Non-cell autonomous communication in senescence and ageing - Extracellular Vesicles

Ageing is a process by which basic cellular functions and tissue homeostasis start to decline and organs become progressively dysfunctional. Aged tissues are enriched in senescent cells which communicate with other neighbouring cells via the senescence-associated secretory phenotype (SASP). This SASP is enriched in soluble factors, metabolites and extracellular vesicles. In our lab, we are interested in understanding how this secretome is regulated and how it influences the surrounding cells and the microenvironment in different conditions. 

On one hand, we have found that small extracellular vesicles (sEV) mediate paracrine senescence in normal proliferating cells (Figure 1A). A comparison analysis show similarities between soluble factor-mediated and sEV-mediated paracrine senescence. However, sEV contained very few soluble SASP factors. An interferon protein, IFITM3, was found to be enriched in sEV from senescent cells and partially responsible for inducing paracrine senescence (Borghesan, Fafian-Labora et al, 2019). Furthermore, we have unveiled that sEV mediate paracrine senescence via activation of the NF-kappa/IKK pathway (Fafian-Labora and O'Loghlen, 2021).

Interestingly, we have found that sEV isolated from young cells present anti-ageing and rejuvenation properties. Importantly, we found that sEV can act as independent metabolic units. Via the glutathione-S-transferase (GST) activity they can ameliorate different features of senescence and ageing both in vitro and in vivo (Figure 1B) (Fafian-Labora et al, 2020a, Fafian-Labora et al, 2020b, Melidoni 2020 and O'Loghlen, 2022).

Identifying senescence regulators is important to stratify cancer patients

Cells undergoing senescence present a characteristic stable cell cycle arrest; thus, senescence is considered to be an intrinsic tumour suppressor mechanism in vivo and in vitro. However, senescent cells display also what has been named as a "Senescence-Associated Secretory Phenotype" or SASP, which mediates both anti- and pro-tumourigenic properties in neighbouring cells. 

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. For this reason, we have performed a functional genome-wide CRISPR/Cas9 genetic screen to identify genes preventing the activation of senescence induced by CDK4/6 inhibitors (Figure 2).

Following this approach, our lab has identified two genes implicated in the coagulation pathway - the coagulation factor IX (F9) and Protein Z Vitamin K Dependent Plasma Glycoprotein (PROZ) - whose loss prevent the induction of senescence. We believe these markers could be used to predict patient response to CDK4/6 inhibitors improving the efficacy of cancer treatments and  overall disease outcome (Carpintero-Fernandez et al, 2022).

Integrins regulate the SASP during senescence

Ageing and cancer are currently a main concern of the developed countries. Although they are primarily different diseases, they both share a common characteristic - the activation of a cellular phenotype called senescence. Senescence is a cellular response to damage. It is characterised by a cell cycle arrest, the production of various secretion factors and the recruitment of inflammatory cells, resulting in tissue remodelling. However, the precise mechanisms for regulating senescence are not well characterised. 

Our lab has identified integrins as regulators of cellular senescence. Integrins are cell surface adhesion receptors formed by an “alpha“ and a “beta” subunit. They identify variations in the extracellular space and mediate intracellular signaling to adapt to changes in the environment. Our lab has characterised how the integrin alpha-v-beta 3 is upregulated during senescence (Figure 3) and induces a TGF-beta rich SASP. Surprisingly, the alpha-v-beta-3 antagonist, cilengitide, prevented the secretion of the SASP, without compromising the proliferative capacity of the senescent cells. Thus, suggesting that cilengitide could be used as an inflammatory chemical with no effect on altering the proliferative effect arrest induced during senescence. Furthermore, the ectopic expression of a vector encoding integrin beta 3 subunit (ITGB3), induces premature senescence in a fashion dependent on the p53/p21CIP pathways. Interestingly, ITGB3 is regulated by the Polycomb Repressive Complex 1 (PRC1) and in particular, the chromobox protein CBX7 regulates this locus during oncogene-induced senescence (by overexpression of H-RasG12V) (Rapisarda et al, 2017, Borghesan and O'Loghlen, 2017).

Integrins as biomarkers of ageing

The number elderly population in first world countries has dramatically increased in the last few decades. In fact, improved life expectancy is one the great triumphs over this last century. However, this comes with a cost, as conditions associated with ageing, such as cancer, cardiovascular and neurodegenerative diseases, are consequently increasing. It is therefore imperative to promote research that will improve our understanding of the mechanisms implicated in the maintenance of health across life. The primarily hallmark of ageing is the decline in the tissue homeostasis. However, the precise basic and cellular mechanisms and the need to identify biomarkers implicated in the ageing process are not well established, hindering the advances in understanding how healthy ageing happens. 

As our previous work shows that the integrin alpha-v-beta-3 regulates senescence, we have developed an interest in understanding whether this integrin could be used as a biomarker of ageing. In fact, we have shown that the endogenous expression levels of alpha-v-beta-3 is increased in fibroblasts derived from old donors in comparison with fibroblasts from young donors. Our results were also confirmed in a subset of tissues derived from old mice when compared to young mice (Figure 4). We are next interested in investigating whether other integrins are also deregulated during ageing and if they play an active role in ageing (Rapisarda et al, 2017, Borghesan and O'Loghlen, 2017).