Driver Mutations of the Adenoma-Carcinoma Sequence Govern the Intestinal Epithelial Global Translational Capacity
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Driver mutations of the adenoma-carcinoma sequence govern the intestinal epithelial global translational capacity Wouter Laurentius Smita, Claudia Nanette Spaana, Ruben Johannes de Boera, Prashanthi Rameshb,c, Tânia Martins Garciaa, Bartolomeus Joannes Meijera, Jacqueline Ludovicus Maria Vermeulena, Marco Lezzerinid, Alyson Winfried MacInnesd, Jan Kostere, Jan Paul Medemab,c, Gijs Robert van den Brinka,f, Vanesa Muncana, and Jarom Heijmansa,g,1 aDepartment of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam University Medical Center, University of Amsterdam, 1105 BK Amsterdam, The Netherlands; bLaboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; cOncode Institute, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; dLaboratory for Genetic Metabolic Diseases, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; eDepartment of Oncogenomics, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; fRoche Innovation Center Basel, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland; and gDepartment of Internal Medicine and Hematology, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands Edited by Napoleone Ferrara, University of California San Diego, La Jolla, CA, and approved August 26, 2020 (received for review July 25, 2019) Deregulated global mRNA translation is an emerging feature of (4), ribosomal proteins and ribosomal RNA (rRNA) (5), and the cancer cells. Oncogenic transformation in colorectal cancer (CRC) is presence of oncogenes such as PIK3CA and c-MYC, that modu- driven by mutations in APC, KRAS, SMAD4, and TP53, known as late translational control (6). Thus, through aberrant signaling of the adenoma-carcinoma sequence (ACS). Here we introduce each pathways that converge on the translational apparatus, specific of these driver mutations into intestinal organoids to show that oncogenes may utilize entire proteomic programs in order to drive they are modulators of global translational capacity in intestinal oncogenic transformation of a normal cell (7). epithelial cells. Increased global translation resulting from loss of In the majority of sporadic CRCs, accumulation of a few driver Apc expression was potentiated by the presence of oncogenic mutations is considered to be sufficient in coordinating many of G12D Kras . Knockdown of Smad4 further enhanced global transla- the cell-intrinsic aspects of oncogenic transformation, which tion efficiency and was associated with a lower 4E-BP1-to-eIF4E epithelial cancer cells must adopt to clonally expand, invade, and ratio. Quadruple mutant cells with additional P53 loss displayed metastasize (8). Although the transcriptomic layers underpinning the highest global translational capacity, paralleled by high prolif- this complex process have been studied extensively, effects on eration and growth rates, indicating that the proteome is heavily specific translational programs, as well as the global translational geared toward cell division. Transcriptional reprogramming facili- capacity itself, are largely unknown. Recently, it has been shown tating global translation included elevated ribogenesis and activa- that Apc-deficient mouse intestinal cells harbor increased global tion of mTORC1 signaling. Accordingly, interfering with the translation rates via mTORC1-mediated modulation of transla- mTORC1/4E-BP/eIF4E axis inhibited the growth potential endowed tion elongation (9). This study clearly demonstrated that inter- by accumulation of multiple drivers. In conclusion, the ACS is char- fering with global translation through the mTORC1/S6K/eEF2K acterized by a strongly altered global translational landscape in axis strongly impaired intestinal epithelial hyperproliferation and epithelial cells, exposing a therapeutic potential for direct target- tumorigenesis caused by deficiency of Apc. Moreover, we have ing of the translational apparatus. Significance global translation | protein synthesis | colorectal cancer | driver mutations Deregulated global mRNA translation is a feature of various he adenoma-carcinoma sequence (ACS) is defined by a set of cancers and considered important in oncogenic transformation. Trecurrent driver mutations in APC, KRAS, SMAD4, and TP53 In colorectal cancer (CRC), the role of the most common driver genes, that accumulate during adenoma formation and pro- mutations in APC, KRAS, SMAD4, and TP53 on the global gression to sporadic colorectal cancer (CRC), often correlating translational capacity are incompletely understood. Here, using with specific stages of the developmental process (1). Since its mouse and human intestinal organoids, we found that each early description by Vogelstein and colleagues, a body of re- mutation governs the global translational capacity of the epi- search has further supported and elaborated on this well- thelial cell. Global translation is linked to known oncogenic established paradigm (2), and the putative oncogenic potential hallmarks, including cell proliferation and growth upon accu- of each mutation has been demonstrated in various genetic rodent mulation of these mutations, posing the translational appara- models (3). It is well known that driver mutations deregulate tus as a potential therapeutic target in CRC. specific cell signaling pathways, but how this ultimately leads to oncogenic transformation of a healthy intestinal cell remains to be Author contributions: W.L.S., A.W.M., G.R.v.d.B., V.M., and J.H. designed research; W.L.S., understood. Over the past decade, mounting evidence has indi- C.N.S., R.J.d.B., P.R., T.M.G., B.J.M., J.L.M.V., and M.L. performed research; J.K. and J.P.M. cated that quantitative and qualitative alterations in mRNA contributed new reagents/analytic tools; W.L.S. and C.N.S. analyzed data; and W.L.S. translation are a prominent feature of various cancers (4). Global wrote the paper. mRNA translation can be defined as the capacity of the cell’s The authors declare no competing interest. translation apparatus to produce nascent polypeptides in order to This article is a PNAS Direct Submission. maintain the cellular proteome. The translational apparatus con- Published under the PNAS license. sists of the mRNA transcripts, ribosomes, translation factors, and 1To whom correspondence may be addressed. Email: [email protected]. aminoacyl-tRNAs. Cancer cells exploit the translational apparatus This article contains supporting information online at https://www.pnas.org/lookup/suppl/ by deregulating these components, which is supported by multiple doi:10.1073/pnas.1912772117/-/DCSupplemental. lines of evidence, including altered expression translation factors First published September 28, 2020. 25560–25570 | PNAS | October 13, 2020 | vol. 117 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1912772117 Downloaded by guest on September 30, 2021 recently shown that heterozygous expression of chaperone GRP78, affected global translation in wild-type cells, we withdrew canon- which leads to repression of p-eIF2α-mediated global translation, ical WNT agonist R-spondin for 24 h from the culture medium of reduced adenoma formation in Apc-deficient mice (10). Interest- wild-type organoids. As expected, when WNT signaling could not ingly, these studies have shown that genetically interfering with be sustained, the rate of global translation declined (SI Appendix, global translation predominantly affected Apc-deficient epithelium, Fig. S1E). with no obvious discernible phenotype of the epithelium during Clinically, human loss-of-function mutations in the APC gene homeostasis (9, 10). This suggests that epithelial cells that exhibit usually precede or coincide with gain-of-function mutations in functional loss of APC rely on ample global translational output the oncogene KRAS, as illustrated by KRAS point mutations during adenoma formation, displaying the importance of transla- present in 40 to 50% of CRCs at early stages (16). The most tional efficiency in transformed cells. Furthermore, efficient mRNA frequent mutation is a base substitution KRASG12D, a conversion translation, which mainly determines the mammalian cellular pro- of glycine (G) to aspartic acid (D), that renders RAS in an active teome (11), may be particularly important for maintaining the on- GTP‐bound state causing continuous activation of RAS signaling cogenic proteome in a cancer cell that must overcome repressed (17). To model this event, we studied whether KrasG12D modu- global translation when exposed to cellular stress (12). lated global translation in wild-type and Apc-deleted epithelium. It remains to be investigated whether the main driver muta- Using inducible heterozygous organoids from Villin-CreERT2- tions associated with CRC development affect the global trans- Kras+/G12D transgenic mice, we generated organoids that grew lational capacity of the intestinal epithelial cell. To address this, independent of culture factor EGF after in vitro recombination we studied the impact of oncogenic mutations in APC, KRAS, (SI Appendix, Fig. S1 A, Middle). Kras+/G12D organoids exhibited SMAD, and TP53 on the global translational capacity of primary an enlarged central lumen with maintained pericentral budding, − − intestinal epithelial cells. Our data reveal a highly altered