Structure of human MDM2 complexed with RPL11 reveals the molecular basis of p53 activation.

The central region of MDM2 is critical for p53 activation and tumor suppression. Upon ribosomal stress, this region is bound by ribosomal proteins, particularly ribosomal protein L11 (RPL11), leading to MDM2 inactivation and subsequent p53 activation. Here, we solved the complex structure of human MDM2-RPL11 at 2.4 Å. MDM2 extensively interacts with RPL11 through an acidic domain and two zinc fingers. Formation of the MDM2-RPL11 complex induces substantial conformational changes in both proteins. RPL11, unable to bind MDM2 mutants, fails to induce the activation of p53 in cells. MDM2 mimics 28S rRNA binding to RPL11. The C4 zinc finger determines RPL11 binding to MDM2 but not its homolog, MDMX. Our results highlight the essential role of the RPL11-MDM2 interaction in p53 activation and tumor suppression and provide a structural basis for potential new anti-tumor drug development.

Identification of a small-molecule RPL11 mimetic that inhibits tumor growth by targeting MDM2-p53 pathway.

BACKGROUND: Targeting ribosome biogenesis to activate p53 has recently emerged as a therapeutic strategy in human cancer. Among various ribosomal proteins, RPL11 centralizes the nucleolar stress-sensing pathway by binding MDM2, leading to MDM2 inactivation and p53 activation. Therefore, the identification of MDM2-binding RPL11-mimetics would be valuable for anti-cancer therapeutics. METHODS: Based on the crystal structure of the interface between RPL11 and MDM2, we have identified 15 potential allosteric modulators of MDM2 through the virtual screening. RESULTS: One of these compounds, named S9, directly binds MDM2 and competitively inhibits the interaction between RPL11 and MDM2, leading to p53 stabilization and activation. Moreover, S9 inhibits cancer cell proliferation in vitro and in vivo. Mechanistic study reveals that MDM2 is required for S9-induced G2 cell cycle arrest and apoptosis, whereas p53 contributes to S9-induced apoptosis. CONCLUSIONS: Putting together, S9 may serve as a lead compound for the development of an anticancer drug that specifically targets RPL11-MDM2-p53 pathway.

Functional role of RRS1 in breast cancer cell proliferation.

RRS1 (human regulator of ribosome synthesis 1), an essential nuclear protein involved in ribosome biogenesis, is overexpressed in some human cancers, yet its role in breast cancer remains unclear. Here, we report a functional analysis of RRS1 in breast cancer and its likely mechanism. Immunohistochemistry (IHC) and RT-qPCR analyses indicated that RRS1 was commonly overexpressed in breast cancer tissues. The copy numbers of RRS1 were higher in tumours compared with those for normal tissues. And there was a significant correlation between copy number and mRNA expression. In addition, RRS1 overexpression was significantly correlated with lymph node metastasis and poor survival. RRS1 mRNA and protein levels were also significantly increased in a panel of human breast cancer cell lines. RRS1 knockdown inhibited proliferation and induced apoptosis and cell cycle arrest in all three cell lines. Furthermore, RRS1 knockdown suppressed the tumour formation and growth of MDA-MB-231 cells in nude mice. Additionally, RRS1 knockdown activated p53 and p21 in MCF-7 cells. A marked increase in the quantity of ribosome-free RPL11 was detected by Western blot. Moreover, co-immunoprecipitation (CoIP) experiments showed that RRS1 knockdown activated p53 by facilitating the direct contact of MDM2 and RPL11/RPL5. Taken together, our results suggest that RRS1 may contribute to breast cancer proliferation through RPL11/MDM2-mediated p53 activation. Therefore, RRS1 may be a promising target for breast cancer therapy.

The role of PICT1 in RPL11/Mdm2/p53 pathway-regulated inhibition of cell growth induced by topoisomerase IIα inhibitor against cervical cancer cell line.

Our previous work showed that a podophyllum derivative (D-3F), named 4-N-(2-Amino-3-fluoropyridine) -4-deoxidation-4'-demethylepipofophyllotoxin, inhibits the activity of topoisomerase II (TOPO II) and then results in DNA damage. Also, D-3F increases the expression of p53 to induce cervical cancer HeLa cell apoptosis by enhancing its stability, due to the translocation of RPL11 to interact with Mdm2 and then consequently causing the blockage of the Mdm2-p53 feedback loop. In present study, we further explored the detailed mechanism of the antitumor activity of D-3F against cervical cancer cell line. Firstly, the decreased level of protein interacting with carboxyl terminus 1 (PICT1) in cervical cancer cell lines (HeLa and SiHa) treated with D-3F, exerted its potent inhibitory effect on cellular proliferation, which was dependent on the inhibition of TOPO IIα activity induced by D-3F in vitro. In addition, the downregulation of PICT1 was required to enhancement of p53 stability, resulted from its promoting the nucleoplasmic translocation of RPL11 to bind to Mdm2 following D-3F treatment. Altogether, it demonstrated that the reduction of PICT1 level in HeLa cell line, as well as SiHa exposed to D-3F, a TOPO IIα inhibitor, may play an essential role in the regulation of RPL11/Mdm2/p53 pathway to induce cell apoptosis. Besides, it suggested the potential of this podophyllum derivative (D-3F) as an alternative agent for therapy in cervical cancer.

Acrolein preferentially damages nucleolus eliciting ribosomal stress and apoptosis in human cancer cells.

Acrolein (Acr) is a potent cytotoxic and DNA damaging agent which is ubiquitous in the environment and abundant in tobacco smoke. Acr is also an active cytotoxic metabolite of the anti-cancer drugs cyclophosphamide and ifosfamide. The mechanisms via which Acr exerts its anti-cancer activity and cytotoxicity are not clear. In this study, we found that Acr induces cytotoxicity and cell death in human cancer cells with different activities of p53. Acr preferentially binds nucleolar ribosomal DNA (rDNA) to form Acr-deoxyguanosine adducts, and induces oxidative damage to both rDNA and ribosomal RNA (rRNA). Acr triggers ribosomal stress responses, inhibits rRNA synthesis, reduces RNA polymerase I binding to the promoter of rRNA gene, disrupts nucleolar integrity, and impairs ribosome biogenesis and polysome formation. Acr causes an increase in MDM2 levels and phosphorylation of MDM2 in A549 and HeLa cells which are p53 active and p53 inactive, respectively. It enhances the binding of ribosomal protein RPL11 to MDM2 and reduces the binding of p53 and E2F-1 to MDM2 resulting in stabilization/activation of p53 in A549 cells and degradation of E2F-1 in A549 and HeLa cells. We propose that Acr induces ribosomal stress which leads to activation of MDM2 and RPL11-MDM2 binding, consequently, activates p53 and enhances E2F-1 degradation, and that taken together these two processes induce apoptosis and cell death.