Unbiased analysis of senescence associated secretory phenotype (SASP) to identify common components following different genotoxic stresses

Abstract

Senescent cells secrete senescence-associated secretory phenotype (SASP) proteins to carry out several functions, such as sensitizing surrounding cells to senesce; immunomodulation; impairing or fostering cancer growth; and promoting tissue development. Identifying secreted factors that achieve such tasks is a challenging issue since the profile of secreted proteins depends on genotoxic stress and cell type. Currently, researchers are trying to identify common markers for SASP. The present investigation compared the secretome composition of five different senescent phenotypes in two different cell types: bone marrow and adipose mesenchymal stromal cells (MSC). We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation, and replicative exhaustion. We took advantage of LC-MS/MS proteome identification and subsequent gene ontology (GO) evaluation to perform an unbiased analysis (hypothesis free manner) of senescent secretomes. GO analysis allowed us to distribute SASP components into four classes: extracellular matrix/cytoskeleton/cell junctions; metabolic processes; ox-redox factors; and regulators of gene expression. We used Ingenuity Pathway Analysis (IPA) to determine common pathways among the different senescent phenotypes. This investigation, along with identification of eleven proteins that were exclusively expressed in all the analyzed senescent phenotypes, permitted the identification of three key signaling paths: MMP2 - TIMP2; IGFBP3 - PAI-1; and Peroxiredoxin 6 - ERP46 - PARK7 - Cathepsin D - Major vault protein. We suggest that these paths could be involved in the paracrine circuit that induces senescence in neighboring cells and may confer apoptosis resistance to senescent cells.

Keywords: mesenchymal stem cells; secretome; senescence.

Figure 1. Induction of senescence in MSC cultures

Left: Representative microscopic fields of acid beta-galactosidase (blue) in treated and control cells are shown. The histogram shows mean percentage value of senescent cells (± SD, n = 3, *p < 0.05; p**<0.01). Right: The graph shows the number of proteins found in secretomes of senescent A MSC and BM MSC. DOXO and H2O2 indicate the doxorubicin- and peroxide-treated MSCs Low (40 mGy) and high (2000 mGy) dose irradiated cells were indicated as IRL and IRH, respectively. Replicative senescent MSCs were called REPs.

Figure 2. Common canonical pathways

The figure shows the pathways common to senescent secretomes. Pathways were grouped according to the classes we identified in the GO analysis. For every experimental condition indicated in the figure with colored dots, IPA analysis allowed the identification of paths that belonged to overlapping networks.

Figure 3. Circuits that mediate paracrine interactions between senescent cells and surrounding environment

According to experimental results and literature data we propose: MMP2 and TIMP2 may play a key role in pathways regulating the effect of senescent secretomes on cancer cells; IGFBP3 and PAI-1 are part of the paracrine circuit that induces senescence in neighboring cells; and PRDX6, ERP46, PARK7, Cathepsin D, and MVP could be part of the common circuit that allows senescent cell survival following stress.