The relevance of the chemokine receptor ACKR3/CXCR7 on CXCL12-mediated effects in cancers with a focus on virus-related cancers.

Recent studies have highlighted the importance of understanding the molecular determinants of CXCL12-mediated effects in cancers. Once previously thought to interact exclusively with CXCR4, CXCL12 also binds with high affinity to CXCR7 (recently renamed ACKR3), which belongs to an atypical chemokine receptor family whose members fail to activate Gαi proteins but interact with ß-arrestins. In addition to its capacity to control CXCL12 bioavailability, ACKR3 can either enhance or dampen CXCR4-mediated signaling and activity. In light of the most recent findings, we have examined the role of ACKR3 in cancer, including a subset of virus-related cancers.

Potential role of estrogen receptor beta as a tumor suppressor of epithelial ovarian cancer.

Ovarian cancer is the gynecological cancer exhibiting the highest morbidity and improvement of treatments is still required. Previous studies have shown that Estrogen-receptor beta (ERß) levels decreased along with ovarian carcinogenesis. Here, we present evidence that reintroduction of ERß in BG-1 epithelial ovarian cancer cells, which express ERα, leads in vitro to a decrease of basal and estradiol-promoted cell proliferation. ERß reduced the frequency of cells in S phase and increased the one of cells in G2/M phase. At the molecular level, we found that ERß downregulated total retinoblastoma (Rb), phosphorylated Rb and phospho-AKT cellular content as well as cyclins D1 and A2. In addition, ERß had a direct effect on ERα, by strongly inhibiting its expression and activity, which could explain part of the anti-proliferative action of ERß. By developing a novel preclinical model of ovarian cancer based on a luminescent orthotopic xenograft in athymic Nude mice, we further revealed that ERß expression reduces tumor growth and the presence of tumor cells in sites of metastasis, hence resulting in improved survival of mice. Altogether, these findings unveil a potential tumor-suppressor role of ERß in ovarian carcinogenesis, which could be of potential clinical relevance for the selection of the most appropriate treatment for patients.

Identification of the chemokine CX3CL1 as a new regulator of malignant cell proliferation in epithelial ovarian cancer.

BACKGROUND: Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX(3)CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX(3)CL1 in EOC. RESULTS: Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX(3)CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX(3)CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX(3)CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX(3)CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX(3)CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX(3)CL1 products. Conversely, CX(3)CL1 promoted through its binding to CX(3)CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX(3)CL1 and faster tumor growth. CONCLUSION: Our findings highlight the previously unappreciated constitutive expression of CX(3)CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients.

CXCL12 expression by healthy and malignant ovarian epithelial cells.

BACKGROUND: CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. METHODS: Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. RESULTS: Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival. CONCLUSION: Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00052468.

Identification of glucocorticoid-induced leucine zipper as a key regulator of tumor cell proliferation in epithelial ovarian cancer.

BACKGROUND: Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation. RESULTS: GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, P < 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, P < 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle. CONCLUSION: The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.

Progesterone reduces the migration of mast cells toward the chemokine stromal cell-derived factor-1/CXCL12 with an accompanying decrease in CXCR4 receptors.

Mast cell recruitment is implicated in many physiological functions and several diseases. It depends on microenvironmental factors, including hormones. We have investigated the effect of progesterone on the migration of HMC-1(560) mast cells toward CXCL12, a chemokine that controls the migration of mast cells into tissues. HMC-1(560) mast cells were incubated with 1 nM to 1 microM progesterone for 24 h. Controls were run without progesterone. Cell migration toward CXCL12 was monitored with an in vitro assay, and statistical analysis of repeated experiments revealed that progesterone significantly reduced cell migration without increasing the number of apoptotic cells (P = 0.0084, n = 7). Differences between progesterone-treated and untreated cells were significant at 1 microM (P < 0.01, n = 7). Cells incubated with 1 microM progesterone showed no rearrangment of actin filaments in response to CXCL12. Progesterone also reduced the calcium response to CXCL12 and Akt phosphorylation. Cells incubated with progesterone had one-half the control concentrations of CXCR4 (mRNA, total protein, and membrane-bound protein). Progesterone also inhibited the migration of HMC-1(560) cells transfected with hPR-B-pSG5 plasmid, which contained 2.5 times as much PR-B as the control. These transfected cells responded differently (P < 0.05, n = 5) from untreated cells to 1 nM progesterone. We conclude that progesterone reduces mast cell migration toward CXCL12 and that CXCR4 may be a progesterone target in mast cells.

Progesterone increases csk homologous kinase in HMC-1560 human mast cells and reduces cell proliferation.

Mast cells proliferate in vivo in areas of active fibrosis, during parasite infestations, in response to repeated immediate hypersensitivity reactions and in patients with mastocytosis. We investigated how progesterone reduces the proliferation of HMC-1(560) mast cells that proliferate spontaneously in culture. Cells were incubated with 1 microM to 1 nM progesterone for 24-48 h. Progesterone (1 microM) reduced the spontaneous proliferation of HMC-1(560) mast cells to half that of cells cultured without hormone. [(3)H] thymidine incorporation was only 50% of control; there were fewer cells in G2/M and more cells in G0/G1. The amounts of phospho-Raf-1 (Tyr 340-341) and phospho-p42/p44 MAPK proteins were also reduced. In contrast progesterone had no effect on MAP kinase-phosphatase-1. The Raf/MAPK pathway, which depends on Src kinase activity, is implicated in the control of cell proliferation. HMC-1(560) cells incubated with the tyrosine kinase inhibitor PP1 proliferated more slowly than controls and had less phospho-Raf-1 (Tyr 340-341) and phospho-p42/p44 MAPK. The Csk homologous kinase (CHK), an endogenous inhibitor of Src protein tyrosine kinases, was also enhanced in progesterone-treated cells. In contrast, progesterone had no effect on the growth of cells transfected with siRNA CHK. We conclude that progesterone increases the amount of csk homologous kinase, which in turn reduces HMC-1(560) mast cell proliferation. This effect parallels decreases in the phosphorylated forms of Raf-1 and p42/44 MAPK, as their production depends on Src kinase activity.

The chemokine SDF-1/CXCL12 contributes to T lymphocyte recruitment in human pre-ovulatory follicles and coordinates with lymphocytes to increase granulosa cell survival and embryo quality.

We investigated the production and the role of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) in pre-ovulatory follicles of women undergoing in vitro fertilization. We detected CXCL12 and its receptor CXCR4 by flow cytometry, western blotting and RT-PCR. We tested cell migration in Transwell experiments. We measured apoptosis using delta psi m-sensitive fluorescent probe DiOC6(3) and we screened apoptosis-related gene expression with macro-arrays. Granulosa cells from follicular aspirates produce CXCL12 that contributes to T lymphocytes recruitment. CXCL12 reduces early apoptosis of granulosa cells. This effect is accompanied by a shift of bcl2/bax ratio, and decreased expression of p53-targeted genes (pig7, pig8, p21, gadd45). Removal of lymphocytes disables CXCL12-mediated anti-apoptotic effect on granulosa cells. Anti-apoptotic activity of CXCL12 is positively correlated to high quality of embryos. In conclusion, CXCL12 is locally produced by luteinizing granulosa cells. It specifically contributes to T lymphocytes recruitment and coordinates with local lymphocytes to increase granulosa cell survival and embryo quality.

CXCL12 and vascular endothelial growth factor synergistically induce neoangiogenesis in human ovarian cancers.

Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.

Lead reduces messenger RNA and protein levels of cytochrome p450 aromatase and estrogen receptor beta in human ovarian granulosa cells.

Exposure to lead causes decreased fertility in women. In the present study, we examined the in vitro effects of lead on cytochrome p450 aromatase (p450 arom) and on estrogen receptor beta (ERbeta), two key proteins for the human ovary. Aromatase is required for the bioconversion of androgen to estradiol; ERbeta mediates estrogen effects in granulosa cells. Granulosa cells were collected from women undergoing in vitro fertilization and then cultured with 10 microM lead acetate. Using atomic absorption spectrometry, we showed that lead accumulated in cells. Aromatase activity as measured by a tritiated water production assay was significantly reduced. Using semiquantitative reverse transcription-polymerase chain reaction and Western blotting procedures, we showed that p450 arom and ERbeta mRNA and protein content were both significantly reduced. Adding 10 microg/ml of cycloheximide, a protein inhibitor, did not eliminate the effects of lead. The present results support the hypothesis that the action of lead on fertility in women may result, in part, from the down-regulation of p450 arom and ERbeta gene transcription in ovarian granulosa.

Hodgkin's disease masquerading as fibrous thyroiditis: potential role of cytokines in in vivo and in vitro studies.

Hodgkin's disease appearing as, or associated with, fibrous thyroiditis has only been described rarely. We report the observation of a patient presenting with a goitre, fibrosis of the thyroid and adjacent structures, and hypothyroidism. The histological examination was compatible with fibrosclerotic thyroiditis. This diagnosis was reviewed 6 months later when the biopsy of a supraclavicular nodule that had subsequently appeared led to the diagnosis of a nodular-sclerosis type of Hodgkin's disease. The plasmatic levels of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) were very high compared to the levels in healthy subjects (12 and 40 IU/l vs. 0.05 and 2.0 IU/l, respectively). These cytokine levels decreased when the initial illness was treated, and their normalization was associated with the disappearance of the cervical and thyroidal fibroses. A parallel in vitro study of these cytokines and of TNF-alpha receptors and IL-13 was performed. The results suggest a possible cause-and-effect relationship between IL-6 and IL-13 produced locally by the tumoral tissue and the development of cervical fibrosis.