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Tumor Biology |
Departments of Gynaecology, Perinatology, and Human Reproduction [S. D., I. N., P. B., E. B., M. G., M. M., G. S.], Experimental Pathology and Oncology [A. A.], Clinical Physiopathology [M. B.], and Human Pathology and Oncology [G. L. T.], University of Firenze, 50134 Firenze, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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On the other hand, the correlation between GTs and several other aspects of tumor progression is still poorly known. The process of tumor progression includes the acquisition of malignant characteristics such as the ability of invading surrounding tissues and the bloodstream. This invasion eventually is critical to the establishment of metastases, which is the major hindrance to cancer therapy. Although most mechanistic studies of tumor invasion in EC have focused on the role of E (8, 9, 10) , it has recently emerged that GTs promote neoangiogenesis both inside tumors (11) and in the chicken chorioallantoic membrane (12) .
This opens the possibility that GTs could directly regulate tumor progression of EC by binding to specific receptors. In fact specific LH/hCG-Rs have been detected in human EC, and their expression is apparently related to the cancer grading (13) . In particular, two isoforms of this receptor arising from alternative splicing of the corresponding gene are documented in EC samples (13) , as well as in neoplastic ovarian tissues (14) .
The LH/hCG-R is a transmembrane glycoprotein receptor, which is a member of the G protein-coupled receptor family (15) . The ligand-receptor binding induces the activation of signaling pathways centered on the cAMP/PKA system as well as on PLC/inositide tris-phosphate pathway (15 , 16) . It has been reported (15) that the prevalence of one or the other signaling pathways depends on the cell type involved and the amount of receptor expressed on the plasma membrane (17) . In endometrial cells of porcine origin, it has been recently reported (18) that LH induces the activation of both cAMP/PKA and PLC/inositide tris-phosphate pathways.
On the basis of these findings, we started a study aimed at determining the effects of LH/hCG system in tumor progression of EC by analyzing the effects of such hormones on the invasion potential of both EC cell lines and primary human EC cells. We report here that hrLH (as well as hCG) induced a significant increase in cell invasiveness through Matrigel-coated porous membranes in the human EC cell line Hec1A, which expresses the LH/hCG-R. This effect turned out to depend on the hrLH binding to its specific receptor and on the following activation of the cAMP/PKA signaling pathway. Moreover, the hrLH-induced increase in Hec1A invasiveness was dependent upon the functional activation of ß1 integrin receptors and the subsequent induction of MMP-2 secretion. Interestingly, these mechanisms were found to be operative in primary EC cells transferred in vitro because hrLH addition produced an increase in cells invasiveness in those primary EC tumors that expressed the LH/hCG-R. Here again, this effect was dependent on PKA activity.
On the whole, data presented in this article show that LH/hCG can regulate EC cell invasiveness, and this provides a rationale for the use of inhibitors of LH secretion such as GnRH analogues in the treatment of EC (19) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) 75 UI (3.4 µg; Serono Laboratories, Geneva, Switzerland) was used at 0.3 UI/ml (6)
. hCG: Profasi 5000 HP (Serono Laboratories, Geneva, Switzerland) was used at 5000 UI/ml. The inhibitor of PKA H89 (N-5-isoquinolinesulfonamide hydrochloride; Sigma Chemical Company, St. Louis, MO) was used at a final concentration of 50 µM. The inhibitor of PLC U73122 (Sigma Chemical Company) was used at a final concentration of 5 µM. Doses of inhibitors were chosen according to various data reported in literature and to preliminary dose response courses.
Cell Culture.
The human endometrial adenocarcinoma cell line Hec1A (American Type Culture Collection, Manassas, VA) was routinely grown in DMEM (Euroclone; Wetherby, West Yorkshire, United Kingdom) supplemented with L-glutamine (DMEM-GLUT) and 10% FCS (Euroclone, Wetherby; complete medium) and incubated at 37°C in 5% CO2. Primary EC cultures were obtained from surgical specimens and prepared as reported previously (19
, 20)
.
Matrigel Invasion Assay.
Hec1A cells, cultured as described above, were harvested by trypsinization and resuspended in DMEM-GLUT containing 250 µg/ml heat-inactivated BSA (DMEM + BSA). Cell suspension (200 µl) containing 600,000 cells was seeded into the upper well of a Boyden chamber (NeuroProbe, Inc., Gaithersburg, MD), separated from the lower compartment (filled with DMEM + BSA) by a porous membrane (8-µm pore diameter) previously overlaid with Matrigel (see above). All of the additions were performed both into the upper and the lower wells. Boyden chambers were incubated at 37°C in humidified atmosphere containing 5% CO2 in air for 24 h. Migrated cells, which remained layering onto the lower face of the porous membrane, were fixed with absolute methanol at 4°C overnight and stained with Diff-Quick staining solution (Dade-Behring Holding GmbH, Liederbach, Germany). Cells were counted on the whole migration field at x40 magnification.
RT-PCR.
Total RNA was extracted from either the Hec1A cells or fresh tissue EC samples by the guanidinium/isothyocyanate method (21)
. cDNA was then synthesized using 1 µg of total RNA in a mix reaction with 2.5 units/µl reverse transcriptase murine leukemia virus-RT (Applied Biosystem, Foster City, CA), 1 mM of each deoxynucleotide triphosphate, and 2.5 µM random exhamers in a 20-µl final reaction volume for 30 min at 42°C and 5 min at 99°C. Ten µl of each resulting cDNA template was then used to perform a nested PCR specific for LH/hCG-R, which consists of a first amplification using 1.25 units AmpliTaq polymerase (Applied Biosystem), 200 µM of each deoxynucleotide triphosphate, 2 mM MgCl2, and 0.15 µM of the specific primers. Ten µl of the initial PCR product were used as template for a second PCR in a reaction mix as above except that different primers, internal to the first ones, were used. In both cases, amplification was subjected to 43 cycles each consisting of 94°C for 1 min, 56°C for 1 min, 72°C for 1 min, and to a 72°C for 5 min cycle for final elongation. The primers used were those described by Lin et al. (12)
. The omission of template in each PCR reaction was performed as negative control.
Integrin Flow Cytometric Analysis.
Hec1A cells were seeded onto Matrigel-coated dishes in DMEM + BSA in the absence or in the presence of 0.3 UI/ml hrLH (see above) and incubated for 24 h as above. Cells were harvested in PBS + EDTA (5 mM) both at time 0 and after 24 h of incubation, washed once in PBS, and then incubated with monoclonal antibodies raised against the ß1 integrin subunit (mAb TS2/16; 30 µg/ml), the ß3 subunit (mAb B212; 30 µg/ml), and the v subunit (mAb L230; 30 µg/ml) for 15' at 37°C. All of the antibodies were kindly gifted by Dr. G. Tarone and Dr. P. Defilippi of the University of Torino (Torino, Italy). Afterward, cells were washed and incubated with an antimouse antibody labeled with FITC (Sigma Chemical Company). For the analysis, 20,000 events were collected, and the fluorescence of samples was acquired in logarithmic scale using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 5-W argon-ion laser.
Adhesion Assay.
Adhesion assays were performed essentially according to Arcangeli et al. (22)
. Briefly, Hec1A cells were radiolabeled for 48 h of exponential growth in complete medium containing 1 µCi/ml methyl-3H-thymidine (specific activity 24 Ci/mmol; Amersham Biosciences Europe GmbH, Freiburg, Germany). After 24 h of labeling, 0.3 UI/ml hrLH were added where needed and cells incubated for an additional 24 h. Cells were then harvested, pelleted, and resuspended in DMEM + BSA; aliquots of cells (1 x 105) were inoculated into each well of 96-well clusters (Corning B.V., Life Sciences, the Netherlands), previously coated with Matrigel (see before). In the appropriate samples, blocking antibodies to FN-R (kindly gifted by Dr. G. Tarone and Dr. P. Defilippi, University of Torino; Ref. 23
) at different dilutions were added at time 0. In any case, cells were incubated at 37°C in 5% CO2 in air; at different times, the medium was aspirated off and adherent cells gently rinsed twice with PBS; the cells were solubilized with 50 µl of 1% SDS in 0.1 N NaOH for 1 h. Radioactive solubilized cells were quantified by scintillation counting and compared on a percentage basis with the radioactivities of the cells inoculated in each well.
Assay of Gelatin-degrading MMPs by Zymography.
The release of MMPs in the medium of cell cultures used for the invasion assay was determined as described by Barletta et al. (24)
. Briefly, concentrated supernatants were electrophoresed on 8% polyacrylamide gel added with porcine gelatin (Sigma Chemical Company). After the run, the gel was incubated at 37°C for 24 h in a solution containing 50 mM Tris-HCl, 200 mM NaCl, and 5 mM CaCl2 and then stained with Coomassie blue. Samples obtained from the medium of HT1080 cell cultures, which are known to express MMP-9 and MMP-2 at high levels were used as controls (24)
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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and 8
), was tested by analyzing the number of cells migrated through Matrigel-covered porous membranes inserted into Boyden chambers. As shown in Fig. 1a
, hrLH increased Hec1A cell invasion through Matrigel, almost doubling the number of cells migrated through the membrane after 24 h of incubation. A similar effect was obtained after addition of hCG (Fig. 1b)
. Interestingly, cells seeded on uncoated porous filters displayed a moderate invading activity, which was almost unaffected by hrLH addition (data not shown).
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), developed in our laboratory, which expresses the LH/hCG-R at high levels (compare Lane 2 with Lane 1 in the inset to Fig. 2a
). On the other hand, hrLH has almost no effect on the invasion of a completely unrelated human epithelial cell line (HEK 293 cells; Fig. 2b
), which lacks the LH/hCG-R (25
, 26)
. We also confirmed the absence of such receptors in these cells by RT-PCR (data not shown).
The dependence of the proinvasion effect exerted by hrLH on Hec1A cells on the signaling mechanisms induced by binding to its specific receptor was therefore tested. It has been reported (15)
that the activation of LH/hCG-R may induce the activation of both the cAMP/PKA and PLC/inositide tris-phosphate signaling pathways. The involvement of both these pathways in the proinvasion effect operated by hrLH on Hec1A cells was therefore analyzed by using specific inhibitors (the PKA inhibitor H89 and the PLC inhibitor U73122). As reported in Fig. 3
, although the addition of H89 almost completely inhibited the increase in cell invasiveness through Matrigel induced by hrLH, the PLC inhibitor U73122 had no significant effect. It can be therefore argued that the effect of hrLH on Hec1A cell invasiveness occurs via the signaling pathway initiated by hrLH binding to LH/hCG-R and centered on the activation of PKA.
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, Hec1A cells treated with hrLH adhere more quickly to Matrigel: this effect is already evident after 30 min of incubation, and the vast majority of cells are firmly adherent to the substratum within 60 min of incubation, instead of after 180 min as in the controls (Fig. 4a)
. Moreover, as shown in Fig. 4b
, the increase in adherent cells operated by hrLH addition after 30 min of incubation onto Matrigel turned out to be completely abolished by cell pretreatment with H89; this suggests that the increase in cell adhesiveness induced by hrLH relies on the activation of the cAMP/PKA signaling pathway. Finally, because the main integrins involved in cell adhesion to Matrigel are those containing the ß1 subunit, the involvement of this integrin subunit in the above reported increase in cell adhesiveness to Matrigel by hrLH was tested. Indeed, Hec1A cell adhesion to Matrigel turned out to be totally dependent on ß1 integrin activity as witnessed by the dose-dependent inhibitory effect of anti-ß1 antibodies (anti-FN-R antibodies; Fig. 4c
).
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, although adhesion to Matrigel induces per se an up-regulation of ß1, hrLH addition does not alter this expression pattern. Interestingly, an up-regulating effect of Matrigel on integrin expression was evident also for two other integrin subunits, namely ß3 and v (Fig. 5
, inset). Therefore, the effect of hrLH on integrins is apparently mediated by an increase in integrin avidity for the substratum more than by an increase in the amount of integrin expression on the plasma membrane.
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, the addition of various amounts of anti ß1 antibodies to hrLH-treated Hec1A cells induced a dose-dependent inhibition of cell invasion through Matrigel (Fig. 6a)
. On the other hand, anti-ß3 antibodies did not affect the hrLH-induced increase in Hec1A cell invasiveness (Fig. 6b)
.
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Therefore, the secretion of MMPs was tested on Hec1A seeded onto Matrigel in the absence or in the presence of hrLH through a zymographic assay. Fig. 7
shows that Hec1A cells are almost unable to secrete MMPs in basal conditions (control), as reported previously (8)
. The addition of hrLH induces the secretion of active MMP-2 (bottom arrow on the left side of Fig. 7
), a MMP widely involved in tumor cell invasion through basement membranes (9
, 29
, 30)
. Interestingly, the induction of MMP-2 secretion by hrLH was completely abolished by pretreatment of cells with the PKA inhibitor H89, here again, stressing the relevance of this signaling pathway in the proinvasiveness activity of hrLH on EC cells. Moreover, preincubation with anti-FN-R (anti-ß1) antibodies abolishes the proteolytic activation of the proenzyme MMP-2, thus leading to an accumulation of its inactive form.
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The possibility that this mechanism could be somehow involved in EC cell invasiveness in vivo was then studied. As a first approach, we tested whether the LH/hCG-R was indeed expressed in primary human EC, as reported by Lin et al. (13)
. Results obtained from 22 cases of primary EC indicated that 45% of tumors indeed expressed the receptor (Table 1
and Fig. 8
). It is worth noting (Fig. 8
, Lane 4) that EC samples expressed mainly the splice variant weighing 153 bp as reported by Lin et al. (13)
.
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displaying different expression of LH/hCG-R, immediately after surgical removal form the uterus. As shown in Fig. 9a
, five of seven cases increased their migratory activity after hrLH addition with an average doubling of the number of migrated cells after 24 h of incubation in the presence of the GT. Interestingly, here again, the hrLH-induced effect was apparently related to the expression of LH/hCG-R by tumor cells. In fact, only those primary tumors that expressed the LH/hCG-R increased their invasion through Matrigel in response to hrLH addition (compare samples in Fig. 9
with those in Table 1
). Interestingly, the proinvasiveness effect of hrLH turned out to be, here again, almost completely inhibited by the PKA inhibitor H89 (Fig. 9b)
.
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In this study, we provide evidence that LH/hCG is involved in tumor progression of EC because it induces an invasive phenotype in the moderately differentiated EC cell line Hec1A. The mechanism for this involves LH/hCG binding to its receptor and inducing the activation of PKA, which in turn activates ß1 integrin adhesiveness to Matrigel and therefore the secretion of active MMP-2. In this light, PKA has been indeed recently reported to positively regulate cell spreading and motility in different cellular models (31 , 32) ; moreover, our data show that the PKA-dependent activation of ß1 integrin appears to be also necessary for the induction of the enzymatic activation of MMP-2.
Data demonstrating that this mechanism is also operative in primary EC are presented. No data regarding a direct effect of LH/hCG on EC cell lines and primary EC invasiveness have been published previously to the best of our knowledge.
Data reported in this article not only stress the role of GTs as tumor progression factors but also lend support to the use of GnRH analogues to inhibit pituitary LH secretion in the therapeutic protocols of LH/hCG-R-expressing tumors (19) because this use could block the invasive features of the tumor and thus permit longer survival, often without interfering with other aspects of neoplastic growth such as proliferation and differentiation as reported by Noci et al. (19) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Takeda Italia (Roma) and by grants from the Associazione Italiana contro le Leucemie (Firenze) and the Ministero dell’Università e Ricerca Scientifica e Tecnologica (Murst, Cofin 2001) (to A. A.). 
2 To whom requests for reprints should be addressed, at Department of Experimental Pathology and Oncology, Viale Morgagni 50, 50134 Firenze, Italy. Phone: 0039-055-4282326; Fax: 0039-055-4282333; E-mail: annarosa.arcangeli{at}unifi.it
3 The abbreviations used are: EC, endometrial cancer; E, estrogen; P, progesterone; GT, gonadotropin, FSH, follicle-stimulating hormone; LH/hCG, luteinizing hormone/human chorionic GT; LH/hCG-R, receptors for the LH/hCG peptide hormone; PLC, phospholiphase C; cAMP, cyclic AMP; PKA, protein kinase A; RT-PCR, reverse transcription-PCR; hr, human recombinant; MMP, matrix metalloproteinase; FN-R, fibronectin receptor; gapdh, glyceraldehyde-3-phosphate dehydrogenase.
Received 1/31/03. Accepted 4/25/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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gene in endometrial carcinoma cells. Int. J. Cancer, 100: 401-406, 2002.[Medline]
(v)ß(3) integrin in the preosteoclastic leukemia cell line FLG 29.1. J. Biol. Chem., 276: 4923-4931, 2001.3ß1 and 6ß4 integrins in tumor invasion. Clin. Exp. Metastasis, 19: 217-223, 2002.[Medline]
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) and of the LH-R-overexpressing clone Hec1A1s () is reported. Conditions were the same as reported in the legend to Fig. 1
= hrLH-treated cells; 
