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.ovarian cancer ͉ Ras ͉ transformation ͉ tumor microenvironment

ABSTRACT

(8,17). Analysis of blood samples from 30 ovarian cancer patients and an equal number of age-matched normal subjects shows significantly increased concentrations of plasma thiobarbituric acid-reactive substances and conjugated dienes in the patient specimens, indicating increased oxidative stress in ovarian cancer (18). However, the same study also shows low levels of SOD, catalase, vitamin C, and vitamin E in the plasma of the patient blood samples, possibly due to their increased utilization in scavenging lipid peroxides as well as their sequestration by tumor cells (18). This is in contrast with the increased serum Mn-SOD observed in another study (17). Decreased Mn-SOD activity and expression have also been reported in certain colorectal carcinomas and pancreatic cancer cells (19,20). These apparent conflicting observations are likely because of the different assays and various cell types used in these studies. Thus, it is important to clarify this issue by further examining the expression levels of SOD in a large number of primary human cancer tissues in comparison with normal tissues. Tissue microarray analysis provides an effective tool for such analyses. This new technique was used in the present study to compare the expression of Mn-SOD and Cu,Zn-SOD in primary human ovarian cancer tissues, benign ovarian lesions, and normal tissues. Although the biochemical activity of Mn-SOD in catalyzing the conversion of O 2 Ϫ to H 2 O 2 in the mitochondria has been well characterized, the potential role of Mn-SOD in cancer development remains to be defined. Because the Mn-SOD level seems decreased in certain cancer cells and forced expression of Mn-SOD appears to suppress malignant phenotypes in certain experimental models, this molecule has been con-* This study was supported in part by Grants CA85563, CA100428, and CA109041 (to P. H.) from the NCI, National Institutes of Health, and RSG-04-028-1-CCE (to J. L.) from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 These authors contributed equally to this work.

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.