Maintenance of p-eIF2 alpha levels by the eIF2B complex is vital for colorectal cancer
Protein synthesis is an essential cellular process highly deregulated in multiple tumour types, where control of several translation factors is hijacked to benefit oncogenic growth. Here we show that colorectal cancer (CRC) is characterized  specifically by elevated levels of phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2alpha). In contrast to its canonical association with reduced translation rates, we reveal that CRC with high p-eIF2alpha has increased protein synthesis rates. Thus, we hypothesize that eIF2B, the sensor of p-eIF2 alpha, plays a central role in cells' inability to relay this inhibitory signal. Using a combination of in cellulo biochemistry, phenotypic assays, and analysis of translation upon modulation of eIF2B subunits alpha and delta, the two eIF2B subunits responsible for sensing p-eIF2 alpha, we demonstrate that CRC cells require an intact eIF2B complex to sense p-eIF2 alpha. Crucially, we show that the alpha subunit of eIF2B is necessary to translate the oncogenic programs driven by APC loss, highlighting its central role in oncogenic transformation. To conclude, we demonstrate that whilst normal cells do not depend on eIF2B alpha, CRC cells require this eIF2B subunit for correct sensing of p-eIF2 alpha and regulation of their proteostasis, thus validating eIF2B alpha as a target for therapeutic intervention in CRC. Overall design: To investigate the role of eIF2B in colorectal cancer, we depleted the 2B alpha (EIF2B1) and 2B delta (EIF2B4) subunits by inducible shRNA, comparing the effects to the induction of a non-targeting control (NTC/shCTRL). Ribo-seq and total cytoplasmic RNA (further mentioned only as Total RNA) sequencing were performed to address the effects on translation as well as transcript levels. This was performed in SW480, a well-established colorectal cancer cell line model. The experiment was performed in four independent experiment (replicates), carried out in separate weeks. Replicates are indicated by _1/_2/_3_c/_4. The replicate _3_c refers to appended sequencing results from two runs of the same library prep due to a partial failed first run of the library.