Shridhar, Viji

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

Abstract Metformin is the most widely used drug for type 2 diabetes. Metformin activates AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism via its upstream kinase LKB1. Activation of AMPK has been shown extensively to cause growth arrest in multiple tumor types by inhibiting the TSC-mTOR (mammalian target of rapamycin) pathway as well as by inducing cell cycle arrest. Our data indicate that metformin exerts an anti-proliferative effect on several chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3 and SKVO3ip, PE01, PE04), which is accompanied by activation of AMPK and cell cycle arrest. Interestingly, we have also determined that metformin is able to inhibit proliferation of ovarian cancer cells when AMPK is silenced by siRNA, where both the expression and activation of AMPK is significantly reduced. Also, metformin was able to attenuate proliferation of AMPK (−/−) mefs, although to a lesser degree in AMPK (+/+) mefs. Additional results indicate that metformin activates p21 and inhibits cyclinD1 activities partially in AMPK (−/) mefs. These data suggest a novel mechanism of metformin action, namely, an AMPK independent pathway which can modulate proliferation and possibly cell cycle regulated events in ovarian cancer. Whether the AMPK independent effects of metformin require LKB1 activity is currently under investigation. Collectively, our preliminary observations indicate that metformin can modulate proliferation and cell cycle arrest in an AMPK independent manner. Citation Information: Cancer Res 2009;69(23 Suppl):C38.

Abstract Ductal carcinoma in situ (DCIS) of the breast is a heterogenous group of proliferative cellular lesions that have the potential to become invasive. Very little is known about the molecular alterations involved in the progression from DCIS to invasive ductal carcinoma (IDC). Heparan endosulfatases HSulf-1 and -2 catalyze or edit sulfate moieties on heparan sulfate proteoglycans and have been implicated in modulating heparin binding growth factor signaling. Several studies have suggested that while expression of HSulf-1 is down-regulated and is considered a tumor suppressor gene, expression of HSulf-2 is over-expressed in primary breast tumors and cell lines. However, the role of HSulf-2 in breast tumorigenesis is poorly understood. Here we have evaluated the effect of HSulf-2 in an in vivo model of breast cancer progression (MCF10DCIS.com). These cells form comedo type DCIS and progress to IDC when transplanted in immune-deficient mice and therefore are an ideal model to study breast cancer progression. Methods: Stable clones depleted of HSulf-2 in MCF10DCIS.com cells were generated and injected into mouse mammary fat pads with non-targeted control shRNA clones (NTC) as controls. HSulf-1, HSulf-2, smooth muscle actin immunostaining and H&E staining was perfromed at weeks 3, 5 and 7. Matrixmetallo Protease (MMP) expression by real time PCR was evaluated at Weeks 3 and 5 in both groups. Results: A significant reduction in tumor growth was observed in HSulf-2 depleted clones compared to NTC clones (p=<0.01). HSulf-2 immunohistochemical staining showed distinct pattern of staining localized to ductal lesions as well as stromal (myoepithelial and myofibroblasts cells). In contrast, the closely related family member, HSulf-1 expression was restricted largely to stromal and myoepithelial layer in ductal lesions. While HSulf-2 staining remained intense from weeks 3 to 7, HSulf-1 expression was diminished during the same time period. In NTC clones, ductal like lesions were observed by week 3 with disruption of the basement membrane and appearance of invasive phenotype by weeks 5 and 7. In contrast, HSulf2 knockdown xenografts showed retention of ductal like structures even at week 5 and retention of basement membrane (p=<0.05). Additionally marked necrotic areas were also observed in HSulf-2 silenced clones. Previous studies have shown that the malignant epithelium induces the development of the stroma necessary for the development of invasive phenotype. Consistent with this observation, we demonstrated decreased MMP expression in xenografts derived from HSulf-2 depleted clones compared with NTC xenografts (p=<0.05). Conclusions: Our data suggest that HSulf-2 may play an important role in the transition from DCIS to an invasive phenotype (IDC) potentially by up regulating MMP expression and activity. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-12.

Abstract It was well accepted for many years that ovarian surface epithelium is the origin of epithelial ovarian carcinomas. However, recent molecular and pathologic evidence indicate that high-grade serous carcinomas of the ovary, which are the most common type of epithelial carcinomas, may also arise from the distal part of fallopian tubes or tubal carcinoma implants. Gene expression profiles of tubal epithelium from BRCA mutation carriers resemble those of high-grade serous carcinomas of the ovary, and ovarian carcinomas and tubal intraepithelial carcinomas have identical p53 mutations. Oviductal glycoprotein 1 (Ogp1) is a glycosylated protein found in the epithelial cells in the oviduct. So far, Ogp1 is the only specific tubal marker for the oviduct. Ogp1 promoter-driven SV40 large T-antigen transgenic mice develop tumors in the female reproductive organs. To characterize these tumors, we did immunohistochemical analysis in frozen and paraffin-embedded sections. Mice were sacrificed at 8 weeks old. Macroscopically the cervico-vaginal region and the fallopian tubes were markedly enlarged. Histologically, three distinct abnormal proliferations were observed; (i) epithelial hyperplasia with cytologic atypia in the oviduct (with endometrioid architectural and cytologic features), (ii) leiomyosarcomatous proliferation in the myometrium, and (iii) squamous carcinoma in situ in the cervix with extensive papillary architecture. SV40 was expressed in these tumor sites. The oviduct demonstrated weak and spotty p53 expression. The expression of Ogp1 and Pax8 (a marker of the salpingeal secretory cells) in the oviduct epithelium showed patchy pattern. Ogp1 expression was not associated with cellular atypia, while Pax8 was expressed in normal cells. Expression of DNA replication licensing factor, Mini-chromosome maintenance protein 7(MCM7), was also mixed. MCM7 expression was higher in neoplastic cells, but atypical cells had less MCM7 expression. A serous marker, WT1, and a Müllerian-specific epithelial marker, Pax2, showed lower expressions in Ogp1-SV40 transgenic mouse compared to wild type control mouse. WT1 and Pax2 expression were uniform. In ovarian surface epithelium, there was no malignant transformation and no different expression of these markers in control and Ogp1-SV40 mice. In conclusion, this study demonstrated that Ogp1-SV40 transgenic mouse develops abnormal oviductal, myometrial, and cervical proliferations with no involvement of ovaries. Weak and spotty p53 expression in the oviduct indicates that p53 pre-dysfunction status in non-malignant precursor lesions. These results indicate that Ogp1-SV40 transgenic mouse is a useful model for studies involving fallopian and low genital tract carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3276.

Abstract Human Sulfatase1 enzyme (HSulf-1) is down-regulated in a majority of ovarian cancer cell lines and primary tumors, but the functional consequence of this downregulation remains unclear. Here, we showed that down-regulation of HSulf-1 by RNA interference in OV202 ovarian cancer cells resulted in an elevated level of phosphorylated AKT. Conversely, over-expression of HSulf-1 in SKOV3 ovarian cancer cells led to decreased phosphorylation of AKT. When cells were treated with CDDP, OV202 cells with knockdown of HSulf-1 displayed more resistance to drug-induced apoptosis, with IC50 value 3-fold higher than cells with non-targeting shRNA, while SKOV3 cells with overexpression of HSulf-1 were more sensitive to drug-induced apoptosis compared to the control cells. Moreover, knockdown of HSulf-1 also endowed ovarian cancer cells with increased resistance to detachment-induced apoptosis (anoikis). Cell proliferation assays revealed that knockdown of HSulf-1 significantly accelerated cell proliferation of ovarian cancer cells in vitro. Importantly, when cells were injected subcutaneously, 7 of 8 (87.5%) nude mice developed tumors with HSulf-1 knockdown cells within 2 weeks. However, none of the mice injected with nontargeted control transduced cells (control group) developed tumors. Four weeks after injection, 8 of 8 (100%) nude mice developed tumors with HSulf-1 down-regulated cells, while only 2 of 5 (40%) mice had tumors in the control group. The average tumor weight was 4.2±2.7g per mice in HSulf-1 knockdown group, significantly higher than 0.6±0.3g per mice in the control group. Collectively, these results strongly suggest that down-regulation of HSulf-1 promotes tumorigenicity of ovarian cancer possibly due to elevated activation of AKT pathway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4998.

Abstract Mini-Chromosome Maintenance protein 7 (MCM7) is involved in replicative licensing and synthesis of DNA. It is previously identified as over-expressed gene in high-grade serous carcinomas compared to serous borderline tumors of the ovary in cDNA microarray studies. In this study, we sought to validate MCM7 expression in ovarian tumors by applying immunohistochemical analysis on tissue microarrays consisting of 445 ovarian tumors. MCM7 expression was quantified as MCM7 labeling index by determining the tumor nuclei positive for MCM7 staining as a percentage of total tumor nuclei. The labeling index scores were independently generated by two methods: PATH score provided by a manual review of each samples by a pathologist and ACIS score provided by Automated Cell Imaging System. Analyses of both scores indicated high degree of concordance in score distribution between PATH and ACIS. MCM7 expression is significantly higher in high-grade serous carcinomas compared to serous borderline tumors, confirming our previous cDNA microarray studies. In both PATH and ACIS analysis, MCM7 expression is also significantly higher in high-grade serous carcinomas compared to other histology subtypes of ovarian cancer. Finally, to identify the clinical significance of MCM7 expression, we determined the association between MCM7 expression and disease-free survival. MCM7 labeling index was dichotomized into two groups (high and low) using median labeling index as cut-off. For both PATH or ACIS-derived scores, univariate analyses indicate significant association of high MCM7 labeling index with better disease-free survival in high-grade serous carcinomas. These results suggest the clinical significance of MCM7 expression in high-grade serous carcinomas of the ovary and the need for further evaluation of MCM7 as a potential theranostic biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4637.

Abstract HSulf-1 modulates the sulfation states of heparan sulfate proteoglycans critical for heparin binding growth factor signaling. In the present study, we show that HSulf-1 is transcriptionally deregulated under hypoxia in breast cancer cell lines. Knockdown of HIF-1α rescued HSulf-1 downregulation imposed by hypoxia, both at the RNA and protein levels. Chromatin immunoprecipitation with HIF-1α and HIF-2α antibodies confirmed recruitment of HIF-α proteins to the two functional hypoxia-responsive elements on the native HSulf-1 promoter. HSulf-1 depletion in breast cancer cells resulted in an increased and sustained bFGF2 (basic fibroblast growth factor) signaling and promoted cell migration and invasion under hypoxic conditions. In addition, FGFR2 (fibroblast growth factor receptor 2) depletion in HSulf-1–silenced breast cancer cells attenuated hypoxia-mediated cell invasion. Immunohistochemical analysis of 53 invasive ductal carcinomas and their autologous metastatic lesions revealed an inverse correlation for the expression of HSulf-1 to CAIX in both the primary tumors (P ≥ 0.0198) and metastatic lesions (P ≥ 0.0067), respectively, by χ2 test. Finally, HSulf-1 expression levels in breast tumors by RNA in situ hybridization showed that high HSulf-1 expression is associated with increased disease-free and overall survival (P ≥ 0.03 and P ≥ 0.0001, respectively). Collectively, these results reveal an important link between loss of HSulf-1 under hypoxic microenvironment and increased growth factor signaling, cell migration, and invasion. Cancer Res; 71(6); 2152–61. ©2011 AACR.

AbstractPurpose: 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 HtrA1 knocked down clones.Results: 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).Conclusions: Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors. Clin Cancer Res; 17(3); 427–36. ©2010 AACR.

Abstract 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.

Abstract 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.