Xiaoping He

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.

IDENTIFICATION OF TUBULINS AS SUBSTRATES OF SERINE PROTEASE HTRA1 BY MIXTURE-BASED ORIENTED PEPTIDE LIBRARY SCREENING

Serine protease HtrA1 belongs to a family of chymotrypsin‐like proteases that were first identified in bacteria and later in mammalian systems. These proteases were identified as components of protein quality control in prokaryotic systems and as regulators of diverse signaling pathways in mammalian systems. In particular, HtrA1 is implicated in trophoblast cell migration and invasion, tumor progression, chemotherapy‐induced cytotoxicity, osteoarthritis, age‐related macular degeneration, and pathogenesis of Alzheimers disease. However, systematic analysis of its potential substrates in biological system is still lacking. Therefore, we performed a mixture‐based oriented peptide library screening to identify putative substrates of HtrA1. We identified [AEGR]‐[LAGR]‐[IAMLR]‐[TVIAL] as consensus residues for P1 to P4 sites. We identified several putative substrates of HtrA1 involved in the pathogenesis of various diseases. In this study, we report on the identification of tubulins as potential substrates of HtrA1, and validated tubulins as in vitro and intracellular substrates of HtrA1. These results provide initial insights into substrate identification and functional characterization of HtrA1 in pathogenesis of various diseases. J. Cell. Biochem. 107: 253–263, 2009.

The serine protease HtrA1 specifically interacts and degrades the tuberous sclerosis complex 2 protein.

Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway. Mol Cancer Res; 8(9); 1248–60. ©2010 AACR.

Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

We recently identified HSulf-1 as a down-regulated gene in ovarian carcinomas. Our previous analysis indicated that HSulf-1 inactivation in ovarian cancers is partly mediated by loss of heterozygosity and epigenetic silencing. Here, we show that variant hepatic nuclear factor 1 (vHNF1), encoded by transcription factor 2 gene (TCF2, HNF1beta), negatively regulates HSulf-1 expression in ovarian cancer. Immunoblot assay revealed that vHNF1 is highly expressed in HSulf-1-deficient OV207, SKOV3, and TOV-21G cell lines but not in HSulf-1-expressing OSE, OV167, and OV202 cells. By short hairpin RNA-mediated down-regulation of vHNF1 in TOV-21G cells and transient enhanced vHNF1 expression in OV202 cells, we showed that vHNF1 suppresses HSulf-1 expression in ovarian cancer cell lines. Reporter assay and chromatin immunoprecipitation experiments showed that vHNF1 is specifically recruited to HSulf-1 promoter at two different vHNF1-responsive elements in OV207 and TOV-21G cells. Additionally, down-regulation of vHNF1 expression in OV207 and TOV-21G cells increased cisplatin- or paclitaxel-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays and this effect was reversed by down-regulation of HSulf-1. Moreover, nude mice bearing TOV-21G cell xenografts with stably down-regulated vHNF1 were more sensitive to cisplatin- or paclitaxel-induced cytotoxicity compared with xenografts of TOV-21G clonal lines with nontargeted control short hairpin RNA. Finally, immunohistochemical analysis of 501 ovarian tumors including 140 clear-cell tumors on tissue microarrays showed that vHNF1 inversely correlates to HSulf-1 expression. Collectively, these results indicate that vHNF1 acts as a repressor of HSulf-1 expression and might be a molecular target for ovarian cancer therapy.

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

HtrA1, a member of serine protease family, has been previously found to be involved in resistance to chemotherapy in ovarian cancer although the underlying mechanism is not clear. Using mixture‐based oriented peptide library approach, previously we identified X‐linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis proteins family, as a potential substrate of HtrA1. The aim of our work is to investigate the link between HtrA1 and XIAP proteins and their relationships with chemoresistance in ovarian cancer. Our results showed that recombinant XIAP was degraded by purified wild‐type HtrA1 but not mutant HtrA1 in vitro. Consistent with the in vitro data, coimmunoprecipitation assays showed that HtrA1 and XIAP formed a protein complex in vivo. Ectopic expression of HtrA1 led to decreased level of XIAP in OV167 and OV202 ovarian cancer cells, while knockdown of HtrA1 resulted in increased level of XIAP in SKOV3 ovarian cancer cells. Furthermore, overexpression of HtrA1 in OV202 cells promoted cell sensitivity to cisplatin‐induced apoptosis that could be reversed by increased expression of XIAP. The cleavage of XIAP induced by HtrA1 was enhanced by cisplatin treatment. Taken together, our experiments have identified XIAP as a novel substrate of HtrA1 and the degradation of XIAP by HtrA1 contributes to cell response to chemotherapy, suggesting that restoring the expression of HtrA1 may be a promising treatment strategy for ovarian cancer.

Loss of HSulf-1 promotes altered lipid metabolism in ovarian cancer

BackgroundLoss of the endosulfatase HSulf-1 is common in ovarian cancer, upregulates heparin binding growth factor signaling and potentiates tumorigenesis and angiogenesis. However, metabolic differences between isogenic cells with and without HSulf-1 have not been characterized upon HSulf-1 suppression in vitro. Since growth factor signaling is closely tied to metabolic alterations, we determined the extent to which HSulf-1 loss affects cancer cell metabolism.ResultsIngenuity pathway analysis of gene expression in HSulf-1 shRNA-silenced cells (Sh1 and Sh2 cells) compared to non-targeted control shRNA cells (NTC cells) and subsequent Kyoto Encyclopedia of Genes and Genomics (KEGG) database analysis showed altered metabolic pathways with changes in the lipid metabolism as one of the major pathways altered inSh1 and 2 cells. Untargeted global metabolomic profiling in these isogenic cell lines identified approximately 338 metabolites using GC/MS and LC/MS/MS platforms. Knockdown of HSulf-1 in OV202 cells induced significant changes in 156 metabolites associated with several metabolic pathways including amino acid, lipids, and nucleotides. Loss of HSulf-1 promoted overall fatty acid synthesis leading to enhance the metabolite levels of long chain, branched, and essential fatty acids along with sphingolipids. Furthermore, HSulf-1 loss induced the expression of lipogenic genes including FASN, SREBF1, PPARγ, and PLA2G3 stimulated lipid droplet accumulation. Conversely, re-expression of HSulf-1 in Sh1 cells reduced the lipid droplet formation. Additionally, HSulf-1 also enhanced CPT1A and fatty acid oxidation and augmented the protein expression of key lipolytic enzymes such as MAGL, DAGLA, HSL, and ASCL1. Overall, these findings suggest that loss of HSulf-1 by concomitantly enhancing fatty acid synthesis and oxidation confers a lipogenic phenotype leading to the metabolic alterations associated with the progression of ovarian cancer.ConclusionsTaken together, these findings demonstrate that loss of HSulf-1 potentially contributes to the metabolic alterations associated with the progression of ovarian pathogenesis, specifically impacting the lipogenic phenotype of ovarian cancer cells that can be therapeutically targeted.

Quinacrine promotes autophagic cell death and chemosensitivity in ovarian cancer and attenuates tumor growth

A promising new strategy for cancer therapy is to target the autophagic pathway. In the current study, we demonstrate that the antimalarial drug Quinacrine (QC) reduces cell viability and promotes chemotherapy-induced cell death in an autophagy-dependent manner more extensively in chemoresistant cells compared to their isogenic chemosensitive control cells as quantified by the Chou-Talalay methodology. Our preliminary data, in vitro and in vivo, indicate that QC induces autophagy by downregulating p62/SQSTM1 to sensitize chemoresistant cells to autophagic- and caspase-mediated cell death in a p53-independent manner. QC promotes autophagosome accumulation and enhances autophagic flux by clearance of p62 in chemoresistant ovarain cancer (OvCa) cell lines to a greater extent compared to their chemosensitive controls. Notably, p62 levels were elevated in chemoresistant OvCa cell lines and knockdown of p62 in these cells resulted in a greater response to QC treatment. Bafilomycin A, an autophagy inhibitor, restored p62 levels and reversed QC-mediated cell death and thus chemosensitization. Importantly, our in vivo data shows that QC alone and in combination with carboplatin suppresses tumor growth and ascites in the highly chemoresistant HeyA8MDR OvCa model compared to carboplatin treatment alone. Collectively, our preclinical data suggest that QC in combination with carboplatin can be an effective treatment for patients with chemoresistant OvCa.

Loss of HSulf-1 expression enhances tumorigenicity by inhibiting Bim expression in ovarian cancer.

The expression of human Sulfatase1 (HSulf‐1) is downregulated in the majority of primary ovarian cancer tumors, but the functional consequence of this downregulation remains unclear. Using two different shRNAs (Sh1 and Sh2), HSulf‐1 expression was stably downregulated in ovarian cancer OV202 cells. We found that HSulf‐1‐deficient OV202 Sh1 and Sh2 cells formed colonies in soft agar. In contrast, nontargeting control (NTC) shRNA‐transduced OV202 cells did not form any colonies. Moreover, subcutaneous injection of OV202 HSulf‐1‐deficient cells resulted in tumor formation in nude mice, whereas OV202 NTC cells did not. Also, ectopic expression of HSulf‐1 in ovarian cancer SKOV3 cells significantly suppressed tumor growth in nude mice. Here, we show that HSulf‐1‐deficient OV202 cells have markedly decreased expression of proapoptotic Bim protein, which can be rescued by restoring HSulf‐1 expression in OV202 Sh1 cells. Enhanced expression of HSulf‐1 in HSulf‐1‐deficient SKOV3 cells resulted in increased Bim expression. Decreased Bim levels after loss of HSulf‐1 were due to increased p‐ERK, because inhibition of ERK activity with PD98059 resulted in increased Bim expression. However, treatment with a PI3 kinase/AKT inhibitor, LY294002, failed to show any change in Bim protein level. Importantly, rescuing Bim expression in HSulf‐1 knockdown cells significantly retarded tumor growth in nude mice. Collectively, these results suggest that loss of HSulf‐1 expression promotes tumorigenicity in ovarian cancer through regulating Bim expression.

Assessment of Resistance to Anoikis in Ovarian Cancer

Anoikis, a form of programmed cell death that occurs due to cell detachment from the extracellular matrix, is a critical mechanism in preventing ectopic cell growth. Acquisition of resistance to anoikis is a prerequisite for epithelial ovarian cancer cells to survive in ascitic fluids before forming metastatic foci. Here we describe a colorimetric method for monitoring the resistance of anoikis of ovarian cancer cells in vitro.

Loss of HSulf-1: The Missing Link between Autophagy and Lipid Droplets in Ovarian Cancer

Defective autophagy and deranged metabolic pathways are common in cancer; pharmacologic targeting of these two pathways could provide a viable therapeutic option. However, how these pathways are regulated by limited availability of growth factors is still unknown. Our study shows that HSulf-1 (endosulfatase), a known tumor suppressor which attenuates heparin sulfate binding growth factor signaling, also regulates interplay between autophagy and lipogenesis. Silencing of HSulf-1 in OV202 and TOV2223 cells (ovarian cancer cell lines) resulted in increased lipid droplets (LDs), reduced autophagic vacuoles (AVs) and less LC3B puncta. In contrast, HSulf-1 proficient cells exhibit more AVs and reduced LDs. Increased LDs in HSulf-1 depleted cells was associated with increased ERK mediated cPLA2S505 phosphorylation. Conversely, HSulf-1 expression in SKOV3 cells reduced the number of LDs and increased the number of AVs compared to vector controls. Furthermore, pharmacological (AACOCF3) and ShRNA mediated downregulation of cPLA2 resulted in reduced LDs, and increased autophagy. Finally, in vivo experiment using OV202 Sh1 derived xenograft show that AACOCF3 treatment effectively attenuated tumor growth and LD biogenesis. Collectively, these results show a reciprocal regulation of autophagy and lipid biogenesis by HSulf-1 in ovarian cancer.