Emerging roles of mitotic autophagy.

Lysosomes exert pleiotropic functions to maintain cellular homeostasis and degrade autophagy cargo. Despite the great advances that have boosted our understanding of autophagy and lysosomes in both physiology and pathology, their function in mitosis is still controversial. During mitosis, most organelles are reshaped or repurposed to allow the correct distribution of chromosomes. Mitotic entry is accompanied by a reduction in sites of autophagy initiation, supporting the idea of an inhibition of autophagy to protect the genetic material against harmful degradation. However, there is accumulating evidence revealing the requirement of selective autophagy and functional lysosomes for a faithful chromosome segregation. Degradation is the most-studied lysosomal activity, but recently described alternative functions that operate in mitosis highlight the lysosomes as guardians of mitotic progression. Because the involvement of autophagy in mitosis remains controversial, it is important to consider the specific contribution of signalling cascades, the functions of autophagic proteins and the multiple roles of lysosomes, as three entangled, but independent, factors controlling genomic stability. In this Review, we discuss the latest advances in this area and highlight the therapeutic potential of targeting autophagy for drug development.

Detection of Nuclear Biomarkers for Chromosomal Instability.

Chromosomal instability (CIN) is a hallmark of cancer, which is characterized by the gain or loss of chromosomes as well as the rearrangement of the genetic material during cell division. Detection of mitotic errors such as misaligned chromosomes or chromosomal bridges (also known as lagging chromosomes) is challenging as it requires the analysis and manual discrimination of chromosomal aberrations in mitotic cells by molecular techniques. In interphase cells, more frequent in the cell population than mitotic cells, two distinct nuclear phenotypes are associated with CIN: the micronucleus and the toroidal nucleus. Several methods are available for the detection of micronuclei, but none for toroidal nuclei. Here, we provide a method to quantify the presence of both nuclear biomarkers for the evaluation of CIN status in non-mitotic cells particularly suited for genotoxicity screens.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.

Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.

Phosphofructokinases Axis Controls Glucose-Dependent mTORC1 Activation Driven by E2F1.

Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. We showed that E2F1 regulates glucose metabolism by increasing aerobic glycolysis and identified the PFKFB3 regulatory enzyme as an E2F1-regulated gene important for mTORC1 activation. Furthermore, PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by substrate accessibility or expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and subsequent mTORC1 activity. Our results support a model whereby a glycolytic metabolon containing phosphofructokinases transiently interacts with the lysosome acting as a sensor platform for glucose catabolism toward mTORC1 activity.

Effect of low doses of actinomycin D on neuroblastoma cell lines.

BACKGROUND: Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis. METHODS: Neuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation. RESULTS: Low-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53(-/-)deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA. CONCLUSIONS: The results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.

V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy.

In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.