Integrating caspase-3 expression and activity in mammary tumor: prognostic significance and therapeutic implications

Abstract

Graphical abstract

Introduction

Breast cancer (BC) is the most commonly diagnosed malignancy in females worldwide and remains the leading cause of cancer-related death despite significant advances in early detection, systemic treatment and surgical techniques [1]. The world burden of the illness continues to rise, especially in low and middle financial gain countries, where treatment opportunities lead to poor outcomes. BC is not a single entity, but a highly heterogeneous disease comprising multiple molecular subtypes and histological subtypes, each with unique clinical trajectories and biological behavior [2].

The central role of caspase-3 in integrating intrinsic and extrinsic apoptotic signaling and its newly discovered non-apoptotic function in mammary tumor progression is summarized in (Fig. 1). A schematic three-dimensional overview illustrating the dysregulation of apoptotic signaling in mammary tumor. Intrinsic subtypes have been known based on hormone receptor status, progesterone receptor (PR), estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), and classification of gene expression profile. A defining feature of cancer development and progression is disruption of normal cellular homeostasis, in which apoptosis plays a central role. Apoptosis is a tightly regulated process critical for immune regulation, tissue development, and clearance of damaged cells [3]. In normal physiology, apoptosis checks a balance between cell proliferation and cell death. Apoptosis resistance is now recognized as one of the core markers of cancer, contributing not only to tumorigenesis but also to treatment resistance and disease progression. At the molecular level, apoptosis is first performed by caspase, a class of cysteine-aspartate proteases that exist in inactive zymogen form and are activated by proteolytic cleavage under specific stimuli [4]. Caspase-8, caspase-9 and effector caspase extrinsic apoptotic pathways are mediated by death ligands binding to their corresponding receptor that triggers activation of caspase-8. In contrast, intrinsic pathways are triggered by intracellular stress signals, leading to mitochondrial outer membrane permeability (MOMP). Both pathways converge on caspase-3, which is responsible for cleaving a variety of cellular substrates. The main executive caspase ultimately leads to the biochemical characteristics and morphological features of apoptosis, including membrane blistering, DNA fragmentation, and cell contraction [4]. Caspase-3 stimulation has historically been considered a marker of effective tumor suppression, reflecting the elimination of malignant cells through apoptosis. As a result, many anti-cancer therapies, including chemotherapy and targeted drugs, have been planned to induce apoptosis through caspase stimulation [5]. Incomplete activation of caspase-3 can lead to activation of signaling pathways that enhance angiogenesis, cell proliferation, and tissue regeneration, including migration, cell adhesion, and the regulation of inflammatory signals [6]. Weak regulation of Caspase-8 expression has been determined in certain types of tumors and is often associated with apoptosis resistance and adverse clinical outcomes. These findings further emphasize the need to reassess the role of caspase beyond its classical function. Adding to this complexity is the interaction between apoptotic pathways and cell cycle machinery. CDK2 is a key regulator of the G1/S phase and is frequently deregulated in BC [7]. In addition to its standard role in cell cycle progression, emerging evidence suggests that CDK2 directly regulates apoptotic signaling by phosphorylating specific serine residues of caspase-3, thereby inhibiting its proteolytic activity [8]. In the context of BC, abnormal CDK2 activity has been associated with adverse clinical outcomes and chemotherapy resistance, suggesting that the CDK2-caspase-3 mechanism represents a critical node linking apoptosis and proliferation [7]. The purpose of this review was to critically analyze the role of caspase-3 in BC, with specific attention to their non-classical functions, and classical, fundamental interaction with cell cycle machinery via CDK2, their contribution in metastasis, invasion, and their relevance as prognostic, diagnostic, and therapeutic targets. The review highlighted the informational opportunities that may advance human and veterinary oncology.

Fig. 1.

Complex roles of caspases in breast cancer. Schematic overview of apoptotic signaling dysregulation in breast cancer. Caspase-3, the central executioner caspase, integrates extrinsic and intrinsic pathways. Although canonical caspase-3 activation induces therapy-driven tumor cell death, context-dependent activity can also promote non-apoptotic processes such as tumor repopulation, migration, and therapy resistance.

Caspases in cancer biology

Classification and activation

Caspases play a central role in the regulation of apoptosis, inflammation, and are generally divided into three wide categories based on their function and structure. Initiator caspases typically have long prodomains containing motifs such as caspase recruitment domains (CARDs), which help their fundamental interaction with adaptor proteins. Activation of caspases happens through proteolytic cellular division at a specific aspartic acid portion, forming an active heterotetrameric enzyme composed of two large and two small subunits. The initiator caspase normally passes through a specialized signaling level rather than by cleavage only. Once activated, they activate downstream effector caspases; initiator caspase cleaves, amplifying apoptotic signaling in a cascade [4].

Intrinsic and extrinsic pathways

Intrinsic pathways are initiated by differences in intracellular stress signals, including oxidative stress, DNA damage, oncogene activation, and growth factor deprivation [3]. Substance binding generates receptor trimerization and adaptor protein recruitment of Fas-associated death domains (FADDs) leads to the gathering of death-inducing signaling complexes (DISCs) in which procaspase-8 is activated through dimerization, recruitment and cleavage. Activated caspase-8 can activate effector caspase and directly cleave, in some cell types, participate in intrinsic pathways, thereby amplifying apoptotic signaling [4].

Non-apoptotic functions

Caspases are increasingly recognized as regulators of a variety of non-apoptotic processes. These include proliferation, cell differentiation, migration, cytoskeletal remodeling, and immune responses [5,9]. Caspase-3 in particular has emerged as a critical mediator of non-apoptotic signaling in cancer. Sublethal activation of caspase-3 may occur during recovery from mild cytotoxic therapy. After treatment, one of the most clinically relevant non-apoptotic effects of Caspase-3 is its engagement in tumor regeneration. It has been proposed that ending tumor cells undergoing caspase-3 activation can release pro-growth factors that advance the proliferation of surviving cancer cells, a phenomenon sometimes mentioned as the “phoenix rise” pathway [5,9].

“Phoenix Rising” mechanism provides a biological statement for its adverse prognostic significance, as summarized in (Fig. 2). The key experimental and clinical studies evaluating caspase-3 expression, biological function, and prognostic significance in tumors are summarized in Table 1. This table integrates findings from mechanistic investigations demonstrating that, although caspase-3 is necessary for apoptosis, its high expression and sublethal activation are frequently associated with aggressive tumor behavior and poor clinical outcomes in mammary tumors.

Fig. 2.

Paradoxical roles of caspase-3 in breast cancer. Caspase-3 acts as the principal executioner of apoptosis, essential for therapy-induced tumor cell death. However, context-dependent activation can promote tumor repopulation, invasion, and therapy resistance via non-apoptotic signaling pathways, including the “Phoenix Rising” mechanism, explaining its adverse prognostic significance.

Table 1.

Expression, biological roles, and clinical significance of caspase-3 in breast cancer.

Study type	Cohort/model	Methodology	Caspase-3 status	Key biological findings	Clinicopathological associations	Survival outcome	Main conclusion	Study
Clinical	107 breast tumors	IHC	Expressed in majority of tumors	Caspase-3 present despite apoptosis resistance	Higher expression in invasive tumors	Not evaluated	Caspase-3 expression does not guarantee apoptosis	[29]
Clinical	123 breast cancer patients	IHC	High expression	Elevated caspase-3 reflects aggressive biology	Associated with high-grade and nodal positivity	Poor OS and DFS	Caspase-3 is an adverse prognostic marker	[30]
Experimental	MCF-7 cell line	Western blot, transfection	Absent → restored	Caspase-3 loss confers chemoresistance	Not applicable	Not applicable	Caspase-3 is required for therapy-induced apoptosis	[31]
Clinical	85 ductal carcinomas	IHC	High expression	Caspase-3 is localized to the cytoplasm	Correlates with higher tumor grade	Not evaluated	Caspase-3 linked to tumor aggressiveness	[32]
Experimental	Mouse models, tumor cells	Functional assays	Activated (sublethal)	Caspase-3 induces PGE₂ release	Promotes tumor repopulation	Not applicable	Caspase-3 drives non-apoptotic tumor regrowth	[33]
Clinical (large cohort)	1902 breast cancers (Nottingham)	IHC + survival analysis	High protein expression	Caspase-3 is involved in non-apoptotic signaling	Stronger effect in ER-positive tumors	Independent poor BC-specific survival	Caspase-3 is a robust adverse prognostic biomarker	[34]
Mechanistic review	Multiple cancer models	Functional studies	Sublethal activation	Promotes migration and invasion	Linked to metastatic potential	Not applicable	Caspase-3 has pro-tumorigenic roles	[35]
Clinical	Breast cancer cohort	IHC (caspase-3 + calpain)	Co-expression	Caspase-calpain synergy	High invasive potential	Poor survival when co-expressed	Protease networks refine prognosis	[36]

This process may contribute to tumor recurrence after chemotherapy, highlighting the paradox that apoptosis-inducing treatments may inadvertently cause tumor regeneration. Caspase-3 is one of the most widely studied effector caspases in BC and is widely expressed in different tumor subtypes. Many studies have failed to distinguish between full-length (inactive) procaspase-3 and its cleaved (active) form, leading to ambiguity in determining whether observed expression reflects apoptotic activity or merely the presence of the protein [10]. Other sources of incompatibility include variation in scoring systems, inter-observer variation, and cohort heterogeneity [11]. Failure to stratify patients by molecular subtype may further mask subtype-specific activity [6,11].

Prognostic significance

The prognostic significance of Caspase-3 expression in BC remains controversial. Several large cohort analyses have demonstrated a contradictory association between high caspase-3 expression and adverse clinical outcomes, including increased risk of recurrence and reduced overall survival. This adverse prognostic significance is particularly evident in hormone receptor-positive and non-basal-like subtypes [6]. Contradictory findings between studies may stem from a variety of factors, including variation in study design, patient populations, and analytical methods [6,11].

Mechanism underlying the paradox

The contradictory relationship between Caspase-3 expression and adverse clinical outcomes prompted research worker to explore mechanisms that separate caspase-3 expression from its functional activity. One prominent mechanism involves post-translational change of CDK2. CDK2 directly phosphorylates the serine 150 position of caspase-3, reducing its enzymatic activity and delaying the performance of apoptosis [8]. This phosphorylation event alters cells to maintain high levels of caspase-3 protein without undergoing efficient apoptosis, thereby decoupling expression from function. Despite convincing experimental information, the clinical relevance of this mechanism in human breast cancer has not been fully proved [9]. Together, these two mechanisms - posttranslational suppression of CDK2 and non-apoptotic signaling - explain how high caspase-3 expression coexists with treatment resistance and poor prognosis.

Therapeutic significance of caspase-3 mediates

Chemotherapy, radiotherapy and targeted drugs are various anti-cancer therapies, but loss of caspase-3 function is connected with treatment resistance. However, high expression does not necessarily predict a better response, which may reflect the dual role of caspase-3 in promoting apoptosis and non-apoptotic processes that support tumor survival and regeneration [12]. Significantly, CDK2-mediated inactivation of caspase-3 may be a key mechanism of treatment resistance, suggesting that a combination of cytotoxic drugs with CDK2 inhibitors may restore apoptotic sensitivity [7,8].

Comparative analysis of caspase-3 and caspase-8 in human and canine breast tumors

Theoretical foundations of comparative oncology

Comparative oncology has become a powerful approach to understanding cancer biology by integrating cross-species discovery. Canine breast tumors (CMTs) share significant similarities with human breast cancer (HBC) in clinical, histopathological, and molecular features, making them a valuable spontaneous model for translational studies [13,14]. Both diseases exhibit comparable hormonal effects, metastatic patterns, molecular subtypes, and response to treatment [15]. Importantly, dysregulation of apoptotic pathways, including altered caspase signaling, has been observed in HBC and CMTs, supporting correlations across species comparisons [16]. In addition, CDK2 dysregulation is well documented in both human and canine tumors, making the CDK2-caspase-3 axis a promising target for cross-species transformation studies [16].

Caspase-3 expression in HBC

In HBC, early studies have shown that caspase-3 quality reflects preserved apoptotic capability; however, more recent large cohort studies have demonstrated a contradictory relationship between poor clinical outcomes and high caspase-3 expression. This poor prognostic significance is particularly noticeable in hormone receptor-positive and non-basal-like subtypes [6]. Mechanically, raised caspase-3 expression is related to non-apoptotic signaling pathways, including enhanced tumor cell survival, and apoptosis-induced proliferation [5]. In addition, CDK2-mediated phosphorylation provides a direct molecular mechanism for caspase-3 inactivation despite high expression levels [8]. Caspase-3 expression in CMTs has also been studied, with results similar to and various from human studies. Immunohistochemical analysis displays that caspase-3 is expressed in both benign and malignant canine breast tissues, and higher expression is often determined in malignant tumors [17,18]. Several canine model studies have suggested that increased caspase-3 expression may be associated with higher apoptotic indices and, in some cases, less aggressive tumor behavior compared to HBC [17]. However, other reports have pointed to associations with high-grade tumors and metastatic potential, highlighting inconsistencies similar to those observed in human studies [19]. The role of CDK2 in the regulation of caspase-3 activity in CMTs remains an important area for future research.

Caspase-8 plays a central role in extrinsic apoptotic signaling and immune surveillance in HBC and CMTs

In HBC, caspase-8 expression is mostly maintained and does not display strong independent prognostic significance [6]. In CMTs, data on caspase-8 are more limited. Existing studies have shown caspase-8 expression but have not shown a clear association with clinical outcome or tumor progression [16]. This interspecies similarity suggests that caspase-8 may play a conserved biological role in apoptosis rather than as a robust prognostic biomarker.

Cell cycle-apoptosis crosstalk across species

The fundamental interaction between CDK2 and caspase-3 represents an important regulatory mechanism in HBC and CMTs. CDK2 overexpression is associated with increased tumor proliferation, poor prognosis, and treatment resistance in both species [16]. In HBC, elevated CDK2 activity is associated with reduced caspase-3 activation and attenuated apoptotic response to chemotherapy [8]. Although less research has been done on CDK2, caspase-3 interactions in CMTs, emerging evidence suggests that similar regulatory networks may be involved in tumor progression [16]. This cross-species conservation supports the use of canine models to study cell cycle-apoptosis crosstalk and evaluate CDK-targeted therapies. CDK2-Caspase-3 crosstalk Cyclin-dependent kinase 2 (CDK2) is a serine/threonine kinase that plays a central role in cell cycle progression, particularly during the G1/S transition. CDK2 forms complexes with cyclin E or cyclin A, phosphorylates key substrates such as retinoblastoma protein (Rb), and promotes the expression of genes required for DNA replication [7]. In addition to its established function in cell cycle control, CDK2 is a direct regulator of apoptosis through its interaction with caspase-3. Biochemical studies have shown that CDK2 phosphorylates serine 150 of caspase-3, a modification that reduces its catalytic activity and delays the initiation of apoptosis [8]. This phosphorylation effect establishes a functional link between proliferation and survival: cells with high CDK2 activity are not only driven to divide but also less sensitive to apoptotic stimuli. In breast cancer, this mechanism may explain the coexistence of high caspase-3 expression with adverse clinical outcomes, as elevated caspase-3 protein levels do not necessarily reflect functional apoptotic capacity. The clinical relevance of CDK2-caspase-3 crosstalk is supported by studies that suggest that CDK2 overexpression is associated with resistance to chemotherapy and radiotherapy in breast cancer patients [7,12]. In addition, pharmacological inhibition of CDK2 has been shown to enhance caspase-3 activation and sensitize breast cancer cells to cytotoxic therapies [8].

Caspase-3, CDK2 and metastasis

Metastasis is the leading cause of death in cancer patients and involves a complex cascade of events: endosmosis, survival in circulation, local invasion, and distant organ colonization [20]. Emerging evidence suggests that both caspase-3 and CDK2 play critical roles beyond the classical functions of apoptosis and cell cycle regulation, and are involved in multiple steps of the metastatic cascade.

Non-apoptotic function of caspase-3 in invasion and migration

Caspase-3 is mainly considered an effector of the extrinsic apoptotic pathway; however, spatially limited activation can upgrade cell migration and invasion. This contradictory effect is mediated by cleavage of specific substrates involved in cytoskeletal dynamics and cell adhesion. Caspase-3-mediated cleavage of focal adhesion kinase (FAK) and paxillin produces fragments that promote cytoskeletal reorganization and enhance cell mobility [21]. Sublethal caspase-3 activation stimulates the Rho-associated protein kinase (ROCK) pathway, resulting in increased actomyosin contractility and cell migration [22]. Caspase-3 cleaves E-cadherin, disrupting cell–cell junctions, facilitating detachment from primary tumors and promoting invasion [23]. In breast cancer, high caspase-3 expression is related to lymph node metastasis and distant repetition, supporting the concept that caspase-3 is involved in metastatic progression independently of its apoptotic function [6,11].

CDK2 in epithelial-mesenchymal transition and invasion

CDK2 has emerged as a key regulator of epithelial-mesenchymal transition (EMT), the process by which epithelial cells acquire mesenchymal characteristics and invasive capabilities. CDK2 directly phosphorylates and stabilizes Snail and Twist, master regulators of EMT, inhibiting E-cadherin expression and inducing stromal markers such as vimentin and N-cadherin [24]. CDK2 activity correlates with increased expression of MMP-2 and MMP-9, enzymes that degrade extracellular matrix components and promote tissue invasion [25]. CDK2 enhances transforming growth factor-beta (TGF-β)-induced EMT by promoting Smad-mediated transcription [26]. Clinical studies have shown that CDK2 overexpression is associated with high tumor grade, lymphovascular invasion, and poor metastasis-free survival in breast cancer patients [27].

Role of CDK2-caspase-3 axis in metastatic progression

The convergence of CDK2 and caspase-3 signaling forms a powerful pro-metastatic network. Table 2 summarizes the three main mechanisms by which the CDK2-caspase-3 axis promotes metastatic progression of breast cancer.

Table 2.

Mechanisms by which CDK2-Caspase-3 axis promotes metastatic progression.

Mechanism	Description	Clinical relevance
Apoptosis evasion	CDK2-mediated phosphorylation inactivates caspase-3, allowing circulating tumor cells (CTCs) to survive anoikis (detachment-induced apoptosis) [8,29]	CTCs with high CDK2 activity are more likely to survive and form distant metastases
Enhanced migration	Sublethal caspase-3 activation promotes cytoskeletal remodeling, while CDK2 drives epithelial-mesenchymal transition (EMT) and matrix metalloproteinase (MMP) expression [21,25]	Co-expression of CDK2 and caspase-3 correlates with an invasive phenotype and lymph node metastasis
Therapeutic resistance	CDK2-mediated caspase-3 inactivation reduces sensitivity to chemotherapy and radiotherapy, enabling metastatic outgrowth [7,12]	Patients with high CDK2 expression and low active caspase-3 have worse clinical outcomes and increased risk of recurrence

Comparative insights from CMTs

CMTs provide a valuable model for studying metastatic progression. Similar to HBC, high CDK2 expression in CMTs is associated with lymph node metastasis and poor prognosis [16]. In addition, caspase-3 expression patterns in CMTs have been shown to be related to high-grade tumors and metastatic potential, although results remain consistent [17,19]. The improvement of these pathways across species supports the use of canine models to evaluate antimetastatic strategies targeting the CDK2-caspase-3 axis.

Therapeutic implications for preventing metastasis

Targeting the CDK2-caspase-3 axis may provide opportunities to delay metastasis. CDK2 selective inhibitors under development may simultaneously inhibit proliferation, reverse EMT, and restore caspase-3 sensitivity, thereby reducing metastatic potential [24]. Combining CDK2 inhibitors with chemotherapy enhances apoptotic clearance and prevents metastatic growth. One of the major challenges is that caspase expression does not necessarily correlate with enzyme activity [17]. The discovery that CDK2-mediated phosphorylation can inactivate caspase-3 without altering its expression level provides a mechanistic explanation for this difference [8]. These methods include the use of fluorescent substrates, activity-based probes, and cleavage-specific antibodies to detect the active form of caspase [28]. Combining these functional assays with assessments of upstream regulators such as CDK2 activity may improve prognostic accuracy. Targeting the CDK2-caspase-3 axis is a promising strategy from a therapeutic perspective. More selective CDK2 inhibitors are currently in clinical development and may provide opportunities to restore caspase-3 sensitivity in treatment-resistant tumors [8]. Combined strategies targeting both cell cycle progression and apoptosis resistance may produce synergistic effects. In addition, there is a growing recognition that a single biomarker is insufficient to capture the complexity of cancer biology. Multi-marker approaches, such as CDK2, cyclin, and checkpoint regulators, as well as EMT combined with invasion markers, may provide a more comprehensive understanding of tumor behavior [7,8,24].

Limitations and future directions

Despite significant development in understanding caspase biology, several limitations have hindered progress. A major situation is the lack of standardized methods for assessing caspase expression, activity, and post-translational modifications. Differences in experimental techniques, including differences in antibodies, scoring systems, and detection conditions, have led to inconsistent results [10]. Another important limitation is reliance on in vitro and preclinical models, which may not fully capture the complexity of human tumors. In particular, the clinical significance of the CDK2-caspase-3 axis and its role in metastasis need to be further verified in a large, well-characterized patient cohort, and correlation analysis between CDK2 activity, caspase-3 phosphorylation status and clinical outcomes (including metastatic recurrence) should be performed [7,8]. Future research should be prioritized; 1. Clinical evaluation of CDK2 inhibitors as antimetastatic agents and chemosensitizers, 2. Characterization of CDK2-caspase-3 axis in circulating tumor cells, 3. Development of phosphorylation-specific antibodies to detect inactive (phosphorylated) versus active caspase-3 in clinical specimens, and Integration of cell cycle, apoptosis and metastasis markers in a multiparametric prognostic model.

Conclusion

Caspase-3 represents a context-dependent biomarker and complex in breast cancer, whose role goes beyond its classical purpose as an extrinsic of apoptosis. Although traditionally associated with tumor suppression, there is increasing evidence that caspase-3 function is regulated by posttranslational modifications, particularly phosphorylation of CDK2, which can inhibit its activity without decreasing expression levels.

CRediT authorship contribution statement

All authors contributed to the study conception and design. FN conceptualized the research project; HN developed the methodology; FN conducted data collection; HN analyzed the data; FN created visualizations; and HN reviewed, edited, approved, and contributed to the final manuscript for publication.

Ethics statement

This study did not involve human participants, animal subjects, or any biological samples requiring ethical approval. Therefore, ethics committee approval was not applicable.

Funding

This study did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sector.

Acknowledgments

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.