Tumor cell plasticity in non-small cell lung cancer: the role of microRNA and implications for diagnosis, prognosis and treatment

Introduction

Lung cancer remains a major cancer burden globally. It leads mortality rate worldwide with 1.23 million deaths in 2022 and 1.57 million new cases (1). At current incidence and mortality rates, the disease burden is projected to rise to 4.62 million new cases and 3.55 million deaths by 2050 (2). However, significant progress has been reported in recent years, with improvements in patient outcomes and survival, including a rising 5-year survival rate and declining mortality rates (3). This improvement is largely the result of early detection, reduced incidence due to decreasing smoking rates, advances in therapy such as targeted therapy, immunotherapy, radiotherapy etc. Late-stage metastatic disease, and disease progression from therapy resistance contribute significantly to non-small cell lung cancer (NSCLC) mortality. NSCLC is the largest pathologic category, comprising 85% of cases, with lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and large cell carcinoma (LCA) making up the major members of this group (4). The presence or absence of molecular drivers due to genetic mutations has driven the treatment of this disease yet an increasing understanding of the molecular biology of NSCLC highlights the importance of a more nuanced approach to diagnostics and management. This review discusses the role of microRNAs (miRNAs) in tumor cell plasticity (TCP), and as a mechanistic driver of NSCLC biology (Table 1). It highlights its function in tumor progression and clinical utility in diagnosis, prognosis and treatment of NSCLC.

TCP

TCP is an important cellular and molecular mechanism underlying disease progression in NSCLC. It is designated as a hallmark of cancer due to its fundamental role in cancer biology (5-7). Hallmarks of cancer were introduced as a biological framework to organizes cellular and molecular processes in cancer into mechanistic concepts. The importance of TCP is underscored by its interaction with multiple hallmarks including invasion and metastasis, inducing vasculature and immune evasion, deregulating cellular energetics, nonmutational epigenetic reprogramming. TCP is a complex of processes whereby cancer cells undergo molecular and phenotypic changes due to microenvironmental stresses, which may arise from stochastic, genetic and epigenetic alterations, and/or therapy-related selective pressures (8,9). These changes are survival adaptations for cancer cells, that generate tumor heterogeneity and lead to therapy resistance. The diverse programs of TCP occur throughout the tumor life cycle from its premalignant state to tumor initiation and evolution, adaptive therapy resistance mechanisms, immune evasion and the acquisition of increasing malignant aggressiveness. A mechanism widely utilized innately in embryonic development and wound healing, cellular plasticity also occurs in terminally differentiated adults cells, particularly when subjected to chronic environmental stress. In cancer, the normal cell’s tool-kit of plasticity is hijacked to facilitate tumor evolution and progression through the survival advantage it confers. Environmental stresses arising from a complex and rapidly evolving tumor microenvironment (TME), constitute a combustible mix of altered metabolic conditions, aberrant signaling molecules, deranged stromal elements and toxic therapeutic agents. Collectively, they create a highly volatile microenvironment that triggers cellular adaptive processes within the cancer cells. These processes involve transcriptional or epigenetic modulations more commonly that genetics changes.

MiRNAs in cancer

MiRNAs are short, non-coding RNA molecules of length 21–25 nucleotide bases. They regulate gene expression through inhibition and degradation of mRNA thus silencing translation. As regulatory molecules with a broad range of normal cellular functions through posttranscriptional control, miRNAs act during embryonic development, in body patterning, stem cell differentiation, and tissue identity (10). Canonical miRNA biosynthesis starts in the nucleus with primary transcription of pri-miRNAs by RNA- polymerase II. Initial processing by the microprocessor complex (Drosha-DGCR8 complex) generates pre-miRNA, which is exported to the cytosol by Exportin 5/RanGTP complex. A miRNA duplex is produced by Dicer, an RNase III enzyme and is loaded onto the Argonaute family of proteins, forming the miRNA-induced silencing complex (miRISC). The mature single stranded miRNA is finally formed by removal of a strand of the initial duplex molecule (11). The mature miRNA functions as a molecular guide, binding complementary RNA targets for degradation or inhibition of its translation (12). In cancer, miRNA functions as tumor suppressors or oncogenic miRNA (oncomirs), acting through its regulation of mRNA throughout cancer development, from tumor development, proliferation, invasion, angiogenesis, apoptosis, immune evasion and metastasis (13-15). Aberrations in miRNA metabolism occur via genetic mutations, methylations, deletions or amplifications of miRNA-coding regions. Their location in fragile or cancer associated genomic regions renders them susceptible to genomic alterations (16-19). Understanding the role of miRNAs in lung cancer, particularly as an epigenetic player, would not only serve in the elucidation of the complexities of lung cancer biology but lay the basis for the development of novel diagnostic, prognostic and therapeutic approaches in the management of this disease (20-26). This review aims to highlight the specific role of miRNAs on lung tumorigenesis, through its regulation of TCP. The strength of this review is its comprehensive overview of the role of miRNAs in TCP in NSCLC. It examines cellular processes, genetic and signaling pathways and their regulation by miRNAs with a view to clinical application. A limitation is some intricate aspects are given limited attention due to the wide-ranging nature of the topic.

Premalignant lesions in NSCLC

Prior to the development of overt malignancy, a succession of histologically defined premalignant phases may result after exposure of a specific cell of origin to predisposing factors. Predisposing conditions like lung injury, lung disease, environmental exposure, most commonly, tobacco exposure, propel tumorigenesis. Premalignant precursor lesions (PML) for LUSC arise when the ciliated glandular epithelium undergo transdifferentiated into squamous epithelium subject to injurious stimuli. The lesions are characterised by increasing genetic abnormalities and progressively altered morphological features including atypical basal and cytological features (27). The most aberrant alterations result in high risk PML such as high-grade intraepithelial neoplasia and carcinoma in situ. These are most associated with invasive LUSC. In LUAD, the adenomatous premalignant lesions have been identified as atypical alveolar hyperplasia. They develop from accumulations of genetic alterations in normal lung tissue from a widespread field of injury, known as a field effect or field carcinogenesis (28,29). Recent experimental evidence implicate alveolar type 2 (AT2) cells and clara cells as potential cells of origin of LUAD, suggesting that variable pathways could converge on a histologically similar carcinoma type (30-32), with the initiating cells influencing the morphology of the resultant tumor. On alveolar injury, the initiating cell undergoes transcriptional reprogramming and enters a transitional state with acquired cellular plasticity and can differentiate into alveolar type I cells. Han et al. comparing single epithelial cells in early LUAD with matched normal lung samples identified an alveolar intermediate cell type with elevated KRT8 expression as possible intermediates to tumor cell transformation of AT2 cells. The cells displayed reduced differentiation and increased plasticity in KRAS mutation driven cells. In mice studies, these cells emerged as PML on tobacco exposure, later acquiring KRAS mutations and persisted in LUAD cells after transformation (33). Cell plasticity also mediates the formation of precancerous lesions arising in the bronchial epithelium, which predisposes to the development of LUSC. In a study, differentiated human bronchial epithelial cells (HBECs) exposed to cigarette smoke, produced morphological changes with deficient epithelial tight junction barrier function. Loss of function in Crb3 gene in adult luminal airway epithelium resulted in activation of transcriptional factors YAP and TAZ mediating basal-like cell growth and aberrant epithelial polarity through ERBB receptor by the Neuregulin-1 growth factor (NRG1) (34). YAP/TAZ are transcriptional regulators in normal development, mediating various processes including lung patterning, morphogenesis and determining basal airway stem cell identity through Hippo signaling (35,36).

MiRNA in premalignant phase of NSCLC

MiRNAs are implicated in premalignant lesions in NSCLC (Figure 1). The initial cell of origin utilize cell plasticity to become precancerous in the early development of LUSC and LUAD. YAP/TAZ transcription regulators define basal airway stem cell identity. Activation of the YAP/TAZ on tobacco exposure leads to basal-like cell growth and abnormal epithelial polarity. MiRNAs regulate YAP/TAZ pathway directly or in a Hippo pathway-dependent manner. miR-345 inhibited cell migration and invasion in NSCLC through its activity on YAP1 and is downregulated in NSCLC (37). MiRNAs implicated in YAP regulation in NSCLC include miR-345, miR-424-3p, miR-26b. Mascaux et al showed a two phase differential pattern of miRNA expression in premalignant lesions (38). In the first phase, there was a global decrease in miRNA expression, oppositive to the pattern seen in normal development. In later stages of bronchial carcinogenesis, an increase in global expression occurred in a reversal of the initial decline. However, specific miRNAs displayed unique expression patterns. MiR-32 and miR-34c, a p53 transcriptional target, had a linear evolution of expression throughout tumor development.

Figure 1 Tumour progression in LUAD and LUSC. MicroRNAs in NSCLC. Upregulated and downregulated microRNA impact cancer progression at all stages of cancer development from premalignant lesions, tumorigenesis, survival, angiogenesis and invasion/metastasis. Reproduced with permission from KEGG (39,40). KEGG, Kyoto Encyclopedia of Genes and Genomes; NSCLC, non-small cell lung cancer.

TCP in tumor evolution

After malignant transformation, NSCLC may progress from a well differentiated phenotype towards more aggressive and malignant phases. The tumor cells acquire increasingly higher grade features, advancing through moderate to poorly differentiated states. The earliest model of cancer progression posited a Darwinian tumor development driven by genetic clonal evolution. In this model, progressive acquisition of driver mutations conferred cancer cells with a survival advantage that lead to clonal dominance (42). However, accumulating evidence demonstrates that factors such as TME and epigenetic alterations influence cell states and facilitate cancer progression (43). Epithelial mesenchymal transition (EMT) is a mechanism of TCP that harnesses epigenetic mechanisms to promote processes such as dedifferentiation, and transdifferentiation for cancer cell survival, growth and progression. EMT is a complex cellular and molecular process in which tumor cells shed their epithelial cell state and adopt a mesenchymal identity. This phenomenon is characterized by disruption of cell-cell adhesion mechanisms, gradual loss of apical polarity, and cytoskeletal reorganization (44). The tumor cells develop increased cell motility and altered interaction with the basement membrance (45,46). EMT confers a high degree of heterogeneity on the tumor as it represents a wide range of cells states in the transition spectrum from epithelial to mesenchymal identity (47). Cells may exist in complete or partial (hybrid) EMT states. EMT is very dynamic and may progress in a reversible bi-directional path, in which cells assume complete or partial features of epithelial or mesenchymal states (48). It may also be multi-directional in which cells assume cell states distinct from these two polar extremities. EMT confers cancers cells with a high degree of plasticity, by which they acquire the ability to reactivate cellular pathways and redirect their phenotypic changes. The incomplete transition of cell state between a well differentiated epithelial identity to a poorly differentiated mesenchymal state in NSCLC with intermediate characteristics confer high malignant potential on the cell. Differentiation shunting tangential to the two polar extremities is called transdifferentiation. For instance, LUAD may activate cell type specific developmental pathways and engineer a squamous cell phenotype or alternatively a small cell neuroendocrine cell state in response to environmental cues (49,50). Other models besides genetic clonal evolution, have been advanced to explain the phenomenon of cell plasticity. Cancer stem cells (CSC) is one such explanatory model (51,52). CSC are a small subpopulation of dedifferentiated cancer cells with the capacity for self-renewal, proliferation and differentiation to replenish the cancer cell population. They display resistance to cell death mechanisms, increased angiogenesis, metastasis, immune suppression and altered metabolism (53,54). Bidirectional transition may occur between non-CSC and CSC states increasing tumor heterogeneity and plasticity (55). These properties imbue CSC with therapy resistance and cancer recurrence. Another model of TCP is emerging from studies in genetically engineered mouse models and human pathological specimen. Marjanovic et al. showed that NSCLC cells develop a high plasticity phenotype with the ability to acquire new states and maintain preceding states throughout tumor evolution. This feature confers a high degree of heterogeneity on the progressing tumor. The tumor cells initiated multiple alternative lung epithelial programs, then proceeded to generate transcriptional programs resembling the primordial gut, finally culminating in cells with a mesenchymal state, characteristic of EMT (56). Though each cell states emerged at specific points in the tumor temporal profile, their new cell identity persisted, resulting in advanced tumors with high cell state diversity. This feature was termed high plasticity cell state (HPCS) with p53 being the guardian of lineage fidelity. Its deletion increased the potential for a wide range of phenotypic states.

Molecular mechanisms of TCP in NSCLC tumor evolution

Signaling pathways that mediate tumor evolution through EMT include fibroblast growth factors (FgF), transforming growth factors beta (TGF-β), bone morphogenetic protein (BMP), receptor tyrosine kinase (RTK), Hedgehog, STAT3, Notch and Wnt pathways (57). They activate EMT by modulating EMT-transcription factors (EMT-TF). TFs such as zinc finger E-box binding homeobox 1 (Zeb1) and Zeb2, Snail, Slug Twist1 are considered the core regulators of EMT with a growing number of additional factors e.g., FOXC2, SOX4 and PRRX1 (58). EMT-TF act by inhibiting epithelial cellular processes, while promoting mesenchymal processes. By the inhibition of epithelial elements like E-cadherin, apicobasal complexes and tight junctions, they restructure cellular systems leading to reorganization of polarity, and changes in intercellular adhesion. Through the enhancement of mesenchymal elements such as fibronectin, vimentin, N-cadherin and the actomyosin network, they reorder the cell shape, its migratory properties and cell adhesion characteristics. EMT-TF also promotes cancer cell renewal through CSC formation by inducing CSC gene expression. They enhance cancer cell survival by anoikis resistance, i.e., resistance to programmed cell death as a result of detachment from the extracellular matrix (ECM) and neighbouring cells. They also modulate histone modification via histone H3 K27 acetylation and H3K27 trimethylation, thus facilitating extensive repression of epithelial genes for EMT during tumor evolution (59). SOX2 activation promotes transdifferentiation from LUAD to LUSC (60) while inactivation of TP53 and RB1 leads to transdifferentiation of LUAD to small cell lung cancer SCLC (61). Other factors also play a role e.g., hypermethylation of NKX2-1 (TTF-1) also enhances transdifferentiation to a squamous cell phenotype while global hypomethylation of lineage plasticity drivers such as ASCL1 and NEUROD1 facilitates transformation to SCLC (62).

MiRNA in EMT in NSCLC

Oncomirs and tumor suppressor miRNAs play an important role in EMT in NSCLC (Figure 2, miRNAs in cancer). The miR200 family is a well-studied suppressor of EMT. They inhibit ZEB1 and ZEB2 TFs, which are negative regulators of the adhesion molecular E-cadherin. Upregulation of miR200 acts to enhance epithelial state and suppress mesenchymal states. Cancer cells in EMT demonstrate downregulation of all five members of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) (63). miR-200c dysregulation predisposed to aggressive, invasive and chemoresistant tumors and shorter overall survival (64,65). miR-145 and miR-497 suppress EMT via inhibition of TGF-β signaling and metadherin (66). In NSCLC, they are downregulated to promote cancer cell migration and invasion. Various oncogenic miRNAs are implicated in NSCLC. miR-21 promotes EMT by repressing the tumor suppressor gene PTEN. Suppression of PTEN activates PI3K/AKT pathway, which promotes invasiveness and increases metastatic potential. miR-15b and miR-27a also promote EMT and therapy resistance in NSCLC, particularly to cisplatin.

Figure 2 Panel 1: in LUAD, cells progress from alveolar and bronchiolar epithelial cells to premalignant atypical adenomatous hyperplasia to primary adenocarcinoma to metastatic adenocarcinoma. In LUSC cells progress from bronchial epithelial cells to bronchial dysplasia, to primary squamous cell carcinoma to metastatic carcinoma. Panel 2: cytoplasmic signal transduction in NSCLC. Oncogenes and tumour suppressor genes interact with multiple pathways including Ras, ErbB, MAPK, PI3K-Akt, Calcium signaling pathway. This leads to proliferation, altered apoptosis, cell survival. Panel 3: within the nucleus, transcriptional activation, p53 signaling leads to tumor progression, G1/S cell cycle progression, uncontrolled proliferation, increased survival, genomic instability. Reproduced with permission from KEGG (39,40). KEGG, Kyoto Encyclopedia of Genes and Genomes; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.

MiRNA in metabolic reprogramming in NSCLC

Metabolic reprogramming is an important hallmark of cancer. It promotes tumorigenesis by providing crucial nutrients, energy sources and bioblocks for cancer growth, survival and proliferation. For instances, the tumor suppressor miRNAs, miR-33b and miR-144 downregulate the glycolysis enzyme LDHA to inhibit glycolysis (67). miR-144 inhibits the glucose transporter GLUT1. These miRNAs are suppressed in NSCLC. The oncogenic miRNAs are implicated in NSCLC metabolic reprogramming. miR-21 plays a role in NSCLC cellular energetics by shifting to aerobic glycolysis through the activation of AMPK/ULK1 signaling pathway. This facilitates cancer processes including cell proliferation, migration and invasion. MiR-31-5b also promotes glycolysis. It acts by suppression FIH, a repressor of the TF HIF-1alpha. Numerous other miRNAs have been implicated in metabolic reprogramming in NSCLC including miR-124, miR-143, miR-206, miR-1, miR-133 (glucose metabolism); miR-33b, miR-21, miR-182 (lipid metabolism); miR-126 (amino acid metabolism) (13) etc.

TCP and metastasis in NSCLC

Metastasis, another cancer hallmark, is a multi-step process involving cell detachment from the parent tumor, digestion and migration through the ECM, intravasation into the vascular system, transportation through the bloodstream or lymphatics to distant site, then extravasation, colonization and growth of the metastatic tumor. Metastasis-initiating cells (MICs) are highly plastic cells implicated as the sole causative cells for metastasis (68). They arise from CSC or dedifferentiated non-CSC. They alter their cell states at different points on the metastatic trajectory, exhibiting varying degrees of stemness, EMT and metabolic plasticity (69). Local invasion occurs frequently with mesenchymal single cells in a full EMT state or less commonly as collective migration in hybrid EMT states with the leading edge having a higher EMT phenotype. Cellular changes such as loss of intercellular adhesion molecules and epithelial polarity, cytoskeletal reorganization and basement membrane (BM) disassembly enhance the invasive phenotype. MIC in circulation as circulating tumor cells (CTC) adopt a more pronounced mesenchymal phenotype with increased resistance to cell death through adherence-independent survival signals (70). Survival of CTC is aided by protective platelet coating, and interactions with white blood cells, endothelial cells and macrophages (71,72). On extravasation, reversal of EMT via mesenchymal-to-epithelial transition (MET) aids dissemination and colonization. Phenotypic changes like altered metabolism protects CTC from oxidative stress during metastatic colonization (73). MIC regain their epithelial phenotype by MET. Acquisition of intercellular adhesion molecules promote metastatic seeding. Seeded cells may remain in dormancy at the metastatic site or may regain an active proliferative capacity. The mechanism of MET activation has not been fully delineated. However the metastatic niche plays an important role, with TME cells providing cytokine signaling, production and modeling of ECM, promoting migration, angiogenesis and TCP (74).

Molecular mechanisms of EMT during metastasis in NSCLC

EMT acts through a multiplicity of processes to aid metastasis (75). The development and maintainance of CSC are part of this key process. EMT-TF mediate stemness through several mechanisms including the epigenetic regulator Bmi1 and PTEN, which activates signaling of PI3K/Akt/GSK-3beta and Notch pathways to promote EMT (76). Hybrid EMT is crucial in the process of metastasis as its high mix of epithelial and mesenchymal properties confer a high degree of heterogeneity and wide range of cellular functions required for the metastatic cascade. Downstream effects of EMT include the downregulation of E-cadherin and upregulation of N-cadherin, the degradation of the adherens junctions and intercellular adhesion, and promotion of cell migration and invasion (77,78). The Notch, VEGF and TGF-β signaling by the metastatic CSC mediates the degradation of the BM and ECM through histone proteases and protein hydrolases such as matrix metalloproteinases (MMPs). The remodeling of the ECM via urokinase-type plasmogen activator (uPA) permits passage through the ECM (79). The cytoskeletal reorganization aids migration through the ECM and diapedesis through the endothelium. After intravasation, expression of thrombin facilitate platelet coating by the SMAD and Notch signaling pathways (80). Travel in vascular system is mediated by interaction of endothelial intercellular adhesion molecules like ICAM1, galactose lectin 3, and selectins with cancer cell adhesion molecules e.g., integrins, CD44, MUC1 (81). On extravasation at a distant site, reversal of EMT occurs via the process of MET. MET-TFs, which include CD1, ZO-1M CLDN4 (encoding claudin-4) and CLDN5 (encoding claudin-5) mediate the reversal of EMT. They inhibit the transcription of mesenchymal genes and stimulate epithelial lineage genes. They are involved in a reciprocal inhibitory loop system with EMT-TF.

miRNAs in cancer stemness regulation and metastasis in NSCLC

Tumor suppressor miRNAs regulate CSC through modulation of CSC pathways such as Wnt/Beta-catenin, Notch and Hedgehog signaling. They may also act indirectly through the network of stemness TFs including OCT4, SOX2 and NANOG (82). MiR-34 family (miR-34a, miR-34b, miR-34c) target p53 as tumor suppressors. They repress tumor growth and metastasis through the inhibition of mRNAs of gene of cell cycles, EMT, metastasis, stemness, apoptosis and senescence (83). MiR-34a inhibits stemness by repressing CD44, a cancer cell adhesion molecules, thus suppressing the self-renewal and tumor initiating ability of CSC. The EMT-TF are modulated by a negative feedback loop in which ZEB is regulated by miR-200 and Snail by miR-34 (75,84). The oncogenic miRNA, miR-410 facilitates stemness in NSCLC through its inhibitory effect on the tumor suppressor GSK3Beta. The resultant activation of the Wnt/Beta-catenin pathway gives rise to increased tumor sphere formation and resistance to chemotherapy.

TCP and immune evasion

EMT enables cancer cells to evade the immune system through several mechanisms including downregulation of antigen presentation, upregulation of immune checkpoint proteins, production of immunosuppressive chemokines and cytokines, and recruitment of immunosuppressive cells and alterations in the ECM (85,86). Immune evasion is a recognized cancer hallmark.

EMT promotes the activation of pro-survival signaling pathways e.g., beta catenin pathway, and DNA repair mechanisms in CSC, endowing them with innate immune evasion properties. It also inhibits antitumor immune response, and enhances immunosuppressive factors such as macrophage stimulating proteins, monocyte chemoattractant protein (MCP-1), TGF-β, C-C motif chemokine ligand 2, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-8, IL-10, etc. (87). These facilitate an immunosuppressive environment. These immunosuppressive molecules together with the increased recruitment of immunosuppressive cells such as regulatory T cells, myeloid derived suppressor cells (MDSC), and tumor associated macrophages (TAM) prevent cancer killing by natural killer cells and cytotoxic CD8⁺ cells (88). EMT results in increased checkpoint molecules (PD-L1/2, PD-1, CTLA-4) expression leading to immune inhibition and immunotherapy resistance (89,90). The non-cellular components of the ECM signals interact with tumor cell receptors to promote proliferation, EMT and stemness (91). A number of LUAD models demonstrate the role of ECM in EMT. A bioengineered model with increased sulfation displayed stimulation of PI3K signaling producing increased invasiveness, cellular growth and CSC. Other studies have shown that cellular components of the ECM e.g., cancer-associated fibroblast interact with cancer cells e.g., via CXCR4/beta-catenin signaling to induce EMT (92).

MiRNA and the TME

TME noncancer cells such as fibroblasts and other immune cells also communicate with cancer cells through miRNAs to support tumor growth, angiogenesis and immune suppression. Cross talk between TME stromal, cellular and noncellular components and cancer cells is mediated by a number of mechanisms including paracrine and exosomal communication. Tumoral exosomes convey and transmit miRNA to other cellular components to reprogram their cellular biology. Exosomal miR-103 extruded from cancer cells in hypoxic states engender M2 macrophage polarization and tumor growth. MiR-1 and miR-101 acted on cancer associated fibroblasts by targeting CXCL12 to negatively regulates NSCLC proliferation and chemoresistance. miR-130a, miR-1207-5p shifted TAM polarization from an M1 to an M2 phenotype through PPARgamma and CSF1 respectively. MiRNAs also affected other immune cells of the TME e.g., regulatory T cells (miR-14 via CXCL1), CD4⁺ lymphocytes (miR-153), CD8⁺ lymphocytes (miR-200b/a/429 via PD-L1), natural killer cells (miR-183* via DAP12) etc. (93).

MiRNAs in immune evasion and immunotherapy resistance

In addition to its activity on the TME, miRNAs mediated immune evasion and immunotherapy resistance through EMT. For instance, expression of members of the miR-200 family are involved in the regulation of PD-L1 expression either by targeting PD-L1 directly or via the suppression of EMT through ZEB1 (94). This promoted T-cell immunosuppression and increased metastasis. miR-3127-5P overexpression upregulates PD-L1, leading to immune escape and immunotherapy resistance.

TCP and chemotherapy resistance

Significant improvements in patient outcomes and survival due to advances in cancer therapies particularly targeted therapy is tempered by the inevitable emergence of drug resistance (95,96). The understanding of acquired cancer therapy resistance is shifting from the simple model of acquired secondary genetic mutations to a model that incorporates a dynamic, often reversible cell state mediated by TCP (97). TCP allows cancer cells to modify their phenotypic and transcriptional profile as an adaptation to drug exposure to gain a survival advantage. In NSCLC, EMT promotes pan-resistance to a wide swath of therapy types including chemotherapy, radiation therapy, immune and targeted therapies. Mechanisms of therapy resistance by TCP include innate EMT resistance, drug-tolerant persistent (DTP) cells, and phenotypic switching.

DTP cells are a subpopulation of the tumor cells that display slow proliferation, metabolic flexibility, TME adaptations and phenotypic plasticity (41,69). Under treatment pressure, DTP enter a quiescent phase with low proliferation through phenotypic reprogramming and survive therapy toxicity. They remerge after drug cessation and resume proliferation but retain drug sensitivity. Entry into the DTP states is mediated by phenotypic reprogramming via epigenetic, transcriptional and translational regulatory processes and intricate interactions in the cancer cell-TME community (98). These processes entail a diapause-like developmental adaptation (99). DTP cells may acquire stable resistance on sustained treatment through adaptive mechanisms and from secondary mutations, diverse mechanisms are implicated in DTP cells (Figure 3) (41). One such mechanism is via Hippo signaling. In a CRISPR screen, the YAP/TEAD axis was identified as a mediator of DTP in epidermal growth factor receptor (EGFR) mutant NSCLC (100). The YAP axis and CDK1 were identified in a mass spectrometry study as mediators of DTP cells through substrates phosphorylation (101).

Figure 3 Characteristics of tumor dormancy include macroenvironment crosstalk, stalled proliferation, chemoresistance, immune evasion, plasticity, stemness, metabolism reduction, microenvironment dependence. Reproduced under the Creative Common License (http://creativecommons.org/licenses/by/4.0/) from (41). EMT, epithelial mesenchymal transition; MET, mesenchymal epithelial transition.

MiRNA in chemotherapeutic resistance

Advances in NSCLC therapeutics, particularly the development of TKI and immune checkpoint inhibitor (ICI) has greatly improved outcomes. Yet resistance to chemotherapy presents a major hurdle in NSCLC management as almost all recipients eventually develop chemoresistance. Drug resistance mechanisms in NSCLC occurs from multiple mechanisms including tumor-intrinsic, microenvironment cross-talk and intercellular communication (96,102). MiRNAs playing an important role in chemoresistance. For instance, the miRNA miR-29ac and 140-5p mediate drug tolerance to TNF-related apoptosis-inducing ligand (TRAIL) in DTP cells by regulating RSK2 expression. RSK2 mRNA is responsible for NF-κB activation, a key player in both innate and acquired TRAIL-resistance (103). miRNAs are particularly potent resistance mechanism due to their intercellular transmission of chemoresistance. Exosomal transmission is a mechanism whereby drug resistance information is communicated from resistant cancer cells to sensitive cells. Exosomal miRNA that mediate chemoresistance may arise from tumor cells or non-tumor cells and act on various cell signaling molecules. In studies on cisplatin, tumor derived miRNAs including miR-100-5p, miR-4443, miR-425-3p target mTOR, METTL3/FSP1 and AKT respectively, to induce chemoresistance (104,105). miR197-5p, miR642a-3p, miR-27b-3p also play a role in cisplatin cancer cell chemoresistance. Exosomal miRNA such as miR-103a-3p, miR130a, miR-193a are derived from cancer associated fibroblasts and bone marrow-derived mesenchymal stem cells. They promote intercellular drug resistance through various mechanisms e.g., miR103a downregulated BAK1 thereby inducing cisplatin resistance (106). miR-155 promotes EMT via exosomal transmission between cancer cells and induces chemoresistance in previously sensitive cancer cells (107). MiR-218 causes a reversion to chemosensitivity to cisplatin in NSCLC by its induction of Slug and Zeb2 (108). MiRNAs are also implicated in targeted therapy resistance. MiR-21 upregulation occurs in gefitinib resistant NSCLC, while its inhibition resensitizes cells to gefitinib (109). Exosomal miR210 leads to EMT and drug resistance to osimertinib, a third generation EGFR tyrosine kinase inhibitor (EGFR TKI). Low levels of miR-200c plays a role in EGFR TKI resistance in wild type EGFR NSCLC (110). Exosomal miRNAs play a role in phenotypic alteration of NSCLC via EMT and histological transformation into SCLC (111).

MiRNAs in lung cancer diagnosis

Numerous studies highlight the power of miRNAs in lung cancer as predictive, diagnostic and therapeutic biomarkers (16). As miRNAs are more specialized than regular gene expression profiling, their occurrence and relative abundance can differentiate tumor types and stage (112). Their physico-chemical stability in body fluids, preservatives and in laboratory processing techniques makes them robust analytes for diagnostic testing (112). Their presence in most body fluids, pleural fluid, blood, colostrum, urine etc. renders them widely accessible as non-invasive analytes. Emerging techniques of circulating cell-free miRNA analysis enables total tumor assessment alleviating the problem of tumor heterogeneity (113). MiRNA that broadly target all aspects of tumor biology have been studied as diagnostic and prognostic biomarkers. For instance, detecting specific miRNA species in tumor samples can differentiate between histologic types. miR-205 has been shown to be specific for squamous cell carcinoma, while miR-124a identifies adenocarcinoma (114). Expression profiling studies of miRNAs showed elevated levels of miR-93, mir-221 and miR-30e in LUSC while LUAD had elevated expression of miR-29b, miR-29C, let-7, miR-100 and miR-125a-5p (115,116). A study comparing metastatic cancers to primary disease identified high expression of miR-126 in metastatic disease, while the primary lung malignancies showed elevated expression of miR-182 (117). An expression signature showing high miR-155 and low let-7a suggested poor survival. MiRNAs could also distinguish high grade pre-neoplastic lesions from low grade and carcinoma in situ (38).

Zhong et al. in their systemic literature review of miRNAs identified predictive and prognostic miRNA biomarkers (14). Blood borne miR-20a, miR-10b, miR-150 and miR-223 were excellent biomarkers for NSCLC. miR-205 had a high specificity for LUSC. Histologic prediction of lung cancer subtype was possible using 38 miRNA in blood and tumor tissue. Prediction for checkpoint inhibition responsiveness could be made with a panel of miRNA biomarkers that included miR-34a, miR-93, miR-106b, miR-181a, miR-193a-3p, miR375. MiRNAs that influenced the expression of ICI biomarkers, PD-L1 and PD-1 expression, were identified and included miR-103a-3p, mir-152, miR-152-3p, miR-15b, miR-16, miR-194, miR-34b and miR-506. These were suggested as potential targets for therapeutic intervention. miRNAs e.g., miR-21, miR-25, miR-27b, miR-19b, miR-152b, miR-164a, and miR-210 were markers for response to platinum-based therapy. These studies show the diagnostic capability of miRNA as a biomarker in cancer diagnosis. Profiling miRNAs that specifically target TCP processes, including EMT, CSC etc. may offer diagnostic insights and therapeutic targets into specific TCP mediated tumor biology. This information could be useful in modeling and predicting tumor evolution, metastasis, therapeutic resistance and other tumor behaviour particularly those resulting from selective pressure from chemotherapy.

MiRNA and potential therapeutic target in lung cancer

Reversing or harnessing the oncogenic or tumor suppressive properties of miRNAs is a potent strategy in cancer therapeutics. They are particularly potent targets as a single miRNA can coordinately regulate numerous genes in different cancer pathways (118). EMT is an important factor in drug resistance. Many miRNAs are involved in regulation of EMT and drug resistance. MiR-26a overexpression reduces proliferation, promotes apoptosis and EMT to MET switch by inhibition of EZH2-dependent EMT induction (119). MiR-130a promotes multidrug resistance, targets MET and reduces TRAIL resistance (120). A number of miRNAs reverse EMT and inhibit specific signaling pathways and/or drug transporters thereby reducing resistance to cisplatin. Examples include miR-164b, which targets protein-tyrosine phosphatase 1b (PTP1B) (121); miR-218 targets Slug/ZEB2 (108); Let-7c inhibits ABCC2-transporter and Bcl-XL (122). Downregulation of miR-205 occurs through activation of SRC and ZEB1 via TDGF1, an EGF-related protein, resulting in EMT stimulation leading to erlotinib resistance in EGFR-mutant NSCLC cells. Thus SRC and EGF may be targets to overcome EGFR TKI resistance (123). Targeting certain signaling pathways may be a mechanism to redirect miRNA EMT and chemoresistance effects. For instance, targeting PRKCA to inhibit FAK/Ras/c-Myc signaling activated miR-296-3p, blocking cisplatin chemoresistance and EMT signaling. In contrast, DDX5/HDGF/beta-catenin signaling inactivated miR-296-3p and promoted metastasis and chemoresistance (124). Experimental evidence has shown that altering the expression of certain miRNAs improves sensitivity to platinum-based therapy. For instance, miR-503 inhibits many resistance related genes such as MDR1, MRP1, ERCC1, survivin, and Bcl-2 leading to reduced drug efflux and improved cisplatin responsiveness (125). However, upregulated miR-196 enhances the activity of these genes resulting in increased drug efflux and cisplatin resistance.

The EMT related miRNAs are potential candidates for targeted therapies in lung cancer. Zhang et al. showed that a panel of miRNAs, miR-193a-3p, miR-210-3p and miR5100 in hypoxic bone marrow stem cell derived exosomes stimulated EMT through the STAT-3 pathway and could be a biomarker for cancer metastasis (126).

Other therapeutic strategies to tackle TCP involve targeting DTP cells. Potential approaches involves adding epigenetic modulators into existing treatment protocols, targeting signaling pathways activated in cancer cells, or targeting microenvironment regulators that promote DTP cells (69). The histone deacetylase (HDAC) can be combined with a TKI in NSCLC to disrupt the repressed chromatin state associated with TKI resistance (127). This approach has been demonstrated to be promising in vitro and in clinical trials (128). The FGFR family (FGFR1/3) are implicated in EGFR TKI resistance. FGFR1 expression promotes survival of mesenchymal-like EGFR-mutated cancer cells. Hence, dual EGFR/FGFR inhibition is a potential resistance-reversal approach and has shown promise in overcoming acquired drug resistance associated with EMT in clinical trials (129). Enhancing the activity of TF ZNF263 improves therapeutic response of LUAD with EGFR-targeted therapies by its effect on DTP (130). Other proposed strategies include treatment with apoptosis inducers, aurora kinases, beta-catenin, AXL, Notch inhibitors etc. (131).

Targeting the TME to make DTP cells more vulnerable may involve disrupting the paracrine support of the TME or inhibiting the pro-cancer TME cells. Promising preclinical models demonstrate that the blocking of the HGF-MET or the IGF-1/IGF-1R axis of TAM and cancer associated fibroblasts promotes reversing DTP states and increases sensitivity of tumor cells to EGFR TKI (106). A third strategy against TCP relies on the targeting of CSCs. Proposed therapeutic strategies include the use of epigenetic drugs that inhibit chromatin modifiers important in the tumorigenicity of CSC e.g., lysine demethylase 1A (LSD1A) (132).

Alternative approaches depend on inhibiting CSC cell surface markers or signaling pathways (69). Bhummaphan et al. demonstrated that Lusianthridin attenuated CSC in lung cancer cell through downregulation of Src-STAT3-c-Myc pathways and reversed the CSC marker CD133 (133). However, CSC targeting poses significant hurdles as they share pathways with important developmental processes resulting in significant toxicity. Furthermore, depleting CSC in tumor disrupts the equilibrium and activates the plasticity of non-CSC to replenish the CSC pool.

Conclusions

The critical role of TCP by miRNAs in NSCLC tumorigenesis is increasing becoming more evident. A comprehensive understanding opens avenues to precisely outline tumor trajectory and its increasing malignant behaviour, and promptly intervene when clinically necessary. Tumor biology such as metastatic capacity, immune evasiveness and therapy resistance can be determined clinically and in real time. The application of this knowledge for precision diagnostics, prognostication and treatment is possible given the highly specialized and cell specific role miRNA plays in every stage of NSCLC carcinogenesis. Temporal and spatial miRNA activity adds an additional layer to prevailing methods in molecular diagnostics. Longitudinal studies of tumor miRNA can illuminate the dynamic life cycle of cancer and give advance notice to alter treatments in response to tumor changes due to plasticity. Non-invasive procedures such as liquid biopsy enables multiple temporal sampling to investigate tumor miRNA status, moving the diagnostic field from a single static time-stamp of tumor status to a dynamic longitudinal picture. The tumor cell-specific and tissue-specific nature of miRNAs, highlighted by tumor heterogeneity, and the paracrine and exosomal cross-talk with cellular components of the TME requires new technologies to properly interrogate the geospatial profile of NSCLC. Such technologies include spatiotemporal profiling and single cell RNA sequencing. These technologies remain to be clinically validated and translated into routine molecular diagnostics. Clinical translation will provide cell specific information in its architectural relationships, giving a finely tuned assessment of tumor behaviour in the context of the TME. Clinical applications of technologies such as cell-free circulating miRNA and exosomal vesicles assessment would produce a global picture of tumor profile. Current methods are typically undermined by tissue sampling bias from tumor heterogeneity. In conclusion, TCP by miRNA holds immense potential for understanding tumor biology, clinical applications and to achieve the aims of precision medicine.

Acknowledgments

None.

Footnote

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2530/prf

Funding: None.

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2530/coif). I.I. serves as an unpaid editorial board member of Translational Cancer Research from August 2025 to June 2027. The author has no other conflicts of interest to declare.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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