Antiproliferative signaling of luteinizing hormone-releasing hormone in human endometrial and ovarian cancer cells through G protein alpha(I)-mediated activation of phosphotyrosine phosphatase.
The signaling pathway through which LHRH acts in endometrial and ovarian cancers is distinct from that in the anterior pituitary. The LHRH receptor interacts with the mitogenic signal transduction of growth factor receptors, resulting in down-regulation of expression of c-fos and proliferation. Only limited data are available on the cross-talk between LHRH receptor signaling and inhibition of mitogenic signal transduction. The present experiments were performed to analyze in endometrial and ovarian cancer cells: 1) whether mutations or splice variants of the LHRH receptor are responsible for differences in LHRH signaling, 2) the coupling of G protein subtypes to LHRH receptor, 3) the phosphotyrosine phosphatase (PTP) activation counteracting growth factor receptor tyrosine kinase activity. For these studies, the well characterized human Ishikawa and Hec-1A endometrial cancer cell lines and human EFO-21 and EFO-27 ovarian cancer cell lines were used, which express LHRH and its receptor. 1) Sequencing of the complementary DNA of the LHRH receptor from position 31 to position 1204, covering the complete coding region (position 56 to position 1042) showed that there are neither mutations nor splice variants of the LHRH receptor transcript in Ishikawa and Hec-1A endometrial cancer cells or in EFO-21 and EFO-27 ovarian cancer cells. 2) All analyzed cell lines except for the ovarian cancer cell line EFO-27 expressed both G proteins, alpha(i) and alpha(q), as shown by RT-PCR and Western blotting. In the EFO-27 cell line only G protein alpha(i), not G protein alpha(q), expression was found. Cross-linking experiments using disuccinimidyl suberate revealed that in the cell lines expressing G protein alpha(i) and G protein alpha(q), both G proteins coupled to the LHRH receptor. Inhibition of epidermal growth factor (EGF)-induced c-fos expression by LHRH, however, was mediated through pertussis toxin (PTX)-sensitive G protein alpha(i). Moreover, LHRH substantially antagonized the PTX-catalyzed ADP-ribosylation of G protein alpha(i). 3) Using a phosphotyrosine phosphatase assay based on molybdate-malachite green, treatment of quiescent EFO-21 and EFO-27 ovarian cancer cells and quiescent Ishikawa and Hec-1A endometrial cancer cells with 100 nM of the LHRH agonist triptorelin resulted in a 4-fold increase in PTP activity (P < 0.001). This effect was completely blocked by simultaneous treatment with PTX, supporting the concept of mediation through G protein alpha(i). As shown by quantitative Western blotting, EGF-induced tyrosine autophosphorylation of EGF receptors was reduced 45-63% after LHRH (100 nM) treatment (P < 0.001). This effect was completely blocked using the PTP inhibitor vanadate (P < 0.001). These results demonstrate that mutations or splice variants of the LHRH receptor in human endometrial and ovarian cancer cells are not responsible for the different signal transduction compared with that in pituitary gonadotrophs. We provide evidence that the tumor LHRH receptor couples to multiple G proteins, but the antiproliferative signal transduction is mediated through the PTX-sensitive G protein alpha(i). The tumor LHRH receptor activates a PTP counteracting EGF-induced tyrosine autophosphorylation of EGF receptor, resulting in down-regulation of mitogenic signal transduction and cell proliferation.
Gründker
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Department of Gynecology and Obstetrics, Georg August University, D-37070 Gottingen, Germany.
Völker
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Emons
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D017398:Alternative Splicing; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects*; D016889:Endometrial Neoplasms / Q000235:genetics / Q000473:pathology*; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos / Q000235:genetics; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology*; D037342:Pertussis Toxin; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry; D011966:Receptors, LHRH / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D012333:RNA, Messenger; D011966:Receptors, LHRH; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D019000:Phosphotyrosine; D007987:Gonadotropin-Releasing Hormone; D004815:Epidermal Growth Factor; D037342:Pertussis Toxin; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go