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Abstract/Summary
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GABARAP belongs to an evolutionary highly conserved gene family that has a fundamental role in autophagy. There is ample evidence for a crosstalk between autophagy and apoptosis as well as the immune response. However, the molecular details for these interactions are not fully characterized. Here, we report that the ablation of murine GABARAP, a member of the Atg8/LC3 family that is central to autophagosome formation, suppresses the incidence of tumor formation mediated by the carcinogen DMBA and results in an enhancement of the immune response through increased secretion of IL-1β, IL-6, IL-2 and IFN-γ from stimulated macrophages and lymphocytes. In contrast, TGF-β1 was significantly reduced in the serum of these knockout mice. Further, DMBA treatment of these GABARAP knockout mice reduced the cellularity of the spleen and the growth of mammary glands through the induction of apoptosis. Gene expression profiling of mammary glands revealed significantly elevated levels of Xaf1, an apoptotic inducer and tumor-suppressor gene, in knockout mice. Furthermore, DMBA treatment triggered the upregulation of pro-apoptotic (Bid, Apaf1, Bax), cell death (Tnfrsf10b, Ripk1) and cell cycle inhibitor (Cdkn1a, Cdkn2c) genes in the mammary glands. Finally, tumor growth of B16 melanoma cells after subcutaneous inoculation was inhibited in GABARAP-deficient mice. Together, these data provide strong evidence for the involvement of GABARAP in tumorigenesis in vivo by delaying cell death and its associated immune-related response.
Gamma (γ)-aminobutyric acid type A receptor (GABAAR)-associated protein (GABARAP) was first identified as a trafficking molecule for GABAARs in neurons.1 We previously reported that GABARAP may function as a candidate tumor suppressor in breast cancer. Introduction of the gene into a breast cancer cell line reduced the growth rate and colony formation in vitro and suppressed tumorigenicity in nude mice. In addition, overexpression of the gene was associated with cytoplasmic vesicle formation,2 a finding supported by the fact that GABARAP has been shown to be involved in autophagy.3 Specifically, it was shown that GABARAP has a crucial role in the autophagic process through mediating membrane hemifusion and to be involved in the maturation of the autophagosome, a key component of autophagy machinery.4, 5 As autophagy is a fundamental mechanism for most cells, it is not surprising that the gene is ubiquitously expressed. Knockout (KO) of the gene, however, did not reveal an obvious pathological phenotype.6 This might be due to the fact that GABARAP belongs to a gene family. Its homolog in yeast, autophagy-related gene 8 (Atg8), is an essential gene and mutants die under starvation.7 In mammals, there are several Atg8 homologs grouped into two subfamilies: microtubule-associated protein-1 light chain 3 (LC3) and GABARAP, of which LC3 is the most well known as it is widely used to monitor autophagic activity.8 The fundamental importance of this gene family in mammals may also be derived from the fact that Atg8 is an ubiquitin-like protein. Thus it is possible that the Atg8 gene family may have a similar relevance for autophagy as ubiquitin has for proteasomal protein degradation.9
Autophagy is an intracellular pathway for bulk degradation of damaged proteins and organelles within the lysosome/vacuole to recycle building blocks for biosynthesis and cellular energy under conditions of stress.10 Its role in cancer is complex and controversial. It may act as a tumor-promoting as well as tumor-suppressive mechanism depending on the cellular context and the genetic background.11 In particular, there is a close relationship between autophagy and apoptosis and some of the molecular constituents in this interplay have already been identified. In most cases, autophagy delays or prevents apoptosis, but in a few circumstances, it may also assist cell death.12
Apart from apoptosis, autophagy is deeply integrated into tumor immunity,13 metabolism14 and the stress response.15 In addition, it has been identified as a potential target of cancer therapy.16 Interestingly, the disruption of Atgs has been associated with immunity and inflammation.17 Specifically, GABARAP KO was shown to increase the secretion of proinflammatory cytokines in the context of sepsis. KO mice were highly susceptible to mortality and revealed increased proinflammatory cytokine secretion through activation of the NOD-like receptor family, pyrin domain containing 3 inflammasome in two sepsis models.18 Tumor-promoting inflammation, avoiding immune destruction and resisting cell death have been identified as hallmarks for tumor initiation and progression.19 It has been proposed that cancer cell-associated autophagy has a key role in subverting anti-tumor immunity.20
The aim of our study was to use a GABARAP KO mouse model to evaluate the potential in vivo role of the gene in tumorigenesis and to clarify whether it acts as a tumor suppressor or enhancer. Our data confirm its relevance for tumor growth with respect to 7,12-dimethylbenz(a)anthracene (DMBA)-induced tumor development as well as syngenic tumor cell inoculation. Unlike our previous in vitro data, we find that GABARAP acts as tumor enhancer in vivo, which seems to be related to the inhibition of apoptosis and antitumor immune response.
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