Inhibitory Effect And Molecular Mechanisms Of Intrabody Against Cyclin D1 On The Growth And Metastasis In Triple Negative Breast Cancer

Breast cancer is the most common cancer among women worldwide.It is also the most common cancer among women in China,which counts for the most morbidity and cancer related mortality.Triple-negative breast cancer(TNBC)is a heterogeneous collection of lacking expression of estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor 2(HER2)amplification.Patients with TNBC have a higher risk of both local and distant invasion and metastases and acquired resistance to drugs,while there was no standard for the choice of therapeutic strategies..TNBC constitutes 20% of all breast cancers and 83% of total breast cancer death.Therefore,it is an urgent problem to develop effective and specific treatments for triple negative breast cancer.Cyclin D1 is one of the cyclins encoded by CCND1,overexpressing in mutilple cancer cases,which is an important target for cancer treatment.However,the studies of the overexpression and prognostic significance of cyclin D1 in TNBC were controversial.An association of expression of cyclin D1 and the outcomes of TNBC remains controversial.In this regard,the exploration of cyclin D1 expression and location would explore the mechanisms of cyclin D1 in TNBC tumorigenesis and therapy.Intrabody technology has the ability to express functional antibodies,which can knock down the target protein at the post-translational level and serve as complementary means of gene knockdown such as RNAi(RNA interference)and small molecule inhibitors.Accumulating researches show that the intrabodies were designed to locatilize target proteins to a different compartment.Thus,intrabodies play a vital role in the treatment and diagnosis of many types of diseases such as viral infection,degenerative diseases and cancers.In our previous work,an ER-retained anti-cyclin D1 intrabody(ER-ADκ)was constructed,which could bind and inhibit cyclin D1 activity in cells.It inhibited the proliferation,migration,and also induced apoptosis in ER+ breast cancer and hepatocellular carcinoma.However,the effect of ER-ADκ on triple negative breast cancer remains unknown.Therefore,this current study was designed to analyze the expression and enriched pathways of cyclin D1 by m RNA microarray of TNBC from GEO database to clarify the transcription of CCND1 and the signals involved in TNBC.Furthermore,series of assays were conducted to clarify the inhibitory role and mechanisms of ER-ADκ on proliferation and migration of MDA-MB-231 and BT-549 cells in vitro and in vivo.The results were as follow:1.The features of cyclin D1 were clarified in TNBC and para-cancer tissues.The transcription and enriched pathways of CCND1 was anaylysed by R language and biological software.The results showed that CCND1 was highly expressed in TNBC tissues.PPI networks were contructed by STRING,and Gene Ontology and KEGG enriched pathways were used for analysis.Cyclin D1 participated in the process of cell cycle,breast cancer,cell senescence,and the signaling pathways related to PI3K-AKT,Wnt,etc.The further assays of immunohistochemical staining in breast cancer tissue chip confirmed the overexpression of cyclin D1 in TNBC cancer tissues,provided a hypothesis of cyclin D1 being a promising target for TNBC therapy.2.ER-ADκ targeting to Cyclin D1 dramatically inhibited the proliferation and migration and promoted the apoptosis of TNBCs.After transfection of ER-ADκ plasmids into MDA-MB-231 and BT-549 TNBC cells,indirect immunofluorescence assay,Western blot and co-IP had been conducted and the results showed that anti-cyclin D1 intrabody(ER-ADκ)was successfully expressed in triple negative breast cancer cell lines such as MDA-MB-231 cells and BT-549 cells and was localized in the endoplasmic reticulum.ER-ADκ could target to cyclin D1 protein directly.Flow cytometry analysis found that ER-ADκ could cause G1/S phase arrest of MDA-MB-231 cells and induce apoptosis of MDA-MB-231 cells.By wound healing and transwell assays,we found that ER-ADκ could inhibit the migration,invasion of MDA-MB-231 cells and BT-549 cells in vitro.The results of the transplanted tumor model of nude mice and the lung metastasis model of tumor cells injected into the tail vein of nude mice showed that ER-ADκ inhibited the growth and proliferation of tripe negative breast cancer cells in vivo,and also suppressed the distant metastasis and lung metastasis of orthotopic breast cancer.These results indicated that ER-ADκ binding to cyclin D1 specifically inhibited the growth,proliferation and metastasis of triple-negative breast cancer cells in vivo.3.The molecular mechanisms of ER-ADκ inhibiting the occurrence and development of TNBC were further investigated.It was found that ER-ADκ binding to cyclin D1,inhibited the phosphorylation of Rb,thereby up-regulated the expression of cell cycle inhibitors such as p21 and p27.ER-ADκ also induced the ER stress to promote the apoptosis.At the same time,ER-ADκ inhibited the EMT-related genes such as N-cadherin,MMP2,MMP9,MMP14,promoted the expression of E-cadherin,and suppressed the translocation of β-catenin from cytoplasm to nucleus,which resulted in the inhibiton of EMT process and the metastasis of TNBC.4.ER-ADκ inhibited the function of cyclin D1 in proliferation and metastasis in vivo.The results showed that ER-ADκ significantly suppressed reduced the weight and volume of MDA-MB-231 cell xenografts in nude mice and induced the ER stress and apoptosis of MDA-MB-231 cells in vivo by the assays of MDA-MB-231 cells injected subcutaneous xenograft tumor in nude mice.Furthermore,ER-ADκ reduced suppressed the formation of lung nodules,it was found that ER-ADκ repressed effectively the ability of metastasis of TNBC effectively cells into the lungs by improving the level of E-cadherin and restraining the β-catenin in the cytoplasm.In summary,cyclin D1 is overexpressed in TNBC tissues and plays a crutical role in the tumorigenesis in of TNBC through promoting cell proliferation,migration and infiltration invasion.By transferring the plasmid of ER-ADκ into MDA-MB-231 cells and BT-549 cell lines,ER-ADκ can be overexpressed and bind to Cyclin cyclin D1 in the endoplasmic reticulum,which inhibits the ability of proliferation and metastasis in vivo and in vitro in for TNBC.Furthermore,molecular mechanisms studies indicate that ER-ADκ may inhibit TNBC proliferation by inhibiting the phosphorylation of Rb,inducing the ER stress mediated apoptosis and restrained β-catenin in the cytoplasm to prevent the EMT process and metastasis.It is the first attempt to knock down cyclin D1 with intrabody in TNBC cells.These data indicate that the intracellular antibodies targeting cyclin D1 can inhibit the growth and metastasis of TNBC significantly and the molecular mechanisms are further explored.This study provides an experimental evidence targeting cyclin cyclin D1 in TNBC therapy.Morever,these results will promote the development of intracellular antibodies as new anti-cancer therapy in future.