E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Increased B-lymphopoiesis by interleukin 7 induces bone loss in mice with intact ovarian function: Similarity to estrogen deficiency

Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss due to stimulated bone resorption by osteoclasts. During our investigations of the pathogenesis of bone loss in estrogen deficiency, we found that OVX selectively stimulates B-lymphopoiesis which results in marked accumulation of B220-positive pre-B cells in mouse bone marrow. To examine the possible correlation between stimulated B-lymphopoiesis and bone loss, 8-week-old female mice were treated with interleukin (IL) 7, which stimulates B-lymphopoiesis in bone marrow. We also examined bone mass in IL-7 receptor-knockout mice that exhibit marked suppression of B-lymphopoiesis in the bone marrow. The increased B-lymphopoiesis induced by IL-7 administration resulted in marked bone loss by stimulation of osteoclastic bone resorption in mice with intact ovarian function. The changes in both B-lymphopoiesis and bone mass in IL-7-treated female mice were similar to those in age-matched OVX mice. In contrast, the trabecular bone volume of the femur was greatly increased in both female and male IL-7 receptor-knockout mice when compared with the respective wild-type and heterozygous littermates. These results show that the perturbation of B-lymphopoiesis in the bone marrow is closely linked to the change in bone mass. We propose here that the increased B-lymphopoiesis due to estrogen deficiency is involved in the mechanism of stimulated bone resorption.

Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1α and its dual block by ovarian steroids

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1α (IL-1α) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1α and repress MMP-1 production by IL-1α-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1α and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1α is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1α, thereby exerting an effective control via a “double-block” mechanism.

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

Luteinizing hormone induction of ovarian tumors: Oligogenic differences between mouse strains dictates tumor disposition

The use of fertility drugs has continued to grow since their introduction in the 1960s. Accompanying this increase has been the speculation that repetitive use of these drugs can cause ovarian tumors or cancer. We recently reported that transgenic mice with chronically elevated luteinizing hormone (LH), an analog of which is commonly used in fertility regimens, develop granulosa cell (GC) tumors. In this report we show that LH induction of these tumors is highly dependent on genetic background. In CF-1 mice, chronically elevated LH invariably causes GC tumors by 5 months of age. However, in hybrid mice generated by crossing CF-1 males with C57BL/6, SJL, or CD-1 females, elevated levels of this same hormone cause a completely different phenotype resembling a luteoma of pregnancy. We also show that three genes likely control these alternative hormonal responses. This clinical correlate of elevated LH reveals remarkably distinct, strain-dependent, ovarian phenotypes. In addition, these results support the rare incidence of GC tumors in the human population, and suggest that the ability of certain fertility drugs to cause ovarian tumors may depend on an individual's genetic predisposition.

Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo

Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protocols for MIS, Chinese hamster ovary cells transfected with the human MIS gene were seeded onto biodegradable polymers of polyglycolic acid fibers and secretion of MIS confirmed. The polymer-cell graft was implanted into the right ovarian pedicle of severe combined immunodeficient mice. Serum MIS in the mice rose to supraphysiologic levels over time. One week after implantation of the polymer-cell graft, IGROV-1 human tumors were implanted under the renal capsule of the left kidney. Growth of the IGROV-1 tumors was significantly inhibited in the animals with a polymer-cell graft of MIS-producing cells, compared with controls. This novel MIS delivery system could have broader applications for other inhibitory agents not amenable to efficient purification and provides in vivo evidence for a role of MIS in the treatment of ovarian cancer.

A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells

Ovarian carcinomas are thought to arise from cells of the ovarian surface epithelium by mechanisms that are poorly understood. Molecules associated with neoplasia are potentially immunogenic, but few ovarian tumor antigens have been identified. Because ovarian carcinomas can elicit humoral responses in patients, we searched for novel tumor antigens by immunoscreening a cDNA expression library with ovarian cancer patient serum. Seven clones corresponding to the homeobox gene HOXB7 were isolated. ELISAs using purified recombinant HOXB7 protein revealed significant serologic reactivity to HOXB7 in 13 of 39 ovarian cancer patients and in only one of 29 healthy women (P < 0.0001). Ovarian carcinomas were found to express HOXB7 at markedly higher levels than normal ovarian surface epithelium, suggesting that immunogenicity of HOXB7 in patients could be associated with its elevated expression in ovarian carcinomas. Overexpression of HOXB7 in immortalized normal ovarian surface epithelial cells dramatically enhanced cellular proliferation. Furthermore, HOXB7 overexpression increased intracellular accumulation and secretion of basic fibroblast growth factor, a potent angiogenic and mitogenic factor. These results reveal HOXB7 as a tumor antigen whose up-regulated expression could play a significant role in promoting growth and development of ovarian carcinomas.

Two genes abrogate the inhibition of murine hepatocarcinogenesis by ovarian hormones.

Hormonal and genetic factors strongly influence the susceptibility of inbred mice to hepatocarcinogenesis. Female C57BR/cdJ (BR) mice are extremely susceptible to liver tumor induction relative to other strains because they are genetically insensitive to the inhibition of hepatocarcinogenesis by ovarian hormones. To determine the genetic basis for the sensitivity of BR mice relative to resistant C57BL/6J (B6) mice, we treated 12-day-old B6BRF1 x B6 and B6BRF1 x B6BRF1 (F2) animals with N,N-diethylnitrosamine (0.1 micromol/g of body weight) and enumerated liver tumors at 32 weeks of age in males and at 50 weeks in females. Genomic DNA samples from backcross and F2 mice were analyzed for 70 informative simple sequence length polymorphism markers. Genetic markers on chromosome 17 (D17Mit21) and chromosome 1 (D1Mit33) cosegregated with high tumor multiplicity in both sexes. Together, these loci [designated Hcf1 and Hcf2 (Hepatocarcinogenesis in females), respectively] account for virtually all of the difference in sensitivity between BR and B6 mice. The Hcf1 locus accounts for a majority of the higher susceptibility of BR mice of both sexes. Backcross female mice heterozygous at both loci (33 +/- 23 tumors per mouse) and at Hcf1 only (17 +/- 18) were 15- and 8-fold more sensitive, respectively, than mice homozygous for the B6 alleles at Hcf1 and Hcf2 (2.2 +/- 3.9). In backcross male mice, the double heterozygotes (35 +/- 22) and Hcf1 heterozygotes (28 +/- 12) were 5.4- and 4.3-fold more sensitive than mice homozygous for B6 alleles at both loci (6.5 +/- 5.4).

Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers.

Monoclonal antibody MAb K1 recognizes a 40-kDa glycoprotein present on the surface of mesothelial cells, mesotheliomas, and ovarian cancers. We have used MAb K1 to isolate a 2138-bp cDNA that encodes this antigen. The cDNA has an 1884-bp open reading frame encoding a 69-kDa protein. When the cDNA was transfected into COS and NIH 3T3 cells, the antigen was found on the cell surface and could be released by treatment with phosphatidylinositol-specific phospholipase C. The 69-kDa precursor is processed to the 40-kDa form. The protein has been named mesothelin because it is made by mesothelial cells. Mesothelin may play a role in cellular adhesion.

Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.

Human neoplasms elicit multiple specific immune responses in the autologous host.

Expression of cDNA libraries from human melanoma, renal cancer, astrocytoma, and Hodgkin disease in Escherichia coli and screening for clones reactive with high-titer IgG antibodies in autologous patient serum lead to the discovery of at least four antigens with a restricted expression pattern in each tumor. Besides antigens known to elicit T-cell responses, such as MAGE-1 and tyrosinase, numerous additional antigens that were overexpressed or specifically expressed in tumors of the same type were identified. Sequence analyses suggest that many of these molecules, besides being the target of a specific immune response, might be of relevance for tumor growth. Antibodies to a given antigen were usually confined to patients with the same tumor type. The unexpected frequency of human tumor antigens, which can be readily defined at the molecular level by the serological analysis of autologous tumor cDNA expression cloning, indicates that human neoplasms elicit multiple specific immune responses in the autologous host and provides diagnostic and therapeutic approaches to human cancer.

The bacterial colicin active against tumor cells in vitro and in vivo is verotoxin 1.

We have identified verotoxin 1 (VT1) as the active component within an antineoplastic bacteriocin preparation from Escherichia coli HSC10 studied over two decades. Recombinant VT1 can simulate the toxicity of anticancer proteins (ACP), and the antineoplastic activity of ACP (and VT1) was abrogated by treatment with anti-VT1 antibody. Similarly, VT1 mimics the protective effect of ACP in a murine metastatic fibrosarcoma model. Prior immunization with VT1 B subunit prevents the effect of VT1 or ACP in this model. The activity of ACP against a variety of human ovarian cell lines was mimicked by VT1, and multidrug-resistant variants were significantly hypersensitive. Primary ovarian tumors and metastases contain elevated levels of globotriaosylceramide compared with normal ovaries, and overlay of frozen tumor sections showed selective VT binding to tumor tissue and the lumen of invading blood vessels. Our contention that VT1 could provide an additional approach to the management of certain human neoplasms is discussed.