Emerging data have indicated that the fibulin (FBLN) family comprising seven members (fibulin-1–7) of widely expressed ECM proteins is associated with lung cancer invasion and metastasis. FBLNs are ECM glycoproteins consisting of EGF-like domain repeats crucial for normal organogenesis and embryonic development (24). They are vital for these biological processes as they regulate cell-to-matrix communication and ECM structure stabilization through intermolecular bridges that bind to several supramolecular structures (25, 26). Besides their structural role, FBLNs are linked to many cellular signaling events and complex biologic processes, including cellular proliferation, adhesion, and migration (25, 27).

Fibulin-1 (FBLN1) expression levels are substantially downregulated in NSCLC (28). The role of FBLNs in regulating the EGFR function is shown in Figure 2 . Harikrishnan et al. used siRNA to knock down FBLN1C and FBLN1D expression in NSCLC Calu-1 cells to examine if FBLN1 isoforms could play a role in controlling EGFR signaling and function (28). Without affecting overall EGFR expression levels, FBLN1C and FBLN1D expression loss significantly increases basal (with serum) and EGF-mediated EGFR activation. Conversely, overexpression of FBLN1D and FBLN1C inhibits EGFR activation, indicating a regulatory crosstalk between the two proteins.

FBLN3’s functions and signaling mechanisms in lung cancer stem cells (CSCs) were investigated (29). Moreover, FBLN3 was downregulated in the lung (30) and nasopharyngeal carcinomas (31). Forced expression of FBLN3 reduces the expression of epithelial-mesenchymal transition (EMT) activators, including N-cadherin and Snail, which inhibit ADC cell invasion and migration. FBLN3 inhibits the stemness activities of ADC cells, as shown by a decline in spheroid formation and the levels of stemness markers, including SRY-like HMG box (Sox2) and β-catenin. FBLN3 effects are mediated by the glycogen synthase kinase-3β (GSK3β)/β-catenin pathway and the upstream regulators of GSK3β such as (PI3K)/AKT and insulin-like growth factor receptor (IGF1R). Furthermore, IGF1R was discovered to be a direct target of FBLN3, which inhibits the action of IGF. Further, FBLN3 inhibits lung CSC and EMT by modulating the IGF1R/PI3K/AKT/GSK3 pathway, and that FBLN3 may be used as a CSC-centered therapeutic alternative (29). FBLN3 could attenuate the invasion of NSCLC A549 cells by inhibiting the transcription of matrix metalloproteinase- (MMP)-7 and MMP-2 (32). Again, Chen et al. revealed the function of FBLN3 and FBLN5 as suppressors of lung cancer invasion and metastasis through the inhibition of Wnt/β-catenin and ERK signaling pathways (33) that, in turn, downregulate MMP-2 and MMP-7 expression (32) and inhibit lung cancer cell survival, proliferation, and metastasis (34, 35). Moreover, FBLN3 overexpression notably decreased the activities of MMP-2 and MMP-9 and repressed the invasion of NSCLC A549 cells; thus, it could be used as a therapeutic strategy for NSCLC (36).

FBLN5 (DANCE), a vascular integrin receptor ligand, is a distinct member of fibulins harboring the RGD (Arg-Gly-Asp) motif associated with endothelial cell adhesion (37). FBLN5 can also depend on RGD to attenuate angiogenesis (38). It interacts directly with elastic fibers in vitro, and its amino-terminal domain serves as a ligand for cell surface integrins αvβ3, α9β1, and αvβ5 (39–41). FBLN5 expression is induced under pathological conditions, including pulmonary hypertension and lung injury (42), and is controlled by transforming growth factor-β (TGF-β) (43). FBLN5 was discovered to be a suppressor of lung cancer invasion and metastasis via inhibiting MMP-7. Indeed, FBLN5 knockdown induces cell invasion and MMP-7 expression. In lung tumors, the expression levels of FBLN5 and MMP-7 are inversely associated. FBLN5 suppresses MMP-7 expression through the ERK pathway, which is mediated by an integrin-binding RGD motif. FBLN5 overexpression in H460 lung cancer cells also prevents metastasis in mice. These findings indicate that epigenetically silenced FBLN5 promotes lung cancer invasion and metastasis by inducing MMP-7 expression through the ERK pathway (44).

Mucins (MUCs) are high M.wt glycoproteins synthesized by many epithelial tissues (45). They are categorized into two major groups: secretory mucins and membrane-bound mucins. There are 11 membrane-bound mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC20, and MUC21) and seven secreted mucins (MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC8, and MUC19) (46). MUCs are involved in the normal development of the lungs and are expressed during the embryonic stages of lung development. The cytoplasmic domain of MUC1-C contains i) a YEKV motif: a substrate for EGFR phosphorylation and a SRC SH2 binding site (47), ii) a YHPM motif: a binding site for PI3K and the AKT pathway activation (48, 49), and iii) a YTNP motif: Upon tyrosine phosphorylation, it interacts with Grb2, which binds MUC1-C to son of sevenless (SOS) and thereby activating the RAS→MEK→ERK pathway (49).

MUC1 is an oncogenic glycoprotein that binds to EGFR, serving as a substrate, and that MUC1 expression can enhance EGFR-dependent signaling. MUC1 expression can prevent degradation of EGFR in breast epithelial cells using overexpression constructs and RNAi-mediated knockdown of MUC1, increasing total cellular pools of EGFR (50). The MAPS (MUC1-associated proliferation signature) includes a cytoplasmic domain of MUC1 (MUC1-CD)-dependent genes, including cyclin B1 (CCNB1), cyclin-dependent kinase inhibitor 3 (CDKN3), cell division cycle protein (CDC2, CDC20), mitotic arrest deficient 2-like protein 1 (MAD2L1), protein regulator of cytokinesis 1 (PRC1), and ribonucleoside-diphosphate reductase subunit M2 (RRM2), which are involved in cell cycle and proliferation regulation and have been linked to poor outcomes in patients with lung adenocarcinoma (51). MUC1 is expressed as MUC1-N and MUC1-C, a non-covalent heterodimer of N-terminal and C-terminal subunits, respectively (46). MUC1 overexpression, in association with MUC1-C, contributes to activation of the nuclear factor Kappa-activated B cells (NF-κB) (52), Wnt/β-catenin/TCF4 (transcription factor 4) (53), and STAT1/3 pathways in NSCLC (54). In NSCLC, the heterodimeric protein MUC1 is abnormally overexpressed, resulting in gene signatures linked to poor patient survival (48). The cytoplasmic domain of MUC1-C is associated with PI3K p85 in NSCLC cells.

Blocking the interaction of MUC1-C with PI3K p85 via cell-penetrating peptides suppresses Akt phosphorylation and its downstream effector mTOR. Treatment of NSCLC cells with GO-203, a MUC1-C peptide inhibitor, results in downregulation of PI3K-Akt signaling, growth inhibition, an increase in reactive oxygen species (ROS), and necrosis induction via a ROS-dependent mechanism. Furthermore, in H1975 (EGFR L858R/T790M) mutant cells and A549 (K-Ras G12S) xenografts developed in nude mice after treatment with GO-203, tumor regressions were observed. These data suggest that MUC1-C is needed for PI3K-Akt pathway activation and survival in NSCLC cells (48). Galectin-3 is a β-galactoside binding protein that has also been linked to human cancer development. Glycosylation of the C-terminal subunit of Asn-36 is necessary for galectin-3 upregulation. Two Sentences have been transferred to section no. 8. Galectin-3 binds to MUC1-C at the glycosylated Asn-36 site. Galectin-3 acts as a bridge between EGFR and MUC1, besides galectin-3 is needed for EGF-mediated interactions between MUC1 and EGFR that support the importance of the MUC1-C-galectin-3 interaction (55).

In EGFR mutant NSCLC, MUC5B-positive patients had significantly longer overall survival and relapse-free survival than MUC5B-negative patients. MUC5B appears to be a novel prognostic biomarker in NSCLC patients with EGFR mutations (56). Lung ADC subtypes, including invasive mucinous adenocarcinoma (IMA) and lepidic predominant adenocarcinoma (LPA) are associated with MUC expression. In this regard, MUC1 is expressed in LPA, whereas MUC5B, MUC5AC and MUC6 are expressed in IMA (57). Also, EGFR and KRAS (Kirsten Rat Sarcoma viral oncogene homolog) mutations and Hnf4α expression may participate in mucin expression profiles in these lung ADC subtypes (57). The overexpression of MUC21 proteins with a particular glycosylation state is implicated in developing EGFR-mutated lung ADCs associated with a high frequency of lymphatic vessels invasion and lymph node metastasis (58). Additionally, MUC5AC is linked to poor prognosis and would be a prospective therapeutic target in lung ADC due to its role in enhancing tumor heterogeneity with mucin production (59). Therefore, developing treatment strategies targeting MUCs’ expression and functions to manage NSCLC progression are under investigation (60).

Fibronectin (FN) is present in multiple isoforms through alternative splicing, where 20 isoforms in humans have been discovered (61) and are involved in mediating many cellular interactions with the ECM (62). It is primarily synthesized by CAFs and polymerized into ECM fibrils that act as scaffolds for ECM binding molecules such as growth factors and cell surface receptors (63). FN is overexpressed in the stroma of NSCLC and can promote cancer cell adhesion, growth, differentiation, migration, invasion, survival, and resistance to chemotherapy (64). FN-dependent molecular pathways can control the tumor cell response to the stromal matrix and represent potential targets for managing chemo-resistant tumors (65). FNIII-1c, a peptide mimetic, can activate Toll-like receptors (TLRs) to promote NF-κB activation and release inflammatory cytokine in fibroblasts ( Figure 3 ) (66, 67). Notably, the PI3K/Akt pathway is the main pathway by which most cytokines and growth factors activate mTOR and its downstream targets. In NSCLC H1838 and H1792 cells, FN induces phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and p70S6K1(two downstream targets of mTOR), and Akt phosphorylation (an upstream inducer of mTOR), whereas it inhibits the tumor suppressor protein phosphatase that antagonizes the PI3K/Akt signal (68). Furthermore, FN inhibits liver kinase B1 (LKB1) mRNA and protein expression, as well as the phosphorylation of AMP-activated protein kinase (AMPK), both of which are known to inhibit mTOR. These data indicate that NSCLC cell proliferation induced by FN is mediated by Akt/mTOR/p70S6K pathway activation and LKB1/AMPK signaling inhibition (68).

Laminins (Lns) are heterotrimeric extracellular glycoproteins found in all BMs. So far, more than 17 Ln isoforms have been identified with a cross-shaped and specific arrangement of α, β, and γ subunits (69). Ln-332 and Ln5 consist of heterogeneous α3, β3, and γ2 chains and serve as BMs’ essential structural constituent. Ln5 plays a crucial role in cellular migration and tumor invasion (70, 71). NSCLC patients with positive Ln5 expression had a slightly lower survival rate than Ln5-negative expression counterparts. Besides, positive Ln5 expression combined with the loss of PTEN, positive active EGFR expression, or positive active Akt expression has a significantly different overall survival. According to Cox regression analysis, the co-expression of Ln5, PTEN, and p-Akt are the three most independent prognostic markers in NSCLC patients. The findings illustrate the intricate tumorigenesis relationship between key signaling pathway molecules and ECM proteins (71) ( Figure 3 ). A Ln receptor, namely integrin α6β4, triggers carcinoma progression through cooperation with various GFRs to facilitate invasion and metastasis (72). Using a lung cancer tissue microarray and immunohistochemistry (IHC), Stewart et al. discovered that SqCC has a higher integrin β4 (ITGB4) expression than ADC, and these data were verified in external gene expression data sets. Overexpression of ITGB4 is also linked to venous invasion and a lower overall patient survival rate. The most highly 50 altered genes related to ITGB4 identified in SqCC were Lns, CD151, collagens, PI3K, and EGFR-associated pathway genes, other recognized signaling partners using cBioPortal. Finally, they show that ITGB4 is overexpressed in NSCLC and is an unfavorable prognostic factor (72). Overall, these data suggest a potential correlation between Lns and EGFR in lung cancer prognosis; however, further studies are still required to profile expression patterns of different Ln types in NSCLC to underscore their clinical relevance.

Fibrinogen is a 350 kDa glycoprotein synthesized mainly by the liver epithelium (73). It comprises two similar sets of three polypeptide chains, including Aα, Bβ, and γ, linked by five symmetrical disulfide bridges (74). Many proteins and cytokines such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) bind to fibrinogen affecting its biological behavior (75, 76). Lungs produce fibrinogen by inflammatory stimuli (77). Fibrinogen changed into insoluble fibrin via activated thrombin considerably affects blood clotting, inflammatory response, wound healing, fibrinolysis and neoplasia. Increased fibrinogen activity considerably affects cancer cell growth, progression, and metastasis (78).

Accumulating evidence indicates a correlation between fibrinogen and EGFR in lung cancer (79–81). A study by Shang et al. discovered a novel serum protein, fibrinogen alpha chain isoform 2 (FGA2), in lung ADC patients with mutated EGFR using microarray data analysis of 41,472 antibodies coupled with mass spectrometry analysis (79). Further, plasma FGA2 levels were remarkably downregulated in EGFR-mutated patients relative to those with the wild-type EGFR (81). In the same study, hyperfibrinogenemia was linked to distant metastasis and lymphatic tissue metastasis. A multivariate model based on fibrinogen and smoking history was also used to predict EGFR mutation status in NSCLC patients (81). Furthermore, Fibrinogen-like protein 1 (FGL1) is significantly overexpressed in the gefitinib-resistant NSCLC cell line PC9/GR more than in the gefitinib-sensitive NSCLC cell line PC9 with an EGFR mutation. However, FGL1 knockdown reduces cell viability, decreases gefitinib IC50, and increases apoptosis in PC9/GR and PC9 cells after gefitinib therapy. FGL1 knockdown in PC9/GR tumor cells increases gefitinib’s inhibitory and apoptosis-inducing effects in a mouse xenograft model. Gefitinib’s possible mechanism for inducing apoptosis in PC9/GR cells includes suppressing FGL1 and activating Poly (ADP-Ribose) Polymerase 1 (PARP1) and caspase 3 pathways. By regulating the PARP1/caspase 3 pathway, FGL1 promotes acquired resistance to gefitinib in the PC9/GR NSCLC cell line. As a result, FGL1 may be a possible therapeutic option for NSCLC patients who have developed resistance to gefitinib (80).

Tenascin-C (TN-C) is a glycoprotein composed of 4 distinct domains interacting with matrix constituents, cell surface proteins, soluble factors, and pathogenic components. TN-C affects pulmonic blood vessel invasions by decreasing apoptosis and promoting cancer cell plasticity, thus, increasing lung metastasis (82). TN-C also binds to more than 25 different molecules, including EGF-L repeats (a low-affinity ligand for the EGFR, MAPK, and phospholipase-C gamma (PLC)-γ signaling). Besides, TN-C binds to FNIII, aggrecan, integrins, and perlecan, along with growth factors such as FGF, platelet-derived growth factor (PDGF), and TGF-β families (83). Again, receptor-type tyrosine-protein phosphatase zeta (PTPRζ1), fibrinogen-like globe (FBG) that can bind to integrins, and TLR4 are TN-C-related molecules (83). These diverse interactions render TN-C a significant driver for many processes such as cell attachment, cell migration, cell spreading, cell survival, focal adhesion, neurite outgrowth, protease, and matrix assembly, and pro-inflammatory cytokine synthesis (83). However, a correlation between TN-C and EGFR has not yet been elucidated in NSCLC.

Periostin (Postn, PN, or osteoblast-specific factor OSF-2) is a vital ECM protein known for its complex role in tumorigenesis (84). It can directly bind to many ECM proteins, including TN-C, FN, collagen, and Postn itself (85). Also, it acts as a ligand for numerous integrins such as α_vβ₃, α_vβ₅, and α₆β₄ to participate in cell adhesion, survival, and migration (85, 86). Postn affects tumor progression by regulating cellular survival, angiogenesis, invasion, and metastasis in epithelial tumors (85). Periostin is overexpressed and enhances metastatic growth in colon cancer by inhibiting stress-induced apoptosis in cancer cells and increasing endothelial cell survival to boost angiogenesis. Although there is no direct association between Postn and EGFR in lung cancer, Postn can regulate EGFR interacting partners or its downstream signaling. Periostin increases cellular survival at the molecular level by activating the Akt/PKB signaling pathway through α_vβ₃ integrins (87). In lung cancer, high Postn expression is positively associated with the EMT markers Snail and Twist and lung cancer stage, according to IHC results. Further, recombinant Postn causes EMT in lung cancer cells through the p38/ERK pathway, and that pretreatment with chemical inhibitors prevents Postn-induced EMT (88). Moreover, the increased Postn expression in the NSCLC A549 cells is one form of cellular response to chemical-mimic hypoxia stress, and this effect can be controlled by hypoxia-inducible growth factors like TGF-α and bFGF, which trigger the RTK/PI3-K pathway leading to upregulation of Postn, and in turn, facilitating the survival of A549 cells in a hypoxic microenvironment via the Akt/PKB pathway (89). Collectively, these data indicate that Postn may serve as a therapeutic target in NSCLC.

Vitronectin (VTN) is a multifunctional glycoprotein found in blood and ECM. It binds collagen, glycosaminoglycans, the urokinase-receptor, and plasminogen and stabilizes plasminogen activation inhibitor-1 (PAI-1)’s inhibitory conformation. VTN can potentially control the ECM proteolytic degradation through its localization in the ECM and binding to PAI-1. VTN also binds to complement, heparin, and thrombin-antithrombin III complexes, suggesting an immune response role and clot formation control (90). VTN is mostly overexpressed in smaller and well-differentiated tumors (91). EGF promotes carcinoma cell metastasis by phosphorylating p130 CAS in an Src-dependent manner, activating Ras-related protein 1 (Rap1), a small GTPase implicated in integrin activation. Src activity induced by EGFR causes phosphorylation of the CAS substrate, required for Rap1 and αvβ5 activation (92). EGFR activation of Src initiates αvβ5-mediated migration in FG (express stably mutational active Y527F (SrcA) pancreatic carcinoma cells. EGF causes cell metastasis and αvβ5-mediated Rap1 activation. Rac1 and Rap1 activity are increased in FG cells plated on anti-β5, but not anti-β1, integrin antibodies after EGF therapy. Rap1 knockdown on vitronectin but not fibronectin prevents EGF-induced cell migration. In the chick CAM model, knocking down integrin β5 expression prevents EGF-induced pulmonary metastasis but not primary tumor weight (92).

Nidogen (NID1 and NID2) are present in the BM and help maintain its stability by connecting COLIV and Ln networks in the ECM (93, 94). The determination of NID2 methylation represents a biomarker for NSCLC diagnosis (95). The lung metastasis of NID1– or 2–deficient mice were studied after being intravenously injected with B16 murine melanoma cells. The authors demonstrated that the depletion of NID2, but not NID1, facilitates melanoma cell lung metastasis. According to histological and ultrastructural examination, the morphology and ultrastructure of BMs, including vessel BMs, are not different in NID1– and 2–deficient lungs. Furthermore, there is no difference in the deposition and distribution of the main BM components between the two mouse strains. These findings indicate that the absence of NID2 can cause subtle changes in endothelial BMs in the lung, allowing tumor cells to move through these BMs more quickly, resulting in a higher risk of metastasis and larger tumors (96). Further, NID2 inhibits liver metastasis in a significant way. NID2 suppresses the EGFR/Akt and integrin/focal adhesion kinase (FAK)/PLC metastasis-related pathways; these data shed light on NID2’s critical tumor metastasis-suppression functions in cancer (97). The roles of NID1 and NID2 in NSCLC have not yet been fully characterized.

Glypicans (GPCs), a heparan sulfate proteoglycan (HSPG) family, consist of core proteins (60- to 70-kDa), heparan sulfate (HS) chains, and a glycosylphosphatidylinositol linkage (110). There are six known GPCs (GPC1-GPC6) in humans. GPCs participate in cell growth by regulating Wnt (111), development by modifying morphogen gradient formation (112), and other multiple signaling pathways. GPCs are abnormally expressed in multiple types of cancer and are crucial for cancer cell growth and progression. The expression of GPC5 is regulated to control cell growth and differentiation throughout mammalian development (113). Also, genetic variations of GPC5 may share in the increased risk of never-smokers (114). GPC5 mRNA and protein levels are overexpressed in A549 and H3255 cells. Using shRNA-mediated knockdown or overexpression of GPC5, the migration rates of A549 and H3255 cells transfected with pRNAT-shRNA-GPC5 are lower than controls employing scratch and transwell assays. Using immunohistochemical staining, the high GPC5 expression level in NSCLC is linked to respiratory symptoms of lung cancer, regional lymph node metastasis, poor differentiation, vascular invasion, and a higher TNM stage. According to the Kaplan-Meier analysis, NSCLC patients with high levels of GPC5 expression have a shorter overall survival time relative to those with low levels of GPC5 expression (115). Conflicting data indicated that GPC5 is downregulated and linked to a poor prognosis in lung ADC tissues. Further, the loss of GPC5 expression is controlled by its hypermethylation, according to de-methylation experiments. GPC5 overexpression inhibits lung cancer cell proliferation, migration, and invasion in vitro and slows tumor growth in vivo, whereas GPC5 knockdown reverses these effects. Moreover, via binding Wnt3a on the cell surface, GPC5 inhibits Wnt/β-catenin signaling, thereby mediating tumor suppressor action (113). Therefore, targeting particular GPCs in the tumor microenvironment that acts as ligands for inducing oncogenic pathways represents an effective cancer therapy strategy (100). Although the functions of GPCs have been assigned in different tumors, including lung (116), colon (117), and breast (118) cancers, esophageal squamous cell (119) carcinoma, and pancreatic ductal adenocarcinoma (120), their interaction with EGFR in NSCLC remains yet to be explored.

The syndecan (SDC) family is a transmembrane protein that possesses HS chains on their extracellular domains (121) and consists of four members (SDC1-SDC4). SDC-1 is frequently misexpressed in cancer and associated with invasion, metastasis, angiogenesis, and dedifferentiation (122–128). SDC-1 acts as a coreceptor for a wide range of growth factors (129), including bFGF and HB-EGF (130). A recent study reported that lung cancer has noticeable SDC-1, yet its expression does not associate with lung cancer patients’ survival rate (121). However, a study by Shah et al. revealed that the expression of either NSCLC subtype classifiers EGFR and SDC-1 determined by tissue microarray is correlated with a 30% reduction in the risk of death. Loss of expression of these histologic classifiers is linked to aggressiveness in lung tumors and a poor prognosis (106). Besides, Zhu and colleagues interestingly reported that NSCLC patients with both a SDC4-ROS1 rearrangement and an activating EGFR mutation might acquire resistance to EGFR-TKIs. Although the coexistence of two driver gene mutations in NSCLC is uncommon, triggering alterations of EGFR, ROS1, ALK, and KRAS have recently been recorded (131, 132).

HA is a plentiful constitute of the pericellular matrix that plays a vital role in regulating tissue homeostasis and cancer progression through its interaction with the cell surface receptor CD44 (133). HA synthesis is controlled by growth factors (e.g., EGF) and cytokines such as IL-1β (133). Three hyaluronan synthases (HAS) isoforms, including HAS1, HAS2, and HAS3, are known. CD44-HA interaction can modulate a variety of intracellular signaling by forming coreceptor complexes with many RTKs (e.g., EGFR) (134) that induce oncogenic pathways involved in cancer cell invasion, migration, and metastasis in the human MCF7 and TamR breast cancer cells (135). HA and CD44 are overexpressed in NHLFs/LCAFs (normal human lung fibroblasts vs. lung cancer-associated fibroblasts), followed by NSCLC cells. In NSCLC cells, exogenous HA somehow rescues the fault in cell proliferation and survival. Further, simultaneous silencing of HAS2 and HAS3 or CD44 suppresses the EGFR/AKT/ERK signaling pathway, cell proliferation, and survival (136, 137). Of note, dual targeting CD44/EGFR by HA-based nanoparticles along with systemic administration of plasmid DNA expressing wild-type (wt-) p53 and microRNA-125b (miR-125b) in a genetically engineered mouse model of lung cancer led to an increase of wild-type p53 and miR-125b gene up to 20-fold associated with elevated caspase-3 and APAF-1 expression-induced apoptosis; thus it may represent an effective gene therapy for NSCLC (138).

Interestingly, treatment with EGF and IL-1β, either alone or combined with TGF-β in ADC, can stimulate HA production in A549 cell line, where treatment with TGF-β/IL-1β changed cell morphology, induced EMT with altered vimentin and E-cadherin gene expression. Also, HAS3 overexpression induces HA synthesis, MMP9 expression, EMT phenotype, and MMP2 activities and increases invasion of epithelial ADC cell line H358 (133). Induction of HA in H358 cells and adding exogenous HA in A549 cells significantly improved resistance to EGFR inhibitor Iressa. These results propose that increased HA production can promote EMT and Iressa resistance in NSCLC (133). Thus, regulating HA expression in NSCLC can be a new therapeutic strategy (133). Again, HA is implicated in EMT through EGF or TGF-β1 signaling in lung cancer cell line A549, where TGF-β1 upregulates HAS1, HAS2, and HAS3 expressions and augments CD44 expression interacts with EGFR, leading to the activation of the downstream signaling AKT and ERK pathways (139). On the contrary, pretreatment with HAS inhibitors such as 4-methylumbelliferone (4-MU) can suppress TGF-β1’s impact on the expression of CD44 and EGFR and inhibit the CD44-EGFR interaction. Collectively, these data indicate that HA/CD44 interaction mediated by TGF-β1 transactivates EGFR signaling, resulting in EMT induction in NSCLC cells (139).

Collagens (COLs) are the major ECM proteins (up to 30% of the total protein mass) in the human body. They are arranged in a relaxed meshwork and possess elasticity to extreme tensile strength owing to their surrounding proteins like elastin and glycoproteins (140). The individual structure of COLs can also create an intricate network that enables them to interact with each other and the surroundings (141). There are 28 known COL types and divided into specific subgroups according to their supramolecular assemblies, including a) fibrillar-forming COLs: the IM significant components such as COL type I, II, III, V, XI, XXVI, XXVII; b) the network-forming COLs: the main components of basement membrane such as type IV, VIII, X, and XVIII COLs (142). Of note, COLI, COLIII, and COLV are predominantly fibroblasts-derived COLs, while COLIV is mainly expressed by epithelial and endothelial cells (143); c) fibril-associated COLs with interrupted triple helices (FACITs) (e.g., IX, XII, XIV, XVI, XIX, XX, XXI, XXII, XXIV); and d) MACITs (membrane-anchored collagens with interrupted triple helices) such as type XIII, XVII, XXIII, and XXV COLs (144–146). COLIV is upregulated in NSCLC stroma, promoting the in vitro impairment of cell apoptosis and multidrug resistance. For example, NSCLC cells expressing COLIV are resistant to cis-platinum (DDP), which is mechanistically attributed to the PI3K pathway (147).

Notably, many studies addressed the significant effect of CAFs in tumorigenesis (148, 149). CAFs are the key players in COL dysregulation and turnover, resulting in desmoplasia (tumor fibrosis), where COLs deposit excessively in the tumor surroundings, crosslink, and linearize, thus increasing tissue stiffness (150). This influences the behavior of the nearby tumor cells and controls cell differentiation, proliferation, migration, gene expression, invasion, metastasis, and survival; thereby, directly affecting the cancer hallmarks (151). Tumor tissue with considerable fibroblast-derived COLs is correlated with poor outcomes (152–154). A study by Li et al. reported that the regulation of autocrine COLI expression for sustaining lung cancer cell growth in 3D cultures with fibroblasts provides a new insight for lung cancer targeted therapy (155). CAFs and other molecules can regulate COLs’ expression in cancer cells, such as transcription factors, mutated genes, receptors, and signaling pathways; these molecules can also affect tumor cell behavior by integrins, RTKs (e.g., EGFR), and discoidin domain receptors (156).

In healthy tissues, the biosynthesis of COLs is highly controlled by a great counterbalance of many enzymes, including MMPs and their inhibitors and lysyl oxidases (LOX) (157, 158). In lung tumors, the stroma comprises a stiffer matrix than normal lung tissues due to more collagen modifications (159) mediated by lysyl hydroxylase-2 or procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) enzyme enhancing cell invasion and metastasis (160). Fibrotic collagen is primarily modified by PLOD2. PLOD2 was elevated in NSCLC specimens and was linked to a poor prognosis in NSCLC patients. PLOD2 directly enhances NSCLC metastasis by promoting migration and indirectly by inducing COL reorganization, evident by gain- and loss-of-function experiments and an orthotopic implantation metastasis model. In addition, PLOD2 regulation is achieved by PI3K/AKT-FOXA1 axis. The transcription factor FOXA1 directly binds to the PLOD2 promoter for the transcription of PLOD2. These findings indicated that the NSCLC metastasis mechanism could be regulated by EGFR-PI3K/AKT-FOXA1-PLOD2 pathway and PLOD2 can be a therapeutic target for NSCLC treatment (161).