Inhibition of 3-phosphoinositide–dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts

Cellular senescence is defined as a stable, persistent arrest of cell proliferation. Here, we examine whether senescent cells can lose senescence hallmarks and reenter a reversible state of cell-cycle arrest (quiescence). We constructed a molecular regulatory network of cellular senescence based on previous experimental evidence. To infer the regulatory logic of the network, we performed phosphoprotein array experiments with normal human dermal fibroblasts and used the data to optimize the regulatory relationships between molecules with an evolutionary algorithm. From ensemble analysis of network models, we identified 3-phosphoinositide–dependent protein kinase 1 (PDK1) as a promising target for inhibitors to convert the senescent state to the quiescent state. We showed that inhibition of PDK1 in senescent human dermal fibroblasts eradicates senescence hallmarks and restores entry into the cell cycle by suppressing both nuclear factor κB and mTOR signaling, resulting in restored skin regeneration capacity. Our findings provide insight into a potential therapeutic strategy to treat age-related diseases associated with the accumulation of senescent cells.

Cellular senescence is defined as a stable, persistent exit from the cell cycle in response to stresses such as telomere shortening, oxidative stress, oncogene activation, and DNA damage (1, 2). A benefit of cellular senescence is prevention of tumorigenesis by blocking proliferation of damaged cells that may undergo malignant transformation (2, 3). However, senescent cells accumulate in tissues during aging and secrete proinflammatory cytokines, which can contribute to aging and age-related diseases, including cancer (2, 3). In studies with animal models, elimination of senescent cells prevents, alleviates, or reverses symptoms of aging (4, 5) and various age-related diseases (6, 7), such as osteoarthritis and atherosclerosis.Cell-cycle arrest alone is not cellular senescence; cellular senescence requires additional signals that convert transient cell-cycle arrest into persistent exit from the cell cycle so that the cells fail to proliferate in response to growth signals, a process called geroconversion (8, 9). Terminally differentiated, nonmitotic cells can also undergo senescence; thus, cell-cycle exit is only one aspect of the senescent phenotype. Cellular senescence is a complex biological mechanism regulated by various signaling pathways (10, 11). Signaling pathways that mediate cellular senescence can be divided into three major categories. The first category includes the pathways that cause cell-cycle arrest in response to DNA damage, such as p53/p21^CIP1 and p16^INK4a/pRb pathways (11–14). The second category consists of the pathways mediating cell growth and energy metabolism, such as PI3K/AKT/mTOR and SIRT1/AMPK pathways (15–18). Activation of mTOR in cells arrested by persistent DNA damage represents a second stimulus that can convert transiently arrested cells into senescent cells that exhibit hypertrophy and an expanded lysosomal compartment (19). The last category consists of the pathways mediating the senescence-associated secretory phenotype (SASP) (3, 10, 20). The SASP is a characteristic feature of senescent cells and reflects their secretion of proinflammatory cytokines and chemokines. These cytokines and chemokines maintain cellular senescence through positive autoregulatory feedbacks, affect nonsenescent nearby cells, and promote aging and age-related diseases, including cancer (3, 21). Nuclear factor κB (NF-κB) activity is important for SASP, and suppression of NF-κB prevents age-related diseases and delays aging in mice (22, 23).Spontaneous reversion from senescence to proliferation is extremely rare, but the reversion through manipulations is not. Some studies reported that senescent cells can reenter the cell cycle (24). The current understanding of senescence is as a dynamic multistep process that is reversible under some conditions (25). About 70 to 90% of cells with low p16^INK4a levels in replicative senescence, which is senescence related to the finite number of divisions a cell can perform before telomeres become too short, resume proliferation following p53 inactivation (26). Inactivation of p53 also enables cells to escape from therapy-induced senescence, caused by the chemotherapeutic agent Adriamycin (27). Cells with oncogene-induced senescence can also escape from the senescent state. For instance, about 50% of mouse embryo fibroblasts with high Ras levels reenter the cell cycle upon inactivation of all three Rb family members (28), and about 70% of the fibroblasts reenter upon activation of H3K9 demethylases (29).Here, we applied a systems biology approach to identify mechanisms underlying cell-cycle arrest, cell growth, and the SASP with the goal of finding inhibitable targets to convert the senescent state to the quiescent state. We studied normal human dermal fibroblasts (NHDFs), which can be experimentally induced into the senescent state (8). We constructed a molecular signaling network of cellular senescence using information in the literature and network databases to identify the relevant molecules, experimental data from time series of phosphorylated proteins in NHDFs to define the input–output relationships that reflect cellular states upon each input condition, and an evolutionary algorithm to determine the regulatory logic of the network (SI Appendix, Fig. S1 A–C). By analyzing the regulatory signaling network, we predicted that PDK1 was an inhibitor target that can convert senescent fibroblasts to quiescent fibroblasts (SI Appendix, Fig. S1D). To validate this prediction, we conducted experiments with NHDFs exposed to PDK1 inhibitors (SI Appendix, Fig. S1E), which eliminated hallmarks of cellular senescence, restored the proliferation of the cells in response to growth factors, and restored skin regeneration capacity in two-dimensional (2D) culture and a three-dimensional (3D) skin equivalent model. Our findings provide insight into a potential therapeutic strategy to treat aging and age-related diseases.