MCF10A/HPVsh vector control cell line. Results are from a representative experiment, which was repeated once with analogous results.</p

Promoter Methylation Status in the MCF10A cell lines.

<p>Candidate CpG islands were identified in the HtrA1 promoter sequence, within the upstream region from bp −561 to −266. DNA was extracted from the various cell lines and analyzed via bisulfite sequencing. The topmost Panel shows this region of interest, which is expanded in the dark strip from F1–R1, with CpG sites indicated by vertical lines (the total of 35 CpGs are marked). Unmethylated CpGs identified by bisulfite sequencing are depicted in the lower panels for the various cell lines, as indicated on the right.</p

Migration and Invasion Assay.

<p>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.</p

HtrA1 Gene Expression in hBC cell lines.

<p>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.</p

Selected significantly altered genes associated with EMT, ATM-DDR, and CSC pathways.

<p>Values represent fold change relative to MCF10A vector control values. All values are significant at p<0.01. * Genes with 2 independent loci measured.</p

Effects of Altered HtrA1 Expression Levels on the miR-200 Cluster.

<p>Two independent analyses were performed, which included the MCF10A vector controls, all 4 different MCF10A/siRNA1-4 cell lines, and the over-expressing MCF10A/HtrA1 cell line. Values for the various cell lines were normalized to the vector controls. We initially focused on miRs showing significant changes (p<0.05) in the MCF10A/siRNA cell lines, of at least 40% magnitude. This initial screening identified a small group of miRs, most of which were members of the miR-200 family. Values for these miRs were then extracted from the data, and all were found to be statistically significantly different at p<0.05 or greater.</p

Characterization of MCF10A cell lines carrying HtrA1 siRNA and overexpression vectors.

<p>A random antisense oligonucleotide library was used to identify optimally accessible sites in HtrA1 mRNA. SiRNAs (short hairpin RNAs) were designed to target these sites, and stably transfected cell lines were developed from MCF10A cells (4 independent cell lines, designated MCF10A/siRNA1-4). A control cell line expressing an irrelevant siRNA (designated MCF10A/HPVsh) was also developed. In parallel, we also developed a cell line over-expressing HtrA1 (designated MCF10A/HtrA1). (A). Target sites empirically identified in HtrA1 mRNA. Identified domains within HtrA1 include: SS, signal sequence; IGFBP, IGF binding site; KI, Kazal Type I protease inhibitor domain; Trypsin-like protease domain; PDZ, PDZ binding domain. (B) Immunoblot analysis for HtrA1 protein. Cytoplasmic and nuclear protein fractions were prepared from the developed cell lines (as indicated), and were probed with a polyclonal antibody preparation directed against a region in the trypsin-like protease domain. As is evident, the reductions in HtrA1 protein levels were >90% compared with the various MCF10A and MCF10A/HPVsh cells. Right panel shows HtrA1 in concentrated culture medium. (C) Cells were plated and growth was measured over a 6 day period. Both of the MCF10A cell lines tested (MCF10A/siRNA1 and siRNA4) grew significantly faster than the control cells (p<0.01 at days 4 and 6). Over-expression of HtrA1 in the MCF10A/HtrA1 cells had no effect on cell growth rate.</p

Cluster Analysis of Whole Genome Gene Expression Data.

<p>Genes showing significant changes (and a minimum of 50% change) in expression levels (at p<0.01) in MCF10A/siRNA cell lines vs. controls (3 independent experiments) were analyzed by Cluster Analysis. Gene expression profiles in MCF10A/siRNA3 and siRNA4 cell lines, and in the MCF10A/HtrA1 over-expressing cell lines, were determined vs. the control cell line. The optimal number of clusters was found to be 22, and the centroids for the various individual clusters are indicated. Clusters 19, 6, and 2 showed the greatest changes in expression levels in the MCF10A/siRNA cell lines which were inversely correlated with HtrA1 expression levels. Clusters 12, 21, and 4 showed the greatest changes in expression levels that were positively correlated with HtrA1 expression levels.</p