Preliminary Study On Molecular Mechanisms Of Lovastatin-Induced Nucleolar Stress In TNBC CSCs

Objective: The study investigates the inhibitory effects of lovastatin(LV)of inducing nucleolar stress on triple-negative breast cancer stem cells(TNBC CSCs)in vitro and the preliminary mechanisms involved.Methods: 1.The effect of LV on post-translational modifications of TNBC CSCs.To elucidate the molecular mechanisms of the selective action of lovastatin on TNBC CSCs,we performed global profiling of lysine acylation,which is the largest group of post-translational modifications that regulate diverse functions of eukaryotic proteins.We initially screened the four common types of lysine acylation,i.e.,acetylation,malonylation,succinylation,and crotonylation,by western blot analysis using antibodies against each type of lysine acylation.Lovastatin treatment showed a greater effect on lysine succinylation(Ksucc)and crotonylation(Kcr)in MDA-MB-231 CSCs than the other two types of modifications.We next performed TMT labeling and affinity enrichment followed by LC-MS/MS to uncover the changes of Kcr modifications and the specific sites.The quality control data indicated that the MS data were consistent with the properties of trypsin-digested peptides,which met the requirement for further analysis.Bioinformatics was performed to annotate the proteins differentially modified by Kcr in response to lovastatin treatment.2.The effect of LV on the change of the nucleolar morphology and volume in TNBC CSCs.TNBC CSCs were treated with LV,and the change of nucleolar morphology and volume was detected by transmission electron microscopy(TEM)and Argyrophilic nucleolar organizer region(Ag NOR).3.The effect of LV on inducing nucleolar stress related proteins in TNBC CSCs.BCSCs were treated with vehicle or LV,the nucleolar stress-related protein level and subcellular localization,such as nucleolar and coiled-body phosphoprotein 1(NOLC1),p53 and RPL3,were revealed by immunofluorescence-confocal microscopy.The protein level of NOLC1 in the nucleus of MDA-MB-231 CSCs was detected by western blot analysis.The levels of the precursor 45 S r RNA and the mature 28 S and 5.8S r RNAs and the transcription of p53 target genes,such as PUMA and p21,were examined by q RT-PCR.4.The effect of LV on protein synthesis pathway in TNBC CSCs.Phosphorylation of m TOR and S6 K which are important factors for protein synthesis was detected by westen blot analysis.5.Target search of statins and molecular docking simulations.Target search for lipophilic and hydrophilic statins was undertaken at Drug Central(http://drugcentral.org/).Venn diagrams were generated using the online Draw Venn Diagram tool(http://bioinformatics.psb.ugent.be/webtools/Venn/).Docking calculations were performed using the Swiss Dock web service(http://swissdock.vital-it.ch)based on EADock DSS.Results: 1.LV dysregulates lysine crotonylation of proteins involved in ribosome biogenesis and nucleolar organization pathways in TNBC CSCs.Lovastatin treatment showed a greater effect on lysine succinylation(Ksucc)and crotonylation(Kcr)in MDA-MB-231 CSCs than the other two types of modifications.We next performed TMT labeling and affinity enrichment followed by LC-MS/MS to uncover the changes of Kcr modifications and the specific sites.We found that lovastatin differentially regulated 50 Kcr sites on 28 proteins in MDA-MB-231 CSCs and 13 Kcr sites on 9 proteins in MDA-MB-453 CSCs.Bioinformatics was performed to annotate the proteins differentially modified by Kcr in response to lovastatin treatment.Kcr-modified proteins were mainly involved in ribosome biogenesis(RPL3,RPL7 A,RPL19,RPL24,RPL26,and RPS10)and nucleolar organization(NOLC1 and SNRPA).2.LV induces the change of morphology and volume of the nucleoli in TNBC CSCs.Lovastatin changed the morphology and increased the area of the nucleoli in MDA-MB-231 CSCs.Similarly,we found an increase in NOR-staining intensity in LV-treated cells.3.LV induces nucleolar stress in TNBC CSCs.Since NOLC1 is one of the two nucleolar organization proteins targeted by lovastatin,we suspect that lovastatin might induce nucleolar stress through disturbance of NOLC1.Indeed,lovastatin increased NOLC1 protein level in the nucleolus of MDA-MB-231 but not MDA-MB-453 CSCs.The protein level of NOLC1 was increased by lovastatin in the nucleus of MDA-MB-231 CSCs.The levels of the precursor 45 S r RNA and the mature 28 S and 5.8S r RNAs were decreased by lovastatin in MDA-MB-231 CSCs.We found an increase in p53 protein level in MDA-MB-231 CSCs.Consistent with its role as a transcription factor,p53 protein enhanced by lovastatin was predominantly in the nucleus.Increased transcriptional activity of p53 was demonstrated by the enhancement of transcription of p53 target genes,PUMA and p21.We investigated whether lovastatin altered the protein level or localization of RPL3,a key component of the protein translation machinery.Surprisingly,lovastatin treatment resulted in reduction of cytoplasmic RPL3 and its nuclear/nucleolar accumulation in MDA-MB-231 CSCs.4.LV causes disturbance of protein synthesis pathway in TNBC CSCs.Diminished protein translation is another functional readout of nucleolar stress.The inhibitory effect of lovastatin on protein translation was revealed by decreased phosphorylation of m TOR and S6 K,factors important for protein synthesis.5.Target search of intracellular target(s)of statins.We searched the direct intracellular target(s)of statins that all lipophilic statins shared two common targets,i.e.,3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR)and nuclear receptor subfamily 1 group I member 2 or 3(NR1I2/NR1I3).However,when the hydrophilic statin pravastatin was analyzed together with the lipophilic statins,only one common target protein,i.e.,HMGCR,was revealed.These results suggest that NR1I2/NR1I3 might be the common target of all lipophilic statins.We next performed molecular docking to simulate the interaction between lovastatin and NR1I2.Coincidently,all the five key amino acid residues in the active site of NR1I2 where lovastatin binds are binding sites for agonist.These data imply that NR1I2 might be the intracellular target responsible for the selective actions of lipophilic statins on TNBC CSCs.Conclusions: 1.Lovastatin induces nucleolar stress in TNBC CSCs.2.Lovastatin-induced nucleolar stress will result in: 1)the change of morphology and increase the area of the nucleoli;2)decreased levels of precursor 45 S r RNA and mature 5.8S and 28 S r RNA;3)intranuclear increase of p53 and enhancement of its transcriptional activity;4)increased nucleolar NOLC1 and RPL3 protein level;and 5)decreased phosphorylation of m TOR and S6 K.