Fight Aging!

A harmful accumulation of lingering senescent cells occurs in all tissues over the course of aging. A cell entering a senescent state ceases to replicate and begins to generate an mix of signals known as the senescence-associated secretory phenotype (SASP). These signals drive chronic inflammation, remodel the surrounding tissue structure, and encourage nearby cells to also become senescent. This can be helpful in the short term, such as following injury, where it can aid in regeneration. When sustained over the long term, it is a cause of aging and age-related disease, however.

Cells become senescent constantly, largely somatic cells reaching the Hayflick limit on cellular replication, but also potentially cancerous, damaged cells. Near all senescent cells either self-destruct via the process of apoptosis or are destroyed by the immune system quite soon after they enter this state. Several different components of the immune system readily attack and destroy errant cells, those that are damaged and potentially cancerous, and those that are senescent. So the question arises as to why any senescent cells survive to linger in tissues for the long term.

While it is true that the immune system declines in effectiveness with age, and there is good evidence for this to degrade its ability to destroy cancerous and senescent cells, some fraction of senescent cells nonetheless still manage to linger past their welcome even in a youthful physiology. In today's open access paper, researchers dig into how exactly this unwanted survival happens, and demonstrate the ability to break the mechanism responsible. The data in the paper is all obtained from cell cultures, but it is nonetheless quite compelling. Given a suitable set of targets in the biochemistry of senescence, it may be possible to enable the immune system to target and destroy the persistent senescent cells that would otherwise manage to evade its attentions, thereby building the basis for a new class of senolytic therapies capable of producing rejuvenation in the old.

Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging

Recent reports in mice show that cellular senescence can also regulate immune processes leading to the elimination of senescent cells (SnCs). In mouse models of hepatocarcinoma and liver fibrosis, restoring p53 function enables senescent cells to be eliminated by natural killer (NK) cells in part via NKG2D detection, while oncogenic RAS-induced senescence of hepatocytes promotes immune responses involving CD4+ T cells, neutrophils, and macrophages that lead to SnC removal. In mouse models of cutaneous wound repair, p53/p21- and p16-proficient SnCs are cleared after healing is complete. Notably, however, p53 and p16 are not required to trigger cellular senescence in human tissues/cells, and many senescence features are p53 or p16 independent, suggesting that additional mechanisms may regulate the interplay between SnCs and the immune system.

Despite evidence of immune surveillance and clearance of SnCs in mice, SnCs accumulate with age in patients and are found in inflamed and damaged tissues, premalignant lesions, and arrested tumors and after chemotherapy or radiotherapy. Persistent SnCs can contribute to age-associated pathologies and tissue dysfunction, including cancer. These effects have been attributed to the senescence-associated secretory phenotype (SASP), which includes inflammatory factors secreted by tissue-resident SnCs.

NKG2D ligands (NKG2D-Ls) are cell surface semaphores that mediate the immune recognition and clearance of cells that are transformed, damaged, stressed, or infected. NKG2D-Ls are mostly absent in healthy tissues. We previously observed an increase in NKG2D-L expression upon senescence induction in vitro in normal human fibroblasts. We measured the expression of NKG2D-L MICA and MICB in tumor samples from 10 patients with prostate cancer before and after mitoxantrone (MIT) treatment, which we previously showed induces cellular senescence based on cell cycle arrest and SASP markers. We found that after senescence-inducing genotoxic chemotherapy, residual tumors expressed significantly higher levels of MICA/B.

These results show that DNA-damaging chemotherapies induce tumors to develop a senescence phenotype associated with elevated levels of NKG2D-Ls. Although this may agree with the notion that SnCs upregulate NKG2D-Ls, it is surprising because NKG2D-Ls should promote the immune detection and clearance of those cells. Thus, other characteristics likely allow these SnCs to elude immune recognition and persist while expressing elevated levels of NKG2D-Ls.

As a first model, we induced cellular senescence by DNA damage or replicative senescence in normal human fibroblasts expressing wild-type p53/p16, or inactivated p53 (p53-), or knocked-down p16 (p16-). Although the p53/p21 and p16/pRb pathways are important effectors of cellular senescence, the upregulation of NKG2D-Ls in fibroblasts occurred regardless of p53 loss before or after senescence-inducing damage, and irrespective of their p16 status. Our other observations in which cells arrested and senesced with high levels of p16 but low levels of NKG2D-Ls, led us to postulate that the induction of NKG2D-L expression by SnCs may depend on the DNA damage response (DDR) but not on cell growth arrest per se.

SnC cycle arrest is carried out by cyclin-dependent kinase inhibitor (CDKI) p16 or p21. To mimic the senescence arrest elicited by these CDKIs, we overexpressed p16 or p21 in fibroblasts. We found that these cells showed limited changes in levels of NKG2D-L. This demonstrates that the expression of NKG2D-Ls is not a consequence of CDKIs' activation or senescence per se, but rather a response to damage that is separable from the growth arrest. Hence, p16 neither establishes nor triggers NKG2D-L expression, and the immunogenic program of cellular senescence can be dissociated from other senescence characteristics, including cell cycle arrest and p16 expression.

To explore how the fate of these different types of SnCs may depend on NKG2D-Ls, we cocultured leukocytes with SnCs or their presenescent counterparts, and measured cytotoxicity. We found that IL-2-preactivated primary natural killer (NK) cells were the main effectors of SnC cytolytic killing. Blocking the NKG2D receptor significantly prevented the killing of SnCs. These results show that NKG2D-Ls are key limiting factors that mediate the immune detection of damaged SnCs and orchestrate the balance between elimination/clearance and survival/persistence of SnCs.

A subset of damaged SnCs actively evades leukocyte recognition and killing. We had initially noticed that the elimination of damaged SnCs in leukocyte cocultures was never complete. So, we treated these persistent SnCs with fresh batches of leukocytes, and scored survival. We found that 70%-80% of the original persistent SnCs remained impervious to killing. Thus, persistent SnCs possessed inherent properties that allowed them to actively evade recognition and cytolysis. To characterize persistent SnCs, we compared NKG2D-L expression in SnCs that had not been exposed (naive) or had been exposed (persistent) to leukocytes. Surprisingly, persistent SnCs expressed equal or greater levels of intracellular NKG2D-L compared with naive cells. However, immunofluorescence showed strikingly diminished levels of NKG2D-Ls on the surface of persistent SnCs relative to naive one.

Since cancer cells can promote their immunoevasion by shedding NKG2D-Ls, SnCs may also shed NKG2D-Ls to elude immune detection and persist. We found that the cell culture media of senescent fibroblasts and epithelial cells contained soluble NKG2D-L MICA and that the media from persistent SnCs contained markedly higher levels of soluble NKG2D-Ls compared with naive counterparts. Thus, SnCs shed NKG2D-Ls regardless of cell type and p53 status, and this was amplified in persistent SnCs that avoided killing.

Because MMP3 was among the most upregulated MMPs across damaged SnCs, we used it as a marker of senescence detectable by immunofluorescence. In contrast to the high but variable MMP3 levels observed among naive SnCs, persistent SnCs consistently displayed intense MMP3 staining. To test the possibility that MMPs inhibition might preserve the cell surface presentation of NKG2D-Ls and thus enhance the killing of persistent SnCs, we used the broad-spectrum MMP inhibitor GM6001. GM6001 effectively blocked MICA shedding in a dose-dependent manner and increased cell surface NKG2D-Ls. Critically, GM6001 treatment of fibroblast and epithelial cancer SnCs prior to coculture with leukocytes markedly decreased SnC survival. Moreover, GM6001 treatment of already-persistent SnCs prior to a second round of leukocytes led to their near complete clearance.

In conclusion, our data show how oncogenic and tumor-suppressive drivers of cellular senescence regulate surveillance processes that can be circumvented to enable SnCs to elude immune recognition but can be reversed by cell surface-targeted interventions to purge the SnCs that persist in vitro and in patients. Since eliminating SnCs can prevent tumor progression, delay the onset of degenerative diseases, and restore fitness; since NKG2D-Ls are not widely expressed in healthy human tissues and NKG2D-L shedding is an evasion mechanism also employed by tumor cells; and since increasing numbers of B cells express NKG2D ligands in NKG2D receptor-deficient mice as they age, we propose that therapeutic interventions designed to increase cell surface presentation of NKG2D-Ls could be effective senolytic strategies to resensitize persistent SnCs to immune detection and rescue their clearance, whether in cancer or aging settings.