Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms
- Division of Hematology/Oncology, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, United States
- Quantitative Biosciences Institute (QBI), University of California San Francisco, United States
- Department of Medicine, Harvard Medical School, United States
- Massachusetts General Hospital Cancer Center, United States
- Department of Cellular and Molecular Pharmacology, University of California San Francisco, United States
- J. David Gladstone Institute, United States
- Department of Surgery and Northwest Liver Research Program, University of Washington, United States
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, United States
- Department of Pharmacology, University of Washington Medical Center, United States
Figures
Figure 1
Recurrent PKA activating somatic alterations in human cancer.
(A) Pathway illustrations of different PKA activating genomic alterations in order from top: PRKACA amplification, DNAJB1-PRKACA fusion, PRKACA activating mutation, and PRKAR1A inactivation or …
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Figure 1—source data 1
Images for Figure 1A.
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Figure 1—source data 2
Table for Figure 1B.
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Figure 1—source data 3
Images for Figure 1D part 1/3.
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Figure 1—source data 4
Images for Figure 1D part 2/3.
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Figure 1—source data 5
Images for Figure 1D part 3/3.
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Figure 2
Kinome profiling to identify signaling nodes downstream of PRKACA.
(A) Global phosphorylation changes in 639V with induction of 3xFLAG-PRKACA or 3xFLAG-PRKAR1AG325D; VASP is shown as a positive control for PKA activation, technical replicates shown. (B) Change in …
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Figure 2—source data 1
Tables for Figure 2A.
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Figure 2—source data 2
Tables for Figure 2B.
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Figure 2—source data 3
Tables for Figure 2C.
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Figure 2—source data 4
Tables for Figure 2D.
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Figure 2—source data 5
Tables for Figure 2E.
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Figure 2—source data 6
Images for Figure 2F.
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Figure 3 with 1 supplement
PKA activity correlates with c-MYC and n-MYC protein levels.
(A) Immunoblots showing the change of PKA activity, as indicated by phospho-PKA substrate, and c-MYC and n-MYC expression in Colo741 and FLX1 cells after treatment with 50 μM forskolin (FSK) and …
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Figure 3—source data 1
Images for Figure 3A.
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Figure 3—source data 2
Tables for Figure 3B.
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Figure 3—source data 3
Images for Figure 3C.
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Figure 3—source data 4
Images for Figure 3D.
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Figure 3—source data 5
Images for Figure 3E.
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Figure 3—source data 6
Images for Figure 3F.
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Figure 3—source data 7
Tables for Figure 3G.
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Figure 3—figure supplement 1
Effects of PKA inhibition on FLX1 cell proliferation.
(A) Impact of individual PRKACA-targeting siRNAs on relative cell confluence in FLX1. Confirmation of knockdown shown on right. Experiment was done in duplicate, the representative results shown …
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Figure 3—figure supplement 1—source data 1
Tables for Figure 3—figure supplement 1A.
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Figure 3—figure supplement 1—source data 2
Tables for Figure 3—figure supplement 1B.
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Figure 4 with 1 supplement
c-MYC alters transcription and proliferation in PKA-dependent cell models.
(A) RNASEQ data from FLX1 cells after 48 hr treatment with four pooled siRNA against PRKACA. PRKACA, ornithine decarboxylase (ODC) and Cyclin D1 (CCND1) are highlighted. (B) Gene set enrichment …
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Figure 4—source data 1
Tables for Figure 4A.
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Figure 4—source data 2
Tables for Figure 4B.
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Figure 4—source data 3
Tables for Figure 4C.
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Figure 4—source data 4
Tables for Figure 4D.
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Figure 4—source data 5
Tables for Figure 4E.
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Figure 4—source data 6
Images for Figure 4E.
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Figure 4—source data 7
Tables for Figure 4F.
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Figure 4—source data 8
Tables for Figure 4G.
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Figure 4—source data 9
Images for Figure 4G.
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Figure 4—figure supplement 1
Data supporting c-MYC effects on transcription and proliferation.
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Figure 4—figure supplement 1—source data 1
Tables for Figure 4—figure supplement 1A.
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Figure 4—figure supplement 1—source data 2
Tables for Figure 4—figure supplement 1B.
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Figure 4—figure supplement 1—source data 3
Images for Figure 4—figure supplement 1B.
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Figure 5 with 2 supplements
AURKA and GSK3B regulate c-MYC in PKA-dependent cell models.
(A) Summary data from the FLX1 cell line treated with 352 kinase inhibitors from an advanced clinical compound library at 2 μM for 120 hr. The targets of selected compounds with a z-score ≥2 are …
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Figure 5—source data 1
Tables for Figure 5A.
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Figure 5—source data 2
Tables for Figure 5B.
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Figure 5—source data 3
Tables for Figure 5C.
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Figure 5—source data 4
Tables for Figure 5D.
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Figure 5—source data 5
Tables for Figure 5E.
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Figure 5—source data 6
Images for Figure 5F.
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Figure 5—figure supplement 1
Signaling effects on c-MYC in PKA-driven cells.
(A) Colo741 cells treated with dose curves of multiple AURKA inhibitors for 72 hr. Relative cell viability was measured by CTG assay vs. untreated control samples. Results are the mean ± SEM of …
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Figure 5—figure supplement 1—source data 1
Tables for Figure 5—figure supplement 1A.
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Figure 5—figure supplement 1—source data 2
Images for Figure 5—figure supplement 1B.
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Figure 5—figure supplement 1—source data 3
Images for Figure 5—figure supplement 1C.
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Figure 5—figure supplement 2
Proteasome-independent PKA effects on c-MYC, (A) immunoblots showing the change of c-MYC protein in FLX1 cells after treatment with 50 μM forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX) and/or 20 μM MG132 for 2 hr.
(B) Immunoblots showing the change of c-MYC level in engineered FLX1 cells with doxycycline (dox)-inducible 3xFLAG- PRKAR1AG325D after dox for 24 hr and/or 20 μM MG132 for 2 hr. (C) Li-Cor western …
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Figure 5—figure supplement 2—source data 1
Images for Figure 5—figure supplement 2A.
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Figure 5—figure supplement 2—source data 2
Images for Figure 5—figure supplement 2B.
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Figure 5—figure supplement 2—source data 3
Images and tables for Figure 5—figure supplement 2C.
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Figure 6
PKA signaling supports translation initiation.
(A) Gene set enrichment analysis (GSEA) of significantly altered phosphoproteins following doxycycline (dox) induction of PKA in proteomics data from this study or chemical PKA stimulation in our …
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Figure 6—source data 1
Tables for Figure 6A.
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Figure 6—source data 2
Images for Figure 6B.
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Figure 6—source data 3
Images for Figure 6C.
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Figure 6—source data 4
Images for Figure 6D part 1/4.
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Figure 6—source data 5
Images for Figure 6D part 2/4.
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Figure 6—source data 6
Images for Figure 6D part 3/4.
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Figure 6—source data 7
Images for Figure 6D part 4/4.
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Figure 7 with 1 supplement
PKA effects on c-MYC are blocked by eIF4A inhibition.
(A) Immunoblots showing the c-MYC protein levels in FLX1 cells after treatment with 100 nM rocaglamide or zotatifin for 24 hr and/or 50 μM forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX) for 4 …
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Figure 7—source data 1
Images for Figure 7A.
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Figure 7—source data 2
Images for Figure 7B.
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Figure 7—source data 3
Images for Figure 7C.
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Figure 7—source data 4
Tables for Figure 7D.
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Figure 7—source data 5
Tables for Figure 7E.
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Figure 7—source data 6
Tables for Figure 7F.
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Figure 7—source data 7
Tables for Figure 7G.
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Figure 7—source data 8
Images for Figure 7H.
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Figure 7—figure supplement 1
Data supporting reversal of PKA effects on c-MYC by eIF4A inhibition.
(A) Immunoblot showing the effect of 48 hr of non-targeting control (NTC) or four pooled PRKACA siRNA on endogenous c-MYC or overexpressed c-MYC lacking a 5’UTR. Doxycycline (dox) treatment was 1 …
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Figure 7—figure supplement 1—source data 1
Images for Figure 7—figure supplement 1A.
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Figure 7—figure supplement 1—source data 2
Images for Figure 7—figure supplement 1B.
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Figure 7—figure supplement 1—source data 3
Tables for Figure 7—figure supplement 1A.
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Author response image 1
PKA effects on cell cycle distribution in FLX1.
(A) FLX1 parental cells were treated with DMSO or 50 μM FSK/IBMX for 4 hours, with BrdU added for the last 20 minutes. Cells were then stained for nuclear content with 7-AAD and active DNA synthesis …
Author response image 2
FLX1 and Colo741 with dox-induced 3xFLAG-c-MYC lacking a 5’UTR and including the stabilizing T58A mutation.
Dox treatment was 1 μG/mL for 40 hours prior to harvest with zotatifin or DMSO for 24 hours. Of note, the MYCT58A allele was toxic in both cells but particularly in FLX1, resulting in reduced …
Author response image 3
FSK/IBMX and zotatifin effects on other translationally regulated transcripts.
(A) Time course of FSK/IBMX in Colo741 and FLX1 showing increased expression of ERBB2; kinetics are somewhat different between the two cell lines. (B) Effect of 24 hour treatment with 100 nM …
Tables
Author response table 1
| CRISPR | ||
|---|---|---|
| PRKACA | MYC | |
| Average | -0.24 | -1.46 |
| 639V | -0.14 | -0.67 |
| Colo741 | -0.35 | -1.38 |
| siRNA | ||
| PRKACA | MYC | |
| Average | -0.15 | -0.48 |
| Colo741 | -1.38 | -0.35 |
Additional files
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Supplementary file 1
Summary of TCGA analysis.
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Supplementary file 2
Individual phosphoproteomic, phosfate, and multiplex inhibitor beads (MIBs) datasets.
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Supplementary file 3
Network propagation results.
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Supplementary file 4
RNASEQ primary data.
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Supplementary file 5
Drug screen in FLX1.
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Supplementary file 6
Drug screen in FLX1 with dox-inducible PRKAR1AG325D.
- https://cdn.elifesciences.org/articles/69521/elife-69521-supp6-v3.xlsx
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Supplementary file 7
siKINOME final results.
- https://cdn.elifesciences.org/articles/69521/elife-69521-supp7-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/69521/elife-69521-mdarchecklist1-v3.docx
