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Rapid growth and unrestrained proliferation is a hallmark of many cancers. To accomplish this, cancer cells re-wire and increase their biosynthetic and metabolic activities, including ribosome biogenesis (RiBi), a complex, highly energy-consuming process. Several chemotherapeutic agents used in the clinic impair this process by interfering with the transcription of ribosomal RNA (rRNA) in the nucleolus through the blockade of RNA polymerase I or by limiting the nucleotide building blocks of RNA, thereby ultimately preventing the synthesis of new ribosomes. Perturbations in RiBi activate nucleolar stress response pathways, including those controlled by p53. While compounds such as actinomycin D and oxaliplatin effectively disrupt RiBi, there is an ongoing effort to improve the specificity further and find new potent RiBi-targeting compounds with improved pharmacological characteristics. A few recently identified inhibitors have also become popular as research tools, facilitating our advances in understanding RiBi. Here we provide a comprehensive overview of the various compounds targeting RiBi, their mechanism of action, and potential use in cancer therapy. We discuss screening strategies, drug repurposing, and common problems with compound specificity and mechanisms of action. Finally, emerging paths to discovery and avenues for the development of potential biomarkers predictive of therapeutic outcomes across cancer subtypes are also presented.

The tumor suppressor protein p53 prevents the malignant transformation of cells by responding to DNA damage, oncogene activation, and abnormal growth signals including ribosome assembly defects. Under normal conditions, p53 activity is controlled by the regulatory proteins MDM2 and MDM4, which suppress its function through ubiquitin-mediated degradation and transcriptional inhibition. A subset of ribosomal proteins initiates the p53 response to impaired ribosome biogenesis. The ability of some ribosomal proteins to control MDM2 and MDM4 activities, and thereby p53, underscores an intriguing aspect of cell biology: proteins primarily known for their roles in ribosome function can exert extra-ribosomal functions. One notable example is the cellular RNA-protein complex involving RPL5, RPL11, and 5S rRNA (5S RNP) which inhibits MDM2 and stabilizes p53. Another RP, RPL22, is frequently mutated in cancers with microsatellite instability and its paralog RPL22L1 is often amplified. Recent studies have revealed that RPL22 directly modulates the alternative splicing of MDM4 to promote p53 activation, suggesting that the ribosomal protein-p53 relationship is more complex than previously thought. Cellular responses to ribosome biogenesis inhibition extend beyond general alterations in transcription and translation to actively determine cancer cell fate by selectively engaging tumor-suppressor pathways. RPL22’s effect on MDM4 and other mRNA splicing events is a striking example. A better understanding of the mechanisms involved could guide the development of improved cancer treatments.

The prognosis for high-grade gliomas, particularly glioblastoma (GBM), remains dismal [...]

Pharmacological inhibition of ribosome biogenesis is a promising avenue for cancer therapy. Herein, we report a novel activity of the FDA-approved antimalarial drug amodiaquine which inhibits rRNA transcription, a rate-limiting step for ribosome biogenesis, in a dose-dependent manner. Amodiaquine triggers degradation of the catalytic subunit of RNA polymerase I (Pol I), with ensuing RPL5/RPL11-dependent stabilization of p53. Pol I shutdown occurs in the absence of DNA damage and without the subsequent ATM-dependent inhibition of rRNA transcription. RNAseq analysis revealed mechanistic similarities of amodiaquine with BMH-21, the first-in-class Pol I inhibitor, and with chloroquine, the antimalarial analog of amodiaquine, with well-established autophagy-inhibitory activity. Interestingly, autophagy inhibition caused by amodiaquine is not involved in the inhibition of rRNA transcription, suggesting two independent anticancer mechanisms. In vitro, amodiaquine is more efficient than chloroquine in restraining the proliferation of human cell lines derived from colorectal carcinomas, a cancer type with predicted susceptibility to ribosome biogenesis stress. Taken together, our data reveal an unsuspected activity of a drug approved and used in the clinics for over 30 years, and provide rationale for repurposing amodiaquine in cancer therapy.

Intratumoral heterogeneity is a characteristic of glioblastomas that contain an intermixture of cell populations displaying different glioblastoma subtype gene expression signatures. Proportions of these populations change during tumor evolution, but the occurrence and regulation of glioblastoma subtype transition is not well described. To identify regulators of glioblastoma subtypes we utilized a combination of in vitro experiments and in silico analyses, using experimentally generated as well as publicly available data. Through this combined approach SOX2 was identified to confer a proneural glioblastoma subtype gene expression signature. SFRP2 was subsequently identified as a SOX2- antagonist, able to induce a mesenchymal glioblastoma subtype signature. A subset of patient glioblastoma samples with high SFRP2 and low SOX2 expression was particularly enriched with mesenchymal subtype samples. Phenotypically, SFRP2 decreased tumor sphere formation, stemness as assessed by limiting dilution assay, and overall cell proliferation but increased cell motility, whereas SOX2 induced the opposite effects. Furthermore, an SFRP2/non-canonical-WNT/KLF4/PDGFR/phospho-AKT/SOX2 signaling axis was found to be involved in the mesenchymal transition. Analysis of human tumor tissue spatial gene expression patterns showed distinct expression of SFRP2 - and S OX2- correlated genes in vascular and cellular areas, respectively. Finally, conditioned media from SFRP2 overexpressing cells increased CD206 on macrophages. Together, these findings present SFRP2 as a SOX2-antagonist with the capacity to induce a mesenchymal subtype transition in glioma cells located in vascular tumor areas, highlighting its role in glioblastoma tumor evolution and intratumoral heterogeneity.

Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET’s tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET’s anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.

Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9). Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site.

Despite several decades of intense research focused on understanding function(s) and disease-associated malfunction of p53, there is no sign of any “mid-life crisis” in this rapidly advancing area of biomedicine. Firmly established as the hub of cellular stress responses and tumor suppressor targeted in most malignancies, p53’s many talents continue to surprise us, providing not only fresh insights into cell and organismal biology, but also new avenues to cancer treatment. Among the most fruitful lines of p53 research in recent years have been the discoveries revealing the multifaceted roles of p53-centered pathways in the fundamental processes of DNA replication and ribosome biogenesis (RiBi), along with cellular responses to replication and RiBi stresses, two intertwined areas of cell (patho)physiology that we discuss in this review. Here, we first provide concise introductory notes on the canonical roles of p53, the key interacting proteins, downstream targets and post-translational modifications involved in p53 regulation. We then highlight the emerging involvement of p53 as a key component of the DNA replication Fork Speed Regulatory Network and the mechanistic links of p53 with cellular checkpoint responses to replication stress (RS), the driving force of cancer-associated genomic instability. Next, the tantalizing, yet still rather foggy functional crosstalk between replication and RiBi (nucleolar) stresses is considered, followed by the more defined involvement of p53-mediated monitoring of the multistep process of RiBi, including the latest updates on the RPL5/RPL11/5 S rRNA-MDM2-p53-mediated Impaired Ribosome Biogenesis Checkpoint (IRBC) pathway and its involvement in tumorigenesis. The diverse defects of RiBi and IRBC that predispose and/or contribute to severe human pathologies including developmental syndromes and cancer are then outlined, along with examples of promising small-molecule-based strategies to therapeutically target the RS- and particularly RiBi- stress-tolerance mechanisms to which cancer cells are addicted due to their aberrant DNA replication, repair, and proteo-synthesis demands.

Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research.