The dysadherin/carbonic anhydrase 9 axis shapes an acidic tumor microenvironment to promote colorectal cancer progression

Abstract

The tumor microenvironment (TME) plays a central role in cancer progression and metastasis. A key feature of the TME is extracellular acidity, which promotes disease progression, immune evasion, and drug resistance. Tumor acidity is increasingly recognized as a critical factor in cancer development and a negative prognostic indicator. Here, we demonstrate that the membrane glycoprotein dysadherin promotes colorectal cancer (CRC) malignancy by modulating TME acidity. Comprehensive bioinformatics and pathological analyses of CRC patient samples revealed that increased tumor acidity is a hallmark of CRC progression and strongly correlates with high expression of dysadherin. Functional studies confirmed that dysadherin enhances malignant traits, particularly under acidic conditions. Mechanistically, dysadherin activates the integrin/FAK/STAT3 signaling pathway, leading to the upregulation of carbonic anhydrase 9 (CA9). CA9 facilitates proton export, contributing to extracellular acidification while maintaining intracellular pH homeostasis, thereby enabling cancer cells to survive and thrive in acidic environments. In a murine liver metastasis model, dysadherin deletion impaired cellular adaptation to the acidic TME and markedly attenuated metastatic colonization, whereas restoring CA9 expression effectively rescued metastatic potential. Overall, our findings identify the dysadherin/CA9 axis as a potential therapeutic target in CRC and provide new insights into how tumors exploit acidosis to drive malignant development and progression.

Fig. 1

An acidic TME is positively associated with dysadherin expression and contributes to CRC progression. a A list of DEGs in tumor tissue compared with normal tissue was obtained from the GEO database (GSE106582) (p < 0.001). The gene list was analyzed via a curated gene set and the GO gene set of GSEA to determine the associated gene signatures. b A UMAP plot of the epithelial cell cluster from CRC patients (GSE144735) was generated. Trajectory analysis was performed via Monocle2, which orders cells on the basis of their malignant progression states. The expression patterns of malignancy-associated and tumor-acidosis–related genes were examined along the inferred trajectory. c Heatmap comparing the relative expression levels of malignancy and tumor-acidosis-related genes in CRC patient tissues (n = 4/group). d IF analysis of dysadherin and LAMP2 expression in CRC patient tissues, with samples grouped according to tumor stage. (S1, n = 45; S2, n = 62; S3, n = 61; total, n = 168). Graphs showing the intensity and correlation of dysadherin and LAMP2 protein expression. e Kaplan–Meier survival analysis was performed on patients with stage II and III CRC. Patients were divided into three groups on the basis of dysadherin and LAMP2 expression: high (n = 52), low (n = 45), and others (n = 26), for a total of 123 patients. Statistical significance was determined by log-rank tests. f IF analysis of dysadherin in the intestines of 24-week-old Apc^Min/+ mice with or without dysadherin (Fxyd5) KO (n = 5/group). The upper violin plot shows the number of visible tumors per mouse. The lower violin plot shows the tumor load, which is calculated as a weighted sum that incorporates both tumor number and size: (small tumors × 1) + (medium tumors × 2) + (large tumors × 3). g pHrodo and HPTS staining of mouse intestinal tumors (n = 5/group). The data are shown as the means ± SEMs. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. Statistical significance was assessed via unpaired two-tailed Student’s t-tests for two-group comparisons and one-way ANOVA with Dunnett’s multiple comparison test for analyses involving three groups. Dys dysadherin, S1 stage 1, S2 stage 2, S3 stage 3

Fig. 2

Dysadherin regulates the malignant phenotypes of CRC cells in the acidic TME. a Proliferation of CRC cells at various pH values was measured by a BrdU assay (n = 3/group). The survival potential and self-renewal properties of dysadherin-OE HCT116 cells (b) and dysadherin KO SW480 cells (c) were measured at pH 7.4 or 6.4 (n = 3/group). d Heatmap comparing the relative expression of genes related to proliferation and apoptosis in dysadherin OE HCT116 cells and dysadherin KO SW480 cells after incubation at pH 7.4 or 6.4 (n = 3/group). The proliferative capacity and apoptotic cell population were assessed after incubation at pH 7.4 or 6.4 (n = 3/group) in dysadherin OE HCT116 cells (e) or dysadherin KO SW480 cells (f). g Immunoblot analyses of apoptosis and proliferation markers after incubation at pH 7.4 or 6.4. Here, and throughout, the numbers under each image are the fold changes in band intensity relative to the initial condition, which were determined via ImageJ. The data are shown as the means ± SEMs. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. Statistical significance was assessed via one-way ANOVA with Dunnett’s multiple comparison test for analyses involving three groups. NTC nontargeting control, KO knockout, EV empty vector, OE overexpression

Fig. 3

Dysadherin regulates the expression of CA9 to promote tumor progression within acidic tumors. a Network analysis via IPA revealed a potential link among dysadherin (FXYD5), the TME, tumor acidosis, cell survival, metastasis, and tumor progression. b IPA was performed on the mRNA sequencing data of NTC and dysadherin-KO SW480 cells. Six candidate genes related to the TME, tumor acidosis, cell survival, metastasis, and tumor progression were identified. Kaplan–Meier survival analysis of the GEO dataset (GSE143985) revealed that CA9 was most strongly associated with poor patient survival. c IF analysis of dysadherin and CA9 expression in CRC patient tissues. Patients were divided into three groups according to tumor stage (S1, n = 45; S2, n = 62; S3, n = 61; total, n = 168). The graphs show the intensity of CA9 and the correlation between dysadherin and CA9. d Kaplan–Meier survival analysis was performed on patients with stage II and III CRC. Patients were divided into three groups on the basis of dysadherin and CA9 expression: high (n = 50), low (n = 49), and others (n = 24), for a total of 123 patients. Statistical significance was determined by log-rank tests. e Immunoblot analyses of dysadherin and CA9 in a panel of human CRC and normal colon cell lines. f qPCR and immunoblotting analysis of dysadherin and CA9 expression in dysadherin-OE HCT116 and dysadherin-KO SW480 cell lines. g IF analysis of dysadherin and CA9 in intestinal tumor tissue from 24-week-old Apc^Min/+; Fxyd5^+/+ and Apc^Min/+; Fxyd5^−/− mice (n = 5/group). The data are shown as the means ± SEMs. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. Statistical significance was assessed via unpaired two-tailed Student’s t-tests for two-group comparisons and one-way ANOVA with Dunnett’s multiple comparison test for analyses involving three groups

Fig. 4

Dysadherin increases the transcriptional activity of CA9 by activating the integrin/FAK/STAT3 axis. a Venn diagram showing five potential transcription factors of CA9. Downstream analysis by IPA revealed that the integrin/FAK (PTK2) axis activated STAT3. b IF analysis of dysadherin, active integrin β1, and p-FAK in dysadherin KO SW480 cells. Antibody clones used for integrin β1 detection: 12G10, which recognizes specific conformational epitopes of human integrin β1. c Immunoblot analyses of dysadherin, p-FAK, t-FAK, p-STAT3, t-STAT3, and CA9 in dysadherin-OE HCT116 cells treated with or without 0.1 µM MK-0429, 1 µM PND-1186 or 1 µM Stattic. d The promoter activity of CA9 upon dysadherin OE with or without MK-0429, PND-1186 or Stattic treatment was tested via a luciferase reporter assay. The total transcript level was normalized to the β-galactosidase transcript level and is presented as the fold change with respect to that in nontreated HCT116 cells (n = 3/group). e Potential binding site for p-STAT3 in the CA9 promoter region identified by a ChIP assay. The CA9 promoter region was fragmented into 7 segments and immunoprecipitated with an anti-p-STAT3 antibody. f The binding affinity of p-STAT3 for the CA9 promoter with or without MK-0429, PND-1186 or Stattic was tested via a ChIP assay (n = 3/group). The data are shown as the means ± SEMs. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. Statistical significance was assessed via unpaired two-tailed Student’s t tests for two-group comparisons and one-way ANOVA with Dunnett’s multiple comparison test for analyses involving three groups

Fig. 5

The dysadherin/CA9 axis promotes CRC cell adaptation and increases cell survival within an acidic TME. The survival potential (a), self-renewal properties (b), proliferative capacity (c), and apoptotic cell population (d) were assessed in NTC or dysadherin KO SW480 cells with CA9 OE at pH 6.4 (n = 3/group). Immunoblot analyses (e) and heatmaps comparing the relative expression (f) of apoptosis and proliferation markers in NTC or dysadherin KO SW480 cells with CA9 OE at pH 6.4 (n = 3/group). The survival potential (g), proliferative capacity (h), and apoptotic cell population (i) were assessed in NTC or dysadherin KO SW480 cells with CA9 OE under lactate-induced acidic conditions. j Left: CA9 expression levels in CRC patient samples from the GEO dataset GSE21510, with patients stratified into high and low groups on the basis of the median CA9 expression value. (CA9^high, n = 74; CA9^low, n = 74). Right: DAVID analysis of DEGs between the CA9-high and CA9-low cohorts. OCR profiles (k, l) and ATP production (m) were monitored in NTC or dysadherin KO SW480 cells with CA9 OE at pH 6.4 (n = 3/group). n Flow cytometric analysis of intracellular ROS production in NTC or dysadherin KO SW480 cells with CA9 OE at pH 6.4 (n = 3/group). The data are shown as the means ± SEMs. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. Statistical significance was assessed via one-way ANOVA with Dunnett’s multiple comparison test for analyses involving three groups

Fig. 6

The dysadherin/CA9 axis facilitates metastasis by promoting CRC cell adaptation to the acidic TME. a Experimental scheme of the intrasplenic injection mouse model. Splenectomy was performed at the time of tumor cell injection. b Top: Representative bioluminescence images of mice bearing luciferase-labeled SW480 cells, comparing NTC controls with dysadherin KO SW480 cells overexpressing CA9 (n = 5/group). Middle: Hematoxylin and eosin-stained liver sections from mice bearing metastatic lesions. Bottom: Quantitative plots depicting photon flux and the metastatic nodule area corresponding to the images shown. c pHrodo and HTPS staining of metastatic nodules in the mouse liver (n = 5/group). The graphs show the intensities of pHrodo and HPTS in the indicated groups. d IF analysis of dysadherin and CA9 expression in the carcinoma in situ and metastatic cancer tissues of CRC patients (n = 10/group). e Schematic illustration summarizing the study, highlighting the contribution of the dysadherin/CA9 axis to CRC progression in acidic conditions. Created with BioRender.com. The data are presented as the means ± SEMs. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. Statistical significance was assessed via two-way ANOVA with Bonferroni’s multiple comparison test or one-way ANOVA with Dunnett’s multiple comparison test for analyses involving more than three groups