Nucleolar Stress Functions Upstream to Stimulate Expression of Autophagy Regulators

Ribosome biogenesis is essential for protein synthesis, cell growth and survival. The process takes places in nucleoli and is orchestrated by various proteins, among them RNA polymerases I–III as well as ribosome biogenesis factors. Perturbation of ribosome biogenesis activates the nucleolar stress response, which classically triggers cell cycle arrest and apoptosis. Nucleolar stress is utilized in modern anti-cancer therapies, however, also contributes to the development of various pathologies, including cancer. Growing evidence suggests that nucleolar stress stimulates compensatory cascades, for instance bulk autophagy. However, underlying mechanisms are poorly understood. Here we demonstrate that induction of nucleolar stress activates expression of key autophagic regulators such as ATG7 and ATG16L1, essential for generation of autophagosomes. We show that knockdown of the ribosomopathy factor SBDS, or of key ribosome biogenesis factors (PPAN, NPM, PES1) is associated with enhanced levels of ATG7 in cancer cells. The same holds true when interfering with RNA polymerase I function by either pharmacological inhibition (CX-5461) or depletion of the transcription factor UBF-1. Moreover, we demonstrate that RNA pol I inhibition by CX-5461 stimulates autophagic flux. Together, our data establish that nucleolar stress affects transcriptional regulation of autophagy. Given the contribution of both axes in propagation or cure of cancer, our data uncover a connection that might be targeted in future.

Nucleophagy contributes to genome stability though TOP2cc and nucleolar components degradation

ABSTRACTThe nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal, healthy cells maintain genome stability avoiding accumulation of nuclear damaged material is less understood. Here we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after Etoposide-induced DNA Double-Stranded Breaks. These nuclear buds and micronuclei co-localize with autophagic proteins BECN1 and LC3 and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and Type II DNA Topoisomerases (TOP2A and TOP2B), or nucleolar protein Fibrillarin, implying they are also targets of nucleophagy. We propose that a basal nucleophagy contributes to genome and nuclear stability and also in response to DNA damage and nucleolar stress.

rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin

The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development.

Quinacrine Induces Nucleolar Stress in Treatment-Refractory Ovarian Cancer Cell Lines

A considerable subset of gynecologic cancer patients experience disease recurrence or acquired resistance, which contributes to high mortality rates in ovarian cancer (OC). Our prior studies showed that quinacrine (QC), an antimalarial drug, enhanced chemotherapy sensitivity in treatment-refractory OC cells, including artificially generated chemoresistant and high-grade serous OC cells. In this study, we investigated QC-induced transcriptomic changes to uncover its cytotoxic mechanisms of action. Isogenic pairs of OC cells generated to be chemoresistant and their chemosensitive counterparts were treated with QC followed by RNA-seq analysis. Validation of selected expression results and database comparison analyses indicated the ribosomal biogenesis (RBG) pathway is inhibited by QC. RBG is commonly upregulated in cancer cells and is emerging as a drug target. We found that QC attenuates the in vitro and in vivo expression of nucleostemin (NS/GNL3), a nucleolar RBG and DNA repair protein, and the RPA194 catalytic subunit of Pol I that results in RBG inhibition and nucleolar stress. QC promotes the redistribution of fibrillarin in the form of extranuclear foci and nucleolar caps, an indicator of nucleolar stress conditions. In addition, we found that QC-induced downregulation of NS disrupted homologous recombination repair both by reducing NS protein levels and PARylation resulting in reduced RAD51 recruitment to DNA damage. Our data suggest that QC inhibits RBG and this inhibition promotes DNA damage by directly downregulating the NS–RAD51 interaction. Additionally, QC showed strong synergy with PARP inhibitors in OC cells. Overall, we found that QC downregulates the RBG pathway, induces nucleolar stress, supports the increase of DNA damage, and sensitizes cells to PARP inhibition, which supports new therapeutic stratagems for treatment-refractory OC. Our work offers support for targeting RBG in OC and determines NS to be a novel target for QC.

The NF-κB Nucleolar Stress Response Pathway

The nuclear organelle, the nucleolus, plays a critical role in stress response and the regulation of cellular homeostasis. P53 as a downstream effector of nucleolar stress is well defined. However, new data suggests that NF-κB also acts downstream of nucleolar stress to regulate cell growth and death. In this review, we will provide insight into the NF-κB nucleolar stress response pathway. We will discuss apoptosis mediated by nucleolar sequestration of RelA and new data demonstrating a role for p62 (sequestosome (SQSTM1)) in this process. We will also discuss activation of NF-κB signalling by degradation of the RNA polymerase I (PolI) complex component, transcription initiation factor-IA (TIF-IA (RRN3)), and contexts where TIF-IA-NF-κB signalling may be important. Finally, we will discuss how this pathway is targeted by aspirin to mediate apoptosis of colon cancer cells.

rRNA Biogenesis Regulates Mouse 2C-like State by 3D Structure Reorganization of Peri-Nucleolar Heterochromatin

Nucleolus is the organelle for ribosome biogenesis and for sensing various types of stress. Its role in regulating stem cell fate is unclear. Here, we present multiple lines of evidence that nucleolar stress induced by interfering rRNA biogenesis can drive 2-cell stage embryo-like (2C-like) transcriptional program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, the nucleolar integrity mediated by rRNA biogenesis maintains the normal liquid-liquid phase separation (LLPS) of nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon rRNA biogenesis defect, the natural LLPS of nucleolus is disrupted, causing dissociation of NCL/TRIM28 complex from PNH and changes of epigenetic states and reorganization of the 3D structure of PNH, which leads to Dux, a 2C program transcription factor gene, to be released from the PNH region and activation of 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are incompatible to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell-to-4-cell transition of murine pre-implantation embryo development.

LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

p53 induces a survival transcriptional response after nucleolar stress.

Accumulating evidence indicate that increased ribosome biogenesis is a hallmark of cancer. It is well established that inhibition of any steps of ribosome biogenesis induces a nucleolar stress characterized by p53 activation and subsequent cell cycle arrest and/or cell death. However, cells derived from solid tumors have demonstrated different degree of sensitivity to ribosome biogenesis inhibition, where cytostatic effects rather than apoptosis are observed. The reason for this is not clear and the p53-specific transcriptional program induced after nucleolar stress has not been previously investigated. Here we demonstrate that blocking rRNA synthesis by depletion of essential rRNA processing factors such as LAS1L, PELP1, and NOP2 or by inhibition of RNA Pol I with the specific small molecule inhibitor CX-5461, mainly induce cell cycle arrest accompanied with autophagy in solid tumor-derived cell lines. Using gene expression analysis, we find that p53 orchestrates a transcriptional program involved in promoting metabolic remodeling and autophagy to help cells survive under nucleolar stress. Importantly, our study demonstrates that blocking autophagy significantly sensitizes cancer cells to RNA Pol I inhibition by CX-5461, suggesting that interfering with autophagy should be considered a strategy to heighten the responsiveness of ribosome biogenesis-targeted therapies in p53-positive tumors.