RPL24: a potential therapeutic target whose depletion or acetylation inhibits polysome assembly and cancer cell growth

Abstract

Partial loss of large ribosomal subunit protein 24 (RPL24) function is known to protect mice against Akt or Myc-driven cancers, in part via translational inhibition of a subset of cap(eIF4E)-dependently translated mRNAs. The role of RPL24 in human malignancies is unknown. By analyzing a public dataset of matched human breast cancers and normal mammary tissue, we found that breast cancers express significantly more RPL24 than matched normal breast samples. Depletion of RPL24 in breast cancer cells by >70% reduced cell viability by 80% and decreased protein expression of the eIF4E-dependently translated proteins cyclin D1 (75%), survivin (46%) and NBS1 (30%) without altering GAPDH or beta-tubulin levels. RPL24 knockdown also reduced 80S subunit levels relative to 40S and 60S levels. These effects on expression of eIF4E-dependent proteins and ribosome assembly were mimicked by 2-24 h treatment with the pan-HDACi, trichostatin A (TSA), which induced acetylation of 15 different polysome-associated proteins including RPL24. Furthermore, HDAC6-selective inhibition or HDAC6 knockdown induced ribosomal protein acetylation. Via mass spectrometry, we found that 60S-associated, but not, polysome-associated, RPL24 undergoes HDACi-induced acetylation on K27. Thus, RPL24 K27 acetylation may play a role in ribosome assembly. These findings point toward a novel acetylation-dependent polysome assembly mechanism regulating tumorigenesis.

Figure 1. RPL24 expression is transcriptionally upregulated during human breast tumorigenesis

RPL24 expression levels were analyzed from the dataset presented in [22]. (a) Box plot of RPL24 expression levels in patient-matched breast carcinoma and normal breast tissues. Lines connect paired data from each patient; and line color reflects relative levels of RPL24 in each paired sample (red: tumor > normal; green: normal > tumor). (b) Differences in RPL24 expression levels between each breast carcinoma and normal breast sample pair. The mean of the differences + SD are shown in red. P-value was obtained using a paired t-test.

Figure 2. RPL24 knockdown reduces breast cancer cell viability while inhibiting cap (eIF4E)-dependent expression of proliferation, survival and genome stability proteins

SKBR3 cells were infected with lentiviruses expressing a GFP control or RPL24-targeting shRNA. After one week of puromycin selection, cells were plated in 96-well plates for viability assays and lysates were taken in parallel for western blots. (a) Western blots were performed on lysates from an equal number of cells using antibodies toward the indicated proteins. Ratios of protein expression normalized to beta-tubulin levels were obtained using ImageJ software. (b) Viability assay readings were taken three hours after plating (day 0) and four days after plating (day 4). The day 4 results were normalized for plating efficiency using the day 0 values. Error bars represent three replicate samples.

Figure 3. RPL24 knockdown reduces 80S and polysome assembly while increasing 60S retention of eIF6

(a,b,d) SKBR3 cells were infected with lentiviruses expressing a GFP control or RPL24-targeting shRNA for three days. (a) Western blots using the indicated antibodies were performed on total cell lysates to assess knockdown efficiency. (b) Lysates were applied to a continuous sucrose gradient (10-50%) and ultracentrifugation followed by fractionation was performed to separate ribosomal subunits and polysomes. (c) Pymol software was used to visualize the location of RPL24 (blue) relative to eIF6 (green) on the previously published structure of the 60S subunit in complex with eIF6 [13]. (d) Western blots using the indicated antibodies were performed on fractions from the 60S peaks using the indicated antibodies.

Figure 4. Ribosomal protein acetylation is induced by pan-HDACi and HDAC6-selective inhibitors

(a-c) SKBR3 cells were treated with the indicated drugs for the indicated period of time. (d) SKBR3 cells were transfected with the indicated siRNAs and allowed to incubate for 72 hours. (a, c, d). The indicated western blots were performed in ribopellets, total cytoplasmic lysates, or nuclear extracts. (b) Mass spectrometry was performed on ribopellets as described in materials and methods and in Figure 6. The fold change in acetylated peptide to total peptide caused by TSA treatment is plotted. Only proteins that underwent at least a two-fold induction upon TSA treatment are shown. Error bars represent the standard error of the mean for three biological replicates.

Figure 5. Like RPL24 knockdown, HDACi reduces 80S assembly while increasing 60S retention of eIF6 and reduces expression of cap (eIF4)-dependently translated proteins

(a,b) SKBR3 cells were treated with TSA (1 μM, 2 h). (a) Polysome profiles were carried out as previously described. (b) Western blots using the indicated antibodies were performed in fractions representing the 60S subunits. (c) SKBR3 cells were treated with TSA for the indicated doses and times, and proteins were identified by western blotting as indicated. Ratios of protein expression normalized to beta-tubulin levels were obtained using ImageJ software.

Figure 6. HDACi enhances lysine (K27) acetylation on 60S, but not polysomal, RPL24

(a) Schematic of mass-spectrometry-based techniques to analyze ribosomal protein acetylation. SKBR3 cells were treated with TSA (1 μM, 2 h or 6 h). To isolate 60S subunits, polysome profiles were performed and 60S fractions were TCA precipitated. Concentrated 60S samples were resolved on 4-12% bis-tris gels and RPL24-containing bands were excised and trypsin digested. In parallel, polysomes were isolated using a discontinuous sucrose gradient as described. Trypsin digestions and acetyl lysine immunoprecipitations were subsequently carried out. Mass spectrometry was performed on 60S-associated RPL24-containing gel bands or polysome-containing acetyl-lysine immunoprecipitations as described in the methods section. (b,c) On 60S-associated and polysome-associated RPL24, the fold induction caused by TSA (1 μM, 2 h) of K27 (b) or K93 (c)-acetylated peptide (normalized to total protein concentration) was plotted. Error bars represent the standard error of the mean for three biological replicates. Note: the data for K93 acetylation of RPL24 K93 is also shown in Figure 4b.

Figure 7. Schematic for modulation of ribosome assembly by RPL24 acetylation

(a) A magnified portion of the RPL24 (blue)-eIF6 (green) interface, visualized with Pymol software, from previous x-ray crystallography data [13], is shown (zoomed out view shown in Figure 3c). T. thermophilia RPL24 residues are labelled and K26, which resides in a region of RPL24 homologous to where human K27 resides, is circled. (b) eIF6 binds to the pre-60S near RPL24 to prevent premature association of the 40S and 60S ribosomal subunits; eIF6 is then released from the mature 60S, allowing it to join with the 40S to form the 80S ribosome. Our model indicates that either RPL24 depletion or TSA (HDACi)-induced RPL24 acetylation on K27 prevents eIF6 release and 80S formation.