The Experts below are selected from a list of 1524 Experts worldwide ranked by ideXlab platform
Jeremy Chien - One of the best experts on this subject based on the ideXlab platform.
-
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
Biology of Ovarian Cancer, 2015Co-Authors: Megan Cooley, Jeremy ChienAbstract:Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA 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.
-
Abstract 2385: Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HTRA1 and deregulated TGFB signaling
Tumor Biology, 2015Co-Authors: Megan Cooley, Jeremy Chien, Alfonso Baldi, Anirban K. Mitra, Andras Czirok, Edina KosaAbstract:Dissemination of ovarian cancer is promoted by deregulated cell adhesion that selectively enhances detachment at primary site and attachment and invasion at secondary sites. Modifications in the extracellular matrix composition within tumor microenvironment can facilitate deregulated cell adhesion, migration, and invasion. We aim to understand how tumor-intrinsic changes affect expression and composition of ECM proteins in disseminated ovarian cancer. The Serine Protease HTRA1 has been shown to inhibit TGFB by targeting TGFB family ligands and receptors for proteolysis. In addition, HTRA1 also degrades ECM proteins, such as decorin, fibronectin, and collagens. We have shown that HTRA1 is down-regulated in ovarian cancer cells with loss contributing to chemotherapy resistance. We propose that loss of HTRA1 in the ovarian tumor microenvironment enhances TGFB signaling that in turn facilitate compositional changes in ECM proteins from stromal cells and promote cancer dissemination. To characterize compositional changes that occur in ECM proteins as a result of increased TGFB signaling, stable isotope amino acid labeling (SILAC) and LC-MS analysis were used. Mesothelial cells were cultured in heavy or light isotopically labeled media to confluence. Tumor cells expressing HTRA1 (NT) or stably transfected with HTRA1-targeting shRNA (sh1) were added to either heavy or light mesothelial cultures with or without TGFB (10 ng/ml). Cells were lifted by EDTA and the remaining matrix proteins were subjected to LC-MS. The data show that ECM compositional changes in fibronectin, laminin and collagen were up regulated in mesothelial cells co-cultured with sh1 compared to NT cells. All extracted proteins with ≥ 4 fold change were analyzed by Ingenuity Pathway Analysis. Proteins up regulated in co-cultures with HTRA1-deficient cancer cells (sh) were determined to be associated with migration, invasion and tumorogenesis whereas proteins upregulated in co-culture with HTRA1-expressing cancer cells (NT) were associated with protein modifications and ER stress. HTRA1-deficient cells show decreased adhesion to collagen I/III matrix and enhanced adhesion to collagen I/VI matrix. HTRA1-deficient cancer cells show morphology characteristics of epithelial-mesenchymal transition and shows higher motility and invasion through mesothelial cell layer. Finally, in clinical samples, decreased HTRA1 expression in primary tumor was significantly associated with higher incidence of carcinomatosis and nodal metastasis in ovarian cancer. We conclude that loss of HTRA1 in ovarian cancer cells results in compositional changes in ECM within the tumor microenvironment, and these changes may contribute to the dissemination of ovarian cancer. Citation Format: Megan Cooley, Jeremy Chien, Anirban Mitra, Alfonso Baldi, Andras Czirok, Edina Kosa. Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HTRA1 and deregulated TGFB signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2015-2385
-
Abstract 1531: HTRA1 sensitizes ovarian cancer cells to cisplatin-induced cytotoxicity by targeting XIAP for degradation
Tumor Biology, 2011Co-Authors: Ashwani Khurana, Jeremy Chien, Jacie L. Maguire, Viji ShridharAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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
-
Abstract 4015: Flavopiridol-induced upregulation of HTRA1 is associated with suppression of its negative transcriptional regulator WT-1 and with enhanced chemosensitivity
Cellular and Molecular Biology, 2011Co-Authors: Tsuyoshi Ohta, Jeremy Chien, Jacie L. Maguire, Ashwani Khurana, Keith C. Bible, Viji ShridharAbstract:We previously identified Serine Protease HTRA1 as a down-regulated gene in epithelial ovarian cancer (EOC) that modulates cisplatin-induced cytotoxicity. However, the mechanism by which HTRA1 sensitizes cells to cisplatin-induced cytotoxicity remained unknown. We have shown previously that the pan-cyclin dependent kinase inhibitor flavopiridol (FP) sensitizes cells to cisplatin-induced cytotoxicity; we hypothesized that flavopiridol might therefore modulate the expression of pro-apoptotic proteins. Microarray data indicated that while flavopiridol treatment alone or in combination with cisplatin up-regulated HTRA1 expression by 2-3 fold (providing a means to upregulate HTRA1 expression), treatment with cisplatin alone had no effect. Interestingly, in searching for underlying mechanisms, we noted that WT-1 transcription factor binding elements that is present in HTRA1 promoter is down-regulated by flavopiridol in our microarray studies. Immunoblot analysis revealed that WT-1 is highly expressed in HTRA1-deficient OV167, OV202, PE04 and Ovcar3 cells but not in HTRA1-expressing SKOV3, TOV21G and PE01 cells, suggesting WT-1 negatively regulates HTRA1 expression in EOC. Immunoblot analysis showed FP treatment resulted in down-regulation of WT-1 with a corresponding increase in HTRA1 protein levels in OV202 cells. Additionally, stable down-regulation of WT-1 by short hairpin RNA (shRNA) in OV202 cells increased HTRA1 expression. Conversely, enhanced expression of full length WT-1 isoform in SKOV3 cells downregulated HTRA1 expression. Reporter assay showed that FP treatment upregulated HTRA1 promoter activity in OV202 cells. Reporter assay and chromatin immunoprecipitation experiments showed that WT-1 is specifically recruited to the HTRA1 promoter. Moreover, downregulation of WT-1 expression in OV202 cells increased cisplatin-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays – an effect reversed by enhanced expression of HTRA1. Collectively, our studies demonstrate a linkage between the effects of flavopiridol on HTRA1 and its regulatory transcription factor WT-1 that appears in part to explain why flavopiridol has chemosensitizing properties 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 4015. doi:10.1158/1538-7445.AM2011-4015
-
Expression and Functional Significance of HTRA1 Loss in Endometrial Cancer
Clinical cancer research : an official journal of the American Association for Cancer Research, 2010Co-Authors: Sally A. Mullany, Jeremy Chien, Takayo Ota, Mehdi Moslemi-kebria, Ramandeep Rattan, Ashwani Khurana, Amy C. Clayton, Andrea Mariani, Karl C. Podratz, Viji ShridharAbstract:Purpose: 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. Experimental design: 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 HtrA-1 knocked down clones. Results: Western blot analysis of HTRA1 in 13 EC cell lines revealed complete loss of HTRA1 expression in all 7 papillary serous EC cell lines. Downregulation of HTRA1 in Hec1A and Hec1B cell lines resulted in a 3-4 fold increase in the invasive potential. Exogenous expression of HTRA1 in Ark 1 and Ark 2 cells resulted in 3-4 fold 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 Ark 2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or Protease deficient (SA mutant) Ark 2 cells. Immunohistochemical (IHC) 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). Conclusions: Collectively, these data indicate loss of HTRA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors.
Viji Shridhar - One of the best experts on this subject based on the ideXlab platform.
-
Abstract 1531: HTRA1 sensitizes ovarian cancer cells to cisplatin-induced cytotoxicity by targeting XIAP for degradation
Tumor Biology, 2011Co-Authors: Ashwani Khurana, Jeremy Chien, Jacie L. Maguire, Viji ShridharAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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
-
Abstract 4015: Flavopiridol-induced upregulation of HTRA1 is associated with suppression of its negative transcriptional regulator WT-1 and with enhanced chemosensitivity
Cellular and Molecular Biology, 2011Co-Authors: Tsuyoshi Ohta, Jeremy Chien, Jacie L. Maguire, Ashwani Khurana, Keith C. Bible, Viji ShridharAbstract:We previously identified Serine Protease HTRA1 as a down-regulated gene in epithelial ovarian cancer (EOC) that modulates cisplatin-induced cytotoxicity. However, the mechanism by which HTRA1 sensitizes cells to cisplatin-induced cytotoxicity remained unknown. We have shown previously that the pan-cyclin dependent kinase inhibitor flavopiridol (FP) sensitizes cells to cisplatin-induced cytotoxicity; we hypothesized that flavopiridol might therefore modulate the expression of pro-apoptotic proteins. Microarray data indicated that while flavopiridol treatment alone or in combination with cisplatin up-regulated HTRA1 expression by 2-3 fold (providing a means to upregulate HTRA1 expression), treatment with cisplatin alone had no effect. Interestingly, in searching for underlying mechanisms, we noted that WT-1 transcription factor binding elements that is present in HTRA1 promoter is down-regulated by flavopiridol in our microarray studies. Immunoblot analysis revealed that WT-1 is highly expressed in HTRA1-deficient OV167, OV202, PE04 and Ovcar3 cells but not in HTRA1-expressing SKOV3, TOV21G and PE01 cells, suggesting WT-1 negatively regulates HTRA1 expression in EOC. Immunoblot analysis showed FP treatment resulted in down-regulation of WT-1 with a corresponding increase in HTRA1 protein levels in OV202 cells. Additionally, stable down-regulation of WT-1 by short hairpin RNA (shRNA) in OV202 cells increased HTRA1 expression. Conversely, enhanced expression of full length WT-1 isoform in SKOV3 cells downregulated HTRA1 expression. Reporter assay showed that FP treatment upregulated HTRA1 promoter activity in OV202 cells. Reporter assay and chromatin immunoprecipitation experiments showed that WT-1 is specifically recruited to the HTRA1 promoter. Moreover, downregulation of WT-1 expression in OV202 cells increased cisplatin-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays – an effect reversed by enhanced expression of HTRA1. Collectively, our studies demonstrate a linkage between the effects of flavopiridol on HTRA1 and its regulatory transcription factor WT-1 that appears in part to explain why flavopiridol has chemosensitizing properties 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 4015. doi:10.1158/1538-7445.AM2011-4015
-
Expression and Functional Significance of HTRA1 Loss in Endometrial Cancer
Clinical cancer research : an official journal of the American Association for Cancer Research, 2010Co-Authors: Sally A. Mullany, Jeremy Chien, Takayo Ota, Mehdi Moslemi-kebria, Ramandeep Rattan, Ashwani Khurana, Amy C. Clayton, Andrea Mariani, Karl C. Podratz, Viji ShridharAbstract:Purpose: 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. Experimental design: 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 HtrA-1 knocked down clones. Results: Western blot analysis of HTRA1 in 13 EC cell lines revealed complete loss of HTRA1 expression in all 7 papillary serous EC cell lines. Downregulation of HTRA1 in Hec1A and Hec1B cell lines resulted in a 3-4 fold increase in the invasive potential. Exogenous expression of HTRA1 in Ark 1 and Ark 2 cells resulted in 3-4 fold 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 Ark 2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or Protease deficient (SA mutant) Ark 2 cells. Immunohistochemical (IHC) 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). Conclusions: 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
Cancer Research, 2010Co-Authors: Takayo Ota, Jeremy Chien, Peng Liu, Viji ShridharAbstract: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.
-
Downregulation of HTRA1 promotes resistance to anoikis and peritoneal dissemination of ovarian cancer cells.
Cancer research, 2010Co-Authors: Takayo Ota, Jeremy Chien, Peng Liu, Viji ShridharAbstract: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. Cancer Res; 70(8); 3109–18. ©2010 AACR.
Alfonso Baldi - One of the best experts on this subject based on the ideXlab platform.
-
Abstract 2385: Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HTRA1 and deregulated TGFB signaling
Tumor Biology, 2015Co-Authors: Megan Cooley, Jeremy Chien, Alfonso Baldi, Anirban K. Mitra, Andras Czirok, Edina KosaAbstract:Dissemination of ovarian cancer is promoted by deregulated cell adhesion that selectively enhances detachment at primary site and attachment and invasion at secondary sites. Modifications in the extracellular matrix composition within tumor microenvironment can facilitate deregulated cell adhesion, migration, and invasion. We aim to understand how tumor-intrinsic changes affect expression and composition of ECM proteins in disseminated ovarian cancer. The Serine Protease HTRA1 has been shown to inhibit TGFB by targeting TGFB family ligands and receptors for proteolysis. In addition, HTRA1 also degrades ECM proteins, such as decorin, fibronectin, and collagens. We have shown that HTRA1 is down-regulated in ovarian cancer cells with loss contributing to chemotherapy resistance. We propose that loss of HTRA1 in the ovarian tumor microenvironment enhances TGFB signaling that in turn facilitate compositional changes in ECM proteins from stromal cells and promote cancer dissemination. To characterize compositional changes that occur in ECM proteins as a result of increased TGFB signaling, stable isotope amino acid labeling (SILAC) and LC-MS analysis were used. Mesothelial cells were cultured in heavy or light isotopically labeled media to confluence. Tumor cells expressing HTRA1 (NT) or stably transfected with HTRA1-targeting shRNA (sh1) were added to either heavy or light mesothelial cultures with or without TGFB (10 ng/ml). Cells were lifted by EDTA and the remaining matrix proteins were subjected to LC-MS. The data show that ECM compositional changes in fibronectin, laminin and collagen were up regulated in mesothelial cells co-cultured with sh1 compared to NT cells. All extracted proteins with ≥ 4 fold change were analyzed by Ingenuity Pathway Analysis. Proteins up regulated in co-cultures with HTRA1-deficient cancer cells (sh) were determined to be associated with migration, invasion and tumorogenesis whereas proteins upregulated in co-culture with HTRA1-expressing cancer cells (NT) were associated with protein modifications and ER stress. HTRA1-deficient cells show decreased adhesion to collagen I/III matrix and enhanced adhesion to collagen I/VI matrix. HTRA1-deficient cancer cells show morphology characteristics of epithelial-mesenchymal transition and shows higher motility and invasion through mesothelial cell layer. Finally, in clinical samples, decreased HTRA1 expression in primary tumor was significantly associated with higher incidence of carcinomatosis and nodal metastasis in ovarian cancer. We conclude that loss of HTRA1 in ovarian cancer cells results in compositional changes in ECM within the tumor microenvironment, and these changes may contribute to the dissemination of ovarian cancer. Citation Format: Megan Cooley, Jeremy Chien, Anirban Mitra, Alfonso Baldi, Andras Czirok, Edina Kosa. Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HTRA1 and deregulated TGFB signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2015-2385
-
Serine Protease HTRA1 associates with microtubules and inhibits cell migration.
Molecular and cellular biology, 2009Co-Authors: Jeremy Chien, Alfonso Baldi, Giovanni Aletti, Takayo Ota, Ravi Shridhar, Mariarosaria Boccellino, Lucio Quagliuolo, Viji ShridharAbstract: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 pattern alterations of the Serine Protease HTRA1 in normal human placental tissues and in gestational trophoblastic diseases.
Histology and histopathology, 2009Co-Authors: Daniela Marzioni, Alfonso Baldi, Teresa Lorenzi, Alexia Quaranta, Manrico Morroni, Caterina Crescimanno, Michele De Nictolis, Paolo Toti, Giovanni Muzzonigro, Antonio De LucaAbstract:HTRA1 is a secreted protein which behaves as a molecular chaperone at low temperatures and as a Serine Protease at high temperatures. When the placenta escapes the normal growth control mechanisms, which are present during normal pregnancy, it may develop trophoblastic diseases, such as hydatidiform mole and choriocarcinoma. The aim of the study is to investigate the expression of HTRA1 in these gestational trophoblastic diseases and evaluate whether different HTRA1 expression might be associated with increasingly severe forms of disease. We used immunohistochemistry to assess the expression of HTRA1 in normal human placenta, hydatidiform mole (partial and complete) and choriocarcinoma. In addition to that we used the western blotting technique to quantify HTRA1 immunoreaction in normal human placentas. The most striking finding of our investigation is the decrease in immunostaining of this Protease with increasing severity of gestational trophoblastic disease. For instance, in partial and complete moles HTRA1 is weakly expressed in the trophoblast. Moreover, absence of immunoreaction for HTRA1 is observable in the choriocarcinoma cells. In conclusion, we suggest that HTRA1 may play an important role in the pathogenesis and progression of hydatidiform moles and choriocarcinomas, and that HTRA1 may play an important role during the normal development of the placenta, as well as in trophoblastic diseases.
-
Expression patterns of two Serine Protease HTRA1 forms in human placentas complicated by preeclampsia with and without intrauterine growth restriction.
Placenta, 2008Co-Authors: Teresa Lorenzi, Alfonso Baldi, Daniela Marzioni, Stefano Raffaele Giannubilo, Angelo Quaranta, C. Crescimanno, A. De Luca, Tullia Todros, Andrea L. Tranquilli, Mario CastellucciAbstract:Abstract 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.
-
Elevated expression of Serine Protease HTRA1 in preeclampsia and its role in trophoblast cell migration and invasion.
American journal of obstetrics and gynecology, 2008Co-Authors: Funminiyi Ajayi, Alfonso Baldi, Viji Shridhar, Nicholas Kongoasa, Thomas Gaffey, Yan W Asmann, William J Watson, Peeyush Lala, Brian Brost, Jeremy ChienAbstract:Aberrant expression of developmentally regulated genes during placental development could affect fetal growth and contribute to preeclampsia. Expression of Serine Protease HTRA1 is developmentally regulated with the highest expression in decidua capsularis, compared with ectoplacental cone, and with the highest expression during later stages of pregnancy, compared with early stages. This study was designed to determine the expression of HTRA1 in placental tissues from control and preeclamptic pregnancies and to determine the effect of HTRA1 expression in trophoblast cell migration and invasion. HTRA1 expression was assessed by immunohistochemistry in placentas from gestational age-matched preeclamptic and control pregnancies. HTRA1 expression in extravillous trophoblast cells, HTR-8/SVneo, was assessed by immunoblotting and immunofluorescence microscopy. Finally, the effect of ectopic expression of HTRA1 on cell migration and invasion was determined in HTR-8/SVneo cells. Higher expression of HTRA1 was detected in placental tissues collected from patients with early-onset preeclampsia, compared with those from gestational age-matched control samples. Moreover, ectopic expression of HTRA1 significantly attenuates HTR-8/SVneo cell migration and invasion. Higher expression of HTRA1 is associated with early-onset preeclampsia and may affect trophoblast cell migration and invasion.
Michael Ehrmann - One of the best experts on this subject based on the ideXlab platform.
-
Inactivation of the Serine Protease HTRA1 inhibits tumor growth by deregulating angiogenesis
Oncogene, 2018Co-Authors: Ralph Klose, Michael Ehrmann, M. Gordian Adam, Eva-maria Weis, Iris Moll, Joycelyn Wüstehube-lausch, Fabian Tetzlaff, Chio Oka, Andreas FischerAbstract:The Serine Protease HTRA1 is involved in several vascular diseases and its expression is often deregulated in cancer. We aimed at identifying how HTRA1 in the vasculature affects tumor growth. Here we report that silencing of HTRA1 in cultured endothelial cells increased migration rate and tube formation, whereas forced HTRA1 expression impaired sprouting angiogenesis. Mechanistically, endothelial HTRA1 expression enhanced Delta/Notch signaling by reducing the amount of the weak Notch ligand JAG1. HTRA1 physically interacted with JAG1 and cleaved it within the intracellular domain, leading to protein degradation. Expression of a constitutive active Notch1 prevented the hypersprouting phenotype upon silencing of HTRA1 . In HTRA1 -deficient mice, endothelial Notch signaling was diminished and isolated endothelial cells had increased expression of VEGF receptor-2. Growth of syngeneic tumors was strongly impaired in HTRA1 ^ −/− mice. The tumor vasculature was much denser in HTRA1 ^−/− mice and less covered with mural cells. This chaotic and immature vascular network was poorly functional as indicated by large hypoxic tumor areas and low tumor cell proliferation rates. In summary, inhibition of HTRA1 in the tumor stroma impaired tumor progression by deregulating angiogenesis.
-
Determinants of amyloid fibril degradation by the PDZ Protease HTRA1
Nature Chemical Biology, 2015Co-Authors: Simon Poepsel, Andreas Sprengel, Barbara Sacca, Farnusch Kaschani, Markus Kaiser, Christos Gatsogiannis, Stefan Raunser, Tim Clausen, Michael EhrmannAbstract:Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved Serine Protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ Protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed. The Serine Protease HTRA1 utilizes a "disintegration" mechanism involving its flexible PDZ domains to first loosen tau amyloid fibrils and subsequently disintegrating the fibrillar core structure for efficient proteolytic degradation.
-
Determinants of amyloid fibril degradation by the PDZ Protease HTRA1
Nature chemical biology, 2015Co-Authors: Simon Poepsel, Andreas Sprengel, Barbara Sacca, Farnusch Kaschani, Markus Kaiser, Christos Gatsogiannis, Stefan Raunser, Tim Clausen, Michael EhrmannAbstract:Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved Serine Protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ Protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed.
-
Human Serine Protease HTRA1 positively regulates osteogenesis of human bone marrow-derived mesenchymal stem cells and mineralization of differentiating bone-forming cells through the modulation of extracellular matrix protein.
Stem cells (Dayton Ohio), 2012Co-Authors: André N. Tiaden, Michael Ehrmann, Gregor Bahrenberg, Ali Mirsaidi, Stephan Glanz, Maike Breiden, Fabienne A. Weber, Paolo Cinelli, Peter J. RichardsAbstract:Mammalian high-temperature requirement Serine Protease A1 (HTRA1) is a secreted member of the trypsin family of Serine Proteases which can degrade a variety of bone matrix proteins and as such has been implicated in musculoskeletal development. In this study, we have investigated the role of HTRA1 in mesenchymal stem cell (MSC) osteogenesis and suggest a potential mechanism through which it controls matrix mineralization by differentiating bone-forming cells. Osteogenic induction resulted in a significant elevation in the expression and secretion of HTRA1 in MSCs isolated from human bone marrow-derived MSCs (hBMSCs), mouse adipose-derived stromal cells (mASCs), and mouse embryonic stem cells. Recombinant HTRA1 enhanced the osteogenesis of hBMSCs as evidenced by significant changes in several osteogenic markers including integrin-binding sialoprotein (IBSP), bone morphogenetic protein 5 (BMP5), and sclerostin, and promoted matrix mineralization in differentiating bone-forming osteoblasts. These stimulatory effects were not observed with proteolytically inactive HTRA1 and were abolished by small interfering RNA against HTRA1. Moreover, loss of HTRA1 function resulted in enhanced adipogenesis of hBMSCs. HTRA1 Immunofluorescence studies showed colocalization of HTRA1 with IBSP protein in osteogenic mASC spheroid cultures and was confirmed as being a newly identified HTRA1 substrate in cell cultures and in proteolytic enzyme assays. A role for HTRA1 in bone regeneration in vivo was also alluded to in bone fracture repair studies where HTRA1 was found localized predominantly to areas of new bone formation in association with IBSP. These data therefore implicate HTRA1 as having a central role in osteogenesis through modification of proteins within the extracellular matrix. STEM Cells2012;30:2271–2282
-
Substrate-induced remodeling of the active site regulates human HTRA1 activity
Nature Structural & Molecular Biology, 2011Co-Authors: Linda Truebestein, Markus Kaiser, Tim Clausen, Annette Tennstaedt, Timon Mönig, T. Krojer, Flavia Canellas, Michael EhrmannAbstract:Crystal structures of the Protease domain of human HTRA1, with or without its PDZ domain, reveal that this enzyme's activity is controlled by substrate binding to its active site, via an induced-fit mechanism. Crystal structures of active and inactive conformations of the human Serine Protease HTRA1 reveal that substrate binding to the active site is sufficient to stimulate proteolytic activity. HTRA1 attaches to liposomes, digests misfolded proteins into defined fragments and undergoes substrate-mediated oligomer conversion. In contrast to those of other Serine Proteases, the PDZ domain of HTRA1 is dispensable for activation or lipid attachment, indicative of different underlying mechanistic features.
Funminiyi Ajayi - One of the best experts on this subject based on the ideXlab platform.
-
Elevated expression of Serine Protease HTRA1 in preeclampsia and its role in trophoblast cell migration and invasion.
American journal of obstetrics and gynecology, 2008Co-Authors: Funminiyi Ajayi, Alfonso Baldi, Viji Shridhar, Nicholas Kongoasa, Thomas Gaffey, Yan W Asmann, William J Watson, Peeyush Lala, Brian Brost, Jeremy ChienAbstract:Aberrant expression of developmentally regulated genes during placental development could affect fetal growth and contribute to preeclampsia. Expression of Serine Protease HTRA1 is developmentally regulated with the highest expression in decidua capsularis, compared with ectoplacental cone, and with the highest expression during later stages of pregnancy, compared with early stages. This study was designed to determine the expression of HTRA1 in placental tissues from control and preeclamptic pregnancies and to determine the effect of HTRA1 expression in trophoblast cell migration and invasion. HTRA1 expression was assessed by immunohistochemistry in placentas from gestational age-matched preeclamptic and control pregnancies. HTRA1 expression in extravillous trophoblast cells, HTR-8/SVneo, was assessed by immunoblotting and immunofluorescence microscopy. Finally, the effect of ectopic expression of HTRA1 on cell migration and invasion was determined in HTR-8/SVneo cells. Higher expression of HTRA1 was detected in placental tissues collected from patients with early-onset preeclampsia, compared with those from gestational age-matched control samples. Moreover, ectopic expression of HTRA1 significantly attenuates HTR-8/SVneo cell migration and invasion. Higher expression of HTRA1 is associated with early-onset preeclampsia and may affect trophoblast cell migration and invasion.
-
Elevated expression of Serine Protease HTRA1 in preeclampsia and its role in trophoblast cell migration and invasion.
American Journal of Obstetrics and Gynecology, 2008Co-Authors: Funminiyi Ajayi, Alfonso Baldi, Viji Shridhar, Nicholas Kongoasa, Thomas Gaffey, Yan W Asmann, William J Watson, Brian Brost, Peeyush K. Lala, Jeremy ChienAbstract:Objective Aberrant expression of developmentally regulated genes during placental development could affect fetal growth and contribute to preeclampsia. Expression of Serine Protease HTRA1 is developmentally regulated with the highest expression in decidua capsularis, compared with ectoplacental cone, and with the highest expression during later stages of pregnancy, compared with early stages. This study was designed to determine the expression of HTRA1 in placental tissues from control and preeclamptic pregnancies and to determine the effect of HTRA1 expression in trophoblast cell migration and invasion. Study Design HTRA1 expression was assessed by immunohistochemistry in placentas from gestational age–matched preeclamptic and control pregnancies. HTRA1 expression in extravillous trophoblast cells, HTR-8/SVneo, was assessed by immunoblotting and immunofluorescence microscopy. Finally, the effect of ectopic expression of HTRA1 on cell migration and invasion was determined in HTR-8/SVneo cells. Results Higher expression of HTRA1 was detected in placental tissues collected from patients with early-onset preeclampsia, compared with those from gestational age–matched control samples. Moreover, ectopic expression of HTRA1 significantly attenuates HTR-8/SVneo cell migration and invasion. Conclusion Higher expression of HTRA1 is associated with early-onset preeclampsia and may affect trophoblast cell migration and invasion.
-
Placental expression of HTRA1 in pregnancies complicated by preeclampsia
American Journal of Obstetrics and Gynecology, 2005Co-Authors: Funminiyi Ajayi, Jeremy Chien, Thomas Gaffey, Brian Brost, William J WatsonAbstract:PREECLAMPSIA FUNMINIYI AJAYI, BRIAN BROST, JEREMY CHIEN, THOMAS GAFFEY, WILLIAM WATSON, Mayo Clinic College of Medicine, Obstetrics and Gynecology, Rochester, Minnesota, Mayo Clinic College of Medicine, Pathology and Laboratory Medicine, Rochester, Minnesota, Mayo Clinic College of Medicine, Anatomic Pathology, Rochester, Minnesota OBJECTIVE: The placenta has a high rate of cell proliferation and undergoes near continuous remodeling with a balance of cell proliferation and death. A Serine Protease, HTRA1, has been shown to be involved with apoptosis, cellular differentiation, and invasiveness (marked downregulation in several cancers). Placental HTRA1 expression is noted to increase as pregnancy progresses. Preeclampsia is a disorder associated with increased villous/ extravillous trophoblast apoptosis and abnormal cytotrophoblast invasion. An association with preeclampsia and HTRA1 has not been evaluated. We aimed to establish if the expression of HTRA1 is altered in the placenta of preeclamptic women. STUDY DESIGN: Qualitative expression of HTRA1 was assessed by immunohistochemical techniques in archived third trimester placental tissues using gestational age-matched normotensive (n=9) and severe preeclamptic women (n=9). Women delivered at term with mild preeclampsia (n=3) were also evaluated. Expression of HTRA1 was rated by a Placental Pathologist on a scale of 0 (no expression) and 3 (marked expression). RESULTS: Visually, no difference in HTRA1 staining of cytotrophoblasts was noted in normotensive or mild preeclamptic placentas. HTRA1 staining was noted throughout the placenta with slightly higher levels of staining in the extravillous cytotrophoblasts of placentas obtained from patients with severe preeclampsia. Placentas of women with severe preeclampsia showed marked increased staining of the villous cytotrophoblasts compared to control placentas. Staining was noted mainly in the cytoplasm; some nuclear staining was also noted. CONCLUSION: HTRA1 is significantly upregulated in the villous cytotrophoblasts of severe preeclamptic women. The extravillous trophoblasts show a smaller increase in baseline staining. Increased expression of HTRA1 during pregnancy could impair cytotrophoblast invasion, survival, and differentiation resulting in placental dysfuction and fetal compromise. Further research is needed to characterize the role of HTRA1 in the pathophysiology of preeclampsia.
