Senescence and its Role in Cancer
The illustration is divided into four parts, which will be explained below.
Part 1: What is senescence, its causes, and associated pathways?
Cellular senescence is a stable cell cycle arrest caused by various internal or external stimuli [1]. Senescence was first reported by Hayflick and Moorhead when they observed the failure of cells to divide after a certain number of doublings in primary cell culture [2]. This was attributed to the gradual telomere attrition over multiple divisions and termed replicative senescence [3]. Senescence can also be induced by other stimuli like oncogene activation, DNA damage, cancer therapy, dysfunctional telomeres, or reactive oxygen species (ROS) [4,5].
Irrespective of the stress stimulus, cell cycle arrest is mediated mainly by p21WAF1/CIP1 (CDKN1A) and p16INK4A (CDKN2A) pathways. Both lead to the hypo-phosphorylation of retinoblastoma protein (RB) which then inhibits the transactivation of the E2F genes involved in nucleotide metabolism and DNA synthesis, resulting in a stable cell-cycle arrest. One thing to keep in mind is that p53-mediated senescence can be reversed by inactivation of p53, but p16-mediated senescence once established cannot be reversed by inactivating p53, pRB, or both [6]. Thus, p16 is a more potent senescent marker. Also, senescence through either pathway cannot be reversed by removing the stress stimulus.
Some examples of pathway activation based on the stress signal are given below:
Oncogenes activate p53-p21 pathway by DNA damage through the ATM-ATR signaling, or without DNA damage through the ARF (CDKN2A locus) transcripts. They can also activate the p16-pRB pathway without DNA damage or p53 involvement. Loss of PTEN can activate the p53-p21 pathway through mTOR without DNA damage. Low doses of chemotherapy and radiotherapy trigger a senescent cell state in human cancer cells, while apoptosis is induced at higher doses [7]. Therapy causes DNA damage activating the p53-p21 pathway. Immunotherapy-induced senescence is still being explored. Dysfunctional telomeres activate p53-p21 axis by DNA damage through the ATM-ATR signaling. Epigenetic modulators can activate both the p53-p21 pathway through ARF without DNA damage or the p16-pRB pathway, whereas ROS activates the p53-p21 pathway through p38.
Part 2: What are the different characteristics or hallmarks of senescence?
Cell cycle arrest may not be the best way to fully define senescence as it is also seen in terminal differentiation and quiescence. Thus, senescence is also characterized by morphological changes, macromolecular damage, deregulated metabolism, and secretory phenotype [8]. Senescent cells (SnCs) exhibit flattened and enlarged cell morphology. They sometimes express surface markers like urokinase-type plasminogen activator receptor (uPAR) and β2 microglobulin (B2M). Cleaved uPAR, could potentially be used in non-invasive approaches to detect senescent cancer cells [9]. Senescence-associated heterochromatin foci (SAHF) form to various extents in SnCs as a gene repressing mechanism. The location of SAHF in the vicinity of E2F target genes imposes stable cell-cycle withdrawal [10]. Loss of the nuclear structural protein laminin B1 compromises the integrity of the nuclear envelope, leading to the production of cytoplasmic chromatin fragments (CCFs), which regulate the secretory phenotype of SnCs [11]. The senescence-associated Alarmin high mobility group box 1 (HMGB1) protein is exported from the nucleus to the extracellular milieu of senescent cells in a p53-dependent manner [12].
The metabolic changes include an increased number of lysosomes showing enhanced lysosomal β-galactosidase activity. This is detected using the senescence-associated β-galactosidase (SA-βGal) assay at pH 6 and is the most widely used marker for the detection of senescence although it is not specific to the senescence phenotype [13]. Lysosomes in SnCs also have higher levels of lipofuscin, composed of insoluble lipid-containing aggregates of lysosomal digestion [14]. Since the production of the senescent secretome is highly energy demanding, SnCs rely heavily on augmented mitochondrial metabolism and glycolysis to meet their ATP needs [15]. Anti-apoptotic pathways are upregulated in SnCs marked by elevated levels of anti-apoptotic BCL2 proteins and reduced levels of pro-apoptotic BCL2-associated protein X (BAX).
The metabolically active SnCs have a complex pro-inflammatory secretory phenotype, called the ‘senescence-associated secretory phenotype, or SASP. SASP includes a plethora of bioactive proteins, including chemokines, cytokines, growth factors, matrix metalloproteinases, and ECM proteins, whose identity is dependent on the cell and tissue type from which a senescent cell arises. SASP can have both protective and deleterious effects based on its origin [8,16,17]. E.g., IL-6 can recruit natural killer (NK) cells but also cause immune suppression and EMT [18,19]. In many ways, the SASP of senescent fibroblasts resembles that of fibroblasts undergoing a wounding response, which entails local remodeling of the tissue structure. It also resembles the secretory phenotype of cancer-associated fibroblasts (CAFs) that are components of the so-called reactive stroma, which facilitates the progression of epithelial cancers [20].
Transient senescence may play an important role in certain developmental or physiological conditions. E.g., transient senescence helps in wound healing [21] and limits fibrosis [22]. However, persistent damage or stress, or during aging, when SnCs accumulate and are not cleared, they lead to age-related disorders like lung fibrosis [23], atherosclerosis [24], diabetes mellitus [25], osteoarthritis [26], neurological disorders [27] and clearance of SnCs delays onset of age-related disorders [28].
Part 3: What is the role of senescent cells or SASP in cancer?
The senescence response may be antagonistically pleiotropic i.e., promoting early-life survival and preventing cancer but eventually limiting longevity and causing age-related diseases including cancer as SnCs accumulate [29]. Senescence was included as a hallmark of cancer in 2022 [30]. It has a dual role in cancer i.e., protective effects when cancer cells become transiently senescent [31] but also tumor-promoting effects when stromal SnCs accumulate in the tumor microenvironment (TME) [32]. Senescent fibroblasts seem to be involved in the active proliferation of preneoplastic epithelial cells [33,34]. Coinjection of breast cancer cells with senescent fibroblasts led to faster growth of cancer masses in an in vivo mouse xenograft model [35]. Senescent CAFs in tumors are tumor-promoting and transfer hallmark capabilities to cancer cells in the TME [16,17,32]. They remodel the TME via their SASP and provide paracrine support for local invasion and distant metastasis of neoplasias or prepare the microenvironment in the distant organ for tumor seeding [16]. Additionally, the SASP of senescent CAFs can recruit innate immune cells that suppress adaptive antitumoral immune responses mediated by CD8 T cells and promote skin tumor growth [18]. They can also induce angiogenesis through vascular endothelial growth factor (VEGF) secretion [36]. Senescent stromal cells including fibroblasts and endothelial cells can enhance proliferation, invasion, and metastasis in breast cancer models [37] and even local invasion and epithelial-to-mesenchymal transition (EMT) [38,39].
Although the SASP of senescent cancer cells is initially tumor suppressive, it is mostly detrimental in the long term. SASP factors can reinforce senescence in an autocrine fashion and influence the tissue microenvironment by paracrine signaling to the adjacent tumor, non-tumor, and immune cells. The positive or tumor-suppressive effects are that some of these cytokines, such as IL-6, IL-8 and CCL2, can recruit NK cells and T cells contributing to immune surveillance. In addition, IL-6 and IL-8 can reinforce cellular senescence in an autocrine fashion through increased ROS production and a sustained DNA damage response. These interleukins can spread senescence to surrounding cancer cells in a paracrine fashion, which further controls tumor growth. Through their SASP, senescent cancer cells can also arrest neighboring cancer cells, improve the vasculature for drug delivery and recruit immune cells that can contribute further to tumor suppression. As the negative or tumor-promoting effect, IL-6 either secreted by senescent cancer cells or released from the extracellular matrix (ECM) by MMPs recruits myeloid-derived suppressor cells (MDSCs), resulting in an immunosuppressive microenvironment. Moreover, the cleaved ECM components release growth factors that can promote further tumor growth and EMT, leading to metastasis. VEGF from the SASP stimulates blood vessel formation that also contributes to metastasis. As a result, it is presently very difficult to predict whether the effects of senescent cancer cells are pro-tumorigenic or anti-tumorigenic [36].
Part 4: How can senescent cells be targeted?
The elimination of SnCs or targeting of SASP is expected to improve patient prognosis and enhance anticancer treatment [40]. Depletion of p16INK4a SnCs in aging mice resulted in reduced tumorigenesis and cancer-associated death [28]. The agents that kill SnCs are called senolytics [40,41]. Examples of senolytics are dasatinib and quercetin (D+Q), Navitoclax or ABT263, an inhibitor of the Bcl-2 family, D-retro-inverso isoform of Foxo4 (Foxo4-DRI) and Hsp90 inhibitors.
Senomorphics, are another class of drugs being developed to suppress aging by targeting the SASP. Since SASPs are so diverse, targeting their master regulators, such as NF-κB and STAT3, has been considered. mTOR inhibition might reduce inflammation and the secretion of inflammatory cytokines by SnCs [42]. However, targeting SASP is a challenge as it can have both anti and pro-tumorigenic effects [17].
To achieve better antitumor responses and to inhibit tumor progression mediated by SASP factors, senescence-inducing therapies can be combined with senolytic treatments [36]. If senescent cancer cell-specific surface antigens are identified, they can be used to deliver immunomodulatory or cytotoxic payloads to senescent cancer cells. Modulating the immunosuppressive SASP can augment the effects of immune checkpoint inhibitors or other antitumor immunotherapies. The development of senescent cell-targeted nanoparticles capable of encapsulating various types of contents and senolytic CAR-T therapies with applications in solid tumors are being explored [43], however, with some challenges in translation.
Future scope and gaps
1. The overall effect of SnCs on cancer progression is still not fully understood due to their heterogeneity based on the time course of the disease and the induction mechanisms. A thorough understanding of SnCs considering cancer type and cancer stage is necessary.
2. There is an unmet need due to the lack of drugs that are highly effective in inducing senescence in a high proportion of cancer cells. Also, most senolytics have variable or off-target effects [44]. Targeting SASP is also challenging due to its dual effects [17].
3. There is a lack of gold standard biomarkers of the senescent state. No single marker can clearly discriminate between senescence and other growth-arrested states.
4. Ablation of SnCs by senolytic therapy in aged individuals needs to be carefully evaluated as there can be a high percentage of normal SnCs in some tissues and this might harm tissue structural integrity or affect vascular endothelial cells, leading to liver and perivascular tissue fibrosis and health collapse.
5. Irreversibility of senescence has been challenged. A small number of cancer cells can be made senescent by cytotoxic therapies but then revert to active proliferation, making senescence reversion a subject of intense debate.
6. Some researchers have used an in vivo on−off switch system, like p16INK4A-ATTAC, for inducible elimination of p16INK4a-positive senescent cells upon drug treatment and p16INK4A-3MR mice, a reporter-ablation mouse model to compare SnCs and non-SnCs, but they are not cell-type-specific [1].
7. Better and efficient tools for detecting senescent cells in vivo are crucial and the optimization of diagnostic probes and sensors capable of targeting SnCs specifically are currently limiting their translatability to human settings [45]. A deeper understanding of the triggers, underlying molecular mechanisms and signaling pathways, as well as how they behave in different cell types and tissues will facilitate the identification and prioritization of diagnostic and targetable biomarkers and the development of novel tools for the detection and monitoring of senescent cells.
8. Identification of specific, or differentially overexpressed, targetable surface markers at the level of the cell membrane (or senescent ‘surfaceome’) is needed to design next-generation senoprobes and antibody-drug conjugates for targeted drug delivery into SnCs. Only a few surface proteins are observed to be overexpressed in SnCs, and are more enriched in particular senescent subtypes rather than being widespread.
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