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Previews will allow such an approach to progress beyond the drawing board. REFERENCES Daly, C.S., Shaw, P., Ordonez, L.D., Williams, G.T., Quist, J., Grigoriadis, A., Van Es, J.H., Clevers, H., Clarke, A.R., and Reed, K.R. (2017). Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium. Oncogene 36, 1793–1803. Kim, I., Pan, W., Jones, S.A., Zhang, Y., Zhuang, X., and Wu, D. (2013). Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes. J. Cell Biol. 200, 419–428.
MacDonald, B.T., and He, X. (2012). Frizzled and LRP5/6 receptors for Wnt/b-catenin signaling. Cold Spring Harb. Perspect. Biol. 4, a007880. Pan, W., Choi, S.C., Wang, H., Qin, Y., VolpicelliDaley, L., Swan, L., Lucast, L., Khoo, C., Zhang, X., Li, L., et al. (2008). Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation. Science 321, 1350– 1353. Saito-Diaz, K., Benchabane, H., Tiwari, A., Tian, A., Li, B., Thompson, J.J., Hyde, A.S., Sawyer, L.M., Jodoin, J.N., Santos, E., et al. (2018). APC inhibits ligand-independent Wnt signaling by the clathrin endocytic pathway. Dev. Cell 44, this issue, 566–581.
Stamos, J.L., Chu, M.L., Enos, M.D., Shah, N., and Weis, W.I. (2014). Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6. Elife 3, e01998. Taelman, V.F., Dobrowolski, R., Plouhinec, J.L., Fuentealba, L.C., Vorwald, P.P., Gumper, I., Sabatini, D.D., and De Robertis, E.M. (2010). Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell 143, 1136–1148. Zhai, L., Chaturvedi, D., and Cumberledge, S. (2004). Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine. J. Biol. Chem. 279, 33220–33227.
FOXO3a Provides a Quickstep from Autophagy Inhibition to Apoptosis in Cancer Therapy Patrice Codogno1,2,* and Etienne Morel1,2,* 1INSERM
U1151-CNRS UMR 8253, Institut Necker Enfants-Malades (INEM), 70014 Paris, France Paris Descartes-Sorbonne Paris Cite´, 70006 Paris, France *Correspondence:
[email protected] (P.C.),
[email protected] (E.M.) https://doi.org/10.1016/j.devcel.2018.02.019 2Universite ´
FOXO3a, a member of the Forkhead transcription factor family, has roles in apoptosis and autophagy. In this issue of Developmental Cell, Fitzwalter et al. (2018) describe how the blockade of FOXO3a turnover, which normally occurs through autophagy, sensitizes cancer cells to apoptosis through FOXO3a-mediated stimulation of pro-apoptotic PUMA/BBC3 expression. Macroautophagy (herein called autophagy) is a lysosomal catabolic pathway for intracellular macromolecules and organelles (Boya et al., 2013). In cancer cells, autophagy can have onco-suppressive or tumor-supporting capacities, depending on the type of tumors and the stage of progression (Amaravadi et al., 2016; Galluzzi et al., 2015). In many cases, autophagy contributes to the resistance to cancer treatment, and autophagy inhibition has been shown to sensitize tumor cells during chemotherapy (Levy et al., 2017; Rebecca and Amaravadi, 2016). The mechanism underlying this beneficial effect is still unclear. Recently, Thorburn and colleagues showed that the pro-apoptotic protein PUMA, a p53-upregulated modulator of apoptosis (also called BBC3), is upregulated upon autophagy inhibition (Thorburn
et al., 2014). The transcription factor FOXO3a was also reported to regulate the expression of PUMA (You et al., 2006) €llgrabe and of autophagy-related genes (Fu et al., 2014). In this issue of Developmental Cell, Fitzwalter and colleagues (2018) experimentally characterize the interplay between the autophagic pathway and the apoptotic cell death program via actions of PUMA and FOXO3a (Figure 1). The authors uncover the mechanism by which autophagy inhibition enhances the activity of pro-apoptotic chemotherapy drugs. They first showed that, in cancer cells, the inhibition of basal autophagy by multiple means—including bafilomycin A1 treatment and CRISPR/Cas9-mediated deletion of genes encoding autophagyrelated proteins (ATG) ATG7 or ATG5—
resulted in an increase in PUMA mRNA levels. They further revealed, by chromatin immunoprecipitation, that this increase is due to direct transcriptional regulation of the PUMA gene. Fitzwalter et al. (2018) hypothesized that PUMA transcriptional regulation is directly controlled by the transcriptional factor FOXO3a, already reported as a PUMA-associated regulator. Indeed, in cells lacking FOX03a (either due to small hairpin RNA-mediated inhibition or CRISPR/Cas9-mediated knockout), the previously described effect of basal autophagy inhibition on PUMA levels was no longer observed. Upon deeper analysis, the authors identified a single Forkhead response element (FHRE) in an intronic region of PUMA that is responsible for this FOXO3a-dependent regulation.
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Furthermore, the authors the toxicities that might result FOXO3a FOXO3a investigated the relationship when autophagy is inhibited between autophagy modulain the presence of the antidegradation degradation AUTOPHAGY AUTOPHAGY tion and FOXO3a activity. By cancer drugs will require MDM2 MDM2 chemical and biological autofurther study. Other questarget target p53 p53 phagy inhibition, they detertions also remain Nutlin Nutlin mined that FOXO3a, previunanswered: If toxicities ously reported to be a result when autophagy inhibisubstrate of proteasometion is coupled with anti-canATGs ATGs associated degradation, cer drugs, can side effects be ATGs ATGs PUMA PUMA could also be targeted to circumvented? What is the and degraded by the lysorelevance of this pathway in PUMA PUMA some, presumably via autop53-negative tumors? What phagosomal delivery, as is the cellular mechanism for CELL SURVIVAL demonstrated by the presthe beneficial effect of autoAPOPTOSIS GROWTH ARREST ence of FOXO3a in autophaphagy in this context? The golysosomal vesicles. same questions must be asked for tumors in which Finally, the authors nicely Figure 1. PUMA- and Autophagy-Dependent FOXO3a Apoptosis PUMA is not the driving force linked this phenomenon with Sensitization in apoptosis induction. The previous results showing an The FOXO3a transcription factor regulates expression of PUMA and some autophagy-related (ATG) genes and is also a cargo for the autophagy work by Fitzwalter et al. enhancement in the activity degradative pathway. Once the final steps of autophagy are inhibited (right (2018) furthers our underof anti-cancer drugs, such panel), FOXO3a escapes lysosomal degradation and induces PUMA transtanding of how and when as doxorubicin and etoposcription, which in turn, through specific targets, allows the sensitization of cancer cells to Nutlin treatment and the shift to from cell growth arrest (left to manipulate autophagy to side, when basal autophagy panel) to apoptosis (right panel). block tumor development was inhibited. Interestingly, and/or progression. Coordithe authors showed that, nation of this line of research whereas autophagy inhibition Autophagy and apoptosis are two pro- with the development of a new generaincreases the FOXO3a-dependent expression of other apoptotic genes (e.g., cesses that determine cell fate by regu- tion of autophagy inhibitors will be fruitful BIM, BNIP3L), the apoptotic effect on lating the balance between survival and for improving cancer therapy. the chemotherapeutic agents was largely death. In most physiological settings, mediated by the FOXO3a-dependent autophagy acts to oppose cell death REFERENCES (Marin˜o et al., 2014). The molecular expression of PUMA. To further interrogate the effect of regulation of autophagy and apoptosis Amaravadi, R., Kimmelman, A.C., and White, E. autophagy modulation on the p53 tran- are intertwined at different levels. This (2016). Recent insights into the function of scriptional program that is known to study from Fitzwalter et al. (2018) dem- autophagy in cancer. Genes Dev. 30, 1913–1930. control PUMA gene expression, the au- onstrates that one molecular link bethors used Nutlin, an anti-cancer drug tween autophagy and apoptosis is Boya, P., Reggiori, F., and Codogno, P. (2013). regulation and functions of autophagy. that increases the levels of p53 by block- through the regulation of the activity of Emerging Nat. Cell Biol. 15, 713–720. ing the activity of the ubiquitin E3 ligase the transcription factor FOXO3a and MDM2, which is responsible for target- its control of the expression of the Fitzwalter, B.E., Towers, C.G., Sullivan, K.D., Andrysik, Z., Hoh, M., Ludwig, M., O’Prey, J., ing p53 for proteasomal degradation. pro-apoptotic PUMA protein, thereby Ryan, K.M., Espinosa, J.M., Morgan, M.J., et al. deciphering the molecular mechanism (2018). Autophagy turnover of FOXO3a mediates They showed in in vitro and in vivo experimental settings that treatment of apoptosis induction when autophagy apoptosis sensitivity and response to cancer therapy through a single genomic site. Dev. Cell 44, with Nutlin in the presence of bafilomy- is inhibited in cancer cells (Figure 1). this issue, 555–565. cin A1 or chloroquine, known inhibitors These findings provide a strong rational €llgrabe, J., Klionsky, D.J., and Joseph, B. (2014). u of autophagy, resulted in increased for the inhibition of autophagy as an FThe return of the nucleus: transcriptional and levels of PUMA mRNA in a human colon adjuvant to cancer therapy in can- epigenetic control of autophagy. Nat. Rev. Mol. cancer cell line (HCT116). Interestingly, cers where autophagy has a tumor-sup- Cell Biol. 15, 65–74. this combination was able, through porting function (Amaravadi et al., 2016; Galluzzi, L., Pietrocola, F., Bravo-San Pedro, J.M., PUMA activation via both p53 and Galluzzi et al., 2015). Knowing whether Amaravadi, R.K., Baehrecke, E.H., Cecconi, F., FOX3a, to switch the Nutlin treatment ef- autophagy has a tumor-supporting Codogno, P., Debnath, J., Gewirtz, D.A., Karantza, V., et al. (2015). Autophagy in malignant fect from cell growth arrest to apoptosis. function in a specific cancer to be transformation and cancer progression. EMBO Consistent with this, xenografted treated is an absolute prerequisite for J. 34, 856–880. HCT116 tumors were responsive to the determining whether to use an auto- Levy, J.M.M., Towers, C.G., and Thorburn, A. combination of Nutlin and chloroquine, phagy inhibitor in combination with (2017). Targeting autophagy in cancer. Nat. Rev. whereas HCT116 cells in which PUMA chemotherapy, so as to avoid possible Cancer 17, 528–542. effects of autophagy Marin˜o, G., Niso-Santano, M., Baehrecke, E.H., lacks the FHRE were not responsive to deleterious inhibition on tumor growth. Similarly, and Kroemer, G. (2014). Self-consumption: the the drugs.
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Previews interplay of autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 15, 81–94. Rebecca, V.W., and Amaravadi, R.K. (2016). Emerging strategies to effectively target autophagy in cancer. Oncogene 35, 1–11.
Thorburn, J., Andrysik, Z., Staskiewicz, L., Gump, J., Maycotte, P., Oberst, A., Green, D.R., Espinosa, J.M., and Thorburn, A. (2014). Autophagy controls the kinetics and extent of mitochondrial apoptosis by regulating PUMA levels. Cell Rep. 7, 45–52.
You, H., Pellegrini, M., Tsuchihara, K., Yamamoto, K., Hacker, G., Erlacher, M., Villunger, A., and Mak, T.W. (2006). FOXO3a-dependent regulation of Puma in response to cytokine/ growth factor withdrawal. J. Exp. Med. 203, 1657–1663.
Did Mitochondria Kill the Frog? Souhir Marsit,1,4 Anne-Marie Dion-Coˆte´,2,3,4 and Daniel A. Barbash2,* de Biologie Inte´grative et des Syste`mes (IBIS), De´partement de Biologie, PROTE´O, Universite´ Laval, Que´bec, Canada of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA 3Department of Ecology and Genetics, Uppsala University, Uppsala, Sweden 4These authors contributed equally *Correspondence:
[email protected] https://doi.org/10.1016/j.devcel.2018.02.020 1Institut
2Department
Genomic divergence can cause reproductive isolation between species. The molecular mechanisms underlying reproductive isolation can thus reveal which genomic features evolve rapidly and become unstable or incompatible in hybrids. In a recent paper in Nature, Gibeaux et al. (2018) report paternal genome instability and metabolic imbalance in hybrids between frog species. Numerous fungi, animal, and plant species originated as hybrids, highlighting the important role of hybridization as a driving force of evolution. However, interspecific hybrids are typically sterile or inviable. Three main mechanisms may lead to hybrid infertility and inviability: activation of the mismatch repair pathway, which limits meiotic recombination between divergent sequences, causing chromosomal non-disjunction and aneuploidy; chromosomal rearrangements, which result in unbalanced gametes following meiotic recombination; and genetic incompatibilities between divergent alleles (Maheshwari and Barbash, 2011; Marsit et al., 2017). In theory, a single, strong genetic incompatibility could be sufficient to cause hybrid sterility or inviability, but the molecular basis of reproductive isolation typically involves a combination of mechanisms. This is especially true for species that diverged millions of years ago. A fascinating property of reproductive isolation is its frequent asymmetry, whereby hybrids may be viable or fertile in one direction of crossing but not in the other. This directionality may result from uni-parental inheritance, because parents contribute unevenly to progeny. Examples include sex chromosomes and maternal transmission of mitochondrial
DNA (mtDNA), as well as maternal provisioning of proteins, mRNAs, and small regulatory RNAs. In a recent paper, Gibeaux et al. (2018) sought to identify the mechanisms underlying asymmetrical reproductive isolation between two species of frog, Xenopus laevis and X. tropicalis. X. laevis is a rare allotetraploid animal that arose through hybridization of two diploid parents followed by whole-genome duplication (Session et al., 2016). X. laevis diverged from the diploid species X. tropicalis 48 million years ago. Hybridization outcome between these species is asymmetric: hybrids produced in crosses with female X. laevis are viable, whereas hybrids arising from crosses with female X. tropicalis are inviable (Figure 1). By leveraging the strength of the Xenopus system in manipulating embryonic development and using state-of-the-art genomics, cytogenetics, and metabolomics, Gibeaux et al. (2018) elegantly tested hypotheses to explain asymmetrical reproductive isolation between these highly diverged species. They first found that inviable hybrid embryos have anaphase defects leading to chromosome mis-segregation and micronuclei formation. This instability apparently results from an interaction between
the paternal genome and the maternal cytoplasm, but not the maternal genome, because eliminating the maternal genome by UV exposure does not suppress the defects. Using whole-genome sequencing, they found that two specific chromosome regions (3L and 4L) are largely absent in the dying embryos (Figure 1). The break points of both chromosomes localize to gaps that remain in the previously assembled genome, which suggests that the deletions correlate with the presence of repetitive sequences. The authors then assessed the cellular and physiological causes by using a combination of metabolomic and transcriptomic approaches. They find that hybrids have perturbations in glycolysis, which takes place in the cytoplasm, and in the tricarboxylic acid (TCA) cycle, which takes place in mitochondria. They further show that defective glycolysis or inhibition of mitochondrial ATP production causes cell lysis or embryo arrest, respectively, at the same developmental stage in pure-species Xenopus as in the inviable hybrids. Therefore, mitochondrial and glycolytic defects produce similar phenotypes in pure-species Xenopus and dying hybrids. How might mito-nuclear incompatibilities contribute to Xenopus hybrid lethality?
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