Tight junction proteins: Gatekeepers turned facilitators in the pathogenesis of gastric adenocarcinoma

Abstract

Gastric cancer (GC) is the fifth most prevalent malignancy worldwide and remains a leading cause of cancer-related mortality. Major risk factors for GC include Helicobacter pylori infection, increasing age, high dietary salt intake, and diets deficient in vegetables and fruits. Due to the often subtle and nonspecific early symptoms, coupled with the lack of routine screening programs, a significant proportion of GC cases are diagnosed at advanced stages. The etiology of GC is multifactorial, and diagnosis is confirmed histologically through endoscopic biopsy, followed by staging via computed tomography, positron emission tomography, staging laparoscopy, and endoscopic ultrasound. Treatment strategies typically involve a multidisciplinary approach including chemotherapy, surgical resection, radiotherapy, and emerging immunotherapeutic options. Despite advances in diagnostic and therapeutic modalities, the prognosis of advanced GC remains poor, with high rates of recurrence and metastasis. In recent years, increasing attention has been given to the role of tight junction (TJ) proteins in the pathogenesis and progression of GC. TJ proteins, critical components of epithelial barrier function, have been implicated in various stages of gastric carcinogenesis, from intestinal metaplasia to invasion and metastasis. Infection and inflammation, particularly due to Helicobacter pylori, disrupt TJ integrity, compromising the gastric mucosal barrier and facilitating neoplastic transformation. This review synthesizes current evidence from PubMed, EMBASE, Google Scholar, ScienceDirect, SpringerLink, and other reputable databases to provide a comprehensive overview of the involvement of TJ proteins in GC. By elucidating the molecular interplay between TJ dysregulation and gastric tumorigenesis, this work aims to highlight the potential of TJ proteins as novel diagnostic biomarkers and therapeutic targets in GC management.

Key Words: Claudins; Gastric cancer; Infection and inflammation; Occludin and zonula occludens; Tight junction

Core Tip: Tight junction (TJ) proteins play a pivotal role in preserving the integrity of the gastric epithelial barrier. Their dysregulation represents a critical early event linking chronic inflammation to gastric carcinogenesis. Inflammatory mediators induce TJ remodeling, thereby increasing mucosal permeability and promoting tumor initiation and progression. Emerging therapeutic strategies targeting TJ proteins either by pharmacologically restoring barrier integrity or modulating aberrant TJ signaling offer promising avenues to suppress inflammation, inhibit tumor growth and metastasis, and ultimately improve clinical outcomes in gastric cancer patients.

INTRODUCTION

Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related mortality worldwide, with a particularly high incidence in East Asia, where it accounts for approximately one in every twelve cancer-related deaths[1]. It is a highly heterogeneous disease, with over one million new cases estimated annually[2,3]. Globally, the lifetime risk of developing GC is approximately 1 in 54 for men and 1 in 126 for women, with men being twice as likely to be affected[2-4]. In the United States, the lifetime risk is about 1 in 95 for men and 1 in 154 for women. India falls into the low-incidence category but shows significant regional variation in disease occurrence[5]. According to GLOBOCAN 2018 data, East Asia, South America and Eastern Europe are the major hotspots for both the incidence and mortality of GC[6]. The disease often begins as precancerous changes in the gastric mucosa and may progress silently to advanced stages. GC presents with a wide range of symptoms, making early diagnosis difficult, and is often resistant to chemotherapy[2,3].

RISK FACTORS

Helicobacter pylori (H. pylori) infection is the most powerful risk factor for GC, promoting carcinogenesis through both direct and indirect mechanisms[2,3]. The cytotoxin-associated gene A protein, a virulence factor of H. pylori, interacts with host proteins that regulate cell growth, movement, and polarity. This interaction leads to morphological changes and increases the likelihood of epigenetic alterations that drive GC development[7]. Smoking is the second most significant risk factor for the onset of GC[2]. The toxins and carcinogens in cigarette fumes cause direct DNA damage, disrupt normal cell growth, and promote malignant transformation of gastric epithelial cells. Both former and current smoking contribute to carcinogenesis[8]. Dietary factors such as smoked and salted foods (pickled vegetables) also play a crucial role being the sources of nitrosamine (dietary carcinogen) that elevate GC risk[2]. Additionally, reactive oxygen species (ROS) generated by inflammation or poor diet cause DNA damage, leading to genetic mutations and cancer development[8]. Alcohol consumption is another contributing factor, its metabolite acetaldehyde, is a recognized human carcinogen that causes DNA damage by inhibiting DNA methylation and by interacting with retinoid metabolism[9]. It acts as a mucosal irritant, further increasing cancer risk[10]. A family history of GC, particularly in first-degree relatives and siblings, is associated with a higher risk, especially in individuals diagnosed before the age of 50[11]. Gastroesophageal reflux disease and obesity are specifically linked to cardia-type GC[12]. Radiation exposure has been associated with an increased risk of gastric lymphomas. Several other conditions are known to elevate the risk of GC, including blood group O, migrants from high- to low-incidence nations, chronic gastric ulcers, adenomatous gastric polyps, and prolonged exposure to H₂-receptor blockers[3].

Gastric adenocarcinoma is the most common type of stomach cancer, originating from the glandular epithelium of the stomach lining. It typically arises in the mucosal layer and often begins as an ulcer or abnormal growth[13,14]. Gastric adenocarcinoma is histologically classified into two main subtypes: Intestinal type and diffuse type[15]. The diffuse type is characterized by a lack of cell cohesion, allowing individual cancer cells to infiltrate and thicken the stomach wall without forming a well-defined mass. This type tends to occur in younger patients, can involve any part of the stomach, and is associated with a poorer prognosis[13]. In contrast, the intestinal type features cohesive neoplastic cells that form gland-like tubular structures. These tumors are often ulcerative and typically develop in the antrum and along the lesser curvature of the stomach. Intestinal-type GC is frequently preceded by a well-defined precancerous sequence, including chronic gastritis, atrophy, intestinal metaplasia, and dysplasia[13].

DIAGNOSIS AND TREATMENT

The diagnosis of GC involves a comprehensive approach, beginning with a detailed clinical history and physical examination, followed by blood investigations and imaging techniques such as ultrasound, barium swallow, computed tomography scan and magnetic resonance imaging to invasive endoscopic biopsy. For accurate staging, advanced imaging modalities like positron emission tomography scans are utilized, followed by staging laparoscopy to assess the extent of disease spread. Early detection remains crucial for improving prognosis, therefore, individuals with risk factors or persistent symptoms should seek prompt evaluation by a gastroenterologist. An endoscopic gastric biopsy is definitive for diagnosing GC.

Although there are various treatment modalities like surgical (partial or total gastrectomy with or without lymph node dissection), chemotherapy, radiation therapy, and immunotherapy, the incidence of metastasis and resistance to therapy is still high. Despite the availability of these treatment modalities, metastatic disease and resistance to therapy remain significant challenges. As such, newer strategies like targeted drug therapy and monoclonal antibody-based treatments are being developed to improve therapeutic outcomes and reduce recurrence. In terms of prevention, dietary modifications play an important role. Increased consumption of fruits and vegetables has been consistently associated with a reduced risk of GC. Their anti-carcinogenic effects are largely attributed to the antioxidant properties of nutrients such as vitamin C and beta-carotene, which help neutralize free radicals and prevent DNA damage.

TIGHT JUNCTION PROTEINS

Tight junctions (TJs) are specialized protein complexes that seal the intercellular space between adjacent epithelial to endothelial cells, playing a critical role in maintaining selective permeability and cellular polarity[16,17]. They are primarily formed by interactions among proteins from three major families: Claudins, zonula occludens (ZO) proteins, and TJ-associated MARVEL proteins, including occludin[16-19]. The gastrointestinal (GI) epithelium provides a clear example of TJ structure and function. It is composed of a single layer of columnar epithelial cells, predominantly absorptive in nature, specialized for nutrient uptake from the intestinal lumen[17,19]. The TJ between these cells not only prevents the unregulated passage of ions, nutrients, and solutes through the paracellular space, but also act as molecular “fences” that maintain the distinction between the apical and basolateral domains of the plasma membrane, ensuring proper cell polarity and function[20,21]. Among TJ proteins, claudins serve as the structural backbone of TJ strands and are integral membrane proteins responsible for regulating paracellular permeability[22]. Under freeze-fracture electron microscopy, claudins appear as continuous, strand-like structures. In contrast, occludin and ZO-1 (a scaffolding protein) are more involved in the regulation, assembly, and signaling associated with TJ integrity. ZO-1 links transmembrane proteins like claudins and occludin to the actin cytoskeleton, facilitating TJ stability and signal transduction[23].

In the gastric epithelium, TJs are vital for preserving mucosal barrier integrity, limiting the translocation of digestive enzymes, microbial toxins, and other harmful substances into the systemic circulation[24]. The coordinated interaction among TJ components (claudins, occludin, ZO proteins, and TJ-associated MARVEL proteins) is essential not only for maintaining epithelial and endothelial barrier integrity but also for modulating intracellular signaling pathways that influence cell proliferation, differentiation, and immune responses[25,26]. Various risk factors contributing to the initiation of GC and progression to advanced metastatic cancer stage are shown in Figure 1.

Figure 1 Factors contributing to the initiation and progression of gastric adenocarcinoma and metastasis. Oxidative stress, free radical induced injury, and Helicobacter pylori infection can trigger chronic inflammation, which in turn causes persistent DNA damage and activates multiple oncogenic pathways that drive carcinogenesis. Moreover, chronic inflammation and ulcer formation can disrupt the balance of gut microbiota, leading to dysbiosis and increased intestinal permeability (‘leaky gut’). This dysregulated microenvironment further promotes epithelial mesenchymal transition, thereby facilitating gastric cancer progression and metastasis. H. pylori: Helicobacter pylori.

ZO

ZO-1 was the first TJ protein identified. It is a cytosolic peripheral membrane protein belonging to the membrane-associated guanylate kinase-like family, functioning as a PDZ domain-containing scaffold[18,27]. Along with ZO-2 and ZO-3, ZO-1 anchors TJs to the actin cytoskeleton and links transmembrane proteins such as claudins, occludin, and junctional adhesion molecule-A (JAM-A) to cytoskeletal and signaling networks[28]. ZO-1 contains three PDZ domains in its N-terminal region: PDZ1 binds claudins (with affinity modulated by claudin phosphorylation), PDZ2 facilitates dimerization, and PDZ3 interacts with JAM-A[18,27,28]. Additional interaction domains SH3, U5, GUK, and an actin-binding region enable binding to other scaffold proteins like afadin and α-catenin. The PDZ3-SH3-GUK-U6 module also drives phase separation and polymerization of ZO proteins during TJ assembly. Functionally, ZO-1 is essential for epithelial barrier integrity, coordinating the structural assembly of TJs and participating in signaling pathways that influence gene expression and epithelial permeability[18,27,28].

The simultaneous deletion of ZO-1 and ZO-2 leads to highly permeable cell monolayers and a complete loss of TJ strands, indicating their redundant roles in TJ stabilization. ZO-1 also mediates communication between TJ components and the actin cytoskeleton[19,29]. Its actin-binding region interacts weakly and transiently with F-actin; both excessively strong or absent interactions can disrupt barrier integrity and increase TJ permeability[30]. Afadin interacts with ZO-1, JAM-A, nectin, and actin, playing a crucial role in initiating TJ formation and cell polarization by linking ZO-1 and JAM-A at adherens junctions. Cingulin and paracingulin form coiled-coil homodimers with a globular head and share an N-terminal ZO-1 interaction motif[19]. Cingulin binds multiple TJ proteins, including JAM-A, ZO-1, and ZO-2, and interacts strongly with both actin and microtubules an interaction regulated by AMP-activated protein kinase phosphorylation[25]. Both cingulin and paracingulin contribute to TJ regulation by suppressing Rho family GTPase activity, thereby downregulating claudin 2 expression and inhibiting cell proliferation[19,28].

TJs are essential for maintaining barrier function, cell polarity, and intracellular signaling pathways[18,27]. Disruption of TJs can result in the loss of cell polarity and an abnormal influx of growth factors, which may stimulate tumorigenic epithelial cells through autocrine and paracrine mechanisms[27-31]. ZO-1 is typically highly expressed in normal mucosa, as well as in carcinoma and metaplastic epithelium. However, its expression is significantly reduced in undifferentiated-type gastric adenocarcinomas, suggesting a correlation between ZO-1 downregulation and tumor progression[27,31].

OCCLUDIN

Occludin is an integral membrane protein of approximately 60 kDa, located in the TJs of epithelial and endothelial cells. It contains four transmembrane domains and, unlike many other TJ proteins, does not exhibit multiple isotypes across species[32]. Occludin plays a key role in maintaining TJ barrier integrity and defense functions. Interestingly, epithelial cells lacking occludin still form well-organized TJ strands, indicating that occludin may play a supportive rather than primary structural role in TJ formation[33,34]. It contributes to maintaining cell polarity and preventing paracellular leakage. Beyond its structural functions, occludin is involved in signaling pathways that regulate TJ assembly and function, as well as in cellular processes such as proliferation, differentiation, and migration[33,34]. Alterations in occludin expression or localization have been associated with disorders involving barrier dysfunction, including inflammatory bowel disease and cancer metastasis. Additionally, occludin enhances intracellular adhesion; its homotypic interactions help organize atypical protein kinase C-PAR3 and PALS1-associated TJ complexes at the leading edge during epithelial migration[35].

CLAUDINS

Claudins are essential transmembrane proteins of TJs, each with four transmembrane domains. The claudin gene family is expressed in a tissue-specific manner, with 24 members identified to date. In normal gastric mucosa, claudin 18 is typically expressed, whereas claudin 4 is not. However, oligonucleotide microarray studies have shown that claudin 4 is upregulated in GC, particularly in the intestinal-type subtype[26]. Elevated claudin 4 expression has been consistently observed in gastric intestinal-type adenocarcinoma (as summarized in Table 1). H. pylori infection has been shown to increase paracellular permeability by disrupting occludin, claudin-4, and claudin-5. Additionally, the transcription factor caudal-related homeobox transcription factor 2 plays a key role in regulating intestinal claudin expression. Notably, reduced claudin 4 expression correlates with higher recurrence rates and poorer survival outcomes in GC patients. Functionally, claudins form the backbone of TJ strands and contribute to paracellular ion selectivity. Certain claudins, such as those in kidney epithelial cells, form channels that selectively permit the passage of ions such as Mg²⁺. Mutations in these claudins can impair ion reabsorption, resulting in excessive urinary Mg²⁺ loss.

Table 1 Dysregulated tight junction proteins and their mechanistic associations with gastric adenocarcinoma.

No.	TJ proteins	Expression level in gastric carcinoma	Functional effect	Ref.
1	ZO	↓ ZO-1 and ZO-2, upregulated or downregulated	Loss of epithelial features. Induce EMT. Promote tumor invasion, metastasis and resistance to apoptosis	[31]
2	Occludins	Decreased expression	Wnt/β-catenin pathway activation. β-catenin pathway accumulation in cytoplasm, translocation to nucleus. Uncontrolled cell proliferation	[31]
3	Claudin 1	Increased	Increased claudin-1 → TJ hyperfunction → alters selective permeability of epithelium. Wnt/β-catenin pathway activation → uncontrolled cell proliferation, promotes EMT	[37]
4	Claudin 2	Increased	Increased intestinal permeability, activation of oncogenic signaling pathway, disruption of normal epithelial architecture, weakened cell-cell adhesion → metastasis	[37]
5	Claudin 3	Increased	Activation of inflammatory pathways (JAK/STAT, NF-κB) → immune invasion, promotes angiogenesis	[19]
6	Claudin 4	Decreased	Invasiveness, metastasis and tumor aggressiveness, poor survival	[31]
		Increased	Wnt/β-catenin pathway activation, PI3K/Akt signaling → enhances cell survival by promoting apoptosis and promotes EMT through TGF-β signaling. Promotes tumor-supportive microenvironment	[37]
7	Claudin 5	Increased	Suppresses apoptosis of gastric cells → uncontrolled proliferation of tumor cells	[38]
8	Claudin 6	Increased	Induces MMP-2 activation through claudin-1 membrane expression → promotes cell migration and invasiveness	[39]
9	Claudin 7	Increased	EMT → tumor invasion, aggression and metastasis	[19,40]
10	Claudin 12	Increased	Through the EMT, regulates apoptosis	[41]
11	Claudin 18	Decreased	Signaling cascade related to chemokines and Wnt signaling pathway	[42,43]
12	Claudin 23	Decreased	Recruits claudin 3, 4 and mediates through it	[44,45]

TJ: Tight junction; ZO: Zonula occludens; EMT: Epithelial mesenchymal transition; Wnt: Wingless related integration site; JAK: Janus kinase; STAT: Signal transducer and activator of transcription; NF-κB: Nuclear factor kappa B; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B; TGF: Tissue growth factor; MMP-2: Matrix metalloproteinase 2.

ROLE OF CLAUDIN IN GC

Dysregulation of claudin expression is frequently observed in tumorigenesis and is closely linked to cancer progression[36]. Claudin 1 expression has been linked to increased invasiveness and metastasis in various cancers, including gastric carcinoma. In hepatocellular carcinoma, claudin 1 promotes epithelial mesenchymal transition (EMT) by upregulating the transcription factors Slug and Zeb[37]. Claudin 4 overexpression has also been found to reduce apoptosis, supporting its role in tumor survival. It is highly expressed in metaplastic epithelium and gastric intestinal-type adenocarcinoma, while showing minimal expression in normal gastric epithelium[37]. Claudin 5 is overexpressed in GC due to its ability to suppress the gastric cells leading to uncontrolled proliferation of cancer cells[38]. Additionally, claudins facilitate tumor cell invasion through the activation of matrix metalloproteinases (MMPs). Specifically, claudin 1 activates MMP-2 via the protein kinase C signaling pathway, and claudin 6 has been shown to trigger MMP-2 activation through its interaction with claudin 1. MMPs not only degrade the extracellular matrix but also modify cell-cell and cell-matrix interactions, thereby promoting EMT and tumor progression. In gastric adenocarcinoma, claudin 1 interacts with extracellular pro-MMP-2 through its extracellular loops, leading to MMP-2 activation via MMP-14. Furthermore, claudin 4 is upregulated during early stages of gastric tumorigenesis, a change strongly associated with DNA hypomethylation[39]. Claudins 3, 4, 5, 12, 18, and 23 are widely expressed throughout the stomach. In particular, claudin 3, claudin 4, and claudin 7 are frequently upregulated in GC, whereas other claudins may be downregulated[40,41]. Unlike esophageal epithelium, the gastric epithelium is adapted to withstand acidic conditions due to its tight TJ network. However, H. pylori infection disrupts this barrier by raising the pH through urease activity, which leads to decreased transepithelial resistance, internalization of occludin, and phosphorylation of the myosin light chain. H. pylori significantly alters both the pore and leak pathways of TJs, contributing to changes in claudin expression that are commonly seen in GC.

The gastric mucosa has more extensive and deeper TJ strands compared to the esophagus, conferring lower water and solute permeability and resistance to gastric acid. Disruptions in claudin expression compromise this defense and contribute to GC development. The stomach-specific claudin 18 is notably downregulated in atrophic gastritis and gastric carcinoma, and its loss is associated with poor prognosis. Claudin 18 plays a crucial role in maintaining cell lineage differentiation and the structural integrity of the gastric mucosa[42,43]. Claudin 23 is decreased in GC, it mediates its action by recruiting claudin 3 and 4[44,45].

INFLAMMATION AND GC

Inflammation represents the body’s natural defense response to injury, infection, or harmful stimuli, involving immune cells, blood vessels, and molecular mediators[46]. Acute inflammation protects the body by eliminating harmful agents and promoting tissue repair[46,47]. However, when inflammation becomes persistent or chronic, it contributes to various pathologies, including cancer. Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukins (ILs) can alter the expression and localization of TJ proteins, compromising the gastric epithelial barrier and increasing susceptibility to malignant transformation[47,48].

H. pylori infection is a well-known risk factor for GC[49]. The bacterium induces chronic gastric inflammation, leading to gastritis and progressive mucosal damage[50]. The persistent inflammation owing to H. pylori infection can disrupt the function and structure of TJs, leading to increased gastric permeability[51]. This allows harmful substances to penetrate the gastric mucosa, contributing to tissue damage and promoting carcinogenesis.

IL-1β

IL-1β is a potent pro-inflammatory cytokine markedly upregulated during H. pylori infection. It plays a central role in sustaining gastric mucosal inflammation and has been strongly associated with an increased risk of GC. IL-1β suppresses gastric acid secretion, creating a favorable environment for H. pylori persistence and chronic inflammation[52]. Beyond immune activation, IL-1β drives gastric carcinogenesis by promoting epithelial proliferation, angiogenesis, and tumor progression[53]. Mechanistically, IL-1β disrupts epithelial barrier integrity by activating the mitogen-activated protein kinase kinase-1-nuclear factor kappa B (NF-κB) signaling cascade[54]. NF-κB activation induces myosin light chain kinase expression, which alters cytoskeletal dynamics and TJ protein organization, ultimately leading to TJ disassembly and loss of barrier function[55]. Genetic polymorphisms in the IL-1β gene, including 511T and 31C variants, are linked to elevated IL-1β production and increased susceptibility to gastric adenocarcinoma, particularly in H. pylori-infected individuals[56]. Moreover, IL-1β promotes carcinogenesis through epigenetic mechanisms, notably aberrant DNA methylation, further underscoring its pivotal role in gastric tumorigenesis[57].

TNF-α

TNF-α is a central mediator of chronic inflammation and a key contributor to gastric carcinogenesis[58]. Secreted mainly by activated macrophages and immune cells, TNF-α fosters a tumor-promoting microenvironment[59]. It activates multiple signaling pathways, including NF-κB and mitogen-activated protein kinases, which regulate genes involved in cell proliferation, angiogenesis, and inhibition of apoptosis[60]. These processes enable gastric epithelial cells to evade growth control, resist cell death, and acquire malignant properties.

During H. pylori infection, TNF-α expression is upregulated as part of the host immune response, perpetuating chronic mucosal inflammation. Persistent TNF-α exposure damages the gastric epithelium, increases ROS generation, and promotes EMT, a key step in invasion and metastasis[61]. Elevated TNF-α levels correlate with aggressive histological subtypes, advanced tumor stages, and poor prognosis[61]. Genetic polymorphisms in TNF-α have also been linked to heightened GC risk in specific populations[61]. Mechanistically, TNF-α enhances GC aggressiveness by increasing cytoplasmic claudin 1 expression, which promotes invasion and cell survival through NF-κB and MMP-7-dependent pathways. In contrast, other TJ proteins such as claudin 4, occludin, and ZO-1 are downregulated in advanced GC, reflecting TJ disruption as a hallmark of tumor progression, although direct regulation of these proteins by TNF-α remains unclear[61,62].

IL-6

IL-6 is a multifunctional cytokine that plays a crucial role in GC progression[62]. Elevated IL-6 levels are consistently detected in the serum and tumor microenvironment of GC patients and are associated with poor prognosis[63]. IL-6 promotes tumor progression primarily through activation of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, which enhances cancer cell proliferation, survival, invasion, and EMT[63]. IL-6 also contributes to epithelial barrier dysfunction by modulating TJ protein expression. Studies in GI epithelial models demonstrate that IL-6 upregulates claudin 2 via the c-Jun N-terminal kinase and phosphatidylinositol 3-kinase/extracellular signal-regulated kinase pathways, increasing paracellular permeability[64]. Although this mechanism is best characterized in the intestinal epithelium, it is likely relevant to the gastric mucosa, particularly under chronic inflammatory conditions such as H. pylori infection. TJ disruption compromises mucosal integrity, facilitates microbial translocation, and perpetuates inflammation, creating a self-sustaining cycle that promotes carcinogenesis. Clinically, IL-6 expression is significantly higher in gastric tumor tissues than in adjacent non-tumor or normal gastric samples. Elevated intra-tumoral and circulating IL-6 levels correlate with advanced disease stage, reduced patient survival, and decline following effective therapy[64,65]. Within the tumor microenvironment, cancer-associated fibroblasts (CAFs) secrete IL-6, which further enhances GC cell growth and invasion through the JAK2/STAT3 pathway[66]. Experimental IL-6 knockout in murine models markedly suppresses gastric tumor formation, underscoring its pivotal role in inflammation-driven gastric carcinogenesis[67].

IL-8

IL-8, also known as C-X-C motif chemokine ligand 8, is a pro-inflammatory chemokine that plays a crucial role in the recruitment of immune cells particularly neutrophils to sites of inflammation[68]. It is frequently upregulated in response to H. pylori infection and is significantly implicated in the progression of GC[69]. IL-8 promotes tumor growth by stimulating angiogenesis, enhancing the supply of oxygen and nutrients to cancerous tissues[69,70]. Additionally, IL-8 recruits neutrophils which release ROS and proteolytic enzymes that can cause DNA damage, facilitating tumor initiation and progression. Beyond its pro-inflammatory role, IL-8 enhances GC cell migration and invasion, contributing directly to metastatic potential[70]. A key pathway for metastasis in GC is via tumor-draining lymph nodes, with lymph node metastasis being a well-established predictor of poor prognosis. The tumor microenvironment comprising CAFs, immune and inflammatory cells, and the extracellular matrix plays a pivotal role in shaping metastatic behavior[71]. CAFs in particular have been identified as a major source of soluble IL-8, contributing to a pro-tumorigenic microenvironment[71].

Recent findings have shown that elevated IL-8 levels can induce programmed death-1 (PD-1) expression on CD8+ T cells, leading to local immunosuppression within both the primary tumor and tumor-draining lymph nodes[72]. Mechanistically, IL-8 activates the JAK2/STAT3 signaling pathway to upregulate PD-1 and simultaneously inhibits PD-1 degradation by downregulating Fbxo38, an E3 ubiquitin ligase involved in PD-1 ubiquitination[71,72]. This dual action fosters an immunosuppressive niche that facilitates immune evasion and promotes lymph node metastasis. Clinically, high levels of soluble IL-8 are closely associated with increased lymph node metastasis and are indicative of a poorer overall prognosis in GC patients. In endothelial cells, IL-8 was shown to enhance permeability by reducing both mRNA and protein levels of occludin, claudin 5, and ZO-1 in a manner dependent on concentration and exposure time. Additionally, it caused redistribution of TJ proteins where occludin shifted from the membrane into the cytoplasm, while membrane-associated ZO-1 was diminished[73]. IL-8 exhibits a complex, context-dependent role in GC. While patient tumor tissues often show reduced claudin 4 expression, elevated IL-8 levels in advanced disease paradoxically upregulate claudin 4. This alteration is closely linked to the induction of EMT, mediated by increased Snail and Twist through extracellular signal-regulated kinase/mitogen-activated protein kinases pathway activation, ultimately emphasizing the significant contribution of IL-8 to GC progression[73].

Oxidative stress

Oxidative stress plays a pivotal role in the initiation and progression of GC by inducing DNA damage, sustaining chronic inflammation, activating oncogenic signaling pathways, and enhancing tumor cell survival[74]. A major source of oxidative stress in the gastric environment is H. pylori infection, a well-established risk factor for GC[75]. H. pylori stimulates the host immune response through neutrophil and macrophage activation, leading to excessive production of reactive oxygen and nitrogen species[75,76]. Persistent generation of these reactive species causes oxidative damage to lipids, proteins, and nucleic acids, contributing to chronic gastritis, intestinal metaplasia, and malignant transformation of gastric epithelial cells. Additionally, ROS activate redox-sensitive transcription factors such as NF-κB and activating protein-1, amplifying pro-inflammatory cytokine expression and perpetuating a cycle of inflammation and oxidative injury[77]. Oxidative stress also disrupts the gastric epithelial barrier by impairing TJ proteins. In gastric epithelial cells, hydrogen peroxide decreases claudin 3 expression and transepithelial electrical resistance, indicating increased permeability. These effects are reversed by the antioxidant rebamipide, demonstrating a direct oxidative stress mediated mechanism of TJ disruption and barrier dysfunction[77].

ROLE OF GASTRIC MICROBIOTA AND DYSREGULATED TJ PROTEINS IN THE DEVELOPMENT OF GC

Under normal conditions, TJs prevent the unregulated entry of gastric and intestinal microbes, their toxins, and antigens into the underlying mucosa. By preserving this selective barrier, TJ proteins maintain immune balance permitting only limited and controlled exposure of microbial components to immune cells (via dendritic cells and specialized epithelial mechanisms) without provoking persistent inflammation. Occludin and claudins determine the sealing and pore-forming properties of the junctions, thereby controlling the size and type of molecules that pass between epithelial cells. ZO-1 connects these proteins to the actin cytoskeleton, providing structural stability and integrating barrier regulation with innate immune signaling. Thus, intact TJ proteins compartmentalize gastric microbiota at the luminal surface, fostering beneficial host-microbe interactions while preventing overgrowth, dysbiosis, or aberrant immune activation. Conversely, disruption of TJ proteins leads to barrier leakage, enabling microbial products such as lipopolysaccharide (LPS) and peptidoglycan to reach deeper tissue layers, where they activate Toll-like receptors, inflammasomes, and cytokine pathways. This cascade drives chronic inflammation and contributes to the progression toward gastric carcinogenesis[78,79].

The gut microbiota plays a fundamental role in maintaining GI health by modulating immune responses, regulating intestinal permeability, and preserving epithelial barrier integrity[80]. It consists of trillions of microorganisms including bacteria, fungi, viruses, and archaea that contribute significantly to the maintenance of TJs[81]. Certain beneficial bacteria, particularly those that produce short-chain fatty acids like butyrate, enhance TJ function[82]. Butyrate specifically strengthens the epithelial barrier by upregulating TJ protein expression and increasing mucus production, which further protects the gut lining[82]. A healthy microbiota also helps modulate local immune responses, thereby preventing chronic inflammation. In contrast, dysbiosis an imbalance in the microbial community can disrupt TJs, increase intestinal permeability, and promote inflammation. Pathogenic or opportunistic bacteria may produce substances that degrade TJ proteins or activate inflammatory signaling pathways, compromising the epithelial barrier[83].

LPS, produced by Gram-negative bacteria, is a key contributor to TJ disruption and inflammation. LPS activates immune cells and induces inflammatory cytokine production, further impairing barrier function[84]. When TJs are compromised, larger molecules such as bacterial endotoxins and undigested food particles can translocate across the gut lining into the systemic circulation a phenomenon often referred to as “leaky gut syndrome” which may trigger systemic inflammation and immune activation[84].

In the gastric environment, reduced acid secretion due to H. pylori infection facilitates the colonization of additional bacterial species. For instance, the genus Lactobacillus is found in higher abundance in GC patients[85]. Microbes from the Lactobacillus and Lactococcus genera produce lactic acid, which may fuel tumor progression by serving as an energy source for cancer cell proliferation and promoting angiogenesis. While these microbes may also play a regulatory role in inflammation, their abundance often decreases during inflammatory conditions[86].

Interestingly, the phylum Nitrospirae has been detected in all GC patients but is absent in individuals with chronic gastritis. Members of this phylum are involved in nitrate and nitrite metabolism[87]. As nitrate consumption is a known risk factor for GC, these bacteria may contribute to the generation of carcinogenic N-nitroso compounds. Several bacterial genera typically found in the oral cavity such as Fusobacterium, Veillonella, Leptotrichia, Haemophilus, and Campylobacter have been detected in higher relative abundance in GC patients. Additionally, Propionibacterium acnes, commonly found on the skin, is often overrepresented in gastric tumor tissues. It is hypothesized that short-chain fatty acids produced by this bacterium may contribute to lymphocytic gastritis[88-90]. The role of inflammation and altered gut microbiota in causing GC is schematically shown in Figure 2.

Figure 2 Schematic illustration of the interplay among tight junctions, inflammation, gut microbiota, and gastric cancer progression. Helicobacter pylori infection triggers gastric inflammation by activating cytokine, nuclear factor kappa B, and signal transducer and activator of transcription 3 mediated inflammatory cascades, leading to tight junction disruption and increased intestinal permeability. The resulting leaky gut permits translocation of microbial toxins and inflammatory mediators, which promote epithelial mesenchymal transition and drive gastric carcinogenesis. IL: Interleukin; TNF: Tumor necrosis factor; NF-κB: Nuclear factor kappa B; STAT3: Signal transducer and activator of transcription 3; H. pylori: Helicobacter pylori; TJ: Tight junction; EMT: Epithelial mesenchymal transition.

TREATMENT APPROACHES IN GC PATIENTS TARGETING TJ PROTEINS

Although several stage-specific treatment modalities are well established such as the FLOT regimen based chemotherapy and gastrectomy with or without lymphadenectomy for locally advanced resectable tumors it is increasingly important to explore novel molecular targets to overcome chemotherapy resistance[91]. TJ proteins have emerged as promising therapeutic targets in this context. Ongoing clinical trials are currently investigating agents directed against TJ proteins, including claudin 4, claudin 8, and claudin 18.2, to evaluate their efficacy and safety in the treatment of GC. Targeting these proteins may therefore represent a novel strategy to overcome treatment resistance and improve patient outcomes. Several ongoing clinical trials are exploring monoclonal antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor (CAR) T-cell therapies directed against TJ proteins such as claudin 4, claudin 8, and claudin 18.2. Among these, claudin 18.2 has emerged as the most promising target, with multiple agents such as zolbetuximab, CMG901, and CAR T-cell therapy CT041[92] showing encouraging results in early- and late-phase trials (as listed in Table 2). These therapies have demonstrated manageable safety profiles and promising antitumor activity, highlighting the therapeutic potential of TJ protein-directed approaches. These TJ protein-targeted therapies represent a rapidly evolving field that may complement or even redefine the current treatment paradigm for GC, particularly in patients who fail to respond adequately to standard chemotherapy-based regimens.

Table 2 Claudin based immunotherapy for gastric cancer patients.

No.	Targeted TJ proteins	Therapeutic agent(s)	Development stages in GC	Effect in GC patients	Ref.
1	Claudin 18.2 mAb	CMG901	Phase I	Demonstrated a manageable safety profile and promising antitumor activity	[93]
2	Claudin 18.2 mAb	IBI343	Phase I	Demonstrated a manageable safety profile with low gastrointestinal adverse events and showed promising efficacy	[94]
3	Claudin 18.2 mAb	CAR T-cell therapy, CT041	Phase II	Demonstrated significant improvement in progression-free survival, with a manageable safety profile	[95]
4	Claudin 18.2 mAb	Zolbetuximab plus mFOLFOX6	Phase III	Significantly prolonged progression-free survival and overall survival compared with placebo	[101]
5	Claudin 18.2 mAb	Zolbetuximab (IMAB362) combined with EOX	Phase II	Provided longer progression-free survival and overall survival compared with EOX alone. The primary endpoint was progression-free survival, while overall survival was assessed as a secondary endpoint	[102]
6	Claudin 18.2 mAb	Zolbetuximab	Phase I	Antitumor activity of IMAB362, both as a single agent and in combination with standard therapeutics, was observed	[103]
7	Claudin 18.2 mAb	Claudiximab	Phase II	Demonstrated significant efficacy and a favorable safety profile	[104]

TJ: Tight junction; GC: Gastric cancer; CAR: Chimeric antigen receptor; EOX: Epirubicin, oxaliplatin and capecitabine.

CLAUDIN 18.2 TARGETED THERAPIES IN GC

CMG901 is a first-in-class ADC comprising a humanized anti-claudin 18.2 monoclonal antibody linked to the microtubule-disrupting agent monomethyl auristatin E. It has been evaluated in patients with gastric and gastroesophageal junction (GEJ) cancers. CMG901 was administered intravenously every three weeks at doses ranging from 0.3-2.4 mg/kg during dose escalation and 2.2-3 mg/kg during dose expansion[93]. The primary endpoints included assessment of dose-limiting toxicities (DLTs) and overall safety. Pancreatitis was identified as a serious DLT at 2.2 mg/kg, establishing the maximum tolerated dose. Other common adverse events included vomiting, reduced appetite, proteinuria, and fatigue. Hematologic toxicities such as leukopenia, neutropenia, lymphopenia, thrombocytopenia, and anemia were also observed. GI symptoms, including nausea, vomiting, and diarrhea, were generally mild and manageable compared with conventional chemotherapy[93].

IBI343 is an ADC composed of a fully humanized monoclonal antibody targeting claudin 18.2, conjugated to the topoisomerase I inhibitor exatecan via site-specific glyco-conjugation using a cleavable linker, yielding a drug-to-antibody ratio of 4. In clinical studies, the recommended phase II dose was 6 mg/kg administered every three weeks. This regimen achieved an objective response and a median progression-free survival of 5.5 months in patients with gastric or GEJ adenocarcinoma exhibiting high claudin 18.2 expression[94]. The treatment demonstrated favorable tolerability and a manageable safety profile. DLTs were observed at 10 mg/kg, consisting of grade 4 myelosuppression and neutropenia, accompanied by grade 3 febrile neutropenia. Severe GI adverse events (grade ≥ 3) were uncommon. Overall, IBI343 monotherapy showed encouraging antitumor activity with acceptable safety. Combination strategies particularly with immunotherapy are under investigation to further enhance therapeutic efficacy. Phase III, multicenter, randomized trials are ongoing to validate its role in claudin 18.2 positive gastric and other solid tumors[94].

CT041 is a claudin 18.2-specific CAR T-cell therapy generated from autologous T cells engineered to express a receptor targeting the claudin 18.2 antigen[95]. Preclinical studies demonstrated potent antitumor activity against claudin 18.2-positive GC cell lines and patient-derived xenograft models. Based on phase I safety evaluations, the data safety monitoring committee recommended a dose of 2.5 × 10⁸ CAR-T cells for expansion cohorts[95]. The most common adverse events were hematologic, related primarily to preconditioning, including leukopenia, neutropenia, anemia, and thrombocytopenia. Notably, no grade ≥ 3 cytokine release syndrome was reported. Tumor regression occurred in most treated patients, with CAR-T cell expansion correlating with reductions in circulating tumor DNA. Collectively, clinical trials of CT041 in advanced gastric and other GI cancers have shown robust antitumor activity with manageable safety profiles[96,97]. Ongoing studies continue to explore next-generation claudin 18.2-targeted approaches, including bispecific antibodies such as IBI389 and universal “modular” CAR-T platforms like IBI345, which aim to broaden applicability and enhance therapeutic outcomes.

More recently, zolbetuximab, a monoclonal antibody selectively targeting claudin 18.2, has emerged as a promising targeted therapy for advanced GC. In phase III trials, the addition of zolbetuximab to chemotherapy significantly improved overall survival compared with chemotherapy alone in HER2-negative patients with claudin 18.2 positive unresectable gastric or GEJ adenocarcinoma[98]. Beyond zolbetuximab, several claudin 18.2-directed immunotherapies including monoclonal antibodies, bispecific antibodies, CAR T-cell therapies, and ADCs are under active clinical development[99,100].

CONCLUSION

TJ proteins are crucial for maintaining gastric mucosal integrity, and their dysregulation is strongly linked to the initiation and progression of GC. Aberrant TJ protein expression increases epithelial permeability, drives chronic inflammation, and promotes tumor growth, invasion, and metastasis. These findings underscore the role of TJ proteins not only in gastric carcinogenesis but also as potential biomarkers and therapeutic targets. While claudin 18.2 based therapies have already entered clinical application, further research is needed to elucidate the mechanisms underlying dysregulation of other TJ proteins. Such efforts may facilitate the development of novel diagnostic tools and precision therapies that improve survival and quality of life in GC patients.