Vitamin A derivative retinoic acid (RA) plays an essential role in human life via its regulation of cell differentiation, proliferation, migration, survival and death. RA-mediated activities impact the transcriptome as well as the kinase cascades through the ubiquitous post-translational modification phosphorylation.
RA has established anti-cancer properties as a result of its antiproliferative activity. However, while some breast cancer cells respond to RA, others are resistant. To date, researchers have not systematically studied the transcriptional pathway alterations and impacted phosphorylation events in RA-resistant cells, which would undoubtedly illuminate RA-mediated signaling events and enhance overall understanding of cancer.
For this reason, Carrier et al. (2016) recently analyzed two human breast cancer cell lines, RA-responsive MCF7 and RA-resistant BT-474, to ascertain whether phosphorylation events that occurred in RA-responsive cells underwent alterations in RA-resistant cells.1 They selected an UltiMate 3000 RSLCnano rapid separation liquid chromatograph coupled with an LTQ Orbitrap Elite mass spectrometer (both Thermo Scientific) for large-scale high-resolution nano liquid chromatography–linear trap quadrupole Orbitrap mass spectrometry (nano-LC-LTQ-Orbitrap MS). To enhance sequence coverage, the team applied two digestion protocols (trypsin/Lys-C and chymotrypsin) to separate samples, and combined the resultant data.
The research team identified 1,222 and 1,452 total proteins in the MCF7 and BT-474 cytosolic extracts, respectively. Looking at the vehicle-treated samples, they detected 135 phosphoproteins (11%) in MCF7 cells and 111 phosphoproteins (8%) in BT-474 cells. When they applied a gene ontology tool, they found the following enriched functional categories: metabolism, cell death, RNA processing, transport, signal transduction and adhesion. They report that the presence of RA only moderately impacted total phosphorylation of cytosolic proteins, as they observed an 80% overlap for phosphoproteins with and without RA treatment. However, RA treatment differentially influenced the cytosolic phosphoproteome for both cell lines. Of note, RA treatment induced serine/threonine protein kinase MST4 phosphorylation in MCF7 cells only, supporting its activation role in the mitogen-activated protein kinase pathway. For BT-474 cells only, RA treatment induced phosphorylation of SMEK1, a serine/threonine-protein phosphatase 4 subunit that upsets the PI3K/Akt pathway, and inhibited phosphorylation of RAB7A. They also reported approximately 30 phosphoproteins unique to each cell line.
The researchers also analyzed nuclear extracts and found 865 and 1,167 total proteins in MCF7 and BT-474 cells, respectively. For untreated cells, they noted 148 phosphoproteins (17%) in MCF7 cells and 343 phosphoproteins (29%) in BT-474 cells. Gene ontology revealed enrichment for transcription DNA-templated chromatin modifications/organization, mRNA processing, and DNA damage and repair. Of the total phosphoproteins, the team observed 80% overlap for MCF7 cells and 34% overlap for BT-474 cells. RA treatment differentially influenced the nuclear phosphoproteome. They saw several mRNA processing proteins and DNA repair proteins phosphorylated only in BT-474 cells. They also noted that the same protein showed an opposite response to RA treatment depending on the cell type.
To detect nuclear transcription regulator RARα, the team required enrichment by immunoprecipitation and thermolysin cleavage before nano-LC-LTQ-Orbitrap MS. They then compared phosphorylated residues and observed a marked increase in phosphorylation at sites S74 and S77 after RA treatment in MCF7 cells only. Treatment with Herceptin partially restored phosphorylation for BT-474 cells, suggesting that the deregulation indicates overexpression/phosphorylation of receptor tyrosine kinase ERB-B2. They also noted that phosphorylation at S74 is dependent on phosphorylation at S77.
Next, the researchers compiled a list of RA-regulated genes for each cell line. They report 40% more RA-regulated genes for MCF7 cells. For each cell type, approximately 80% of the RA-regulated genes were non-overlapping. The overlapping genes (20% or 70 genes) demonstrated opposite regulation, depending on cell type. Notably, the research team observed an RA-dependent increase in TGF1β expression in MCF7 cells only and in BMP and activin membrane-bound inhibitor (BAMBI), a negative regulator of the TGF1β signalling pathway, in BT-474 cells only. Overall, they indicate that RA resistance is a deregulation of RA-target genes. Further, the team indicates that this deregulation in BT-474 cells correlates with decreased recruitment of RARα as a result of deficient phosphorylation at site S77.
Carrier et al. offer this study as evidence of the role phosphorylation plays in RA response. They suggest the results here set the stage for further investigation of deregulation of RA response as a result of disease, including cancer.
1. Carrier, M., et al. (2016) “Phosphoproteome and transcriptome of RA-responsive and RA-resistant breast cancer cell lines,” PLoS ONE, 11(6) (e0157290), doi:10.1371/journal.pone.0157290.