Studies On The Roles Of GPCRs In Mammary Development And Tumorigenesis

GPCR(G protein-coupled receptor)family comprises proteins with seven trans-membrane domains that function as the receptors to sense the stimuli outside the cells and activate the inside signaling pathway.They are widely involved in cell proliferation,differentiation,migration and so on,which regulate various types of normal and abnormal physiological activity.There are a lot of drugs based on GPCR have been developed.In this study,we investigated three GPCRs(GPR48,GPR54 and GPR124)functions separately in breast stem cells,breast development,breast cancer,and tumor angiogenesis?1.Gpr48 regulates mammary development and mammary stem cell self-renewal through Sox2;Gpr48 delays breast cancer progression and lung metastasis.GPR48,also known as leucine-rich repeat-containing G protein-coupled receptor 4(LGR4),belongs to the leucine rich G protein couple receptor family(LGRs).Members of this family have multiple leucine rich repeat at N terminal and a GPCR trans-membrane domain.Members of the family have been reported to mark stem cell and regulate stem cell renewal in several tissues.In this study,Gpr48 gene trap mutant mice was used to explore the biological function and the mechanism of Gpr48 in mammary gland development,mammary stem cell and breast cancer progression and metastasis.We report here that genetic ablation of Gpr48 causes mammary stem cell depletion and functional impairment.There was a dramatically decrease in the number of functional mammary stem cells in Gpr48-/-mammary glands as determined by mammary regeneration following limiting dilution transplantation,and the mammosphere formation ability of Gpr48-/-MECs diminished rapidly after three passages,indicating a crucial role for Gpr48 in mammary stem cell self-renewal.Branching morphogenesis was impaired in the absence of Gpr48,resulting in a persistent decrease in ductal tree side branching in Gpr48-/-mice.Mechanistically,Gpr48 loss resulted in decreased expression of Wnt target genes,especially Sox2,a transcriptional factor that is also a key factor in cell reprograming.Sox2 overexpression in Gpr48-/-mammary cells restored mammary regenerative potential,demonstrating Sox2 as a critical effector of Gpr48 in mammary stem cell self-renewal.We also found that LEF binding and CRE binding to the Sox2 promoter was decreased inGpr48-/-mammary cells,and that treatment with Wnt3a rescued the impaired organoid branching morphogenesis of Gpr48-/-mammary cells.Altogether,our data support a model which Gpr48 regulation of Sox2 through both the Wnt/?-catenin/Lef signaling and the cAMP/PKA/CRE signaling to control mammary stem cell self-renewal and functions.We used MMTV-PyMT transgenic mouse model and crossed Gpr48+/-mice with MMTV-PyMT mice.By H&E staining for 6,9,13 and 15 weeks old mice tissue,we found that breast cancer progression was delayed in both PyMT&Gpr48+/-and PyMT&Gpr48-/-mice compared to the wild-type allele.Moreover,Gpr48 haploinsufficiency inhibited breast cancer cell proliferation.The data of tumor free curve showed that the Gpr48+/-delayed tumor progression and significantly reduced the number of tumors.Also,we found that Gpr48 haploinsufficiency inhibited lung metastasis.Furthermore,by using shRNA control and shRNA GPR48 infected breast cancer cells we found that knockdown of GPR48 can inhibite breast cancer cell migration and invasion.2.Gpr54 haploinsufficiency delays breast tumor initiation,progression and lung metastasisActivation of KiSSl receptor(KiSS1R or GPR54)by its ligands(kisspeptins)regulates a diverse function both in normal physiology and pathophysiology.In cancer,KiSSl-induced GPR54 signaling is known to inhibit tumor angiogenesis and metastasis.However,roles of KiSSl and GPR54 in earlier stages of tumor progression and metastasis in vivo are still unknown.In this study,we demonstrate a critical role for GPR54 in early stages of tumor progression using mouse tumor models.PyMT/Gpr54 mice with different Kiss lr genotypes were obtained by crossing MMTV-PyMT transgenic mouse with Gpr54 heterozygous mouse(Gpr54+/-).Gpr54 heterozygosity attenuated breast tumor initiation,growth,latency,multiplicity and metastasis in MMTV-PyMT/Gpr54 mouse models.To confirm the effects of Gpr54 in tumor progression and limit any effect of endogenous hormones,we isolated primary tumor cells from PyMT/Gpr54+/+ or PyMT/Gpr54+/-mice and performed in vitro and in vivo tumorigenesis assays.Gpr54 heterozygosity inhibited PyMT-induced in vitro tumorigeneity and in vivo tumor growth in NOD.SCID/NCr mice.To understand the underlying mechanism,we showed that activation of Gpr54 by kisspeptin-10 led to RhoA-activation and RhoA-dependent gene expression through Gaq-p63RhoGEF signaling pathway.Furthermore,anchorage-independent growth was tightly linked to the dosage-dependent regulation of RhoA by Gpr54.When MCF10A cells overexpressing H-Rasv12 were subjected to in vitro tumorigenesis assays,knockdown of GPR54 or inactivation of RhoA in MCF10A cells reduced Ras-induced anchorage-independent growth,similar to our data obtained from PyMT/Gpr54+/-mouse models.Altogether,we conclude that Gpr54 haploinsufficiency delays breast tumor initiation,progression and metastasis through its downstream Gaq-p63RhoGEF-RhoA signaling pathway.3.GPR124 in endothelial cells regulates VEGF-induced tumor angiogenesisG protein-coupled receptor 124(GPR124;also called tumor endothelial marker 5,TEM5)is highly expressed in tumor angiogenic vessels.While recent studies have revealed GPR124 role in vasculogenesis,its function in tumor angiogenesis is still unknown.Here,we demonstrate that GPR124 is required for VEGF-induced tumor angiogenesis.GPR124 is highly expressed in tumor angiogenic vessels in situ and in cultured vessel cells such as human umbilical vein endothelial cells(HUVEC)and human microvessel cells(HMEC).In xenograft mouse tumor growth assay,GPR124 silencing in endothelial cells inhibited tumor angiogenic vessel formation and tumor growth.In the in vitro studies,GPR124 regulated VEGF-induced tumor angiogenic processes such as cell-cell interaction,permeability,migration,invasion,and tube formation with less effect on proliferation.Furthermore,VEGF regulated GPR124 expression and a cleavage of its extracellular region.N-terminal fragment of GPR124 containing RGD motif inhibited cell migration and tube formation.GST-RGD derived from GPR124 N-termini induced endothelial cell attachment and blocked integrin ?V?3-or ?5?1-mediated cell interaction,indicating that N-terminal fragment of GPR124 serves as a ligand of integrins.Altogether,our study concludes that GPR124 positively regulates VEGF-induced tumor angiogenesis.