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Beta 1-integrin–c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes

Receptor tyrosine kinases (RTKs) and integrins cooperate to stimulate cell migration and tumour metastasis. Here we report that an integrin influences signalling of an RTK, c-Met, from inside the cell, to promote anchorage-independent cell survival. Thus, c-Met and β1-integrin co-internalize and become progressively recruited on LC3B-positive ‘autophagy-related endomembranes' (ARE). In cells growing in suspension, β1-integrin promotes sustained c-Met-dependent ERK1/2 phosphorylation on ARE. This signalling is dependent on ATG5 and Beclin1 but not on ATG13, suggesting ARE belong to a non-canonical autophagy pathway. This β1-integrin-dependent c-Met-sustained signalling on ARE supports anchorage-independent cell survival and growth, tumorigenesis, invasion and lung colonization in vivo. RTK–integrin cooperation has been assumed to occur at the plasma membrane requiring integrin ‘inside-out' or ‘outside-in' signalling. Our results report a novel mode of integrin–RTK cooperation, which we term ‘inside-in signalling'. Targeting integrin signalling in addition to adhesion may have relevance for cancer therapy.

There are a few minor issues that need to be addressed. The most serious is the assignment of the integrin as a scaffold linking Shc to c-Met. The data show modulation of c-Met/Shc interaction by integrin but it is clearly not essential for co-IP, nor is the biochemistry sufficiently detailed to reach this interpretation. The authors need to be more cautious in their conclusions. Where are the images quantified on the right? It's a little confusing to link the images on the left to a different quantification on the right.
Fig 2e,f,g. It is hardly surprising that tumor growth is dependent on integrins. The authors need to be cautious in claiming that this is entirely due to c-met synergy.  There is a dramatic, perhaps 10 fold or more, upregulation of Shc in the integrin b1 YYFF cells that makes these results hard to interpret. In fact, the change in Shc co-IP with c-met is rather subtle. This result points toward a modulatory role for integrin signaling but is not consistent with an essential adapter or scaffolding function.

Reviewer #2 (Remarks to the Author)
The authors have satisfactorily addressed my concerns from the previous round of review. In particular, they added new data characterizing better the relationship between autophagy and Met/β1-integrin co-signaling (showing that Met activation or β1-integrin levels do not affect basal autophagy, and that autophagy does not contribute to Met degradation). Moreover, additional data using ATG13 depletion indeed argue that canonical autophagy does not underlie the observed phenomenon and generation of ARE. At this stage I do recommend publication of this manuscript. In my view, it documents well an intriguing case of signaling emanating from intracellular compartments due to the cross-talk between two classes of transmembrane receptors.
The authors have constructively revised the manuscript, which now represents a very impressive body of work that makes an important point with many novel aspects.
We thank the reviewer for acknowledging the improvements of this manuscript and recommending publication.
There are a few minor issues that need to be addressed. The most serious is the assignment of the integrin as a scaffold linking Shc to c-Met. The data show modulation of c-Met/Shc interaction by integrin but it is clearly not essential for co-IP, nor is the biochemistry sufficiently detailed to reach this interpretation. The authors need to be more cautious in their conclusions .   Fig 1b. Where are the images quantified on the right? It's a little confusing to link the images on the left to a different quantification on the right. We have removed this graph, as it was redundant with the quantification shown below the pictures.
Fig 2e,f,g. It is hardly surprising that tumor growth is dependent on integrins. The authors need to be cautious in claiming that this is entirely due to c-met synergy. We do not claim that β1-integrin dependent tumour growth is entirely due to c-Met synergy. As we use tumorigenesis (Figure 2e) and invasion (Figure 2g) models driven by c-Met activity, our results indicate that the c-Met dependent tumorigenesis and invasion require β1-integrin, at least in part. There is also some active ERK1/2 in the cytoplasm but at a lower intensity. The picture has been contrasted to allow a good visualisation of active ERK1/2 on endomembranes. These cells express a constitutive active c-Met-GFP construct, which therefore must dynamically triggers the activation of pools of ERK1/2 on endomembranes. In our view, activated ERK1/2 do not remain on the endomembranes but a pool of active ERK1/2 is always detected on endomembranes. We have modified the text in the result part to reflect this view. The graphs in Figure 5l and m represent the percentage of P-ERK1/2 colocalisation with c-Met-LC3B in c-Met-GFP cells knocked down for β1-integrin and in β1-YYFF cells compared to the colocalisation, in the respective controls (control knock down or β1A cells), set as 100%. The legend has been modified to clarify this .   Fig 6d. There is a dramatic, perhaps 10 fold or more, upregulation of Shc in the integrin b1 YYFF cells that makes these results hard to interpret. In fact, the change in Shc co-IP with c-met is rather subtle. This result points toward a modulatory role for integrin signaling but is not consistent with an essential adapter or scaffolding function. Although the level of expression of p52 Shc is increased in β1A-YYFF versus β1A cells, p52 Shc co-immunoprecipitation with c-Met is reduced compared to β1A cells, in basal conditions and upon HGF stimulation. Moreover, there is no significant increase in the co-immunoprecipitation upon HGF versus no HGF in β1A-YYFF cells while it is significantly increased in β1A cells. These results, together with results presented Figure 5lm are consistent with a scaffolding role of β1-integrin to our point of view. However, we have modified the text in the manuscript and only suggest this role. We have removed the scaffold function from the abstract and only mention signalling.
Reviewer #2 (Remarks to the Author): The authors have satisfactorily addressed my concerns from the previous round of review. In particular, they added new data characterizing better the relationship between autophagy and Met/β1-integrin co-signaling (showing that Met activation or β1-integrin levels do not affect basal autophagy, and that autophagy does not contribute to Met degradation). Moreover, additional data using ATG13 depletion indeed argue that canonical autophagy does not underlie the observed phenomenon and generation of ARE. At this stage I do recommend publication of this manuscript. In my view, it documents well an intriguing case of signaling emanating from intracellular compartments due to the cross-talk between two classes of transmembrane receptors.
We thank the reviewer for acknowledging the improvements of this manuscript and recommending publication.