Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression

https://www.nature.com/articles/s41467-017-02787-4

Silvia Márquez-Jurado, Juan Díaz-Colunga, Ricardo Pires das Neves, Antonio Martinez-Lorente, Fernando Almazán, Raúl Guantes & Francisco J. Iborra

Chemotherapies often leave a proportion of cancer cells behind. Even genetically identical cells grown in vitro show this effect, suggesting that there exists some level of non-genetic heterogeneity in cancer cells. 

Two well-known pathways are able to induce cell death: the intrinsic and extrinsic pathways. The intrinsic pathway (which does not involve signalling from outside of the cell) directly involves mitochondria. In contrast, the extrinsic pathway may be activated via the binding of specific ligands to cell death receptors on the plasma membrane of the cell and does not directly involve mitochondria. However, several proteins may participate in both the intrinsic and extrinsic pathways, meaning that these pathways have cross-talk. 

TNF-related apoptosis-inducing ligand (TRAIL) is a protein which may induce the extrinsic apoptosis pathway. When cells are treated with TRAIL, the authors observe that the fraction of cells which are killed saturates at 35% with the concentration of TRAIL, and that there is heterogeneity in the time to death at every concentration of TRAIL. The authors also observed (as noted previously by other authors) that sister cells tended to have the same fate and very similar times to death (Pearson correlation >0.8).

The authors investigated how mitochondrial content affected cell death propensity. They found that:

• Cells with higher mitochondrial mass (as determined by Mitotracker Green with 24 hr live-cell imaging) were more likely to die under TRAIL (as well as other cell death inducing drugs such as CHX and DRB).
• Cells show a weak correlation between time to death and mitochondrial mass (rho = -0.47) for intermediate TRAIL concentrations.
• After sorting cells into mito-high and mito-low fractions, (a fold-change of ~x5 between fractions), mito-high fractions had ~x3 more RNA than mito-low.
• Mitochondrial mass contributed to around 50% of the total variability observed in proteins which participate in apoptosis (both pro-apoptotic and anti-apoptotic).
• Including the observed correlation between mitochondrial mass and protein levels of apoptosis genes in a pre-existing mathematical model of the extrinsic cell death pathway in HeLa cells was able to recapitulate many of the authors' experimental observations.

The authors also investigated whether these results hold in real tumours, as opposed to cell culture conditions where environmental noise is minimised. The authors stained sections from colon cancer biopsies with antibodies against Aconitase 2 (for mitochondrial mass) and various cell death proteins. Whilst the mitochondrial contribution to variability for some apoptotic proteins was lost, others were retained (where pro-apoptotic proteins tended to have a higher correlation with mitochondrial mass than anti-apoptotic proteins).
 
Thoughts:
Does cell volume confound any of these observations?