The Wnt Target Protein Peter Pan Defines a Novel p53-independent Nucleolar Stress-Response Pathway.

Proper ribosome formation is a prerequisite for cell growth and proliferation. Failure of this process results in nucleolar stress and p53-mediated apoptosis. The Wnt target Peter Pan (PPAN) is required for 45 S rRNA maturation. So far, the role of PPAN in nucleolar stress response has remained elusive. We demonstrate that PPAN localizes to mitochondria in addition to its nucleolar localization and inhibits the mitochondrial apoptosis pathway in a p53-independent manner. Loss of PPAN induces BAX stabilization, depolarization of mitochondria, and release of cytochrome c, demonstrating its important role as an anti-apoptotic factor. Staurosporine-induced nucleolar stress and apoptosis disrupt nucleolar PPAN localization and induce its accumulation in the cytoplasm. This is accompanied by phosphorylation and subsequent cleavage of PPAN by caspases. Moreover, we show that PPAN is a novel interaction partner of the anti-apoptotic protein nucleophosmin (NPM). PPAN depletion induces NPM and upstream-binding factor (UBF) degradation, which is independent of caspases. In summary, we provide evidence for a novel nucleolar stress-response pathway involving PPAN, NPM, and BAX to guarantee cell survival in a p53-independent manner.

Novel Methionine Aminopeptidase 2 Inhibitor M8891 Synergizes with VEGF Receptor Inhibitors to Inhibit Tumor Growth of Renal Cell Carcinoma Models.

N-terminal processing by methionine aminopeptidases (MetAP) is a crucial step in the maturation of proteins during protein biosynthesis. Small-molecule inhibitors of MetAP2 have antiangiogenic and antitumoral activity. Herein, we characterize the structurally novel MetAP2 inhibitor M8891. M8891 is a potent, selective, reversible small-molecule inhibitor blocking the growth of human endothelial cells and differentially inhibiting cancer cell growth. A CRISPR genome-wide screen identified the tumor suppressor p53 and MetAP1/MetAP2 as determinants of resistance and sensitivity to pharmacologic MetAP2 inhibition. A newly identified substrate of MetAP2, translation elongation factor 1-alpha-1 (EF1a-1), served as a pharmacodynamic biomarker to follow target inhibition in cell and mouse studies. Robust angiogenesis and tumor growth inhibition was observed with M8891 monotherapy. In combination with VEGF receptor inhibitors, tumor stasis and regression occurred in patient-derived xenograft renal cell carcinoma models, particularly those that were p53 wild-type, had Von Hippel-Landau gene (VHL) loss-of-function mutations, and a mid/high MetAP1/2 expression score.

The Wnt/ß-Catenin Pathway is Activated as a Novel Nucleolar Stress Response.

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/ß-Catenin pathway regulates ribosome formation. Here, we demonstrate that induction of nucleolar stress by different strategies stimulates the Wnt/ß-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/ß-Catenin signaling in a ß-Catenin-dependent manner and stabilizes ß-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/ß-Catenin targets, which is followed by the p53 target CDKN1A (p21). As PPAN expression is induced by Wnt/ß-Catenin signaling, our data establish a novel feedback mechanism and reveal that nucleolar stress over-activates the Wnt/ß-Catenin pathway, which most likely serves as compensatory mechanism to sustain ribosome biogenesis.

SHP2 Inhibition Influences Therapeutic Response to Tepotinib in Tumors with MET Alterations.

Tepotinib is an oral MET inhibitor approved for metastatic non-small cell lung cancer (NSCLC) harboring MET exon 14 (METex14) skipping mutations. Examining treatment-naive or tepotinib-resistant cells with MET amplification or METex14 skipping mutations identifies other receptor tyrosine kinases (RTKs) that co-exist in cells prior to tepotinib exposure and become more prominent upon tepotinib resistance. In a small cohort of patients with lung cancer with MET genetic alterations treated with tepotinib, gene copy number gains of other RTKs were found at baseline and affected treatment outcome. An Src homology 2 domain-containing phosphatase 2 (SHP2) inhibitor delayed the emergence of tepotinib resistance and synergized with tepotinib in treatment-naive and tepotinib-resistant cells as well as in xenograft models. Alternative signaling pathways potentially diminish the effect of tepotinib monotherapy, and the combination of tepotinib with an SHP2 inhibitor enables the control of tumor growth in cells with MET genetic alterations.

Loss of Peter Pan protein is associated with cell cycle defects and apoptotic events.

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.

Physiotherapy in obstetrics through the eyes of pregnant women: a qualitative study / Fisioterapia em obstetrícia pelos olhos das gestantes: um estudo qualitativo

Abstract Introduction: Physiotherapy in women's health emerged with the aim of promoting and maintaining physical and emotional health from the beginning to the end of pregnancy, through preventive measures against possible injuries that may occur during pregnancy, childbirth and the postpartum. Access to knowledge about obstetric physiotherapy among pregnant women is increasing, which may contribute to a search for trained professionals. Objective: To analyze the perception of pregnant women about obstetric physiotherapy. Methods: This is a qualitative study carried out at the Materno Infantil Center, in Capanema, Parana state (PR), using a semistructured interview. Content analysis used the Bardin method to categorize the thematic axes. Results: Seven pregnant women aged between 18-24 years (57%), 23-32 weeks of gestation (42%), multiparous (71%), with secondary education (42%) and income between 1-3 monthly minimum wages (86 %) were included in the study. It was found that participants' perception of physiotherapy was inaccurate, because none of the women had accessed this treatment. The women stated that physiotherapists reduce labor pain, but they have no knowledge of how these professionals work in the postpartum. Conclusion: It is concluded that it is necessary to increase information on the contribution of physiotherapists during prenatal care, delivery and postpartum.

Resumo Introdução: A fisioterapia em saúde da mulher é uma área que surgiu com o objetivo de promover e manter a saúde física e emocional do início ao final da gestação, exercendo um trabalho preventivo para os possíveis agravos que venham a ocorrer durante a gestação, parto e puerpério. O acesso ao conhecimento das gestantes sobre a atuação do fisioterapeuta na área de obstetrícia vem aumentando a cada dia e isso pode contribuir para uma procura por profissionais capacitados. Objetivo: Analisar a percepção das gestantes sobre a atuação da fisioterapia em obstetrícia. Métodos: Trata-se de um estudo com abordagem qualitativa, realizado no centro Materno Infantil, em Capanema, PR, configurado na forma de entrevista semiestruturada. Utilizou-se análise do conteúdo por meio do método de Bardin para categorizar os eixos temáticos. Resultados: Foram incluídas sete gestantes com idade entre 18 e 24 anos (57%), 23-32 semanas de gestação (42%), multigestas (71%), com ensino médio (42%) e renda de 1 a 3 salários mínimos (86%). Verificou-se que o contato com a fisioterapia é somente imaginável, porque nenhuma das participantes do estudo acessou esse desdobramento das políticas de saúde da mulher gestante. As gestantes afirmaram que o profissional de fisioterapia atua promovendo a diminuição da dor durante o trabalho de parto, porém não possuem conhecimento sobre como o fisioterapeuta atua no puerpério. Conclusão: Conclui-se que é necessário ampliar a informação sobre a atuação do fisioterapeuta durante o pré-natal, parto e pós-parto.

Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2.

The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.