Human endometrial stromal cell-trophoblast interactions: mutual stimulation of chemotactic migration and promigratory roles of cell surface molecules CD82 and CEACAM1.

We have previously shown that the presence of trophoblast cells enhances invasiveness of decidualizing human endometrial stromal cells. The metastasis suppressor CD82, which has antimigratory function in tumor cells, is up-regulated in decidualizing endometrial stromal cells. CEACAM1 is expressed in trophoblast cells at the invasion front in early placenta and is considered proinvasive. Here, we investigate the role of CD82 and CEACAM1 in cocultures of the endometrial stromal cell line T-HESC and AC-1M88 trophoblast cells. In transwell migration assays, chemotaxis of AC-1M88 cells was stimulated by coplated T-HESC in the lower compartment or by the combination of heparin-binding EGF-like growth factor (HB-EGF), interleukin-1 beta (IL-1beta), and leukemia inhibitory factor (LIF), local factors present at the time of implantation. In an implantation model of AC-1M88 trophoblast spheroids on a monolayer of T-HESC, spheroid expansion was enhanced in the presence of HB-EGF/IL-1beta/LIF. Silencing of CEACAM1 in AC-1M88 blunted this response. Chemotactic migration of T-HESC was stimulated by trophoblast secretions or HB-EGF/IL-1beta/LIF. These responses were suppressed by CD82 depletion in T-HESC. Proteome profiling revealed the presence of platelet-derived growth factor (PDGF)-AA in trophoblast supernatant. Chemotaxis of T-HESC toward PDGF-AA was significantly inhibited by CD82 silencing. Neutralization of PDGF-AA in AC-1M88 conditioned media reduced the chemotactic effect on T-HESC. In summary, we demonstrate a mutual stimulation of chemotactic migration between trophoblast and endometrial stromal cells and promigratory roles for the cell surface molecules CEACAM1 and CD82, which may serve to support tissue remodeling at the implantation site.

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors.

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site.

Expansion of human trophoblastic spheroids is promoted by decidualized endometrial stromal cells and enhanced by heparin-binding epidermal growth factor-like growth factor and interleukin-1 ß.

Successful pregnancy in humans depends on deep invasion of the maternal decidua by extravillous trophoblast cells (EVTs), a process regulated by autocrine and paracrine signals in the decidual-trophoblast microenvironment. Here we examined whether trophoblast invasion is affected by decidual differentiation of endometrial stromal cells (ESC) and modulated locally by cytokines and growth factors. Trophoblast spheroids were generated from the EVT-derived cell line AC-1M88 and placed onto monolayers of either undifferentiated or decidualized ESC, or directly onto tissue culture surface. Co-cultures were treated with epidermal growth factor (EGF), hepatocyte growth factor, heparin-binding EGF-like growth factor (HB-EGF), interleukin-1ß (IL-1ß) and leukaemia inhibitory factor (LIF). Expansion of spheroids over 2-3 days was significantly enhanced by a monolayer of undifferentiated ESC compared with tissue culture surface and further increased if ESC had been decidualized. HB-EGF and IL-1ß, alone or in combination with LIF, stimulated spheroid expansion but only on undifferentiated ESC. CEACAM1, an adhesion molecule implicated in trophoblast invasion, was up-regulated in AC-1M88 cells by conditioned medium from decidualized ESC, and by HB-EGF, IL-1ß and LIF in combination. Treatment of ESC with HB-EGF or IL-1ß increased the level of the tetraspanin CD82, a metastasis suppressor found in decidual cells at the implantation site. We suggest that decidualized ESC support trophoblast invasion by paracrine signals that may include HB-EGF, IL-1ß and LIF.

Osteopontin (OPN) expression in thyroid carcinoma.

BACKGROUND: Osteopontin (OPN) and its interacting partner CEA-cell adhesion molecule (CEACAM1) mediate similar biological functions and have been expressed in several types of cancer. Here we investigated the prognostic significance of OPN in thyroid tumours and correlated our findings with the expression of CEACAM1. MATERIALS AND METHODS: 297 human thyroid samples were collected in a tissue microarray and as fresh frozen samples to perform immunohistochemistry and western blotting for OPN and to compare these data with our previously published data on CEACAM1 expression. RESULTS: Nearly all normal samples were negative for OPN. Some thyroid adenomas were weakly OPN positive whereas many carcinomas were strongly positive. In contrast to CEACAM1, which was preferentially expressed in metastatic papillary carcinomas, no associations were found between OPN expression and patient age, gender and tumour size. CONCLUSION: These results may have future implications for the diagnosis and management of patients with thyroid cancers.

Transforming growth factor-{beta} coordinately induces suppressor of cytokine signaling 3 and leukemia inhibitory factor to suppress osteoclast apoptosis.

Local release of TGF-beta during times of high bone turnover leads to elevated levels within the bone microenvironment, and we have shown that TGF-beta suppresses osteoclast apoptosis. Therefore, understanding the influences of TGF-beta on bone resorbing osteoclasts is critical to the design of therapies to reduce excess bone loss. Here we investigated the mechanisms by which TGF-beta sustains suppression of osteoclast apoptosis. We found TGF-beta rapidly increased leukemia inhibitory factor (LIF) expression and secretion by phosphorylated mothers against decapentaplegic-dependent and -independent signaling pathways. TGF-beta also induced suppressor of cytokine signaling 3 (SOCS3) expression, which was required for TGF-beta or LIF to promote osteoclast survival by. Blocking LIF or SOCS3 blocked TGF-beta promotion of osteoclast survival, confirming that LIF and SOCS3 expression are necessary for TGF-beta-mediated suppression of osteoclast apoptosis. Investigation of the mechanisms by which LIF promotes osteoclast survival revealed that LIF-induced expression of Bcl-X(L) and repressed Bcl-2 interacting domain expression by activating MAPK kinase, AKT, and nuclear factor-kappaB pathways. Suppression of Janus kinase/signal transducer and activator of transcription signaling further increased Bcl-X(L) expression and enhanced osteoclast survival, supporting that this pathway is not involved in prosurvival effects of TGF-beta and LIF. These data show that TGF-beta coordinately induces LIF and SOCS3 to promote prosurvival signaling. This alters the ratio of prosurvival Bcl2 family member Bcl-X(L) to proapoptotic family member Bcl-2 interacting domain, leading to prolonged osteoclast survival.

DC-SIGN and SRCL bind glycans of carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1): recombinant human glycan-binding receptors as analytical tools.

Members of the family of carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) belonging to the immunoglobulin (Ig) superfamily are expressed in a variety of normal and malignant human tissues. As components of the cell membrane, these glycoproteins can make contact with adjacent cells. CEACAM1 and CEACAM5 (CEA) express Lewis(x) (Le(x)) structures. As shown by mass spectrometry in conjunction with enzymatic digestion, CEACAM1 contains at least seven Le(x) residues. Fucosyltransferase IX is the main fucosyltransferase responsible for attachment of terminal fucose, the key feature of the Le(x) structure, to CEA and CEACAM1. The Le(x) residues of both, CEACAM1 and CEA, interact with the human Le(x)-binding glycan receptors DC-SIGN and SRCL. Since subpopulations of human macrophages express DC-SIGN or SRCL, Le(x)-carrying CEACAMs may modulate the immune response in normal tissues such as the human placenta or in malignant tumours, for example in colorectal, pancreatic or lung carcinomas.

Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b.

BACKGROUND: Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC) decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. METHODS: Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. RESULTS: St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82). Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. CONCLUSION: St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural progesterone. St-T1b cells therefore serve as a useful model for primary ESC.

CEACAM1 expression in pancreatic endocrine tumors.

The aim of this study was to examine the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in pancreatic endocrine tumors (PETs) and to correlate it with clinicopathologic parameters. Sixty-nine PETs were examined for tumor size, necrosis, local peripancreatic invasion and lymphovascular invasion, lymph node, and liver metastasis. The mitotic count, expressed per 10 high-power fields (HPF) and MIB1 index were assessed and tumors were classified according to the World Health Organization classification. A tissue microarray was constructed and stained with an extensive panel of endocrine markers and CEACAM1. Twenty-nine tumors were from males and 40 from females, age range: 23 to 80 years (mean 52.4 y), tumor size ranged from 0.8 to 11 cm (mean 3.5 cm), 8 patients had multiple endocrine neoplasia 1 syndrome, and 1 had von Hippel-Lindau disease. Twenty tumors demonstrated local invasion, 32 had lymphovascular invasion, 16 had lymph node metastasis, and 10 had liver metastasis. CEACAM1 was positive in 47 cases and negative in 22 cases (31.9%). Ninety percent of the CEACAM1-negative cases had a MIB1 index 2% (P=0.02). 86.4% of the CEACAM1-negative PETs had a mitotic count 2/10 HPF. In addition, 80% of tumors >or=2 cm in diameter were CEACAM positive (P<0.05). CEACAM1-positive tumors were more frequently insulin negative (9 of 10 cases) (P=0.005) and vasoactive intestinal peptide-positive PETs were all CEACAM1 immunopositive (7 of 7 cases) (P=0.005). Benign tumors and PETs of uncertain malignant behavior were more frequently CEACAM1-negative and low-grade malignant cases were CEACAM1 positive (27 of 29 cases) (P=0.001). In addition, CEACAM1-positive tumors were statistically correlated with cytokeratin 19-positive tumors (P<0.05). PETs showing CEACAM1 positivity have a statistically significant correlation with several of the pathologic parameters of aggressive behavior and its overexpression is seen in those cases with increased invasiveness.

Osteopontin stimulates invasion of NCI-h295 cells but is not associated with survival in adrenocortical carcinoma.

Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin alphavbeta3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin alphavbeta3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI-h295 cells (>six-fold increase, p < 0.001). Transfection with integrin alphavbeta3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti-integrin beta3 antibody, indicating a functional relationship of OPN and integrin alphavbeta3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan-Meier analysis including 111 patients with primary tumours graded for OPN staining and follow-up data available. In conclusion, our in vitro data indicate that OPN and integrin alphavbeta3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue.

Correlations between reduced expression of the metastasis suppressor gene KAI-1 and accumulation of p53 in uterine carcinomas and sarcomas.

Kangai (KAI)-1 (CD82) is a metastasis suppressor gene, which belongs to the family of tetraspanin proteins. A loss of KAI-1 expression is associated with the advanced stages of many human malignancies. The present study was designed to investigate the expression pattern of KAI-1 in the normal endometrium and uterine tumors and to correlate it with the expression of tumor suppressor protein p53. KAI-1 could be found in the normal endometrium throughout the menstrual cycle. Thirteen of 42 endometrial carcinomas demonstrated moderate KAI-1 expression, but low expression of p53. Twenty-nine of 42 endometrial carcinomas showed reduced or absent KAI-1 expression, which correlated with strong expression of p53 (p < 0.001). There were significant correlations between KAI-1 expression and histological type, e.g., 93% of endometrioid carcinomas displayed a low or moderate immunostaining for KAI-1, whereas nearly all of the serous/clear cell carcinomas were KAI-1 negative (p < 0.001); tumor grading, e.g., 73% of high grade tumors showed no KAI-1 expression (p < 0.001). Most of the investigated uterine sarcomas were negative for KAI-1, whereas they displayed a strong immunostaining for p53. In conclusion, KAI-1 and p53 show inverse expression. The reduced KAI-1 expression may be the result of dysregulated p53 function and could be an important step in the endometrial carcinogenesis.

Role of Fra-2 in breast cancer: influence on tumor cell invasion and motility.

Fra-2 (Fos-related antigen 2) is a member of the Fos family of AP-1 transcription factors which is often up-regulated in mammary carcinomas. Previous results suggested that it might be involved in the regulation of breast cancer invasion and metastasis. In order to analyze the role of Fra-2 in breast cancer cells, it was silenced in the highly invasive MDA-MB231 cells using RNA interference. On the other hand, stable transfectants of the weakly invasive MCF7 cell line were established in order to analyze the effects of Fra-2 overexpression. In both approaches, cell proliferation was not or only weakly influenced by Fra-2. In contrast, the invasive potential of the cells was increased, and a weaker effect on motility was observed. By cDNA microarray analysis of the MCF7 transfectants followed by validation on a protein level, we identified several Fra-2 target genes which might be involved in cell invasion and migration, i.e., ALCAM and connexin 43. Additionally, mRNA expression levels of various genes which are associated with a more malignant behavior of the tumors in vivo were up- or downregulated, i.e., members of the MAGE family, S100P, TIMP2, IL24 etc. These results show that Fra-2 overexpression is associated with a more aggressive tumor phenotype and is probably involved in breast cancer progression in vivo.

Ulex europeus agglutinin-I binding as a potential prognostic marker in ovarian cancer.

BACKGROUND: Ovarian cancer represents the malignant tumour of the female genital tract with the worst prognosis, mainly caused by early intraperitoneal spread. Cell-to-cell and cell-to-matrix interactions play a functionally important role in this spread and are both mediated by the cell membrane. Changes in the glycosylation of the cell membrane, as detected by lectin histochemistry, are sometimes associated with a poor prognosis. PATIENTS AND METHODS: The expression of lectin binding of 164 ovarian cancer patients was analysed and the staining results were correlated with the clinical data of the patients. RESULTS: The univariate and multivariate statistical analysis revealed an independent prognostic significance for Ulex europeus agglutinin-I (UEA-I) binding. CONCLUSION: These findings indicate that UEA-I binding can serve as a prognostic factor in ovarian cancer.

Expression of the metastasis suppressor KAI1 in decidual cells at the human maternal-fetal interface: Regulation and functional implications.

At the human maternal-fetal interface, the decidua forms a dense matrix that is believed to limit trophoblast invasion. We investigated whether the metastasis suppressor KAI1 (CD82) is expressed at the maternal-fetal interface. Immunohistochemistry showed strong expression of KAI1 in decidual cells, whereas trophoblast cells were negative for KAI1. In luteal phase endometrium, KAI1 was present in decidualizing endometrial stromal cells. We investigated whether KAI1 expression in endometrial stromal cells is regulated by the decidualizing stimuli cAMP and progesterone or by the cytokine interleukin (IL)-1beta. Western blot analysis revealed induction of KAI1 protein by cAMP analog, but not by progesterone, in a delayed fashion. In contrast, IL-1beta rapidly stimulated KAI1 expression at the transcript level and at the protein level. Cultured decidual cells from term placenta expressed a basal level of KAI1 protein that was elevated on cAMP stimulation. Silencing of KAI1 by RNA interference attenuated expression of decorin, a decidual product implicated in limiting trophoblast invasion. This study shows for the first time the expression of KAI1 in decidual cells at the human maternal-fetal interface, where the metastasis suppressor might participate in intercellular communication with trophoblast cells and the control of trophoblast invasion.

Expression pattern of osteopontin in endometrial carcinoma: correlation with expression of the adhesion molecule CEACAM1.

Osteopontin (OPN) and CEACAM1 have diverse biological functions in the uterus and placenta throughout the estrous cycle and pregnancy and have been shown to interact with integrin beta3. OPN is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. Recently we showed the expression pattern of OPN in gestational trophoblastic tumors. CEACAM1 is an adhesion molecule of the carcinoembryonic antigen family that we have recently found to be expressed in endometrial cancer and that has been shown to be down-regulated in colorectal, prostate, and breast cancer. In this study, immunohistochemistry and immunofluorescence with specific antibodies were performed on a series of 20 normal endometrial samples, 17 endometrial hyperplasias, and 43 endometrial carcinomas (28 endometrioid, 10 serous, and 5 clear cell carcinomas) to investigate the expression pattern and cell-type specific localization of OPN and to correlate it with the expression of CEACAM1. In addition, Western blot was performed on normal human endometrium and endometrial neoplasia. Strong OPN expression with a consistent cytoplasmic localization in epithelial glandular cells was observed in the normal human endometrium in 80% of the samples of the proliferative and secretory phase (score 8-12). Similar results could be found in endometrial hyperplasias. Strong expression of OPN could be observed in 29 (67.4%) of the 43 analyzed endometrial carcinomas. Of the 43 analyzed tumors, 18 (41.8%) were in the high score (8-12) category with a strong OPN expression level; 11 of 43 (25.5%) showed a moderate score (4-7) category. In endometrioid carcinoma with increasing malignancy grade, increasing areas with low OPN expression level or complete loss of OPN expression could be observed. In contrast, serous tumors showed a strong OPN expression level. Similar results could be found in Western blot analysis. CEACAM1 showed similar results and could be found to be coexpressed with OPN in normal human endometrium and in endometrial neoplasia as we showed using immunofluorescence. In this study, the different expression patterns of OPN in endometrial tumors could additionally support the biological diversity of endometrioid and serous carcinomas together with other markers. We suggest that OPN might play a different role in the pathogenesis of endometrial cancer (possibly as a functional complex with CEACAM1) and could be relevant for invasive growth of such lesions.

Corticotropin-releasing hormone modulates human trophoblast invasion through carcinoembryonic antigen-related cell adhesion molecule-1 regulation.

Abnormalities in the process of trophoblast invasion may result in abnormal placentation. Both the embryonic trophoblast and maternal decidua produce corticotropin-releasing hormone (CRH), which promotes implantation. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is expressed in extravillous trophoblasts (EVTs) of normal human placenta, may also function in tro-phoblast/endometrial interactions. We investigated whether locally produced CRH plays a role in trophoblast invasion, primarily by regulating CEACAM1 expression. We examined cultures of freshly isolated human EVTs, which express CEACAM1, and an EVT-based hybridoma cell line, which is devoid of endogenous CEACAM1. CRH inhibited EVT invasion in Matrigel invasion assays, and this effect was blocked by the CRH receptor type 1 (CRHR1)-specific antagonist antalarmin. Additionally, CRH decreased CEACAM1 expression in EVTs in a dose-dependent manner. After transfection of the hybridoma cell line with a CEACAM1 expression vector, the invasiveness of these cells was strongly enhanced. This effect was inhibited by addition of blocking monoclonal antibody against CEACAM1. Furthermore, blocking of endogenous CEACAM1 in EVTs inhibited the invasive potential of these cells. Taken together these findings suggest that CRH inhibits trophoblast invasion by decreasing the expression of CEACAM1 through CRHR1, an effect that might be involved in the pathophysiology of clinical conditions, such as preeclampsia and placenta accreta.

Stimulation of CEACAM1 expression by 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 in endometrial carcinoma cells.

Downregulation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), a cell adhesion molecule with tumor suppressing properties has been observed in a high percentage of carcinomas of the endometrium and other malignancies. The mechanisms for the dysregulation and the role of hormones and cytokines on the expression of CEACAM1 in endometrial carcinomas is unknown. We therefore studied the effect of estradiol, medroxyprogesterone acetate (MPA), RU486, gamma-interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 on the expression in the non-expressing endometrial tumor cell lines Hec1B and Skut1B, respectively. No induction of CEACAM1 expression was observed in Hec1B endometrial adenocarcinoma cells in response to hormones and cytokines whereas treatment with TPA and calcium ionophore A23187 resulted in the strong expression of endogenous CEACAM1 on the mRNA and protein levels. In contrast, no induction of CEACAM1 expression was observed in endometrial mixed mesenchymal Skut1B cells. Studies of other members of the CEACAM family revealed that the re-expression in Hec1B carcinoma cells is restricted to CEACAM1 suggesting a cell type-specific and cell type-independent mechanism of CEACAM1 activation via the protein kinase C (PKC) pathway. Induction of CEACAM1 expression was dependent on protein kinase C protein synthesis and luciferase reporter assays with CEACAM1 promoter constructs demonstrated that the re-expression of CEACAM1 is regulated at the transcriptional level. This is the first report demonstrating that activators of PKC are able to specifically induce the expression of CEACAM1 in human carcinoma cells and our findings may provide a basis for the therapeutic inhibition of tumor growth in malignancies in which CEACAM1 is downregulated.

Expression of the high-mobility group protein HMGI(Y) in gestational trophoblastic diseases.

The high-mobility group protein HMGI(Y) is a member of a family of nonhistone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes, and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. Recently we could show that HMGI(Y) is expressed in the normal human placenta, where it is localized in the nuclei of villous cytotrophoblast, in the anchoring villi at the implantation site and in extravillous (intermediate) trophoblast invading the maternal decidua. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. The purpose of this study was to investigate the expression pattern of HMGI(Y) in gestational trophoblastic diseases (GTD), which has not been studied so far. To analyze the expression of HMGI(Y), we performed immunohistochemistry on a total of 29 cases of GTD, including 21 hydatidiform moles and 8 choriocarcinomas. Hydatidiform moles showed a positivity for HMGI(Y) in villous cytotrophoblast and in areas of the trophoblast proliferations on the villous surface; villous syncytiotrophoblast was negative. The choriocarcinomas showed strong immunoreactivity in all cases. The expression pattern of HMGI(Y) in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD and might be potentially useful as an additional diagnostic marker for such lesions.

Inhibition of casein kinase I delta alters mitotic spindle formation and induces apoptosis in trophoblast cells.

The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.

Expression pattern of the activating protein-1 family of transcription factors in gestational trophoblastic lesions.

The human placenta is an endocrine tissue with a unique capacity for rapid, but tightly controlled, proliferation and invasion. Gestational trophoblastic diseases (GTDs) are placental pathologies with endocrine activity and partially malignant potential and include hydatidiform moles, placental site nodules, and tumors such as placental site trophoblastic tumor and choriocarcinomas. The activating protein-1 (AP-1) family of transcription factors is composed of the cellular homologs of the Jun and Fos oncoproteins, which are immediately involved in cellular proliferation, differentiation, and invasion processes and in the regulation of endocrine genes. The expression pattern of the AP-1 family in the normal human placenta has been recently described, where most of the factors were found in the extravillous (invasive) trophoblast. Their systematic expression in GTD has not been studied thus far. For this reason in this study, we investigated the expression pattern of the AP-1 family in GTDs and compared it with the expression in normal placenta using immunohistochemistry with specific polyclonal antibodies against all members of the AP-1 family (JunB, JunD, c-Jun, c-Fos, FosB, Fra-1, Fra-2). Immunohistochemistry was performed on normal human placentas (positive control) and on 28 cases of GTD including 7 choriocarcinomas and 21 hydatidiform moles. In the normal placenta and in hydatidiform molar samples, most AP-1 factors (especially c-Jun, JunD, and Fra2) were expressed in the intermediate (extravillous) trophoblast. In addition, in molar lesions, strong expression was found in trophoblasts proliferating from the surface of villi. There was only a weak expression of JunB and Fra2 in small fractions of villous cyto- and syncytiotrophoblast nuclei. In choriocarcinomas, there was a strong expression for c-Jun, JunD, Fra1, and Fra2. The specific localization to extravillous trophoblasts and their expression pattern in GTDs indicate that the AP-1 family of transcription factors might be implicated in regulating proliferation and invasion of trophoblasts and play a role in the pathogenesis of GTDs.

Osteopontin is colocalized with the adhesion molecule CEACAM1 in the extravillous trophoblast of the human placenta and enhances invasion of CEACAM1-expressing placental cells.

CONTEXT: The human placenta is a complex tissue and possesses, through its capacity to proliferate and to invade maternal tissue, qualities that are usually found in malignant tumors. Osteopontin (OPN) and CEACAM1 may regulate these processes. OBJECTIVE: The present study was designed to investigate the expression pattern of OPN in the human placental components and to correlate it with CEACAM1 expression and function in placental cell invasiveness. DESIGN: Immunohistochemistry with an OPN-specific antibody and immunofluorescence were performed on normal placental samples to investigate the expression pattern of OPN and CEACAM1 in the human placenta. Extravillous trophoblast (EVT) hybridoma cells transfected with CEACAM1 and stimulated with OPN were studied using the Matrigel invasion assay. RESULTS: All placentae presented very strong expression of OPN in the EVT at the invasion front, where it colocalized with CEACAM1. In addition, OPN was also present in the villous trophoblast, with strongest expression in the cytotrophoblast of the first trimester. Transfection with CEACAM1 followed by stimulation with OPN resulted in increased invasiveness of EVT hybridoma cells. CONCLUSION: The present study shows the first systematic analysis of OPN expression pattern in the human placenta showing strong expression in the EVT at the invasion front. Colocalization of OPN with CEACAM1 in the EVT indicates that they might act together to regulate invasiveness at the maternal-fetal interface. Using an in vitro model, we also demonstrated increased cellular invasiveness after OPN treatment. We speculate that OPN and CEACAM1 may act as a functional complex involved in the regulation of placental invasiveness.