Breast cancer (BC) is the condition wherein some cells in the breast develop abnormally, leading to uncontrolled cell division, and ultimately the formation of tumours. It can be a devastating condition for millions of patients and their families worldwide [1]. Overall, BC is the most commonly occurring cancer and the most frequently diagnosed cancer in women [2]. Despite advances in detection and improved treatment strategies, about 30% of patients with early-stage BC have recurrent disease [3]. The cancer cells in these women have already spread to distant sites by the time they are diagnosed. While overall cancer survival rates have improved, the survival rates for patients with metastases have not [4]. A growing problem encountered in the clinic are tumours that have intrinsic or acquired resistance to targeted therapies. It is thus essential to understand the mechanisms of drug resistance and identify therapies that prevent or bypass resistance.

The development of drug resistance accounts for up to 90% of BC deaths [3]. There are two predominant forms of resistance—intrinsic and acquired resistance, each accounting for 50% of patients with drug resistance [5].

Intrinsic resistance describes resistance that the patient possesses prior to exposure to drugs. This can be due to: (a) a pre-existing genetic mutation present in the tumour cells; (b) heterogeneity in tumours in which insensitive subclones are selected by drug treatment; or (c) activation of intrinsic pathways as defence mechanisms to certain drugs, such as drug efflux pumps, DNA damage repair (DDR) pathways and epigenetic modification.

Acquired resistance refers to the gradually declining efficacy of drug treatment. This can occur by: (a) activation of a second proto-oncogene that becomes the new driver gene; (b) mutation or altered expression levels of a drug target, rendering it insensitive to the drug; or (c) alterations of the tumour microenvironment (TME) [5].

Prolonged periods of targeted and chemotherapeutic treatment have been found to induce phenotypes resembling cellular senescence [47]. Cellular senescence is defined as a permanent or temporary cell cycle arrest. Senescent cells possess a number of hallmarks, including enhanced beta-galactosidase activity, senescence-associated secretory phenotype (SASP), cellular enlargement, and senescence associated heterochromatin foci (SAHF) [48–50]. SASP is characterized by a widespread change in protein expression in senescent cells, rendering it arrested in cell proliferation but still metabolically active.

Coppé et al. [51] have extensively characterized the SASP in human epithelial cells, and human and mouse fibroblasts. According to their work, the SASP can be classified into early events (alarm signals and mediators of tissue repair), and late events (anti-apoptotic proteins and inflammatory signals), which both negatively and positively regulate tumour progression, aging, and other pathologies. The wide impact of SASP can result in inhibition of cell division via genetic instability, oncogene inactivation, and promotion of immune clearance in cells [52].

Senescent cells also differ in shape and size to a normal cell. They show increased protein content, an elevation in the amount of anti-apoptotic proteins, increased lysosomal hydrolase activity [presented through senescence-associated β-galactosidase (SA-βGal)], DNA damage, and telomere associated foci (TAFs) [53]. Some notable factors associated with SASP expression changes include pro-inflammatory interleukins (ILs; IL-6, IL-7, IL-1α, IL-1β, IL-13, IL-15), chemokines (GRO-α, GRO-β, MCP-1, MCP-2, eotaxin-3), growth factors (EGF, bFGF, HGF, VEGF), proteases (MMP-1, MMP-3, MMP-10, MMP-12, MMP-13, MMP-14, capthesin B), and ECM elements like fibronectin and altered collagen (Figure 1) [51, 53].

Senescence is increasingly recognized as an important mechanism of therapeutic resistance. In the following sections, we will provide an overview of the current understanding of the role of senescence in BC.

Mitochondrial dysfunction correlates strongly with cellular senescence and is accompanied by accumulation of intracellular iron (a nearly 30-fold increase in intracellular iron) [83, 84].

Recently, Li et al. [84] reported a direct correlation between increased intracellular iron levels, mitochondrial dysfunction, and senescence. Intracellular iron assists in the generation of hydroxyl radicals through Fenton’s reaction. Fenton’s reaction involves the oxidation of ferrous iron to ferric iron in the presence of peroxide. It generates hydroxyl and hydroperoxyl radicals, which are unstable/reactive oxygen pollutants. With the critical role that iron plays in the generation of hydroxyl radicals, it is unsurprising that the induction of senescence impacts the expression levels of iron regulatory proteins like transferrin, transferrin receptor (TfR), and ferritin (Figure 2).

TfR is an integral membrane protein responsible for the uptake of iron and its expression is tightly controlled in normal cells. It transports iron-bound transferrin into the cell and is often overexpressed in BC to accommodate the increased iron demand for catalysis and biogenesis [85]. Elevated TfR levels have been observed in mouse embryonic fibroblasts induced to undergo IR-induced senescence [83, 86].

Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted protein that can bind iron and plays a role in intracellular iron trafficking in mammals. It is correlated with tumour progression and is overexpressed in BCs with poor prognosis [87]. NGAL has been identified as a SASP factor in etoposide-induced senescence BC and mouse models of spinal cord injury [88, 89]. Furthermore, NGAL is upregulated in BC cells exposed to conditioned media from TIS and OIS cells [90].

Ferritin is a protein complex that captures intracellular ferrous iron (Fe²⁺) and converts it into ferric iron (Fe³⁺) through intrinsic ferroxidase activity, in order to reduce intracellular damage caused by ROS (Figure 2) [91]. FTH1 (ferritin-heavy chain) is the active component of this protein complex and directly correlates with ferritin activity [92]. FTH1 levels are higher in senescent cells and protect against ferroptosis, an iron-dependent cell death pathway [83].

Ferroportin is a transmembrane exporter responsible for the release of Fe²⁺ from the cell. It is downregulated in BC cells as a means to retain a larger amount of iron inside the tumour cells [86]. High levels of ferroportin have been associated with impaired tumour progression [93], and senescent cells display deregulated expression and altered subcellular localization [94]. Ferroportin seems to localise to intracellular compartments away from the plasma membrane, likely preventing it from partaking in iron efflux [83]. This impaired localization suggests an elevation in inactive transporters in senescent cells, which is consistent with senescence associated lysosomal degradation.

Another protein involved in the regulation of oxidative stress and ferroptosis is the nuclear factor-E2-related factor 2 (Nrf2). Nrf2 is a key transcription factor that regulates intracellular oxidative stress damage, and in turn, ferroptosis. It is a basic Leucine Zipper (bZIP) transcription factor that regulates the expression of proteins such as glutathione S-transferase, heme oxygenase 1, and NADPH quinone oxidoreductase [95]. Nrf2 activation is regulated by Kelch-like ECH-associate protein 1 (Keap1). Keap1 binds to non-phosphorylated Nrf2 in the cytoplasm, promoting its ubiquitination and proteasomal degradation [96]. NF-κB, when stimulated by inhibitor of NF-κB kinase (IKK), activates Nrf2 via phosphorylation, leading to the dissociation of Keap1 and its translocation into the nucleus [94, 97]. This protein complex then binds to apoptotic/electrophilic response elements, promoting transcription of cytoprotective genes [96, 98–101]. TfR can activate IKK and thus play a role in NF-κB signaling and activation of Nrf2 [102]. With senescent cells demonstrating a strong anti-apoptotic tendency despite the expression of apoptotic proteins, the levels of pNrf2 are a prominent indicator of senescent activity. Interestingly, silencing of Nrf2 has been associated with premature replicative senescence in human embryonic fibroblasts [80, 98].

In conclusion, TIS impacts iron metabolism in BC cells and affects iron transporters such as NGAL and TfR. It also affects the iron carriers such as ferroportin and ferritin, and the corresponding stress response pathways [103].

A crucial element of membrane trafficking is the endosomal recycling pathway (ERP), which plays an important role in regulating the composition of the plasma membrane. This involves the internalization of cargo from the cell surface by a process called endocytosis. Endocytosed cargo is delivered to an intracellular organelle called the early endosome, where its fate is decided. It can either be returned to the cell surface via the ERP, or sent to lysosomes for degradation [104]. There are two main recycling pathways, based on the rate at which the process is carried out. In the fast recycling pathway, cargo is directly transported to the surface from the early endosome; in the slow recycling pathway the cargo is transported back to the plasma membrane via a perinuclear localized endosomal recycling compartment [105, 106].

A number of studies report that disruption of endocytosis results in the induction of cellular senescence [107–109]. For instance, the disruption of clathrin-mediated endocytosis (CME) caused by the destabilization of lysosomal membranes, results in DNA damage and changes in mitogenic signaling, leading to centrosome overduplication and induction of senescence [108].

There are a number of different mechanisms by which surface proteins can be internalized into the cell including CME, and caveolin-mediated endocytosis. Clathrin is a molecular scaffold that mediates the formation of vesicles at the plasma membrane [103], and caveolin is a cholesterol-binding protein that assists in the formation of cave-like structures in the plasma membrane, called caveolae [110].

The differences in the morphology and functional properties of senescent cancer cells have led to many efforts to identify vulnerabilities that might be exploited to treat drug resistant cancers. One of the reported benefits of senescence in cancer is the restriction in tumour progression. It has also been reported that senescent properties can spread from one cancer cell to another [62, 125].

The detrimental effects of senescence induction via therapy include the promotion of EMT and concomitant increase in invasiveness of pre-malignant epithelial cells through the IL-6 and CXCL8-dependant pathways [126].

The SASP provides an opportunity for development of a “one-two punch” strategy in treatment of cancers with the use of senotherapeutics, therapeutic agents that target cellular senescence. Senomorphics (also known as senostatics) are a class of molecules that suppress some senescent properties and may even reverse the senescent phenotype. The field of senotherapeutic research is a growing area of drug discovery that encompasses targeted approaches, reverse-pharmacology, drug repurposing and other methodologies [127].

Senolytics target and kill senescent cells [53, 127] (Figure 3). Senomorphics suppress SASP, and in turn, decrease inflammation. The drawback of using senomorphics is that SASP is critical for wound healing [53].

It was first hypothesised that senescent cells upregulate anti-apoptotic and pro-survival pathways. This was confirmed by the continued survival of these cells upon upregulation of pro-apoptotic pathways, indicating the presence of factors that can counteract this cell death pathway. This led to the development of senolytics, which specifically target these anti-apoptotic pathways in senescent cells [128, 129]. Senescent cell anti-apoptotic pathways (SCAPs) possess similar properties to the anti-apoptotic pathways in B cell lymphoma and leukocytic leukaemia cells, as they release tissue destructive proapoptotic factors, but avoid undergoing apoptosis themselves [130, 131]. Senolytic compounds were considered more potent if they could target multiple SCAP nodes at the same time, as targeting single-node SCAPs like BCL2 pathway inhibitors [e.g., N(ABT-263), A1331852, A1155463] tended to affect a restricted range of senescent cells, and also increase risk of toxicity through off-target effects such as thrombocytopenia and neutropenia [130–132]. Targeting multiple SCAPs would allow them to attack a wider range of senescent cell types [133, 134].

Recent studies have identified the COPI pathway as a promising senolytic target in drug therapy-induced senescent cells [135]. Disrupting COPB2, a component of the COPI complex, results in Golgi dispersal and induction of apoptosis in senescent cells, indicating that this transport pathway may be a vulnerability in senescent cells [135]. The guanine-nucleotide exchange factor GBF1 is required for the activation of Arf GTPases. Conventional GBF1 inhibitors like brefeldin A selectively killed cells undergoing OIS, however, they have poor pharmacological properties and are not clinically approved. N-myristoylation inhibitors (NMTi), a clinically approved treatment for parasitic protozoan infections, were shown to phenocopy COPI inhibitors and act as potent senolytics [135, 136]. These work by preventing the addition of a myristoyl group to the amino terminus of Arf GTPases. Lack of this key lipid modification leads to reduction in the levels of Arfs 1, 3, 5 and 6 and a corresponding downregulation of the COPI pathway.

Ideal senolytics should target several SCAPs, be administered orally, and be pre-approved by the United States Food and Drug Administration (FDA) [137]. A wide-ranging list of senotherapeutics have been identified to date, such as dasatinib, quercetin, fisetin, luteolin, curcumin and navitoclax, and are in varying stages of clinical development [128, 133, 134, 138–140].

This review explores the various facets of therapy resistance, and drug TIS stands out as a major drug resistance mechanism in BC. It involves the induction of irreversible cell cycle arrest and the development of SASP in cancer cells in response to chemotherapeutic agents and targeted therapies.

One of the key advantages of TIS is its potential to restrict tumour progression by halting the proliferation of cancer cells. However, in contrast to apoptosis, which results in cell death, senescent cells remain metabolically active and can influence the TME through the SASP. With both tumour suppressive and tumour promoting properties, senescence can be a double-edged sword in cancer treatment.

Challenges remain in effectively harnessing the therapeutic potential of TIS. Senescent cells can develop resistance to apoptosis, allowing them to persist within TMEs, and potentially contribute to disease relapse. Additionally, the SASP generated can have diverse impacts on tumour progression complicating therapeutic strategies.

Substantial effort is currently focussed on developing senotherapeutics which selectively target senescent cells, or modulate SASP. These include senolytics, which induce apoptosis in senescent cells, and senomorphics which suppress SASP. These represent a promising approach towards eliminating therapy-resistant cancer cells and enhancing treatment efficacy.

Several studies have explored the impact of senescence on iron metabolism, and the role that endocytosis of iron and iron transporters plays in this. These pathways could provide new targets for developing novel strategies to overcome drug TIS.

In conclusion, targeting drug TIS holds promise for overcoming drug resistance in BC. TIS has the potential to restrict tumour progression and enhance treatment efficacy through the induction of cell cycle arrest and modulating the TME. However, further research is required to better understand the impact of senescence on tumour progression and to optimize TIS-based therapeutic strategies while mitigating any potential adverse effects.