Rpl24^Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

Abstract

Increased protein synthesis supports the rapid cell proliferation associated with cancer. The Rpl24^Bst mutant mouse reduces the expression of the ribosomal protein RPL24 and has been used to suppress translation and limit tumorigenesis in multiple mouse models of cancer. Here, we show that Rpl24^Bst also suppresses tumorigenesis and proliferation in a model of colorectal cancer (CRC) with two common patient mutations, Apc and Kras. In contrast to previous reports, Rpl24^Bst mutation has no effect on ribosomal subunit abundance but suppresses translation elongation through phosphorylation of eEF2, reducing protein synthesis by 40% in tumour cells. Ablating eEF2 phosphorylation in Rpl24^Bst mutant mice by inactivating its kinase, eEF2K, completely restores the rates of elongation and protein synthesis. Furthermore, eEF2K activity is required for the Rpl24^Bst mutant to suppress tumorigenesis. This work demonstrates that elevation of eEF2 phosphorylation is an effective means to suppress colorectal tumorigenesis with two driver mutations. This positions translation elongation as a therapeutic target in CRC, as well as in other cancers where the Rpl24^Bst mutation has a tumour suppressive effect in mouse models.

Introduction

Tumour cells require rapid protein synthesis to acquire sufficient biomass in order to divide and, as such, protein synthesis is directly regulated by many oncogenic signalling pathways (Proud, 2019; Robichaud et al., 2019; Smith et al., 2021). As well as exploiting protein synthesis to drive cell division, cancers use translation to selectively synthesise a proteome geared towards proliferation, survival, and immune evasion. For example, in colorectal cancer (CRC) translation of the mRNA encoding the proto-oncogene c-MYC is selectively upregulated by eIF4E and mTORC1 signalling (Knight et al., 2020a). Likewise, independent reports have shown that the expression of the immune suppressive ligand PD-L1 is maintained on tumour cells by the activity of the translation factors eIF4A and eIF5B as well as phosphorylation of eIF4E and eIF2α (Suresh et al., 2020; Xu et al., 2019; Cerezo et al., 2018).

APC is the most commonly mutated gene in CRC, followed by TP53 and then KRAS (Guinney et al., 2015). We have previously shown that Apc-deficient mouse models of CRC are dependent on fast translation elongation, a process which can be suppressed by rapamycin leading to near complete reversal of tumorigenesis (Faller et al., 2015). This approach has had clinical success, where rapamycin (sirolimus) regressed APC-deficient polyps of familial adenomatous polyposis patients in two independent clinical trials (Yuksekkaya et al., 2016; Roos et al., 2020). Clinical data also suggest that CRCs increase translation elongation to potentiate proliferation, exemplified by the lower expression of the inhibitory kinase eEF2K correlating with worse patient survival (Ng et al., 2019). However, the regulation of translation elongation in CRC is complex, notably being influenced by specific cancer-associated mutations. We have shown that mutation of Kras drives resistance to rapamycin in Apc-deficient models both in terms of its effect on elongation and on proliferation (Knight et al., 2020a). This is consistent with KRAS-mutant CRCs being resistant to rapalogues, and other therapeutics (Ng et al., 2013; Spindler et al., 2013; DeStefanis et al., 2019) and highlights the unmet need for effective therapies against KRAS-mutant cancers. Indeed, a recently developed compound covalently targeting the Kras^G12C mutation has shown remarkable potency against this specific mutation (Hong et al., 2020).

Evidence suggests that targeting translation in KRAS-mutant CRC can be effective. As well as our recent study re-sensitising Kras-mutant CRCs to rapamycin by targeting translation initiation (Knight et al., 2020a), we have demonstrated that Kras-mutant models of CRC depend upon the transporter SLC7A5 to maintain protein synthesis by facilitating the influx of amino acids (Najumudeen et al., 2021). These data support protein synthesis as a tractable target in CRC, with the discovery of additional factors regulating these pathways only improving the potential to target protein synthesis in the clinic (Knight and Sansom, 2021).

In this study, we analyse the previously characterised Rpl24^Bst mutation in models of CRC with Apc deletion and Kras mutations. This spontaneously arising four nucleotide deletion in the Rpl24 gene, which encodes RPL24 (a component of the 60S ribosomal subunit also called large ribosomal protein subunit eL24), disrupts splicing of its mRNA, effectively resulting in a Rpl24 heterozygous animal (Oliver et al., 2004). Animals present with impaired dorsal pigmentation and malformed tails, among other defects, leading to the designation of a belly spot and tail (Bst) phenotype and the Rpl24^Bst designation. This tool has been used to suppress overall protein synthesis in genetically engineered mouse models of c-MYC-driven B-cell lymphoma, Pten-deficient T-cell acute lymphoblastic leukaemia, T-cell-specific Akt2 activation and a carcinogen-driven model of bladder cancer (Barna et al., 2008; Signer et al., 2014; Hsieh et al., 2010; Jana et al., 2021). In these studies, tumorigenesis increased total protein synthesis, which was rescued by the Rpl24^Bst mutation. Suppression of protein synthesis was sufficient to slow tumorigenesis, with some Rpl24^Bst/+ mice surviving over three times longer than the median survival of tumour model mice wild-type for Rpl24. However, the means by which the Rpl24^Bst mutation suppresses protein synthesis was not addressed in these studies, instead deferring to the original observation that there is likely a defect in ribosome production (Oliver et al., 2004).

Here, we show that decreased expression of RPL24 suppresses proliferation and extends survival in an Apc-deficient Kras-mutant pre-clinical mouse model of CRC. Importantly, we find that reduced RPL24 does not alter the available pool of ribosomal subunits, as previously suggested, but instead alters signalling that regulates a translation factor. Specifically, we observe increased phosphorylation of eEF2, an event that inhibits translation elongation. We directly measure translation elongation to show that the Rpl24^Bst mutation suppresses protein synthesis at the elongation step, consistent with increased phosphorylation of eEF2. Reducing P-eEF2 by inactivating its inhibitory kinase, eEF2K, completely restores translation elongation and protein synthesis rates as well as reversing the beneficial effect of Rpl24^Bst mutation in our tumour models. Interestingly, we find that the Rpl24^Bst mutation has no effect in Kras wild-type models. We attribute this to a specific requirement for physiological RPL24 in Kras-mutant cells, which may provide additional mechanisms to target these cells clinically.

Finally, we provide evidence from transcriptomic and proteomic analyses of patient tissue that supports the signalling pathways uncovered in our pre-clinical models being altered in the human disease. Altogether this work demonstrates that the Rpl24^Bst mutation is tumour suppressive in Kras-mutant CRC and elucidates an unexpected mode of action underlying its impact on protein synthesis. This has implications for targeting translation elongation in cancer and provides mechanistic insight to supplement the previously published efficacy of the Rpl24^Bst mouse in models of cancer and other diseases.

Results

Rpl24^Bst mutation does not alter intestinal homeostasis but suppresses the rate of translation

Prior to addressing the role of RPL24 in intestinal tumorigenesis we first analysed whether the Rpl24^Bst mutation had any effect on normal intestinal homeostasis (Figure 1A). We observed a reduction in RPL24 expression (Figure 1B), but no differences in intestinal architecture, proliferation shown by BrdU incorporation (Figure 1B) or abundance of stem cells (Olfm4), Paneth cells (lysozyme), or goblet cells (AB/PAS) (Figure 1—figure supplement 1A). Similarly, homeostasis in the colons of Rpl24^Bst mutant mice was unaffected, exemplified by no change in proliferation scored by BrdU incorporation (Figure 1—figure supplement 1C). In accordance with these in vivo observations, ex vivo organoids made from the small intestines of Rpl24^Bst/+ mice established in culture and grew comparably to wild-type controls (Figure 1C and Figure 1—figure supplement 2A). Surprisingly, we measured a >40% reduction in total protein synthesis, by ³⁵S-methionine labelling, in Rpl24^Bst/+ organoids compared to wild-type counterparts (Figure 1D). Therefore, despite no change in homeostasis, Rpl24^Bst mutation had a dramatic effect on protein synthesis. This indicates a resistance to reduced protein synthesis in the wild-type intestine, which appears to function normally despite a dramatic reduction in protein output.

Figure 1. Rpl24^Bst mutation slows translation elongation but does not affect homeostasis in the intestinal epithelium.

(A) Schematic representation of experimental procedure. Intestines from wild-type or Rpl24^Bst/+ mice were analysed by histology or processed to make intestinal organoids. (B) Staining for H&E, BrdU, RPL24, and P-eEF2 T56 in sections from the small intestines of wild-type and Rpl24^Bst/+ mice. Red brackets in P-eEF2 staining indicate crypts and villi, corresponding to quantification to the right. Bars represent 50 µm. Graphs on the right show scoring for BrdU-positive cells, and H-score calculated for RPL24 and P-eEF2 T-56, plotted ± standard error of the mean (SEM). Significance was determined by one-tailed Mann–Whitney U test. (C) Micrographs of small intestinal organoids generated from wild-type or Rpl24^Bst/+ mice. (D) Protein synthesis rate quantified by ³⁵S-methionine incorporation in wild-type or Rpl24^Bst/+ organoids (n = 3), expressed relative to the wild-type protein synthesis rate (=1). Data are from three biologically independent organoid lines for each genotype represented ± SEM with significance determine by Mann–Whitney U test. (E) Representative polysome profiling from wild-type or Rpl24^Bst/+ organoids. Average polysome:subpolysome ratios from three independent organoid lines per genotype are shown above each profile ± SEM. (F) Western blotting from protein lysates generated from three biologically independent organoid lines for each genotype. Values for RPL24 expression relative to β-actin and P-eEF2 T56 relative to eEF2 are shown under each lane. The average of the wild-type lanes in both cases has been set to 1. There is a 47% reduction in RPL24 and a 66% increase in P-eEF2 T56 in the Rpl24^Bst/+ organoids. (G) Schematic of the potential role of RPL24 in regulating protein synthesis via eEF2. All scale bars are 50 μm.

Figure 1—figure supplement 1. Rpl24^Bst mutation does not affect intestinal homeostasis.

(A) Representative images of staining for intestinal lineages and translation associated phosphorylation sites – P-4E-BP1 T37/46, P-RPS6 S240/4, and P-eIF2α S51 of intestinal sections from wild-type and Rpl24^Bst/+ mice. Olfm4 defines stem cells, lysozyme for Paneth cells, and AB/PAS (Alcian blue/periodic acid-Schiff) for goblet cells. Bars represent 50 µm. (B) H-Score quantification of P-RPS6 S240/4, P-4E-BP1 T37/46, and P-eIF2α S51 from the same experiment as in (A). (C) BrdU staining of the medial colon from wild-type and Rpl24^Bst mice, top, with scoring of BrdU-positive cells per half crypt/villus axis below. Scores are from 3 mice per genotype, each plotted as the average of at least 20 axes. All scale bars are 50 μm.

Figure 1—figure supplement 2. Rpl24^Bst mutation does not alter proliferation in organoids or the 60S:40S ratio, but does suppress regeneration post irradiation.

(A) Organoids either wild-type of mutant for Rpl24^Bst were grown over 4 days and the change in growth plotted relative to day 1. Triplicate independent lines were used for each genotype, with the average of these plotted on the graph ± standard error of the mean (standard error of the mean, SEM). Lack of significance was determined by Mann–Whitney U test. (B) Area under the curve for 40S and 60S ribosomal subunits was determined from the traces in Figure 1E. Data are from three independent biological replicates ± SEM. (C) Wild-type or Rpl24^Bst mice were given 10 Gy of irradiation then sampled 72 hr later. The number of regenerative crypts was quantified from at least six cross sections and the average plotted relative to wild-type regeneration set as 1 (n = 4 per genotype). Micrograph insets show representative sections of intestine for wild-type (black box) and Rpl24^Bst/+ (green box) mice. Red arrows indicate regenerating crypts. Bars represent 50 µm. Data are represented as the mean ± SEM with significance determined by Mann–Whitney U test. All scale bars are 50 μm.

We then investigated how Rpl24^Bst mutation suppresses translation. Performing sucrose density gradients to quantify the number of ribosomes engaged in active translation we observed an increase in ribosomes bound to mRNAs in polysomes in Rpl24^Bst/+ organoids, particularly the heavy polysomes (Figure 1E). This appears to contradict the reduction in global protein synthesis observed in Figure 1D. However, we and others have previously observed increased polysomes in conjunction with reduced protein synthesis in model systems where translation elongation is reduced (e.g. Knight et al., 2015; Faller et al., 2015). In these instances, slowed translation elongation increased the abundance of polysomes via changes in the phosphorylation of the elongation factor eEF2. We therefore assayed the regulatory phosphorylation of eEF2 (threonine 56/T56) in wild-type and Rpl24^Bst mutant samples. This phosphorylation event excludes eEF2 from the ribosome thereby impairing the translocation step of translation elongation, reducing protein synthesis (Ryazanov and Davydova, 1989; Carlberg et al., 1990).

In small intestinal tissue assayed by immunohistochemistry (IHC) we observed an increase in P-eEF2 T56, specifically in the proliferating crypt and transit amplifying zone of Rpl24^Bst/+ mouse intestines (Figure 1B). Likewise, we observed a 66% increase in P-eEF2 T56 in lysates generated from Rpl24^Bst/+ organoids compared to wild-type organoids (Figure 1F). These organoids also showed a 47% reduction in RPL24 expression by western blot (Figure 1F). Thus, in these two proliferative settings (intestinal crypts in situ and ex vivo organoids) Rpl24^Bst mutation increases the phosphorylation of eEF2, which is known to suppress translation. Surprisingly, we observed that in the differentiated villus, Rpl24^Bst mutation suppressed P-eEF2 T56 (Figure 1B). The reasons for this are unclear but may relate to different cell functions in the two compartments. This effect on P-eEF2 T56 is specific, as we observed no effect on the phosphorylation of other translation related proteins, 4E-BP1 or RPS6 (at S240/S244), readouts for modulation of signalling downstream of mTORC1 (Figure 1—figure supplement 1A, B). Similarly, we saw no change in the phosphorylation of the translation stress marker eIF2α in Rpl24^Bst mutant mice (Figure 1—figure supplement 1A, B).

Altogether, this analysis of wild-type tissue indicates that physiological RPL24 expression is not required for proliferation or function, but reduction of RPL24 expression reduces the rate of translation. Interestingly, this involves regulation of translation elongation and correlates with increased P-eEF2 (Figure 1G). Previously the Rpl24^Bst mutation had been suggested to suppress ribosome biogenesis, resulting in uneven 40S and 60S ratios. However, we observed no alteration in the relative levels of the 40S and 60S subunits in sucrose density gradients (Figure 1—figure supplement 2B), consistent with previous reports that RPL24 deletion has negligible effect on ribosome biogenesis (Barkić et al., 2009).

The wild-type mouse intestine regenerates following γ-irradiation, dependent on Wnt and mitogen-activated protein kinase (MAPK) signalling pathways. These pathways are often deregulated in colorectal tumours, such that this intestinal regeneration acts as a surrogate for oncogenic potential, with reduced regenerative capacity indicative of reduced tumorigenic proliferation (Faller et al., 2015). We observe that mutation of Rpl24 restricts regeneration of the small intestine (Figure 1—figure supplement 2C). Thus, RPL24 expression enables regeneration, which may correlate with effects in tumorigenesis.

RPL24 is required for proliferation in Apc-deficient Kras-mutant intestinal tumours

Next, we analysed the effect of the Rpl24^Bst mutation on a model of CRC driven by tamoxifen inducible Villin^CreER-mediated deletion of Apc and activation of KRAS with a G12D mutation. This model can be used with homozygous deletion of Apc (Apc^fl/fl) where intestinal hyperproliferation generates a short-term (3–4 days) model or with heterozygous deletion of Apc (Apc^fl/+) where intestinal adenomas form following spontaneous loss of the second copy of Apc. The recombination of a lox-STOP-lox allele at the endogenous Kras locus expresses a constitutively active G12D mutant form of the protein. Mice were generated with the Apc^fl/fl and Kras^G12D alleles with and without the Rpl24^Bst mutation (Figure 2A). Hyperproliferation in the Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ mutant mice was significantly suppressed compared to Apc^fl/fl Kras^G12D/+ control mice (Figure 2B, C). Reduced RPL24 expression was confirmed by IHC (Figure 2C) and coincided with increased P-eEF2 throughout the proliferative crypt area of the Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ intestine (Figure 2C and Figure 2—figure supplement 1). Hyperproliferation in the colon mirrored that of the small intestine, with reduced proliferation in mice mutant for Rpl24 compared to those expressing wild-type levels of RPL24 (Figure 2—figure supplement 1). In parallel, organoids were derived from the small intestines of the same genotypes (Apc^fl/fl Kras^G12D/+ and Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+) and their growth compared. Rpl24^Bst mutation resulted in significantly less proliferation ex vivo (Figure 2D), consistent with the in vivo experiment shown in Figure 2B.

Figure 2. Rpl24^Bst mutation suppresses proliferation and extends survival in an Apc-deficient Kras-mutant mouse model of colorectal cancer (CRC).

(A) Schematic representation of experimental protocols. Villin^CreER Apc^fl/fl Kras^G12D/+ or Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Tissue was taken for histological analysis or processed into intestinal organoids. (B) Quantification of BrdU incorporation in small intestinal crypt/villus axes following deletion of Apc and activation of Kras, with (n = 4) or without (n = 3) Rpl24^Bst mutation. Data are represented as the mean number of BrdU-positive cells per half crypt/villus from >20 axes per mouse ± standard error of the mean (SEM). Significance was determined by Mann–Whitney U test. (C) Representative images of intestines from the same experiment as in (B), stained for H&E, BrdU, P-eEF2 T56, and RPL24. The red bar on the H&E images indicates the extent of the proliferative zone. Bars represent 50 µm. (D) Apc^fl/fl Kras^G12D/+ organoids with or without Rpl24^Bst mutation were grown for 4 days and growth relative to day 1 determined by Cell-Titer Blue assay. Data show the mean ± SEM of n = 3 independent organoid lines. Significance was determined by one-tailed Mann–Whitney U test. (E) Top: schematic of experimental protocol. Villin^CreER Apc^fl/+ Kras^G12D/+ or Villin^CreER Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ mice induced with 80 mg/kg tamoxifen then monitored until clinical endpoint. Survival plot for these genotypes for the days post-induction that they reached endpoint. The median survival and n number for each cohort are shown and significance determined by Mantel–Cox test. Censored subjects were removed from the study due to non-intestinal phenotypes. All scale bars are 50 μm.

Figure 2—figure supplement 1. Rpl24Bst mutation leads to increased eEF2 phosphorylation.

(A) Top: Schematic representation of experimental approach. Villin^CreER Apc^fl/fl Kras^G12D/+, Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+, or Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Bottom: Staining for P‐4E‐BP1 T37/46, P‐RPS6 S240/4, and P‐eIF2α S51 from the genotypes above. Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A intestines were not stained for P‐eIF2α S51. Right: H‐score quantification for P‐eEF2 T56 (matched to images shown in Figures 2C and 5C), P‐4E‐BP1 T37/46, P‐RPS6 S240/4, and P‐eIF2α S51 . Small intestines from at least 3three animals from each genotype (Apc^fl/+ Kras^G12D/+, Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+, and Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A) were stained and the intensity quantified from the proliferative crypt region. Data are plotted ± standard error of the mean (standard error of the mean, SEM). Significance was determined by one‐way analysis of variance (ANOVA) analysis with Tukey’s multiple comparison. (B) Left: Representative micrograph images of each genotype stained for BrdU in the medial colons of Villin^CreER Apc^fl/fl Kras^G12D/+ and Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ mice. Right: Scores for BrdU positivity from 4 mice per genotype, each plotted as the average of at least 20 half crypt/villi. Significance was determined by Mann– Whitney U test.

Figure 2—figure supplement 2. Rpl24^Bst mutation suppresses proliferation in cells from colorectal cancer models.

(A) Top: schematic showing the generation of colonic adenoma cultures, where Villin^CreER Apc^fl/+ Kras^G12D/+ and Villin^CreER Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ mice were induced and aged until colonic tumours were present. Individual adenomas were then excised and cells isolated. Bottom: Growth of these cultures over 4 days, plotted relative to day 1. Biologically independent triplicate organoid lines were used, with the averages plotted ± SEM. Significance was tested by Mann– Whitney U test. (B) Tumour number (left) and total tumour volume (right) for indicated tumour models. Each point represents and individual mouse. From left to right n = 16, 11, 21, 13, 19, and 8 mice.

In the tumour model, where a single copy of Apc is deleted, Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ mice lived on average 32 days longer than Apc^fl/+ Kras^G12D/+ controls, an extension of survival of 45% (Figure 2E). Furthermore, Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ organoids derived from the adenomas in this tumour model grew more slowly than controls (Figure 2—figure supplement 2). There was no significant difference in the number or volume of tumours at experimental endpoint (Figure 2—figure supplement 2), indicating that adenomas can form but take longer to reach a clinically significant burden. Therefore, RPL24 enables proliferation in Apc-deficient, KRAS-activated cells within the intestinal epithelium of the mouse.

RPL24 maintains translation elongation in Apc-deficient Kras-mutant intestinal tumour models

The suppression of tumorigenesis in the Apc-deficient Kras-mutant model correlated with increased phosphorylation of eEF2 (Figure 2C and Figure 2—figure supplement 1). Increased P-eEF2 was not accompanied by increased P-eIF2α, which controls translation initiation in response to various stress signals (Figure 2—figure supplement 1). This indicates that stress signalling to eIF2α was not influenced by the Rpl24^Bst mutation, highlighting the specificity in the RPL24-dependent regulation of eEF2. To investigate this further we used three methods to measure the rate of translation: polysome profiling, ³⁵S-methionine labelling, and harringtonine run-off assays (Figure 3A). Polysome profiling from extracted crypts from Apc^fl/fl Kras^G12D/+ and Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ mice showed an increase in polysomes with the Rpl24 mutation (Figure 3B), and notably a significant increase in the quantity of heavy polysomes (Figure 3C). Intestinal organoids of the same genotype showed a 35% reduction in ³⁵S-methionine incorporation (Figure 3D). These same organoids had a greater than 40% decrease in elongation rate measured by harringtonine run-off (Figure 3E and Figure 3—figure supplement 1A). Together these data provide compelling evidence that normal RPL24 expression is required to maintain translation elongation in this CRC model. Polysome profiles and protein synthesis rate measurements from Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ adenoma cultures also showed more polysomes and lower protein synthesis compared to control adenoma cultures (Figure 3—figure supplement 1B, C). The reduced protein synthesis rate does not correlate with differences in free ribosomal subunit availability as the ratio of 40S to 60S subunits is unchanged by the Rpl24 mutation in Apc-deficient Kras-mutant cells (Figure 3—figure supplement 1D). To investigate this further we assayed selected ribosomal protein abundances in Apc^fl/fl Kras^G12D/+ and Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids by western blot. RPL10 expression was increased by the Rpl24 mutation, RPL22 was decreased, while RPS6 levels were unchanged (Figure 3—figure supplement 1E). While the significance of these individual changes is unknown, the data show that there is not a global suppression of ribosomal protein expression in Rpl24 mutant cells, consistent with no change in free ribosomal subunit levels.

Figure 3. Rpl24^Bst mutation slows translation elongation in Apc-deficient Kras-mutant mouse models of colorectal cancer (CRC).

(A) Schematic representation of experimental approach. Villin^CreER Apc^fl/fl Kras^G12D/+ or Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Intestines were enriched for crypt epithelium for sucrose density analysis or processed into intestinal organoids. (B) Representative sucrose density polysome profiles generated from Apc^fl/fl Kras^G12D/+ intestinal extracts with or without the Rpl24^Bst mutation. Subpolysomal components (40S, 60S, and 80S) and polysomes are labelled, with the polysomes also split pictorially into light and heavy. (C) Quantification of the heavy:light polysome ratio from the experiment in (B). Data show the mean of analysis from three mice ± standard error of the mean (standard error of the mean, SEM) with significance determined by one-tailed Mann–Whitney U test. (D) Relative protein synthesis rate quantified by ³⁵S-methionine incorporation in Apc^fl/fl Kras^G12D/+ three biologically independent organoid lines either wild-type or mutant for Rpl24^Bst. Data are represented ± SEM with significance determine by Mann–Whitney U test. (E) Ribosome run-off rate determined in Apc^fl/fl Kras^G12D/+ small intestinal organoid lines either wild-type or mutant for Rpl24^Bst (n = 3 per genotype). Data are represented as the mean of three biological replicates ± SEM with significance determine by Mann–Whitney U test. Raw data are available in Figure 3—figure supplement 1A. (F) Schematics of the regulation of protein synthesis and tumour proliferation downstream of RPL24. Smaller RPL24 in bottom scheme represents reduced RPL24 expression. ‘P’ represents phosphorylation of eEF2.

Figure 3—figure supplement 1. The effect of Rpl24^Bst mutation on translation and ribosome composition.

(A) Representative polysome profiles from Apc^fl/fl Kras^G12D/+ or Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ small intestinal organoid cultures, pre-treated with harringtonine for 5 min/300 s (H300) or untreated (H0). These traces were analysed for the run-off rates shown in Figure 3E. (B) Representative polysome profiles from Apc^fl/+ Kras^G12D/+ or Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ colonic adenoma cultures (left) and quantification of the polysome to subpolysome ratio from these (right). Two biologically independent lines were analysed per genotype and plotted ± standard error of the mean (SEM). Scheme above denotes the generation of adenoma cultures from distinct colonic tumours in aged Villin^CreER Apc^fl/+ Kras^G12D/+ and Villin^CreER Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ mice. (C) Relative protein synthesis rates quantified by ³⁵S-methionine incorporation in the colonic adenoma cultures described in (B) with n = 3. The average protein synthesis rates were plotted relative to Apc^fl/+ Kras^G12D/+ controls (=1) for three organoid lines per genotype ± SEM. Significance was determined by Mann–Whitney U test. (D) 60S to 40S ratio from sucrose density gradients from lysates generated from the indicated genotypes. Data show the mean ± SEM. Representative traces are shown in Figure 3B and Figure 4—figure supplement 2B. (E) Western blotting from protein lysates generated from three biologically independent organoid lines for each genotype. Mean values for RPL24, RPL10, RPL22, and RPS6 expression relative to β-actin and P-eEF2 T56 relative to eEF2 are shown next to the relevant blot ± SEM. The average of the Villin^CreER Apc^fl/+ Kras^G12D/+ lanes in all cases has been set to 1.

Figure 3—figure supplement 2. Assocation of ribosomal proteins with the ribosomes in Rpl24^Bst mutant and wild-type organoids.

Top: protein purified from sucrose density gradients performed on Villin^CreER Apc^fl/+ Kras^G12D/+ and Villin^CreER Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ organoids was resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot performed for ribosomal proteins and β-tubulin, as a non-ribosomal control. Fractions within the gradient are annotated as non-ribosomal (non-ribo), 40S, 60S, 80S, and polysomes. Data are representative of three independent biological replicates. These gradients were also used in the analysis shown in Figure 3A, E, with these being the H0 replicates from that experiment. Bottom: quantification of RPL24 and RPL10 abundances, as a percentage of total cytoplasmic protein, across gradients from Villin^CreER Apc^fl/+ Kras^G12D/+ and Villin^CreER Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ organoids. Circles show individual values from three replicates with bars plotting the mean of these ± standard error of the mean (standard error of the mean, SEM).

The reduced expression of RPL24, but maintained ribosomal subunit stoichiometry, in Rpl24^Bst/+ mice raises the possibility of heterogeneous ribosomes (reviewed by Gay et al., 2021), with some potentially lacking RPL24. To address this, we purified protein from sucrose density gradients from Apc^fl/fl Kras^G12D/+ and Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids and analysed ribosomal protein expression within subpolysomes and polysomes (Figure 3—figure supplement 2). The large subunit protein RPL10 showed little change in distribution between the two genotypes, consistent with no change in free ribosomal subunit levels and a shift from light to heavy polysomes, but similar overall polysome number. Importantly, RPL24 can incorporate into 60S, 80S, and polysomes in Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids. The distribution of RPL24 appeared altered, with less in the 60S and more in the polysomes, although this was not significant. It is difficult to interpret absolute amounts of protein in each fraction and to compare this between genotypes. However, considering that Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids express less RPL24 (Figure 3—figure supplement 1E) the reduction in RPL24 in 60S subunits is even more striking. Thus, our data do not rule out the possibility of ribosome heterogeneity in that some 60S subunits in Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids may lack RPL24.

Interpreting this molecular analysis in conjunction with the effects on tumorigenesis leads to the conclusion that RPL24 expression maintains translation elongation and protein synthesis rates, which in turn maintain tumour-related proliferation (Figure 3F). Suppressing RPL24 expression increases P-eEF2, an effect also seen in lysates from Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids (Figure 3—figure supplement 1E), which decreases the rate of elongation and overall protein synthesis and correlates with suppressed tumorigenesis and proliferation in vivo.

Rpl24 mutation has no effect in CRC models expressing wild-type Kras

In parallel to analysing the effect of Rpl24 mutation in Apc-deficient Kras-mutant intestinal tumours, we also assessed its role in Apc-deficient models wild-type for Kras. We have previously shown that these are dependent on signalling from mTORC1 to maintain low levels of P-eEF2, and that rapamycin induces P-eEF2 to great therapeutic benefit (Faller et al., 2015). We observed that the hyperproliferation in the small intestine or colon of Apc^fl/fl mice was not reduced by the Rpl24^Bst mutation (Figure 4A, B and Figure 4—figure supplement 1A). Furthermore, in both germline Apc^Min/+ and inducible Apc^fl/+ models of Apc deficiency we see no benefit of the Rpl24^Bst mutation, with no difference in survival or tumour development (Figure 4C and Figure 4—figure supplement 1B). In agreement, Apc^fl/fl organoids with the Rpl24^Bst mutation grew at an identical rate to those wild-type for Rpl24 in culture (Figure 4D). Together these data demonstrate that reduced RPL24 expression does not limit tumorigenesis in Apc-deficient CRC models with wild-type Kras.

Figure 4. Rpl24^Bst mutation does not suppress proteins synthesis or proliferation in Apc-deficient Kras wild-type mouse models of colorectal cancer (CRC).

(A) Schematic representation of experimental approach. Villin^CreER Apc^fl/fl or Villin^CreER Apc^fl/fl Rpl24^Bst/+ mice were induced by two intraperitoneal injection of tamoxifen at 80 mg/kg on days 0 and 1 then intestinal tissue analysed on day 4. Intestines were analysed histologically or intestinal organoids generated. (B) Top: representative micrographs showing proliferation as BrdU positivity and extent of proliferation as a red bar in H&E image. RPL24 and P-eEF2 T56, staining is also shown for each genotype. Bars represent 50 µm. Below: BrdU scoring from Apc^fl/fl or Apc^fl/fl Rpl24^Bst/+ mouse intestines and H-scores for RPL24 and P-eEF2 T56 protein levels. For BrdU scoring BrdU was administered 2 hr before sampling and at least 20 half crypt/villus axes were scored per animal and the mean plotted ± standard error of the mean (standard error of the mean, SEM). (C) Apc^Min/+ tumour model survival curve, for mice with and without Rpl24^Bst mutation. Lack of a significant difference was determined by Mantel–Cox test. (D) Relative growth of Apc^fl/fl and Apc^fl/fl Rpl24^Bst/+ small intestinal organoids over 3 days, measure by Cell-Titer Blue assay. The average change in proliferation is plotted from three independent biological replicates per genotype. (E) Relative protein synthesis rates quantified from ³⁵S-methionine incorporation into Apc^fl/fl, Apc^fl/fl treated with 250 nM rapamycin for 24 hr and Apc^fl/fl Rpl24^Bst/+ small intestinal organoids. Significant changes were calculated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. N = 3 per genotype with the mean protein synthesis rate for each genotype plotted ± SEM. (F) Relative expression of ribosomal protein mRNAs in Villin^CreER Apc^fl/fl and Villin^CreER Apc^fl/fl Kras^G12D/+ whole intestine samples, where wild-type tissue has been normalised to 1. The fold increase in expression from Apc^fl/fl to Apc^fl/fl Kras^G12D/+ samples for Rpl24 and the average of all other RP mRNAs are shown. Statistical analysis was by one sample t-test of the other RP mRNA fold changes using the fold change for Rpl24 mRNA as the hypothetical mean. All scale bars are 50 μm.

Figure 4—figure supplement 1. Rpl24^Bst mutation has no benefit in a model of colorectal cancer (CRC) with wild-type Kras.

(A) Left: staining for BrdU in the medial colons of Villin^CreER Apc^fl/fl and Villin^CreER Apc^fl/fl Rpl24^Bst/+ mice. Bottom: scores are from 3 and 5 mice per genotype, each plotted as the average of at least 20 half crypt/villi. Lack of significance was determined by Mann–Whitney U test. (B) Left: schematic of experiment, Villin^CreER Apc^fl/+ mice with or without Rpl24^Bst mutation were induced then aged until showing signs of intestinal tumours. Right: survival curve from the Villin^CreER Apc^fl/+ tumour model, with and without Rpl24^Bst mutation. Censored subjects were sampled for health reasons not relating to the intestine. Lack of a significant difference was determined by Mantel–Cox test. All scale bars are 50 μm.

Figure 4—figure supplement 2. Rpl24^Bst mutation has no effect on polysomes or some signaling pathways.

(A) Schematic representation of experimental approach. Villin^CreER Apc^fl/fl or Villin^CreER Apc^fl/fl Rpl24^Bst/+ mice were induced by two intraperitoneal injection of tamoxifen at 80 mg/kg on days 0 and 1 then intestinal tissue analysed on day 4. Intestines analysed histologically or by epithelial extraction for sucrose density gradient analysis. (B) Left: representative polysome profiles generated from Apc^fl/fl intestinal extracts with or without the Rpl24^Bst mutation. Subpolysomal components and polysomes are labelled. Right: quantification of the polysome:subpolysome ratio across three biologically independent replicates for each genotype. Data show the mean ± standard error of the mean (SEM). (C) Staining of small intestinal tissue for P-4E-BP1 T37/46, P-RPS6 S240/4, and P-eIF2α S51 from Villin^CreER Apc^fl/fl or Villin^CreER Apc^fl/fl Rpl24^Bst/+ mice alongside H-score quantification from the proliferative zones of the intestines of at least 3 mice per genotype. (D) Relative ribosomal protein mRNA abundances from RNA sequencing of wild-type, Villin^CreER Apc^fl/fl and Villin^CreER Apc^fl/fl Kras^G12D/+ whole small intestinal tissue. Three independent biological samples were analysed per genotype with the averages used in this analysis. RNA sequencing reads for Villin^CreER Apc^fl/fl and Villin^CreER Apc^fl/fl Kras^G12D/+ tissue was normalised to wild-type tissue set to 1. Values are shown horizontally scaled as fold changes to the wild-type tissue for each genotype. All scale bars are 50 μm.

Despite no effect on proliferation, we observed an increase in P-eEF2 in Apc^fl/fl Rpl24^Bst/+ intestines compared to Apc^fl/fl (Figure 4B), showing that P-eEF2 is consistently increased in the intestinal crypts of Rpl24^Bst/+ mice. However, we detected no change in the ratio of polysomes to subpolysomes in Apc^fl/fl Rpl24^Bst/+ intestines compared to Apc^fl/fl (Figure 4—figure supplement 2A, B) and no change in protein synthesis rate between organoids of these same genotypes (Figure 4E). In contrast, rapamycin treatment significantly reduces protein synthesis in Apc^fl/fl organoids treated in parallel (Figure 4E). From these data, we conclude that the change in P-eEF2 does not limit the rate of protein synthesis which allows efficient tumorigenesis in these Kras wild-type models of CRC. There was no alteration in the phosphorylation status of 4E-BP1, RPS6, or eIF2α in the Rpl24^Bst mutants in the Apc^fl/fl model (Figure 4—figure supplement 2C), indicating that translation promoting mTORC1 signalling remains high while translation stress signalling to eIF2α is unchanged.

We hypothesised that the reason for the KRAS specificity seen with the Rpl24^Bst mutation may relate to expression levels between the different genotypes analysed. Using unbiased RNA sequencing data from wild-type, Apc^fl/fl and Apc^fl/fl Kras^G12D/+ small intestinal tissue we observed a consistent increase in ribosomal protein expression following Apc deletion, then again following KRAS activation (Figure 4—figure supplement 2D). This is consistent with previous reports (Smit et al., 2020), and a requirement to increase protein synthesis as a direct consequence of KRAS activation. Indeed, the mRNAs for all ribosomal proteins with sufficient reads were increased on average nearly 1.5-fold by KRAS activation in the small intestine (Figure 4F). In contrast, the Rpl24 mRNA was only increased by 1.14-fold following Kras mutation, despite a nearly twofold increase following deletion of Apc (Figure 4F). This manifests as a significant difference in the RNA expression of Rpl24 compared to the other ribosomal proteins. Therefore, RPL24 expression may be sufficient in Rpl24^Bst mice in Apc-deleted models, but then becomes limiting following Kras mutation due to the limited upregulation of Rpl24 expression accompanying KRAS activation.

Genetic inactivation of eEF2K completely reverses the anti-proliferative benefit of Rpl24 mutation

Thus far we have demonstrated a correlation between the increase in P-eEF2 and the slowing of translation elongation following Rpl24^Bst mutation. To test whether the slowing of elongation caused by Rpl24^Bst mutation was dependent on P-eEF2, we used a whole-body point mutant of eEF2K, the kinase that phosphorylates eEF2, which almost completely inactivates its kinase activity (Gildish et al., 2012). We crossed this Eef2k^D273A/D273A allele to the Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ mice to generate Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A mice. In the short-term hyperproliferation model, the inactivation of Eef2k completely reversed the suppression of proliferation seen in Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ small intestines (Figure 5A–C), and the medial colon (Figure 5—figure supplement 1A). The kinase inactive Eef2k allele resulted in P-eEF2 being undetectable (Figure 5C and Figure 2—figure supplement 1), and we previously reported no difference in hyperproliferation between Apc^fl/fl Kras^G12D/+ and Apc^fl/fl Kras^G12D/+ Eef2k^D273A/D273A models (Knight et al., 2020a). The reversal in proliferation rate with Eef2k and Rpl24^Bst mutations was also seen in intestinal organoid growth after 3 days (Figure 5D). Furthermore, inactivation of eEF2K reverted the survival benefit of the Rpl24^Bst mutation in the Apc^fl/+ Kras^G12D/+ tumour model (Figure 5E). This experiment also shows the lack of effect of the Eef2k^D273A/D273A mutation on tumorigenesis. Indeed, Eef2k^D273A/D273A had no impact on tumorigenesis in inducible Apc^fl/+ and germline Apc^Min/+ models of Apc-deficient intestinal tumorigenesis (i.e., expressing wild-type Kras) (Figure 5—figure supplement 1B, C). Consistent with this, the Eef2k^D273A/D273A allele had no effect on proliferation in the Apc^fl/fl hyperproliferation model, either alone or in combination with Rpl24^Bst mutation in both the small intestine and colon (Figure 5—figure supplement 1D). Heterozygous inactivation of Eef2k resulted in a slight reversal of the extension of survival associated with Rpl24^Bst mutation in the Apc^fl/+ Kras^G12D/+ tumour model (Figure 5—figure supplement 2A).

Figure 5. Genetic inactivation of Eef2k reverses the reduced tumorigenesis following Rpl24^Bst mutation in Apc-deficient Kras-mutant models of colorectal cancer (CRC).

(A) Schematic representation of experimental approach. Villin^CreER Apc^fl/fl Kras^G12D/+, Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+, or Villin^CreER Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Intestines were analysed histologically or processed into intestinal organoids. (B) BrdU incorporation quantified from within small intestinal crypt/villus axes following deletion of Apc and activation of Kras, either wild-type of mutant for Rpl24, or mutant for Rpl24 and Eef2k. Data are represented as the mean of at least 20 crypt/villi per mouse ± standard error of the mean (SEM) with significance determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. N = 3 per genotype. (C) Representative images of H&E, BrdU, and P-eEF2 T56 staining of intestines from the same experiment as (B). Red bar on H&E indicates extent of proliferative zone. Bars represent 50 µm. (D) Organoids deficient for Apc and with activated Kras with or without Rpl24^Bst mutation, or mutant for both Rpl24 and Eef2k were grown for 3 days and growth relative to day 1 determined by Cell-Titer Blue assay. Data show the mean ± SEM of n = 3 biologically independent organoid lines. Significance was determined by one-tailed Mann–Whitney U test. (E) Survival plot for Apc Kras ageing mice with or without the Rpl24^Bst mutation, Eef2K mutation and with both Rpl24 and Eef2k mutations. Median survival and n numbers for each cohort are shown and significance determined by Mantel–Cox test. Censored subjects were removed from the study due to non-intestinal phenotypes. All scale bars are 50 μm.

Figure 5—figure supplement 1. Eef2k^D273A/D273A mutation has no effect on tumorigenesis in Kras wild-type models.

(A) Scoring for BrdU incorporation in the medial colons of Apc^fl/fl Kras^G12D/+, Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+, and Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A mice. Scores are from 3, 4, and 3 mice, respectively, each plotted as the average of at least 20 half crypt/villi. Significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. (B) Survival curve from the Apc^fl/+ tumour model for mice bearing the Eef2k^D273A/D273A mutation or wild-type for Eef2k. Lack of a significant difference was determined by Mantel–Cox test. (C) Apc^Min/+ tumour model survival curve, for mice with wild-type Eef2k or the inactivating mutation, Eef2k^D273A/D273A. Lack of a significant difference was determined by Mantel–Cox test. (D) Scoring for BrdU-positive cells in the proximal small intestine and medial colons of Apc^fl/fl, Apc^fl/fl Rpl24^Bst/+, Apc^fl/fl Eef2k^D273A/D273A, and Apc^fl/fl Rpl24^Bst/+ Eef2k^D273A/D273A mice. Each point on the graphs is plotted as the average of at least 20 half crypt/villi.

Figure 5—figure supplement 2. Heterozygous mutation of Eef2k^D273A/+ partially suppresses the effects of Rpl24^Bst mutation in the tumour model.

(A) Survival curve from the Apc^fl/+ Kras^G12D/+ tumour model for mice bearing the Rpl24^Bst mutation and/or one copy of the inactivating Eef2k mutation. Median survival in days and n numbers are shown for each genotype. Significance test was performed by Mantel–Cox test. Apc^fl/+ Kras^G12D/+ and Apc^fl/+ Kras^G12D/+ Rpl24^Bst/+ survival curves are reused from Figure 5E. (B) Tumour number (top) and total volume (bottom) scored macroscopically from Apc^fl/+ Kras^G12D/+ mice with or without Rpl24^Bst mutation and with no, one, or two copies of the inactivating Eef2k^D273A mutation. Each point is an individual mouse with the bars depicting the mean ± standard error of the mean (standard error of the mean, SEM). From left to right n = 20, 11, 14, 9, 18, and 11 mice.

Therefore, mutation of Rpl24 requires functional eEF2K to suppress proliferation and extend survival in this model of CRC. There was no alteration in the number or cumulative size of tumours in the ageing model at endpoint (Figure 5—figure supplement 2B), again identifying tumour cell proliferation, rather than tumour initiation, as the principal factor regulated by RPL24 and eEF2K. These data also confirm that the effect of Rpl24^Bst mutation on the tumour phenotype is entirely dependent upon eEF2K activity.

Rpl24^Bst mutation suppresses translation exclusively via eEF2K/P-eEF2

Next, we addressed the molecular consequences of inactivation of eEF2K downstream of Rpl24^Bst mutation. The reduction in protein synthesis that results from Rpl24^Bst mutation is completely reversed by inactivating eEF2K (Figure 6A), with Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A organoids having an almost identical translation capacity as Apc^fl/fl Kras^G12D/+ controls. Similarly, the rate of ribosome run-off was also reverted in Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A compared to Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids, again to the same rate as controls with wild-type Rpl24 (Figure 6B and Figure 6—figure supplement 1A). In agreement, crypt fractions from Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A mice have a reduced, although not significant, the number of heavy polysomes compared to Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ crypt cells (Figure 6—figure supplement 2B), indicating faster translation elongation following eEF2K inactivation.

Figure 6. Genetic inactivation of Eef2k restores translation rates following Rpl24^Bst mutation.

(A) ³⁵S-methionine incorporation to determine relative protein synthesis by in Apc^fl/fl Kras^G12D/+ small intestinal organoids wild-type or mutant for Rpl24 or with both Rpl24 andConsistent with this, the Eef2k mutations. Data are represented ± standard error of the mean (standard error of the mean, SEM) with significance determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. N = 3 per genotype, each representing an independent organoid line. (B) Ribosome run-off rate determined in Apc^fl/fl Kras^G12D/+ small intestinal organoids mutant or wild-type for Rpl24 or with both Rpl24 and Eef2k mutations. Data are the mean of three biologically independent organoid lines represented ± SEM with significance determined by Mann–Whitney U test. Raw data are available in Figure 6—figure supplement 1A. The run-off rate for Apc^fl/fl Kras^G12D/+ control organoids is reproduced from Figure 3E. (C) Schematic representation of findings in Apc-deficient Kras-mutant mouse and organoid models. Top: RPL24 expression maintains translation and proliferation by suppressing the phosphorylation of eEF2 by limiting eEF2K activity. Middle: reduced expression of RPL24 activates eEF2K, increasing P-eEF2, reducing translation elongation and suppressing tumorigenesis and proliferation. Bottom: inactivation of eEF2K reverts the phenotype in Rpl24^Bst cells, due to the inability to phosphorylate and suppress eEF2. Elevated elongation rates correlate with increased proliferation following inactivation of eEF2K.

Figure 6—figure supplement 1. Inactivation of Eef2k restores translation elongation speed in Rpl24^Bst mutant mice.

(A) Polysome profiles from sucrose density gradients of Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ or Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ Eef2k^D273A/D273A small intestinal organoid cultures, pre-treated with harringtonine for 5 min/300 s (H300) or untreated (H0). These traces are representative of those analysed for the run-off rates shown in Figure 6B. (B) Representative sucrose density profiles generated from Apc^fl/fl Kras^G12D/+ intestinal extracts with or without the Rpl24^Bst mutation and inactivation of eEF2K. Subpolysomal components and polysomes are labelled, and the polysomes have been split pictorially into light and heavy. To the right of this is quantification of the heavy:light polysome ratio. Data show the mean ± standard error of the mean (standard error of the mean, SEM) of 5, 3, and 3 mice reading from left to right. Significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison.

Figure 6—figure supplement 2. Rpl24^Bst mutation has no effect on P-ERK or P-ACC.

(A) Immunohistochemistry (IHC) staining for P-ERK T202/T204 in intestinal tissue form Apc^fl/fl Kras^G12D/+ or Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ mice. Representative images from each genotype are shown on the right and H-score quantification from three animals per genotype on the left. (B) Western blotting on lysates from Apc^fl/fl Kras^G12D/+ or Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ organoids, for P-ACC S79, ACC, and β-actin as a sample control. All scale bars are 50 μm.

This has important implications for the function of RPL24, showing that ribosomes in Rpl24^Bst mutant cells can elongate efficiently despite the reduction in RPL24 expression. However, reduced RPL24 increases P-eEF2 which inhibits elongation, with an absolute requirement for eEF2K for this (Figure 6C). Importantly, the combination of the Rpl24 and eEF2K mutants shows that when P-eEF2 is abolished elongation occurs at normal speed, despite reduced expression of RPL24.

eEF2K activity is regulated by several upstream signalling pathways, as well as factors such as calcium ion and oxygen levels (Ballard et al., 2021). Given that the Rpl24^Bst mutation increases phosphorylation of P-eEF2, we assessed what effect the mutation had on multiple kinases upstream of eEF2K in Apc^fl/fl Kras^G12D/+ and Apc^fl/fl Kras^G12D/+ Rpl24^Bst/+ small intestinal tissue and organoids. mTORC1 suppresses eEF2K activity via p70 S6K. However, we saw no difference in the mTORC1-regulated phosphorylation of RPS6 (S240/4) and 4E-BP1 (T37/46) (Figure 2—figure supplement 1). Similarly, MAPK signalling also suppresses eEF2K, via the RSK pathway. There was no difference in signalling through this pathway in Rpl24 mutant mice, as assayed via P-ERK1/2 (T202/Y204) (Figure 6—figure supplement 2A). AMPK directly phosphorylates and activates eEF2K in response to changes in intracellular ATP:AMP ratio. Using phosphorylation of the canonical AMPK target acetyl-CoA carboxylase (P-ACC S79) as a read-out of AMPK activity, Rpl24 mutation had no effect on AMPK signalling (Figure 6—figure supplement 2B). Thus, we find no evidence for RPL24 expression levels affecting the activity of three kinases that in turn regulate eEF2K.

The expression of RPL24, EEF2K, and EEF2 is indicative of fast elongation in human CRC

Using pre-clinical mouse models, we have demonstrated that physiological RPL24 expression maintains low eEF2K-mediated phosphorylation of eEF2 (Figure 7A). Hypo-phosphorylated eEF2 then ensures rapid protein synthesis enabling tumour proliferation in vivo. We next sought to position this pre-clinical work in the context of clinical studies of the human disease. Using publicly available datasets for RNA expression in normal and cancerous colon tissues we observe increased RPL24 and EEF2 expression in conjunction with reduced EEF2K expression (Figure 7B). This mirrors the signalling pathways in our mouse models and ensures high expression of active eEF2. Elevated EEF2 message and reduced EEF2K message levels in these clinical samples are consistent with conservation of this signalling in the clinic. Similar results are seen for the protein expression of RPL24, eEF2K, and eEF2 from colon adenocarcinoma samples (Figure 7—figure supplement 1A), and for the three mRNAs in rectal adenocarcinoma (Figure 7—figure supplement 1B). These expression analyses highlight the conservation of the proliferative tumour-associated signalling pathways characterised in our pre-clinical mouse models and patient samples.

Figure 7. Expression of RPL24, EEF2K, and EEF2 is consistent with increased eEF2 activity in colorectal cancer (CRC) tumours.

(A) Schematic of the findings presented here from pre-clinical mouse models. KRAS activation requires RPL24 expression to maintain low eEF2 phosphorylation. This occurs via a double negative regulation of eEF2K, whereby RPL24 suppresses eEF2K, which suppresses eEF2. eEF2 activity correlates with protein synthesis and proliferation rates. Dashed lines indicate indirect or undefined regulatory pathways. (B) RNA expression levels of RPL24, EEF2K, and EEF2 between normal colon and colon adenocarcinoma samples using data extracted from The Cancer Genome Atlas by TNMplot. Relative expression changes are annotated, as well as p values for each transcript.

Figure 7—figure supplement 1. Expression of RPL24, EEF2K, and EEF2 suggest increased eEF2 activity in colorectal cancer (CRC) tumours.

(A) Protein levels of RPL24, eEF2K, and eEF2 in normal colon and colon adenocarcinoma collated from the Clinical Proteomic Tumor Analysis Consortium by UALCAN. Numbers of samples and p values are shown for each protein. (B) RNA expression levels of RPL24, EEF2K, and EEF2 between normal rectum and rectum adenocarcinoma samples using data extracted from The Cancer Genome Atlas by TNMplot. Relative expression changes are annotated, as well as p values for each transcript.

Discussion

The original characterisation of the Rpl24^Bst mutation identified a defect in ribosome biogenesis affecting the synthesis of 60S subunits (Oliver et al., 2004). The evidence to support this was limited to analyses of fasted/refed mouse livers for nascent rRNAs and by polysome profiles purporting to show reductions in 28S rRNA precursors and mature 60S subunits, respectively. In contrast, RNAi depletion of RPL24 in human cell lines had no effect on ribosome biogenesis, or on the relative abundance of 40S and 60S subunits (Barkić et al., 2009; Wilson-Edell et al., 2014). Furthermore, two reports have identified RPL24 as an exclusively cytoplasmic protein, leading to the hypothesis that it is assembled into mature ribosomes in the cytoplasm after rRNA synthesis and processing has already occurred (Barkić et al., 2009; Saveanu et al., 2003). In agreement with this, in the hindbrain of E9.5 embryos, the Rpl24^Bst mutation had no effect on nucleolar architecture, indicating that it is not required for nucleolar function (Herrlinger et al., 2019). Our data agree with these later examples, as we fail to see any effect of the Rpl24^Bst mutation on 60S:40S ratio in wild-type or transformed mouse intestines. Of further importance to the field, we also demonstrate in our tumour model that the translation defect in Rpl24^Bst mutant mice is restored to normal levels following inactivation of eEF2K in Rpl24^Bst mutant mice, showing that the defect is dependent on eEF2K. From this, we conclude that although the ribosomes produced in Rpl24^Bst/+ mice do allow protein synthesis to proceed at near physiological levels, this process is restricted via signalling through eEF2K/P-eEF2.

RPL24 depletion suppresses tumorigenesis in Apc-deficient Kras-mutant (APC KRAS) mouse models of CRC, but not in Apc-deficient Kras wild-type (APC) models. In line with this, protein synthesis is reduced following depletion of RPL24 in our APC KRAS models, but not in APC models, despite induction of P-eEF2 in both cases. We previously showed that suppression of translation elongation via mTORC1/eEF2K/P-eEF2, using rapamycin, in the same APC models dramatically suppressed proliferation and extended survival (Faller et al., 2015). Thus, while RPL24 depletion or rapamycin treatment of Apc-deficient intestines each induce P-eEF2, only rapamycin treatment suppresses protein synthesis. The effect on protein synthesis appears to be the differential driving this divergence, with proliferation only impaired when protein synthesis is reduced. Crucially, RPL24 deficiency does not suppress mTORC1 activity since phosphorylation of the mTORC1 effectors 4E-BP1 and RPS6 (at S240/S244) is not reduced in Apc-deficient Rpl24^Bst/+ intestines, providing an explanation of how proliferation and protein synthesis are maintained in Apc-deficient Rpl24^Bst mutant animals. We also provide evidence that Rpl24 expression is lower than that of other ribosomal protein mRNAs following Kras mutation, which could explain why diminished RPL24 expression specifically suppresses proliferation in Kras-mutant models.

In view of the mechanisms we have described here, it is interesting to reflect on previous work with the Rpl24^Bst mutant mouse. The Rpl24^Bst mutation suppresses tumorigenesis in three mouse models of different types of blood cancer, and in a model of bladder cancer (Signer et al., 2014; Barna et al., 2008; Hsieh et al., 2010; Jana et al., 2021). In each case, the Rpl24^Bst mutation was found to decrease translation, although how the translation was suppressed was not fully determined. Barna et al. demonstrated a reduction in cap-dependent translation using a luciferase reporter in Rpl24^Bst MEFs (Barna et al., 2008). Furthermore, Rpl24^Bst mutation dramatically suppressed translation following MYC activation, consistent with Rpl24^Bst slowing tumorigenesis via reduced translation. We have shown that in mouse models of CRC the molecular mechanism by which normal levels of RPL24 maintain translation is via eEF2K and P-eEF2, therefore implicating this pathway in these previously studied blood cancer models.

The Rpl24^Bst mutation has also been used to analyse brain development from neural progenitor cells and explored as a model for retinal degenerative disease (Riazifar et al., 2015; Herrlinger et al., 2019). These studies found a revertant phenotype in neural progenitor cells overexpressing LIN28A and a defect in subretinal angiogenesis in Rpl24^Bst/+ mice. The role of translation elongation should now be analysed with respect to these phenotypes. eEF2K, and the many upstream pathways it integrates, are potential targets for intervention in these in vivo models. It will also be of interest to determine how the inactivation of eEF2K affects the whole-body phenotypes of the Rpl24^Bst mouse, such as the coat pigmentation and tail defects.

eEF2K has a pleiotropic effect in tumorigenesis, acting akin to a tumour suppressor or promoter dependent on the context (Knight et al., 2020b). In some cancers, eEF2K promotes tumorigenesis. For example, under nutrient deprivation eEF2K acts as a pro-survival factor in transformed fibroblasts and tumour cell lines by suppressing protein synthesis to ensure survival (Leprivier et al., 2013). Here, we show that eEF2K inactivation does not modify intestinal tumorigenesis. However, eEF2K is required for the suppression of tumorigenesis and protein synthesis following mTORC1 inhibition in APC cells (Faller et al., 2015) or Rpl24^Bst mutation in APC KRAS cells (this work). Therefore, although eEF2K does not directly drive tumorigenesis, low eEF2K activity ensures there is no blockade of translation or proliferation in tumour cells. Furthermore, eEF2K expression is required for drug or signalling responses that suppress tumorigenesis, giving it tumour suppressive activity. In accordance, CRC patients with low eEF2K protein expression suffer a significantly worse prognosis (Ng et al., 2019). Furthermore, we present mRNA and protein expression data showing that eEF2K is reduced in clinical CRC samples compared to normal tissue. This agrees with a model where low eEF2K allows rapid translation elongation to promote proliferation.

Using the same clinical datasets we demonstrate that both RPL24 and eEF2 are elevated in CRC, consistent with their roles in promoting translation and proliferation. This presents the possibility of directly targeting either RPL24 or eEF2 for anti-cancer benefit. In agreement with this, RNAi against RPL24 in human breast cancer cell lines dramatically reduced proliferation (Wilson-Edell et al., 2014). Similarly, inhibiting eEF2, using a compound reported to slow its exit from the ribosome and thus the rate of protein synthesis, reduces cell line and patient derived organoid growth (Stickel et al., 2015; Keysar et al., 2020). These reports agree with the data presented here suggesting that targeting of RPL24 or eEF2 would be beneficial in CRC. Identifying the mechanistic link between RPL24 and eEF2K is part of our ongoing work, but here we have ruled out mTORC1, MAPK, and AMPK signalling as contributing factors.

This work uncovers an unexpected role for the ribosomal protein RPL24 in the regulation of translation elongation, acting via eEF2K/P-eEF2. We demonstrate that depletion of RPL24 suppresses tumorigenesis in a pre-clinical mouse model of a CRC. We provide genetic evidence supported by molecular assays of translation elongation to demonstrate that RPL24 depletion activates eEF2K to elicit tumour suppression in our models. We also speculate as to the role of eEF2K in the previously published blood cancer models where RPL24 depletion was beneficial. This work provides additional evidence for the anti-tumorigenic role of eEF2K in CRC, highlighting the potential for targeting translation elongation for this disease.

Materials and methods

Key resources table.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Mus musculus)	Tg(Vil1-cre/ ERT2)23Syr	el Marjou et al., 2004	RRID:MGI:3053826
Genetic reagent (Mus musculus)	Apctm1Tno	Shibata et al., 1997	RRID:MGI:1857966
Genetic reagent (Mus musculus)	Krastm4Tyj	Jackson et al., 2001	RRID:MGI:2429948
Genetic reagent (Mus musculus)	Rpl24Bst	Oliver et al., 2004	RRID:MGI:1856685
Genetic reagent (Mus musculus)	Eef2kD273A	Gildish et al., 2012
Genetic reagent (Mus musculus)	ApcMin	Moser et al., 1990	RRID:MGI:1856318
Cell line (Mus musculus)	VillinCreER Apcfl/fl KrasG12D/+ small intestinal organoids	This study
Cell line (Mus musculus)	VillinCreER Apcfl/fl small intestinal organoids	This study
Cell line (Mus musculus)	VillinCreER Apcfl/fl KrasG12D/+ Rpl24Bst/+ small intestinal organoids	This study
Cell line (Mus musculus)	VillinCreER Apcfl/fl Rpl24Bst/+ small intestinal organoids	This study
Antibody	BrdU(mouse monoclonal)	BD Biosciences #347,580	RRID:AB_400326	IHC: (1:250)
Antibody	P-eEF2 T56(rabbit polyclonal)	Cell Signaling Technology #2,331	RRID:AB_10015204	WB: (1:2000)IHC: (1:100)
Antibody	eEF2(rabbit polyclonal)	Cell Signaling Technology #2,332	RRID:AB_10693546	WB: (1:2000)
Antibody	P-4E-BP1(rabbit monoclonal)	Cell Signaling Technology #2,855	RRID:AB_560835	IHC: (1:250)
Antibody	P-RPS6 S240/4(rabbit monoclonal)	Cell Signaling Technology #5,364	RRID:AB_10694233	IHC: (1:100)
Antibody	P-eIF2α S51(rabbit monoclonal)	Cell Signaling Technology#3,398	RRID:AB_2096481	IHC: (1:50)
Antibody	P-ERK T202/ Y204(rabbit polyclonal)	Cell Signaling Technology #9,101	RRID:AB_331646	IHC: (1:400)
Antibody	Lysozyme(rabbit polyclonal)	Dako A0099	RRID:AB_2341230	IHC: (1:300)
Antibody	RPS6(mouse monoclonal)	Cell Signaling Technology #2,317	RRID:AB_2238583	WB: (1:2000)
Antibody	RPL10(rabbit polyclonal)	Novus NBP1-84037	RRID:AB_11007661	WB: (1:2000)
Antibody	RPL22(rabbit polyclonal)	Abcam ab111073	RRID:AB_10863642	WB: (1:2000)
Antibody	Acetyl-CoA carboxylase(rabbit monoclonal)	Cell Signaling Technology #3,676	RRID:AB_2219397	WB: (1:1000)
Antibody	P-acetyl-CoA carboxylase S79(rabbit monoclonal)	Cell Signaling Technology #3,661	RRID:AB_330337	WB: (1:1000)
Antibody	β-Actin(mouse monoclonal)	Sigma-Aldrich #A2228	RRID:AB_476697	WB: (1:10,000)
Antibody	β-Tubulin(mouse monoclonal)	Cell Signaling Technology #2,128	RRID:AB_823664	WB: (1:4000)
Antibody	Goat Anti-Mouse Immunoglobulins/ HRP(goat polyclonal)	Dako #P0447	RRID:AB_2617137	WB: (1:2000)
Antibody	Goat Anti-Rabbit Immunoglobulins/ HRP(goat polyclonal)	Dako #P0448	RRID:AB_2617138	WB: (1:2000)
Sequence- based reagent	Olfm4 RNAScope	ACD #311,838	RNAScope
Peptide, recombinant protein	Recombinant Murine Noggin	Peprotech #250-38		100 ng/ml
Peptide, recombinant protein	Animal-Free Recombinant Human EGF	Peprotech #AF-100-15		50 ng/ml
Peptide, recombinant protein	Recombinant Mouse R-Spondin 1 Protein	R&D Systems #3474-RS		500 ng/ml
Commercial assay or kit	Vectorstain Elite ABC-HRP	Vector Laboratories PK-6102	RRIDs:AB_2336820
Commercial assay or kit	Vectorstain Elite ABC-HRP	Vector Laboratories PK-PK-6101	RRIDs:AB_2336821
Commercial assay or kit	Cell Proliferation Labelling Reagent	Amersham Bioscience RPN201
Commercial assay or kit	Cell-Titer Blue	Promega #G8080
Chemical compound, drug	Rapamycin	LC Laboratories #R-5000
Chemical compound, drug	Harringtonine	Santa Cruz sc-204771
Chemical compound, drug	EasyTag EXPRESS 35S Protein Labeling Mix	Perkin Elmer #NEG772002MC
software, algorithm	Image J	Rueden et al., 2017	RRID:SCR_003070
software, algorithm	G*Power	Faul et al., 2009	RRID:SCR_013726

Materials availability

The mouse strains used will be made available on request. However, this may require a Materials Transfer Agreement and/or a payment if there is potential for commercial application. We ourselves are limited by the terms of Materials Transfer Agreements agreed to by the suppliers of the mouse strains.

Mouse studies

Experiments with mice were performed under licence from the UK Home Office (licence numbers 60/4183 and 70/8646). All mice used were inbred C57BL/6J (Generation ≥8) and were housed in conventional cages with a 12 hr light/dark cycle and ad libitum access to diet and water. Mice were genotyped by Transnetyx in Cordova, Tennessee. Experiments were performed on mice between the ages of 6 and 12 weeks; both male and female mice were used. Sample sizes for all experiments were calculated using the G*Power software (Faul et al., 2009) and are shown in the figures or legends. Researchers were not blinded during experiments. The Villin^CreER allele (el Marjou et al., 2004) was used for intestinal recombination by intraperitoneal (IP) injection of tamoxifen in corn oil at a final in vivo concentration of 80 mg/kg. The Apc flox allele, Apc^Min, Kras^G12D lox-STOP-lox allele, Eef2k^D273A and Rpl24^Bst alleles were previously described (Jackson et al., 2001; Shibata et al., 1997; Gildish et al., 2012; Oliver et al., 2004; Moser et al., 1990). Tumour model experiments began with a single dose of tamoxifen after which mice were monitored until they showed signs of intestinal disease – paling feet from anaemia, weight loss and hunching behaviour. Tumours were scored macroscopically by counting and recording diameter after fixation of intestinal tissue. Tumour volumes were calculated from the tumour diameters assuming a spherical tumour shape. For short-term experiments, mice wild-type for Kras were induced on consecutive days (days 0 and 1) and sampled 4days after the first induction (day 4). Mice bearing the Kras^G12D allele were induced once and sampled on day 3 post-induction. Where indicated, 250 μl of BrdU cell proliferation labelling reagent (Amersham Bioscience RPN201) was given by IP injection 2 hr prior to sampling. BrdU-positive cells were scored from crypt base to villus tip (a half crypt/villus axis) from the proximal small intestine or medial colon and represented as the mean number of positive cells from at least 20 axes per mouse. For the regeneration experiments, mice were exposed to 10 Gy of γ-irradiation at a rate of 0.423 Gy/min from a caesium 137 source. They were then sampled 72 hr after irradiation and regenerative crypts scored from H&E stained sections as previously described (Faller et al., 2015). Batch correction was used by normalising each experiment to relevant wild-type controls.

Histology and IHC

Tissue was fixed in formalin and embedded in paraffin. IHC staining was carried out as previously (Faller et al., 2015), using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099). The IHC protocol followed the Vector ABC kit (mouse #PK-6102, rabbit #PK-6101). RNAScope analysis was conducted according to the manufacturer’s guidelines (ACD) using a probe to murine Olfm4 (#311838). For all staining, a minimum of three biological replicates were stained and representative images used throughout. For BrdU scoring in short-term model experiments tissue was fixed in methanol:chloroform:acetic acid at a ratio 4:2:1 then transferred to formalin and embedded in paraffin.

Intestinal organoid culture

Crypt cultures were isolated and then maintained as previously described (Knight et al., 2020a). In all crypt culture experiments, multiple biologically independent cultures were generated and analysed from different animals of the shown genotypes. DMEM/F12 medium (Life Technologies #12634-028) was supplemented with 5 mM HEPES (Life Technologies #15630-080), 100 U/ml penicillin/streptomycin (Life Technologies #1540-122), 2 mM L-glutamine (Life Technologies #25030-024), 1× N2, 1× B27 (Invitrogen #17502-048 and #12587-010), 100 ng/ml noggin (Peprotech #250-38), and 50 ng/ml EGF (Peprotech #AF-100-15). Wild-type cultures were also supplemented with 500 ng/ml R-spondin (R&D Systems #3474-RS). For ex vivo growth assays, cells were plated in technical triplicate in 96-well plates, and proliferation measured using Cell-Titer Blue (Promega #G8080) added to previously untreated cells each day for up to 4 days. Rapamycin (LC Laboratories R-5000) was dissolved in DMSO and administered for 24 hr, comparing to DMSO vehicle-treated cells.

Western blotting

Samples were lysed (10 mM Tris [pH 7.5], 50 mM NaCl, 0.5% NP40, 0.5% SDS supplemented with protease inhibitor cocktail [Roche #11836153001] PhosSTOP [Roche #04906837001] and benzonase [Sigma-Aldrich #E1014]) on ice and then the protein content estimated by BCA assay (Thermo Fisher Scientific #23225). 20 μg of protein were denatured in loading dye containing SDS then resolved by 4–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Invitrogen #NP0336BOX). For western blots from gradients, equivalent volumes were loaded from each gradient fraction. Protein was transferred to nitrocellulose membranes, blocked with excess protein and immunoblotted overnight at 4°C using the following antibodies; P-eEF2 T56 (CST #2331), eEF2 (CST #2332), RPL24 (Sigma-Aldrich HPA051653), RPS6 (CST #2317), RPL10 (Novus NBP1-84037), RPL22 (Abcam ab111073), ACC (CST #3676), P-ACC S79 (CST #3661), β-tubulin (CST #2128), and β-actin (Sigma-Aldrich #A2228) as a sample control. One-hour incubation at room temperature with secondary antibodies (horseradish peroxidase [HRP]-conjugated anti-mouse secondary [Dako #P0447]; HRP-conjugated anti-rabbit secondary [Dako #P0448]) was followed by exposure to autoradiography films or a ChemiDoc MP imager (BioRad) with ECL reagent (Thermo Fisher Scientific #32106). Quantification was performed using Image J (Rueden et al., 2017).

Sucrose density gradients

Cells were replenished with fresh medium for the 6 hr before harvesting. This medium was then spiked with 200 µg/ml cycloheximide (Sigma-Aldrich #C7695) 3 min prior to harvesting on ice. Crypt fractions from mice were isolated by extraction of the epithelium from 10 cm of proximal small intestine. Each data point plotted represents an individual animal. PBS-flushed linearly opened small intestines were incubated in RPMI 1640 medium (Thermo Fisher Scientific #21875059) supplemented with 10 mM EDTA and 200 µg/ml cycloheximide for 7 min at 37°C with regular agitation to extract villi. Crypts were isolated by transferring remaining tissue to ice-cold PBS containing 10 mM EDTA and 200 µg/ml cycloheximide for a further 7 min, again with agitation. The remaining tissue was discarded. Samples were lysed (300 mM NaCl, 15 mM MgCl2, 15 mM Tris pH 7.5, 100 µg/ml cycloheximide, 0.1% Triton X-100, 2 mM DTT, and 5 U/ml SUPERase.In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) on ice and post-nuclear extracts placed on top of 10–50% wt/vol sucrose gradients containing the same buffer (apart from no Triton X-100, DTT, or SUPERase.In). These were then spun in an SW40Ti rotor at 255,000 rcf for 2 hr at 4°C under a vacuum. Samples were then separated through a live 254 nM optical density reader (ISCO). Polysome to subpolysome (P:S), heavy to light polysome (H:L), or 60S to 40S (60S:40S) ratios were calculated using the manually defined trapezoid method. For harringtonine run-off assays, cultures were prepared in duplicate for each genotype. One was pre-treated with 2 µg/ml harringtonine (Santa Cruz sc-204771) for 5 min (300 s) prior to cycloheximide addition. This was then processed as above. Run-off rates were calculated as previously described (Knight et al., 2015). Trichloroacetic acid protein precipitation from sucrose density gradients was performed as before (Knight et al., 2013).

³⁵S-methionine incorporation assay

Organoids were replenished with medium 6 hr prior to analysis while in the optimal growth phase post-splitting. Technical triplicates were used from organoids from three different animals per experiments. ³⁵S-methionine (Perkin Elmer #NEG772002MC) was used at 30 µCi/ml for 30 min. Samples were lysed using the same buffer described for Western blotting. Protein was precipitated in 12.5% (wt/vol) trichloroacetic acid onto glass microfiber paper (Whatmann #1827-024) by use of a vacuum manifold. Precipitates were washed with 70% ethanol and acetone. Scintillation was read on a Wallac MicroBeta TriLux 1450 scintillation counter using Ecoscint scintillation fluid (SLS Ltd #LS271) from these microfiber papers. In parallel the total protein content was determined by BCA assay using unprecipitated sample. Protein synthesis rate was expressed as the scintillation normalised to the total protein content (CPM/µg/ml protein), which was then changed to a relative value compared to relevant controls for each experiment.

RNA sequencing

This was performed as previously described (Knight et al., 2020a), using three animals per genotype. The reads for each ribosomal protein mRNA were then averaged and the fold change compared to wild-type tissue calculated and plotted. Source data for this analysis are available linked to Figure 4F.

Publicly available clinical data analysis

The TNMplot and UALCAN web portals were used to analysis publicly available dataset from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium. Details of these portals are available in these publications (Bartha and Győrffy, 2021; Chandrashekar et al., 2017).

Statistical analyses

All statistical analyses are detailed in the relevant figure legends. In all cases, calculated p values less than or equal to 0.05 were considered significant. N numbers for each experiment are detailed within each figure, as individual points on graphs or within figure legends.

Funding Statement

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

• Cancer Research UK A17196 to Owen J Sansom.

• Cancer Research UK A24388 to Owen J Sansom.

• Cancer Research UK A21139 to Owen J Sansom.

• H2020 European Research Council 311301 to Owen J Sansom.

• Wellcome Trust 201487 to Giovanna R Mallucci, Tobias von der Haar, Christopher Mark Smales, Anne E Willis, Owen J Sansom.

• National Health and Medical Research Council to Christopher Proud.

Data availability

Source data for Figure 1F, Figure 3 - figure supplement 1, Figure 3 - figure supplement 2, Figure 4F and Figure 6 - figure supplement 2 have been uploaded.