Ambra1 is a novel autophagy tumour suppressor gene

Macroautophagy is the major regulated catabolic mechanism used by eukaryotic cells to degrade long-lived proteins and organelles. It involves the formation of cytosolic doublemembrane vesicles, called autophagosomes, that sequester portions of cytoplasm and then they fuse with lysosomes to form autolysosomes. Autophagy has a well-documented role in the maintenance of tissue homeostasis and in the response to stressful environments; moreover, this process is often dysregulated in various human diseases, such as neurodegeneration and cancer. In this context, autophagy has been identified as a crucial process in oncogenesis and in tumour progression. Allelic loss of the essential autophagy gene Beclin 1 occurs in human cancers and renders mice tumour-prone. The regulation of the Beclin 1/Vps34 complex lipid kinase activity is a critical step in autophagy signaling pathway. Ambra1 (Activating molecule in Beclin 1-regulated autophagy) has been shown to be an important member of this complex and to be involved in autophagosome formation. This evidence prompted us to investigate a possible role for Ambra1 as a haplo-insufficient tumour suppressor gene. We show that monoallelic deletion of Ambra1 promotes tumorigenesis. We found that Ambra1+/gt mice have a significantly higher probability than Ambra1+/+ mice to develop a malignancy, showing approximately three folds increase of spontaneous tumorigenesis in a number of organs, such as liver, spleen, lymphonodes, and lung. In lung, Ambra1 hemizygous tumours show traits of lung papillary adenocarcinoma. We have previously shown that Ambra1 deficiency during embryogenesis in vivo and in vitro induces an increase in cell proliferation. Therefore, we have also investigated whether the observed tumours could be related to a direct impairment of cell growth control by autophagy. The first evidence that we found was a general increase of the organ size, especially of liver, kidney, heart and spleen of the heterozygous animals in comparison with the wt mice, thus suggesting a role for Ambra1 in cell growth control. Moreover, we elucidate this aspect also in vitro by analysing the proliferation rate and the markers of cell cycle in Ambra1 defective systems. In principle, the demonstration of a haplo-insufficient tumour suppressor phenotype for Ambra1’s reduced function in mice may have direct implications for analysing the molecular pathogenesis of human cancer. Therefore, we isolated mouse embryonic fibroblast (MEFs) from embryos wt, heterozygous and knockout for the Ambra1 gene trap mutation and we evaluated the cell growth rate by BrdU incorporation assay and by cell counting. Both analyses revealed a marked increase in the proliferation rate of Ambra1+/gt and Ambra1gt/gt MEFs when compared with wild-type cells, indicating that the loss of the only one allele of Ambra1’s alleles is sufficient to increase cellular proliferation. Since the molecular mechanism responsible for these results could be due to a deregulation of the cell cycle, we decided to examine the main cell cycle regulators. First of all we focussed our attention on analysing the levels of the four main cyclins: D, E, A, B. During our analysis we have found that in Ambra1 defective systems, unlike the cyclin D and E, high levels of the cyclin A and B were present. These proteins, called mitosis cyclins, are more expressed in the S- and M-phase of cell cycle. Therefore, our finding correlates with the hyperproliferative phenotype specific for Ambra1 deficient cells. Moreover the transcription of the cyclin A gene is under the control of p107 hyperphosphorylation (Zerfass et al., 1996), a protein belongs to the Retinoblastoma protein family. The increased levels of cyclin A could explain the hyperphoshorylate state of p107 that we found in our experimental systems. Moreover, since activities of cyclin/CDK complexes is also mediated by their binding to other proteins, we decided to examine whether the Ambra1 dosage was correlated to the expression of these proteins. In particular, we analyzed the levels of two main members of the Cip/Kip family, p21 and p27. In our studies we found a deregulation of a number of cell cycle regulatory proteins: A and B cyclins, p107, p21 and p27. This may explain the hyperproliferative phenotype observed in Ambra1-defective systems in vitro. In summary, we identified a novel haplo-insufficient tumour suppressor gene. This strongly support for the idea that Ambra1 could play an important role in the regulation of tumour development and that its activity is tightly regulated in coordination with cell growth. The detailed mechanism by which Ambra1 contributes to tumour suppression is still unknown.