The components and regulation of the Hippo pathway and its relationships with the progression and treatment of Non-small cell lung cancer (NSCLC)

Abstract

Background

Lung cancer is one of the most common malignant tumours, with non-small cell lung cancer (NSCLC) being the predominant type. The morbidity and mortality rates of lung cancer are increasing worldwide. The Hippo signalling pathway is one of the eight classical human cancer signalling pathways, forming a complex signalling network regulated by intracellular and extracellular signals. Under physiological conditions, it plays a crucial role in inhibiting tumor development.

Main text

Inhibition of the Hippo signalling pathway leads to abnormal activation of its downstream core components, YAP/TAZ, which in turn enhances tumor cell proliferation, invasion, and migration. Recent studies have identified numerous small-molecule inhibitors that target key proteins within the Hippo signalling pathway, which is highly important for the treatment of tumors.

Conclusion

This paper reviews the activation mechanisms of the Hippo signalling pathway, its crosstalk with other pathways, and potential therapeutic targets to provide new therapeutic avenues.

1. Introduction

Lung cancer remains a formidable adversary in the realm of public health, tragically leading the charge among causes of cancer-related mortality globally, with an estimated annual toll of 1.6 million lives lost. The World Health Organisation (WHO) divides this malignancy into two principal classifications—small cell lung cancer and non-small cell lung cancer (NSCLC)-on the basis of distinct pathological characteristics, therapeutic responsiveness, and prognostic outcomes. NSCLC, comprising a staggering 80–85% of all lung cancer diagnoses, stands out as the dominant variant. Recent data from the American Cancer Society heralded a silver lining, reporting a five-year survival rate increase to 60% for NSCLC patients—a testament to progress. However, despite the commendable strides made over the last two decades in elucidating the intricate biology and natural history of NSCLC, coupled with advancements in early detection methodologies and a diversification of therapeutic strategies, the overarching cure rates and long-term survival statistics for NSCLC continue to lag behind expectations. This is particularly true in scenarios where the disease has escalated to the metastatic stage. Consequently, there is a pressing imperative to delve deeper into the multifaceted etiology of NSCLC, with a keen focus on identifying pivotal molecular alterations and discernible biomarkers that catalyse disease progression, a frontier that has become a relentless scientific exploration.

Against this backdrop, the Hippo signalling pathway emerges as a beacon of interest, assuming a preeminent position among the 8 major pathways controlling tumorigenesis. Its critical role in orchestrating organ dimensions, cellular proliferation, regenerative capabilities, and tumorigenesis has garnered significant attention. Over the past few years, a plethora of groundbreaking investigations have illuminated the profound interplay between the Hippo signalling axis and the myriad facets of carcinogenesis—including tumor initiation, enhanced aggressiveness, recurrence patterns, metastatic dissemination, tumor stem cell attributes, and the emergence of therapeutic resistance. This review endeavours to meticulously dissect the latest scientific advancements concerning the Hippo signalling pathway within the pivotal domain of NSCLC. Specifically, these findings underscore the pathway’s involvement in the oncogenic transformation of NSCLC, its influence on therapeutic outcomes, the genesis of drug resistance, and its potential as a novel avenue for targeted therapeutic interventions. By synthesising the extant literature, we endeavour to provide a panoramic view of the intricate pathophysiological underpinnings of NSCLC, thereby laying the groundwork for the future development of efficacious therapeutic modalities.

2. The Hippo signalling pathway: an architect of tumour regulation and therapeutic potential

The Hippo signaling pathway was first discovered in Drosophila melanogaster and has become an important regulator of tumor development. This pathway is composed of STE20-like kinases MST1/2, Salvador homologue SAV1, MOB kinase activator 1 A/B(MOBKL1A/B), Yes-associated protein (YAP) and WW domain-containing transcriptional regulator 1(TAZ), and TEAD family members(Transcriptional Enhancers of Activators). The Hippo pathway not only controls tumors, but also plays a key role in cell growth, proliferation, differentiation, embryonic development, tissue regeneration, organ homeostasis, and wound healing. Recent studies have shown that upstream signal regulation is very complex, involving cell polarity, mechanical stress, density, soluble factors and environmental stress [1].

The Hippo pathway is a highly conserved pathway in the evolutionary process of species, maintaining a balance between cell growth and death, and is essential for embryonic development, tissue regeneration, organ repair, and wound healing. Its imbalance can lead to tissue overgrowth, leading to human diseases, especially cancer. YAP and TAZ promote tumor growth, while MST1/2 and LATS1/2 inhibit tumor. Disruption of Hippo pathway is associated with tumor formation, metastasis and drug resistance, and is a potential target for cancer therapy. Studying this pathway is expected to provide a theoretical basis for finding new potential therapeutic targets for cancer patients [2–4] (Fig. 1).

Fig. 1.

Two states of the Hippo signalling pathway

Hippo ON (healthy cell homeostasis): Under normal physiological conditions, the Hippo pathway maintains tight regulation through a kinase cascade reaction. MST1/2 binds to the scaffolding protein SAV1 to form a complex, which further phosphorylates and activates the downstream kinase LATS1/2 (requires MOB1 assistance). Activated LATS1/2 then phosphorylates the transcriptional co-activator YAP/TAZ, triggering its binding to 14-3-3 proteins for retention in the cytoplasm or degradation via the ubiquitination pathway. YAP/TAZ, which does not enter the nucleus, is unable to bind to the nuclear transcription factor TEAD1-4, thereby maintaining the balance between cell proliferation, apoptosis, and tissue homeostasis.

Hippo OFF (pathway dysregulation in cancer): During tumorigenesis, the Hippo pathway is inactivated by abnormal upstream signaling (e.g., loss of cell polarity, altered mechanistic microenvironment) or mutation of key components. The phosphorylation process of the kinase cascade (MST1/2-LATS1/2) is blocked, and YAP/TAZ escapes the cytoplasmic lag in a dephosphorylated form, translocates to the nucleus and binds to TEAD1-4 to form a transcription complex. Activation of this complex drives unlimited proliferation, metastasis and drug resistance of tumor cells, ultimately promoting cancer progression.

2.1 Upstream molecules of the Hippo signalling pathway

2.1.1 FERM family proteins

In 2006, Hamaratoglu et al. identified two FERm-domain-containing proteins in mammals - merlin and expansion (FRMD6 homologous proteins) - that play key regulatory roles in the Hippo signaling pathway [5].

In NSCLC, the FERM protein family shows significant regulatory duality: On the one hand, FRMPD1 (FRMD2) specifically binds to WWC3 through its FERM domain, activates Hippo pathway kinase LATS1 and induces phosphorylation and inactivation of YAP [6]; On the other hand, PLEKHH2 binds to β-arrestin1 through the FERM domain, relieves its spatial inhibition of FAK, activates the FAK/PI3K/Akt phosphorylation hierarchy, and promotes the malignant progression of NSCLC [7]. This functional paradox reveals that the carcinogenic/tumor suppressor properties of FERM protein depend on its interacting partner - it enhances the Hippo tumor suppressor signal when binding to WWC3, and activates the pro-cancer pathway when binding to β-arrestin1, providing a new molecular stratification basis for targeted therapy of NSCLC.

This context-dependent duality is further exemplified by recent studies showing that FRMD6 has oncogenic properties in lung cancer. Its overexpression in squamous lung cancer and lung adenocarcinoma was significantly associated with poor patient prognosis. Mechanistically, FRMD6 promotes tumor proliferation, migration, and tumorigenesis in vivo by enhancing the interaction between mTOR and S6K, thereby activating the downstream pS6K/pS6 signaling axis. Notably, this pro-tumorigenic effect contrasts with the tumor suppressor function of FRMD6 observed in other cancers, such as renal cell carcinoma, highlighting its critical dependence on tissue-specific microenvironmental cues [8].

2.1.2 Angiomotin (AMOT)

AMOT is involved in regulating angiogenesis, cell polarity and migration, and plays a central role in cancer development. It regulates molecules such as NF2 and LATS in the Hippo signaling pathway, affecting tissue growth and organ size homeostasis.

The multifaceted nature of the functionality of AMOT is exemplified by its interactions with multiple molecules. For example, the CC domain of AMOT, when unphosphorylated, is occupied by the small G protein Rho. This occupancy effectively hinders NF2 binding to AMOT, consequently diminishing Hippo pathway activity during embryogenesis. Furthermore, the AMOT-LATS1 complex highlights the positive regulatory role of AMOT in the Hippo signalling pathway [9]. Gene knockout studies have revealed that the deletion of AMOT leads to abnormal delay in the nuclear localization of YAP in late embryos, and in mutant embryos (AMOT-/- or AmotY/Y), YAP cannot complete extraconuclear translocation after the E32 phase, confirming the key role of AMOT in the dynamic regulation of YAP and the activation of the Hippo pathway [10–12]. In addition, the deubiquitinating enzyme DUB3 can stabilize the AMOT/LATS complex by reducing the ubiquitination level of AMOT, thereby inhibiting YAP activity. This process depends on the activity of the DUB3 enzyme and interacts directly with LATS2 [13].

In NSCLC, AMOT is a key component of the Hippo signaling pathway, and its expression is influenced by the factor inhibiting HIF (FIH). When FIH is absent, AMOT expression is upregulated, resulting in increased phosphorylation of YAP, preventing nuclear translocation of YAP, and inhibiting the carcinogenic activity of YAP/TAZ. AMOT regulates cell localization of YAP by interacting with YAP, thus acting as a potential tumor suppressor in NSCLC. In addition, high expression of AMOT was associated with decreased cell proliferation and increased apoptosis in NSCLC. These findings reveal the tumor suppressive effect of AMOT in the Hippo signaling pathway and its regulatory mechanism [14, 15]. Furthermore, clinical evidence clearly demonstrates that AMOT p130 isoforms are associated with lung adenocarcinoma (LUAD) progression. In patients with lung adenocarcinoma, reduced AMOT p130 expression was significantly associated with poor prognosis and was an independent predictor of decreased survival. Patients with low expression levels had significantly shorter overall survival compared to patients with normal AMOT p130 expression [16]. It is worth noting that although AMOT family proteins have been reported to have both oncogenic and oncostatic roles in various types of cancers, the above findings strongly support that AMOT p130 exerts an oncostatic function in lung adenocarcinoma. On the contrary, the role of AMOT p130 in lung squamous cell carcinoma (LUSC) remains controversial, and whether its mechanism of action is opposite to that in lung adenocarcinoma needs to be verified by subtype-specific studies.

2.1.3 RAS-Association domain family proteins (RASSFs)

RASSFs, recognised as tumour suppressors, participate in cancer suppression by regulating the core components of the Hippo pathway. Among them, the C-RASSF subclass maintains its phosphorylation status by binding to MST kinase and promotes the activation of the Hippo pathway (for example, RASSF1A drives the nuclear aggregation of YAP/p73 through the Fas-MST2-LATS1 cascade and activates the transcription of pro-apoptotic genes). Its function is evolutionarily conformed, and drosophila homologues regulate pathway activity by competitively binding to Hpo kinase. In tumorigenesis, the absence of RASSF can cause the YAP binding chaperone to shift from p73 to the oncogenic transcription factor TEAD/RunX2, promoting malignant transformation [17–19]. In addition, RASSF can induce hypermethylation of the promoter of LATS2 by binding to it [20], or promote the retention of YAP in the cytoplasm through GTPase interaction, thereby inhibiting cell proliferation and inducing apoptosis [21].

In NSCLC, the RASSF protein family exerts tumor suppressive effects by multi-dimensionally regulating key oncogenic signaling pathways. Notably, while RASSF-mediated tumor suppression has been established in NSCLC, no statistically powered study has yet investigated its functional heterogeneity between major histological subtypes or molecular subgroups. Specifically: RASSF10 inhibits the invasion and metastasis ability of NSCLC cells by specifically suppressing the phosphorylation at the T1479 site of low-density lipoprotein receptor-related protein 6 (LRP6) and blocking the abnormal activation of the Wnt/β-catenin signaling pathway [22]; As a specific inhibitor of wild-type EGFR tumors, RASSF3 regulates the endocytic circulation process of EGFR, reduces the membrane localization stability of EGFR, and ultimately induces attenuation of EGFR-dependent pro-survival signals [23]. RASSF8 targets both the Wnt and NF-κB oncogenic pathways simultaneously - on the one hand, it inhibits the nuclear translocation of β-catenin by isolating it; on the other hand, it inhibits the transcriptional activity of NF-κB by blocking the degradation of IκBα, achieving a synergistic anti-cancer effect [24].

Notably, structural biology studies have revealed the core role of the RASSF protein’s coiled-coil domain in its functional execution. This domain not only acts as a molecular scaffold to mediate the interaction between RASSF and effector proteins such as LRP6 and EGFR, but also can expose its C-terminal SARAH domain through conformational changes, thereby enhancing the binding ability with downstream effector molecules. This feature has been confirmed in two independent studies to be the key structural basis for RASSF family members to inhibit the malignant phenotype of tumors [22].

2.1.4 WW Domain-Containing proteins

As a key upstream regulator of the Hippo signaling pathway, the WWC family of scaffolding proteins (comprising the WW and C2 domains) precisely regulates cell proliferation kinetics and organ developmental homeostasis through a cascade of reactions, and WWC3 can activate the core cascade of the Hippo pathway by specifically binding to the LATS1 kinase, thereby suppressing the tumor progression of NSCLC. The molecular mechanism study further revealed that FRMPD1, as a novel regulator, enhanced the stability of the WWC3-LATS1 complex to significantly increase the activity of the Hippo pathway and exerted tumor suppressor effects [6]; WBP2, by competitively occupying the LATS1-binding interface of WWC3, formed a molecular cascade of antagonistic effects, which led to the inactivation of the Hippo pathway and produced a pro-tumorigenic effect [25].

In summary, the complex regulatory network of the Hippo signaling pathway consists of multiple upstream molecules acting synergistically: FERM family proteins (e.g., FRMPD1/PLEKHH2) activate the LATS1 oncogenic pathway or FAK/PI3K/Akt oncogenic pathway by binding to WWC3 or β-arrestin1, respectively; AMOT regulates YAP nuclear localization through phosphorylation-dependent Rho/LATS1 binding switch regulates YAP nuclear localization, and its stability is regulated by DUB3 and FIH to enhance oncogenic function; the RASSF family utilizes the convoluted helix and SARAH structural domains to integrate signals from Wnt, EGFR, and other pathways, and promotes apoptosis through activation of the MST kinase cascade; and WW structural domain proteins (e.g., WWC3) competitively bind to LATS1 with WBP2, determining the Hippo pathway activity status.

2.2 Central kinase complex of the Hippo signalling pathway

Upon activation of the Hippo signalling pathway, a cascade of events in which MST1/2, along with its scaffold protein Salvador 1(SAV1), undergo phosphorylation, is initiated. This phosphorylation event is swiftly followed by the phosphorylation of LATS1/2 and their respective scaffold proteins, MOB1A/1B. The phosphorylation of LATS1/2 by the MST1/2-SAV1 complex results in the inhibition of YAP and TAZ, preventing their translocation to the nucleus and subsequent interaction with TEAD transcription factors. Consequently, the transcriptional coactivators YAP and TAZ are retained in the cytoplasm and are unable to activate TEAD-mediated gene transcription.

Conversely, when the Hippo pathway is suppressed, YAP/TAZ are no longer phosphorylated and are restrained by LATS1/2, allowing them to migrate to the nucleus. Once in the nucleus, YAP/TAZ bind to TEAD, initiating the transcription of target genes. This nuclear translocation of YAP/TAZ, facilitated by Hippo pathway inhibition, underscores their pivotal role as effector molecules within this signalling cascade. By binding to TEAD, YAP/TAZ promotes the expression of downstream target genes, orchestrating a range of cellular responses, including proliferation, differentiation, and survival.

In essence, YAP/TAZ function as critical effectors of the Hippo pathway, serving as molecular switches that toggle between cytoplasmic retention and nuclear translocation on the basis of the pathway’s activity status. Their ability to modulate gene expression through interaction with TEAD highlights the capacity of the Hippo pathway to fine-tune cellular functions and maintain tissue homeostasis. Understanding the precise mechanisms governing YAP/TAZ regulation by the Hippo pathway is crucial for elucidating the role of the Hippo pathway in various physiological and pathological processes, including cancer and developmental disorders [26].

2.2.1 The role of MST

The MST family includes the prototypic MST1/2 and their paralogous homologs MST3/4. MST1/2 belong to the GCKII subfamily, which regulate apoptosis as stress-responsive factors, and are upstream kinases that control cell growth and apoptosis. They act as core components of the Hippo pathway and regulate cell differentiation, apoptosis, adhesion and migration by mediating phosphorylation, dimerization and nuclear localization [27]. In mammals, activation of MST1/2 phosphorylates LATS1/2, which contributes to the degradation of YAP/TAZ to inhibit cell overproliferation.

In NSCLC, MISP protein inhibits homodimerization and autophosphorylation of MST1/2 kinase by binding to it, leading to sustained activation of the downstream effector YAP. Activated YAP in turn upregulates the expression of the transporter protein SLC7A11 and protects cells from iron death [28]. In addition, mouse model studies also revealed that MST1/2-deficient lung adenocarcinomas upregulate PKM2, the rate-limiting enzyme of glucose metabolism, thereby promoting tumor aggressiveness [29]. Taken together, the above studies reveal that MST1/2, as a highly conserved gene, plays an important protective role in suppressing tumorigenesis and development.

2.2.2 The role of LATS

LATS1/2 is a highly conserved oncogene belonging to the NDR kinase family encoding serine/threonine kinases [1]. Its kinase activity is achieved through autophosphorylation and NDR1/2 regulation [30]. Activated LATS1/2 phosphorylates YAP/TAZ, which is retained in the cytoplasm and degraded by ubiquitin, thereby inhibiting downstream target gene expression. This process precisely controls cell proliferation through phosphorylation and conformational regulation, and the absence of activity can lead to malignant proliferation.

The core functions of LATS1/2 include the regulation of cell differentiation, proliferation, apoptosis and migration, and the maintenance of genome stability [31]. In NSCLC, the expressions of LATS1 and LATS2, the core kinases of the Hippo signaling pathway, are significantly downregulated. Although both have conserved structures and functional redundancy, their expression ratios (such as high LATS1/LATS2) are closely related to cisplatin resistance in lung adenocarcinoma. However, the low ratio (LATS2/LATS1) enhances chemotherapy sensitivity; Regulating the proportion of LATS through genetic intervention can reverse cisplatin resistance, suggesting that targeting the protein balance of LATS may become a potential therapeutic strategy [32]. However, LATS-targeted drugs have not been well studied in NSCLC and have been reported in other diseases. LATS1 inhibitors have therapeutic effects in CML by promoting apoptosis [33].

2.2.3 The role of YAP/TAZ

The mode of activation of YAP and TAZ is dependent on synergistic interactions with other signaling pathways, including G protein-coupled receptors (GPCRs), integrin β3, the Wnt pathway, the NOTCH pathway, the RAS, Akt, and metabolic enzymes. Among these pathways, YAP/TAZ can be activated downstream or act synergistically with pathway components.In addition, YAP/TAZ can directly bind a variety of transcription factors, such as the SMAD family, RUNX, p63, p73 and the AP-1 factor JunD. In lung cancer, YAP plays a key role in driving tumor progression and treatment resistance. It has been found that YAP promotes the generation of residual foci and drug-resistant persistent cells (DTP) through the FAK-YAP signaling axis during the targeting of oncogenic drivers such as EGFR, ALK, and KRAS, and that the combination of inhibition of FAK-YAP and first-line targeting therapy can effectively eliminate drug-resistant cells and enhance tumor regression [34]. Meanwhile, YAP is also an important promoter of radiotherapy resistance, and its complex with TEAD4 activates downstream effector molecules, such as NRP1, to promote the proliferation, metastasis, and resistance of cancer cells to radiotherapy, and targeting this axis is expected to enhance the efficacy of radiotherapy [35]. Further mechanistic studies revealed that the oncogenic activity of YAP/TAZ is dependent on its binding to the SRCAP complex, which mediates the deposition of histone variant H2A.Z in the promoter region, thereby activating the oncogenic transcriptional program of YAP/TAZ. In lung cancer models and patients, SRCAP/H2A.Z was significantly up-regulated and positively correlated with YAP expression, advanced stage, and poor prognosis, and depletion or blockade of SRCAP inhibited H2A.Z deposition, oncogene expression, and tumor growth, suggesting that the SRCAP-H2A.Z axis is a key effector of YAP/TAZ oncogenesis, and providing a novel to overcome YAP-driven lung cancer progression diagnostic and therapeutic targets [36]. In addition to promoting tumorigenesis, sustained activation of YAP/TAZ mediates resistance to long-term drug therapy [37].

2.3 Effector molecules and target genes of the Hippo signalling pathway

TEAD1-4 is a major target factor for YAP/TAZ, a YAP/TAZ-TEAD complex that binds and activates gene promoters critical to cell function, promoting tissue homeostasis and response to environmental signals. The specific interaction of YAP/TAZ with TEAD and its recognition of cis-regulatory elements ensure that Hippo pathway accurately regulates gene expression. This regulation is essential for maintaining balanced tissue growth, as dysregulation of the Hippo pathway has been linked to a variety of diseases such as cancer and developmental disorders.

2.3.1 CCN family proteins

The CCN protein family is the main transcriptional activating receptor downstream of the Hippo signalling pathway. It plays an important regulatory role in basic life activities such as cell proliferation, survival, migration, differentiation, and apoptosis and is also central to a variety of disease states, such as cancer, fibrosis, ovaria-related diseases, and the cardiovascular system.

The CCN protein family consists of six members, namely, CCN1 (Cyr61), CCN2 (CTGF), CCN3 (NOVI), CCN4 (WISP1), CCN5 (WISP2), and CCN6 (WISP3). When the Hippo signalling pathway is activated, YAP remains in the cytoplasm and cannot enter the nucleus to bind to TEAD transcription factors. This prevents the transcriptional activation of CCN proteins and other downstream target genes. Conversely, when the Hippo signalling pathway is inhibited, nonphosphorylated YAP/TAZ can freely enter the nucleus, form a complex with TEAD, initiate the expression of genes, including CCN proteins, and promote cell proliferation and survival.

In NSCLC, members of the CCN family, CYR61 (CCN1), CTGF (CCN2), and WISP-1 (CCN4), affect tumor progression by regulating key oncogenic pathways. Compared with normal lung tissue, the expressions of CYR61 and CTGF in NSCLC were significantly downregulated [38]. Among them, CYR61 was clearly identified as a matrix signaling molecule with tumor suppressor function [39]. Its mechanism of action presents multi-dimensional characteristics: CYR61/CCN1 inhibits the expansion of malignant clones of lung cancer by activating the p53/p21 pathway, causing the cell cycle to stagster at the G1/S phase [40], and inducing an increase in the activity of aging-related β-galactosidase (SA-β-GAL); CYR61 antagonizes Wnt/β-catenin signaling and reduces the transcriptional activity of c-Myc, thereby relieving the inhibitory effect of c-Myc on p53. Eventually, it forms a positive feedback regulatory loop of β-catenin-c-myc-p53, significantly inhibiting the proliferation of NSCLC cells [41]. Epigenetic studies have shown that abnormal hypermethylation in the promoter region of CYR61 is the main mechanism for its expression silencing, while DNA methyltransferase inhibitors (such as 5-aza-dC) can restore its expression and reverse the tumor phenotype [42].

It is worth noting that although WISP-1 also belongs to the CCN family, its specific role in NSCLC remains controversial and may show a bidirectional regulatory feature dependent on tissue subtypes. These findings reveal the significant biological significance of CCN family proteins in regulating the progression of lung cancer through the cross-talk mechanism.

2.3.2 Ten-Eleven translocation 1 (TET1)

TET1 is a DNA demethylase and an important target gene of Hippo signalling. TET1 forms a positive feedback loop with YAP and TEAD proteins and participates in the regulation of DNA methylation, thus affecting cell proliferation and tumorigenesis.

First, TET1 can regulate cellular plasticity and promote the acquisition of cellular plasticity in hepatocellular luminal cells during tissue injury or organ formation by activating the YAP/Hippo and ErbB/MAPK signalling pathways [43]. TET1 affects cell proliferation, migration, and regeneration through DNA demethylation, especially by regulating key components of the Hippo pathway, such as WW Domain Transcription Regulator (WWTR1)/TAZ and TEAD1, as well as related genes in the ErbB/MAPK pathway. Dynamic regulation of DNA methylation. TET1, an important DNA demethylase, is involved in regulating gene expression, especially in response to injury or tissue regeneration. For example, TET1-mediated hydroxylated DNA methylation is necessary for cell activation in hepatocellular luminal cells [44]. In addition, TET1 affects key components of the YAP/Hippo pathway, and a change in its activity status directly affects the phosphorylation level of YAP, thereby affecting the expression of genes related to cell proliferation and apoptosis and promoting or inhibiting the development of tumours [44, 45].

In recent years, the regulatory role of the LncRNA-TET1 axis in NSCLC and its interaction mechanism with immune checkpoints have attracted much attention. Studies have shown that specific lncrnas inhibit malignant tumor progression by up-regulating the DNA demethylase TET1. The mechanism involves restoring the expression of tumor suppressor genes, blocking EMT, and potentially regulating immune-related pathways [46, 47]. It is notable that EGFR mutations (such as L858R) have no significant association with TET1 expression, suggesting that the two may independently drive tumor progression. This provides a theoretical basis for the combination of EGFR-targeted therapy and epigenetic regulation [48]. Meanwhile, the correlation between TET1 and PD-L1 shows the possibility of bidirectional regulation: TET1 may directly activate PD-L1 expression through demethylation or indirectly reduce PD-L1 by inhibiting oncogenic signals, thereby affecting the state of the immune microenvironment. This complexity suggests that the combined application of TET1 activators and immune checkpoint inhibitors requires precise assessment of the regulatory direction. In the future, the molecular network of LncRNA-TET1-PD-L1 needs to be further analyzed, and the potential of TET1 as a prognostic marker or a predictor of immunotherapy response needs to be verified through clinical cohorts. The molecular typing system integrating EGFR mutations, TET1 expression and PD-L1 levels may optimize the individualized treatment strategy for NSCLC and promote the clinical transformation of epigenetic and immunotherapy combination therapy [49]. TET1 directly or indirectly regulates key factors in the Hippo signalling pathway, such as LATS1 and YAP, by influencing the methylation status of DNA. Thus, it plays a key role in cell proliferation, apoptosis, tissue repair, and tumor development. These studies highlight the complex regulatory mechanisms by which TET1 maintains tissue homeostasis and participates in disease progression.

2.3.3 Binding Immunoglobulin protein (BIP)/HSPA5

As a core regulatory molecule of endoplasmic reticulum stress (ERS), BIP (HSPA5) has been revealed its regulatory function in tumors such as breast cancer, although it has not been well studied in NSCLC. Recent studies have shown that BIP unexpectedly promotes the migration and invasive ability of tumor cells while inhibiting tumor proliferation by directly binding to YAP and blocking its nuclear localization, a paradoxical effect that highlights the double-edged nature of YAP in triple-negative breast cancer (TNBC) progression [50]. Notably, MFN2, a key protein in the regulation of mitochondrial dynamics, was found to play a transorganelle regulatory role through the YAP-Hippo signaling axis: overexpression of MFN2 enhanced the transcriptional activity of YAP, whereas a YAP-specific inhibitor completely abrogated the maintenance of oxidative stress damage and calcium homeostasis by MFN2, suggesting that this pathway may be an important molecular basis [51]. These findings not only revealed the cross-regulatory mechanism of the endoplasmic reticulum-mitochondria-Hippo signaling network, but also established the biological status of YAP as a core hub of cellular stress response, which not only provides a new direction for the development of therapeutic strategies for cancer and neurological diseases, but also lays a theoretical foundation for the exploration of novel molecular targets for NSCLC.

2.3.4 Transcription factors

In the development of NSCLC, multiple transcription factors play the role of coregulators and are profoundly involved in key processes such as tumor proliferation, invasion, metastasis, and treatment resistance by influencing the gene expression program. In-depth study of the functions of these transcription factors and their regulatory mechanisms is essential for understanding the pathophysiology of NSCLC and developing novel therapeutic strategies.

SMAD3’s dual role:: In NSCLC studies, the role of SMAD proteins is mainly focused on SMAD3. As a core transcription factor of the TGF-β signaling pathway, SMAD3 directly represses pro-cell cycle gene expression, induces cellular senescence and exerts tumor suppressor effects in lung epithelial cells by forming a transcriptional complex with ATOH8. Notably, the deletion of ATOH8 is regarded as one of the key mechanisms for the transformation of SMAD3 from a tumor suppressor to a pro-oncogenic factor. However, SMAD3 also exhibits pro-oncogenic properties in lung cancer progression: histone methyltransferase SMYD2 promotes the expression of SMAD3 by decreasing the level of H3K4me1 modification in the promoter region of SMAD3 to drive tumor invasion the expression of SMAD3 to block the TGF-β signaling pathway, and ultimately inhibits the invasive ability of NSCLC cell invasion ability [58]. The above evidence suggests that multiple oncogenic factors mediate the pro-oncogenic effects of SMAD3 by regulating its expression and function [52].

The RUNX family: environment-dependent regulators: Another important class of transcription factors is the RUNX family of proteins, which are highly conserved transcription factors in postnatal animals and play key regulatory roles in development. Members of this family are involved in the development of many cancers through multiple dysregulated molecular mechanisms, and different RUNX proteins exhibit tumor type-specific functions. Notably, RUNX proteins can act as tumor promoters or suppressors depending on the cellular microenvironment. In NSCLC, chemotherapy-resistant cells showed significant down-regulation of miR-129-5P, accompanied by elevated expression of SOX4 and RUNX1; restoration of miR-129-5P expression effectively induced apoptosis and DNA damage, and significantly inhibited tumor cell proliferation [53]. Further studies revealed that Chb-M’, a RUNX family DNA binding inhibitor, induced apoptosis in p53 mutant NSCLC cells and inhibited tumor progression [54]. In addition, RUNX1 could promote the proliferation and migration ability of NSCLC cell lines by activating the mTOR signaling pathway [55].

P73: a pro-oncogenic member of the p53 family: Unlike the classical oncogenic protein p53, P73, as one of the major members of the p53 family of proteins, mainly plays a pro-oncogenic role in normal physiological processes and tumor development, although its function also involves many aspects of cellular life activities. In NSCLC, P73α, one of the common isoforms of P73, can regulate stearoyl coenzyme A desaturase-1 (SCD-1) to mediate lipid metabolism, which in turn affects tumor cell growth [56]. In addition, MCL1 can interact with P73 and down-regulate its transcriptional activity, which provides a theoretical basis for the combination of MCL1 inhibitors with platinum-based chemotherapeutic agents [57].

FOXO family: interaction with the Hippo pathway: In contrast to the pro-carcinogenic effects of P73, FOXO family transcription factors (e.g., FOXO1/3), as key tumor suppressors, can inhibit the progression of NSCLC through the modulation of biological processes, such as apoptosis, cell-cycle blockade, and anti-oxidative stress. Recent studies have shown that the Hippo pathway interacts closely with the FOXO family of transcription factors, as shown in the following: YAP/TAZ can restrict the transcriptional activity of FOXO through competitive binding, which promotes cell survival and enhances drug resistance [58]; at the same time, the inactivation of the Hippo pathway leads to the accumulation of YAP in the nucleus, which accelerates the progression of NSCLC via the ubiquitination pathway (PMID: 38843589IF: 7.5 Q1 B2). At the same time, inactivation of the Hippo pathway leads to the accumulation of YAP in the nucleus, which accelerates the degradation of FOXO proteins through the ubiquitination pathway, thereby impairing its tumor suppressor function []. In NSCLC, the imbalance between YAP and FOXO expression is not only significantly associated with chemoresistance, but also with poor patient prognosis, therefore, targeting the YAP-FOXO axis may be a potential therapeutic strategy to reverse the drug-resistant phenotype of NSCLC.

Finally, we attempt to summarize the upstream regulators of Hippo signalling pathway using a molecular mechanism diagram (Fig. 2).

Fig. 2.

Regulation of Hippo signalling pathway

Cytosolic signaling

Mechanical signaling receptors CD44, FAT4 and GPCRs at the plasma membrane collaborate to sense mechanical stimuli in the microenvironment; GPCRs activate downstream effector molecules through G proteins to regulate cytoskeletal reorganization and transmission of mechanical signals; the RASSF family of proteins may affect the efficiency of signaling by binding to microtubules or by regulating the MAPK pathway.

Cytoplasmic signaling integration and regulation: Mechanical and GPCR signals converge in the cytoplasm to the core kinase cascade of the Hippo pathway: the SAV1-MST1/2 complex phosphorylates and activates LATS1/2, which further phosphorylates the transcriptional coactivator YAP/TAZ, which contributes to its cytoplasmic retention and formation of complexes with scaffolding proteins (AMOT, FRMD6, KIBRA, Merlin), inhibiting their function.SIK1-3 may be involved in the energy stress response by regulating LATS1/2 activity or the AMPK pathway.Rho GTPases indirectly affect LATS1/2 activity and YAP/TAZ subcellular localization by modulating F-actin assembly and actin contractility.BIP/HSPA5, as endoplasmic reticulum chaperone proteins, may be activated through the BIP/HSPA5, as endoplasmic reticulum chaperone proteins, may regulate overall signaling homeostasis through unfolded protein responses.

Intranuclear transcriptional regulation and feedback

after entering the nucleus, dephosphorylated YAP/TAZ binds to TEAD1-4 transcription factors to activate target gene transcription and drive cell proliferation, migration and survival. Meanwhile, YAP/TAZ forms complexes with Smad proteins (TGF-β pathway effector molecules), Runx, p73, etc., to synergize or antagonize the expression of specific genes. TET1 may enhance transcriptional accessibility by modifying target gene promoters through DNA demethylation. In addition, target gene products may feedback regulate upstream Rho activity or GPCRs expression, forming a dynamic regulatory loop.

3. Interactions between the Hippo signalling pathway and other pathways

Various studies have shown that interactions between the Hippo pathway and other pathways correlate with elevated levels of YAP/TAZ and TEAD protein expression in the nucleus, which are associated with poor prognosis and increased treatment resistance. These abnormal YAP/TAZ levels can be explained by the cross-activation of the Hippo signalling pathway with protumor signalling pathways.

3.1 PI3K/Akt signalling pathway

The Phosphoinositide 3-Kinase/Protein Kinase B (PI3K/Akt) signalling pathway is a quintessential intracellular cascade that is intricately involved in critical cellular processes such as growth, metabolism, survival, and apoptosis. Upon stimulation by extracellular cues, such as growth factors, this pathway is activated through receptor binding, activating PI3K. PI3K catalyses the generation of phosphatidylinositol (3,4,5)-trisphosphate, activating Akt. Akt subsequently influences several downstream targets, including mTOR, Glycogen Synthase Kinase 3 Beta (GSK-3β), and the Forkhead Box O (FoxO) family of transcription factors. The crosstalk between the Hippo signalling pathway and the PI3K/Akt pathway directly or indirectly modulates proteins, thereby regulating cell cycle progression and apoptosis and playing a pivotal role in controlling cell growth, survival, migration, and tumorigenesis.

First, the PI3K/Akt and Hippo signalling pathways synergistically regulate cell proliferation and apoptosis. The PI3K/Akt pathway influences YAP/TAZ, the central molecules of the Hippo pathway, directly or indirectly, affecting cellular proliferation and survival. Several studies have demonstrated that an activated PI3K/Akt pathway directly activates YAP, inhibiting its phosphorylation and degradation and promoting cell growth and proliferation [60]. Another line of research indicates that the PI3K/Akt pathway indirectly activates YAP/TAZ by suppressing upstream kinases of the Hippo pathway, such as MST1/2 and LATS1/2 [61]. Conversely, the Hippo pathway can also modulate the PI3K/Akt pathway, as YAP/TAZ can directly activate PI3K transcription, establishing a positive feedback loop that enhances cell proliferation [62].

The interplay between the PI3K/Akt and Hippo signalling pathways is equally significant in the context of disease progression. Aberrant activation of the PI3K/Akt pathway and inactivation of the Hippo pathway are frequently observed in numerous cancers, acting in concert to drive tumorigenesis and disease progression. For example, in cancers of the breast, stomach, and lung, the activation of PI3K/Akt leads to the nuclear accumulation of YAP, which promotes tumor cell growth and migration [63]. Conversely, studies have shown that inhibiting the PI3K/Akt pathway can restore Hippo signalling activity, curtailing tumor progression [64].In NSCLC, the key mediators of cross-regulation of the PI3K/Akt and Hippo pathways are LncRNAs. e.g., the pan-cancer marker MIR4435-2HG forms a ceRNA network through competitive binding of 20 miRNAs, simultaneously activating pro-survival signaling by PI3K/Akt and transcriptional activity of YAP/TAZ-TEAD in the Hippo pathway to drive tumor proliferation and drug resistance. Clinical analysis showed that high MIR4435-2HG expression was significantly associated with poor prognosis in NSCLC patients and predicted the risk of multiple cancers, and it was also involved in regulating the therapeutic effects of drugs such as cisplatin and resveratrol [65]. Another study revealed that LINC00973 promotes NSCLC cell migration and angiogenesis through a ceRNA network that regulates pathways such as PI3K/Akt, Hippo and TGF-β. The core genes of this network (e.g. RTKN2, NFIX) were identified as independent prognostic factors that may exacerbate tumor progression by activating YAP or mTOR. These findings provide potential therapeutic strategies for targeting the LncRNA-pathway crossover network [61].

Moreover, the complex interplay between these two pathways during tissue damage repair, inflammatory responses, and stress has been documented. For example, in models of myocardial infarction, compounds such as ursolic acid and 3,3′-diindolylmethane synergistically inhibit myocardial apoptosis and promote repair by suppressing the PI3K/Akt pathway and activating the Hippo pathway [66]. Additionally, YAP/TAZ participates in the regulation of immune cells through the PI3K/Akt pathway in inflammatory reactions, influencing the expression of inflammatory cytokines and the immunological microenvironment [67].

Given the crucial roles of the PI3K/Akt and Hippo pathways in disease, they represent prime targets for therapeutic intervention in cancer and other disorders. Considerable efforts have been directed toward developing various natural compounds and small molecule inhibitors, such as ursolic acid, 3,3′-diindolylmethane, and luteolin, to maintain the equilibrium between these pathways, with potential benefits in inhibiting tumor growth and facilitating tissue repair [68–70]. However, the efficacy of targeted therapies must account for the possibility that the balance between these two pathways may shift under different pathological conditions, necessitating further clinical validation of combined targeted approaches.

3.2 ERK signalling pathway

The Extracellular Signal-Regulated Kinase (ERK) signalling pathway, a constituent of the Ras/MAPK (mitogen-activated protein kinase) family, is triggered by extracellular signals, notably growth factors, through a cascade of phosphorylation events mediated by a series of protein kinases, including Ras, Raf, and MEK. This sequential activation culminates in the phosphorylation and activation of ERK1/2, which subsequently activates a plethora of downstream transcription factors. These transcription factors influence gene expression, driving cellular proliferation, survival, and differentiation. Dysregulation of the ERK signalling pathway is implicated in a multitude of diseases, ranging from malignancies to neurodegenerative disorders and cardiovascular conditions [27, 71].

A bidirectional regulatory mechanism exists between the ERK and Hippo signalling pathways. Numerous studies have shown that the ERK signalling pathway can either positively or negatively modulate the Hippo pathway. ERK-mediated dephosphorylation and subsequent nuclear localisation of YAP/TAZ promote cell proliferation and confer resistance against apoptosis [72–77]. Conversely, ERK can also influence tissue growth by activating components of the upstream Hippo signalling pathway, such as MST1/2, thereby impacting YAP/TAZ activity. Dysfunctional interactions between ERK and the Hippo signalling pathway have been linked to tumor progression in various cancer types. For example, the promotion of tumor cell growth and survival by YAP through the PI3K/Akt signalling pathway is exacerbated by sustained ERK activation, intensifying oncogenic effects [78, 79].

It was found that in NSCLC, inhibition of ERK1/2 could suppress tumor cell invasion and migration by down-regulating the activity of the Hippo pathway, suggesting that targeting ERK1/2 or components of the Hippo pathway may become a new anti-tumor strategy. However, this regulatory network has a complex feedback mechanism: YAP1, as an effector molecule downstream of the Hippo pathway, can reverse activate the ERK signaling pathway. By inhibiting the expression of autophagy-related proteins, it weakens osimertinib-induced tumor cell death, thereby driving targeted therapy resistance. This bidirectional regulatory loop of ERK-HIPPO-YAP1 not only highlights the key role of pathway crossover in tumor progression, but also provides new ideas for overcoming drug resistance - for example, combining ERK inhibitors with YAP1 antagonists or autophagy activators may reverse osimertinib resistance and inhibit metastasis [77]. In the future, it is necessary to further analyze the spatial dynamic regulation of the ERK/Hippo/YAP1 axis and its association with the EGFR mutation status in order to optimize the precise treatment strategy.

3.3 AMP-Activated protein kinase (AMPK)

Under energy stress conditions, such as glucose starvation or metabolic stress, both the AMPK and Hippo signalling pathways are activated, inhibiting YAP activity. AMPK acts as a cellular energy sensor that is activated when energy is deficient and regulates energy metabolism by phosphorylating multiple downstream targets. As an effector of the Hippo signalling pathway, YAP activity is regulated by the kinase LATS1/2. When phosphorylated, YAP is retained in the cytoplasm and cannot enter the nucleus to activate gene expression. These findings suggest that AMPK not only directly inhibits the transcriptional activity of YAP by phosphorylating specific sites, such as S61 and S94 but also indirectly activates the Hippo pathway by increasing the level of angiomotin family proteins. In addition, energy activation can independently activate LATS1/2, and the Hippo signalling pathway can still be activated even in the case of AMPK inactivation, which proves that there are dual AMPK-dependent and AMPK-independent regulatory mechanisms for LATS1 activation [80].

In glucose metabolism, glucose metabolism status is an upstream regulator of the Hippo pathway. In liver cells, AMPK is activated when glucose levels decrease, which, in turn, controls YAP activity by regulating the Hippo pathway. This finding reveals a potential link between glucose metabolism and the Hippo pathway in tissue maintenance and cancer prevention, specifically the role of YAP in promoting glycolysis and glucose uptake [80].

It has been reported in the literature that Sestrin2 inhibits YAP activation in the Hippo pathway by promoting AMPK phosphorylation in NSCLC [81]. Activated AMPK can block the interaction of YAP with the transcription factor TEAD, thereby downregulating the expression of the pro-carcinogenic gene FOXM1 and inhibiting the proliferation, migration, invasion and EMT of NSCLC cells.The regulation of the AMPK-YAP/FOXM1 axis provides a novel mechanism for targeting NSCLC progression, suggesting that AMPK activation may exert an anti-tumor effect by inhibiting YAP signaling. The interaction between the two is relevant to the mechanism of drug resistance in NSCLC: In NSCLC with KRAS mutations (e.g., KRAS; LKB1 or KRAS; TP53), YAP1 up-regulation is a key factor in acquired drug resistance [82, 83].YAP1 promotes drug resistance by enhancing tumor cell adaptation (e.g. secretome reprogramming) and resistance to targeted therapies (e.g., TBK1/MEK inhibitors) resistance, promoting drug resistance. Furthermore, YAP1 activation is associated with H3K27 promoter acetylation, which can be reversed by BET inhibitors, suggesting that epigenetic interventions can restore therapeutic sensitivity.

AMPK activators combined with Hippo pathway inhibitors may overcome drug resistance in KRAS-mutant lung cancer through dual inhibition of YAP activity. Drug combinations based on the AMPK-YAP regulatory axis have shown synergistic anti-tumor effects in preclinical models, providing new ideas for NSCLC treatment.

Taken together, the interaction between the AMPK and Hippo signalling pathways affects cell and tissue functions at multiple levels, including energy metabolism, cell growth, liver health, and the progression of kidney disease. These findings not only deepen our understanding of these two critical signalling pathways but also provide a theoretical basis for developing novel therapeutic strategies for multiple diseases.

3.4 Wnt/β-catenin signalling pathway

The Wnt/β-catenin signalling pathway is among the most influential pathways in embryonic development and organogenesis and plays a critical role in regulating cellular proliferation, differentiation, polarisation, migration, and apoptosis across diverse cell types. Extensive research has explored the interplay between the Wnt and Hippo signalling pathways, particularly focusing on their collaborative regulation of myocardial cell proliferation and heart size [84]. Evidence suggests that the Hippo signalling pathway restrains Wnt signalling, thereby limiting cardiac expansion. This regulation is mediated through the actions of Yes-associated protein (YAP), which serves as a molecular bridge between the two pathways.

Nonphosphorylated YAP interacts with β-catenin, a pivotal component of the Wnt signalling pathway, in the nucleus, whereas phosphorylated YAP binds to DVL2, hindering the nuclear translocation of β-catenin [85]. Azzolin et al. revealed that YAP can interfere with the β-catenin destruction complex, enabling YAP/TAZ to accumulate in the nucleus and activate Wnt/YAP/TAZ-dependent biological effects when the Wnt pathway is active. Conversely, when the Wnt pathway is inactive, YAP/TAZ contributes to β-catenin inactivation. In the context of the Wnt pathway being active, the dissociation of the YAP/TAZ complex facilitates Wnt/β-catenin signalling [86]. In the mammalian intestine, YAP sustains the proliferation of crypt stem cells and activates the Wnt pathway; in vitro experiments have demonstrated that YAP downregulation reduces β-catenin-dependent transcription. The cell type dictates which transcriptional coactivator with which YAP initially associates [87].

Moreover, the nuclear localisation of YAP significantly impacts its regulatory effect on β-catenin. Piezo1, a crucial mechanosensor for bone homeostasis, stimulates YAP expression and nuclear localisation, subsequently increasing β-catenin expression and nuclear localisation. Within the nucleus, YAP directly interacts with β-catenin to form the YAP-β-catenin transcription complex [88]. The Wnt pathway also governs YAP through nuclear accumulation, suggesting a reciprocal mechanism between Hippo and Wnt signalling: YAP activity potentiates Wnt signalling [86]. However, contradictory findings exist, potentially owing to cell type-specific differences. In neural stem cells, YAP-β-catenin binding has a more pronounced influence on neuronal differentiation than TEAD binding does, in contrast with previous results obtained in mesenchymal stem cells [89]. Consequently, additional research is warranted to clarify the underlying mechanisms governing the interplay between the Wnt and Hippo signalling pathways [90].

In recent years, the interactive regulation of the Wnt and Hippo pathways in NSCLC has become a research hotspot. Key cross-molecules such as ZMIZ2 and WWC3 can simultaneously activate Wnt/β-catenin and inhibit the Hippo pathway, promoting the nuclear translocation of YAP/TAZ and the expression of proliferation-promoting genes [91, 92]. KCTD11 upregulates LATS1 by antagonizing the β-catenin/TCF complex and inhibits the characteristics of tumor stem cells. In addition, the Wnt component DVL collaboratively regulates EMT-related genes with YAP1, accelerating invasion and metastasis [93]. The Wnt-Hippo interaction mediates the resistance of EGFR inhibitors and chemotherapy by maintaining the plasticity of tumor cells. Targeting cross-node or combined pathway inhibitors may overcome the resistance. In the future, it is necessary to deeply analyze the dynamic mechanism and its role in molecular typing and the immune microenvironment.

3.5 JAK‒STAT signalling pathway

The Janus kinase-signal transducers and activators of transcription (JAK-STAT) signalling pathway constitutes a central mechanism for cellular responses to a wide array of cytokines, including interferons and interleukins. Activation ensues upon the binding of ligands (such as interferons) to receptors, triggering the activation of receptor-associated JAK kinases (JAK1, JAK2, JAK3, and TYK2), leading to receptor phosphorylation and subsequent STAT protein phosphorylation [94]. Aberrant activation of the JAK-STAT pathway is frequently observed in hematological malignancies and solid tumors, including myelodysplastic syndromes, multiple myeloma, specific lymphomas, and various solid tumors [95–97]. Given its pivotal role in disease pathology, the JAK-STAT pathway represents a critical target for antitumour and anti-inflammatory therapeutic strategies.

In the tumor microenvironment, the activation of cancer-associated fibroblasts (CAFs) is intricately linked to the JAK-STAT pathway’s regulation by the Hippo pathway. CAFs promote JAK-STAT pathway activation by secreting cytokines such as IL-6, which in turn promotes tumor cell growth and migration [98]. Conversely, C-terminal TAZ (CTAZ), a mutant of the Hippo pathway, was found to inhibit JAK-STAT signalling, reducing STAT1/2 nuclear localisation and interferon-stimulated gene (ISG) expression [99]. These findings suggest that the Hippo pathway may negatively regulate the JAK-STAT pathway under certain conditions through CTAZ and other factors, thereby impacting tumor progression.

Interaction of Hippo with the JAK-STAT3 signaling pathway in NSLCL often tumor resistance is closely related Gefitinib induces tetraploidization of NSCLC cells through activation of the YAP-MKK3/6-p38 MAPK-STAT3 signaling axis, leading to genomic instability and multidrug resistance [100]. p38 MAPK inhibitors such as losmapimod) can block this process and restore gefitinib sensitivity, suggesting that targeting the YAP-p38 axis is a novel strategy to overcome drug resistance. In EGFR-mutant NSCLC, STAT3 and Src-YAP1 signaling are dually activated during EGFR inhibitor treatment to promote cell survival [101]. Co-targeting of EGFR, STAT3 and Src synergistically inhibited tumor growth, and high STAT3/YAP1 expression was significantly associated with shorter progression-free survival in patients. Upon inhibition of RTK pathways such as EGFR, TNF-NF-κB, STAT3, and YAP signaling are activated, driving adaptive resistance [102]. Prednisone significantly enhances the efficacy of EGFR inhibitors by shutting down bypass RTK signaling through broad inhibition of inflammatory pathways, including YAP and STAT3, suggesting the need for combined multi-pathway intervention.

In conclusion, the complex interplay of the JAK-STAT signalling pathway with the Hippo pathway is multifaceted and influences inflammation, tumor progression, and tissue regeneration. Understanding the nuances of this interaction provides insights into the development of targeted therapies for a range of diseases, from cancer to inflammatory disorders.

3.6 Dachsous-Fat signalling pathway

The Dachsous-Fat signalling pathway, initially characterised in Drosophila melanogaster, plays a pivotal role in the development of wings and the regeneration of cricket legs. This pathway is intricately connected to the Hippo signalling pathway, which significantly influences cell proliferation and tissue homeostasis.

Studies have revealed that Ft signalling is relayed to Dachsous, forming a critical link between the Ft pathway and the Hippo pathway [103]. This connection is further underscored by the discovery of Vamana, a protein that colocalises with Ds, indicating its integral role within the fat signalling pathway in Drosophila [104]. Vamana facilitates the transmission of Ft signals, thereby modulating Hippo pathway activity.

Genetic screening efforts have identified the MSN gene as a target of Yki, the transcriptional coactivator downstream of the Hippo pathway. MSN functions downstream of the Ft pathway and can regulate the Hippo signalling pathway in a negative feedback loop, demonstrating the intricate cross-talk between these pathways [105].

Furthermore, the fat-regulated binding protein Drosophila FAT also participates in the negative feedback regulation of the Hippo pathway, reinforcing the complex network of interactions governing cellular proliferation and differentiation. This research also highlights how the scaffold protein Mats influences Hippo pathway activity through competitive binding with Wts, the kinase that phosphorylates and inactivates Yki. This competitive binding mechanism adds another layer of complexity to the regulation of the Hippo pathway, illustrating the multifaceted nature of the signalling cascades involved in controlling cell growth and tissue patterning [106].In NSCLC, FAT Atypical Calcineurin 4 (FAT4) has been identified as an oncogene in lung cancer. The herbal extract Fraxinus excelsior A (JUA) directly binds to and activates FAT4, subsequently activating FAT4-HIPPO signaling and inhibiting YAP nuclear translocation. It inhibited lung cancer development, prolonged mouse survival in vivo, and suppressed NSCLC cell activity through cell cycle arrest, proliferation inhibition, stemness inhibition, and senescence promotion [107]. Another study found that FAT1 regulates the activation of ER stress pathway through YAP signaling and affects the susceptibility of NSCLC cells to VCP inhibitors [108].

3.7 Transforming growth factor-beta (TGF-β)/Small mothers against decapentaplegic (SMAD) signalling pathway

The TGF-β/SMAD signalling pathway is a highly conserved mechanism that triggers a cascade of intracellular events, primarily through the engagement of TGF-β superfamily ligands (including TGF-β1, β2, and β3) with cell surface receptors [109]. This process involves the formation of a complex between TGF-β receptor types I and II (TβRI and TβRII), which activates the kinase activity of TβRI and promotes the intracellular phosphorylation of SMAD2 and SMAD3. Subsequently, phosphorylated SMAD2/3 forms a complex with SMAD4, which translocates to the nucleus, binds to DNA, and regulates the transcription of target genes. This pathway is involved in a multitude of biological processes, including the inhibition of cell growth, apoptosis, EMT, and immune regulation. Dysregulation of the TGF-β/SMAD signalling pathway, either through abnormal activation or inhibition, is implicated in various diseases, notably cancer, and contributes to uncontrolled cell proliferation and facilitates tumor invasion and metastasis [110].

The interplay between the TGF-β/SMAD signalling pathway and the Hippo pathway manifests at multiple levels. Initially, TGF-β indirectly modulates the function of YAP/TAZ by activating SMAD2/3. Consequently, TGF-β-induced SMAD2/3 competes with YAP/TAZ for binding to TEAD transcription factors, influencing YAP/TAZ-mediated gene expression. This mechanism is crucial for regulating cell growth and EMT [111]. Additionally, activation of the TGF-β signalling pathway promotes extracellular matrix (ECM) remodelling, which alters cell morphology and mechanical sensing, subsequently influencing the activity of the Hippo pathway [112]. Changes in ECM stiffness, for example, modulate the phosphorylation state and nuclear localisation of YAP/TAZ by regulating the activities of the MST1/2 and LATS1/2 kinases. Furthermore, YAP/TAZ, an effector molecule of the Hippo pathway, directly or indirectly regulates the expression and activity of TGF-β. YAP/TAZ promotes the expression of the TGF-β ligand UPD3 and influences cell proliferation and differentiation via the JAK/STAT signalling pathway [113].

In the context of cancer, the interplay between the TGF-β/SMAD and Hippo signalling pathways is particularly intricate and significant. While TGF-β promotes the EMT of tumor cells, enhancing their migratory and invasive capabilities, dysregulation of the Hippo pathway often results in hyperactivation of YAP/TAZ, accelerating tumor growth and metastasis [114]. Conversely, under certain conditions, such as specific cellular microenvironments or signal stimuli, the activated Hippo signalling pathway can inhibit the expression of TGF-β signalling components, such as by repressing SNAIL1, which suppresses ECM remodelling and angiogenesis, thereby curtailing tumor progression [115]. Targeting the interaction between these two signalling pathways could thus present innovative therapeutic strategies for cancer treatment, such as the use of small molecule inhibitors to block TGF-β signalling or the inhibition of YAP/TAZ activity through the activation of the Hippo pathway to impede tumor growth and metastasis [111].In recent years, there has been an increase in studies regarding the interaction between the TGF-β/Smad pathway and the Hippo pathway in NSCLC. It has been found that the interaction between YAP and TGF-β can regulate Serpin family E member 1 (SERPINE1, an important regulator significantly associated with poor prognosis of lung cancer), a serine protease inhibitor, in mesenchymal lung cancer cells [116]. In addition, TGF-β also promotes YAP-dependent AXL induction in MSCs [117]. Pefani et al. found that RASSF1A expression regulates TGF-β-induced YAP1/SMAD2 interactions and leads to SMAD2 cytoplasmic retention and inefficient transcription of TGF-β target genes. Furthermore, RASSF1A limited TGF-β-induced invasion, providing a new framework for how RASSF1A affects YAP1 transcriptional output and triggers its tumor suppressor function [117].

3.8 Notch signalling pathway

The Notch signalling pathway is a highly conserved evolutionary mechanism that governs cell fate decisions primarily through direct cell-to-cell contact. Central to this pathway are the Notch receptors and their ligands, predominantly from the Jagged and Delta families [118, 119]. Upon ligand interaction with Notch receptors on adjacent cells, the Notch receptors undergo internalisation and cleavage, releasing the Notch intracellular domain (NICD). This NICD migrates to the nucleus, where it binds to transcription factors such as Recombination Signal Binding Protein for Immunoglobulin kappa J region (RBPJ) and Mastermind-like proteins (MAML), subsequently engaging with Hes and Hey family transcriptional regulators [120]. This interaction activates the transcription of target genes, including members of the Hes and Hey families, thereby controlling the proliferation, differentiation, apoptosis, and self-renewal capacity of stem cells.

Within the realms of stem cell and cancer biology, the interplay between the Notch and Hippo signalling pathways is crucial for cell differentiation. For example, during lung and kidney development, the synergistic interaction between YAP and the Notch signalling pathway promotes cell proliferation and differentiation [119]. Moreover, in intestinal stem cells, RASSF1A influences HES1 stability, indicating that the crosstalk between the Hippo and Notch pathways governs the maintenance and differentiation of stem cells [121]. Dysregulated activation of both the Notch pathway and the Hippo pathway is frequently associated with carcinogenesis, tumor invasion, and drug resistance in various cancers. YAP has been demonstrated to augment Notch signalling by increasing the expression of Notch ligands, such as Jagged 1 (JAG1), thus promoting cancer progression. Conversely, the Notch pathway can also positively modulate YAP expression and activity, establishing a positive feedback loop that accelerates tumour cell growth. Consequently, the convergence of these two pathways emerges as a potential therapeutic target, for instance, by inhibiting YAP or Notch activity to suppress tumor growth.

Notably, In EGFR-mutated NSCLC, YAP can regulate the generation of lung cancer stem cells by activating the Notch signaling pathway [122]. Another study showed that specific microRNAs can synergistically activate the Hippo pathway while inhibiting Notch signaling, thereby promoting malignant progression of NSCLC [123].

3.9 Role of noncoding RNAs in regulating Hippo signalling pathways

Noncoding RNAs, notably long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), have emerged as critical regulators in the modulation of hippo signalling pathways, revealing intricate mechanisms that govern the progression of NSCLC. These versatile RNA molecules function by serving as molecular decoys for microRNAs (miRNAs), directly suppressing the activity of transcription factors, and engaging in specific interactions with core components of the hippo signalling pathways. They employ multilevel regulatory strategies to precisely control the activity states of pivotal proteins such as YAP/TAZ, exerting a significant influence on disease progression [124–126].

Specific lncRNAs, including Hotair [127], Linc00973 [61], and Linc00243 [128], have been identified as potent modulators of the hippo signalling pathway. Through their unique molecular mechanisms, these lncRNAs can either suppress or stimulate pathway activity, consequently promoting or inhibiting the oncogenic process in NSCLC. These discoveries significantly increase our understanding of the molecular regulatory network underlying NSCLC, laying a robust theoretical foundation for developing innovative therapeutic strategies that leverage the power of lncRNAs and circRNAs.

Research into the specific roles of noncoding RNAs in regulating hippo signalling pathways has progressively revealed the complexities of molecular regulation in NSCLC. This knowledge provides a novel framework for identifying diagnostic markers and therapeutic targets related to specific RNA molecules. Ongoing investigations in this field are anticipated to provide more precise and personalised treatment options for NSCLC patients. Moreover, this research contributes to the broader field of tumour biology, expanding the potential for noncoding RNA-based therapies in the evolving landscape of cancer treatment [129, 130].

Disruptions in the hippo signalling pathway are central to the malignant transformation of NSCLC cells. By influencing key biological processes, such as cell proliferation, apoptosis, and metastasis, these disruptions critically impact the prognosis of NSCLC patients. A comprehensive understanding of how the hippo signalling pathway interacts with other pathways and the role of noncoding RNAs in this interplay is indispensable for developing more effective therapeutic strategies against NSCLC [130].

Future studies should prioritise further elucidation of the regulatory mechanisms underlying the Hippo signalling pathway, validation of its potential as a therapeutic target, and exploration of strategies to overcome therapeutic resistance in NSCLC. These efforts are expected to catalyse significant advancements in personalised medicine, leading to breAkthroughs in the treatment of NSCLC and contributing to the wider field of oncology.

3.10 Regulation of the tumor microenvironment

Recent studies have found that the core effector molecules of the Hippo pathway, YAP/TAZ, play a key role in tumor immune escape and inflammatory responses by regulating the functions of immune cells and matrix interactions in the tumor microenvironment. Its mechanism of action mainly includes the following aspects: YAP/TAZ affects the anti-tumor immune response by regulating the differentiation balance of T lymphocyte subsets. TAZ can specifically enhance the activity of the transcription factor RORγt, promote the differentiation of pro-inflammatory Th17 cells, and simultaneously inhibit the expression of the key regulatory factor Foxp3 in Treg cells, thereby disrupting the Th17/Treg balance and forming an inflammatory microenvironment conducive to the activation of CD8 + T cells YAP, on the other hand, up-regulates the secretion of IL-6 and TGF-β by tumor-associated fibroblasts (CAFs) through the activin signaling pathway (SMAD2/3 dependent), further inducing Th17 polarization [131]. It is worth noting that this Th17 advantage may have a double-edged sword effect: on the one hand, it enhances CD8 + T cell-mediated tumor killing; on the other hand, it may also accelerate tumor progression through pro-inflammatory factor storms.

In the regulation of tumor-associated macrophages (TAMs), the activation of YAP/TAZ can significantly promote the secretion of chemokines such as CCL2/5 by NSCLC cells and recruit monocyte-derived macrophages expressing CCR2. These TAMs present an M2-like immunosuppressive phenotype, characterized by abnormally high expression of PD-L1 and arginase-1 (Arg-1), resulting in impaired toxic function of CD8 + T cells and being closely related to the poor prognosis of patients [132]. Mechanologically, the YAP-TEAD complex directly binds to the promoter region of macrophage colony-stimulating factor (CSF1), promoting its transcriptional expression and thereby maintaining the survival and function of TAMs It has been reported that the Hippo signaling pathway YAP/TAZ is closely related to the expression regulation of the immune checkpoint molecule PD-L1. In EGFR-mutated NSCLC, the accumulation of YAP in osimertinib-resistant cells can drive TEAD and STAT3 to collaboratively bind to the PD-L1 promoter, resulting in upregulation of PD-L1 expression, and then mediate adaptive immune escape through the PD-1/PD-L1 axis [131]. This mechanism provides a theoretical basis for the combined targeting of the Hippo pathway and immune checkpoint inhibitors.

Preclinical stut the cdies have shown thaombination of the YAP/TAZ inhibitor vitopporfin and anti-PD-1 therapy can significantly restore the function of CD8 + T cells and deplete Treg cells, achieving long-term tumor regression [133]. Currently, a phase I clinical trial is evaluating the efficacy of the TEAD inhibitor IK-930 combined with pembrolizumab in patients with advanced NSCLC with abnormal Hippo pathways, aiming to enhance the treatment response by reversing the immunosuppressor status of the microenvironment.

We have integrated a molecular mechanism diagram of the crosstalks between the Hippo signalling pathway and other classical signalling pathways (Fig. 3).

Fig. 3.

Schematic diagram of cell signalling mechanisms

PI3K-Akt signaling pathway can act on YAP/TAZ.Activation of Akt may affect the downstream effects of the Hippo signaling pathway by phosphorylating YAP/TAZ and altering its intracellular localization and activity.The Erk signaling pathway starts from Tyrosine kinase, and activates Erk via SOS, RAS, and MAPK.Activated Erk may affect gene expression and is associated with the Hippo signaling pathway, either indirectly or directly by regulating the expression of related genes. The activated Erk can affect gene expression and is associated with the Hippo signaling pathway, which may indirectly or directly affect the function of the components of the Hippo signaling pathway by regulating the expression of the relevant genes.In the JAK - STAT signaling pathway, JAK kinase phosphorylates and activates STAT3, and the activated STAT3 can act on YAP/TAZ, which may regulate the transcriptional activity of YAP/TAZ and affect its downstream effects. In the AMPK energy-activated pathway, AMP binds to AMPK and activates it, and activated AMPK can phosphorylate multiple targets, such as mTOR, ULK1, HMG-CoA Reductase, ACC, etc. This pathway can regulate cellular energy metabolism. This pathway regulates cellular energy metabolism and affects the activity of the Hippo signaling pathway through the regulation of related molecules, which may interact with key nodes such as LATS1/2, etc. In the Wnt signaling pathway, GSK-3, Axin, β-catenin, etc. are involved. In the Wnt signaling pathway, GSK-3, Axin, β-catenin, etc. This pathway can act on YAP/TAZ, for example, by regulating the stability and activity of β-catenin, it connects with YAP/TAZ of the Hippo signaling pathway, and affects the regulation of downstream gene expression. After binding to TEAD and TCF, YAP/TAZ regulates the expression of a series of downstream genes, including Ld1, Ld2, Axin, CyclinD, Slug, C-myc, etc., which affects the process of cell proliferation and differentiation.

4. Nexus between the Hippo signalling pathway and NSCLC development

Recent scientific investigations have shown that the aberrant expression of the YAP/TAZ proteins and the suppression of Hippo signalling pathway activity are pivotal contributors to oncogenesis. YAP and TAZ, which serve as central regulatory molecules within the Hippo pathway, are subject to meticulous regulation by a multilayered and multidimensional network of factors. This underscores the intricate complexity of the signalling cascade, wherein the activities of YAP and TAZ are finely tuned by a plethora of regulatory elements, ensuring precise control over cellular processes.

Furthermore, a plethora of studies have shown that inactivation of the Hippo signalling pathway is intricately linked to the malignant behavior of tumors. This includes the increase in tumor size, the increase in malignancy, the alteration of cellular senescence, and the development of resistance to antitumour therapies. These discoveries not only enrich our comprehension of the molecular underpinnings of cancer but also underscore the importance of the Hippo signalling pathway as a prospective therapeutic target. These findings provide a robust scientific foundation for the exploration of innovative anticancer strategies aimed at harnessing the Hippo pathway for therapeutic benefit.

The deregulation of the Hippo pathway, particularly the overactivation of YAP/TAZ, has emerged as a common theme in NSCLC progression. In NSCLC, the loss of Hippo pathway components, such as MST1/2 and LATS1/2, or gain-of-function mutations in YAP/TAZ contribute to uncontrolled cell proliferation and tumor growth. Moreover, dysregulation of the Hippo pathway can lead to increased cellular plasticity, enabling cancer cells to evade apoptosis, resist chemotherapy, and acquire invasive properties, thus facilitating metastasis.

The intricate interplay between the Hippo pathway and NSCLC underscores the potential of targeting this pathway for therapeutic intervention. Strategies aimed at reactivating the Hippo pathway or inhibiting YAP/TAZ activity hold promise for mitigating tumor growth and improving patient outcomes. For example, small molecule inhibitors that specifically target YAP/TAZ or upstream regulators of the Hippo pathway could serve as novel therapeutics. Additionally, combinatorial approaches that integrate Hippo pathway modulators with existing chemotherapy or immunotherapy regimens might increase treatment efficacy and overcome drug resistance.

In conclusion, dysfunction of the Hippo signalling pathway is intimately tied to the development and progression of non-small cell lung cancer. The growing body of evidence highlighting the role of this pathway in cancer biology underscores its potential as a therapeutic target. Continued research into the mechanisms governing the Hippo pathway and its interaction with other signalling cascades will undoubtedly unveil new avenues for cancer treatment, offering hope for improved clinical outcomes in patients with NSCLC.

4.1 Fundamental framework of the Hippo signalling pathway and its role in NSCLC

Upon activation of the Hippo pathway, a finely tuned regulatory mechanism ensues: the kinase activity of LATS1/2 is amplified, leading to the precise phosphorylation of YAP and TAZ. This interaction curtails YAP/TAZ nuclear entry and promotes their degradation, effectively quelling their transcriptional activator potential and decelerating cell proliferation, thus maintaining a delicate equilibrium between cell growth and apoptosis.

However, this intricate regulatory mechanism collapses when the Hippo signalling pathway is disrupted or inactivated. YAP and TAZ, which are unbound by phosphorylation, freely traverse the nuclear membrane, where they enter the nucleus, where they bind to TEAD family transcription factors to form a potent transcriptional complex. This complex markedly elevates the expression of genes involved in cell proliferation, anti-apoptosis, and metabolic reprogramming, igniting a “fire” of cell proliferation. This unbridled cell growth accelerates tumour formation and sets the stage for disease progression, potentially culminating in malignancy. Dysregulation of the Hippo signaling pathway plays a key role in tumorigenesis, progression and metastasis. Numerous preclinical and clinical studies have confirmed that in NSCLC, MST and LATS, the core kinases of the Hippo pathway, promote the binding of YAP/TAZ to the 14-3-3 protein through phosphorylation, thereby inhibiting the entry of YAP/TAZ into the nucleus and exerting its cancer-suppressive function [134]. Notably, both LATS1/2 gene fusion events as well as low expression levels of LATS1 have been reported to be significantly associated with poor prognosis in NSCLC patients [135, 136]. On the other hand, aberrant activation of YAP/TAZ as a downstream effector of the pathway is an important oncogenic driver of Hippo pathway dysregulation. Several studies have found that YAP/TAZ gene amplification has the second highest frequency in squamous lung cancer; also, the overexpression level of YAP/TAZ protein is closely associated with poor survival outcomes in NSCLC patients.

The TEAD family of transcription factors is a major downstream target of YAP/TAZ and is widely expressed in human tissues [137, 138]. Notably, TEAD alone is largely devoid of autonomous transcriptional activity, and its function is highly dependent on transcriptional co-activators (YAP/TAZ) to drive the expression of target genes [139]. Specifically, YAP/TAZ binds to the C-terminus of TEAD through its N-terminal structural domain to form the stable YAP/TAZ-TEAD transcriptional complex, which constitutes the core nuclear transcriptional module of the Hippo pathway [1]. In addition, the transcriptional activity of TEAD is finely regulated by its nuclear/cytoplasmic localization [140] as well as palmitoylation modifications [141, 142]. However, more in-depth studies are still needed to elucidate the specific effects of TEAD nuclear expression levels on the prognosis of NSCLC patients, as well as its potential clinical application as a novel biomarker for NSCLC.

4.2 Differential activation and regulation of the Hippo signalling pathway in NSCLC

Extensive scientific data suggest that the expression of key Hippo signalling pathway regulators is perturbed in NSCLC: inhibitors such as WW domain-containing E3 ubiquitin protein ligase 2(WWP2) [25] and Regulator of G protein signalling 20 (RGS20) [143] are aberrantly upregulated, whereas critical pathway activators LATS1/2 are downregulated, limiting their function. This imbalance contributes directly to the nuclear accumulation of the transcriptional coactivators YAP and TAZ, amplifying their oncogenic activity and driving uncontrolled growth, increased invasiveness, and metastatic spread in tumor cells. An illustrative example is the mechanism by which the WBP2 protein impedes the normal activation of the Hippo pathway through competitive binding to WW domain-containing protein 3 (WWC3) and LATS1, setting off a cascade of events that facilitate NSCLC progression and revealing subtleties in tumor microenvironment control [25].

Additionally, the role of circular RNA molecules such as circ-100,395 in modulating the Hippo pathway is intriguing. circ_100395, through its unique “molecular sponge” effect, efficiently adsorbs and neutralises miR-141-3p, leading to the re-expression of LATS1 [144]. This process indirectly reactivates the suppressed Hippo pathway, effectively restraining NSCLC development. This discovery expands our understanding of the role of circRNAs in cancer control and provides a foundation for investigating novel NSCLC treatment strategies.

Collectively, these findings illuminate the complex regulatory dynamics of the Hippo signalling pathway in the malignant transformation of NSCLC, underscoring its paramount role as a central regulatory hub that orchestrates tumor cell behavior and disease progression. These findings contribute significantly to our scientific comprehension of the molecular mechanisms of NSCLC and offer crucial insights for developing innovative targeted therapies against the Hippo signalling pathway in the future.

In the study of clinical cases, the key components of the Hippo pathway, YAP and TAZ, show significant abnormal expression characteristics in NSCLC. Multiple studies have shown that the level of YAP protein increases in 66.3%−87.8% of lung adenocarcinoma (LAC) tissues. The enhancement of its nuclear localization is closely related to advanced TNM, lymph node metastasis, EGFR amplification and poor prognosis [145]. It is worth noting that the YAP1 gene was amplified in 23% of NSCLC tissues, and the missense mutation of R331W was found to be associated with the susceptibility to lung adenocarcinoma [83]. TAZ was overexpressed in 66.8% of NSCLC and was significantly associated with tumor invasion, metastasis and shortened survival period [146], and the prognosis was worse when combined with YAP [145]. Conversely, the tumor suppressor LATS1/2 shows promoter hypermethylation and down-regulation in NSCLC [147], and its low expression is associated with a better prognosis and longer survival period [32, 148]. These findings suggest that components of the Hippo pathway may serve as important prognostic indicators and potential therapeutic targets for NSCLC.

4.3 The Hippo signalling pathway and therapeutic strategies for NSCLC

Given the prominent oncogenic impact of the Hippo signalling pathway in NSCLC, targeting this pathway has become a focal point in cancer research. Numerous compelling studies have demonstrated the potential of both natural compounds and novel chemically synthesised molecules in modulating the Hippo pathway. Extracts from Taxus chinensis var. mairei, notably water-soluble ones, alongside synthetic molecules such as trans-3,5,4’-trimethoxystilbene, have been shown to activate the Hippo signalling pathway and inhibit the nuclear translocation of YAP [149]. These molecules significantly curb NSCLC proliferation and metastasis, expanding the resource pool for antitumour drug discovery and development while opening new avenues in synthetic chemistry.

Studies have shown that the Hippo effector molecule YAP/TAZ can activate downstream EGFR signals or mediate EGFR-TKI resistance [150]. Preclinical experiments have shown that YAP inhibitors (such as verteporfin) combined with osimertinib can significantly inhibit the growth of drug-resistant tumors. Hippo and immune checkpoints: The aggregation of YAP nuclei caused by the inactivation of the Hippo pathway can up-regulate the expression of PD-L1, suggesting that the combination of YAP inhibitors and PD-1/PD-L1 antibodies may enhance the immune efficacy [151]. Cross-molecules such as ZMIZ2 can collaboratively regulate Hippo/YAP and Wnt/β-catenin signals. Targeting such nodes may achieve dual-pathway inhibition [92].

Moreover, innovative strategies are emerging to indirectly curtail YAP activity by precisely regulating key upstream Hippo pathway molecules, including WWC3, LATS1/2, and circular RNAs (circRNAs). Such approaches offer a fresh perspective on NSCLC treatment. Targeted upregulation of CircPACRGL expression, for example, subtly diminishes YAP stability and activity, effectively thwarting its tumorigenic functions [152]. Similarly, the development and application of small molecule inhibitors such as GA-017 seek to accurately restore the dysregulated activity of the Hippo signalling pathway. Early experimental outcomes suggest that this compound holds substantial promise in NSCLC therapy [143].

These pioneering studies not only elucidate diverse strategies for inhibiting YAP activity through direct activation of the Hippo signalling pathway or indirect regulation of its critical components but also introduce novel and promising interventions for the clinical management of NSCLC [153]. These findings not only deepen our understanding of the molecular mechanisms underlying NSCLC but also lay a robust theoretical and experimental groundwork for more precise and efficacious personalised treatments. By harnessing the regulatory potential of the Hippo pathway, researchers are paving the way for advanced therapeutic modalities that could revolutionise NSCLC care, offering renewed hope to patients facing this challenging disease.

In summary, the pivotal role of the Hippo signalling pathway in NSCLC pathogenesis presents a rich landscape for therapeutic exploration. The identification of natural and synthetic modulators, coupled with the discovery of novel regulatory mechanisms, opens multiple fronts for attacking the disease. These advancements, from the utilisation of traditional plant-derived compounds to the design of sophisticated small-molecule inhibitors, collectively represent a significant advance in NSCLC treatment strategies. As research progresses, the prospect of tailored therapies that specifically target the Hippo pathway has become increasingly feasible, heralding a new era in cancer therapy characterised by greater specificity, fewer side effects, and improved patient outcomes [154–157].The following are several relatively common inhibitors of the Hippo signaling pathway.

4.3.1 YAP-TEAD inhibitors: verteporfin

Originally utilised in photodynamic therapy for age-related macular degeneration under the brand name Visudyne, verteporfin was serendipitously discovered to inhibit YAP function even in the absence of light. Its mechanism primarily disrupts the interaction between YAP and TEAD family transcription factors, consequently suppressing YAP-mediated gene transcription. This characteristic has positioned verteporfin as a promising YAP inhibitor, demonstrating therapeutic potential in various disease models. Verteporfin inhibits tumor cell growth, migration, and invasion; influences fibrosis; dampens inflammatory responses; and promotes tissue repair [158].

At the cellular level, verteporfin diminishes the concentrations of YAP and its phosphorylated form(S127), reducing the nuclear localisation of YAP. In nephropathy models, verteporfin inhibits angiogenesis by downregulating components of the Hippo pathway and YAP signalling, preventing the dedifferentiation of renal tubular epithelial cells and suggesting its indirect role in modulating cell fate decisions [159]. In diabetic neuropathy, it mitigates lipid peroxidation and apoptosis and restrains vascular endothelial cell dedifferentiation by suppressing YAP activity, highlighting its multifaceted effects in complex pathologies [160]. In terms of molecular mechanisms, verteporfin regulates YAP activity through a dual mechanism: Firstly, it promotes the binding of YAP to 14-3-3σ by up-regulating the expression of 14-3-3σ protein, thereby isolating YAP from the cytoplasm and targeting proteasome degradation. The second is to activate the expression of 14-3-3σ through the p53-dependent pathway, revealing that the functional state of p53 is the key molecular basis for the therapeutic effect of verteporfin. This p53/14-3-3σ/YAP regulatory axis not only expands the mechanism of action of verteporfin, but also provides potential biomarkers for precise screening of the p53 wild-type patient population to optimize its clinical application [161].

The aberrant activation of YAP is intricately linked to tumor progression, invasion, and metastasis in several malignancies, notably breast, melanoma, pancreatic, and hepatocellular carcinomas. By hindering the formation of the YAP-TEAD complex, verteporfin reduces the expression of YAP target genes, including those involved in fibrosis, angiogenesis, and immune cell recruitment (e.g., CTGF, CCN1, and CCN2). This leads to decreased tumor cell proliferation and migration and augmented sensitivity to chemotherapeutic agents. In melanoma, the antiproliferative and migratory effects of verteporfin correlate with reduced YAP concentrations and enhanced apoptosis, particularly in YAP-overexpressing cell lines, underscoring the pivotal role of YAP as a target for the antitumour activity of verteporfin [67, 162–164].

Emerging evidence suggests that verteporfin may exert YAP-independent antitumour effects, such as regulating cell proliferation and senescence by inhibiting the focal adhesion kinase (FAK) signalling pathway and enhancing TP53-dependent apoptosis by curtailing Murine Double Minute 2 (MDM2)-mediated TP53 degradation in glioblastoma [165]. These additional modes of action may confer a unique therapeutic edge to verteporfin in combating drug-resistant cancers.

In addition to oncology, the potential of verteporfin in treating other diseases is increasingly recognised. In diabetic nephropathy, it reduces podocyte loss while increasing renal tubular epithelial cell apoptosis, highlighting the need for precise dosage and target modulation. In hepatic fibrosis, verteporfin effectively curbs fibrosis and inflammation, improving liver function by inhibiting YAP/TAZ activity [159]. In heterotopic ossification, it reduces trauma-induced ectopic bone formation by suppressing the YAP/coactivator signalling axis, suggesting potential applications in skeletal disorders [166].

Although verteporfin has demonstrated multiple effects such as anti-tumor and anti-fibrosis in multi-disease models, its clinical transformation is limited by issues such as delivery efficiency, off-target toxicity and drug resistance. In contrast, the novel inhibitor MYF-01-37 achieves more efficient inhibition of oncogene transcription by precisely targeting the palmitoylation pocket of TEAD (the key site C359), competitively blocking the formation of the YAP-TEAD complex through hydrophobic interaction and hydrogen bond networks, and interfering with the palmitoylation modification dependent on the stability of TEAD. This design breaks through the traditional upstream kinase regulation mode. By directly intervening in the functional dependent domain of TEAD, it significantly improves selectivity and reduces toxicity in normal tissues, providing a new idea for overcoming YAP-dependent tumor drug resistance. In the future, it is necessary to further verify its in vivo delivery efficiency, the synergistic effect with immunotherapy, and develop precise typing strategies based on TEAD mutation profiles or YAP activity to promote the transformation of Hippo targeted therapy from mechanism innovation to clinical benefits.

4.3.2 YAP inhibitors: compound CA3

Compound CA3 (CA3) has garnered considerable interest as a recently discovered YAP inhibitor because of its unique biological properties and therapeutic potential, particularly in neuroscience and myocardial regeneration.

YAP plays a critical role in neuronal development, synaptic plasticity, learning, memory, and emotion regulation. Recent studies have shed light on the impact of YAP modulation within the hippocampal CA3 region. For example, Takeda G protein-coupled receptor 5 (TGR5) was found to influence depression-like behaviours by regulating neuronal activity in the CA3 area, specifically through enhanced feed-forward inhibition (FFI) of CA3 parvalbumin-positive interneurons (PV + INs). This mechanism supports the reorganisation of neural networks, facilitating the development of cortical neuron populations during memory consolidation. Although direct evidence linking CA3 and the YAP inhibitor CA3 is scarce, it is hypothesised that the regulation of YAP activity indirectly influences neuroplasticity within the CA3 region, impacting memory formation and emotional states [167].

Furthermore, CA3’s significance in pattern separation and completion has been established. Considering the role of YAP in promoting cell proliferation and tissue repair, CA3 modulation by YAP inhibitors may indirectly influence these cognitive functions by affecting neuroplasticity within the CA3 area [168]. By reversing the abnormal excitability of the CA3 region, YAP inhibitors could restore youthful neuronal activity patterns, enhancing memory function in ageing organisms.

In cardiac regeneration, YAP activation has been shown to stimulate CM proliferation. Studies have explored the effects of YAP activators on the proliferation of adult human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiac progenitor cells, underscoring the crucial role of YAP in myocardial regeneration [169]. While direct investigations into the application of CA3 in myocardial regeneration remain limited, the theoretical utility of the YAP inhibitor CA3 in controlling excessive cardiomyocyte proliferation or fine-tuning myocardial regeneration processes is evident [170]. By inhibiting overactive YAP, CA3 might aid in regulating hiPSC-CM proliferation, particularly in high-density culture conditions where YAP activity is naturally reduced, suggesting the potential inhibitory effect of CA3 under specific circumstances.

Research into neurodegenerative diseases, notably Alzheimer’s disease (AD), has pinpointed synaptic dysfunction in the CA3 region as a key factor in disease progression. Studies have demonstrated that amyloid-beta (Aβ) inhibits NMDAR currents in CA3‒CA1 synapses in wild-type mice, an effect not observed in tau protein knockout mice, implicating tau protein and Aβ in mediating synaptic sensitivity. Although the direct effects of the YAP inhibitor CA3 were not assessed, these findings highlight the vulnerability of the CA3 region to neurodegeneration and provide a foundation for future explorations into the role of YAP in neuroprotection and disease intervention [171, 172].

Compound CA3 shows promising potential in neuroscience and cardiac regeneration. Despite the scarcity of direct experimental data concerning CA3, the literature indirectly underscores the importance of YAP activity in regulating neuronal plasticity, memory formation, emotion regulation, and CM proliferation. Future studies should delve deeper into the specific mechanisms underlying the effects of CA3, particularly those focused on neural network dynamics, synaptic plasticity, and cardiac regeneration in the context of YAP inhibition. Given the multifaceted roles of YAP across different cell types and disease models, developing more selective YAP inhibitors to finely tune YAP activity represents a crucial direction for future therapeutic strategies. In the context of cardiac health, further insights into the potential value of YAP are needed.

4.3.3 YAP inhibitor K-975: targeting the YAP/TAZ-TEAD interaction

The YAP inhibitor K-975, a novel compound that specifically targets the YAP/TAZ-TEAD protein‒protein interaction, has garnered significant interest in molecular medicine. Owing to its substantial therapeutic potential, it holds promise for treating a variety of diseases, including malignant mesothelioma, Chronic Obstructive Pulmonary Disease (COPD)-related respiratory inflammation, and liver regeneration [173].

K-975 was developed to target a conserved cysteine residue within the palmitoylation-binding pocket of the TEAD protein, inhibiting the YAP1/TAZ-TEAD protein‒protein interaction through covalent binding. This mechanism was validated via X-ray crystallography [174].

In malignant mesothelioma cell lines lacking NF2 expression, K-975 exhibited potent antitumour activity, significantly reducing the size of subcutaneously implanted tumours in mice and extending model survival. When combined with chemotherapeutic agents such as pemetrexed or cisplatin, K-975 demonstrated greater antitumour effects than did cisplatin alone, highlighting its potential in mesothelioma therapy. The weak inhibition of NF2-expressing cell lines suggests that NF2 status could serve as a biomarker for K-975 responsiveness in clinical trials [174].

In addition to its oncological applications, K-975 has shown therapeutic potential in nonmalignant conditions. In adult retinal pigment epithelial cells, K-975 suppressed connective tissue growth factor (CTGF) expression and disrupted YAP/TAZ-TEAD signalling, suggesting its potential in treating fibrotic eye diseases [175]. Furthermore, K-975 stabilises β-catenin complexes and inhibits airway inflammation and hyperreactivity induced by toluene diisocyanate in asthma models, indicating its potential as a novel therapy for treating steroid-resistant asthma [176].

As a potent YAP/TAZ-TEAD PPI inhibitor, K-975 offers unique advantages in cancer treatment and has broad therapeutic potential for nonneoplastic diseases such as ocular fibrosis and respiratory inflammation. Its covalent binding mechanism and selectivity open new avenues for Hippo signalling pathway-targeted therapies. However, prior to clinical implementation, the long-term safety of K-975, its synergistic effects with other drugs, and strategies to overcome potential resistance must be thoroughly investigated. Understanding the complex regulatory mechanisms of the YAP/TAZ-TEAD signalling pathway in various tissues and diseases will advance precision medicine, offering patients more personalised and efficacious treatments.

The development and study of K-975 and its analogues will deepen our understanding of cellular signalling regulation, potentially revealing novel insights into regulatory mechanisms and their clinical implications. This work aims to uncover new dimensions of regulation and their correlation with therapeutic benefits.

4.3.4 A novel YAP-TEAD binding inhibitor: MYF-01-37

Recently, a novel YAP-TEAD binding inhibitor, MYF-01-37, has been developed and exerts its effect by specifically targeting the palmitoylated pocket (the key cysteine C359 site) of the TEAD protein. This molecule precisely empresses into the conserved domain of TEAD1 through hydrophobic interactions and hydrogen bond networks, competitive blocking the binding of the PPxY motifs of YAP to the Ω-loop of TEAD, thereby inhibiting the formation of the YAP-TEAD complex and the transcriptional activation of downstream oncogenes (such as CTGF, CYR61). Meanwhile, MYF-01-37 may interfere with the palmitoylation modification process of TEAD, further weakening its stability and function. Compared with traditional Hippo pathway inhibitors, its design of directly targeting TeAD-dependent modifications has both high selectivity and potential low toxicity, providing a new strategy for overcoming YAP-dependent tumor drug resistance. In the future, its in vivo efficacy and potential for combined treatment need to be further verified [177].

We analyzed and summarized small molecule inhibitors related to the Hippo pathway, listed in Table 1.

Table 1.

Targeted therapy of the Hippo signalling pathway

Inhibitor name	Target protein	Disease model	Main mechanism of action	Therapeutic effect
Verteporfin	YAP-TEAD interaction	Tumour, diabetic nephropathy, liver fibrosis, ectopic ossification	YAP binding to TEAD was blocked, YAP concentration and nuclear localisation were reduced, and YAP target gene expression was inhibited	Reduce tumour growth, migration, invasion; Affects fibrosis, inflammation and tissue repair; Protects kidneys and neurons
CA3	YAP	Cardiac regeneration, neurodegenerative diseases such as Alzheimer’s disease	Inhibition of over-activated YAP, regulation of hiPSC-CM proliferation, may affect neuronal network dynamics, synaptic plasticity and cardiac regeneration	Control of cardiomyocyte overproliferation with potential implications for neuroprotection and disease intervention
K-975	YAP/TAZ-TEAD PPI	Malignant mesothelioma, COPD related respiratory inflammation, liver regeneration	Specifically targeted YAP/TAZ interaction with TEAD to inhibit YAP-mediated gene transcription	Treat a variety of diseases, including malignancies and organ inflammation
MYF-01-37	YAP-TEAD interaction	NSCLC	Inhibition of BMF by YAP/TEAD/Slug	Promote cancer cell apoptosis

4.4 Clinical research on drugs targeting the Hippo signaling pathway

Despite compelling preclinical evidence implicating Hippo pathway dysregulation (particularly YAP/TAZ hyperactivity) in NSCLC pathogenesis and treatment resistance, direct clinical translation remains nascent. While four anti-cancer drugs targeting the Hippo signaling pathway in the recruitment stage of Phase I clinical trials: IAG933 targets advanced mesothelioma and solid tumors with NF2/LATS1/LATS2 gene variations or YAP/TAZ fusions by inhibiting the YAP-TEAD interaction; IK-930, BPI-460372BBB and VT3989 all target the palmitoylation modification of TEAD, and their indications cover malignant mesothelioma, epithelioid hemangioendothelioma and solid tumors with Hippo pathway abnormalities such as NF2 deletion, YAP/TAZ fusion and LATS1/2 mutation. All trials focused particularly on patients with advanced or metastatic tumors who did not respond to standard treatment and are currently in the process of recruiting subjects [178]. However, their absence in dedicated NSCLC trials underscores key challenges: pathway redundancy, on-target toxicity risks (due to Hippo’s role in organ homeostasis), and the limited druggability of transcriptional co-activators.

Future translation of Hippo-targeted therapies requires: (1) Development of safer, brain-penetrant YAP/TAZ-TEAD inhibitors optimized for NSCLC subtypes; (2) Validation of Hippo activity biomarkers (e.g., CTGF serum levels, YAP/TAZ nuclear localization) to enrich patient selection in early trials; (3) Rational combination studies with standard-of-care agents (e.g., TKIs, immunotherapy, platinum-doublets) guided by resistance mechanisms; and (4) Leveraging advanced models (e.g., PDX organoids) to predict efficacy and bypass compensatory signaling. Collaborative initiatives like the NCI MATCH platform could accelerate testing of Hippo modulators in biomarker-selected NSCLC populations. While clinical maturity is evolving, the Hippo pathway represents a high-potential frontier for overcoming persistent therapeutic challenges in NSCLC.

5. The Hippo pathway: a nexus connecting drug resistance and lung cancer therapy

The abnormal activation of the Hippo signaling pathway shows pleiotropic regulatory characteristics in the field of tumor drug resistance. Studies have shown that the upregulation of FZD10 mediated by N6-methyladenosine significantly enhances the resistance of hepatoma cells to lenvatinib by cooperatively regulating the Wnt and Hippo signaling axes, revealing a new mechanism of the interaction between epigenetic modifications and signaling pathways [179]. It is notable that this multi-pathway synergistic regulatory pattern is widespread in various tumor drug resistations: In breast cancer, the deletion of the FAT1 tumor suppressor gene drives CDK4/6 inhibitor resistance by releasing the inhibition of YAP by the Hippo pathway [180, 181]; Preclinical studies of pancreatic cancer have confirmed that irbesartan can restore chemosensitivity by blocking the Hippo/c-Jun signaling axis [182]. In rapidly changing chronic myeloid leukemia, abnormal FLT3-TAZ signal transduction has been confirmed as a key target for drug resistance [183]. The new mechanism by which Fusobacterium nucleatum in colon cancer inhibits pyroptosis through the Hippo pathway further expands the dimension of the role of this pathway in mediating drug resistance in the tumor microenvironment [184]. These cross-cancer studies jointly reveal the pivotal position of the Hippo signaling network in the regulation of drug resistance - it can not only serve as an independent driver but also form a dynamic regulatory network with epigenetic modifications, tumor stem cell characteristics, and the microbiome. Based on this core discovery, in-depth exploration is urgently needed: During the drug resistance process of NSCLC, how does the Hippo pathway regulate the drug resistance phenotype through unique molecular interaction interfaces?

Drug resistance in NSCLC manifests predominantly in two forms: acquired resistance, often triggered by prolonged exposure to EGFR tyrosine kinase inhibitors (TKIs), and primary resistance, typically associated with KRAS gene mutations. Among these, the emergence of the T790M mutation in the epidermal growth factor receptor (EGFR) stands out as a principal cause of TKI resistance in lung cancer patients [151].Studies on drug resistance and the Hippo signaling pathway have found that inhibition of the MAPK signaling pathway in lung cancer leads to mislocalization of the polarity protein Scribble, which inhibits the Hippo pathway, contributing to the activation of YAP into the nucleus, which in turn up-regulates the expression of its target, MRAS. Induced expression of MRAS activated MAPK signaling by binding to SHOC2 feedback, driving resistance to KRAS G12C inhibitors, while blocking YAP activation or MRAS expression significantly enhanced the efficacy of the inhibitors in vivo [185].

The research has revealed the multi-dimensional regulatory mechanism of the YAP signaling axis in the drug resistance of targeted therapy for non-small cell lung cancer. The core discovery shows that YAP mediates EGFR-TKI resistance by activating downstream AXL receptors, and its expression level directly affects the sensitivity of tumor cells - YAP silencing can disintegrate the drug resistance phenotype, while overexpression significantly weakens the drug response. It is worth noting that AXL, as a YAP-dependent effector molecule, its activity inhibition can effectively reverse drug resistance, which provides a key entry point for targeted intervention [186]. Further studies have shown that YAP1 also enhances the activity of the EGFR/MEK pathway by inducing resistance to ROS1 inhibitors and synergistic down-regulation of DUSP1 by YY1, while the YAP1 inhibitor CA3 significantly sensitizes the efficacy of the third-generation EGFR-TKI by restoring DUSP1 expression and activating autophagy [77]. Furthermore, CDK4/6 amplification has been confirmed to serve as a predictive biomarker for primary drug resistance in patients with EGFR mutations [187]. The discovery that the active ingredients of traditional Chinese medicine enhance dridity and chemosensitivity by activating the Hippo/p53 signaling pathway has expanded the feasibility of the combined treatment strategy [188, 189]. These breakthroughs not only analyzed the complexity of the drug resistance mechanism, but also laid the foundation of translational medicine for the development of combination therapies based on the regulation of the YAP signaling axis (such as EGFR/YAP dual targeting).

The Hippo signalling pathway, particularly the overexpression of YAP, a key molecule within this pathway, has emerged as a predictive marker for reduced sensitivity to TKIs among patients. Conversely, TAZ overexpression has been implicated in decreased resistance to gefitinib, with TAZ knockdown resulting in the opposite effect. Similarly, YAP overexpression has been shown to augment resistance to EGFR-targeted therapies. Notably, silencing YAP restores sensitivity in EGFR-TKI-resistant cells, highlighting the potential of targeting the Hippo pathway to overcome drug resistance in NSCLC [77].

6. Conclusions

The incidence and mortality of lung cancer have been ranked first among various malignant tumors, and most of its pathological types (about 85%−90%) are NSCLC, while systemic spread due to tumor invasion and metastasis is the main cause of death for about 80% of patients [190, 191]. Despite the progress of targeted therapy and immunotherapy in recent years, the prognosis of advanced patients is still unsatisfactory. Therefore, revealing the molecular mechanisms of lung cancer invasion and metastasis and searching for new markers of the biological behavior of lung cancer are not only beneficial for the prognostic assessment of lung cancer, but also provide experimental bases and new targets for the development of effective targeted therapeutics, which is of great theoretical and practical significance. And Hippo signaling pathway is a highly conserved signaling pathway during biological development. This review provides an overview of the basic architecture of the Hippo signaling pathway and summarizes that dysregulation of the Hippo signaling pathway plays a central role in the occurrence, metastasis and therapeutic resistance of NSCLC. This review systematically elucidates the molecular mechanism by which YAP/TAZ drives the malignant phenotype of tumors through the regulation of downstream target genes and interacting pathways and reveals its dual roles in TME immunoregulation.

First, the development of targeted therapeutic agents against Hippo signaling pathway for NSCLC has high clinical value. It provides a key therapeutic strategy for NSCLC: ① Direct targeting of YAP/TAZ-TEAD complexes such as vitexpofol and K-975 significantly inhibits tumor growth and reverses PD-L1-mediated immune escape by blocking the interaction of YAP/TAZ with TEAD. Inhibitors targeting TEAD palmitoylated modifications exhibit high selectivity and avoid off-target toxicity of conventional kinase inhibitors [177]. ② The development of targeted drugs has been equally beneficial for combination therapy, and when drugs targeting the Hippo signaling pathway are used in combination with EGFR-TKIs, YAP inhibitors can overcome ositinib resistance, especially in subpopulations with LATS1/2 deletion or YAP nuclear clustering [133]. In addition, when combined with immune checkpoint inhibitors, preclinical models showed that TEAD inhibitors synergized with anti-PD-L1 antibodies to enhance CD8 + T cell infiltration and deplete Tregs.③ Several studies have demonstrated that epigenetic modifications of key proteins in the Hippo signaling pathway affect the activity of the pathway. Targeting Non-Coding RNAs Provides New Avenues for Low-Toxicity Therapies by Activating LATS1 or Inhibiting YAP Stability [61].

Although numerous studies have revealed the important role of the Hippo signaling pathway in the development of NSCLC, there are still several key scientific questions that need to be addressed in this field. First, the heterogeneity of the regulatory network of the pathway has not been elucidated: in the two major NSCLC subtypes, EGFR-mutant and KRAS-mutant, members of the Hippo pathway show significant differential expression characteristics. In addition, the spatial distribution pattern of YAP/TAZ in different tumor microenvironments was negatively correlated with the degree of immune cell infiltration, suggesting that it may form an immunosuppressive microenvironment by regulating immune checkpoint molecules such as PD-L1. Notably, the aberrant activation of TEAD4 in KRAS-mutant tumors may promote tumor progression through metabolic reprogramming pathways, which provides new ideas for subtype-specific therapeutic strategies. Second, the molecular basis of the resistance mechanism has not been fully resolved. Although in vitro experiments have demonstrated that YAP/TAZ nuclear translocation induces EGFR-TKI resistance, there is insufficient research on the correlation between EGFR-TKI resistance and the Hippo signaling pathway as reflected in clinical cases. Moreover, the process of drug resistance may involve changes in the Hippo pathway and the specific mechanisms causing the changes remain unclear. In addition, differences in YAP/TAZ activity thresholds due to tumor stem cell heterogeneity may affect drug sensitivity, which needs to be dynamically validated in a three-dimensional microenvironment through organoid models. Finally, clinical translation faces multiple challenges. Currently conducted clinical trials of Hippo-targeted therapies generally suffer from unclear enrollment criteria and imperfect efficacy assessment systems. This requires precise dosing regimen design guided by biomarkers. A dynamic monitoring system can be established by using circulating tumor DNA combined with imaging histology, while exploring the treatment optimization model with the sequence of immune checkpoint inhibitor use.

Recent studies have further revealed that YAP, a core effector molecule of the Hippo pathway, exhibits significant cell/tissue environment-dependent dual functions in tumorigenesis, and its role may be fundamentally reversed by cancer type, molecular microenvironment, or disease stage. The conventional view is that YAP exerts its proto-oncogene function by promoting cell proliferation and inhibiting apoptosis, a mechanism that has been widely validated in hepatocellular carcinoma, breast cancer, and triple-negative breast cancer. However, in colon cancer and hematologic malignancies, YAP exhibits paradoxical tumor suppressor effects. For example, in CRC, deletion or inactivation of YAP unexpectedly enhances tumor cell invasion and metastasis by deregulating the JNK-c-Jun signaling axis [192]; Similarly, in AML models, high YAP expression inhibits leukemic stem cell self-renewal through activation of the pro-differentiation pathway, while its silencing leads to accelerated disease progression [193]. Thus, while targeting the Hippo signaling pathway is beneficial for cancer treatment, the normal function of YAP/TAZ in the homeostasis of human tissues may raise concerns about the safety of YAP/TAZ-TEAD-targeted therapy. Of particular concern is the risk of nephrotoxicity: animal experiments have shown that podocyte-specific YAP gene deletion triggers progressive kidney injury through activation of apoptotic pathways and irreversible cell number reduction [194]. This mechanism suggests that YAP/TAZ-TEAD inhibitors not only carry their own risk of nephrotoxicity, but may also amplify the renal damaging effects of existing therapeutic regimens-especially in a population of conventionally treated patients with a well-defined risk of renal injury, such as those receiving radiotherapy [194]. Notably, inhibition of this signaling axis may trigger multisystemic risks. At the level of immune regulation, the modulatory effect of YAP/TAZ on immune homeostasis implies that its inhibitors may exacerbate the state of immunosuppression in immunodeficient patients. At the level of tissue repair, since YAP/TAZ is involved in the repair of multiple organ injuries through the regulation of stem cell renewal, inhibition of this signaling axis may disrupt the dynamic balance between tissue regeneration and maintenance of function. Typical examples can be seen in the intestinal system: when intestinal stem cells are depleted by radiotherapy, the key role of the YAP signaling pathway in epithelial regeneration makes its inhibitors likely to exacerbate treatment-associated intestinal mucosal injury [195].

Based on the multidimensional regulatory properties of Hippo signaling pathway in NSCLC, we make a prospect for future research. First, to establish a multi-omics-driven precise typing system: by integrating single-cell sequencing, spatial transcriptome and phosphorylated proteome data, we resolved the dynamic activation characteristics of the Hippo pathway in different driver gene subtypes such as EGFR/KRAS/ALK. Using the organoid drug sensitivity screening platform, we systematically map the synergistic effects of YAP/TAZ inhibitor vitexoporfin with MEK inhibitors and other targeted drugs, and then establish an individualized drug decision-making model based on the pathway activity score. Second, the development of nano-delivery system: for the existing Hippo-targeted drugs with low bioavailability and off-target toxicity, multi-functional nanocarriers responsive to the tumor microenvironment can be designed. To realize the precise delivery of targeted drugs. Finally, innovative combined mechanics-immunity intervention strategy: on the one hand, it is necessary to elucidate the mechanics mechanism of YAP/TAZ by regulating the formation of pre-metastatic ecological niche of immunosuppressed cells, and develop cell-based therapy combined with mechanosensitive drugs; on the other hand, the use of cutting-edge technology to build a bionic tumor mechanics microenvironment, screen small molecule compounds targeting the axis of the mechanical stress-Hippo pathway and achieve dynamic mechanics microenvironment by means of a biodegradable hydrogel. On the other hand, cutting-edge technology was used to construct a biomimetic tumor mechanical microenvironment, screen small molecule compounds targeting the mechanical stress-Hippo pathway axis, and realize dynamic regulation of the mechanical microenvironment through biodegradable hydrogel. These breakthroughs will promote the development of Hippo pathway research from molecular mechanism to clinical translation.

Abbreviations

AD

Alzheimer’s disease

Akt

Protein Kinase B

AMOT

Angiomotin family proteins

AMPK

Adenylate activated protein kinase

APAP

Acetaminophen

BIP

Binding Immunoglobulin Protein

CAFs

Cancer-associated fibroblasts

CCN1

Cyr61

CCN2

CTGF

CCN3

NOVI

CCN4

WISP1

CCN5

WISP2

CCN6

WISP3

circRNAs

Circular RNAs

CML

Chronic Myeloid Leukemia

COPD

Chronic Obstructive Pulmonary Disease

CRB3

Crumbs3

DUB3

Deubiquitylating Enzyme 3

EC

Endothelial cells

ECM

Extracellular matrix

EGFR

Epidermal growth factor receptor

EMT

Epithelial-to-mesenchymal transition

ER

Endoplasmic reticulum

ErbB

Erythroblastic Leukemia Viral Oncogene Homologue

ERK

Extracellular Signal-Regulated Kinase

FAK

Focal adhesion kinase

Fas

Fas Cell Surface Death Receptor

FERM

Four-point-one, ezrin, radixin, moesin

FFI

Feed-forward inhibition

FoxO

Forkhead Box O

GC

Gastric cancer

GPCRs

G-protein coupled receptors

GSK-3

Glycogen Synthase Kinase 3 Beta

GTase

Guanine Nucleotide Binding Protein

hiPSC-CMs

Human-induced pluripotent stem cell-derived cardiomyocytes

HSC

Hepatic stellate cells

IL-6

Interleukin 6

IL-8

Interleukin 8

ISG

Interferon-stimulated gene

JAG1

Jagged 1

JAK

Janus Kinase

KIBRA

Kidney and Brain protein

LATS1

Large Tumor Suppressor Kinase 1

lncRNAs

Long noncoding RNAs

LPA

Phosphatidic acid

MAML

Mastermind-like proteins

MAPK

Mitogen-Activated Protein Kinase

MDM

Murine Double Minute 2

MFN2

Mitofusin 2

miRNAs

MicroRNAs

MOBKL1A/B

MOB kinase activator 1 A/B

MST1/2

Mammalian STE20-like protein kinases 1/2

mTOR

Mammalian Target of Rapamycin

NEDD4L

Neural precursor cell expressed developmentally downregulated 4-like

NF2

Neurofibromatosis type 2 protein

NICD

Notch intracellular domain

P53

Tumor Protein 53

P73

P53 Family Member

PI3K

Phosphoinositide 3-Kinase

PKC

Protein Kinase C

PLC

Phospholipase C

PTPN14

Protein Tyrosine Phosphatase Nonreceptor Type 14

Raf

Rapidly Accelerated Fibrosarcoma

Ras

Rat Sarcoma Viral Oncogene Homologue

RASSFs

RAS-Association Domain Family Proteins

RBPJ

Recombination Signal Binding Protein for Immunoglobulin kappa J region

RGS20

Regulator of G protein signalling 20

RunX2

Runt-Related Transcription Factor 2

S1P

sphingosine-1-phosphate

SAV1

Salvador 1

SLE

Systemic lupus erythematosus

SMAD

Mothers Against Decapentaplegic”

SIRT1

Silent Information Regulator 2 Homologue 1

STAT

Signal Transducers and Activators of Transcription

TAZ

WW domain-containing transcriptional regulator 1

TRII

TGF- receptor types II

TRI

TGF- receptor types I

TEAD

Transcriptional Enhancer of Activator

TET1

Ten-Eleven Translocation 1

TGF-

Transforming Growth Factor-beta

TGR5

Takeda G protein-coupled receptor 5

TKIs

Tyrosine kinase inhibitors

TNBC

Triple-negative breast cancer

Wnt

Wingless-type MMTV Integration site Family Member

WTS

Warts

WW45

WW Domain Containing Protein 45

WWC3

WW domain-containing protein 3

WWP2

WW domain-containing E3 ubiquitin protein ligase 2

WWTR

WW Domain Transcription Regulator

YAP

Yes-associated protein

YKI

Yorkie

Author contributions

YHF and XTG searched and summarised all published studies, prepared the tables and figures and wrote the original draft. QH and XZR were responsible for the critical revision of the manuscript. All authors approved the final version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China and Sponsored by Tianjin Health Research Project (grants 81902986 to Qiang Han, grants 82003119 to Xuezhu Rong, grants TJWJ2023QN004 to Shulei Sun).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All the authors provided consent for the publication of this study.

Competing interests

The authors declare no competing interests.