Autophagy and receptor tyrosine kinase signalling: A mTORC2 matter

KEYWORDS:

• AKT
• autophagy
• c-MET
• ERK
• mTORC2
• protein phosphorylation
• RTK

This article refers to:

Autophagy is a tightly regulated catabolic pathway that contributes to tissue homeostasis and the response to stress. Through a multistep process, autophagy gives rise to double membrane vacuoles (autophagosomes), which mediate the degradation of cytoplasmic long-lived proteins and organelles into lysosomes. Signalling cascades downstream to growth factor receptor tyrosine kinases (RTKs), changes in nutrient availability as well as energy levels have been shown to control autophagy.¹ Vice versa, our knowledge on the role of autophagy and/or ATG proteins in regulating cell signalling independently of their role in cargo degradation is still limited. Recent findings have suggested a role for autophagy in the control of cell signalling and protein phosphorylation.^2-5

Our group has recently established a novel role of basal autophagy in regulation of RTK phosphorylation in colorectal cancer cell lines. Specifically, mTORC1-independent basal autophagy was found to positively modulate phosphorylation levels of several RTKs, such as c-MET, c-RET and Dtk. It was additionally shown that genetic suppression of basal autophagy decreased mTORC2-mediated activation of AKT at S473 but did not alter mTORC1 activity. We decided to focus on c-MET given its established role in cancer pathogenesis and found that autophagy positively mediates its phosphorylation via regulation of mTORC2 since reduced mTORC2 activation in autophagy-impaired cells was responsible for the defect in RTK phosphorylation. Specifically, genetic inhibition of mTORC2 as well as pharmacological inhibition of both mTORC1/2 led to decreased c-MET phosphorylation in autophagy proficient but not autophagy-impaired cells.³ Recently, a non-canonical autophagy pathway was found to regulate intracellular c-MET signalling in breast and lung cancer cell lines upon detachment, following HGF stimulation. However, in these settings genetic suppression of autophagy genes did not affect c-MET phosphorylation.⁵

Bernard et al., described autophagy as an activator of mTORC2 signalling, employing a different model system. Using starvation, a classical inducer of autophagy, this study demonstrated the importance of the autophagic response in triggering mTORC2 activation. It was found that increased autophagic flux under prolonged starvation in fibroblasts was followed by increased phosphorylation of AKT at S473 whereas genetic as well as pharmacological blockage of autophagy reduced AKT phosphorylation, with implications for myofibroblast differentiation.⁴

Other recent studies implicated autophagy in regulation of cell signalling.^2,5 Specifically, it was found that ERK1/2 is positively regulated by autophagy following EGFR stimulation in liver explants and brown adipose tissue. Interestingly, this effect was independent from phosphorylation levels of MEK.² A non-canonical autophagy pathway has been also suggested to control ERK1/2 activation downstream of intracellular c-MET signalling in breast and lung cancer cell lines upon detachment. Barrow-McGee et al., has shown that β1-integrin promotes c-MET-dependent ERK1/2 activation through sustained p52^Shc signalling, following HGF stimulation. ATG5 siRNA reduced p52^Shc phosphorylation upstream to ERK1/2 activation without affecting c-MET protein levels or its phosphorylation. Beclin1 siRNA reduced ERK1/2 activation while pERK1/2 was unchanged following ATG13 siRNA.⁵ In this respect, our unpublished data also indicate that basal autophagy positively regulates phosphorylation of ERK1/2 protein in selected colorectal cancer cell lines. Further investigation is needed to unravel whether autophagy-regulated ERK1/2 phosphorylation is controlled in an RTK-dependent or independent manner.

Our work along with the studies mentioned above unravel a complex bidirectional relationship between well-described autophagy-master regulator kinases and autophagy-related proteins. However the underlying mechanisms remain to be fully elucidated. One possibility is that autophagosomes or autophagy-related endomembranes act as subcellular scaffolding compartments or signalling platforms for regulation of cell signalling. In fact, EGF stimulation has been shown to increase colocalisation of MEK and ERK with LC3B-positive autophagosomal membranes. Specifically, ERK2 was suggested to directly interact with autophagosomal membranes through its substrate-binding domains.² Furthermore, Barrow-McGee et al., showed that c-MET and β1-integrin colocalise with LC3B and/or Beclin1-positive compartments and that a pool of phosphorylated ERK1/2 localise along with c-MET in autophagy-related endomembranes, following HGF stimulation. However, knockdown of autophagy genes did not affect c-MET phosphorylation suggesting that autophagy-related endomembranes act as scaffolds for c-MET downstream signalling cascade and ERK1/2 activation.⁵ Our group has additionally found, c-MET to partially colocalise with LC3B-positive perinuclear vesicles. Unlike the study by Barrow-McGee et al., c-MET association with LC3-positive structures may affect its phosphorylation, since CQ-mediated accumulation of autophagosomes increased phosphorylation of c-MET only in autophagy-proficient conditions. Therefore, autophagic-like vesicles may represent signalling platforms whereby c-MET phosphorylation is controlled through mTORC2.³ Interestingly, two recent studies demonstrated that mTORC2 kinase activity is associated with intracellular membrane compartments and regulation of AKT inside the cell.^6,7 Ebner et al., observed endogenous mTORC2 activity at the plasma membrane as well as at intracellular compartments such as outer mitochondrial membranes, and a subpopulation of early and late endosomes. mTORC2 activity at early and late endosomes was dependent on the presence of functional PI3K whereas at plasma membrane was PI3K-independent. The Pleckstrin homology (PH)-domain of Sin1, component of mTORC2, was shown to mediate the association of mTORC2 with membranes. Sin1 was localising on the surface and not in the lumen of late endosomes.⁶ Arias et al., observed that the lysosomal-associated mTORC2/PHLPP1/AKT axis modulates Chaperone-mediated autophagy (CMA) activity by regulating the assembly of the CMA-translocation complex at the lysosomal membrane. Specifically, it was shown that AKT activity could be regulated at a subpopulation of lysosomes via either mTORC2-kinase activity or PHLPP1-phosphatase activity. mTORC2 protein components as well as PHLPP1 and AKT were found to localise at the cytosolic side of the lysosomal membrane.⁷

Undoubtedly, a new chapter describing a novel role of autophagy in cell signalling regulation has opened. However, further work is now needed not only to resolve the precise mechanistic nature orchestrating such regulation but also its functional consequences. Given the controversial role of autophagy in cancer initiation and progression, it would be important future studies to shed light on the functional implications of autophagy-mediated signalling axis on cancer biology, invasion and metastasis, metabolic adaptation, immunity and/or interaction with the microenvironment. Therefore, further work is warranted to answer whether autophagy-mediated signalling axis could become a therapeutic target in conditions whereby elevated autophagy contributes to cancer development and/or progression.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

DH was supported by a program grant from Cancer Research UK C2259/A16569.