SB431542 is a selective inhibitor of the TGF-$\beta$ type I receptor (ALK5), which effectively blocks the TGF-$\beta$/Smad2/3 signaling cascade [1]. SB431542 has been widely used in studies of cell differentiation and reprogramming. It facilitates the maintenance of pluripotency and promotes directed differentiation by modulating TGF-$\beta$ signaling, as demonstrated in embryonic stem cells [2, 3]. In the context of cancer biology, SB431542 has been used to study the role of TGF-$\beta$ signaling in tumor progression and epithelial-to-mesenchymal transition (EMT). For example, Halder et al. [4] showed that SB431542-mediated inhibition of TGF-$\beta$ suppresses EMT and reduces invasiveness in mammary tumor cells. Beyond its classical roles, SB431542 has also been shown to modulate transcription factor networks. Notably, it induces TAp63 expression in human embryonic stem cells [5]. Furthermore, a recent study demonstrated that SB431542 upregulates TAp63 in HaCaT keratinocytes carrying two mutant p53 alleles (R282Q and H179Y), highlighting its broader role in activating the p63 pathway under mutant p53 conditions [6].

p63 exists in multiple isoforms, some with transactivation domains (TAp63) and others without ($\mathrm{\Delta }$Np63), which have differing or even opposing functions [7, 8]. TAp63, a transactivation-competent isoform of p63, functions as a tumor suppressor and shares structural and functional similarities with p53, a member of the p53 family with an N-terminal transactivation domain that can induce canonical p53 target genes [9, 10]. TAp63 plays a critical role in inducing cell cycle arrest, apoptosis, and the DNA damage response in p53-mutant or dysfunctional cells and exhibits p53-like tumor-suppressive activity [11]. Restoration or activation of TAp63 has been shown to suppress tumor growth and metastasis in mouse models by inducing senescence and activating DNA repair genes [12]. Therefore, pharmacological activation of TAp63 represents a promising therapeutic strategy for mutant p53 tumors [13, 14]. Studies indicate that doxorubicin induces TAp63 expression in some cancer cell lines, suggesting a compensatory tumor suppressor role in the absence of wild-type p53 [15, 16]. Specifically, TAp63 enhances chemosensitivity by promoting apoptosis in response to doxorubicin, thereby inhibiting tumor progression [16].

In this research, we investigated the potential anticancer effects of SB431542, a selective ALK5 inhibitor, in combination with doxorubicin in mutant p53 breast cancer cells. SB431542 effectively inhibits TGF$\beta$/Smad signaling, which plays a central role in tumor progression and metastasis [4]. Moreover, doxorubicin, while a cornerstone of breast cancer therapy, is often limited by resistance and cardiotoxicity [17, 18, 19]. We hypothesized that SB431542 would enhance doxorubicin efficacy in mutant p53 cells, which frequently exhibit chemoresistance [20]. This approach has the potential to overcome drug resistance and improve outcomes in aggressive breast cancers.

Our findings demonstrate that SB431542 enhances the cytotoxic and apoptotic effects of doxorubicin in breast cancer cells harboring mutant p53, particularly through mechanisms involving TAp63 activation. In p53-mutated breast cancer cells, combination treatment with SB431542 and doxorubicin significantly reduced cell viability, as evidenced by CCK-8 assay results (Fig. 2), and yielded a CDI value of 0.54–0.63 (Table 1), indicating a synergistic interaction. Apoptosis was markedly increased in both MDA-MB-231 and T47D cells upon combination treatment, as indicated by enhanced caspase-3/7 activity (Figs. 3,4). These results are consistent with prior reports that TGF-$\beta$ pathway inhibition can sensitize cancer cells to chemotherapeutic agents by enhancing apoptotic signaling [30].

Mechanistically, our data suggest that the enhanced apoptosis induced by SB431542 and doxorubicin is at least partially mediated through the activation of p63. Immunofluorescence staining revealed that p63 expression was upregulated in T47D and MDA-MB-231 cells treated with either SB431542 or doxorubicin, with further enhancement observed upon combination treatment (Figs. 5,6). Since TAp63 has been shown to promote apoptosis, cell cycle arrest, and DNA damage response in p53-deficient contexts [16, 31], these findings support the hypothesis that SB431542 may enhance doxorubicin-induced cytotoxicity by upregulating TAp63. This is further supported by the reversal of cytotoxicity upon co-treatment with PET$\alpha$, a p53 family inhibitor (Fig. 7), implicating the p53 family, likely TAp63, as a key mediator of the observed antitumor effects.

Our results also indicate that apoptosis is the primary mode of cell death induced by the combination treatment, as evidenced by caspase dependency. Co-treatment with a pan-caspase inhibitor (Z-VAD-FMK) significantly attenuated the cytotoxic effect of SB431542 and doxorubicin in both T47D and MDA-MB-468 cells (Fig. 8), consistent with caspase-3/7 activation data. This suggests that the observed cell death is caspase-mediated and apoptotic in nature. Previous studies have demonstrated that activation of TAp63 can trigger mitochondrial apoptosis pathways, particularly in the context of p53 mutation or loss [16]. Our findings align with these observations and extend them by identifying a novel combination—SB431542 plus doxorubicin—that effectively activates this alternative apoptotic pathway in mutant p53 breast cancer models.

This research provides compelling evidence that SB431542 enhances the antitumor efficacy of doxorubicin in breast cancer cells with mutant p53, through a mechanism that involves activation of TAp63 and caspase-mediated apoptosis. This is of clinical interest as TP53 mutations are present in over 30% of breast cancers, particularly in aggressive subtypes such as triple-negative breast cancer (TNBC), and are often associated with poor response to chemotherapy and increased recurrence [32]. Targeting the function of mutant p53 may pave the way for novel treatments in aggressive TNBC [33]. In this study, two cell lines—MDA-MB-231, and MDA-MB-468—were TNBC cells. This means our findings are particularly relevant to TNBC, a breast cancer subtype that currently lacks targeted therapies and is often resistant to conventional chemotherapy. The observed enhancement of doxorubicin efficacy by SB431542 suggests that this combination could represent a promising therapeutic strategy specifically for TNBC patients harboring TP53 mutations. By activating alternative tumor-suppressive pathways such as TAp63 and promoting caspase-mediated apoptosis, this approach may help overcome the limitations imposed by mutant p53 and improve clinical outcomes in this challenging breast cancer subtype.

A key mechanistic insight from our work is the observed upregulation of p63 expression, both at baseline in SB431542-treated cells and in combination with doxorubicin (Figs. 5,6). TAp63, a structural homolog of p53, has been shown to compensate for p53 loss by activating similar downstream pro-apoptotic targets such as BAX, PUMA, and NOXA [34, 35]. The reversal of cytotoxicity upon treatment with the p53 family inhibitor PET$\alpha$ (Fig. 7) further confirms the central role of p63 in mediating this chemosensitizing effect. Given that doxorubicin also induces p63 expression, it is plausible that SB431542 acts as a TAp63 amplifier, enabling more robust transcriptional activation of apoptotic genes.

T47D (harboring the p53 L194F mutation) and MDA-MB-231 (with the p53 R280K mutation) breast cancer cell lines carry aggregated forms of p53 mutations [36]. Both cell lines exhibited higher resistance to the chemotherapeutic drug doxorubicin than MDA-MB-468 cells, which carry the non-aggregated p53 R273H mutation and demonstrated a better response to doxorubicin [37]. Our combination treatment produced significantly synergistic effects in all three cell lines, as indicated by coefficient of drug interaction (CDI) values below 0.7. This suggests a synergistic interaction regardless of the p53 mutation aggregation status. Therefore, this drug pairing shows potential therapeutic benefit in breast cancers with either aggregated or non-aggregated types of p53 mutations.

In addition, emerging evidence suggests that TGF-$\beta$ pathway inhibition may also indirectly modulate the tumor immune microenvironment. Although our current in vitro data do not directly address immune interactions, the TGF-$\beta$ blocking antibody or SB431542 has been reported to enhance anti-tumor immunity in vivo by reducing immunosuppressive signaling and promoting cytotoxic T-cell activity [38, 39, 40]. Therefore, combining SB431542 with doxorubicin may yield both direct cytotoxic effects and immunomodulatory benefits, which could be further explored in immunocompetent animal models.

Further analysis shows that p63 expression is consistently and markedly reduced in breast cancer compared with normal tissue (Fig. 10B). Interestingly, this pronounced downregulation was highly cancer-type specific, occurring prominently in breast cancer and prostate adenocarcinoma but not in most other malignant tissues (Fig. 10A), indicating a context-dependent regulatory function.

Within breast cancer, lower p63 expression was strongly associated with more aggressive clinicopathological features, including HER2-positive and triple-negative subtypes (Fig. 10C). These subtypes are characterized by higher proliferative indices and poorer outcomes, suggesting that loss of p63 may contribute to tumor progression or reflect dedifferentiation of epithelial cells. Similarly, the decrease in p63 expression observed in stage IV disease supports the notion that p63 reduction may correlate with metastatic capability or advanced tumor biology (Fig. 10D).

A particularly notable finding is the association between p53 mutation and reduced p63 expression in breast cancer (Fig. 10E). We demonstrated that p63 expression can be successfully restored in TP53-mutant breast cancer cells using SB431542, particularly in combination with doxorubicin. This pharmacologic induction of p63 suggests that, despite the presence of mutant p53, the TAp63 pathway remains responsive to external modulation. The ability of SB431542 plus doxorubicin to elevate p63 levels and reactivate downstream apoptotic signaling highlights a potential therapeutic strategy to counteract the loss of p53 function and improve treatment responsiveness in these otherwise chemoresistant tumors.

Doxorubicin induces DNA damage and activates p53 family stress signaling [16, 41], leading to induction of TAp63 activation. In contrast, SB431542 blocks TGF-$\beta$/SMAD signaling, which normally suppresses p63 [42, 43]. By relieving this repression, SB431542 enhanced TAp63 expression. When combined, both agents converge on the same p63-dependent apoptotic pathway, amplifying pro-apoptotic gene expression and overcoming compensatory survival mechanisms. As a result, SB431542 and doxorubicin produce a synergistic cytotoxic effect through coordinated activation of TAp63-mediated apoptosis (Fig. 11).

Fig. 11.

Proposed mechanism underlying the synergistic effects of SB431542 and doxorubicin via convergent activation of the p63-dependent apoptotic pathway. Doxorubicin induces DNA damage, leading to activation of p53-family stress signaling and activation of TAp63. Concurrently, SB431542 inhibits TGF-$\beta$/SMAD signaling, relieving TGF-$\beta$–mediated repression of p63 and its downstream pro-apoptotic targets. When combined, the two drugs converge on a common apoptotic axis, leading to amplified TAp63 activation and enhanced expression of apoptotic genes, ultimately driving synergistic cell death.

Our findings provide a strong mechanistic rationale for combining the TGF-$\beta$ inhibitor SB431542 with doxorubicin to treat mutant p53 breast cancer. This approach effectively bypasses the dysfunctional p53 pathway by engaging the TAp63-dependent cell death mechanism. The strategy is highly feasible, supported by the existence of a clinical-grade TGF-$\beta$ inhibitor (e.g., Galunisertib), which can translate our findings. The current major constraint is the absence of in vivo data. Future studies to validate the efficacy and safety of this combination using a clinically relevant TGF-$\beta$ inhibitor in animal models will be imperative before proceeding to clinical trials

Our findings identify a novel drug combination—SB431542 plus doxorubicin—that effectively activates an alternative apoptotic pathway in mutant p53 breast cancer models. Our findings highlight a previously underexplored therapeutic strategy: targeting the TGF-$\beta$/TAp63 axis to restore apoptosis in mutant p53 cancers. This approach may be particularly effective in tumors with compromised p53 function and resistance to conventional chemotherapeutics. Future studies will focus on validating this strategy in vivo, exploring combination regimens with immune checkpoint inhibitors, and identifying predictive biomarkers of TAp63 responsiveness to guide patient selection.

The data are available from the corresponding authors on reasonable request.

YLK, CCC and BHC conceived and designed the study. YLK, YJL, TCH, KYW and STC performed experiments. JYC, KYL, HHW and YTC performed the statistical analysis. CCC and BHC received fundings to support research. BHC wrote the manuscript. CCC and BHC conducted review and editing. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

We would like to acknowledge the technical support given by the Basic Medical Core Laboratory, I-Shou University College of Medicine.

This research was supported by the Medical Student Research and Development Scholarship Program [EDAHS112038], and the grant of E-DA Hospital [EDAHJ114001].

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBL45389.