Introduction Relaxin levels are increased in cases of human breast cancer and has been shown to promote cancer cell migration in carcinoma cells of the breast, prostate gland and thyroid gland. In oestrogen receptor alpha-negative MDA-MB-231 human breast cancer cells, relaxin was shown to downregulate the metastasis-promoting protein S100A4 (metastasin), a highly significant prognostic factor for poor survival in breast cancer patients. The cellular mechanisms of relaxin exposure in breast cancer cells are not fully understood. The aim of this study was to investigate short-term and long-term effects of relaxin on cancer cell motility and S100A4 expression and to determine the long-term effects of relaxin on in vivo tumour growth in an oestrogen-independent context.

Relaxin and INSL3 are novel autocrine/paracrine insulin-like hormones in tumor biology. Both effectors can bind to and activate the leucine-rich G-protein coupled receptors LGR7 relaxin receptor) or LGR8 (relaxin/INSL3 receptor). These relaxin-like ligand-receptor systems modulate cellular functions and activate signaling cascades in a tumor-specific context leading to changes in tumor cell proliferation, altered motility/migration and enhanced production/secretion ofpotent proteolytic enzymes. Matrix-metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP) and acid hydrolases such as cathepsins can facilitate tissue degradation and represent important proteolytic mediators of relaxin-like actions on tumor cell invasion and metastasis. This review presents recent new findings and emphasises the important functions of the relaxin/INSL3 ligand-receptor system as novel autocrine/paracrine effectors influencing tumor progression and tissue invasiveness.

The functional role of INSL3 and its receptor RXFP2 in carcinogenesis is largely unknown. We have previously demonstrated (pro-)cathepsin-L as a target of INSL3 in human thyroid cancer cells facilitating penetration of tumor cells through elastin matrices. We demonstrate the expression of RXFP2 in human thyroid tissues and in mouse follicular thyroid epithelial cells using Cre-recombinase transgene driven by Rxfp2 promoter. Recombinant and secreted INSL3 increased the motility of thyroid carcinoma (TC) cells in an autocrine/paracrine manner. This effect required the presence of RXFP2. We identified S100A4 as a novel INSL3 target molecule and showed that S100A4 facilitated INSL3-induced enhanced motility. Stable transfectants of the human follicular TC cell line FTC-133 expressing and secreting bioactive human INSL3 displayed enhanced anchorageindependent growth in soft agar assays. Xenotransplant experiments in nude mice showed that INSL3, but not EGFP-mock transfectants, developed fast-growing and highly vascularized xenografts. We used human umbilical vein endothelial cells in capillary tube formation assays to demonstrate increased 2-dimensional tube formations induced by recombinant human INSL3 and human S100A4 comparable to the effect of vascular endothelial growth factor used as positive control. We conclude that INSL3 is a powerful and multifunctional promoter of tumor growth and angiogenesis in human thyroid cancer cell xenografts. INSL3 actions involve RXFP2 activation and the secretion of S100A4 and (pro-)cathepsin-L.Comprising 1% of all malignancies, thyroid cancer is the most common carcinoma of endocrine glands and displays the highest increase in incidence of all malignancies in the United States over the time interval 1975-2000 (http://seer. cancer.gov/csr/1975_2004/; www.cancer.ca). 1 There are 4 types of thyroid carcinoma (TC) that comprise >98% of all thyroid malignancies: papillary (PTC), follicular (FTC), anaplastic, undifferentiated (UTC) and medullary TC (MTC). We showed previously that the insulin-like peptide hormone, insulin-like peptide 3 (INSL3) and a novel INSL3 splice form are present in human hyperplastic thyroid adenoma and thyroid cancer. 2 INSL3 is a member of the relaxin family and signals through the type C leucine-rich repeat G proteincoupled receptor RXFP2, also named GREAT and LGR8. [3][4][5][6][7] Activation of RXFP2 causes an increase in cAMP levels and, via the actions of the small G-proteins Ga s and Ga oB , activates and negatively modulates adenylate cyclase activity, respectively, which affects cAMP-response element transcriptional activity. [8][9][10] Deletion of the gene for INSL3 or the INSL3 receptor causes impaired transabdominal testis descent and cryptorchidism in rodents and boys. [11][12][13][14] Both INSL3 and the homologous peptide relaxin are found in tumor tissues but little information is currently available on the functional role of both the INSL3-RXFP2 and the relaxin-RXFP1 system in cancer cells. 15 In prostate cancer, increasing evidence from cell an...

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Relaxin increases cell motility and in vitro invasiveness in human thyroid carcinoma cells but the underlying molecular mechanisms of this action are largely unknown. In the present study, we show that relaxin transcriptionally upregulates the calcium-binding protein S100A4 (metastasin) and increases the cytosolic 10-kDa monomer and the 20-kDa dimer form of S100A4 in human thyroid carcinoma cells. The relaxin-induced increase in cell motility was blocked completely when S100A4 expression was diminished using an S100A4 small interfering RNA knockdown approach. We have shown previously the expression of the insulin-like family member relaxin in human thyroid carcinoma tissues but not in benign thyroid tissues. Human thyroid carcinoma tissues expressing relaxin also stained positive for S100A4. In nude mouse experiments, human thyroid carcinoma cell transfectants with constitutive expression of relaxin generated large and fast-growing tumors with significantly increased numbers of proliferating cells. We provide evidence in our cell model that the relaxin target protein S100A4 secreted by the thyroid carcinoma transfectants may not only enhance tumor cell motility but also promote xenograft angiogenesis as determined by the higher density of tumor microvessels and the angiogenic potential of S100A4 in in vitro tube formation assays. In conclusion, we have identified S100A4 as a major mediator of the actions of relaxin in thyroid carcinoma cell motility and in vivo thyroid tumor angiogenesis.