VSIR, also called VISTA, is a type I transmembrane protein of the B7/CD28 family. Immunologists have long studied it for its immunosuppressive functions within the tumor microenvironment, where it attenuates effector T cell activation, fosters regulatory T cell expansion, and supports the suppressive activity of myeloid-derived suppressor cells and tumor-associated macrophages. But recent work has documented frequent upregulation of VSIR on tumor cells themselves across multiple cancer types, and in melanoma, tumor-cell-specific VSIR expression has been shown to promote tumor onset in preclinical models. In gastric cancer, elevated VSIR correlates with advanced stage, lymph node metastasis, and poor clinical outcomes. These observations raised an provocative question: could VSIR, beyond its checkpoint role, be driving tumor progression through mechanisms intrinsic to the cancer cell? The research team, led by Jinshan Liu, Guoquan Huang, and colleagues at institutions affiliated with Chongqing Medical University, set out to answer it by searching for the signaling pathways that VSIR might control.

Their attention soon turned to AXL, a receptor tyrosine kinase of the TAM family that has emerged as a central regulator of tumor progression, metastasis, and therapeutic resistance across many cancers. When bound by its ligand GAS6, AXL activates the PI3K/AKT, MAPK/ERK, and STAT3 pathways, enhancing cell survival, epithelial-mesenchymal transition, and immune evasion. In gastric cancer, AXL overexpression has been documented in both cell lines and patient specimens and is associated with aggressive phenotypes including peritoneal dissemination. Mining The Cancer Genome Atlas stomach adenocarcinoma cohort through the GEPIA2 platform, the researchers confirmed that AXL expression climbs steadily with tumor stage and that patients with high AXL levels suffer significantly worse overall survival, with a hazard ratio of 1.4. The clinical signal was unmistakable, but the regulatory circuitry that produces AXL overexpression in an inflammatory setting had not been mapped.

The team’s first mechanistic clue came from transcriptome sequencing of gastric cancer cells. When HGC-27 gastric cancer cells were engineered to overexpress VSIR and then stimulated with LPS to mimic bacterial inflammation, AXL mRNA levels rose dramatically. The effect was strikingly conditional: without LPS, VSIR overexpression alone failed to induce AXL, but once the inflammatory stimulus was applied, VSIR markedly enhanced AXL transcription. Conversely, silencing VSIR with two independent small interfering RNAs abolished the LPS-driven induction of AXL at both the mRNA and protein levels. The results established VSIR as both necessary and sufficient, within the inflammatory context, to promote AXL expression, forging a direct molecular link between innate immune signaling and AXL-driven tumor progression.

To identify the transcription factor mediating this effect, the researchers cross-referenced four independent prediction databases—hTFtarget, CHEA, FIMO_JASPAR, and ENCODE—and found that two candidates, STAT3 and EGR1, were consistently predicted to bind regulatory regions of the AXL gene. Chromatin profiling from the Cistrome Database revealed strong enrichment of STAT3-binding signals at an enhancer upstream of AXL, coinciding precisely with the active histone marks H3K27ac and H3K4me1, the biochemical signatures of functional enhancer elements. Chromatin immunoprecipitation followed by quantitative PCR in HGC-27 cells confirmed that STAT3 physically occupies this enhancer region. The evidence became causal when the team cloned the enhancer fragment into a luciferase reporter: co-transfection with a STAT3 expression plasmid dramatically boosted reporter activity, while site-directed mutation of the predicted STAT3-binding motif, changing the sequence CAT CTG GAA AG to ACC ACA ACC CA, abolished that activation. Knocking down STAT3 with siRNA reduced endogenous AXL mRNA and protein, and parallel experiments in mouse bone marrow-derived macrophages showed that LPS-induced AXL upregulation was completely eliminated when STAT3 was depleted. Together, these experiments established STAT3 as the essential transcriptional mediator linking LPS stimulation to AXL expression in both myeloid and cancer cells.

But how does VSIR, a membrane checkpoint protein, activate STAT3? The answer, the researchers found, lies in the IL-6 pathway. Under LPS stimulation, VSIR-overexpressing cells produced substantially more IL-6 mRNA, and western blotting revealed enhanced phosphorylation of STAT3—without any measurable change in the phosphorylation of p65 or JAK1, indicating a specific rather than generalized activation of inflammatory signaling. When the team silenced TLR4, the VSIR-driven boost in IL-6 production vanished, placing VSIR functionally downstream of the TLR4 receptor. The emerging picture is a coherent cascade: LPS engages TLR4, VSIR potentiates IL-6 release, IL-6 activates STAT3, and STAT3 directly switches on AXL transcription.

Remarkably, transcription proved to be only half the story. Co-immunoprecipitation experiments revealed that VSIR physically interacts with the AXL protein itself, and immunofluorescence microscopy confirmed the colocalization of the two molecules in both HGC-27 and MKN-45 gastric cancer cells. Cycloheximide chase assays, which track protein decay over time, demonstrated that VSIR overexpression significantly prolongs the half-life of AXL. The proteasome inhibitor MG132 erased the difference in AXL levels between VSIR-proficient and VSIR-deficient cells, pointing to the ubiquitin-proteasome pathway as the route of degradation, and direct ubiquitination assays showed that VSIR overexpression markedly attenuates the polyubiquitination of endogenous AXL. In other words, VSIR both writes more AXL mRNA and protects the resulting protein from destruction, a dual mechanism that ensures robust, sustained AXL signaling precisely when inflammatory conditions would otherwise favor tumor growth.

Clinical validation came from multiplex immunofluorescence staining of patient tissues, including samples from twelve gastric cancer patients treated at the First Affiliated Hospital of Chongqing Medical University. Tumor samples showed a significant positive correlation among VSIR, AXL, and phosphorylated STAT3, mirroring the in vitro findings. More striking still, when the team compared HER2-positive tumors from patients resistant to HER2-targeted therapy, treatment-naive HER2-positive tumors, HER2-negative tumors, and normal gastric mucosa, the fraction of cells simultaneously positive for VSIR, AXL, and HER2 was markedly highest in the resistant HER2-positive group. Because elevated VSIR and AXL were also present in treatment-naive HER2-positive tumors, the authors conclude that this co-expression pattern is a general feature of HER2-positive gastric cancer rather than a specific marker of acquired resistance—implying that these tumors depend on dual engines of growth: HER2-driven proliferation and VSIR-driven, AXL-mediated survival.

The therapeutic implications were tested directly in a subcutaneous xenograft model in nude mice, with animals randomized into five treatment groups. VSIR overexpression robustly accelerated tumor growth, and while the HER2 inhibitor tucatinib alone produced only modest suppression, the AXL inhibitor AXL-IN-13 sharply curtailed VSIR-driven tumor expansion, reducing tumor volume and weight to levels comparable to or below controls. The dual blockade of HER2 and AXL produced the most potent anti-tumor effect of all, nearly abolishing the pro-tumorigenic activity of VSIR, with Ki-67 immunohistochemistry confirming sharply reduced proliferation in the treated tumors. Rescue experiments in vitro reinforced the causal chain: when VSIR was knocked down in MKN-45 cells, migration and invasion declined sharply, but re-expressing AXL restored these metastatic capabilities. Taken together, the study recasts VSIR as a tumor-intrinsic oncogenic amplifier that operates independently of its immune checkpoint function, positions the VSIR-AXL axis as a druggable vulnerability in an inflammation-associated malignancy, and supplies a strong preclinical rationale for combining AXL inhibitors with HER2-directed therapy in patients whose tumors currently escape single-agent blockade.

Subject of Research: The role of VSIR in regulating AXL expression and promoting gastric cancer proliferation through inflammatory STAT3 signaling

Article Title: Mechanistic insights into VSIR-mediated AXL regulation and gastric cancer proliferation

Article References: Liu, J., Huang, G., Li, Y., Tan, Y., Qin, B., Hao, C., Peng, J., Zhu, H., Zhang, B., Cheng, Y., & Qian, K. (2026). Mechanistic insights into VSIR-mediated AXL regulation and gastric cancer proliferation. iScience, 29(10), Article 117580. https://doi.org/10.1016/j.isci.2026.117580

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117580

Keywords: gastric cancer, VSIR, VISTA, AXL, STAT3, IL-6, TLR4, Helicobacter pylori, HER2, immune checkpoint, protein stability, targeted therapy