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Simple Summary Ribosome biogenesis takes place in nucleoli and is essential for cellular survival and proliferation. In case this function is disturbed, either due to defects in regulatory factors or the structure of the nucleolus, nucleolar stress is provoked. Consequently, cells classically undergo cell cycle arrest and apoptosis. Induction of nucleolar stress is known to eliminate cells in the background of cancer therapy and paradoxically is also associated with increased cancer formation. Recent reports demonstrated that nucleolar stress triggers autophagy, a conserved pathway responsible for recycling endogenous material. Thus, it was suggested that autophagy might serve as compensatory pro-survival response. However, the mechanisms how nucleolar stress triggers autophagy are poorly understood. Here we show that induction of nucleolar stress by depleting ribosome biogenesis factors or by interfering with RNA polymerase I function, triggers expression of various key autophagy regulators. Moreover, we demonstrate that RNA pol I inhibition by CX-5461 correlates with increased ATG7 and ATGL16L1 levels, essential factors for generating autophagosomes, and stimulates autophagic flux. Abstract 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.

The nucleolus is a subnuclear compartment, which governs ribosome biogenesis. Moreover, it functions as hub in the stress response by orchestrating a variety of processes, such as regulation of cell cycle progression, senescence and apoptosis. Emerging evidence links the nucleolus also to the control of genomic stability and the development of human malignancies. Peter Pan (PPAN) is an essential ribosome biogenesis factor localized to nucleoli and mitochondria. We earlier showed that PPAN depletion triggers p53-independent nucleolar stress and apoptosis. In this study we investigated the precise localization of nucleolar PPAN during cell cycle and its function in cell cycle regulation. We show that PPAN knockdown impairs cell proliferation and induces G0/G1 as well as later G2/M cell cycle arrest in cancer cells. Although PPAN knockdown stabilizes the tumor suppressor p53 and induces CDKN1A/p21, the proliferation defects occur largely in a p53/p21-independent manner. We noticed a reduced number of knockdown cells entering cytokinesis and an elevation of binucleation. PPAN knockdown is also associated with increased H2A.X phosphorylation (γH2A.X) in cancer cells. We evaluated a potential signaling axis through the DNA damage response kinases ATM and ATR and alternatively apoptosis as a potent driver of γH2A.X. Interestingly, PPAN knockdown does not involve activation of ATM/ATR. Instead, γH2A.X is generated as a consequence of apoptosis induction in cancer cells. Strikingly, PPAN depletion in human fibroblasts did neither provoke apoptosis nor H2A.X phosphorylation, but recapitulated p53 stabilization. In summary, our data underline the notion that the PPAN-mediated, p53-independent nucleolar stress response has multiple facets.

Ribosomes are mandatory for growth and survival. The complex process of ribosome biogenesis is located in nucleoli and requires action of the RNA polymerases I–III, together with a multitude of processing factors involved in rRNA cleavage and maturation. Impaired ribosome biogenesis and loss of nucleolar integrity triggers nucleolar stress, which classically stabilizes the tumor suppressor p53 and induces cell cycle arrest and apoptosis. Nucleolar stress is implemented in modern anti-cancer therapies, however, also emerges as contributor to diverse pathological conditions. These include ribosomopathies, such as the Shwachman Bodian Diamond Syndrome (SBDS), which are not only characterized by nucleolar stress, but paradoxically also increased cancer incidence. Wnt signaling is tightly coupled to cell proliferation and is constitutively activated in various tumor types. In addition, the Wnt/β-Catenin pathway regulates ribosome formation. Here, we demonstrate that induction of nucleolar stress by different strategies stimulates the Wnt/β-Catenin pathway. We show that depletion of the key ribosomopathy factor SBDS, or the nucleolar factors Nucleophosmin (NPM), Pescadillo 1 (PES1) or Peter Pan (PPAN) by si RNA-mediated knockdown or CRISPR/Cas9 strategy activates Wnt/β-Catenin signaling in a β-Catenin-dependent manner and stabilizes β-Catenin in human cancer cells. Moreover, triggering nucleolar stress by the chemotherapeutic agents Actinomycin D or the RNA polymerase I inhibitor CX-5461 stimulates expression of Wnt/β-Catenin targets, which is followed by the p53 target CDKN1A (p21). As PPAN expression is induced by Wnt/β-Catenin signaling, our data establish a novel feedback mechanism and reveal that nucleolar stress over-activates the Wnt/β-Catenin pathway, which most likely serves as compensatory mechanism to sustain ribosome biogenesis.