IGF2 mRNA binding protein-2 is a tumor promoter that drives cancer proliferation through its client mRNAs IGF2 and HMGA1
- Massachusetts General Hospital, United States
- Harvard Medical School, United States
- Broad Institute of Harvard and MIT, United States
- University of Oxford, United Kingdom
Figures
Figure 1
The IMP2 gene is amplified and overexpressed in many cancers and drives proliferation.
(A) The frequency of IMP1,2,3 gene copy number amplification in various cancers. Data from TCGA. (B) IMP1, 2 and 3 mRNA levels in various cancers. Data from TCGA. (C) IMP2 overexpression enhances …
Figure 2
Overexpression of the Grb14 and IGFBP2 polypeptides is mostly responsible for the slowed proliferation of Imp2−/− MEFs.
(A) Imp2−/− MEFs exhibit increased abundance of the IGFBP2, Grb14 and P21Cip1 polypeptides as compared with Imp2+/+ MEFs but unaltered levels of IMP1 and IMP3. (B) Time course of shRNA-mediated …
Figure 3
IGFBP2 polypeptide overabundance in Imp2−/− MEFs is due to increased gene transcription whereas Grb14 polypeptide overabundance is due to markedly slower polypeptide degradation.
(A) Igfbp2, Grb14 and P21 mRNA abundance in Imp2+/+ and Imp2−/− MEFs. RNA was extracted from Imp2+/+ and Imp2−/− MEFs and the abundance of Grb14, Igfbp2, P21, Gapdh and Actb mRNA was determined by …
Figure 4
The ability of IMP2 to drive the proliferation of human cancer cells is mostly due to IMP2 regulation of IGF2, Grb14 and IGFBP2 abundance.
(A) CRISPR mediated deletion of IMP2 from human cancer cell lines causes markedly increased abundance of IGFBP2 and Grb14. Extracts prepared from the six cell lines shown in Figure 1D were subjected …
Figure 5 with 3 supplements
A decreased level of Hmga1, an IMP2 client mRNA, causes the upregulation of IGFBP2 and Grb14 abundance and the slowed proliferation of IMP2 deficient MEFs and RD cells.
(A) IMP2 binds Hmga1 mRNA. IPs were prepared from extracts of Imp2+⁄+ and Imp−⁄− MEFs (left pair) and from RD cells (right pair) with anti-IMP2 antibody (black) or nonimmune IgG (orange). The …
Figure 5—figure supplement 1
RNAseq indicates that IMP2 binds HMGA1 mRNA predominantly at the 3’UTR.
. The read density track over the Hmga1 transcript is shown; the input RNA is below, and the IMP2-IP is the upper track, showing significant enrichment across the 3’UTR. The sequence of murine Let7a …
Figure 5—figure supplement 2
Depletion of IGFBP2 does not affect the abundance of Grb14.
. Doxycycline-inducible shRNA directed at Igfbp2 was stably expressed in MEFs. Extracts were prepared before and 1, 2 and 3 days after addition of doxycycline (100 ng/ml) and immunoblotted for the …
Figure 5—figure supplement 3
Depletion of HMGA1 prolongs the half-life of the Grb14 polypeptide.
. Doxycycline-inducible shRNAs directed against Hmga1 or GFP were stably expressed in MEFs. Forty eight hours after addition of doxycycline (100 ng/ml), cycloheximide (250 μM) was added and extracts …
Figure 6
The ability of IMP2 to stimulate MEF proliferation and reduce IGFBP2 and Grb14 abundance requires an acidic charge at IMP2 residue 164.
(A) The ability of IMP2 to drive MEF proliferation requires an acidic charge at IMP2 residue 164. Imp2−/− MEFs were engineered to express doxycycline-inducible cDNAs encoding Flag tagged IMP2 …
Figure 7
IMP2 promotes proliferation by increasing IGF2 polypeptide and by stabilizing HMGA1 mRNA, whose polypeptide product suppresses the abundance of IGFBP2 and Grb14, inhibitors of IGF2 action.
https://doi.org/10.7554/eLife.27155.012
Author response image 1
IGF1R signaling is impaired in Imp2-/- MEFs.
Author response image 2
Depletion of HMGA1 reduces InsR mRNA whereas reduction or elimination of IMP2, although reducing HMGA1 abundance, does not alter InsR abundance.
Additional files
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Supplementary file 1
RNA seq of total RNA and of IMP2 immunoprecipitates from Imp2+/+ and Imp2−/− MEFs.
- https://doi.org/10.7554/eLife.27155.013
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Supplementary file 2
Proteomic analysis of Imp2+/+ and Imp2−/− MEFs left tab = summary middle tab = all data right tab = polypeptide ratios: Imp2−/−/Imp2+/+.
- https://doi.org/10.7554/eLife.27155.014
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Supplementary file 3
Proteomic analysis of Imp2−/− MEFs stably expressing empty Flag vector (designated C) or Flag-IMP2, wildtype (Ser162/Ser164) or with Ser162 and Ser164 substituted with either Ala or Asp: WT/AD/DD/DA/AA/.
- https://doi.org/10.7554/eLife.27155.015
