Downregulation Of HtrA1 Research Articles

Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration.

We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced both by HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism: SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration

Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration

We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism:SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.

Inorganic arsenic exposure promotes malignant progression by HDAC6-mediated down-regulation of HTRA1.

Inorganic arsenic (iAs) has been a human health concern and is associated with intestinal malignancies. However, the molecular mechanisms of the iAs-induced oncogenic process in intestine epithelial cells remain elusive, partly because of the known hormesis effect of arsenic. Here, we established that six-month exposure to iAs at a concentration similar to those found in contaminated drinking water could promote malignant characteristics, including enhanced proliferation and migration, resistance to apoptosis, and mesenchymal-like transition in Caco-2 cells. Transcriptome analysis and mechanism study revealed that key genes and pathways involved in cell adhesion, inflammation and oncogenic regulation were altered during chronic iAs exposure. Specifically, we uncovered that down-regulation of HTRA1 was essential for the iAs-induced acquisition of the cancer hallmarks. Further, we evidenced that the loss of HTRA1 during iAs-exposure could be restored by HDAC6 inhibition. Caco-2 cells with chronic exposure to iAs exhibited enhanced sensitivity to WT-161, a specific inhibitor of HDAC6, when used alone than in combination with a chemotherapeutic agent. These findings provide valuable information for understanding the mechanisms of arsenic-induced carcinogenesis and facilitating the health management of populations in arsenic-polluted areas.

TGF-β/Smad Signalling Activation by HTRA1 Regulates the Function of Human Lens Epithelial Cells and Its Mechanism in Posterior Subcapsular Congenital Cataract.

Congenital cataract is the leading cause of blindness among children worldwide. Patients with posterior subcapsular congenital cataract (PSC) in the central visual axis can result in worsening vision and stimulus deprivation amblyopia. However, the pathogenesis of PSC remains unclear. This study aims to explore the functional regulation and mechanism of HTRA1 in human lens epithelial cells (HLECs). HTRA1 was significantly downregulated in the lens capsules of children with PSC compared to normal controls. HTRA1 is a suppression factor of transforming growth factor-β (TGF-β) signalling pathway, which plays a key role in cataract formation. The results showed that the TGF-β/Smad signalling pathway was activated in the lens tissue of PSC. The effect of HTRA1 on cell proliferation, migration and apoptosis was measured in HLECs. In primary HLECs, the downregulation of HTRA1 can promote the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway and can significantly upregulate the TGF-β/Smad downstream target genes FN1 and α-SMA. HTRA1 was also knocked out in the eyes of C57BL/6J mice via adeno-associated virus-mediated RNA interference. The results showed that HTRA1 knockout can significantly upregulate p-Smad2/3 and activate the TGF-β/Smad signalling pathway, resulting in abnormal proliferation and irregular arrangement of lens epithelial cells and leading to the occurrence of subcapsular cataract. To conclude, HTRA1 was significantly downregulated in children with PSC, and the downregulation of HTRA1 enhanced the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway, which led to the occurrence of PSC.

HtrA1 expression and the prognosis of high-grade serous ovarian carcinoma: a cohort study using digital analysis

BackgroundThe expression of high temperature requirement factor A1 (Htra1) has been reported to be decreased in ovarian carcinoma, but its prognostic effect remains undetermined.MethodsWe evaluated the impact of HtrA1 downregulation in tumoral tissues on cancer progression and death in women with serous ovarian carcinoma. HtrA1 staining was performed on tissue microarrays (TMA) comprised of tumor samples from a cohort of 106 women who were diagnosed with primary high-grade serous ovarian carcinoma and receiving standard treatment at the Québec University Hospital between 1993 and 2006. HtrA1 expression was assessed visually (percentage of positive nuclei) and by digital image analysis (percentage of positive area). Cox regression multivariate models included standard prognostic factors and were used to estimate adjusted hazard ratios (aHR) for progression or death in the cohort.ResultsBy visual analysis, a low percentage of HtrA1-positive nuclei (< 10% vs ≥10%) tend to be associated with a lower risk of progression (aHR = 0.71; 95% Confidence interval (CI) = 0.46–1.09; P = 0.11) and mortality (aHR = 0.65; 95% CI = 0.41–1.04; P = 0.07). Low nuclear HtrA1 expression assessed by digital image analysis (< median % vs ≥ median %) showed a significant association with lower risk of progression (aHR = 0.62; 95% CI = 0.40–0.95; p = 0.03) and death (aHR = 0.60; 95% CI = 0.38–0.95; p = 0.03).ConclusionAltogether, our results demonstrate that nuclear downregulation of HtrA1 is associated with a better prognosis in women with high grade serous ovarian carcinoma.

Inhibition of cell proliferation, migration and apoptosis in blue-light illuminated human retinal pigment epithelium cells by down-regulation of HtrA1.

To investigate the effect of HtrA1 on the proliferation, migration and apoptosis of human retinal pigment epithelium (RPE) cells in the light injured model, as well as the expression of the apoptosis related molecules. The human RPE cell line ARPE-19 was exposed to blue light to establish the light injured model. The cells were transfected with HtrA1 siRNA to knockdown HtrA1 expression. Subsequent expression of HtrA1 was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. Changes in cell proliferation, migration and apoptosis were assessed by cell counting kit-8 (CCK-8), Transwell assay and flow cytometry respectively, as well as changes in the mRNA and protein levels of Bax, Caspase-3 and Bcl-2 expression. HtrA1 was highly expressed in ARPE-19 cells after blue light irradiation. Knockdown of HtrA1 expression inhibited the proliferation, migration and apoptosis of the blue-light-irradiated ARPE-19 cells (P<0.05). Bax and Caspase-3 expression were significantly reduced both at mRNA and protein levels (P<0.05) after siRNA treatment. Bcl-2 expression significantly increased in blue-light-irradiated ARPE-19 cells after siRNA interference (P<0.05). Silence of HtrA1 may inhibit the proliferation, migration and apoptosis of ARPE-19 cells in light injured model. Moreover, HtrA1 suppression in blue-light-irradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3, and up-regulation of Bcl-2 expression.

The expression of serine protease HtrA1 in human periodontal ligament tissue and the effect of HtrA1 on the proliferation of human periodontal ligament cells

To observe the expression of serine protease HtrA1 in human periodontal ligament tissue and to explore the effect of HtrA1 on the proliferation of human periodontal ligament cells (hPDLC). Six human premolars and three human third molars(patient's ages ranging from 12 to 25, with intact root, without caries and/or periodontitis) were obtained in the Department of Maxillofacial Surgery of Wuhan University Hospital of Stomatology. Reverse transcription-PCR(RT-PCR) and immunohistochemistry analysis were applied to investigate the expression of HtrA1. Primary hPDLC were obtained by tissue-culture method in vitro. The proliferation of hPDLC was determined by methyl thiazolytetrazolium(MTT). Lentivirus-mediated over-expression and reduction of HtrA1 level was performed. An empty vector was used as negative control. On days 1, 3, 5, 7 and 9, the growth of hPDLC was characterized using cell counting kit-8(CCK-8) assay. RT-PCR data indicated that HtrA1 mRNA was expressed in human periodontal ligament tissue. Immunohistochemistry analysis showed HtrA1 was expressed in human periodontal ligament, mainly in the cytoplasm of hPDLC and the extracellular matrix. The MTT result suggested that the growth curve was consistent with the growth characteristics of hPDLC. The stable over-expression and knockdown cell lines was successfully established by lentivirus with more than 90% transfection efficiency. CCK-8 assay showed that HtrA1 over-expression inhibited the proliferation of hPDLC(0.897±0.060, 0.890±0.083, 1.631±0.038, 1.111±0.041, 1.110±0.189), while cell proliferation increased after down-regulation of HtrA1(0.329±0.021, 0.529±0.044, 0.973±0.056, 1.626±0.102, 2.344±0.198)(P<0.05). HtrA1 is expressed in human periodontal ligament tissue at both mRNA and protein levels, and may play an important role in regulating the proliferation of hPDLC.

HtrA1 regulates epithelial–mesenchymal transition in hepatocellular carcinoma

Background and aimsEpithelial–mesenchymal transition (EMT) is involved in the development and progression of cancer. HtrA1 had been showed to play a modulatory role in metastasis of hepatocellular carcinoma (HCC). The relationship between HtrA1 and EMT in HCC was investigated in the present study. MethodsThe HtrA1 expression in human HCC tumor tissues and cells was determined by real-time PCR. SiRNA-HtrA1 and pcDNA-HtrA1 were respectively transfected into HepG2 and MHCC97H cells to observe their effects on cell migration and expression of EMT-associated markers Vimentin and E-cadherin. The relationship between HtrA1 and EMT in 60 HCC patients was also investigated. ResultsHtrA1 expression of tumor tissues was down-regulated with the increasing of number in lymph nodes metastasis in HCC patients. HtrA1 down-regulation led to the significant increase of cell migration, Vimentin expression and decrease of E-cadherin expression, while HtrA1 overexpression resulted in an opposite function. The HtrA1 expression was positively related to the E-cadherin level (R2 = 0.5903, P < 0.001) and negatively correlated with Vimentin level (R2 = 0.6067, P < 0.001) in tumor tissues of HCC, respectively. ConclusionHtrA1 expression was closely related to EMT, which might be a potential mechanism underlying metastasis of HCC.

Abstract POSTER-BIOL-1310: Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer

Abstract Tumor dormancy is an important research topic because of its potential implications in metastasis and acquired resistance to chemotherapy. Dormant tumor cells at the secondary site may persist after chemotherapy and regrow as metastatic recurrence. Seminal studies by Dr. Bissell and Dr. Hendrix have shown that the tissue microenvironment can reprogram tumor cells into dormancy. Extracellular matrix (ECM) proteins are key components of tumor microenvironment and may regulate dormancy, and disruption of ECM homeostasis may cause recurrence and metastatic growth. A major cytokine that regulates ECM remodeling is tumor growth factor-beta (TGF-beta), and it is implicated in modulating tumor cell dormancy. In the tissue microenvironment, serine protease HtrA1 was shown to inhibit TGF-beta signaling by targeting TGF-beta family ligands and receptors for proteolysis. Our lab showed that HtrA1 is down-regulated in ovarian cancer cells, and HtrA1 loss promotes resistance to anoikis and in vivo metastasis. Loss of HtrA1 is also associated with increased incidence of carcinomatosis. Finally, previous studies have shown that HtrA1 degrades ECM proteins. Therefore, we hypothesize that loss of HtrA1 in the ovarian tumor microenvironment enhances TGF-beta signaling and promotes ECM protein production by stromal cells, thereby contributing to ECM remodeling and tumor dormancy. Ovarian cancer cells with endogenous HtrA1 were transduced with HtrA1-targeting shRNA (sh1) to evaluate the effect of HtrA1 downregulation on invasion, tumor dormancy, and ECM remodeling. Cells transduced with non-targeting shRNA (NT) served as a control. Cancer cells (NT and sh1) were plated over confluent mesothelial cell layer with or without 10 nM TGF-beta for 24 hours and 48 hours. No significant difference in cell invasion was observed between those cells cultured with or without TGF-beta. In order to test which matrix proteins are critical for cell proliferation versus cell cycle arrest, MicroMatrix 36 arrays were used to culture NT and sh1 cells. Cells were cultured for 48 hours on matrix slides, then fixed and stained with anti Ki67. It was determined matrix containing combinations of fibronectin showed cells that stained positive for Ki67, when compared to those not containing fibronectin or collagen together. Changes in intracellular signaling in the presence and absence of matrix were also investigated using kinase and MAPK arrays. Mesothelial cells were cultured until confluent, and cells were removed from culture plates using 2.0 mM EDTA in PBS. Matrix produced from mesothelial cells remained on the plates. NT and sh1 cells were then plated in the presence and absence of matrix and were allowed to culture until cell attachment was observed (3 hours at 37oC, 5% CO2). Cells were lysed and lysates were cultured on arrays according to manufactures instructions. ERK and Akt pathways were observed to be activated in cells plated with matrix compared to those without matrix. Taken together the evidence thus far suggests that matrix composition is critical for cell proliferation. Currently, Stable Isotope Amino Acid labeling is being employed to investigate changes in matrix remodeling on a global scale. In addition, Imaging Mass Spectrometry will also be used to evaluate formalin fixed paraffin embedded tissue samples from patients to identify at a molecular level changes in extracellular matrix composition between tumor and normal tissues. Citation Format: Cooley, Megan K., Chien, Jeremy. Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1310.

HtrA1 Downregulation Induces Cisplatin Resistance in Lung Adenocarcinoma by Promoting Cancer Stem Cell‐Like Properties

Cisplatin (CDDP) resistance usually develops during lung adenocarcinoma (LAC) therapy. However, the comprehensive mechanisms remain largely unclear. In this study, we first established a CDDP-resistant LAC cell line-A549/CDDP from its parental cell line-A549. The results showed that CDDP resistance in A549/CDDP cells correlates with acquirement of cancer stem cell-like properties (increased percentage of CD133-expressing subpopulation, sphere formation and levels of some pluripotency-associated markers). HtrA1 expression at both mRNA and protein levels was reduced in CDDP-resistant A549/CDDP cells compared with that in A549 cells. Ectopic expression of HtrA1 in A549/CDDP cells reversed cancer stem cell-like properties and CDDP resistance. In A549 cells, stable knockdown of HtrA1 expression promoted cancer stem cell-like properties and CDDP insensitivity, however, these effects were blocked by inhibition of PI3K/Akt pathway using LY294002. Furthermore, HtrA1 knockdown could significantly stimulate PI3K/Akt signaling in A549 cells. In vivo studies, HtrA1 knockdown promoted tumorigenesis and conferred CDDP resistance in xenograft A549 tumors, which were reversed by intraperitoneal injection of LY294002. In conclusion, these results indicate that HtrA1 downregulation confers CDDP resistance by inducing cancer stem cell-like properties via PI3K/Akt-dependent pathway in A549 cells. Therefore, HtrA1 may be a potential target for overcoming CDDP resistance in LAC.

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer

HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p = 0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR = 0.45 [CI 0.23–0.90], p = 0.023; HR = 0.55 [CI 0.32–0.94], p = 0.028, respectively), with even more pronounced impact in node-positive patients (HR = 0.21 [CI 0.07–0.63], p = 0.006; HR = 0.29 [CI 0.13–0.65], p = 0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n = 2809): HR = 0.79 [CI 0.7–0.9], log-rank p = 0.0003; for OS (n = 971): HR = 0.63 [CI 0.48–0.83], log-rank p = 0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.

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.

Abstract 1531: HtrA1 sensitizes ovarian cancer cells to cisplatin-induced cytotoxicity by targeting XIAP for degradation

Abstract We have previously identified serine protease HtrA1 as a down-regulated gene in ovarian cancer and loss of HtrA1 may contribute to cell resistance to cisplatin treatment. However, the exact mechanism by which HtrA1 regulates chemosensitivity in ovarian cancer is currently unknown. Here, we used mixture-based oriented peptide library approach and identified XIAP, an important member of the inhibitor of apoptosis (IAP) protein family, as one possible substrate of HtrA1. Recombinant wild type (WT) HtrA1 but not protease-inactive SA mutant HtrA1 can cleave XIAP protein in vitro. Over-expression of HtrA1 led to decreased level of XIAP in OV167 and OV202 ovarian cancer cells, while down-regulation of HtrA1 resulted in increased level of XIAP in SKOV3 ovarian cancer cells. CDDP treatment enhanced the cleavage of XIAP by HtrA1. Immuno-precipitation assay revealed that HtrA1 binds to XIAP directly both in vitro and in vivo. Increased cell resistance to cisplatin treatment was observed in SKOV3 and TOV-21G cells after stable knockdown of HtrA1 with shRNA. In contrast, over-expression of HtrA1 in OV202 cells promotes cell sensitivity to cisplatin treatment which can be reversed by increased expression of XIAP. In conclusion, our experiments reveal XIAP as a novel substrate of HtrA1 and the mechanism by which HtrA1 confers cell sensitivity to cisplatin treatment may be partially through its degradation of XIAP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1531. doi:10.1158/1538-7445.AM2011-1531

Expression and Functional Significance of HtrA1 Loss in Endometrial Cancer

The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines. Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA1 knocked down clones. Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all seven papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a three- to fourfold increase in the invasive potential. Exogenous expression of HtrA1 in Ark1 and Ark2 cells resulted in three- to fourfold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark2 cells. Immunohistochemical analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (P = 0.016). Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors.

Downregulation of HtrA1 Promotes Resistance to Anoikis and Peritoneal Dissemination of Ovarian Cancer Cells

We previously identified serine protease HtrA1 as a downregulated gene in epithelial ovarian cancer (EOC), but the functional consequence of loss of HtrA1 in EOC remains largely unclear. Here, we report that loss of HtrA1 attenuates anoikis--a critical physiologic barrier for tumor metastasis. In response to loss of anchorage, HtrA1 expression was upregulated in SKOV3 cells, resulting in autocatalytic activation of HtrA1. Stable knockdown of HtrA1 in SKOV3 and TOV21G cells resulted in resistance to anoikis due to enhanced activation of epidermal growth factor receptor (EGFR)/AKT pathway. In suspended SKOV3 cells, enhanced expression of HtrA1 inhibited EGFR/AKT pathway, leading to increased cell death, whereas protease-inactive mutant HtrA1 failed to result in either the inhibition of EGFR/AKT pathway or increased cell death, suggesting the requirement of HtrA1 protease activity in regulating anoikis. Immunoprecipitation and immunofluorescence assays revealed that HtrA1 interacted with EGFR not only on the cell membrane but also in the nucleus. Most importantly, downregulation of HtrA1 significantly enhanced the peritoneal dissemination of SKOV3ip1 cells in nonobese diabetic/severe combined immunodeficient mice, with increased phospho-EGFR level in corresponding tumor nodules compared with that in xenografts originated from the control cells. Taken together, these data reveal for the first time a novel function of HtrA1 in promoting anoikis by attenuating activation of EGFR/AKT pathway that may contribute to its metastasis suppression capacity, thus providing a possible explanation for the aggressive nature of human ovarian tumors with downregulated HtrA1.

Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration

HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified alpha- and beta-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of alpha-, beta-, and gamma-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in the regulation of cell motility by modulating microtubule stability.

Expression of human HtrA1, HtrA2, HtrA3 and TGF-β1 genes in primary endometrial cancer

The HtrA family of serine proteases takes part in cellular stress response including heat shock, inflammation and cancer. Downregulation of human HtrA1 and HtrA3 genes has been reported in some cancers, including endometrial cancer (EC), suggesting a tumor-suppressor role for both genes. The mechanism of the HtrA function is not known, however, evidence exists showing that both HtrA1 and HtrA3 regulate biological processes by modulating TGF-beta signaling. In the presented study the expression of human HtrA1, HtrA2, HtrA3 and TGF-beta1 genes was examined in 124 endometrial tissue specimens including 88 cancers and 36 normal endometria. The expression of the tested genes was evaluated at mRNA and protein levels by semi-quantitative RT-PCR and western blotting methods, respectively. Our results showed significant decrease of HtrA1 and HtrA3 mRNA and protein levels in EC compared to normal tissues. The most dramatic decrease was found for HtrA3 at both mRNA and protein levels (3.2- and 5.6-fold, respectively). Moreover, the HtrA3 protein (short isoform) was not detected in 19% of the cancers, and its level decreased from the premenopausal to the postmenopausal group. The HtrA2 protein levels were significantly lower in EC tissues compared to normal tissues. We also found a significant increase of the TGF-beta1 protein level in EC as well as a significant negative correlation between HtrA1/2/3 and TGF-beta1 relative protein levels. Our results showing downregulation of HtrA1 and HtrA3 gene expression support previous studies suggesting a tumor suppressor role for these genes. Furthermore, our data suggest that HtrA2 may be involved in EC development as well as suggest the involvement of HtrA1, HtrA2 and HtrA3 in the inhibition of TGF-beta signaling in endometrial tissues.

Expression Patterns of Two Serine Protease HtrA1 Forms in Human Placentas Complicated by Preeclampsia with and without Intrauterine Growth Restriction

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of ∼30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE–IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE–IUGR placentas. In conclusion, we suggest that the ∼30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Changes in mRNA and protein levels of human HtrA1, HtrA2 and HtrA3 in ovarian cancer

Objectives Expression of human HtrA1, HtrA2, HtrA3 and TGF-β1 genes was examined in ovarian tissue specimens including 19 normal ovaries, 20 benign tumors, 7 borderline tumors, 44 cancers and 8 Krukenberg tumors. Design and methods mRNA and protein levels were evaluated by semi-quantitative RT-PCR and Western-blotting methods, respectively. Results A statistically significant decrease of HtrA1 and HtrA3 expression in ovarian tumors comparing to normal tissues was observed. A dramatic decrease of HtrA3 mRNA and protein levels in all tumor tissue groups, and a loss of HtrA3 protein in 30% malignant tumors were found. A significant decrease of HtrA1 mRNA, and of HtrA3 mRNA and protein in malignant tumors compared to benign tumors was revealed. HtrA2 expression in tumor tissues was slightly decreased. Expression of TGF-β1 in tumor tissues was not significantly different compared to control tissues. Conclusions Our results show downregulation of HtrA1 and HtrA3 genes' expression in different types of ovarian tumors and give additional evidence that these genes may function as tumor suppressors.

Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity

Resistance to chemotherapy presents a serious challenge in the successful treatment of various cancers and is mainly responsible for mortality associated with disseminated cancers. Here we show that expression of HtrA1, which is frequently downregulated in ovarian cancer, influences tumor response to chemotherapy by modulating chemotherapy-induced cytotoxicity. Downregulation of HtrA1 attenuated cisplatin- and paclitaxel-induced cytotoxicity, while forced expression of HtrA1 enhanced cisplatin- and paclitaxel-induced cytotoxicity. HtrA1 expression was upregulated by both cisplatin and paclitaxel treatment. This upregulation resulted in limited autoproteolysis and activation of HtrA1. Active HtrA1 induces cell death in a serine protease-dependent manner. The potential role of HtrA1 as a predictive factor of clinical response to chemotherapy was assessed in both ovarian and gastric cancer patients receiving cisplatin-based regimens. Patients with ovarian or gastric tumors expressing higher levels of HtrA1 showed a higher response rate compared with those with lower levels of HtrA1 expression. These findings uncover what we believe to be a novel pathway by which serine protease HtrA1 mediates paclitaxel- and cisplatin-induced cytotoxicity and suggest that loss of HtrA1 in ovarian and gastric cancers may contribute to in vivo chemoresistance.