Functional Modulation Of Melanoma By M3 Muscarinic Acetylcholine Receptor

BackgroundMelanoma is characterized with multiple genetic background,complex cellular signal transduction and plastic biological behaviors.At present,the treatment on melanoma is intractable since its high malignancy,easy-metastasis and poor prognosis.Melanoma cell plasticity and phenotype plasticity maintain the adaption of cancer cells to the microenvironment during the process of growth,migration and metastasis.The plasticity is responsible for the cancer cell biological function switch and metastatic development as well.At present,melanoma cell plasticity is not fully understood.Various factors might be involved the process,including phenotype heterogeneity,dynamic morphologies and growth patterns,metastatic biological behaviors biological behaviors(such as proliferation,affinity,and invasion).Nevertheless,the plasticity provides a novel chance to understand the cancer cell property and therapeutic strategy against malignant melanoma developments.M3 muscarinic choline receptors belong to the G protein coupled receptor family.At present,five mAChR subtypes(M1-M5)have been cloned and further divided into two functional groups according to the different binding ability of these receptors to G protein.MACh R,as a G protein coupled receptor,may regulate cell biological behavior through a variety of intracellular signal mechanisms mediated by different subtypes,and participate in the proliferation and invasion of tumor cells.Among them,the function of M3 receptor involved in the proliferation of other malignant tumors are still obscure.Our previous research results suggest that M3 may activate the ERK pathway after activating PLC and intracellular calcium,which is involved in the regulation of melanoma proliferation.p75 neurotrophin receptor(p75NTR,CD271)belongs to a member of TNF receptor family,is the low affinity receptor common to several mature neurotrophins.A series of studies suggest the contributions of M3 mAchR and p75NTR in melanoma.However,the molecular mechanism remains unclear.AimsThe present study is designed to reveal the interactions and biologcal functions of M3 mAchR and p75NTR in melanoma development.The results might provide therapeutic strategy against malignant melanoma developments.MethodsBy pathological analysis of melanoma tissues and in vitro cell cultures,we study the morphological property and switch.A series of methods in molecular biology,gene interference,we investigate the expressions and functions of muscarinic acetylcholine receptor M3 in morphological switches,proliferation and invasion.Further immunoflurencent staining and co-immunoprecipitation assay show the intercation of M3 and p75 neurotrophin receptor(p75NTR).Results1.Human melanoma tissue and cell culture indicate the ability of morphological switch of melanoma cellsMelanoma cell morphological phenotypes are analyzed in HE tissue samples from the different stages including in situ melanoma confined to the epidermis,superficial spreading melanoma,nodular melanomas in vertical growth phase,and highly aggressive melanoma in lymph node metastases phase.The cancer cells are highly morphological heterogeneity form Pagetoid cell in the epidermis to epithelioid melanocytes with abundant cytoplasm,vesicular nuclei often a brisk mitotic activity to spindle-shaped,hyperchromatic melanocytes that may lack pigment production,and to multinucleated melanoma cells with dense nuclear chromatin and unapparent nucleoli.Statistical analysis show that,from primary tumor to distant metastatic stages,epithelial cells rather than mesenchymal cells dominate human melanoma tissue.We further compare melanoma cells in patient tissue and cultured melanoma cell lines WM793 B,451Lu and A2058.Morphological characterizations of melanoma in patients can be mimicked by in WM793 B and 451 Lu.Differently,highly invasive A2058 cell line derived from lymph node metastases transmitting from skin melanoma characterize in the spindle-shaped mesenchymal cells2.Invasiveness-triggered state transition(ITST): a novel model of melanoma cell plasticityIn the scratch assay,A2058 cells migration into the scratch maintained spindle-shaped mesenchymal tumour cells at 12 h,24 h,48 h,indicating no morphological transition during superficial migration.In the transwell test,the original spindle-shaped A2058 melanoma cells show epithelioid morphology after vertical invading through 8 μm-diameter micropore.These results indicate the morphological transitions during malignant melanoma cells vertical invasion and metastasis.In general,at least two major stages of ITST are involved.The first stage(within 72 h)is invading melanoma cells transit to epithelial cells maintained for about three days after vertical invasion.The following stage(after 96h)is the morphological recovering process from epithelial cells to spindle-shaped mesenchymal cells.Therefore,we established a cellular model of invasiveness-triggered state transition(ITST)that the morphological changes induced by melanoma cell transwell invasion.ITST is potential mechanism to maintain the epithelial cells state during invasion and metastasis into deep tissue.Immunofluorescent staining is applied to detect the expressions of epithelial mesenchymal transition(EMT)biomarkers,including E-cadherin,desmoplakin,N-cadherin,vimentin in A2058 melanoma cells.EMT-related protein E-cadherin is highly expressed in epithelial cells and vimentin is highly expressed in mesenchymal cells.Although desmoplakin and N-cadherin are detectable in A2058 melanoma cells by immunofluorescent staining,the dynamic changes are slight from epithelial to mesenchymal cells.These results support that morphological transition during melanoma cancer cells invasion are associated with the dynamic changes of EMT-related proteins.E-cadherin and vimentin are valuable biomarker for morphological switch.3.Muscarinic acetylcholine receptor M3 modulates melanoma cell proliferation,invasion and ITSTIt was largely unknown on the mechanism of muscarinic acetylcholine receptors in melanoma.Here,we focus on M3 muscarinic acetylcholine receptor.Western blot suggests the most significant inhibitory effect of siRNA 1 and the minor effects of siRNA 2 and siRNA 3.MTT test is to examine the effects of M3 siRNA on melanoma cell proliferation.The inhibitory effect is 48.1%,22.3% and 20.1% at 24 h,48h and 72 h,respectively.The peak suppressive role at 24 h gradually decreases after siRNA treatment.Transwell invasion assay is applied for melanoma cell invasion and morphological changes.The results show that M3 siRNA dramatically decreased the total number of invaded melanoma cells and morphological state transition.The cell number and percentage of epithelial cells are decreased after siRNA treatment.On the country,the cell number and percentage of spindle-shaped mesenchymal cell are increased after siRNA treatment.4.Targets and signaling pathway of muscarinic acetylcholine receptor M3We used protein chips and siRNA techniques toidentifed the targets and signaling pathways of muscarinic acetylcholine receptor M3.In the siRNA treated group,17 out 157 phosphorylation proteins expressions were increased,6 proteins were decreased and 134 proteins was not changed.Further KEGG pathway and STRING analysis showed that M3 siRNA could affect multiple cancer-related signaling pathways and molecules including nerve growth factor receptor.These results indicated the potential interactions of M3 and p75 neurotrophin receptor(p75NTR)and p75NTR was the pontential targets of muscarinic acetylcholine receptor M3.5.Interactions of M3 and p75 neurotrophin receptor(p75NTR)Double immunofluorescence staining shows that M3 and p75NTR intensively co-expressed in cytoplasm and cell membrane of spindle-shaped mesenchymal cells.However,M3 not overlapping with p75NTR is present in cell membrane of epithelial tumour cells.The results suggest that the co-expression of M3 and p75NTR might be correlated with melanoma cell morphology.Co-IP experiments are performed to test the interaction of M3 and p75NTR.Pharmacology test showed that M3 agonist Pilocarpine enhanced melanoma cell proliferation and invasion and p75NTR antagonist NSC49652 suppressed these respones.Western blot showed that M3,p75NTR,p-CDK1 and p-AKT proteins were increased by by Pilocarpine and suppressed by NSC49652.These data indicated the interaction of M3 and p75NTR involved in melanoma development.6.CDK1 as one of the downstream of M3-p75NTR signaling pathwayProtein chips showed the decreased expression of p-CDK1 in M3 siRNA treatment.Bioinformation analysis showed that CDK1 was one of the downstream molecules of M3-p75NTR signaling pathway.Western blot data further supported that M3 siRNA indeed suppressed the protein expressions of P75 NTR and p-CDK1.Based on these observations,we provided a novel cellular singling pathway in melanoma cell regulation: M3-p75NTR-CDK1 signaling pathway.7.Roles of muscarinic acetylcholine receptor M3 and signaling pathways in melanoma in vivoConstruction of cell line showed stable expression of A2058-m Cherry.The effects of M3 siRNA on the fluorescence density of A2058-m Cherry cells were detected.The results showed that the relative intensity of m Cherry and DAPI were suppressed by M3 siRNA.In vivo effects of M3 siRNA were tested in the tumor volume in null mice 1-6 weeks after A2058-m Cherry cells xenograft.The tumor volume(mm~3)of control mice was 0,0,89.2,822.8,1463.0,2128.0.The tumor volume(mm~3)of M3 siRNA group was 0,0,37.2,104.6,192.0,246.7.These results suppoted the suppressive effects of M3 siRNA on melanoma growth in vivo.In vivo melanoma imagings showed that,6 weeks ater A2058-m Cherry cells xenograft in null mice,the bioluminescence(p/sec/cm~2/sr/m W/cm~2×10~9)was 7.8 and 3.2 in control and M3 siRNA groups.Epithelial cells are prominent in A2058-m Cherry xenograft in null mice.Double immunofluorescent staining showed that,compared with the non specific expressions of EMT markers Vimentin,E-cadherin and N-cadherinin in extracellular matrix of melanoma tissue,M3 mAChR was a better indicator for melanoma cell proliferation in vivo.Immunofluorescent staining showed the expressions of M3,p75NTR,p-CDK1 in A2058-m Cherry melanoma cells xenograft in null mice.Western blot was used to check the proteins expressions in melanoma tissue.The relative expression of GAPDH was 1 and 1.07,the relative expression of M3 mAChR was 1 and 0.40,the relative expression of p75NTR was 1 and 0.23;the relative expression of p-CDK1 was 1 and 0.51 in control group and M3 siRNA groups,respectively.These results showed that M3 siRNA not only suppressed the M3 mAChR expression,but also affected its downstream targets p75NTR and p-CDK1.The results supported the contribution of M3-p75NTR-CDK1 signaling pathway in melanoma malignancy.ConclusionMelanoma cell has ability of morphological switch during metastatic invasion.We established a cellular model of invasiveness-triggered state transition(ITST)that the morphological changes induced by melanoma cell transwell invasion.ITST might be the biological basis maintaining the epithelial cells state during invasion and metastasis.Muscarinic acetylcholine receptor M3 modulates melanoma cell proliferation,invasion and ITST.Interaction of M3 and p75 neurotrophin receptor(p75NTR)in M3-p75NTR-CDK1 signaling pathway is the potential mechanism for these biological functions.M3 inhibition suppressed p75NTR and CDK1 expressions,decreased melanoma cell proliferation and invasion,impaired in vivo growth and development.Epithelial cells are prominent in melanoma cells xenograft in null mice.Compared with the non-specific expressions of EMT markers Vimentin,E-cadherin and N-cadherinin in extracellular matrix of melanoma tissue,M3 mAChR was a better indicator for melanoma cell proliferation in vivo.The results supported the contribution of M3-p75NTR-CDK1 signaling pathway in melanoma malignancy.M3 provides a novel chance to understand the cancer cell property and therapeutic strategy against malignant melanoma developments.