FL3 Targeting PHB1 To Inhibit The Malignant Progression Of Castration-resistant Prostate Cancer And Its Molecular Mechanism

BackgroundProstate cancer is the second most common malignant tumor in men in the world,and it is also a common tumor of the male reproductive system in China.In recent years,the incidence of prostate cancer has increased rapidly with the changes in people’s lifestyles,the aging of the population and the promotion of serum prostate specific antigen(PSA)screening.At present,androgen deprivation therapy(androgen deprivation therapy,ADT)is the standard treatment for localized prostate cancer,but most patients will experience biochemical recurrence or develop into more malignant castration-resistant prostate cancer(CRPC).However,the standard treatment regimen for CRPC(enzalutamide with abiraterone)has limited efficacy,accompanied by gastric and hematological side effects,and eventually drug resistance.Therefore,it is urgent to find new methods to treat CRPC.Prohibitin(PHB,also known as PHB1,UniProtKB-P35232)is abnormally expressed in many types of tumors.The function of PHB1 protein is closely related to its cell location.Recent studies have shown that membrane-localized PHB 1 plays an important regulatory role in the MAPK signaling pathway and is necessary for K-RAS-mediated C-Raf activation,nuclear-localized PHB 1 can act as a transcriptional co-regulator that can inhibiting the transcription of AR and its downstream target genes.FL3 is extracted from natural products Chinese plant,which has been shown to have antitumor activity without toxicity to normal tissues.Studies have shown that after FL3 binds to PHB1,it can inhibit the activation of the MAPK signaling pathway and affect the cellular localization of the PHB1 protein,thereby exerting anti-cancer activity.Up to now,no studies have reported whether FL3 exerts a tumor suppressor effect and its mechanism in prostate cancer cells and tissues.Research purposes1)Explore the relationship between PHB 1 expression and prostate cancer progression2)Explore the role of PHB 1 in the malignant progression of prostate cancer3)Explore whether PHB1 small molecule ligand FL3 has a therapeutic effect in prostate cancer4)Explore the molecular mechanism of FL3 inhibiting PHB1 in the treatment of CRPCResearch method1.The relationship between PHB1 expression and prostate cancer progression1)Database analysis of the relationship between PHB1 and the malignant progression of prostate cancer2)Immunohistochemical detection of PHB 1 expression in prostate cancer tissues with different Gleason scores3)RT-qPCR and Western blot to detect the basic expression of PHB1 in prostate cancer cell line2.The role of PHB 1 in the malignant progression of prostate cancer1)Use PHB1 siRNA and PHB1 expression plasmid to transiently transfect prostate cancer cells,and measure the transfection efficiency by RT-qPCR and Western blot2)Carry out cell function experiments,including MTS,Transwell invasion and metastasis experiments,plate clone formation experiments,to detect the effect of PHB 1 on the proliferation,migration and invasion of prostate cancer cells3.Preliminary study on the molecular mechanism of PHB 1 involved in castration resistance of prostate cancer1)Relationship between PHB1 and Androgen receptor(AR)signaling pathway.LNCaP/C4-2B cells were cultured in hormone-deprived conditions for 3 days.After androgen depletion,DHT stimulation was added to detect the changes in AR and PHB1 expression.2)Whether the subcellular distribution of PHB1 can be changed under the condition of hormone deprivation.LNCaP and C4-2B cells were cultured in CSS and FBS,respectively,and the effect of hormone deprivation on the subcellular localization of PHB 1 was detected by nucleoplasma separation and immunofluorescence assay4.PHB1 small molecule ligand FL3 is combined with ENZ to enhance the therapeutic effect of ENZLNCaP/C4-2B/PC3 cells were treated with different concentrations of FL3,and cell proliferation ability was detected by MTS1)LNCaP/C4-2B cells were treated with ENZ and FL3 alone or combined with ENZ and FL3,and the proliferation ability of LNCaP/C4-2B cells was detected by MTS2)The effect of FL3 on the treatment of prostate cancer in vivo was detected in the tumor-bearing experiment of C4-2B cell mice5.Molecular mechanism of FL3 inhibiting PHB1 in CRPC1)After LNCap/C4-2B cells were treated with FL3,the changes of PHB1 subcellular localization were detected by nucleoplasma separation and immunofluorescence assay2)After LNCap/C4-2B cells were treated with FL3,the changes of c-Raf/MEK/ERK phosphorylation cascading activation were detected by Western blot3)After LNCaP/C4-2B cells were treated with FL3,the transcription levels of AR and its downstream target genes were detected by RT-qPCRResearch results1.In castration-resistant prostate cancer,the expression of PHB1 is significantly up-regulatedImmunohistochemistry and database mining revealed that PHB1 was up-regulated in prostate cancer tissues with high Gleason scores and CRPC prostate cancer models.Basic expression testing showed that PHB 1 was highly expressed in prostate cancer cell lines compared with normal prostate epithelial cells.The expression of PHB 1 in CRPC cell line was significantly higher than that in ADPC cell line.2.PHB1 promotes the proliferation,migration and invasion of prostate cancer cells and participates in castration resistanceCell function experiment was carried out,and MTS experiment showed that:In LNCap,overexpression of PHB1 significantly improved cell proliferation ability compared with the control group,and the growth inhibition of LNCap cells caused by CSS-conditioned culture could be partially reversed by overexpression of PHB1.In C4-2B/PC3 cells,the cell proliferation ability transfected with si-PHB 1 was significantly inhibited compared with the control group.Similarly,The proliferation of C4-2B cells cultured under CSS condition was inhibited to a higher degree than that cultured under FBS condition.Transwell experiments showed that PHB1 overexpression promoted the invasion and migration ability of LNCap cells,on the contrary,PHB1 knockout significantly inhibited the invasion and migration ability of C4-2b/PC3 cells.The above results proved that PHB1 promoted the proliferation,invasion and migration of prostate cancer cells,reduced the dependence of prostate cancer cells on androgens,and participated in castration resistance.3.Preliminary study on the molecular mechanism of PHB1 involved in castration resistance of prostate cancer1)PHB1 is an androgen inhibitory geneLNCaP/C4-2B cells were cultured under CSS for a period of time,and DHT was added to stimulate cell growth by setting a time gradient and a concentration gradient.The results showed that after DHT stimulation,AR expression increased,and PHB1 mRNA and protein expression decreased in a time and concentration dependent manner.2)Hormone deprivation affected the subcellular distribution of PHB1After 7 days of culture of LNCaP/C4-2B cells under CSS,nucleoplasmic separation and immunofluorescence experiments showed that PHB1 translocated from the nucleus to the cytoplasm compared with control cells.4.PHB1 small molecule ligand FL3 is combined with ENZ to enhance the therapeutic effect of ENZMTS results showed that FL3 could significantly inhibit the proliferation of prostate cancer cells,and the inhibition ability of FL3 in CRPC phenotype C4-2B/PC3 cells was significantly higher than that of LNCaP cells.Combination of FL3 and ENZ can enhance the efficacy of ENZ.In vivo studies in the C4-2B xenograft model also confirmed the efficacy of FL3 in the treatment of prostate cancer.5.Molecular mechanism of FL3 inhibiting PHB1 in CRPC1)FL3 inhibits the proliferation of prostate cancer cells by affecting the subcellular localization of PHB 1.LNCaP cells were treated with FL3 40nM for 48h/C4-2B cells were treated with FL3 20nM for 48h,and the nucleoplasma separation and immunofluorescence assay were performed.2)FL3 inhibits the activation of MAPK signaling pathway After LNCaP/C4-2B cells were treated with FL3,Western blot results showed that the phosphorylation cascade of Ras/c-Raf/MEK/ERK was decreased.3)FL3 inhibits AR signaling pathwayAfter LNCaP/C4-2B cells were treated with FL3,RT-qPCR results showed that AR and its downstream target genes were inhibited.Research conclusionsPHB1 is significantly upregulated in CRPC and is involved in castration resistance to promote the malignant progression of prostate cancer.Its ligand drug FL3 inhibits the overactivation of MAPK signaling pathway and the transcription of AR and its downstream target genes by affecting the subcellular distribution of PHB1,thus inhibiting the proliferation of castration-resistant prostate cancer cells.Meanwhile,FL3 is combined with ENZ.It can improve the therapeutic effect of ENZ,thus reducing the dosage and side effects.