A research team led by Takako Kihara of the Department of Diagnostic Pathology at Hyogo Medical University School of Medicine in Japan, working with colleagues in Japan and China, now reports evidence that a single gap junction protein may help explain the poor prognosis of small intestinal GISTs. In a study published in Pathology and Oncology Research, the investigators show that Connexin 43, or Cx43, a four-pass transmembrane protein that assembles into hemichannels and gap junctions, enhances the liver metastatic ability of GIST cells in living animals. The work builds on the team’s earlier finding that most small intestinal GISTs express high levels of Cx43, while gastric GISTs show minimal expression of this protein.

Cx43 is the most ubiquitously expressed and extensively studied connexin in human tissues. Six Cx43 molecules form a hemichannel, and two paired hemichannels create a gap junction, a structure essential for transmitting ions, small molecules, and metabolites between cells. In cancer biology, Cx43 plays a dual role that varies by tumor type and stage. In colorectal and pancreatic cancer it acts as a tumor suppressor, whereas in breast cancer, gastric cancer, melanoma, and ovarian cancer it is considered a tumor promoter, with metastatic lesions showing elevated Cx43 expression at both protein and mRNA levels compared with primary lesions. In breast cancer, Cx43 staining is absent in non-invasive ductal carcinoma but appears in the cytoplasm and membrane of invasive tumors, and it intensifies further in lymph node metastases. A recent study in colorectal cancer found that Cx43 on the cell membrane suppresses tumor progression, but its translocation to the nucleus is associated with metastatic spread.

To test whether Cx43 drives the metastatic behavior characteristic of small intestinal GISTs, the researchers compared two versions of the GIST-T1 cell line. The original GIST-T1 cells, derived from a metastatic pleural tumor of a gastric GIST in a 47-year-old Japanese woman with a heterozygous KIT exon 11 mutation, express minimal Cx43 and represent gastric GISTs. The team engineered a second line, GIST-T1-Cx43, by transfecting full-length Cx43 cDNA into these cells and selecting stable clones with Zeocin, creating cells with high Cx43 expression that model small intestinal GISTs. Western blot analysis confirmed that Cx43 protein levels were significantly higher in the engineered cells.

The centerpiece of the study was a newly developed in vivo liver metastasis xenograft model. Because GISTs are typically indolent tumors, establishing a liver metastasis model has proven challenging, and no such model had previously been documented. Thirteen female NOG mice, an immunodeficient strain lacking functional T, B, and natural killer cells, were anesthetized with isoflurane, and 2 million GIST cells were injected into the spleen with a 29-gauge needle. Cells entering the splenic vein travel through the portal circulation to the liver, seeding metastatic foci. Four weeks after intrasplenic injection, the livers were excised, sectioned at 2-millimeter intervals, and examined histologically with hematoxylin and eosin staining, counting any cluster of three or more tumor cells as one metastatic focus.

The results were striking. Mice transplanted with GIST-T1-Cx43 cells developed 2.53 times more hepatic metastatic foci than mice receiving the original GIST-T1 cells, a difference that reached statistical significance at p = 0.010. A single outlier in each group was identified with Grubbs’ test and excluded after confirming data normality. This in vivo finding provides direct evidence that elevated Cx43 expression enhances the liver metastatic potential of GIST cells, corroborating the clinical observation that small intestinal GISTs, which naturally express high Cx43, carry a higher risk of distant metastasis.

Conventional in vitro experiments painted a more nuanced picture of how Cx43 reshapes tumor cell behavior. When seeded at low density and incubated for seven days, GIST-T1-Cx43 cells grew significantly more slowly than the original cells, with suppressed proliferation at p < 0.001. Yet the same cells showed a 3.43-fold increase in migration through uncoated transwell membranes (p < 0.001) and a 1.70-fold increase in invasion through Matrigel-coated chambers (p = 0.036). Cx43 thus appears to trade proliferative capacity for motility, a hallmark of the metastatic phenotype.

The team then examined how Cx43 affects the interaction between tumor cells and vascular endothelium, a critical step in the metastatic cascade. In a static adhesion assay, Calcein-AM-labeled GIST-T1-Cx43 cells adhered to confluent monolayers of human umbilical vein endothelial cells 1.67 times more than the original cells (p < 0.001). In a transendothelial migration assay, the engineered cells migrated through the endothelial monolayer 1.56 times more (p < 0.001). These findings parallel the team’s earlier work on CADM1, another surface molecule predominantly expressed in small intestinal GISTs, which similarly promoted endothelial adhesion and transmigration while suppressing proliferation.

Western blot analysis of KIT downstream signaling revealed a mechanistic clue. MAPK phosphorylation was activated, while AKT phosphorylation was suppressed, in GIST-T1-Cx43 cells compared with the original line, indicating significant activation of the RAS-RAF-MAPK pathway. This pathway is crucial for tissue remodeling and cell migration, both essential for tumor invasion and metastasis, and its aberrant activation in cancer promotes tumor growth and metastatic spread. The findings suggest that Cx43 may enhance liver metastasis of GIST cells by shifting signaling toward this pro-migratory cascade.

The study’s implications extend to therapy. The authors propose that an antibody targeting Cx43 could inhibit small intestinal GIST metastasis by suppressing migration, invasion, tumor-endothelial adhesion, and transendothelial migration. Their recent work showed that an antibody-drug conjugate targeting CADM1 exhibits significant antitumor effects on GIST cells with high CADM1 expression, and they suggest that ADCs targeting both Cx43 and CADM1 may offer even greater efficacy. The study has limitations: the in vivo model addressed liver metastasis but not peritoneal dissemination, the effect of Cx43 on other surface adhesion molecules was not thoroughly investigated, and the role of Cx43 in angiogenesis and immune evasion within the tumor microenvironment remains unexplored. Nevertheless, by establishing the first documented GIST liver metastasis model and validating it in vitro, the team has identified Cx43 as a potential novel therapeutic target for inhibiting metastasis in the GIST subtype that needs it most.

Subject of Research: Oncology

Article Title: Connexin 43 enhances liver metastatic ability of GIST cells in vivo

Article References: Kihara, T., Yuan, J., Yamasaki, T., Yoshida, M., Ohkouchi, M., Nakaya-Hashikura, Y., Kimura, N., Hyodo, K., Ohe, C., & Hirota, S. (2026). Connexin 43 enhances liver metastatic ability of GIST cells in vivo. Pathology & Oncology Research, 32, Article 1612383. https://doi.org/10.3389/pore.2026.1612383

Image Credits: AI Generated

DOI: 10.3389/pore.2026.1612383

Keywords: GIST, Connexin 43, liver metastasis, small intestinal tumors, gap junctions, RAS-RAF-MAPK, cancer metastasis, therapeutic target, Connexin, enhances, liver, metastatic