Mutation of the Conserved Threonine 8 within the Human ARF Tumour Suppressor Protein Regulates Autophagy.

Rosa Fontana,Girolama La Mantia,Daniela Guidone,Viola Calabrò,Tiziana Angrisano,Alessandra Pollice,Maria Vivo

doi:10.3390/biom12010126

Rosa Fontana, Girolama La Mantia + Show 5 more

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https://doi.org/10.3390/biom12010126

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Abstract

Background: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. Methods: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. Results: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells’ inability to elicit autophagosomes formation upon T8D expression. Conclusions: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein’s ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

Highlights

The protein LC3-I is recruited on the phagophore membrane and converted to its lipidated form, LC3-II, which induces its growth and its closure in the mature autophagosome
Immunoblot analysis show that the amount of LC3-II in ARF silenced cells was reduced to 50% compared with control cells, showing that the autophagic pathway was impaired, in line with previous data in other cell contexts [13,15]
8 (T8A mutant) does not affect ARF ability to restrain cell proliferation, the opposite muT8D ARF mutant confers a proliferative advantage when overexpressed in HeLa cells

Summary

Introduction

ARF activates the transcription factor p53 that triggers the expression of apoptosis inducers and cell cycle inhibitory genes. This function is accomplished through the inhibition of the functions of the MDM2 oncoprotein (mouse double minute 2, HDM2 in human), allowing p53 stabilization and the activation of downstream pathways [5]. We provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells’ inability to elicit autophagosomes formation upon T8D expression. Our results indicate that ARF phosphorylation controls protein’s ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression

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Conclusion