Cancer is a paramount societal, public health and economic challenge in the twenty-first century, accounting for nearly 16.8% of global deaths (one in six deaths) and 22.8% of deaths attributable to non-communicable diseases (1). The disease is responsible for 3 in 10 premature deaths from non-communicable diseases worldwide (30.3% among individuals aged 30-69 years), ranking amongst the three principal causes of mortality within this demographic across 177 of 183 nations (2). In the United States in 2025, there will be ~2,041,910 new cancer diagnoses, equating to ~5,600 cases daily (3). Oncological research has predominantly focused on intrinsic characteristics of neoplastic cells, with comparatively limited attention to extracellular stromal components. The extracellular matrix (ECM) constitutes a crucial element of tumor stroma. Alterations in ECM protein expression represent significant mechanisms influencing tumor cell proliferation, migration and invasion, with Fibulin2 serving as a typical protein. Even intracellular modifications in Fibulin2 expression may have a substantial impact on the ECM due to the secretory properties of Fibulin2. Furthermore, Fibulin2 is considered to be a promising biomarker in multi-tumor research contexts (4-7).

Fibulin2 was initially characterized by Pan et al (8), who identified it in a murine fibroblast cDNA library using the Fibulin1 cDNA probe. Fibulin2 is expressed in humans and mice (Fig. 1A and B). In human physiology, Fibulin2 is highly expressed in cardiac tissue, placenta, testes and the urinary bladder, but expressed at low levels in brain tissue, splenic parenchyma and bone marrow (Fig. 1A). Fibulin2 exhibits aberrant expression across malignancies of diverse organs (9), such as colorectal cancer (4) and lung cancer (10). The protein structures of human and murine Fibulin2 exhibit remarkable homology (8,11) (Fig. 2A and B), providing a robust theoretical basis for using murine cellular systems and animal models as surrogates for human studies in oncological research. In the Na, I, II and III domains, the amino acid sequence identity is ~90%, whereas the Nb domain shows only 62% sequence identity (8,11). Human Fibulin2 contains 18 exons (11) (Fig. 3), while mouse Fibulin2 contains 17 exons (8). Surface plasmon resonance and/or solid-phase microplate analyses have shown that Fibulin2 has high binding affinity for Perlecan (12,13), laminin5 (14,15), Fibronectin (16), collagen XVIII (17), Aggrecan, Versican, Brevican lectin domains (18), the C-terminal region V of Perlecan (19), and the short arms of Laminin-5 and Laminin-1 (20), which helps maintain the ECM (21). Consequently, ECM stabilization mediated by Fibulin2 represents a pivotal mechanism underlying tumor suppression.

Nevertheless, Fibulin2 does not universally exert inhibitory effects across all malignancies; rather, it acts as a promoter in certain neoplastic contexts. For instance, increased Fibulin2 expression inhibits cell proliferation in nasopharyngeal carcinoma (22), Kaposi's sarcoma (23) and breast cancer (24). Conversely, suppression of Fibulin2 expression effectively attenuates lung adenocarcinoma progression (10). Therefore, the present review summarizes the diverse roles of Fibulin2 across various malignancies, while examining underlying mechanisms, alongside comprehensive analysis of the potential of Fibulin2 as a biomarker. Regarding the mechanistic influence of Fibulin2 on tumor development and its utility as a tumor marker, several critical issues persist. The mechanisms governing Fibulin2 function across numerous malignancies require further elucidation. The role and mechanistic basis of the effects of Fibulin2 on tumor stroma remain poorly understood. Furthermore, its clinical application as a biomarker is not yet mature. Therefore, the present overview provides guidance for the clinical translation of Fibulin2 as both a therapeutic target and predictive marker in oncological diseases.

Fibulin2 acts as an upstream regulator of the Ras-MEK-ERK1/2 pathway in hepatocellular carcinoma and the TGF-β/ Smad2/TGFB induced factor homeobox 2 (TGIF2) pathway or β-catenin in gastric cancer (25-27). Differences in Fibulin2 expression between tumor tissues and adjacent normal tissues lead to the activation of these pathways (25,26). However, to the best of our knowledge, the factors causing altered Fibulin2 expression in liver and gastric cancer, as well as its upstream regulators, remain unclear. In p53-null immortalized mouse keratinocytes and RasV12-transformed mouse keratinocytes, which are skin cancer models, Fibulin2 is regulated by the upstream molecule Integrin α3β1 (28). Changes in Fbln2 expression in lung adenocarcinoma cells alter the expression of downstream Integrin genes, including Itga1, Itga2, Itga10 and Itgb1 (10). A reciprocal regulatory relationship may exist between Fibulin2 and certain Integrin proteins, which warrants further investigation. In nasopharyngeal carcinoma, Fbln2 acts as an upstream inhibitor of VEGF-165, VEGF-189 and MMP-2. The upstream factors regulating Fbln2 expression in nasopharyngeal carcinoma remain to be elucidated (22). Given that Fibulin2 is a secreted protein, it likely exerts autocrine or paracrine effects on its producing cells or neighboring cells, potentially binding to receptors such as Integrin proteins to establish feedback loops. However, such feedback mechanisms have not received sufficient attention in current tumor-related studies, and further research is needed to demonstrate their existence across various tumor types. In the extracellular space, Fibulin2 interacts with multiple proteins: Integrin β1 in non-small cell lung cancer (29), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 and ADAMTS-12 in breast cancer (30), Nidogen1 in colorectal cancer (CRC) (31), and mucin 4 (MUC4) in pancreatic cancer (32). These are protein-protein interactions rather than upstream-downstream regulatory relationships (Fig. 4). In breast cancer, ADAMTS-5 cleaves Fibulin2, and this role can be inhibited by ADAMTS-12 (30). Differences in mechanisms of Fibulin2 regulating tumor development across tumor types are shown in Table I.

In the enteric nervous system of patients with colorectal carcinoma, Ndrg4 expression is absent or markedly reduced (33), while enteric nerve cells concurrently secrete higher levels of Fibulin2. Fibulin2 and Nidogen1 promote human colorectal carcinoma cell proliferation and migration. A study substantiated its conclusions by stimulating cells with Fibulin2 and Nidogen1 proteins, then assessing cell proliferation and migration (31). However, the study failed to demonstrate the isolated effects of Fibulin2 on CRC cells and mechanisms (31). In an experiment investigating subcutaneous tumor formation in lung adenocarcinoma, tumors were generated in mice by injecting tumor cells in PBS subcutaneously into the right flank. Tumors formed by Fbln2-knockdown cells exhibited reduced size, weight and tissue consistency compared with those formed by normal cells (10). In vitro experiments revealed that Fbln2-knockdown cells, compared with normal cells, exhibited markedly attenuated type I collagen adhesion capacity alongside markedly reduced tumor cellular pseudopodia (10). Itga1, Itga2, Itga10 and Itgb1 were notably reduced following Fbln2 depletion in lung adenocarcinoma cells (10). These genes encode Integrin proteins α1β1, α2β1, α10β1 and α11β1, respectively, which can bind to collagen (34). In summary, Fibulin2 is closely associated with other ECM proteins in lung adenocarcinoma. Aberrant Fibulin2 expression leads to abnormal changes of other ECM proteins such as integrin proteins and collagen (10). Immunohistochemistry revealed Fibulin2 upregulation in lung cancer tissues compared with adjacent normal tissues. In vitro validation relied exclusively on Fbln2 knockdown. Incorporating overexpression experiments would enhance methodological rigor (10). In p53-null immortalized mouse keratinocytes and RasV12-transformed mouse keratinocytes, knockdown of Fbln2 diminished cell invasion; however, subcutaneous injection of Fbln2-knockdown cells failed to produce a notable reduction in tumor growth volume compared with controls (28). Fibulin2 expression was regulated by Integrin α3β1 and promoted the invasion of immortalized/transformed keratinocytes (28). The research was limited by overly simplistic mechanistic exploration, necessitating further investigation of more specific mechanisms. In mouse non-small cell lung cancer tissue sections, ECM-expressed Fibulin2 interacts with cell membrane-expressed Integrin β1. The interaction between Integrin β1 and Fibulin2 mediates tumor cellular adhesion to the ECM while conferring chemotherapy drug resistance (29). The study employed immunofluorescence double-labeling experiments to demonstrate partial colocalization of Integrin β1 and Fibulin2, subsequently inferring disease mechanisms (29). Additional experiments are necessary, including experiments verifying whether Integrin β1 is a receptor of Fibulin2 and assessing its impact on tumors. In human hepatocellular carcinoma cells, Fibulin2 promotes carcinoma cell proliferation while inhibiting apoptosis through Ras-MEK-ERK1/2 signaling pathway activation (25). In in vitro experiments on liver cancer cells, Fbln2 knockdown and overexpression were performed, and the expression changes of key proteins in the Ras-MEK-ERK1/2 signaling pathway were detected by western blotting (WB) (25). Fibulin2 exerts tumor-promoting effects in both in vitro and in vivo experiments (25). However, this merely establishes an association between Fibulin2 and the Ras-MEK-ERK1/2 signaling pathway rather than a direct regulatory relationship, as indirect mechanisms may exist. Future research could use glutathione S-transferase (GST) pull-down to further verify the direct interactions between Fibulin2 and these pathway proteins. Fibulin2 expression alterations within neoplastic tissues and corresponding modifications in tumor cell proliferation, migration and invasion (demonstrating the pro-carcinogenic properties of Fibulin2) are shown in Table II.

Fibulin2 is associated with Integrin proteins in mechanistic investigations of lung adenocarcinoma (10), non-small cell lung carcinoma (29), p53-null immortalized murine keratinocytes and RasV12-transformed murine keratinocytes (28). In a hepatocellular carcinoma study, the authors did not study Integrin proteins (25), the Ras-MEK-ERK1/2 signaling pathway constitutes a crucial downstream pathway of Integrin according to previous investigations (35-37). Integrins, as principal ECM cell-surface receptors, represent essential mediators of cellular communication with the tumor microenvironment (TME) (35,38,39). Integrins comprise a family of 24 heterodimeric receptors (40). Integrins consist of two subunits: α and β subunits, which have extensive extracellular domains and short cytoplasmic domains (41,42). These interact with ECM proteins outside the cell membrane while connecting to downstream pathways, including the Ras-MEK-ERK1/2 and FAK/SFK pathways in the cytoplasm (35). When ECM protein, including Fibulin2, Nidogen, Collagen and Laminin, or Integrin expression is altered, fibrosis or remodeling can change tissue stiffness, and downstream Integrin pathways such as the Ras-MEK-ERK1/2 and FAK/SFK pathways may be activated, affecting tumor migration, invasion and proliferation (35,43,44) (Fig. 5).

Alterations in Fibulin2 expression in tumor tissues and corresponding changes in tumor cell proliferation, migration and invasion (demonstrating the anti-carcinogenic properties of Fibulin2) are summarized in Table III. In human gastric cancer, Fibulin2 expression in malignant tissues is reduced compared with that in adjacent normal tissues, and is negatively associated with β-catenin (26). β-catenin is a bifunctional protein with both cell adhesion and signal transduction activities, and was initially identified through a study of the cell adhesion molecule E-cadherin (63). β-catenin is an essential component of the Wnt/β-catenin signaling pathway, serving a pivotal role in gastric carcinoma pathogenesis and progression (64). A study has established direct or indirect regulatory relationships between the Fibulin family and β-catenin, with both substantially influencing gastric carcinoma development (65). β-catenin is typically localized in the cytoplasm, with its expression suppressed through ubiquitin/proteasome-mediated protein degradation (66). Following construction of Fbln2 overexpression plasmids and transfection of them into AGS and SGC-790 human gastric cancer cell lines, WB analysis has confirmed that Fibulin2 overexpression downregulated β-catenin and its downstream c-myc and cyclin D1, thereby inhibiting the proliferation of gastric cancer cells (26). Immunohistochemistry demonstrated downregulation of Fibulin2 in gastric cancer tissues compared with adjacent normal tissues (26). In vitro validation relied exclusively on Fbln2 overexpression, and incorporation of knockdown experiments would enhance methodological rigor (26). Another study has confirmed that reduced Fibulin2 expression in gastric carcinoma tissues promotes tumor cell proliferation and metastasis through activation of the TGF-β/Smad2/TGIF2 pathway (27). The study used normal and Fbln2-knockdown human gastric cancer cell lines for RNA sequencing, demonstrating upregulation of the TGF-β signaling pathway in Fbln2-knockdown gastric cancer cells (27). Following Fbln2 knockdown and overexpression in gastric carcinoma cells, WB analysis revealed corresponding increases and decreases in Smad2 and TGIF2 phosphorylation (27). Addition of TGF-β signaling pathway inhibitor reversed these expression changes (27). The specific mechanism by which decreased Fbln2 expression in cells alters the activity of the TGF-β pathway requires further exploration. This establishes an association rather than a direct regulatory relationship between Fibulin2 and the TGF-β/Smad2/TGIF2 pathway, as indirect mechanisms cannot be excluded. Future research could use GST pull-down to demonstrate direct interactions between Fibulin2 and pathway proteins.

Kaposi's sarcoma, which originates from human vascular endothelial cells, is characterized by vascular proliferation caused by HIV infection of endothelial cells (23). In cutaneous microvascular endothelial cells after 10 days of infection, Fibulin2 protein and mRNA expression are decreased by 50- and 26-fold, respectively. Simultaneously, mRNA levels of the ECM-binding partners fibronectin and tropoelastin are decreased 5- and 25-fold, respectively. This weakens the binding of Fibulin2 to ECM proteins, including fibronectin and tropoelastin, compromising basement membrane (BM) stability and promoting tumor progression (23). Fibronectin is crucial for numerous cell functions, including migration, proliferation and differentiation (67). Transcriptional downregulation of fibronectin has been associated with highly metastatic breast carcinoma cells in murine models (68). Tropoelastin is the soluble precursor of elastin, inducible by ultraviolet irradiation and degradable by MMP-12 (69). Loss of tropoelastin causes developmental tissue disorders, including aneurysms, atherosclerosis and reduced skin elasticity (70). Tropoelastin and fibronectin are essential ECM proteins critical for wound healing (71). The study investigating Kaposi's sarcoma failed to establish causative relationships between Fibulin2 alterations and changes in fibronectin or tropoelastin (23). Future research should include cellular Fbln2 knockdown and overexpression for validation.

In astrocytoma research, U251 cells (a human glioma cell line) exhibited reduced migration and invasion following Fibulin2 overexpression. The conclusion that Fibulin2 inhibits astrocytoma was confirmed by comparing Fbln2-overexpressing U251 cells with negative controls transfected with empty vectors (7). A previous study revealed that across different astrocytoma grades, Fibulin2 inhibits tumor development (7). The authors suggested an ECM-stabilizing function of Fibulin2; however, experimental verification is required (7). During pancreatic carcinoma development, MUC4 protein expression is markedly upregulated on pancreatic carcinoma cell surfaces and MUC4 binds to Fibulin2 (32). This binding interferes with the normal interaction between Fibulin2 and the G1 domain of Nidogen (NIDO), disrupting BM integrity (32) (Fig. 6). Tumor cells consequently breach the BM more readily, facilitating invasion and metastasis. In pancreatic cancer tissue sections, immunofluorescence double-labeling experiments have shown colocalization of MUC4 and Fibulin2 (32). However, immunofluorescence double-labeling experiments merely suggest potential connections between these proteins and do not convincingly demonstrate the roles of MUC4 and Fibulin2 in pancreatic carcinoma (32). Future studies may use conditional knockout of MUC4 and Fibulin2 in murine models for further validation. Additionally, numerous previous articles and reviews have erroneously characterized Fibulin2 as an oncogenic protein in pancreatic carcinoma when citing the conclusions of this study about pancreatic carcinoma. The original study provides no evidence to suggest that Fibulin2 promotes pancreatic carcinoma metastasis and invasion (32).

Breast carcinoma is a prevalent malignancy in women with a high incidence (72). In a breast carcinoma study, transfection of Fbln2 plasmids into human breast carcinoma cell lines reduced tumor cell migration and invasion compared with those of negative control lentiviral vector groups (24). Fibulin2 expression is reduced in breast carcinoma tissues compared with adjacent normal tissues (73). Knockdown of Fbln2 is associated with disruption of the type IV collagen sheath surrounding breast cells in vitro (74); Ibrahim et al (74) hypothesized that its downregulation may be associated with BM disruption and early invasion. However, the BM composition is complex, precluding exclusion of compensation by other Fibulin family members (such as Fibulin1 and Fibulin5) or ECM proteins, which requires demonstration (75). A study involving 272 patients with breast carcinoma from a Norwegian hospital found a positive association between elevated perivascular Fibulin2 expression in breast carcinoma and survival rates (76). Elevated Fibulin2 expression was associated with luminal breast carcinoma, pronounced elastic tissue hyperplasia in the tumor stroma and favorable prognosis, while reduced expression was associated with the basal-like phenotype, triple-negative breast carcinoma, interval breast carcinoma, vascular invasion and poor prognosis (76). The study lacked mechanistic exploration and only reported observed associations.

The TME is currently being investigated as a novel therapeutic target for cancer. ADAMTS is secreted by carcinoma and stromal cells, potentially modifying the TME through various mechanisms, thereby promoting or inhibiting tumors (77). ADAMTS-12 is a secreted metalloproteinase that has functions in tissue remodeling and cell migration or adhesion (78,79). In 293-EBNA cells, yeast two-hybrid screening and co-immunoprecipitation experiments demonstrated that the carboxyl-terminal region of Fibulin2 interacts with the spacer region of ADAMTS-12 (80). When Fibulin2 and ADAMTS-12 were concurrently overexpressed in breast cancer cells, they reduced cell invasion and migration in vitro, inhibited cell migration on relevant matrices, decreased mammosphere unit formation, and suppressed tumor growth in vivo (80). Fibulin2 and ADAMTS-12 expression in breast cancer tissue was negatively associated with histopathological tumor stage, with patients exhibiting the best prognosis when both were highly expressed (80). ADAMTS-4 and ADAMTS-5 are members of the ADAMTS secreted metalloproteinase family (81). A previous study has indicated that both ADAMTS-5 and Fibulin2 are expressed in the stromal and epithelial components of breast cancer tissue, with immunofluorescence double staining showing partial colocalization (30). ADAMTS-4 and ADAMTS-5, predominantly ADAMTS-5, can specifically cleave Fibulin2, enhancing breast carcinoma cell migration and invasion, while increasing the tumorigenic potential (30) (Fig. 7). The study compared the invasion of breast cancer cell lines with simultaneous overexpression of ADAMTS-5 and Fibulin2, breast cancer cell lines with overexpression of ADAMTS-5 or Fibulin2 alone and the control group (30). The invasion capacity of MCF-7 cells was increased by the simultaneous overexpression of Fibulin-2 and ADAMTS-5 compared with the control group and overexpression of Fibulin2 alone (30). However, the invasion capacity of MCF-7 cells was decreased by the simultaneous overexpression of Fibulin-2 and ADAMTS-5 compared with overexpression of ADAMTS-5 alone (30). ADAMTS-5-mediated Fibulin2 degradation is inhibited by ADAMTS-12 (Fig. 7) (30). However, in vivo experiments only showed partial colocalization of ADAMTS-5 or ADAMTS-12 with Fibulin2 via immunofluorescence or immunohistochemistry, which is insufficient to establish their relationship with breast cancer (30). Future research using conditional gene-knockout mice to substantiate the relationship between these proteins and breast cancer would be more compelling. When mammary fibroblasts are cultured in medium containing excess Fibulin2 and ADAMTS-5, the expression levels of α-smooth muscle actin (α-SMA) increase. Stimulation with excess Fibulin2 alone can also increase α-SMA expression levels, as observed by WB (the authors did not conduct statistical analysis), indicating that Fibulin2 promotes breast fibroblast activation (30). Breast fibroblasts are a key component of the tumor stroma (82). This indicates that changes in Fibulin2 expression in breast cancer cells can activate stromal fibroblasts and potentially drive their differentiation into CAFs, a phenomenon closely related to the secretory properties of Fibulin2 (30). Similar mechanisms may occur in other tumor types; however, they have received limited attention to date and warrant further investigation.

Due to the opposing roles of different ADAMTS subtypes in breast cancer, developing highly specific inhibitors that selectively block the tumor-promoting activities of ADAMTS subtypes without affecting their potential tumor-suppressive effects remains challenging. Small-molecule inhibitors targeting aggrecanases, including ADAMTS-4 and ADAMTS-5, have advanced to a phase III clinical trial for orthopedic applications (83). This provides valuable insights for the development of ADAMTS-targeted therapeutics for breast cancer treatment.

Cancer progression is accompanied by uncontrolled tumor growth, local invasion and metastasis; processes that depend heavily on the proteolytic activities of multiple MMPs. These enzymes affect tissue integrity by degrading ECM components such as Fibulin2 (84). In a nasopharyngeal carcinoma study involving samples from 30 patients, Fbln2 levels in carcinoma tissues substantially exceeded those in normal tissues, as detected by gene chip technology (22). In nasopharyngeal cancer cell lines, overexpression of Fbln2s, which encodes the short isoform of Fibulin2, downregulated the expression levels of VEGF-165, VEGF-189 and MMP-2 compared with those in the control group (22). Sustained angiogenesis is indispensable for both tumor growth and metastasis (85). MMP-2 is an effective gelatinase capable of cleaving protein components of the ECM and is involved in the invasion and metastasis process of tumor cells (86). Evidence has indicated that Fibulin2 is cleaved by MMP-2 (87), and inhibits nasopharyngeal carcinoma cell migration and invasion by strictly regulating MMP-2 expression (22). However, the study could not establish a direct regulatory relationship between Fbln2 and VEGF-165, VEGF-189 or MMP-2, because there may be unknown molecules or signaling pathways mediating the interaction between Fbln2 and VEGF-165, VEGF-189 or MMP-2. WB analysis of osteosarcoma cell line lysates has indicated multiple Fibulin2 fragments (87). Gelatinase MMP-2 facilitates Fibulin2 degradation through MMP-dependent mechanisms in osteosarcoma cell lines (87). Following addition of an MMP-2 inhibitor to the cell culture medium, WB indicated no change in the cleaved Fibulin2 fragments, indicating that unidentified mechanisms require exploration (87). This study using an osteosarcoma cell line relied entirely on in vitro experiments, and in vivo experiments are required for verification, as the in vivo environment is more complex than the in vitro cell culture environment (87).

Most documented MMP inhibitors exhibit non-specific binding and have diminished efficacy, attributable to their pronounced sequence homology with other MMPs (88). To date, no effective MMP inhibitor has successfully completed clinical trials and secured regulatory approval from the Food and Drug Administration for tumor treatment (84,88).

Studies on Fibulin2 as a biomarker are summarized in Table IV. For case-control studies involving human populations in Table IV, according to the 2011 Evidence Hierarchy of the Oxford Centre for Evidence-based Medicine, the evidence level of most studies was 4 (102).

Although Fibulin2 shows considerable potential as a therapeutic target and biomarker, several challenges and limitations remain. Addressing these impediments is crucial for successful clinical translation.

The oncogenic or suppressive effects of altered Fibulin2 expression in malignant cells vary markedly across diverse tumors. The contributing factors include distinct mechanisms, including the activation of different signaling pathways and the complex relationships with BM or other ECM proteins, tumor development stage, species and tumor origin. Although current mechanistic studies help clarify aspects of pathogenic processes, several outstanding issues remain to be resolved. These include validating the upstream and downstream molecules of Fibulin2, confirming the existence of feedback loops, exploring alternative pathways, and clarifying the relationship between Fibulin2 and stromal fibroblasts. These considerations are crucial to improve the safety and effectiveness of future relevant drug research. The use of Fibulin2 alone as a marker for tumor diagnosis or staging remains not well-established. Future research should further explore methods to enhance specificity, such as identifying novel and specific markers to be used in combination with Fibulin2 as a dual-marker system.