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Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor

Abstract

Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene1, that suppresses metastases of human melanomas2 and breast carcinomas3 without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named ‘metastin’. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.

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Figure 1: Purification of metastin.
Figure 2: Pharmacological characterization.
Figure 3: Cell migration and invasion.
Figure 4: Formation of focal adhesions and stress fibres.
Figure 5: Quantitative RT-PCR analyses of KiSS-1 and hOT7T175 transcripts in human multiple tissue cDNA panels and matched cDNA pairs (Clontech).

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Accession codes

Accessions

GenBank/EMBL/DDBJ

Data deposits

The nucleotide sequence data reported here will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB051065 (hOT7175) and AB051066 (rOT7T175).

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Acknowledgements

We thank R. Sasada, M. Mori, T. Henta, M. Harada, K. Oda and Y. Horikoshi for technical advice and assistance.

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Correspondence to Tetsuya Ohtaki.

Supplementary information

Figure A

Figure A (JPG 41 KB)

The effect of metastin on pulmonary metastases. a, Number of macroscopic metastases (n = 4 in each group). b—d, Total number of microscopic metastases found in 10 slices from each lung (n = 4) is shown for each category: b, colonies over 300 µm or 100 cells; c, colonies of 20—100 cells; d, colonies of 1—20 cells. Typical metastasis in each category is shown below. Bars, 200 µm; **, p < 0.01; *, p < 0.05 (Student’s t test).

METHODS. Spontaneous pulmonary metastasis assay was done as described in the Methods section. After tumor foci were counted macroscopically, lungs were fixed with Mildform 10N (Wako Pure Chemical Ind.), immersed in sucrose (12% for 4 h, 15% for 8 h and 18% for 24 h), and frozen rapidly at –80 °C. Slices (17 µm) were cut by every 150 mm thickness on a cryostat at –16 °C, thaw-mounted onto Super frost slide glasses (Matsunami Glass Ind., Ltd.), dehydrated, and mounted with CANADA BALSAM (SIGMA CHEMICAL CO.). Microscopic metastases were counted using a Nikon ECLIPSE E800 microscope. Sum of metastases in 10 slices was obtained. A single metastasis distributed over two slices was not counted double.

Figure B

Figure B (JPG 14 KB)

The effect of metastin on tumor growth. Tumor size (mean±SE) of saline-treated B16-BL6/h175 melanomas (open circle), metastin-treated B16-BL6/h175 melanomas (closed circle), saline-treated B16-BL6/mock melanomas (open square), and metastin-treated B16-BL6/mock melanomas (closed square). N = 7 for each group.

METHODS. B16-BL6/h175 or B16-BL6/mock cells (3 x 106 cells/100 µl) were inoculated subcutaneously into the left flank of female C57 BL/6 mice (9 weeks old) on day 0. On day 6, an Alzet pump (Model 1002, Alza) containing 1 mM metastin (100 µl in saline) was implanted subcutaneously in the mice. Tumor measurements were made in two directions using calipers, and tumor volume was calculated by using a2b*1/2, where "a" is the width and "b" is the length of tumor.

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Ohtaki, T., Shintani, Y., Honda, S. et al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411, 613–617 (2001). https://doi.org/10.1038/35079135

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