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© The Author 2007. Published by Oxford University Press.
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Requirement of KISS1 Secretion for Multiple Organ Metastasis Suppression and Maintenance of Tumor Dormancy
Affiliations of authors: Departments of Pathology (KTN, PAP, DRH, KSV, ARF, DRW), Medicine–Genetic and Translational Medicine (MAAL), Cell Biology (ES, DRW), Medicine–Hematology/Oncology (MAAL, JCK), and Pharmacology and Toxicology (DRW), University of Alabama, Birmingham, AL; Department of Pathology and Immunotherapy Center, Medical College of Georgia, Augusta, GA (JMN, SCP); Comprehensive Cancer Center, University of Alabama, and Center for Metastasis Research, National Foundation for Cancer Research, Birmingham, AL (DRW)
Correspondence to: Danny R. Welch, PhD, 1670 University Blvd., VH-GO19, Birmingham, AL 35294-0019 (e-mail: danwelch{at}uab.edu).
Background: The KISS1 protein suppresses metastasis of several tumor models without blocking orthotopic tumor growth, but the mechanism remains elusive. For its role in human sexual maturation, KISS1 protein is secreted and processed to kisspeptins, which bind to the G protein–coupled receptor GPR54. We tested the hypothesis that KISS1 secretion is required for metastasis suppression via GPR54.
Methods: KISS1 containing an internal FLAG epitope with (KFM) or without (KFM
SS) a signal sequence was transfected into C8161.9 human melanoma cells, which do not express endogenous KISS1. Whole-cell lysates and conditioned medium from C8161.9KFM and C8161.9KFMSS cells were collected and analyzed for kisspeptins by immunoprecipitation and enzyme-linked immunosorbent assay. GPR54 levels were measured using real-time reverse transcription–polymerase chain reaction. The ability of conditioned medium from C8161.9KFM and C8161.9KFMSS cells to stimulate calcium mobilization in GPR54-expressing Chinese hamster ovary cells (CHO-G) and in C8161.9 cells was evaluated. Metastasis was monitored in athymic mice (groups of 10 per experiment) that were injected with C8161.9KFM or C8161.9KFMSS cells labeled with enhanced green fluorescent protein. Survival of mice injected with C8161.9 or C8161.9KFM cells was analyzed by Kaplan–Meier methods.
Results: Full-length KFM and KFMSS were detected in whole-cell lysates of C8161.9KFM and C8161.9KFMSS cells, respectively, but kisspeptins were detected only in conditioned medium of C8161.9KFM cells. In vivo, C8161.9KFM, but not C8161.9KFMSS, cells were suppressed for metastasis to lung, eye, kidney, and bone, with corresponding differences in mouse survival (median > 120 versus 42 days). C8161.9KFM cells seeded mouse lungs but did not form macroscopic metastases. Conditioned medium from C8161.9KFM, but not C8161.9KFMSS, cells stimulated calcium mobilization in CHO-G cells. GPR54 expression was low in C8161.9 cells, which were not stimulated by conditioned medium from C8161.9KFM cells.
Conclusions: KISS1 secretion was required for multiple organ metastasis suppression and for maintenance of disseminated cells in a dormant state. The absence of GPR54 expression in C8161.9 cells (whose metastatic spread was suppressed by KFM) suggests that metastasis suppression is not mediated through this receptor. The results imply the existence of another KISS1 receptor and/or paracrine signaling. The findings raise the possibility that soluble KISS1, kisspeptins, or mimetics could be used to maintain tumor dormancy, rendering treatment of already disseminated tumor cells (i.e., micrometastases) a legitimate target.
| CONTEXT AND CAVEATS Prior Knowledge The KISS1 gene encodes a secreted protein that, in the context of sexual maturation, is processed to peptides known as kisspeptins. Kisspeptins exert effects on sexual development by binding to the G protein–coupled receptor GPR54. KISS1 was initially identified as a metastasis suppressor, but whether KISS1 secretion, processing, and GPR54 binding are necessary for suppression of metastasis is not known. Study Design A human melanoma cell line that does not express KISS1 was transfected with an easily detectable tagged form of KISS1 that was either unaltered or altered so that it could not be secreted. Transfected cells were analyzed in vitro and in mice. Contributions Only cells transfected with the secretable tagged form of KISS1 secreted kisspeptins to the medium. Such cells showed less metastatic spread in vivo than the cells transfected with the nonsecretable form of KISS1. GPR54 was expressed at a very low level in the melanoma cell line used in the study. Implications KISS1 metastasis suppressor activity requires secretion and processing of the protein, but kisspeptin binding to GPR54 may not be necessary. Kisspeptins may have the potential to modify metastasis in vivo. Limitations A single cell line was used. Whether metastasis of the transfected line in mice accurately reflects its behavior in humans is not known. The ability of exogenously supplied kisspeptins to suppress metastasis in vivo was not tested.
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Manuscript received July 13, 2006; revised December 7, 2006; accepted January 4, 2007.
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J Natl Cancer Inst 2007 99: 257.
J Natl Cancer Inst 2007 99: 977.
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