When DNA is damaged, cells are equipped with mechanisms to repair the DNA. However, when these repair mechanisms fail to function, cells resort to independent stress-response mechanisms like apoptosis and cellular senescence. During apoptotic events, damaged cells are permanently removed. However, when cells undergo senescence, damaged cells are taken out of the cell cycle and develop a persistent pro-inflammatory phenotype called senescence-associated secretory phenotype (SASP). SASP expression is often beneficial early in life for wound healing, but overtime can accumulate with age. As a result of this accumulation, aging may become accelerated and age-related diseases may be induced.
Therefore, Baar et al. and colleagues identified mechanisms on the generation of senescent cells and how they affect tissue homeostasis. By identifying these mechanisms, Baar et al. provides a possible therapeutic solution to treat damages from medical treatments and accelerated aging. In order to first identify the mechanism of senescent cell generation, Baar et al. and colleagues took normal human IMR90 cells and IMR90 senescent cells and compared the expression profiles of both. They expected that the IMR90 senescent cells would have lower levels of pro-apoptotic proteins and higher levels of anti-apoptotic proteins in comparison to the control. However, they observed that the IMR90 senescent cells produced the exact opposite result. Based on this finding, it was suggested that the IMR90 senescent cells are primed for apoptosis, but a transcription regulator may be inhibiting the process. One transcription regulator that was specifically focused on was FOXO4, a member of the FOXO family.
Although a less prominent regulator compared to its siblings, FOXO1 and FOXO3, FOXO4 was found to be up-regulated during senesce. To determine what role FOXO4 played in senescent cells, a lentiviral shRNA was used to inhibit its expression in cells prior to senescent induction. Through inhibition, Baar et al. and colleagues saw that the cells did not become senescent and had undergone apoptosis instead. Furthermore, in cells already senescent, they saw that the inhibition of FOXO4 had decreased the cell viability and density. These results suggested that FOXO4 plays a key role in the generation of senescent cells.
To better understand the role of FOXO4 in senescent cells, the authors first focused on FOXO4’s pattern during senescence. During senescence development, FOXO4 was found to reside in promyelocytic leukemia (PML) bodies that were adjacent to 53BP1-containing DNA-SCARS. Localized to DNA-SCARS in senescent cells was also p53. As both FOXO4 and p53 co-localized to these structures and FOXOs have been known to bind p53, it was proposed that FOXO4 interacts with p53.
Thus, the authors focused on interfering with this binding to possibly induce apoptosis. In order to interfere with this binding, Baar et al. created a D-retro inverso (DRI)-isoform that corresponded to the reverse sequence of the FOXO4 domain involved with binding to p53. This DRI isoform was utilized as previous DRI-modified peptides produced new chemical properties that improved potency in vitro and in vivo. Furthermore, previous DRI-modified peptides were discovered to be therapeutically effective in clinical trials.
After creating FOXO4-DRI, the peptide was then fused with a HIV-TAT peptide to facilitate cellular uptake. What Baar et al. and colleagues noticed was that FOXO4-DRI outcompeted FOXO4 in binding to p53 as it had a higher affinity. This binding of FOXO4-DRI to p53 presented similar results to FOXO4 shRNAs. Nuclear exclusion of p53 occurred, which allowed for it to interact with mitochondria to cause the release of cytochrome c and induce apoptosis. In addition to observing FOXO4-DRI in senescent cells, the authors also observed the effects of FOXO4-DRI in normal cells. They also discovered that in the normal cells, FOXO4-DRI did not produce any damaging effects.
Based on the results that FOXO4-DRI could induce apoptosis in vitro, Baar et al. and colleagues wanted to also look at its effects on senescent cells in vivo. Additionally, they wanted to see if FOXO4-DRI could restore tissue homeostasis in damaged tissues. To do so, they used a fast-aging XpdTTD/TTD mouse model, a naturally aged mouse model for a control, and chemotherapy-treated animals. Each of these models all consisted of a high number of senescent cells. When treated with FOXO4-DRI, several parameters were observed to determine whether tissue homeostasis was restored. These parameters were exploratory behavior, voluntary running wheel activity, standard tissue function tests specifically for the liver and kidney, and fur density. From observing these parameters across all three treated models, significant functional improvements were seen.
This paper by Barr et al. offers new insight into understanding senescent cells. The observations made by the authors on FOXO4’s involvement in inhibition of apoptosis allowed for the discovery of a new possible therapeutic agent, FOXO4-DRI. Although further studies still need to be conducted to better understand its pharmacokinetic properties, Barr et al. and colleagues have opened a new door for restoring tissue homeostasis in patients undergoing chemotherapy treatment or suffering from age-related disease.
