Role of PAI-1 in the progression and treatment resistance of non-small cell lung cancer.

1
Introduction and background
Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for approximately 1.8 million deaths annually [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases. The standard treatment for early-stage NSCLC is surgical resection combined with perioperative chemotherapy, resulting in a reported 5-year overall survival (OS) rate of 75–85 % [2,3]. Furthermore, perioperative immunotherapy using immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1) (such as nivolumab and pembrolizumab) or programmed death-ligand 1 (PD-L1) (such as atezolizumab) has been shown to improve OS [[4], [5], [6]]. Recent studies have shown that perioperative immunotherapy with ICIs improves overall survival (OS) in patients with early-stage NSCLC. For example, the phase 3 KEYNOTE-671 trial demonstrated that adding perioperative pembrolizumab to chemotherapy improved 3-year OS from 64 % to 71 % (HR 0.72, 95 % CI 0.56–0.93; p = 0.0052) [6]. Concurrent chemoradiotherapy (CRT) followed by immunotherapy is the current standard of care for patients with locally advanced NSCLC who are ineligible for surgery. Despite this approach, the 5-year OS rate remains at 43 %, and the number of patients who are able to achieve a cure is limited [7]. In advanced-stage disease, targeted therapies have demonstrated efficacy in patients harboring driver mutations such as epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase rearrangements. Nonetheless, acquired resistance inevitably develops in most cases, and curative outcomes remain rare [8]. In patients without identifiable driver mutations, ICIs targeting PD-1, PD-L1, or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (such as ipilimumab and tremelimumab) are administered in combination with chemotherapy. However, the median OS in these patients remained approximately 15–22 months [[9], [10], [11]]. Therefore, developing novel therapeutic strategies to improve survival is warranted.
Small-cell lung cancer (SCLC) accounts for approximately 15 % of all lung cancer cases. Compared to NSCLC, SCLC is characterized by more rapid progression and is often diagnosed at an inoperable stage. In patients with locally advanced SCLC, concurrent chemoradiotherapy followed by immunotherapy, including durvalumab, yielded a 3-year OS rate of approximately 57 % [12]. However, curative outcomes are rare. In extensive-stage SCLC, the current standard first-line therapy combines chemotherapy and immunotherapy (a platinum agent (cisplatin or carboplatin) plus etoposide, in combination with either atezolizumab or durvaluma); however, the median OS remains limited to 12–13 months [13,14]. Therefore, development of novel and effective treatments for SCLC is the requirement of the hour.
Regarding the background of poor prognosis in lung cancer treatment, plasminogen activator inhibitor-1 (PAI-1) has been increasingly implicated not only in tumor progression but also in the development of resistance to radiotherapy, chemotherapy, immunotherapy, and targeted therapies in NSCLC [[15], [16], [17], [18]]. Consequently, PAI-1 has emerged as a promising novel therapeutic target, and its potential clinical applications warrant further investigation. Because limited evidence is available regarding its role in SCLC, this review focuses on the findings reported to date in NSCLC.

2
Involvement of PAI-1 in the progression of NSCLC
During cancer progression, including lung cancer, the plasminogen activator (PA) of the fibrinolytic system promotes cancer cell migration, invasion, and metastasis by degrading the extracellular matrix (ECM) and suppressing cell adhesion. Consequently, PAI-1, which inhibits PA, was initially considered as a suppressor of cancer progression [19,20]. However, subsequent studies demonstrated that PAI-1 does not act as a suppressor. Instead, by regulating ECM remodeling through the inhibition of urokinase-type uPA activity and by binding to vitronectin to disrupt the interaction between the cell surface uPA–integrin complex and vitronectin, PAI-1 actively contributes to cancer cell invasion and metastasis [[21], [22], [23], [24]].
Recent in vitro and in vivo studies using lung cancer cells have elucidated the involvement of PAI-1 in lung cancer progression and the underlying molecular mechanisms. PAI-1 enhances the proliferative capacity of lung cancer cells by inducing epithelial–mesenchymal transition (EMT) [17]. Regarding the mechanism of EMT induction, PAI-1 binds to a protein inhibitor of activated STAT3, an inhibitory regulator of STAT3, leading to STAT3 activation and subsequent upregulation of EMT-related gene expression [25].
Moreover, PAI-1 has been implicated in modulating the tumor microenvironment, particularly through its effects on tumor angiogenesis and cancer-associated fibroblasts (CAFs) activation. Masuda et al. reported that PAI-1 promotes the migration of vascular endothelial cells in response to vascular endothelial growth factor (VEGF) stimulation and that tumor angiogenesis and tumor progression are significantly suppressed in PAI-1 knockout mice compared with wild-type controls in a murine lung cancer model [26]. Additionally, PAI-1 has been shown to inhibit apoptosis in CAFs and to promote their transdifferentiation into myofibroblast-like phenotypes; the latter will subsequently secrete TGF-β and PAI-1, thereby enhancing the proliferative capacity of surrounding cancer cells [17].

3
Association between tissue and plasma PAI-1 expression with NSCLC progression
While previous in vitro and in vivo studies have demonstrated the involvement of PAI-1 in lung cancer progression, emerging evidence suggests a relationship between PAI-1 expression levels in either tumor tissues or plasma and the progression and prognosis of lung cancer. We previously reported that PAI-1 expression was significantly associated with T factor and TNM stage in lung adenocarcinoma tissues [17], as defined by the seventh edition of the TNM Classification proposed by the IASLC Lung Cancer Staging Project [27]. Furthermore, other studies have shown that in NSCLC, higher levels of PAI-1 expression in tumor tissues are correlated with poorer prognosis [[28], [29], [30]]. These studies measured PAI-1 protein levels in surgically resected NSCLC specimens using an enzyme-linked immunosorbent assay (ELISA). In one study focusing on 99 patients with lung adenocarcinoma, patients were stratified based on the median PAI-1 level, and those in the high PAI-1 group had significantly worse overall survival (p = 0.04; hazard ratio [HR] 1.62, 95 % confidence interval [CI]: 1.02–2.56) [28]. Similarly, another study that included 118 patients with NSCLC, comprising a higher proportion of squamous and large cell carcinoma, showed a trend toward poorer prognosis in the high PAI-1 group. However, the result was not statistically significant (p = 0.16) [29]. This discrepancy may be attributed to differences in patient backgrounds, histological subtypes, methods of PAI-1 measurement, and cut-off values used to define the high and low expression groups. Prospective observational studies with larger cohorts are needed to standardize the assay method for PAI-1 and establish clinically relevant cut-off values for prognostic stratification.
The association between plasma PAI-1 levels and tumor progression was also investigated. A previous study showed that plasma PAI-1 levels significantly correlated with the size of the primary tumor [31]. Moreover, several reports have indicated a relationship between genetic polymorphisms (SNPs), such as 4G/5G and A15T, that affect PAI-1 stability, function, and tumor progression or prognosis. Notably, the PAI-1 A15T polymorphism is significantly associated with OS, with carriers of variant alleles demonstrating a worse prognosis [32]. This polymorphism has also been suggested to increase plasma PAI-1 levels in individuals carrying the T allele, potentially contributing to disease progression [33].

4
PAI-1 and therapeutic resistance
4.1
Resistance to radiotherapy
Radiotherapy is widely used as a curative treatment for locally advanced lung cancer and as a palliative therapy for metastases to the brain, bone, and other sites. A preclinical study has demonstrated that radioresistant NSCLC cells secrete higher levels of PAI-1 than radiosensitive cells, thereby promoting the survival and malignant transformation of radiosensitive cancer cells [15]. Specifically, radiation-induced hypoxic conditions within the tumor microenvironment upregulate PAI-1 expression from radioresistant cancer cells via reactive oxygen species generation and activation of hypoxia-inducible factor-1α. Elevated PAI-1 levels subsequently activate the AKT and ERK1/2 signaling pathways, leading to the inhibition of apoptosis and enhancement of radioresistance. Moreover, these cells exhibit TGF-β-induced EMT characteristics and increased migratory capacity [Fig. 1A and Table 1] [15].

4.2
Resistance to chemotherapy
Chemotherapy using cytotoxic anticancer agents remains an essential therapeutic modality for lung cancer management. PAI-1 is believed to contribute to chemotherapy resistance through multiple mechanisms, with a particular emphasis on its role in EMT induction. Masuda et al. reported that PAI-1 activates CAFs and promotes their differentiation into myofibroblast-like phenotypes, which subsequently express high levels of PAI-1 and TGF-β, leading to the attenuation of cisplatin efficacy against lung cancer cells through TGF-β-mediated EMT [Fig. 1B and Table 1] [17]. A similar mechanism has also been reported in esophageal squamous cell carcinoma, where cisplatin-induced PAI-1 secretion from CAFs exerts paracrine effects that promote tumor progression and chemoresistance [34].
Clinical studies have also explored the relationship between PAI-1 levels and the efficacy of chemotherapy. Niki et al. demonstrated that in patients with advanced NSCLC, plasma PAI-1 levels were elevated, along with other inflammation- and thrombosis-related biomarkers. They further investigated the correlation between these markers and the chemotherapy outcomes. PAI-1 strongly correlated with platelet-derived microparticles (PDMPs) and high-mobility group box 1 (HMGB1), and patients with high levels of all three factors had significantly shorter OS and disease-free survival. These findings suggest that elevated PAI-1 levels are independent prognostic factors associated with poor outcomes [35].

4.3
PAI-1 in resistance to molecular targeted therapy
The involvement of PAI-1 in resistance to molecular targeted therapies has been reported in the context of EGFR tyrosine kinase inhibitors (TKIs), such as osimertinib. As shown in [Fig. 1C and Table 1], one study demonstrated that EGFR-mutant cancer cells that persisted after EGFR-TKI treatment exhibited high PAI-1 expression. This elevated PAI-1 expression promotes EMT through the integrin but not TGF-β signaling pathway, characterized by decreased E-cadherin and increased vimentin and N-cadherin expression, ultimately leading to the acquisition of resistance to EGFR-TKIs [18]. Additionally, Thu et al. reported that PAI-1 overexpression plays a central role in the acquired resistance to MET-TKIs, specifically crizotinib, in NSCLC patients with MET amplification. In crizotinib-resistant cell lines derived from EBC-1 cells, PAI-1 gene expression was markedly upregulated, and inhibition of PAI-1 using either the small-molecule inhibitor tiplaxtinin or shRNA-mediated knockdown restored crizotinib sensitivity [36]. These two studies provide important evidence that PAI-1 may serve as a novel therapeutic target for overcoming resistance to EGFR-TKIs and MET inhibitors, supporting the potential clinical application of combination strategies in the future.

4.4
Resistance to immunotherapy
4.4.1
Mechanisms behind the contribution of PAI-1 to the tumor immune microenvironment (TIME)
PAI-1 has been implicated in shaping TIME through its effects on tumor-associated macrophages (TAMs). High PAI-1 expression has been correlated with increased TAM infiltration in various cancers [37]. Mechanistically, PAI-1 enhances macrophage motility [[38], [39], [40]] and facilitates macrophage polarization toward an M2-like phenotype by interacting with the C-terminal domain of urokinase-type PA [37,41]. These M2-polarized TAMs secrete immunomodulatory chemokines such as CXCL10 and CCL22, which suppress lymphocyte infiltration into tumor tissues [42]. As tumor-infiltrating lymphocytes (TILs) are positively associated with the efficacy of PD-1 blockade in NSCLC, the TAM-mediated exclusion of TILs may compromise the therapeutic benefits of ICIs [43,44]. Moreover, TAMs have been reported to induce PD-L1 expression in tumor cells [[45], [46], [47]], thereby contributing to T cell apoptosis and reinforcing immune evasion. These findings suggest that PAI-1 promotes resistance to ICI therapy by fostering the immunosuppressive TIME through TAM recruitment and activation [Fig. 2A and Table 1].
In addition, high levels of PAI-1 in the TIME induce tumor angiogenesis [Fig. 2A]. Tumor blood vessels are frequently disorganized and lack normal hierarchical architecture. Endothelial cell–cell junctions are weakened and pericyte coverage is reduced, leading to increased vascular permeability and plasma leakage, which elevates interstitial pressure. These abnormalities contribute to vessel compression and irregular, inefficient blood flow, ultimately impairing the delivery of therapeutic agents and the infiltration of immune cells into the tumor microenvironment [48]. Therefore, such aberrant angiogenesis can act as a mechanism of resistance to ICI therapy.

4.4.2
PAI-1 in resistance to immunotherapy
PAI-1 enhances PD-L1 expression in cancer cells and promotes the formation of an immunosuppressive TIME, thereby reducing the efficacy of immunotherapy [49]. Masuda et al. have shown that PAI-1 plays a central role in the acquisition of ICI resistance. In a murine NSCLC model, tumor cells that persisted after anti-PD-1 antibody treatment exhibited high expression of PAI-1, accompanied by the induction of TGF-β-induced EMT and establishing an immunosuppressive TIME. Furthermore, combination therapy with the PAI-1 inhibitor, TM5614, markedly suppressed the expression of the EMT-related genes TGF-β and PD-L1 as well as the infiltration of TAMs and tumor angiogenesis. This combination significantly increased the number of CD8-positive TILs and enhanced the antitumor effects of the treatment [Fig. 2B]. In addition, the analysis of human NSCLC surgical specimens revealed that the tumors exhibited markedly elevated PAI-1 expression following anti-PD-1 antibody therapy. To our knowledge, this is the first study to demonstrate that PAI-1 may serve as a therapeutic target in the context of resistance to anti-PD-1 antibody therapy in NSCLC [16].

5
Therapeutic potential of PAI-1 inhibitors
The therapeutic efficacies of PAI-1 inhibitors, including TM5614, SK-216, and tiplaxtinin, have been investigated in preclinical and clinical studies. Kang et al. demonstrated that, in an experimental radiotherapy model, combination treatment with radiotherapy and tiplaxtinin suppressed radioresistance and enhanced radiotherapy efficacy [15]. Tokumo et al. reported that in a murine model of EGFR-mutant lung cancer, combination therapy with osimertinib and SK-216 resulted in greater antitumor efficacy than osimertinib monotherapy [18]. Masuda et al. are conducting a phase II investigator-initiated clinical trial to evaluate the effectiveness and safety of nivolumab and TM5614 combination therapy in patients with NSCLC who have completed standard treatment [50].

6
Future directions
Future studies should focus on these two areas. First, further elucidation of the molecular mechanisms involving PAI-1 is required, which may be achieved using advanced technologies such as spatial transcriptomics, patient-derived organoids, and sophisticated computational modeling. Secondly, establishing reliable biomarkers for identifying patients likely to benefit from PAI-1 inhibition is critical. Additionally, as the current research has primarily focused on NSCLC, the role of PAI-1 in SCLC should be explored in future studies.

7
Conclusion
PAI-1 plays an important role in lung cancer progression and the development of resistance to radiotherapy, chemotherapy, immunotherapy, and molecular-targeted therapies. Therefore, therapeutic strategies targeting PAI-1 hold great promise for overcoming the limitations of existing treatments and significantly improving the prognosis of patients with lung cancer.

Declaration of AI in the writing process
While conducting this study, the authors did not use generative AI or AI-assisted technologies.

Funding
This research did not receive any specific grant.

Declaration of competing interest
None.