Suppression of relaxin receptor RXFP1 decreases prostate cancer growth and metastasis.

Relaxin (RLN) is a small peptide hormone expressed in several cancers of reproductive and endocrine organs. Increased expression of RLN in prostate cancer correlates with aggressive cancer. RLN G-protein-coupled receptor (RLN family peptide receptor 1, RXFP1) is expressed in both androgen receptor (AR)-positive and -negative prostate cancers as well as in prostate cancer cell lines. RLN behaves as a cell growth factor and increases invasiveness and proliferation of cancer cells in vitro and in vivo. The objective of this study is to determine whether downregulation of RXFP1 expression using small interfering RNA (siRNA) reduces cancer growth and metastasis in a xenograft model of prostate cancer. We used two well-characterized prostate adenocarcinoma cell lines, AR-positive LNCaP cells and AR-negative PC3 cells. The tumors were established in nude male mice by s.c. injections. Intratumoral injections of siRNAs loaded on biodegradable chitosan nanoparticles led to a downregulation of RXFP1 receptor expression and a dramatic reduction in tumor growth. In LNCaP tumors, the siRNA treatment led to an extensive necrosis. In PC3 xenografts treated with siRNA against RXFP1, the smaller tumor size was associated with the decreased cell proliferation and increased apoptosis. The downregulation of RXFP1 resulted in significant decrease in metastasis rate in PC3 tumors. Global transcriptional profiling of PC3 cells treated with RXFP1 siRNA revealed genes with significantly altered expression profiles previously shown to promote tumorigenesis, including the downregulation of MCAM, MUC1, ANGPTL4, GPI, and TSPAN8. Thus, the suppression of RLN/RXFP1 may have potential therapeutic benefits in prostate cancer.

INSL3 has tumor-promoting activity in thyroid cancer.

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 anchorage-independent 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.

Transabdominal testicular descent is disrupted in mice with deletion of insulinlike factor 3 receptor.

BACKGROUND: Several factors are implicated in transabdominal testicular descent, including insulinlike factor 3 (INSL3) hormone and Müllerian-inhibiting substance (MIS). A transgene insertional mutation found on chromosome 5 in the mouse, known as crsp, causes deletion of a transmembrane G protein-coupled receptor gene, Great, which is highly expressed in the gubernaculum. The authors describe here a detailed analysis of the testicular descent and gubernacular development in crsp mice to determine the role of the Great gene in this process. METHODS: Homozygous (crsp/crsp) mutant and wild-type heterozygous (crsp/+) mice were examined at birth (D 0) and at days 10 (D 10) and 30 (D 30) postnatally. Serial sagittal or coronal sections were analyzed for position of the gonads and cremaster sac development. RESULTS: Transabdominal testicular descent was absent at D 0 in crsp/crsp homozygous mice with no swelling reaction in the gubernacula. By D 10 the cremaster sac was significantly thinner (P <.05) and contained less collagen in the mutants than in the wild-type controls. On D 30 the cremaster sacs of mutant males were similar in thickness to those in females. CONCLUSIONS: Disruption of the Great gene causes failure of the transabdominal phase of descent, identical to that seen in the Insl3-deficient mutants, consistent with the recent data suggesting that Great gene encodes the Insl3 transmembrane receptor. No differences between D 30 mutant males and females were found in the gubernacula, suggesting that Insl3/Great signaling regulates gubernacular development.

Mutations of the GREAT gene cause cryptorchidism.

In humans, failure of testicular descent (cryptorchidism) is one of the most frequent congenital malformations, affecting 1-3% of newborn boys. The clinical consequences of this abnormality are infertility in adulthood and a significantly increased risk of testicular malignancy. Recently, we described a mouse transgene insertional mutation, crsp, causing high intraabdominal cryptorchidism in homozygous males. A candidate gene Great (G-protein-coupled receptor affecting testis descent), was identified within the transgene integration site. Great encodes a seven-transmembrane receptor with a close similarity to the glycoprotein hormone receptors. The Great gene is highly expressed in the gubernaculum, the ligament that controls testicular movement during development, and therefore may be responsible for mediating hormonal signals that affect testicular descent. Here we show that genetic targeting of the Great gene in mice causes infertile bilateral intraabdominal cryptorchidism. The mutant gubernaculae fail to differentiate, indicating that the Great gene controls their development. Mutation screening of the human GREAT gene was performed using DHPLC analysis of the genomic DNA from 60 cryptorchid patients. Nucleotide variations in GREAT cDNA were found in both the patient and the control populations. A unique missense mutation (T222P) in the ectodomain of the GREAT receptor was identified in one of the patients. This mutant receptor fails to respond to ligand stimulation, implicating the GREAT gene in the etiology in some cases of cryptorchidism in humans.