The concerted action of oncogenic driver mutations directs global translation in intestinal epithelial cells

ABSTRACT

Oncogenic transformation of colorectal cancer cells is driven by a set of mutations that cause aberrant signaling of growth factor–receptor pathways. Using organoids, we demonstrate that the most frequent driver mutations in APC, KRAS, SMAD4, and TP53 are enhancers of the global mRNA translational capacity, which is linked to intestinal cell growth in an mTOR-dependent manner.

Global translation, the conversion rate of mRNA to protein, is an energy costly process that predominantly controls cellular protein abundance.¹ It is thus tightly regulated according to cellular demands, and plays a crucial role in biological processes such as cell proliferation and differentiation. In cancer, these processes are disturbed, and cancer cells are known to exhibit features of dysregulated translation, such as altered expression of key translation factors and a higher abundance of ribosomal RNA.² Although translational control is considered to be important for cancer development and progression, in colorectal cancer, the role of dysregulated translation in oncogenic transformation of epithelial cells remains to be fully understood.

Colorectal carcinogenesis (CRC) is a complex pathological process in which intestinal epithelial cells acquire genetic mutations that hyperactivate key signal transduction pathways. This orchestrates cellular hallmarks in cancer, such as uncontrolled proliferation and cell growth. Classical CRC is characterized by adenoma formation and progression toward an invasive carcinoma that may then metastasize. Vogelstein et al. observed that these developmental stages are histologically associated with a number of frequently prevalent mutations.³ These so-called driver mutations in the genes Adenomatous polyposis coli (APC), Kirsten ras (KRAS), Mothers against decapentaplegic homolog 4 (SMAD4), and Tumor protein p53 (TP53) are collectively referred to as the adenoma-carcinoma sequence (ACS). Additional genetic screening and functional studies in various rodent models have firmly proven the capacity of these mutations to initiate and promote oncogenic transformation.

Recent studies have shed more light on the involvement of altered global translation in intestinal cells during adenoma formation. Loss of tumor-suppressor APC increased the global translational capacity by accelerating the translation elongation rate.⁴ This was controlled by active mammalian target of rapamycin complex 1 (mTORC1) signaling by altering global mRNA translation in an Eukaryotic elongation factor 2 (eEF2) dependent manner, which was proven to be essential for adenoma formation in mice.⁴ Our group has shown that haploinsufficiency of enteric chaperone Glucose-Regulated Protein 78 (GRP78) leads to activation of the unfolded protein response, a cellular stress response pathway that triggers immediate and transient global translational arrest, resulted in the development of fewer adenomas in mice.⁵ These studies indicate that dysregulated translation is a key aspect of CRC cells, which may be related to the presence of driver mutations.

In a recent study, we aimed to better understand the relationship between ACS driver mutations and the cellular global translational capacity, by introducing specific mutations in Apc, Kras, Smad4, and Tp53 in mouse intestinal epithelial organoids.⁶ We then monitored the individual and combined impact of these mutations on global translation using ³⁵S-methionine incorporation, on proliferation using 5-Ethynyl-2ʹ-deoxyuridine (EdU) incorporation, and on growth rates by microscopic quantification of organoid size. We first noticed that the addition of oncogenic KRAS synergistically enhanced the elevated global translation rate as a results of APC deficiency. Surprisingly, a further rise in the global translation rate was observed in APC-KRAS mutant cells upon additional loss of SMAD4 and p53 (abbreviated AKSP organoids), exposing a remarkable residual capacity of intestinal epithelial cells to boost translation rates to very high levels. Similar patterns were observed in genetically engineered human and tumor-derived organoids. Furthermore, these changes in global translation corresponded well with increased cell proliferation and outgrowth rates of mature organoids from a single cell.

Microarray analysis in order to identify the underlying biological pathways, indicated an involvement of ribosome biosynthesis and Eukaryotic Translation Initiation Factor 4E (eIF4E) signaling which is downstream of mTOR. Subsequent analysis revealed that accumulating driver mutations indeed gradually increased downstream mTOR activity. Importantly, mTOR signaling is known to integrate an array of metabolic and environmental cues to coordinate cell proliferation and growth, thereby maintaining cellular homeostasis. This mainly occurs at the level of global mRNA translation by the downstream effectors Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and P70-S6 Kinase 1 (S6K). APC and KRAS are functionally linked to mTORC1 activity, but involvement of SMAD4 remains entirely elusive.

Interestingly, findings from two recent studies have demonstrated that the SMAD4 protein is able to bind the promoter region of the mouse and human 4EBP1 gene, stimulating global translation initiation by disinhibiting eIF4E function.^7,8 In line with these results, we found significantly lower mRNA and protein levels of 4EBP1 and a smaller fraction bound to the CAP-binding complex, upon SMAD4 depletion. We hypothesized that a concerted action of functional SMAD4 loss combined with oncogenic effects resulting from mutated APC and KRAS could fully optimize global translation efficiency in a synergistic manner dependent on eIF4E-mediated translation initiation (Figure 1). To test this, we inhibited the mTORC1-eIF4E axis using various inhibitors including rapamycin, torin-1, and ribavirin. Although cells with multiple ACS mutations become rapamycin resistant, we found that triple (AKS) and quadruple (AKSP) mutant organoids remained strongly sensitive to both torin-1 and ribavirin, displaying overt growth reduction.

Figure 1.

Deregulation of global mRNA translation by mutations in the adenoma-carcinoma sequence of CRC

Shown are the currently understood mechanisms through which oncogenic mutations of the adenoma-carcinoma sequence are able to influence global mRNA translation.mTORC1; Mammalian target of rapamycin complex 1, S6K; P70-S6 Kinase 1, eEF2K; Eukaryotic elongation factor-2 kinase, eEF2; Eukaryotic elongation factor-2, ERK; extracellular signal-regulated kinase, MNK; MAPK-interacting protein kinase, eIF4E; Eukaryotic translation initiation factor 4E, 4EBP1; Eukaryotic translation initiation factor 4E-binding protein 1.

Discrepant effects that we observed between inhibition of mTORC1 using rapamycin and torin-1 may stem from the much higher potency of the latter drug. More specifically, we speculate that the ability of torin-1 to suppress rapamycin-resistant mTORC1 functions along the mTORC1-4EBP1-eIF4E axis could be crucial.⁹ The antiviral compound ribavirin, through a different molecular mechanism, also attenuates mTOR-eIF4E signaling and causes impaired CAP-dependent translation.¹⁰ Future work must delineate the mechanistic aspects of drugs such as ribavirin on cancer cell behavior, and will reveal whether the course of in vivo adenoma formation can be altered.

In summary, we and others have highlighted that dysregulated global translation is a key feature of intestinal epithelial cells and that intervening with global translational control affects adenoma formation of APC-deficient cells. In our recent work, we demonstrated that in addition to deletion of APC, mutations in KRAS, SMAD4, and TP53 are modulators of global translation.⁶ This poses the translational apparatus and an interesting potential target for the development of treatment strategies for CRC.

Funding Statement

This work was supported by the KWF Kankerbestrijding (NL) [11053/2017- 1].