Effects Of G Protein-coupled Estrogen Receptor Agonist G-1 On Mantle Cell Lymphoma

Mantle cell lymphoma(MCL)is a rare and aggressive form of B-cell lymphoma,accounting for 3-7%of non-Hodgkin’s lymphoma(NHL).90%MCL patients carry translocation t(11;14)(q13;q32)that results in overexpression of cyclin D1 and increased cell proliferation.Additional genomic alterations,which are involved in cell cycle,DNA damage,signal transduction,and apoptosis,are also found to contribute to MCL progression and resistance to conventional chemotherapy.Recent advances in MCL therapy,including the development of Bruton’s tyrosine kinase(BTK)inhibitor such as Ibrutinib and the application of immunomodulatory imide drugs such as lenalidomide to MCL,have established chemo-free regimes as a promising new direction of MCL therapy.However,several challenges remain.For example,primary and acquired resistance to ibrutinib is common,leading to poor prognosis.Thus,there is an urgent need to identify new targeted therapeutic options for MCL.MCL has a high male/female ratio,which is up to 2.5-3.5:1,and male gender is considered an independent negative prognostic factor.In addition to conventional estrogen receptors ER-α and ER-β,estrogens also mediate rapid signaling pathways via G-proteincoupled estrogen receptor(GPER).GPER is involved in non-genomic estrogenic signaling including calcium mobilization and generation of cyclic AMP,and stimulation of GPER activates matrix metalloproteinases,epidermal growth factor receptor(EGFR),ERK pathways.Studies have revealed that GPER exhibits an important function in various pathological processes including malignant diseases.G-1 is a GPER-selective agonist.It has been shown that activation of GPER with G-1 inhibit several cancers,such as hepatocellular carcinoma and colorectal cancer.However,reports on GPER’s involvement in MCL are rare,and its functions and underlying mechanisms in lymphoma remain to be addressed.Methods1.Data of GPER protein and mRNA expression in lymphoma was retrieved from Human Protein Atlas(https://www.proteinatlas.org)and Oncomine(https://www.oncomine.org).GPER protein expression was analyzed in lymphoma biopsies from NHL patients and in 6 normal lymph node biopsies with immunohistochemistry method,as well as in 4 MCL cell lines with Western blot.2.GPER antagonists G-36 and G-15 were used to inhibit GPER in MCL cells,and cell proliferation and apoptosis were analyzed with Cell Counting Kit(CCK-8)and via detecting Annexin V/PI using flow cytometry.In addition,GPER expression of MCL cells was reduced via siRNA transfection,followed by proliferation and apoptosis analysis.To activate GPER,MCL cells were treated with GPER agonist G-1,and then various analysis were performed including:cell cycle via PI staining and flow cytometry,Mitochondrial membrane potential via JC-1 staining and flow cytometry,reactive oxygen species(ROS)via flow cytometry tracking the conversion of H2DCFDA to fluorescent DCF,DNA damage via Western blot detecting DNA damage marker y-H2A.X,apoptosis via Western blot examining caspase activation,NF-κB signaling via Western blot observing the phosphorylation level of P65.3.To observe the in vivo effects of G-1 on MCL,MCL-xenografted mice were established by injecting MCL cells into SCID mice.These MCL mice were randomly split into two groups,one was treated with G-1 and the other with vehicle control.Tumor growth was observed and recorded.The expression of DNA damage marker γ-H2A.X and proliferation marker Ki67 in the tumors were detected with immunofluorescent and immunohistochemistry methods.The number and area of lymphatic vessels in tumor tissues were obtained by analyzing PDPN and LYVE-1 labeling.To observe the metastasis of MCL,the MCL model in lymph nodes was established by injecting MCL cells into the inguinal lymph node of male SCID mice.Results1.The expression of GPER is decreased in patients with NHLAccording to the data in Human Protein Atlas and Oncomine database,GPER is expressed in normal lymph nodes diffusely but decreases in lymphoma.We collected and analyzed 11 MCL and 6 normal lymph nodes biopsies and observed similar pattern.GPER was negative in 2 MCL tissues,yet it was expressed in most of the biopsies from MCL patients and in all 4 MCL cell lines.To determine sex difference of GPER expression in lymphoma tissues,we collected 15 DLBCL tissues,10 FL tissues,5 MALT tissues,4 SLL tissues,and 11 MCL tissues,and observed similar GPER positive rate in female(13/20,65%)and male patients(16/25,64%).2.GPER specific antagonists or knockdown of GPER expression did not inhibit the proliferation of MCL cellsTreatment with up to 10 μM of GPER specific antagonist G-36 did not affect the proliferation and apoptosis of MCL cells,and neither did another GPER specific antagonist G-15.Reducing GPER expression in MCL cells via siRNA transfection slightly increased cell proliferation while cell apoptosis was not affected.3.GPER specific agonist G-1 inhibited MCL cell proliferation and promoted apoptosisIn vitro studies with MCL cell lines indicated that activating GPER with GPER specific agonist G-1 inhibited cell proliferation and promoted apoptosis.We found that G-1 induced G2/M cell cycle arrest,mitochondrial membrane potential reduction and caspase activation in MCL cells.In addition,G-1 induced ROS generation,DNA damage and apoptosis of MCL cells;Pretreatment of MCL cells with ROS scavenger N-acetyl-L-cysteine(NAC)or NADPH oxidase(NOX)1 antagonist ML171 reduced G-1-induced DNA damage and apoptosis,indicating that G-1 caused DNA damage and apoptosis at least partly by NOX1mediated ROS generation.G-1 inhibited NF-κB P65 phosphorylation implying that regulating NF-κB pathway contributed to G-1’s inhibition of MCL cell proliferation.4.G-1 reduced tumor growth and lymph node metastasis in MCL-xenografted miceWe established MCL-xenografted models with SCID mice and treated the mice with either vehicle control or G-1.We found that G-1 significantly slowed tumor growth,the DNA damage marker γ-H2A.X was presented in G-1 treated tumors,and G-1 treated tumors had less Ki67 expression than the control group.By detecting lymphatic markers podoplanin(PDPN)and LYVE-1,we found abundant lymphatic vessels in the tumors of control group,while decreased lymphangiogenesis was observed in G-1 group.In the lymph nodes of MCL mice without G-1 treatment,we found MCL cells spreading to the ipsilateral axillary lymph nodes,but G-1 treatment reduced the spreading.ConclusionWe found that GPER expression was decreased in NHL,and for the first time we demonstrated that GPER specific agonist G-1 inhibited MCL both in vitro and in vivo.GPER activation with selective agonist G-1 induced cell cycle arrest,DNA damage,mitochondria membrane potential abnormality,and eventually apoptosis of MCL cell lines.We found that G-1 induced DNA damage and apoptosis of MCL cells by promoting reactive oxygen species via nicotinamide adenine dinucleotide phosphate oxidase,and inhibited MCL cell proliferation by inactivation of NF-κB signaling.In addition,G-1 exhibited anti-tumor functions and inhibited metastasis in MCL xenografted mice.