Senescence induction contributes to cancer therapy responses and is crucial for p53-mediated tumor suppression. However, whether p53 inactivation actively suppresses senescence induction has been unclear. Here we demonstrate that E2F1 overexpression, due to p53 or p21 inactivation, promotes expression of human oncoprotein CIP2A, which in turn, by inhibiting PP2A activity, increases stabilizing serine 364 phosphorylation of E2F1. Several lines of evidence demonstrate that increased activity of E2F1-CIP2A feedback renders breast cancer cells resistant to senescence induction. Importantly, mammary tumorigenesis is impaired in a CIP2A deficient mouse model, and CIP2A deficient tumors display markers of senescence induction. Moreover, high CIP2A expression predicts for poor prognosis in a subgroup of breast cancer patients treated with senescence-inducing chemotherapy. Together these results implicate E2F1-CIP2A feedback loop as a key determinant of breast cancer cell sensitivity to senescence induction. It also constitutes a promising pro-senescence target for therapy of cancers with inactivated p53-p21 pathway.
(Macro)Autophagy is a phylogenetically conserved membrane-trafficking process that functions to deliver cytoplasmic cargoes to lysosomes for digestion. The process is a major mechanism for turnover of cellular constituents and is therefore critical for maintaining cellular homeostasis. Macroautophagy is characteristically distinct from other forms of autophagy due to the formation of double-membraned vesicles termed autophagosomes which encapsulate cargoes prior to fusion with lysosomes. Autophagosomes contain an integral membrane-bound form (LC3-II) of the microtubule-associated protein 1 light chain 3 β (MAP1LC3B), which has become a gold-standard marker to detect accumulation of autophagosomes and thereby changes in macroautophagy. Due to the role played by macroautophagy in various diseases, the detection of autophagosomes in tissue sections is frequently desired. To date, however, the detection of endogenous LC3-II on paraffin-embedded tissue sections has proved problematic. We report here a simple, optimized and validated method for the detection of LC3-II by immunohistochemistry in human and mouse tissue samples that we believe will be a useful resource for those wishing to study macroautophagy ex vivo.
autophagy; LC3; tissue sections; immunohistochemistry
Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis.
autophagy; cancer; invasion; migration; organotypic model
Macro(autophagy) is a cellular mechanism which delivers cytoplasmic constituents to lysosomes for degradation. Due to its role in maintaining cellular integrity, autophagy protects against various diseases including cancer. p53 is a major tumor suppressor gene which can modulate autophagy both positively and negatively. p53 induces autophagy via transcriptional activation of damage-regulated autophagy modulator (DRAM-1). We report here that DRAM-1 encodes not just one mRNA, but a series of p53-inducible splice variants which are expressed at varying levels in multiple human and mouse cell lines. Two of these new splice variants, termed SV4 and SV5, result in mature mRNA species. Different from ‘full-length’ DRAM-1 (SV1), SV4 and SV5 do not localize to lysosomes or endosomes, but instead partially localize to peroxisomes and autophagosomes respectively. In addition, SV4 and SV5 can also be found co-localized with certain markers of the endoplasmic reticulum. Similar to SV1, SV4 and SV5 do not appear to be inducers of programmed cell death, but they do modulate autophagy. In summary, these findings identify new autophagy regulators that provide insight into the control of autophagy downstream of p53.
DRAM-1; mRNA splice variants; p53; cell death; autophagy
Autophagy is a catabolic membrane-trafficking process that leads to sequestration and degradation of intracellular material within lysosomes. It is executed at basal levels in every cell and promotes cellular homeostasis by regulating organelle and protein turnover. In response to various forms of cellular stress, however, the levels and cargoes of autophagy can be modulated. In nutrient-deprived states, for example, autophagy can be activated to degrade cargoes for cell-autonomous energy production to promote cell survival. In other contexts, in contrast, autophagy has been shown to contribute to cell death. Given these dual effects in regulating cell viability, it is no surprise that autophagy has implications in both the genesis and treatment of malignant disease. In this review, we provide a comprehensive appraisal of the way in which oncogenes and tumour suppressor genes regulate autophagy. In addition, we address the current evidence from human cancer and animal models that has aided our understanding of the role of autophagy in tumour progression. Finally, the potential for targeting autophagy therapeutically is discussed in light of the functions of autophagy at different stages of tumour progression and in normal tissues.
(Macro)autophagy is a membrane-trafficking process that serves to sequester cellular constituents in organelles termed autophagosomes, which target their degradation in the lysosome. Autophagy operates at basal levels in all cells where it serves as a homeostatic mechanism to maintain cellular integrity. The levels and cargoes of autophagy can, however, change in response to a variety of stimuli, and perturbations in autophagy are known to be involved in the etiology of various human diseases. Autophagy must therefore be tightly controlled. We report here that the Drosophila cyclindependent kinase PITSLRE is a modulator of autophagy. Loss of the human PITSLRE ortholog, CDK11, initially appears to induce autophagy, but at later time points CDK11 is critically required for autophagic flux and cargo digestion. Since PITSLRE/CDK11 regulates autophagy in both Drosophila and human cells, this kinase represents a novel phylogenetically conserved component of the autophagy machinery.
PITSLRE; CDK11; cyclin-dependent kinase; autophagy; human; Drosophila
The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.
p53; SUMO-2/3; sumoylation; MDM2; ARF; L11
The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms.
LIMK; RhoC; p53; DNA damage; actin; cofilin; cytoskeleton
The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIMK2 are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments which were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms.
LIMK; RhoC; p53; DNA damage; actin; cofilin; cytoskeleton
Autophagy is an evolutionarily conserved, catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Autophagy is regulated via a group of genes called AuTophaGy-related genes and is executed at basal levels in virtually all cells as a homeostatic mechanism for maintaining cellular integrity. The levels and cargos of autophagy can be modulated in response to a variety of intra- and extracellular cues to bring about specific and selective events. Autophagy is a multifaceted process and alterations in autophagic signalling pathways are frequently found in cancer and many other diseases. During tumour development and in cancer therapy, autophagy has paradoxically been reported to have roles in promoting both cell survival and cell death. In addition, autophagy has been reported to control other processes relevant to the aetiology of malignant disease, including oxidative stress, inflammation and both innate and acquired immunity. It is the aim of this review to describe the molecular basis and the signalling events that control autophagy in mammalian cells and to summarize the cellular functions that contribute to tumourigenesis when autophagy is perturbed.
The analysis of DNA tumor viruses has provided landmark insights into the molecular pathogenesis of cancer. A paradigm for this field has been the study of the adenoviral E1a protein which has led to identification of proteins such as p300, p400 and members of the retinoblastoma family. Through binding Rb family members, E1a causes deregulation of E2F proteins – an event common to most human cancers and a central pathway in which oncogenes, including E1a, sensitize cells to chemotherapy-induced programmed cell death. We report here, however, that E1a not only causes deregulation of E2F, but importantly that it also causes the post-transcriptional up-regulation of E2F1 protein levels. This effect is distinct from deregulation of E2F1, however, as mutants of E2F1 impaired in pRb binding are induced by E1a and E2F1 induction can also be observed in Rb-null cells. Analysis of E1a mutants selectively deficient in cellular protein binding revealed that induction of E2F1 is instead intrinsically linked to p400. Mutants unable to bind p400, despite being able to deregulate E2F1, do not increase E2F1 protein levels and they do not sensitize cells to apoptotic death. These mutants can, however, be complemented by either knockdown of p400, resulting in restoration of the ability to induce E2F1, or by over expression of E2F1, with both events re-enabling sensitization to chemotherapy-induced death. Due to the frequent deregulation of E2F1 in human cancer, these studies reveal potentially important insights into E2F1-mediated chemotherapeutic responses that may aid the development of novel targeted therapies for malignant disease.
E2F1; E1a; apoptosis; protein-stability; p400
The p53-inducible BH3-only protein PUMA is a key mediator of p53-dependent apoptosis, and PUMA has been shown to function by activating Bax and mitochondrial outer membrane permeabilization. In this study we describe an ability of PUMA to induce autophagy that leads to the selective removal of mitochondria. This function of PUMA depends on Bax/Bak and can be reproduced by overexpression of Bax. The induction of autophagy coincides with cytochrome c release, and taken together the results suggest that PUMA functions through Bax to induce mitochondrial autophagy in response to mitochondrial perturbations. Surprisingly, inhibition of PUMA or Bax-induced autophagy dampens the apoptotic response, suggesting that under some circumstances the selective targeting of mitochondria for autophagy can enhance apoptosis.
PUMA; Bax; autophagy
transcription factor NF-κB is most clearly linked to the inhibition of
extrinsic apoptotic signals such as TNFα by upregulating known anti-apoptotic genes, NF-κB has also been proposed to be required for
p53-induced apoptosis in transformed cells. However, the involvement of NF-κB in this process is poorly understood. Here we investigate this mechanism and show that in
transformed MEFs lacking NF-κB (p65-null cells) genotoxin-induced cytochrome c release is
compromised. To further address how NF-κB contributes to apoptosis, gene
profiling by microarray analysis of MEFs was
performed, revealing that NF-κB is required for
expression of Noxa, a pro-apoptotic BH3-only protein that is induced by
genotoxins and that triggers cytochrome c release. Moreover, we find
that in the absence of NF-κB, genotoxin treatment cannot induce Noxa
mRNA expression. Noxa expression had been shown to be regulated directly by
genes of the p53 family, like p73 and p63, following genotoxin treatment.
Here we show that p73 is activated after genotoxin treatment only in the
presence of NF-κB and that p73 induces Noxa gene
expression through the p53 element in the promoter. Together our data
provides an explanation for how loss of NF-κB abrogates
apoptosis; p73; NF-κB B; Noxa
Adenoviruses for gene or oncolytic therapy are under development. Notable among these strategies is adenoviral delivery of the tumor suppressor p53. Since all therapeutics have limitations in certain settings, we have undertaken retroviral suppressor screens to identify genes conferring resistance to adenovirus-delivered p53. These studies identified the tumor antigen LRRC15, which is frequently overexpressed in multiple tumor types, as a repressor of cell death due to adenoviral p53. LRRC15, however, does not impede p53 function per se but impedes adenoviral infection. Specifically, LRRC15 causes redistribution of the coxsackievirus-adenovirus receptor away from the cell surface. This effect is manifested in less adenoviral binding to the surfaces of LRRC15-expressing cells. This discovery, therefore, not only is important for understanding adenoviral biology but also has potentially important implications for adenovirus-based anticancer therapeutics.
The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP — a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.
Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest, functions which have been shown to be separable activities of p53. We have characterized a series of p53 mutants with amino acid substitutions at residue 175 and show that these mutants fall into one of three classes: class I, which is essentially wild type for apoptotic and cell cycle arrest functions; class II, which retains cell cycle arrest activity but is impaired in the induction of apoptosis; and class III, which is defective in both activities. Several residue 175 mutants which retain cell cycle arrest function have been detected in cancers, and we show that these have lost apoptotic function. Furthermore, several class II mutants have been found to be temperature sensitive for apoptotic activity while showing constitutive cell cycle arrest function. Taken together, these mutants comprise an excellent system with which to investigate the biochemical nature of p53-mediated apoptosis, the function of principal importance in tumor suppression. All of the mutants that showed loss of apoptotic function also showed defects in the activation of promoters from the potential apoptotic targets Bax and the insulin-like growth factor-binding protein 3 gene (IGF-BP3), and a correlation between full apoptotic activity and activation of both of these promoters was also seen with the temperature-sensitive mutants. However, a role for additional apoptotic activities of p53 was suggested by the observation that some mutants retained significant apoptotic function despite being impaired in the activation of Bax- and IGF-BP3-derived promoters. In contrast to the case of transcriptional activation, a perfect correlation between transcriptional repression of the c-fos promoter and the ability to induce apoptosis was seen, although the observation that Bax expression induced a similar repression of transcription from this promoter suggests that this may be a consequence, rather than a cause, of apoptotic death.