The Effects And Molecular Mechanisms By Which SPIN1 Promotes Cancer Cell Proliferation Through Negative Regulation Of P53 Stability

Background and PurposeThe well-documented tumor suppressor p53,referred as“the guardian of the genome”,is tightly controlled in cells.While loss of wild type p53 causes fatal damages to the genome,it is not surprising that the TP53 gene is mutated in more than 50%human cancers,and the functions of p53 are often impeded through various mechanisms in the remainder.One predominant negative regulator of p53 is the E3ubiquitin ligase MDM2,while MDM2 is a transcriptional target gene of p53,forming a negative feedback loop.A plethora of cellular stress or regulators could stabilize p53by blocking the MDM2-p53 feedback loop,such as p19^ARF.Accumulating evidence has continuingly verified this ribosomal proteins?RPs?-MDM2-p53 pathway as a new emerging mechanism for boosting p53 activation in response to ribosomal stress over the past decade.Ribosomal stress,also called nucleolar stress,is often triggered by aberrant ribosome biogenesis caused by inhibition of rRNA synthesis,or malfunction of ribosomal proteins or nucleolar proteins,or chemical insult.Upon ribosomal stress,a series of ribosomal proteins,including RPL5,RPL11,RPL23 and et al,will translocate from the nucleolus to the nucleoplasm and bind to MDM2,blocking its ability to ubiquitinate p53 and consequently stabilizing p53 to maintain cellular homeostasis.Although there are a few proteins that have been identified to regulate this RPs-MDM2-p53 pathway,such as PICT-1 inhibition of RPL11 to regulate MDM2-p53pathway and SRSF1 forms a complex with RPL5 and MDM2 to activation of RPL5,it still remains to determine if there are more proteins that can regulate the RPs-MDM2-p53 pathway.This study aims to clarify if SPIN1 promotes cancer cell proliferation by negatively regulating the p53 pathway.We will use molecular and cellular biological methods,clinical specimens and bioinformatic tools to illustrate how SPIN1 modulates p53 expression and activity,and reveal the exact influence of SPIN1 on tumor growth in human cancer.Completion of this project will not only uncover a new mechanism for unlimited proliferation in cancer cells,but also provide potential therapeutic strategies for cancer patients.MethodsPart I.SPIN1 is a binding partner of RPL5.1.The RPL5–MDM2–p53 pathway is a key cellular defense mechanism against aberrant cell proliferation stemming from enhanced oncogenic activities.In order to identify the upstream regulators mediated the activity of this RPL5-MDM2-p53pathway,we performed co-immnunoprecipitation?co-IP?using HEK293 cells that stably expressing Flag-RPL5,and the co-immunoprec-ipitated proteins were cut out for mass spectrometry?MS?analysis.Among abundant candidates,SPIN1?Spindlin 1?attracted our interest due to its critical role in rDNA transcription and tumorigenesis;2.Construction of eukaryotic expression plasmids?Myc-SPIN1,Flag-RPL5,Flag-SPIN1 and GFP-RPL5?by using gene subcloning technology,then co-immnunoprecipitation?co-IP?were performed to detect if SPIN1 could bind to RPL5exogenously and endogenously;3.To clarify the exact mechanism underlying SPIN1-RPL5 binding,we constructed prokaryotic expression plasmids?GST-SPIN1 and GST-RPL5,full length and fragments?,as well as purified His-SPIN1 and His-RPL5 protein from bacteria.Then the GST pull down assays were utilized to verify if SPIN1 could bind RPL5directly and further to confirm the binding site.Part II.The role of SPIN1 on cancer cell proliferation and itsunderlying mechanism1.To determine if SPIN1 has any role in regulation of the p53 pathway,we utilized siRNA to silence the SPIN1 expression in wild-type p53-containing cells,including osteosarcoma U2OS,lung cancer H460 and colon cancer HCT116 cells,then Western blotting and quantitative Real-time PCR?qRT-PCR?were performed to detect the changes of p53 pathway upon SPIN1 knockdown.2.In order to determine if depletion of SPIN1 might affect p53-dependent cellular outcomes,we constructed the interfering shRNA targeting SPIN1,and generated both HCT116^p53+/+and HCT116^p53-/-cell lines that express scramble shRNA or SPIN1shRNA.Then the CCK-8 assay,colony formation assay and FACS analysis were conducted to evaluate the biological outcomes of SPIN1 knockdown;Moreover,western blotting was also performed to determine the protein levels of p53,p21 and PUMA after SPIN1 silencing.3.In translate the above-described cellular funcions of SPIN1 into more biological significance,we established a xenograft tumor model by inoculating aforementioned HCT116 cell lines that expressed scramble shRNA or SPIN1 shRNA into NOD/SCID mice,and moditored tumor growth for 21 days.Then western blotting and qRT-PCR were performed to detect the changes of p53 pathway in vivo upon SPIN1 knockdown.4.To translate the knowledge gained from the above cell-based and animal studies to the clinic,we examined the protein levels of SPIN1 in primary colon cancer samples and normal colon tissues by Western blotting.We further searched some available online databases,such as Oncomine,for SPIN1 expression in several human cancers.Part III.Deciphering the role and molecular mechanism of SPIN1 in regulating p53 stability1.To investigate the molecular mechanism underlying SPIN1's regulation of p53,we utilized cycloheximide-chase experiments to determine if SPIN1 could regulate the stability of p53 protein.Moreover,we performed in vivo ubiquitination assay to determine if SPIN1 overexpression could affect the MDM2-mediated p53ubiquitination and degradation.Furthermore,we also transfected plasmids encoding Flag-SPIN1 and p53 into MEF^{p53-/-;MDM2-/-}cells to validate if the induction of p53degradation by SPIN1 was MDM2-dependent.2.To confirm if SPIN1 negatively regulates p53 stability by inhibiting the interaction between RPL5-MDM2,we conducted the Co-IP assay to detect the binding of RPL5 to MDM2 upon SPIN1 overexpression.In addition,we also utilized immunofluorescence?IF?assay to detect the co-localization of SPIN1 and RPL5 in cells.3.To investigate if SPIN1 depletion could induce ribosomal stress by interrupting rRNA synthesis,consequently leading to p53 activation,we conducted q RT-PCR to check the expression of rRNA genes.Then ribosome profiling was used to detect the levels of free forms of RPL5 and RPL11 upon SPIN1 knockdown.Furthermore,immunoprecipitation?Co-IP?was performed to observe the interation between free form-RPL5/RPL11 and MDM2.ResultsPart I.SPIN1 is a binding partner of RPL5.1.The IP-MS?Immunoprecipitation mass spectrometry?results reveals a number of candidates that might bind to RPL5,some of which have been previously described as p53 binding and regulatory proteins.Among which,SPIN1 attracted our interest due to its critical role in rRNA synthesis and tumorignenesis.2.Our Co-IP results showed that SPIN1 could specifically bind to RPL5,but not RPL11 or RPL23 exogenously,moreover,the endogenous SPIN1-RPL5 was observed as RPL5 was co-immunoprecipitated with SPIN1 by using anti-SPIN1 antibody.3.The GST pull down assays have demonstrated that SPIN1 and RPL5 could bind each other directly.Further analysis showed that the second Tudor like domain of SPIN1 was responsible for RPL5 binding,and both the N-and C-termini of RPL5 were found to bind to SPIN1.Part II.The role of SPIN1 in cancer cell proliferation and its underlying mechanism1.SPIN1 depletion dramatically elevated the protein levels of p53 and its target genes p21,PUMA and MDM2 in several wild-type p53 containing cells,including U2OS,H460 and HCT116 cells.In addition,silencing SPIN1 in HCT116 cells increased the mRNA levels of p21 and PUMA,without affecting TP53 mRNA expression.Conversely,overexpression of SPIN1 in HCT116 cells decreased the protein levels of p53 and its targets,such as p21 and PUMA,and the mRNA levels of these targets genes,without affecting the TP53 mRNA level.2.SPIN1 knockdown significantly repressed cell viability and colony formation ability,as well as induced cell apoptosis in HCT116 cells,which was more predominantly in p53-containing HCT116 cells(HCT116^p53+/+)than that in p53-null cells(HCT116^p53-/-).3.Our xenograft experiments further confirmed that SPIN1 silencing retarded tumor growth in vivo,consistently,the western blotting and qRT-PCR assays validated that SPIN1 impedes xenograft tumor growth predominantly by activating the p53 and its target genes,such as PUMA and p21,although SPIN1 might also possess p53-independent functions in regulation of cell growth and survival.4.Analysis of clinical samples showed that SPIN1 is significantly highly expressed in colon cancer tissues compared with normal colon tissues.More intriguingly,the expression profiles of SPIN1 obtained from Oncomine database has demonstrated that SPIN1 is extensively upregulated in a panel of human cancers,including gastric cancer,breast cancer,melanoma and et al.Part III.Deciphering the role and molecular mechanism of SPIN1 in regulating p53 stability1.The cycloheximide-chase experiments have shown that SPIN1 knockdown prolonged p53's half life while ectopic expression of SPIN1 shortened p53's half life.Moreover,SPIN1 overexpression accelerated the MDM2-mediated p53 ubiquitination and degradation in a dose-dependent manner.Interestingly,the induction of p53degradation by SPIN1 was MDM2-dependent,as overexpression of SPIN1 failed to repress ectopic p53 protein expression in Trp53 and MDM2 double knockout MEF^p53-/-;MDM2-/-cells.2.Our co-immunoprecipitation results showed that ectopic SPIN1 dramatically reduces the amount of RPL5 co-immunoprecipitate with MDM2 in a dose-dependent manner,although SPIN1 overexpression did not alter the interactions between RPL11and MDM2.Furthermore,the immunofluorescence results showed that SPIN1 and RPL5 were clearly co-localied in the nucleolus.3.Our qRT-PCR results showed that SPIN1 depletion suppressed rRNA synthesis.The western blotting analysis after sucrose gradient fractionation assay showed that the levels of RPL5 and RPL11 in the soluble and ribosome-unbound fraction were markedly increased in SPIN1-depeletion cells,which in turn increasing the endogenous RPL5/RPL11-MDM2 interaction,accompanying with elevated p53 and MDM2 protein levels.More interestingly,the reduction of RPL5 or RPL11 abrogated SPIN1knockdown-induced p53 levels.Conclusions1.SPIN1 is identified as a novel RPL5-binding partner,the SPIN1 Tudor 2 domain is responsible for RPL5 binding and SPIN1 binds to both of the N-and C-termini of RPL5 of RPL5.2.The in vitro and in vivo experiments show that SPIN1 depletion suppresses cancer cell proliferation by predominantly activating p53.Elevated protein expression of SPIN1 is observed in a panel of human colon tumor samples and the analysis of Oncomine database suggest that SPIN1 is extensively overexpressed in a number of human cancers;3.The molecular mechanisms underlying SPIN1 promotes cancer cell proliferation by negatively regulating p53 activity include two different ways:first of all,SPIN1 overexpression may retain RPL5 in the nucleolus,thereby preventing RPL5from suppression of MDM2 activity and resulting in p53 degradation,favoring tumor growth;moreover,disruption of SPIN1 causes ribosomal stress,the release of free forms RPL5/RPL11 to bind MDM2 may be also responsible for p53 activation.