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

Connexin 36 (Cx36) forms electrical synapses that allow direct cytoplasmic continuity between adjacent retinal neurons, enabling the rapid exchange of ions and small metabolites. In the outer retina, Cx36-containing gap junctions mediate rod-cone coupling and regulate visual signal integration, while in the inner retina, they contribute to electrical coupling between retinal ganglion cells and amacrine cells. These channels are essential for temporal precision in visual processing, enhancing signal-to-noise ratio and firing synchrony. However, under pathological conditions such as excitotoxicity, the same gap junctions that normally support healthy signal transmission can become conduits for the spread of toxic signals—including calcium overload and pro-apoptotic molecules—from injured neurons to their healthy neighbors.

The research team, led by Adrian Smędowski at the Medical University of Silesia in Katowice, Poland, used immortalized 661W murine photoreceptor cells transfected with GFP-tagged Cx36 to visualize the protein’s intracellular distribution under controlled stress conditions. The 661W cell line, derived from mouse cone photoreceptors, expresses cone-specific markers and retains light responsiveness through cGMP modulation, making it a physiologically relevant model for studying retinal photoreceptor biology. The researchers chose this model because it provides a more relevant retinal phenotype compared to other neuron-like cell lines, though they acknowledge that findings from an immortalized cell line must ultimately be validated in intact retinal tissue.

To probe the degradation pathways responsible for Cx36 turnover, the team employed two complementary pharmacological approaches. First, they used bafilomycin A1, a macrolide antibiotic that disrupts autophagic flux by blocking V-ATPase-dependent acidification of lysosomes and inhibiting autophagosome-lysosome fusion. Second, they exposed the cells to N-methyl-D-aspartate (NMDA) to induce excitotoxic stress, mimicking the glutamate-mediated overactivation that contributes to neuronal damage in retinal neurodegenerative diseases. By applying these interventions separately and in combination with Cx36 overexpression, the researchers could dissect the relative contributions of the autophagy-lysosome pathway and the ubiquitin-proteasome system (UPS) to connexin proteostasis.

The results revealed a striking pattern of proteostatic reorganization. Following bafilomycin A1 treatment, immunofluorescence analysis showed intracellular accumulation of the autophagy adaptor protein SQSTM1/p62, with signal intensities increasing approximately 1.5- to 2-fold compared to controls (n = 3, p < 0.05). Under normal conditions, p62 selectively binds polyubiquitinated protein aggregates and delivers them to autophagosomes via interaction with LC3, facilitating their degradation upon fusion with lysosomes. When this pathway is blocked, p62 itself accumulates because it can no longer be cleared. The researchers also observed a marked upregulation of PSMA1, a structural subunit of the 20S proteasome core particle, suggesting a potential compensatory shift toward proteasomal degradation when lysosomal clearance is compromised.

Perhaps most intriguingly, the team detected elevated levels of RAB11A, a small GTPase associated with the recycling endosome pathway. RAB11A controls the return of internalized receptors from recycling endosomes to the plasma membrane and contributes to vesicular trafficking processes underlying endosomal sorting. Its increased expression may reflect an adaptive cellular response to impaired protein degradation, potentially contributing to altered intracellular trafficking and redistribution of proteins—including Cx36—under stress conditions. Notably, the researchers did not observe changes in other endosomal markers such as EEA1, RAB5A, or RAB9A, suggesting that the recycling pathway specifically, rather than the broader endocytic machinery, is recruited in response to lysosomal blockade.

When the researchers turned to the excitotoxicity model, they found that NMDA treatment was associated with increased ubiquitin accumulation, particularly in Cx36-overexpressing cells. Ubiquitin serves as a molecular tag that marks damaged or misfolded proteins for degradation by the 26S proteasome through a cascade involving E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. The enhanced ubiquitin signal in Cx36-expressing cells exposed to NMDA suggests that excitotoxic conditions compromise Cx36 stability and promote its ubiquitination, indicating enhanced proteostatic stress specifically in cells bearing higher loads of the gap junction protein. Interestingly, Cx36 immunoreactivity remained predominantly cytoplasmic with a punctate distribution pattern, and no major redistribution was observed following NMDA treatment.

The implications of these findings extend beyond basic cell biology. Previous studies have demonstrated that blocking gap junctions under excitotoxic or ischemic conditions can improve retinal ganglion cell survival by up to 70 percent, underscoring the critical role of intercellular communication in propagating neuronal injury. Genetic knockout studies have further shown that eliminating Cx36 significantly reduces retinal ganglion cell death under excitotoxic stress. The current work adds a new dimension to this understanding by suggesting that modulating Cx36 turnover or stabilization—rather than simply upregulating or downregulating its expression—may represent a more effective neuroprotective strategy. By targeting the proteostatic machinery that controls connexin degradation, it may be possible to fine-tune gap junction abundance and coupling strength in ways that preserve normal visual signal transmission while limiting the pathological spread of toxic signals.

The study’s authors are careful to note several important limitations. The immunofluorescence-based approach, while providing unique spatial resolution for assessing intracellular distribution, does not allow precise quantification of total protein expression. Definitive confirmation of impaired autophagic flux would require LC3-II turnover analysis, which was not performed in this study. Similarly, the observed PSMA1 upregulation suggests but does not prove functional proteasome involvement. The use of Cx36 overexpression itself may influence intracellular proteostasis, and the cytoplasmic distribution of the tagged protein likely reflects overexpression-driven accumulation within the endoplasmic reticulum or vesicular compartments rather than functional gap junction localization at the plasma membrane. Future work incorporating Western blotting, LC3-II flux assays, proteasome activity measurements, live-cell imaging, and validation in retinal explants or animal models will be essential to confirm and extend these preliminary observations.

Nevertheless, the study opens a compelling new avenue for investigating retinal neurodegeneration. Both glaucoma and age-related macular degeneration are characterized by progressive dysfunction of proteostatic mechanisms—whether through oxidative stress in the retinal pigment epithelium, mitochondrial dysfunction in retinal ganglion cells, or chronic neuroinflammation. If connexin proteostasis proves to be a modifiable node in these degenerative cascades, targeted pharmacological interventions aimed at specific degradation pathways could offer a precision approach to preserving vision. The researchers suggest that better understanding of these subcellular mechanisms will potentially allow the design of precisely targeted treatments in the future, transforming our approach to diseases that currently rob millions of people worldwide of their sight.

Subject of Research: Proteostatic regulation of the neuronal gap junction protein connexin 36 in photoreceptor cells under excitotoxic stress

Article Title: Gap junction protein connexin 36 proteostatic mechanisms in photoreceptor cells subjected to excitotoxic stress

Article References: Katan, M., Mróz, K., Pacwa, A., Lewin-Kowalik, J., & Smędowski, A. (2026). Gap junction protein connexin 36 proteostatic mechanisms in photoreceptor cells subjected to excitotoxic stress. Acta Biochimica Polonica, 73, Article 16091. https://doi.org/10.3389/abp.2026.16091

Image Credits: AI Generated

DOI: 10.3389/abp.2026.16091

Keywords: connexin 36, gap junctions, photoreceptors, excitotoxicity, autophagy, ubiquitin-proteasome system, retinal degeneration, glaucoma, age-related macular degeneration, p62/SQSTM1, RAB11A, proteostasis

The protein in question is connexin 26, or Cx26, encoded by the gene GJB2. In healthy tissue, six connexin subunits assemble into hemichannels that dock with neighboring cells to form gap junctions, pores through which small molecules and ions pass freely. Cx26 was long considered a tumor suppressor, yet accumulating evidence has linked its overexpression to metastasis and poor prognosis in several cancers. Earlier work had noted that Cx26 accumulates in the cytoplasm of colorectal cancer cells rather than sitting on the plasma membrane, but whether this mislocalization was merely a passive hallmark of aggressiveness or an active driver of it remained unresolved.

To answer that question, the team led by researchers at Nanjing University turned to a uniquely informative pair of cell lines: SW480, isolated from a primary colon adenocarcinoma, and SW620, derived a year later from a lymph node metastasis in the same patient. The metastatic SW620 cells expressed far more Cx26 than their primary counterparts, yet their gap junction function was weaker, not stronger. Surface-level Cx26 was nearly absent in SW620 cells. Instead, fractionation and immunofluorescence experiments revealed that the protein had accumulated in an unexpected destination: the mitochondria. When the researchers induced epithelial-mesenchymal transition, or EMT, in SW480 cells with TGF-β, Cx26 levels rose and the protein shifted toward mitochondria in step with the cells’ acquisition of invasive traits.

The pattern held beyond the laboratory dish. In a human colorectal cancer tissue microarray, multiplex immunofluorescence showed that Cx26 overlapped strongly with the mitochondrial marker TOM20 in metastatic lesions of the liver, lung, and ovary, while primary tumors retained substantial membrane-localized Cx26 with little mitochondrial colocalization. A parallel comparison of the mouse melanoma sublines B16F1 and B16F10—which share a genetic background but differ sharply in metastatic capacity—reproduced the same relationship: the highly metastatic B16F10 cells carried less membrane Cx26 and more mitochondrial Cx26. Mitochondrial translocation of Cx26, the authors conclude, is a defining feature of the aggressive, metastatic phenotype.

What steers the protein to mitochondria? Signal peptide predictions pointed to the first forty amino acids of Cx26, but deleting them changed nothing. The answer emerged from a co-immunoprecipitation screen coupled to mass spectrometry, which identified PIMT—protein L-isoaspartyl/D-aspartyl O-methyltransferase, encoded by PCMT1—among 118 candidate interacting proteins. PIMT is a conserved repair enzyme that methylates isoaspartyl residues created by the spontaneous deamidation of asparagine, a chemical decay process that kinks protein backbones and undermines stability. In the metastatic SW620 cells, the interaction between PIMT and Cx26 was far stronger than in SW480 cells, and silencing PIMT pushed Cx26 back toward the cell membrane, restored gap junction communication, and drained the protein out of mitochondria.

Mass spectrometry then pinpointed the chemistry: PIMT methylates Cx26 at asparagine 176, a residue within the extracellular loop that normally helps dock hemichannels between adjacent cells. Mutating that residue to alanine shortened the protein’s half-life, reduced its mitochondrial abundance, and increased its cytoplasmic pool. PIMT overexpression, by contrast, protected wild-type Cx26 from degradation while leaving the N176A mutant untouched. The team showed that Cx26 is degraded by the ubiquitin-proteasome system, and that PIMT suppresses Cx26 ubiquitination through its enzymatic activity. In short, PIMT acts as a chaperone of sorts—repairing Cx26’s conformation, stabilizing it, and licensing its journey to the mitochondrial compartment.

Inside the mitochondria, Cx26 proved functionally potent. Overexpression of Cx26 increased mitochondrial calcium uptake, measured with a genetically encoded calcium sensor, while the N176A mutation blunted that uptake. Electron microscopy and co-immunoprecipitation revealed that Cx26 strengthens contacts between the endoplasmic reticulum and mitochondria by bridging the calcium release channel IP3R with the mitochondrial channel VDAC1. The consequences for tumor behavior were dramatic: Cx26 overexpression accelerated EMT in SW480 cells, enlarged xenograft tumors, and multiplied pulmonary metastatic burden in mouse models, while Cx26 knockdown in SW620 and B16F10 cells did the opposite. Blocking mitochondrial calcium uptake with ruthenium red or the MCU inhibitor MCU-i4 reversed the EMT phenotype, establishing calcium flux as the mechanistic link between mitochondrial Cx26 and metastatic capacity.

The therapeutic twist came from an unbiased screen. Querying the Genomics of Drug Sensitivity in Cancer databases across 23 colorectal cancer cell lines, the researchers found that higher GJB2 expression generally predicted resistance to standard chemotherapeutics, including 5-fluorouracil and oxaliplatin. Bortezomib, a 26S proteasome inhibitor used as first-line therapy for multiple myeloma and mantle cell lymphoma, broke the pattern: cells with high GJB2 expression were more sensitive, showing lower IC50 values. In direct comparisons, bortezomib killed metastatic SW620 cells more effectively than primary SW480 cells, while the conventional colorectal cancer drugs showed no such preference. Cx26 overexpression sensitized SW480 cells to the drug, confirming the connection.

Curiously, the mechanism had little to do with the proteasome. Cellular thermal shift assays, molecular docking, differential scanning fluorimetry, and surface plasmon resonance all converged on an unexpected target: PIMT itself. Bortezomib bound PIMT directly, raised its melting temperature by 5.6 degrees Celsius, and enhanced its methyltransferase activity in vitro. Treatment with the drug strengthened the PIMT-Cx26 interaction, drove more Cx26 into mitochondria, and triggered a catastrophic surge of intracellular calcium. That calcium flood generated reactive oxygen species and activated ERK signaling, producing PARP cleavage and LC3-II accumulation—hallmarks of apoptosis and autophagy. Scavenging reactive oxygen species, blocking mitochondrial calcium uptake, inhibiting ERK with PD0325901, or knocking down Cx26 or PIMT all blunted bortezomib’s killing of SW620 cells, mapping the pathway end to end.

The study reframes Cx26 mislocalization from a passive biomarker into an organelle-specific engine of malignancy, and it challenges the assumption that solid tumors are inherently resistant to proteasome inhibitors. Bortezomib, in this context, would not work as a proteasome poison but as a molecular lever that amplifies the very PIMT-Cx26 axis on which metastatic cells have come to depend, converting their addiction into a death signal. The authors caution that mitochondrial Cx26 localization must be validated as a survival predictor in larger clinical cohorts, and that direct biochemical confirmation of the N176 modification with purified substrates is still needed. Even so, the work offers a concrete strategy for a disease state with few options: identify tumors addicted to mitochondrial Cx26, and turn that dependency against them with a drug already sitting on the pharmacy shelf.

Subject of Research: PIMT-mediated mitochondrial translocation of connexin 26 in metastatic colorectal cancer and its therapeutic targeting by bortezomib

Article Title: Mitochondrial Localization of Cx26 Promotes Colorectal Cancer Malignancy and Exposes a Fatal Vulnerability to Drug Targeting

Article References: Wang, J., Chen, L., Qin, J., Wang, X., Ye, S., An, N., Bai, M., Shen, Y., Wu, X., & Xu, Q. (2026). Mitochondrial Localization of Cx26 Promotes Colorectal Cancer Malignancy and Exposes a Fatal Vulnerability to Drug Targeting. Advanced Science, Article e78117. https://doi.org/10.1002/advs.78117

Image Credits: AI Generated

DOI: 10.1002/advs.78117

Keywords: colorectal cancer, connexin 26, Cx26, PIMT, mitochondria, metastasis, EMT, bortezomib, calcium signaling, gap junctions, drug sensitivity, Advanced Science