SPIN1 promotes tumorigenesis by blocking the uL18 (universal large ribosomal subunit protein 18)-MDM2-p53 pathway in human cancer
- Tulane University School of Medicine, United States
- School of Dentistry at Case Western University, United States
- Xavier University of Louisiana, United States
- Ochsner Clinical Foundation, United States
- The First Affiliated Hospital of Nanchang University, China
Figures
Figure 1 with 1 supplement
SPIN1 binds to uL18, but not uL5, or uL14.
(A) Identification of SPIN1 as a candidate of uL18 binding protein by immunopurification and mass spectrometric analysis. Lysates from HEK 293 cells were immunoprecipitated with the anti-Flag …
Figure 1—figure supplement 1
SPIN1 interacts with uL18 in HEK293 cells.
HEK293 cells were transfected with plasmids encoding Myc-SPIN1 and Flag-uL18, and 48 hr later cell lysates were collected for IP-WB analysis using anti-Flag antibody.
Figure 2 with 1 supplement
SPIN1 knockdown inhibits cell proliferation and induces apoptosis.
(A) SPIN1 knockdown induces protein levels of p53 and its target genes. U2OS, H460 and HCT116p53+/+ cells were transfected with scramble siRNA (scr-siRNA) or SPIN1 siRNA and harvested 48 hr …
Figure 2—figure supplement 1
SPIN1 knockdown increase p53 and its targets protein levels in a dose-dependent manner.
HCT116 p53+/+ (A) and U2OS (B) cells were transfected with a titrated concentration of SPIN1 siRNA (20 nM, 40 nM and 60 nM). And cells were harvested 48 hr after transfection and followed by WB …
Figure 3
SPIN1 reduces p53 stability by enhancing MDM2-mediated ubiquitination.
(A) and (B) p53-half-life is increased by SPIN1 knockdown. (A) HCT116p53+/+ cells transfected with scramble or SPIN1 siRNA for 48 hr, were treated with 100 μg/ml of cycloheximide (CHX), and …
Figure 4 with 1 supplement
SPIN1 blocks uL18-MDM2 interaction by sequestering uL18 in the nucleolus.
(A) SPIN1 overexpression attenuates p53 activation induced by ectopic uL18. U2OS cells were co-transfected with plasmids encoding Flag-uL18 or Myc-SPIN1 for 36 hr and harvested for WB analysis with …
Figure 4—figure supplement 1
SPIN1 does not bind to MDM2, and SPIN1 and uL18 co-localize in the nucleolus.
(A) SPIN1 does not bind to MDM2. HCT116p53-/- cells were transfected with combination of plasmids encoding Myc-SPIN1 and HA-MDM2, followed by IP-WB analysis with indicated antibodies. (B) SPIN1 and …
Figure 5 with 2 supplements
SPIN1 depletion increases ribosome-free uL18 and uL5.
(A) Knockdown of SPIN1 releases free forms of uL18 and uL5. HCT116p53+/+ were transfected with scramble or SPIN1 shRNA for 36 hr and subjected to sucrose gradient fractionation assay followed by WB …
Figure 5—figure supplement 1
SPIN1 knockdown reduces rRNA expression and SPIN1-Y170A mutant retains activity to repress p53.
(A) Scramble siRNA or SPIN1 siRNA was introduced into U2OS cells. RNA levels were analyzed using Q-PCR (*p value < 0.05; **p value < 0.01 by tailed t-test; n = 6). (B). SPIN1-Y170A inhibits p53. …
Figure 5—figure supplement 2
Mapping of domains responsible for uL18-SPIN1 and uL18-MDM2 binding.
(A) uL18 interacts with the second Tudor like domain of SPIN1. Purified GST-tagged SPIN1 fragments, including aa 1-262(FL), aa 50–120, aa 121–262, aa 121–193, aa 194–262 and GST protein alone were …
Figure 6 with 4 supplements
SPIN1 knockdown retards tumor growth more dramatically by inducing p53 activity.
(A) and (B) Growth curves of xenograft tumors derived from HCT116p53+/+ cells and HCT116p53-/- cells that expressed scramble or SPIN1 shRNA. Data are represented as mean ± SEM, n = 6. (C) The images …
Figure 6—figure supplement 1
Quantification of protein expression analyzed from xenograft tumors by Image J software.
All data were presented as mean ± SEM, n = 6, *p<0.05, **p<0.01 by two-tailed t-test.
Figure 6—figure supplement 2
High expression of SPIN1 is detected in multiple cancers and associated with poor prognosis in cancer patients.
(A) TCGA database was utilized, and the data were modified from the cBioPortal for Cancer Genomics (http://www.cbioportal.org/). (B) The expression profile of SPIN1 in cancers and normal tissues was …
Figure 6—figure supplement 3
Western blotting analyses of human colon cancer tissues (n = 22) and normal colon tissue (n = 20) and quantification of SPIN1 expression (Mean ± SEM, p<0.05).
https://doi.org/10.7554/eLife.31275.016
Figure 6—figure supplement 4
Expression of genes involved in p53 pathway is correlated with SPIN1 expression.
mRNA expression levels of 664 colorectal tumors were retrieved from Genomic Data Commons (https://portal.gdc.cancer.gov/). In the data set, gene expression levels were measured with FPKM (Fragments …
Figure 7
A model for SPIN1 regulation of the uL18-MDM2-p53 pathway in cancer.(see text in the Discussion for details).
https://doi.org/10.7554/eLife.31275.018
Author response image 1
SPIN1-dT2 fails to inhibit p53.
U2OS cells were transfected with wild type and Tudor 2 domain deletion mutant (FLAG-dT2) SPIN1 and cells were harvested 48 hrs after transfection for Western Blot (Left panel) and qRT-PCR (Right …
Tables
Table 1
uL18-associated polypeptides identified from mass spectrometry analysis of proteins as shown in Figure 1A.
https://doi.org/10.7554/eLife.31275.004| Gel slice number | Protein | Accession | Molecular weight | Score |
|---|---|---|---|---|
| P1 | Myb-binding protein 1A (MYBBP1A) | gi|6959304 | 149727 | 149 |
| P5 | Protein arginine N-methyltransferase 5 (PRMT5) | gi|2323410 | 72685 | 121 |
| P7 | 60S ribosomal protein L5 uL18(RPL5) | gi|14591909 | 34569 | 1014 |
| P11 | Serine/arginine-rich splicing factor 1 (SRSF1) | gi|5902076 | 27746 | 75 |
| P11 | Spindlin 1 (SPIN1) | gi|5410330 | 29602 | 95 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (human) | SPIN1 | National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/gene/10927) | Gene ID: 10927; Accession number: NM_006717; UniPro ID: Q9Y657 | |
| Gene (human) | RPL5/uL18 | National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/gene/6125 | gene ID: 6125; Accession number: NM_000969; UniPro ID: P46777 | |
| Gene (human) | RPL11/uL5 | National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/gene/6135 | gene ID: 6135; Acctssion number: NM_000975; UniPro ID: P62913 | |
| Gene (human) | RPL23/uL14 | National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/gene/9349 | gene ID: 9349; Accession number: NM_000978; UniPro ID: P62829 | |
| Gene (human) | TP53 | National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/gene/7157 | gene ID: 7157; Accession number: NM_000546; UniPro ID: P04637 | |
| Gene (human) | p21/CDKN1A | National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/gene/1026 | gene ID: 1026; Accession number: NM_000389; UniPro ID: Q42580 | |
| Gene (human) | PUMA/BBC3 | National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/gene/27113 | gene ID: 27113; Accession number: NM_001127240; UniPro ID: Q9BXH1 | |
| Gene (human) | MDM2 | National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/gene/4193 | gene ID: 4193; Accession number: NM_001145337; UniPro ID: Q00987 | |
| Strain, strain background (mouse) | NOD-SCID | Jackson Laboratories https://www.jax.org/strain/001303 | Stock No: 001303 | |
| Cell line (human) | 293 | ATCC https://www.atcc.org/Products/All/CRL-1573.aspx | Catalog number: ATCC CRL-1573; RRID:CVCL_0045 | |
| Cell line (human) | H1299 | ATCC https://www.atcc.org/Products/All/CRL-5803.aspx | Catalog number: ATCC CRL-5803; RRID: CVCL_0060 | |
| Cell line (human) | U2OS | ATCC https://www.atcc.org/Products/All/HTB-96.aspx | Catalog number: ATCC HTB-96; RRID:RRID:CVCL_0042 | |
| Cell line (human) | H460 | ATCC https://www.atcc.org/Products/All/HTB-177.aspx | Catalog number: ATCC HTB-177; RRID:CVCL_0459 | |
| Cell line (human) | HCT116 p53+/+ | from Dr. Bert Vogelstein at the John Hopkins Medical institutes | ||
| Cell line (human) | HCT116 p53-/- | from Dr. Bert Vogelstein at the John Hopkins Medical institutes | ||
| Cell line (human) | MEF (Mdm2-/-; p53-/-) | from Dr. Guillermina Lozano from MD Anderson Cancer Center, the University of Texas. | ||
| Antibody | Mouse anti-human Flag monoclonal antibody | Sigma-Aldrich | Catalog number: F1804; RRID: AB_262044 | Applications: WB; Immunofluoresce |
| Antibody | Mouse anti-human Myc monoclonal antibody | Santa Cruz Technology | Catalogue number: sc-40 | Applications: WB; Immunofluoresce |
| Antibody | Mouse anti-human GFP monoclonal antibody | Santa Cruz Technology | Catalogue number: sc-9996; RRID: AB_627695 | Applications: WB; Immunofluoresce |
| Antibody | Mouse anti-human GST monoclonal | ProteinTech | Catalogue number: HRP-66001; RRID: AB_10951482 | Applications: WB |
| Antibody | Rabbit anti-bacterial His polyclonal antibody | ProteinTech | Catalogue number:10560–1-lg; RRID: AB_1607770 | Applications: WB |
| Antibody | Rabbit anti-human SPIN1 polyclonal antibody | ProteinTech | Catalogue number:12105–1-AP; RRID: AB_2196111 | Applications: WB |
| Antibody | Mouse anti-human p53 monoclonal antibody | Santa Cruz Technology | Catalogue number: sc-126; RRID: AB_628082 | Applications: WB |
| Antibody | Mouse anti-human p21 monoclonal antibody | Neomarkers, Fremont, | Catalogue number: MS-891-P0; RRID:AB_143907 | Applications: WB |
| Antibody | Rabbit anti-human PUMA polyclonal antibody | ProteinTech | Catalogue number:55120–1-AP; RRID:AB_10859944 | Applications: WB |
| Antibody | Mouse anti-human β-actin monoclonal antibody | Santa Cruz Technology | Catalogue number: sc-47778; RRID:AB_2714189 | Applications: WB |
| Antibody | Rabbit anti-human GAPDH polyclonal antibody | Proteintech | Catalogue number:10494–1-AP; RRID:AB_2263076 | Applications: WB |
| Chemical compound, drug | Cycloheximide | Sigma-Aldrich | Catalogue number: 66-81-9 | |
| Chemical compound, drug | MG-132 | Sigma-Aldrich | Catalogue number:474787 | |
| Chemical compound, drug | 5-FU | Sigma-Aldrich | Catalogue number:51218 | |
| Chemical compound, drug | Actinomycin D (Act D) | Sigma-Aldrich | Catalogue number: 50-76-0 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.31275.019
