Chromatin damage generated by DNA intercalators leads to degradation of RNA Polymerase II.

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53.

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

Loss of nucleolar histone chaperone NPM1 triggers rearrangement of heterochromatin and synergizes with a deficiency in DNA methyltransferase DNMT3A to drive ribosomal DNA transcription.

Nucleoli are prominent nuclear structures assembled and organized around actively transcribed ribosomal DNA (rDNA). The nucleolus has emerged as a platform for the organization of chromatin enriched for repressive histone modifications associated with repetitive DNA. NPM1 is a nucleolar protein required for the maintenance of genome stability. However, the role of NPM1 in nucleolar chromatin dynamics and ribosome biogenesis remains unclear. We found that normal fibroblasts and cancer cells depleted of NPM1 displayed deformed nucleoli and a striking rearrangement of perinucleolar heterochromatin, as identified by immunofluorescence staining of trimethylated H3K9, trimethylated H3K27, and heterochromatin protein 1γ (HP1γ/CBX3). By co-immunoprecipitation we found NPM1 associated with HP1γ and core and linker histones. Moreover, NPM1 was required for efficient tethering of HP1γ-enriched chromatin to the nucleolus. We next tested whether the alterations in perinucleolar heterochromatin architecture correlated with a difference in the regulation of rDNA. U1242MG glioma cells depleted of NPM1 presented with altered silver staining of nucleolar organizer regions, coupled to a modest decrease in H3K9 di- and trimethylation at the rDNA promoter. rDNA transcription and cell proliferation were sustained in these cells, indicating that altered organization of heterochromatin was not secondary to inhibition of rDNA transcription. Furthermore, knockdown of DNA methyltransferase DNMT3A markedly enhanced rDNA transcription in NPM1-depleted U1242MG cells. In summary, this study highlights a function of NPM1 in the spatial organization of nucleolus-associated heterochromatin.

Targeted inactivation of Mdm2 RING finger E3 ubiquitin ligase activity in the mouse reveals mechanistic insights into p53 regulation.

It is believed that Mdm2 suppresses p53 in two ways: transcriptional inhibition by direct binding, and degradation via its E3 ligase activity. To study these functions physiologically, we generated mice bearing a single-residue substitution (C462A) abolishing the E3 function without affecting p53 binding. Unexpectedly, homozygous mutant mice died before E7.5, and deletion of p53 rescued the lethality. Furthermore, reintroducing a switchable p53 by crossing with p53ER(TAM) mice surprisingly demonstrated that the mutant Mdm2(C462A) was rapidly degraded in a manner indistinguishable from that of the wild-type Mdm2. Hence, our data indicate that (1) the Mdm2-p53 physical interaction, without Mdm2-mediated p53 ubiquitination, cannot control p53 activity sufficiently to allow early mouse embryonic development, and (2) Mdm2's E3 function is not required for Mdm2 degradation.

Transcription factor PROX1: its role in development and cancer.

The homeobox gene PROX1 is critical for organ development during embryogenesis. The Drosophila homologue, known as prospero has been shown to act as a tumor suppressor by controlling asymmetric cell division of neuroblasts. Likewise, alterations in PROX1 expression and function are associated with a number of human cancers including hematological malignancies, carcinomas of the pancreas, liver and the biliary system, sporadic breast cancer, Kaposiform hemangioendothelioma, colon cancer, and brain tumors. PROX1 is involved in cancer development and progression and has been ascribed both tumor suppressive and oncogenic properties in a variety of different cancer types. However, the exact mechanisms through which PROX1 regulates proliferation, migration, and invasion of cancer cells are by large unknown. This review provides an update on the role of PROX1 in organ development and on its emerging functions in cancer, with special emphasis on the central nervous system and glial brain tumors.

Small molecule-mediated disruption of ribosome biogenesis synergizes with FGFR inhibitors to suppress glioma cell growth.

BACKGROUND: High-grade gliomas are malignant brain tumors characterized by aggressiveness and resistance to chemotherapy. Prognosis remains dismal, highlighting the need to identify novel molecular dependencies and targets. Ribosome biogenesis (RiBi), taking place in the nucleolus, represents a promising target as several cancer types rely on high RiBi rates to sustain proliferation. Publicly available transcriptomics data of glioma patients revealed a positive correlation between RiBi rates and histological grades. We, therefore, hypothesized that glioma cells could be susceptible to RiBi inhibition. METHODS: Transcriptomics data from glioma patients were analyzed for RiBi-related processes. BMH-21, a small molecule inhibitor of RNA pol I transcription, was tested in adult and pediatric high-grade glioma cell lines and a zebrafish transplant model. Cellular phenotypes were evaluated by transcriptomics, cell cycle analysis, and viability assays. A chemical synergy screen was performed to identify drugs potentiating BMH-21-mediated effects. RESULTS: BMH-21 reduced glioma cell viability, induced apoptosis, and impaired the growth of transplanted glioma cells in zebrafish. Combining BMH-21 with TMZ potentiated cytotoxic effects. Moreover, BMH-21 synergized with Fibroblast Growth Factor Receptor (FGFR) inhibitor (FGFRi) Erdafitinib, a top hit in the chemical synergy screen. RiBi inhibition using BMH-21, POLR1A siRNA, or Actinomycin D revealed engagement of the FGFR-FGF2 pathway. BMH-21 downregulated FGFR1 and SOX2 levels, whereas FGF2 was induced and released from the nucleolus. CONCLUSIONS: This study conceptualizes the implementation of RiBi inhibition as a viable future therapeutic strategy for glioma and reveals an FGFR connection to the cellular response upon RiBi inhibition with potential translational value.

Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

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.

p53 at the crossroad of DNA replication and ribosome biogenesis stress pathways.

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.

Targeting Ribosome Biogenesis in Cancer: Lessons Learned and Way Forward.

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.

Uncoupling of the ERα regulated morphological phenotype from the cancer stem cell phenotype in human breast cancer cell lines.

The CD24(low/-)CD44(+)EpCAM(+) phenotype is associated with breast cancer initiating cells. To investigate if these putative breast cancer stem cell markers are regulated by estrogen receptor alpha (ERα) we have determined the expression levels of EpCAM, CD44 and CD24 in several well characterized breast cancer cell lines. The expression levels of the three adhesion proteins were quantitatively different in the cell lines but the composite CD24(low/-)CD44(+)EpCAM(+) breast cancer stem cell phenotype was shown to exist as a small fraction, between 0.1% and 1.2%, in all breast cancer cell lines tested. Experimental silencing of ERα resulted in a reduced epithelial appearance and partial reduction of CD24 mRNA, while levels of CD44 and EpCAM were unaltered. Moreover, knockdown of ERα led to a change in the morphology of the cells similar to the epithelial to mesenchymal transition phenotype and was associated with decreased E-cadherin expression. Our findings offer new insights into the regulation of the breast cancer stem cell phenotype by ERα and suggest that treatments targeting the breast cancer stem cell adhesion molecules and the ERα pathway may be complementary.

SFRP2 induces a mesenchymal subtype transition by suppression of SOX2 in glioblastoma.

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 SOX2-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.

The exon-junction complex helicase eIF4A3 controls cell fate via coordinated regulation of ribosome biogenesis and translational output.

Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3's tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance. EIF4A3 depletion induces cell cycle arrest through impaired RiBi checkpoint-mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following eIF4A3 depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (MDM2) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3's unexpected role in RiBi, with implications for cancer pathogenesis and treatment.

Cancer-associated mutations in the MDM2 zinc finger domain disrupt ribosomal protein interaction and attenuate MDM2-induced p53 degradation.

The p53-inhibitory function of the oncoprotein MDM2 is regulated by a number of MDM2-binding proteins, including ARF and ribosomal proteins L5, L11, and L23, which bind the central acidic domain of MDM2 and inhibit its E3 ubiquitin ligase activity. Various human cancer-associated MDM2 alterations targeting the central acidic domain have been reported, yet the functional significance of these mutations in tumor development has remained unclear. Here, we show that cancer-associated missense mutations targeting MDM2's central zinc finger disrupt the interaction of MDM2 with L5 and L11. We found that the zinc finger mutant MDM2 is impaired in undergoing nuclear export and proteasomal degradation as well as in promoting p53 degradation, yet retains the function of suppressing p53 transcriptional activity. Unlike the wild-type MDM2, whose p53-suppressive activity can be inhibited by L11, the MDM2 zinc finger mutant escapes L11 inhibition. Hence, the MDM2 central zinc finger plays a critical role in mediating MDM2's interaction with ribosomal proteins and its ability to degrade p53, and these roles are disrupted by human cancer-associated MDM2 mutations.

The antimalarial drug amodiaquine stabilizes p53 through ribosome biogenesis stress, independently of its autophagy-inhibitory activity.

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.

Identification of functionally distinct and interacting cancer cell subpopulations from glioblastoma with intratumoral genetic heterogeneity.

BACKGROUND: Glioblastomas display a high level of intratumoral heterogeneity with regard to both genetic and histological features. Within single tumors, subclones have been shown to communicate with each other to affect overall tumor growth. The aim of this study was to broaden the understanding of interclonal communication in glioblastoma. METHODS: We have used the U-343 model, consisting of U-343 MG, U-343 MGa, U-343 MGa 31L, and U-343 MGa Cl2:6, a set of distinct glioblastoma cell lines that have been derived from the same tumor. We characterized these with regard to temozolomide sensitivity, protein secretome, gene expression, DNA copy number, and cancer cell phenotypic traits. Furthermore, we performed coculture and conditioned media-based experiments to model cell-to-cell signaling in a setting of intratumoral heterogeneity. RESULTS: Temozolomide treatment of a coculture composed of all 4 U-343 cell lines presents a tumor relapse model where the least sensitive population, U-343 MGa 31L, outlives the others. Interestingly, the U-343 cell lines were shown to have distinct gene expression signatures and phenotypes although they were derived from a single tumor. The DNA copy number analysis revealed both common and unique alterations, indicating the evolutionary relationship between the cells. Moreover, these cells were found to communicate and affect each other's proliferation, both via contact-dependent and -independent interactions, where NOTCH1, TGFBI, and ADAMTS1 signaling effects were involved, respectively. CONCLUSIONS: These results provide insight into how complex the signaling events may prove to be in a setting of intratumoral heterogeneity in glioblastoma and provide a map for future studies.

Thermal Proteome Profiling Identifies Oxidative-Dependent Inhibition of the Transcription of Major Oncogenes as a New Therapeutic Mechanism for Select Anticancer Compounds.

Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial step for drug development but remains a challenging task despite recent advances in technology. In this study, we applied multidimensional proteomics, sensitivity correlation analysis, and transcriptomics to identify a common MoA for the anticancer compounds RITA, aminoflavone (AF), and oncrasin-1 (Onc-1). Global thermal proteome profiling revealed that the three compounds target mRNA processing and transcription, thereby attacking a cancer vulnerability, transcriptional addiction. This led to the preferential loss of expression of oncogenes involved in PDGF, EGFR, VEGF, insulin/IGF/MAPKK, FGF, Hedgehog, TGFß, and PI3K signaling pathways. Increased reactive oxygen species level in cancer cells was a prerequisite for targeting the mRNA transcription machinery, thus conferring cancer selectivity to these compounds. Furthermore, DNA repair factors involved in homologous recombination were among the most prominently repressed proteins. In cancer patient samples, RITA, AF, and Onc-1 sensitized to poly(ADP-ribose) polymerase inhibitors both in vitro and ex vivo These findings might pave a way for new synthetic lethal combination therapies.Significance: These findings highlight agents that target transcriptional addiction in cancer cells and suggest combination treatments that target RNA processing and DNA repair pathways simultaneously as effective cancer therapies.

Emerging functions of ribosomal proteins in gene-specific transcription and translation.

Ribosomal proteins have remained highly conserved during evolution presumably reflecting often critical functions in ribosome biogenesis or mature ribosome function. In addition, several ribosomal proteins possess distinct extra-ribosomal functions in apoptosis, DNA repair and transcription. An increasing number of ribosomal proteins have been shown to modulate the trans-activation function of important regulatory proteins such as NF-kappaB, p53, c-Myc and nuclear receptors. Furthermore, a subset of ribosomal proteins can bind directly to untranslated regions of mRNA resulting in transcript-specific translational control outside of the ribosome itself. Collectively, these findings suggest that ribosomal proteins may have a wider functional repertoire within the cell than previously thought. The future challenge is to identify and validate these novel functions in the background of an often essential primary function in ribosome biogenesis and cell growth.

Expanding the scope of candidate prognostic marker IGFBP2 in glioblastoma.

Glioblastoma is the most common malignant brain tumor in adults. Unfortunately, it has a very poor prognosis and no cure. In a recent paper by Yuan et al. (Bioscience Reports (2019), DOI:10.1042/BSR20190045) RNAscope was used to detect insulin-like growth factor binding protein 2 (IGFBP2) mRNA in glioblastoma biopsies. The study revealed that patients with high levels of IGFBP2 mRNA had shorter survival and that IGFBP2 transcript level was an independent prognostic factor. It is also of value to determine the prognostic effect of IGFBP2 on established biomarkers such as isocitrate dehydrogenase (IDH1) mutations or telomerase reverse transcriptase (TERT) promoter mutation. In the present study, the combination of having a TERT promoter mutation, and at the same time a high level of IGFBP2 mRNA, was associated with very poor survival rates. It was concluded that IGFBP2 predicts the survival of the patients with TERT promoter mutation. This finding may have important implications for glioblastoma prognosis. IGFBP2 re-emerges as a candidate biomarker and potential therapeutic target in glioma. Further research into its functional roles during glioma progression may provide additional insights into this deadly disease.

Nucleolus as an emerging hub in maintenance of genome stability and cancer pathogenesis.

The nucleolus is the major site for synthesis of ribosomes, complex molecular machines that are responsible for protein synthesis. A wealth of research over the past 20 years has clearly indicated that both quantitative and qualitative alterations in ribosome biogenesis can drive the malignant phenotype via dysregulation of protein synthesis. However, numerous recent proteomic, genomic, and functional studies have implicated the nucleolus in the regulation of processes that are unrelated to ribosome biogenesis, including DNA-damage response, maintenance of genome stability and its spatial organization, epigenetic regulation, cell-cycle control, stress responses, senescence, global gene expression, as well as assembly or maturation of various ribonucleoprotein particles. In this review, the focus will be on features of rDNA genes, which make them highly vulnerable to DNA damage and intra- and interchromosomal recombination as well as built-in mechanisms that prevent and repair rDNA damage, and how dysregulation of this interplay affects genome-wide DNA stability, gene expression and the balance between euchromatin and heterochromatin. We will also present the most recent insights into how malfunction of these cellular processes may be a central driving force of human malignancies, and propose a promising new therapeutic approach for the treatment of cancer.

Reduced Expression of PROX1 Transitions Glioblastoma Cells into a Mesenchymal Gene Expression Subtype.

The homeodomain transcription factor PROX1 has been linked to several cancer types, including gliomas, but its functions remain to be further elucidated. Here we describe a functional role and the prognostic value of PROX1 in glioblastoma. Low expression of PROX1 correlated with poor overall survival and the mesenchymal glioblastoma subtype signature. The latter finding was recapitulated in vitro, where suppression or overexpression of PROX1 in glioma cell cultures transitioned cells to a mesenchymal or to a nonmesenchymal glioblastoma gene expression signature, respectively. PROX1 modulation affected proliferation rates that coincided with changes in protein levels of CCNA1 and CCNE1 as well as the cyclin inhibitors CDKN1A, CDKN1B, and CDKN1C. Overexpression of SOX2 increased PROX1 expression, but treatment with a CDK2 inhibitor subsequently decreased PROX1 expression, which was paralleled by decreased SOX2 levels. The THRAP3 protein was a novel binding partner for PROX1, and suppression of THRAP3 increased both transcript and protein levels of PROX1. Together, these findings highlight the prognostic value of PROX1 and its role as a regulator of glioblastoma gene expression subtypes, intratumoral heterogeneity, proliferation, and cell-cycle control.Significance: These findings demonstrate the role and prognostic value of PROX1 in glioblastomas; low PROX1 levels correlate with a mesenchymal gene expression subtype and shorter survival in glioblastoma tumors. Cancer Res; 78(20); 5901-16. ©2018 AACR.