Nucleolar Stress Response via Ribosomal Protein L11 Regulates Topoisomerase Inhibitor Sensitivity of P53-Intact Cancers

Abstract

Nucleolar stress response is caused by perturbations in ribosome biogenesis, induced by the inhibition of ribosomal RNA processing and synthesis, as well as ribosome assembly. This response induces p53 stabilization and activation via ribosomal protein L11 (RPL11), suppressing tumor progression. However, anticancer agents that kill cells via this mechanism, and their relationship with the therapeutic efficiency of these agents, remain largely unknown. Here, we sought to investigate whether topoisomerase inhibitors can induce nucleolar stress response as they reportedly block ribosomal RNA transcription. Using rhabdomyosarcoma and rhabdoid tumor cell lines that are sensitive to the nucleolar stress response, we evaluated whether nucleolar stress response is associated with sensitivity to topoisomerase inhibitors ellipticine, doxorubicin, etoposide, topotecan, and anthracyclines. Cell proliferation assay indicated that small interfering RNA-mediated RPL11 depletion resulted in decreased sensitivity to topoisomerase inhibitors. Furthermore, the expression of p53 and its downstream target proteins via western blotting showed the suppression of p53 pathway activation upon RPL11 knockdown. These results suggest that the sensitivity of cancer cells to topoisomerase inhibitors is regulated by RPL11-mediated nucleolar stress responses. Thus, RPL11 expression may contribute to the prediction of the therapeutic efficacy of topoisomerase inhibitors and increase their therapeutic effect of topoisomerase inhibitors.

Keywords: RPL11; drug sensitivity; nucleolar stress response; p53; topoisomerase inhibitors.

Figure 1

RPL11-mediated nucleolar stress response regulates sensitivity to topoisomerase inhibitors in p53 wild-type malignant rhabdoid tumor JMU-RTK-2 cells. Cells were transfected with scramble, RPL11#1, and RPL11#2 siRNAs and treated with the indicated concentrations of drugs ((A) Ellipticine, (B) Etoposide, (C) Topotecan, (D) Siremadlin). After 3 days of culture, the number of surviving cells was measured using the MTT assay. * p < 0.05 vs.RPL11#1 siRNA group; ^# p < 0.05 vs. RPL11#2 siRNA group. Results represent independent five experiments.

Figure 2

Effect of nucleolar stress response on topoisomerase inhibitor-mediated survival in the p53 wild-type rhabdomyosarcoma cells. RMS-YM cells were transfected with scramble, RPL11#1, and RPL11#2 siRNAs and treated with the indicated concentrations of drugs ((A) Ellipticine, (B) Etoposide, (C) Topotecan, (D) Siremadlin). After 3 days of culture, the number of surviving cells was measured using the MTT assay. * p < 0.05 vs.RPL11#1 siRNA group; ^# p < 0.05 vs. RPL11#2 siRNA group. Results represent independent five experiments.

Figure 3

Reduced nucleolar stress response suppresses topoisomerase inhibitor-mediated activation of the p53 pathway in malignant rhabdoid tumor JMU-RTK-2 cells. Cells were transfected with scramble, RPL11#1, and RPL11#2 siRNA and treated with drugs (3 µM Ellipticine, 30 µM Etoposide, 1 µM Topotecan, 1 µM Siremadlin). After 24 h of culture, the cell lysate was subjected to immunoblotting with antibodies against p53, MDM2, p21, RPL11, and actin. Actin was used as a loading control. The numbers below the bands indicate the ratio of the protein/actin expression determined using LuminoGraphII Image Analyzer with CS Analyzer 4 Software. Results represent independent four experiments.

Figure 4

Nucleolar stress response regulates p53 pathway activation by topoisomerase inhibitors in rhabdomyosarcoma cells. RMS-YM cells were transfected with scramble, RPL11#1, and RPL11#2 siRNA and treated with indicated drugs (5 µM Ellipticine, 30 µM Etoposide, 1 µM Topotecan, 0.3 µM Siremadlin). After 4 h of culture, the cell lysate was subjected to immunoblotting with antibodies against p53, MDM2, p21, RPL11, and Actin. Actin was used as a loading control. The numbers below the bands indicate the ratio protein/actin expression determined using LuminoGraphII Image Analyzer with CS Analyzer 4 Software. Results represent independent four experiments.

Figure 5

Effect of nucleolar stress response on sensitivity to anthracycline and p53 pathway activation by anthracycline drugs. JMU-RTK-2 cells were transfected with scramble, RPL11#1, and RPL11#2 siRNAs and treated with the indicated concentrations of drugs ((A) Epirubicin and (B) Doxorubicin). After 3 days of culture, the number of surviving cells was measured using the MTT assay. (C) JMU-RTK-2 cells were transfected with scramble, RPL11#1, or RPL11#2 siRNA and treated with 0.3 µM Epirubicin, or 0.1 µM Doxorubicin. After 24 h of culture, the cell lysate was subjected to immunoblotting with antibodies against p53, MDM2, P21, RPL11, and Actin. Actin was used as a loading control. The numbers below the bands indicate the ratio of protein/actin determined using LuminoGraphII Image Analyzer with CS Analyzer 4 Software. * p < 0.05 vs.RPL11#1 siRNA group; ^# p < 0.05 vs. RPL11#2 siRNA group. Results represent independent three experiments.

Figure 6

Nucleolus stress response may be one of the determinants of topoisomerase inhibitor sensitivity in cancer. (A) When RPL11 is normally expressed, topoisomerase inhibitors induce RPL11 to bind to MDM2 and block ubiquitination-dependent degradation of p53. Consequently, cancer cells inhibit cell growth and induce apoptosis. (B) When the expression of RPL11 is low, RPL11 is unable to induce sufficient inhibition of MDM2, resulting in the destabilization of p53 and resistance to topoisomerase inhibitors in cancer cells.