Stress-responsive Protein RBM3 Inhibits Prostate Cancer Bone Metastasis Progression By Upregulating CTNNB1 M~6A Methylation

Objective:Prostate cancer is one of the most common malignant tumors in men worldwide.It is an invasive cancer,which is composed of prostatic epithelial cells with secretory function and can have a variety of histomorphology with basal cell loss.The lesions mainly occur in the peripheral region of the prostate.At present,it is believed that the risk factors of prostate cancer include inflammation,infection,smoking,obesity,alcohol,genetic susceptibility and so on.At the molecular level,gene mutation,promoter methylation and epigenetic modification are all considered to be involved in the development of prostate cancer.The development of bone metastasis in prostate cancer is a staged process that includes(1)the colonization of disseminated tumor cells(DTCs).(2)DTCs are dormant in the bone microenvironment.(3)the reactivation and development of DTCs.(4)remodeling of bone microenvironment by DTCs.Wnt signaling plays an important role in this series of processes.The β-catenin in prostate cancer tissues was highly expressed in 55%of primary prostate cancer and 85%of bone metastatic prostate cancer tissues.Bone microenvironment is not only suitable for the survival of disseminated DTCs,but also endows tumor cells with the ability to improve stemness and secondary metastasis.N6-methyladenosine(m6A)is the most common,abundant and conserved RNA modification in mammals.m6A modification is written by m6A methyltransferases(such as METTL3/14/16,RBM15/15B,VIRMA,KIAA1492,ZC3H3,etc.).It is read by m6Abinding proteins(such as YTHDF1/2/3,YTHDC1/2,HNRNPA2B1,IGF2BP1/2/3,etc.)and eventually removed by demethylases(such as ALKBH5,FTO).RNA m6A modification can occur in different types of RNA,such as mRNA,tRNA,rRNA,circRNA,miRNA,lncRNA,etc.,and plays an important role in regulating RNA alternative splicing,translation,translocation,and stability.Most of the m6A sites were found near the stop codon and contained in the conserved motif DRACH(D=G/A/U,R=G/A,H=A/U/C).m6A regulation also plays an important role in prostate cancer.For example,most aggressive prostate cancers are accompanied by elevated total levels of m6A due to the presence of CNVs events(DNA copy number loss)in FTO and ALKBH5,as well as high expression of m6A-related "Reader" and "Writer"[1].The total level of m6A will gradually increase with the progression of prostate cancer,which is related to high Gleason score.RBM3 is a nuclear RNA binding protein and a cold stress protein.It mainly exists in the Sertoli cells of the mammalian testis and participates in the regulation of a variety of cell functions,especially cell adaptation and neuroprotection under low temperature environment.RBM3 is up-regulated in a variety of cancer types and is closely related to the prognosis and development of prostate cancer.Previous studies from our group have shown that RBM3 expression is significantly decreased during thermotherapy in prostate cancer cells,which may partly increase the sensitivity of prostate cancer to thermotherapy.Furthermore,we found that RBM3 overexpression attenuated the stemness characteristics of prostate cancer cells by interfering with the alternative splicing of CD44.The apparent downregulation of RBM3 expression in bone metastatic prostate cancer compared with orthotopic tumors,together with the enhanced stemness characteristic of bone metastatic prostate cancer,led us to be interested in investigating the role of RBM3 in bone metastatic prostate cancer.Results:1.CLIP-seq results showed that the RNA-binding protein RBM3 was closely related to β-catenin,a key protein of Wnt canonical signaling,and RBM3 interacted with CTNNB1(β-catenin mRNA),and overexpression of RBM3 inhibited β-catenin Expression of downstream target genes.Compared with in situ prostate cancer,RBM3 protein expression was decreased in bone metastatic prostate cancer and was accompanied by increased β-catenin protein expression.2.Overexpression of RBM3 inhibits the effect of osteoblast co-culture on the enhancement of prostate cancer cell stemness by inhibiting the activation of β-catenin.3.RBM3 can bind to the full length of CTNNB1,but it is more inclined to the 3’UTR region.Overexpression of RBM3 can down-regulate the level of total m6A in prostate cancer cells and selectively increase the level of methylation at CTNNB1 sitel,thereby down-regulating the stability of CTNNB1.4.The overall m6A level of prostate cancer cells increased under osteoblast co-culture,and the expression of CTNNB1 increased after co-culture,accompanied by obvious extranuclear export,and overexpression of RBM3 could still inhibit the methylation modification of CTNNB1 m6A sitel by enhancing During this process,the nuclear-localized RBM3 showed a clear phenomenon of co-exporting with CTNNB1.5.The bone metastasis of prostate cancer was simulated by injecting prostate cancer cells into the bone marrow cavity of the femur of mice.After 6 weeks of feeding,it was found that the bone metastases formed by prostate cancer cells overexpressing RBM3 were smaller than those of the control group,and by immunohistochemistry we found that the level of β-catenin protein in the RBM3overexpressing group was lower than that in the control group,reflecting a trend consistent with in vitro experiments.Conclusion:RBM3 is closely correlated with Wnt/β-catenin signaling and bone metastasis in prostate cancer.Overexpression of RBM3 inhibits the activation of Wnt/βcatenin signaling by binding to CTNNB1 and selectively methylation at the 3’UTR of CTNNB1.These results leading the loss of the stemness of prostate cancer cells under osteoblast co-culture and seriously affects the growth of prostate cancer cells in the bone microenvironment.