The propensity for proteins to undergo LLPS is further influenced by environmental factors, including temperature, pH, ionic strength, and macromolecular crowding agents. These factors primarily influence multivalent interactions underpinning condensate formation, such as π-π stacking, cation-π interactions, electrostatic forces, hydrophobic interactions, and hydrogen bonding. For instance, the Bub3-interacting and GLEBS-containing protein Z (BuGZ) undergoes LLPS, predominantly driven by hydrophobic interactions, exhibiting pronounced temperature sensitivity [21]. In contrast, superoxide dismutase 1 (SOD1) and microtubule-associated protein Tau form condensates governed by both electrostatic and hydrophobic forces, rendering them highly sensitive to salt concentrations due to electrostatic shielding effects [27, 28]. Notably, LLPS in most proteins results from the cooperative modulation of multiple factors. This is exemplified by nuclear ribonucleoprotein A1 (hnRNPA1), whose LLPS is modulated by its low-complexity domain (LCD) and aromatic amino acid residues. Consequently, hnRNPA1 condensate formation is critically dependent on multiple factors, including ionic strength, pH, and temperature [29]. Furthermore, macromolecular crowding agents promote condensate formation by mimicking the intracellular milieu and enhancing local protein concentrations [29,30,31].
LLPS is a fundamental biological process governing diverse cellular functions in eukaryotic cells. While essential for critical physiological roles in normal cells, dysregulation of LLPS underlies various pathological states, including tumorigenesis. Accumulating evidence indicates that oncogenesis is driven by aberrant gene expression patterns concomitant with dysregulated phase separation. This section examines the mechanisms by which LLPS regulates key cellular processes in lung cancer and the pathological implications of its dysregulation for tumor development (Fig. 2).
In response to starvation, hypoxia, drug exposure, or viral infection, cells rapidly form SGs to reduce the synthesis of non-essential proteins and adjust their energy metabolism. Many macromolecules dynamically assemble into SGs to coordinate physiological activities including mRNA localization, translation regulation, degradation, antiviral responses, and tumorigenic signaling pathways [32, 33]. Component analyses reveal that SG formation coincides with translational repression. Consequently, SGs contain abundant translation initiation factors, RNA-binding proteins (RBPs), and untranslated mRNAs [34, 35]. SGs are widely recognized as condensates [36,37,38]. The Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2) serve as core scaffold proteins essential for driving SG formation. Expression of either protein alone is sufficient to initiate SG condensation [37].
Accumulating evidence suggests that SGs function as regulatory hubs influencing the progression of lung cancer. Mutation or dysregulation of SG regulators impairs therapeutic efficacy, immune responses, and clinical prognosis in NSCLC [34]. Mechanistically, the tumor suppressor tripartite motif-containing protein 72 (TRIM72, also known as MG53) regulates NSCLC proliferation and migration by modulating G3BP2 activity [9]. Conversely, in NSCLC cells, the oncogene ETS variant transcription factor 4 (ETV4) suppresses hexokinase-1 (HK1) activity, releases the inhibition of histone deacetylase 6 (HDAC6) and G3BP2 expression, and promotes formation of the lysosomal-TSC2 complex. Collectively, these events attenuate signaling through the mechanistic target of rapamycin complex 1 (mTORC1) and enhance cellular stress adaptation [33]. Furthermore, SGs can regulate SARS-CoV-2 infection mediated by the viral nucleocapsid (N) protein under stress conditions [32].
Nuclear paraspeckles are membraneless, spherical subnuclear bodies predominantly involved in transcriptional regulation and alternative splicing [39]. Initially identified by Archa in 2002 [40], their core structural components comprise the proteins non-POU domain-containing octamer-binding protein (p54/nrb), splicing factor proline- and glutamine-rich (SFPQ), paraspeckle component 1 (PSPC1), and the essential long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1). NEAT1 specifically localizes to paraspeckles, serving as an essential structural scaffold for their assembly. Paraspeckle formation is directly dependent on NEAT1 expression levels. Furthermore, NEAT1 orchestrates the recruitment and localization of p54/nrb and SFPQ to paraspeckles [41]. In NSCLC, the transcription factor octamer-binding transcription factor 4 (OCT4) binds the NEAT1 promoter, inducing its upregulation. Elevated NEAT1 expression correlates positively with larger tumor size, advanced clinical stage, increased metastatic potential, vascular invasion, and poor postoperative survival [42]. Notably, a clinical study from northern China showed an association between NEAT1 polymorphism rs2230905 and an increased risk of lung adenocarcinoma [43].
Mechanistically, NEAT1 functions as a competitive endogenous RNA (ceRNA) by sequestering miR-let-7a. This sequestration relieves let-7a-mediated repression of its downstream target insulin-like growth factor 2 (IGF-2), thereby promoting NSCLC cell proliferation and metastasis [12]. In LUAD, high NEAT1 expression inhibits miR-335 function, leading to derepression of hepatocyte growth factor receptor (c-MET) and contributing to sorafenib resistance [44]. Additionally, epigallocatechin-3-gallate (EGCG) enhances reactive oxygen species (ROS) generation, downregulates ERK1/2 signaling, and upregulates both NEAT1 and the copper transporter 1 (CTR1). This NEAT1 upregulation promotes cisplatin sensitivity [14]. NEAT1 also interacts with DNA methyltransferase 1 (DNMT1) to inhibit the tumor suppressor p53 and suppress cyclic GMP-AMP (cGAMP) synthesis. Consequently, this interaction inhibits the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, facilitating tumor immune evasion from T cell-mediated surveillance [13].
Autophagy is a conserved, degradative cellular process that facilitates the recycling of cytoplasmic components. This process enables the clearance of damaged organelles, degradation of macromolecules, and elimination of intracellular pathogens. Accumulating evidence indicates that LLPS serves as a critical regulatory mechanism in autophagy, modulating autophagic flux and influencing cell fate. For instance, calcium transients at the endoplasmic reticulum (ER) surface induce LLPS of focal adhesion kinase family-interacting protein of 200 kDa (FIP200). This phase transition recruits components of the Unc-51-like autophagy activating kinase (ULK) complex, thereby spatially organizing autophagy initiation [45]. Additionally, p62/SQSTM1, a key scaffold protein for autophagosome formation, forms LLPS-driven condensates that concentrate ubiquitinated cargo. These p62/SQSTM1 condensates selectively recruit ubiquitinated substrates through specific non-covalent multivalent interactions and facilitate their subsequent encapsulation into autophagosomes. These findings collectively demonstrate that LLPS orchestrates autophagosome assembly site formation, modulates the kinetics of autophagy initiation, and governs substrate selectivity during autophagic encapsulation, highlighting its pivotal regulatory role in autophagy [46, 47].
The regulation of autophagy is primarily orchestrated by three key signaling pathways: the AMPK/mTOR, PI3K/AKT, and MAPK/ERK1/2 cascades. Agents such as lactoferrin (LTF) and gitogenin induce AMPK phosphorylation, leading to activation of the AMPK/mTOR pathway [48,49,50,51]. This activation triggers autophagy, suppresses cell proliferation, and promotes apoptosis and radioresistance. Conversely, activation of the PI3K/AKT/mTOR pathway by factors like microRNA-199a-5p and C-C motif chemokine ligand 2 (CCL2) inhibits autophagy, enhances cell proliferation, migration, and invasion, and reduces both apoptosis and drug resistance [52, 53]. Similarly, activation of the MAPK/ERK1/2/mTOR pathway promotes tumorigenesis by inhibiting autophagy [54].
However, the precise role of autophagy in lung cancer pathogenesis remains contentious. For instance, ubiquitin-specific peptidase 15 (USP15) reportedly induces autophagy via the TNF receptor-associated factor 6 (TRAF6)-Beclin 1 (BECN1) signaling axis, suppressing lung cancer progression [55]. In contrast, fucosyltransferase 2 (FUT2)-mediated induction of autophagy in LUAD inhibits apoptosis [56]. Furthermore, autophagy inhibition may also enhance drug resistance [57]. Additionally, autophagy can degrade the transcription factor SRY-box transcription factor 2 (SOX2), reducing cancer stemness and promoting tumor cell differentiation [58]. Nonetheless, some evidence suggests that autophagy may facilitate the self-renewal of lung cancer stem cells by degrading ubiquitinated p53 [59]. Collectively, these findings highlight that the functional consequences of autophagy are highly context-dependent, varying with the specific activating stimuli and cellular milieu.
Cajal bodies are localized within the nucleoplasm of eukaryotic cells. WD repeat domain 79 (WDR79), a WD-repeat protein and essential scaffolding component for Cajal body assembly, is highly expressed in NSCLC [60,61,62]. Depletion of WDR79 significantly inhibits NSCLC cell proliferation and induces apoptosis [8].
Mechanistically, WDR79 interacts with ubiquitin-specific protease 7 (USP7), which reduces ubiquitination of the E3 ligase mouse double minute 2 homolog (MDM2) and its substrate p53. This stabilization extends the half-life of both proteins, thereby promoting proliferation [63]. Additionally, WDR79 protects ubiquitin-like protein containing PHD and RING finger domains 1 (UHRF1) against polyubiquitination-mediated degradation. This facilitates UHRF1-dependent DNA methylation and histone modifications, consequently enhancing NSCLC tumorigenesis [64].
Nuclear speckles compartmentalize diverse mRNA splicing factors and protein processing factors essential for regulating protein synthesis and assembly. The speckle-type POZ protein (SPOP), a major component of nuclear speckles, localizes to these structures. Functioning as a tumor suppressor, SPOP expression is frequently suppressed in NSCLC via methylation of its promoter region [65, 66]. This suppression consequently impairs SPOP-mediated regulation of the NF-κB pathway [67, 68].
Additionally, the deubiquitinating enzyme USP42 localizes to SC-35-positive nuclear speckles in an enzyme activity-dependent manner via its C-terminal positively charged residues. USP42 recruits the alternative splicing component pleiotropic regulator 1 (PLRG1), promoting its LLPS and integration into nuclear speckles. This process modulates mRNA alternative splicing, thereby significantly influencing tumor cell growth [10].
Beyond the previously characterized condensates, cells harbor numerous uncharacterized condensates. Aberrantly expressed proteins or dysregulated nucleic acids can induce the formation of these condensates, ultimately drives tumor progression and confers therapeutic resistance in NSCLC.
Biomolecular condensates play significant roles in the pathogenesis of lung cancer. For instance, the upregulation of lncRNAs MELTF-AS1 and MNX1-AS1 promotes tumorigenesis and progression by inducing phase separation of downstream target proteins [69, 70]. Furthermore, post-translational modifications (PTMs) such as deubiquitination and myristoylation contribute directly or indirectly to condensate formation, facilitating tumor development [71, 72]. Condensates involving echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), specificity protein 1 (SP1), and Src homology 2 (SH2) proteins potently activate downstream oncogenic signaling pathways [73,74,75].
Additionally, condensates contribute to therapeutic resistance mechanisms. The oncogene c-MYC activates MYLK-AS1 transcription, which subsequently promotes interleukin enhancer binding factor 3 (ILF3) phase separation; the resulting ILF3 condensates stabilize glutamate dehydrogenase 1 (GLUD1) mRNA, enhancing mitochondrial glutamine metabolism and conferring resistance to tyrosine kinase inhibitors (TKIs) [76]. Similarly, interactions between forkhead box P1 (FOXP1) and the specificity protein 8 super-enhancer (SP8-SE) form transcriptional condensates that upregulate SP8 expression. This promotes homologous recombination repair in SCLC, leading to increased chemoresistance [77]. Likewise, retinoid X receptor gamma (RXRγ) condensates enhance the transcription of target genes, promoting tumor stemness and metastasis, ultimately leading to chemoresistance in SCLC [78].
Biomolecular condensates also modulate anti-tumor immunity. Exposure to Interferon-gamma (IFNγ) induces formation of lysine acetyltransferase 8-interferon regulatory factor 1 (KAT8-IRF1) condensates that bind to the programmed death-ligand 1 (PD-L1) promoter, augmenting its transcription and enabling tumor cells to evade immune surveillance [79]. Collectively, condensates exert multifaceted roles in tumor initiation, progression, and therapeutic resistance (Table 1).