Gong Yang

A genetically defined model for human ovarian cancer.

Disruptions of the p53, retinoblastoma (Rb), and RAS signaling pathways and activation of human telomerase reverse transcriptase (hTERT) are common in human ovarian cancer; however, their precise role in ovarian cancer development is not clear. We thus introduced the catalytic subunit of hTERT, the SV40 early genomic region, and the oncogenic alleles of human HRAS or KRAS into human ovarian surface epithelial cells and examined the phenotype and gene expression profile of those cells. Disruption of p53 and Rb pathway by SV40 early genomic region and hTERT immortalized but did not transform the cells. Introduction of HRASV12 or KRASV12 into the immortalized cells, however, allowed them to form s.c. tumors after injection into immunocompromised mice. Peritoneal injection of the transformed cells produced undifferentiated carcinoma or malignant mixed Mullerian tumor and developed ascites; the tumor cells are focally positive for CA125 and mesothelin. Gene expression profile analysis of transformed cells revealed elevated expression of several cytokines, including interleukin (IL)-1β, IL-6, and IL-8, that are up-regulated by the nuclear factor-κB pathway, which is known to contribute to the tumor growth of naturally ovarian cancer cells. Incubation with antibodies to IL-1β or IL-8 led to apoptosis in the ras-transformed cells and ovarian cancer cells but not in immortalized cells that had not been transformed. Thus, the transformed human ovarian surface epithelial cells recapitulated many features of natural ovarian cancer including a subtype of ovarian cancer histology, formation of ascites, CA125 expression, and nuclear factor-κB-mediated cytokine activation. These cells provide a novel model system to study human ovarian cancer.

The chemokine growth-regulated oncogene 1 (Gro-1) links RAS signaling to the senescence of stromal fibroblasts and ovarian tumorigenesis

Epithelial–stromal interactions play a critical role in tumor initiation and progression; cancer-associated stroma, but not normal stroma, is known to be tumor-promoting. However, the molecular signal used by epithelial cancer cells to reprogram normal stroma to a tumorigenic stroma is not known. Here, we present evidence to suggest that the chemokine growth-regulated oncogene 1 (Gro-1) may be one such signaling molecule. We showed that the expression of Gro-1 is activated by RAS and is vital for cell survival and the malignant transformation of ovarian epithelial cells. Surprisingly, we found that Gro-1 is a potent inducer of senescence in stromal fibroblasts and that this effect depends on functional p53. Senescent fibroblasts induced by Gro-1 can promote tumor growth whereas abrogation of senescence through immortalization results in loss of such tumor promoting activity. We also demonstrated that stromal fibroblasts adjacent to epithelial cancer cells are senescent in human ovarian cancer specimens and in heterografts from RAS-transformed human ovarian epithelial cells and ovarian cancer cells. Moreover, Gro-1 was expressed at significantly higher amounts in ovarian cancer than in normal tissues and was higher in serum samples from women with ovarian cancer than in serum from women without ovarian cancer. These findings provide strong evidence that RAS-induced Gro-1 can reprogram the stromal microenvironment through the induction of senescence of fibroblasts and thus can promote tumorigenesis. Therefore, Gro-1 may be a therapeutic target as well as a diagnostic marker in ovarian cancer.

Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: Role in cell proliferation and response to oxidative stress

Superoxide dismutases (SODs) are important antioxidant enzymes responsible for the elimination of superoxide radical (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document}). The manganese-containing SOD (Mn-SOD) has been suggested to have tumor suppressor function and is located in the mitochondria where the majority of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} is generated during respiration. Although increased reactive oxygen species (ROS) in cancer cells has long been recognized, the expression of Mn-SOD in cancer and its role in cancer development remain elusive. The present study used a human tissue microarray to analyze Mn-SOD expression in primary ovarian cancer tissues, benign ovarian lesions, and normal ovary epithelium. Significantly higher levels of Mn-SOD protein expression were detected in the malignant tissues compared with normal tissues (p < 0.05). In experimental systems, suppression of Mn-SOD expression by small interfering RNA caused a 70% increase of superoxide in ovarian cancer cells, leading to stimulation of cell proliferation in vitro and more aggressive tumor growth in vivo. Furthermore, stimulation of mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} production induced an increase of Mn-SOD expression. Our findings suggest that the increase in Mn-SOD expression in ovarian cancer is a cellular response to intrinsic ROS stress and that scavenging of superoxide by SOD may alleviate the ROS stress and thus reduce the simulating effect of ROS on cell growth.

HER2 signaling modulates the equilibrium between pro- and antiangiogenic factors via distinct pathways: Implications for HER2-targeted antibody therapy

We determined the impact of HER2 signaling on two proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and on an antiangiogenic factor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 and T-47D breast cancer cells that endogenously express low levels of HER2 resulted in elevated expression of VEGF and IL-8 and decreased expression of TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody (trastuzumab, or Herceptin®) or a retrovirus-mediated small interfering RNA against HER2 (siHER2) decreased VEGF and IL-8 expression, but increased TSP-1 expression in BT474 breast cancer cells that express high levels of HER2. These in vitro results were further evaluated by treatment of BT474 xenografts in immunosuppressed mice with trastuzumab. Trastuzumab inhibited growth of BT474 xenografts and decreased microvascular density associated with downregulation of VEGF and IL-8 and with upregulation of TSP-1 expression. Inhibiting the PI3K-AKT pathway decreased VEGF and IL-8 expression. AKT1 overexpession increased VEGF and IL-8 expression, but did not increase TSP-1 expression. A p38 kinase inhibitor, SB203580, instead blocked TSP-1 expression and a p38 activator, MKK6, increased TSP-1 expression. Trastuzumab stimulated sustained p38 activation and SB203580 attenuated the TSP-1 upregulation induced by trastuzumab. HER2 signaling therefore influences the equilibrium between pro- and antiangiogenic factors via distinct signaling pathways. Trastuzumab inhibits angiogenesis and tumor growth, at least in part, through activation of the HER2-p38-TSP-1 pathway and inhibition of the HER2-PI3K-AKT-VEGF/IL-8 pathway.

Activation of Antioxidant Pathways in Ras-Mediated Oncogenic Transformation of Human Surface Ovarian Epithelial Cells Revealed by Functional Proteomics and Mass Spectrometry

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic rasV12 allele. Of ∼2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

The role of constitutively active signal transducer and activator of transcription 3 in ovarian tumorigenesis and prognosis

Signal transducer and activator of transcription 3 (Stat3), which is a latent transcription factor that participates in the transcriptional activation of apoptosis and cell cycle progression, has been implicated as an oncogene in several neoplastic diseases. However, the specific role of Stat3 in ovarian carcinogenesis remains poorly understood. The objectives of the current study were to examine the effect of Stat3 activation on the phenotypic transformation of an immortalized, nontumorigenic ovarian epithelial cell line and to evaluate the expression of tyrosine‐activated Stat3 (pStat3) in tissue microarrays from 303 ovarian carcinomas to determine its prognostic relevance and to correlate its expression with several upstream oncogenes of Stat3 and with the oncogenes involved in apoptosis and proliferation.

CXCR2 Promotes Ovarian Cancer Growth through Dysregulated Cell Cycle, Diminished Apoptosis, and Enhanced Angiogenesis

Purpose: Chemokine receptor CXCR2 is associated with malignancy in several cancer models; however, the mechanisms involved in CXCR2-mediated tumor growth remain elusive. Here, we investigated the role of CXCR2 in human ovarian cancer. Experimental Design: CXCR2 expression was silenced by stable small hairpin RNA in ovarian cancer cell lines T29Gro-1, T29H, and SKOV3. Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, flow cytometry, electrophoretic mobility shift assay, and mouse assay were used to detect CXCR2, interleukin-8, Gro-1, cell cycle, apoptosis, DNA binding of NF-κB, and tumor growth. Immunohistochemical staining of CXCR2 was done in 240 high-grade serous ovarian carcinoma samples. Results: Knockdown of CXCR2 expression by small hairpin RNA reduced tumorigenesis of ovarian cancer cells in nude mice. CXCR2 promoted cell cycle progression by modulating cell cycle regulatory proteins, including p21 (waf1/cip1), cyclin D1, CDK6, CDK4, cyclin A, and cyclin B1. CXCR2 inhibited cellular apoptosis by suppressing phosphorylated p53, Puma, and Bcl-xS; suppressing poly(ADP-ribose) polymerase cleavage; and activating Bcl-xL and Bcl-2. CXCR2 stimulated angiogenesis by increasing levels of vascular endothelial growth factor and decreasing levels of thrombospondin-1, a process likely involving mitogen-activated protein kinase, and NF-κB. Overexpression of CXCR2 in high-grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival (P < 0.001) and of early relapse (P = 0.003) in the univariate analysis. Conclusions: Our data provide strong evidence that CXCR2 regulates the cell cycle, apoptosis, and angiogenesis through multiple signaling pathways, including mitogen-activated protein kinase and NF-κB, in ovarian cancer. CXCR2 thus has potential as a therapeutic target and for use in ovarian cancer diagnosis and prognosis. Clin Cancer Res; 16(15); 3875–86. ©2010 AACR.

IFITM1 plays an essential role in the antiproliferative action of interferon- γ

Interferon-γ (IFN-γ) is a pleiotropic cytokine involved in antiproliferative and anti-virus responses, immune surveillance and tumor suppression. These biological responses to IFN-γ are mainly mediated by the regulation of gene expression. It has been reported that growth-inhibitory role of IFN-γ is dependent on activation of signal transducers and activators of transcription 1 (STAT1); however, the molecular basis downstream of STAT1 remains unclear. Here, we report that an IFN-γ-induced gene, interferon-induced transmembrane protein 1 (IFITM1), plays a key role in the antiproliferative action of IFN-γ. Overexpression of IFITM1 negatively regulated cell growth, whereas suppression of IFITM1 blocked the antiproliferative effect of IFN-γ, accelerated the cell growth rate and conferred tumorigenicity to a non-malignant hepatocyte in nude mice. Further, IFITM1 could inhibit the activity of extracellular signal-regulated kinase, enhance the transcriptional activity of p53 and stabilize the p53 protein by inhibiting p53 phosphorylation on Thr55. Suppression of p53 reduced the growth-inhibitory capacity of both IFITM1 and IFN-γ. Therefore, these findings indicated that the antiproliferative action of IFN-γ requires the induction of IFITM1, and provided a crosstalk between two well-known signaling mediators, STAT1 and p53, both of which play critical roles in tumor suppression.

Cyclin E expression is correlated with tumor progression and predicts a poor prognosis in patients with ovarian carcinoma

Cyclins, cyclin dependent kinases (cdks), and their inhibitors act in combination to regulate progression through the cell cycle and often are dysregulated in carcinoma. The authors hypothesized that cyclin E plays an important role in ovarian carcinogenesis and that its overexpression may be an indicator of a poor prognosis.

Stanniocalcin 1 and Ovarian Tumorigenesis

BACKGROUND Stanniocalcin 1 (STC1) is a secreted glycoprotein hormone. High expression of STC1 has been associated with several cancers including ovarian cancer, but its role in the development of ovarian cancer is not clear. METHODS We used five human ovarian epithelial cancer cell lines (OVCA420, OVCA432, OVCA433, SKOV3, and HEY), immortalized human ovarian surface epithelial cells (T29 and T80), ovarian cancer tissues from 342 patients, serum from 73 ovarian cancer patients and from58 control subjects, and 116 mice, with six or eight per group. Protein expression was assessed. Cells overexpressing STC1 protein were generated by ectopic expression of human STC1 cDNA. STC1 expression was silenced by using small interfering RNA against STC1. Cell proliferation, migration, colony formation, and apoptosis were assessed. Xenograft tumor growth in mice was studied. Neutralizing anti-STC1 antibody was used to inhibit STC1 function. All statistical tests were two-sided. RESULTS STC1 protein expression was higher in all human ovarian cancer cell lines examined than in immortalized human ovarian epithelial cell lines, higher in ovarian cancer tissue than in normal ovarian tissue (P < .001), and higher in serum from ovarian cancer patients than from control subjects (P = .021). Ovarian cancer cells with STC1 overexpression, compared with corresponding control cells, had increased cell proliferation, migration, and colony formation in cell culture and increased growth of xenograft tumors in mice. These activities in normal or malignant ovarian cells with STC1 overexpression, compared with control cells, were also accompanied by increased expression of cell cycle regulatory proteins and antiapoptotic proteins but decreased cleavage of several caspases. Within 24 hours of treatment, apoptosis in cultures of HEY ovarian cancer cells treated with neutralizing anti-STC1 monoclonal antibody was higher (17.3% apoptotic cells) than that in cultures treated with mouse IgG control cells (4.4%) (12.9% difference, 95% confidence interval = 11.6% to 14.2%). CONCLUSIONS STC1 protein may be involved in ovarian tumorigenesis.