Molecular implications of MUC5AC-CD44 axis in colorectal cancer progression and chemoresistance

Abstract

Background: Differential expression of mucins has been associated with several cancers including colorectal cancer (CRC). In normal physiological conditions, secretory mucin MUC5AC is not expressed in the colonic mucosa, whereas its aberrant expression is observed during development of colon cancer and its precursor lesions. To date, the molecular mechanism of MUC5AC in CRC progression and drug resistance remains obscure.

Methods: MUC5AC expression was determined in colon tissue microarray by immunohistochemistry. A RNA interference and CRISPR/Cas9-mediated system was used to knockdown/knockout the MUC5AC in CRC cell lines to delineate its role in CRC tumorigenesis using in vitro functional assays and in vivo (sub-cutaneous and colon orthotopic) mouse models. Finally, CRC cell lines and xenograft models were used to identify the mechanism of action of MUC5AC.

Results: Overexpression of MUC5AC is observed in CRC patient tissues and cell lines. MUC5AC expression resulted in enhanced cell invasion and migration, and decreased apoptosis of CRC cells. MUC5AC interacted with CD44 physically, which was accompanied by the activation of Src signaling. Further, the presence of MUC5AC resulted in enhanced tumorigenesis and appearance of metastatic lesions in orthotopic mouse model. Additionally, up-regulation of MUC5AC resulted in resistance to 5-fluorouracil (5-FU) and oxaliplatin, and its knockout increased sensitivity to these drugs. Finally, we observed that up-regulation of MUC5AC conferred resistance to 5-FU through down-regulation of p53 and its target gene p21 and up-regulation of β-catenin and its target genes CD44 and Lgr5.

Conclusion: Our findings suggest that differential expression of secretory mucin MUC5AC results in enhanced tumorigenesis and also confers chemoresistance via CD44/β-catenin/p53/p21 signaling.

Keywords: 5′-fluorouracil; CD44; Chemoresistance; Colon cancer; Colorectal cancer; MUC5AC; Mucins; β-Catenin.

Fig. 1

Elevated expression of MUC5AC in colorectal cancer patients and cell lines. a Expression of MUC5AC in colorectal cancer (CRC) patients (N = 287) and normal (N = 41) cases was analyzed in The Cancer Genome Atlas Program (TCGA) colorectal adenocarcinoma (COAD) dataset. A statistically significant difference was observed between normal vs cancer (^***p < 0.001). b Immunohistochemistry staining of MUC5AC in a commercially available CRC tissue microarray (US Biomax, Rockville, United States). MUC5AC-positive staining is represented as a composite score. Representative images of differential MUC5AC staining in normal (N = 10), adenocarcinoma (N = 34, ^**p < 0.01), and metastatic adenocarcinoma (N = 20, ^**p < 0.01). c High expression of MUC5AC is associated with a decrease in overall and disease-free survival in CRC patients. d and e Quantitative real-time PCR and immunoblotting images showing decreased levels of MUC5AC expression in HCT-8 and LS174T cells. f Confocal images showing reduction in the MUC5AC expression in Sh5AC cell lines of both HCT-8 and LS174T by using MUC5AC primary antibody and stained with Alex Fluor secondary antibody (Red). Nuclear staining was carried out with DAPI (blue)

Fig. 2

MUC5AC-expressing colon cancer cells exhibit a higher percentage of stem cells or side population. a Schematic depicting the location of gRNAs (at exon 2) of CRISPR/Cas9 used for knocking out the MUC5AC gene. b and c Immunoblot showing deletion of MUC5AC in clones of CRC cell lines (HCT-8 and LS174T). d and e Side or stem cell population (SP) was isolated in the presence of an ATP-binding cassette inhibitor (verapamil). A higher SP percentage was observed in HCT-8 and LS174T parental cells as compared to MUC5AC knockout tumor cells (^*p < 0.05 and ^**p < 0.01)

Fig. 3

MUC5AC expression results in the maintenance of a stem-like phenotype in colon cancer cells. a Side or stem cell population (SP) cells were isolated based on Hoechst exclusion staining via FACS and expanded by culturing the cells in the specific stem cell growth media. Western blot analysis showed an increase in MUC5AC and CD44 levels in parental SP cells. b and c Morphology analyses of SP and non-side population (NSP) of parental and KO clones isolated from HCT-8 and LS174T cells. d and e Formation of colon spheres/tumor spheres in parental and KO clones (Clone-1 and -2) from HCT-8 and LS174T after 10 days. A significant decrease in spheroid formation was observed in KO clones as compared to parental cells (*p < 0.05)

Fig. 4

MUC5AC physically interacts with CD44 to accelerate cellular signaling via Src. a Schematic representation of conditioned media (CM) collected from parental and KO clones (Clone-1 and -2) of HCT-8 and LS174T cell lines. b MUC5AC concentration is measured before or after CM preparation by ELISA. c and d Immunoblotting of CD44 from KO clones (Clone-1 and -2) treated with CM collected from parental cell lines (HCT-8 and LS174T) showed higher expression of CD44 in the presence of CM compared with untreated cells. No difference in CD44 expression was observed in KO clones supplemented with conditioned medium from KO clones. e Significantly higher expression of CD44 was observed in tumor patients (N = 275) compared with healthy controls (N = 41) in TCGA-COAD (^*p < 0.05). f MUC5AC and CD44 correlated positively in the TCGA-COAD dataset. g Immunoblot showed a physical interaction of MUC5AC-CD44 in CRC cell lines. h Both HCT-8 and LS174T cell lines co-stained with MUC5AC and CD44 antibodies. FITC-labelled and Alexa Fluor-labelled secondary antibodies were used for MUC5AC (Green) and CD44 (Red) staining, respectively. DAPI was used for nuclei staining. i Interaction of MUC5AC with CD44 mediates activation of various down-stream signaling molecules assessed in whole cell lysates isolated from parental and KO clones (Clone-1 and -2) of HCT-8. j Transient knock-down of CD44 altered its down-stream signaling molecules

Fig. 5

Decreased tumor burden in mouse model injected subcutaneously with MUC5AC knockdown cell line. a Schematic representation of subcutaneous injection of HCT-8 scrambled (Scr) and MUC5AC knockdown (Sh5AC) cells in athymic nude mice. b and c Mice injected with Sh5AC cell lines exhibited decreased tumor volume and weight as compared to Scr animals. d Immunoblotting of various signaling molecules associated with MUC5AC-CD44 interaction in subcutaneous tumor tissue lysate. e Expression of transmembrane mucin MUC4 was lower in colon cancer patients compared with healthy controls. f Correlation between MUC4 and MUC5AC mRNA expression in colon cancer patients (TCGA-COAD dataset). g and h In vitro knockdown and knockout of MUC5AC showed an increase in MUC4 expression (analyzed by anti-MUC4 8G7 antibody). Tumor tissues from mice subcutaneously injected with Sh5AC cells also expressed higher MUC4 as compared to Scr group

Fig. 6

MUC5AC expression increases tumor burden in orthotopic mouse model. a Schematic representation of colon orthotopic mouse model. Approximately 2 × 10⁶ cells (HCT-8 parental and MUC5AC knockout, labelled with luciferase) were injected in the serosa of the caecal region. b and c In vivo tumor growth was imaged using the IVIS imaging system after intraperitoneal injection of D-luciferin (150 mg/kg) at days 20 and 50. Photon counts from both parental and KO clones were quantified 20 and 50 days post-implantation. d Representative hematoxylin and eosin stain of parental and KO cells injected orthotopically in athymic nude mice. Higher tumor burden was observed in the colon of mice injected with parental cells

Fig. 7

Presence of MUC5AC mediates resistance to 5-FU. a 5-FU treatment at 1 and 5 μM for 48 h increased expression of MUC5AC in HCT-8 and LS174T cell lines. b Similarly, in the presence of oxaliplatin both cell lines showed upregulation of MUC5AC expression. c DNA content in different phases of the cell cycle was assessed by propidium iodide staining of parental and MUC5AC knockout clones (Clone-1 and -2) followed by FACS analysis. Cell cycle analysis showed that in the absence of MUC5AC, cells were arrested at G2/M phase. d The presence of MUC5AC facilitates 5-FU resistance via the β-catenin/p53/p21 axis and via upregulation of β-catenin target genes Lgr5 and CD44. e Schematic diagram showing MUC5AC mediates CRC cell survival, migration, and invasion and confers 5-FU resistance via Src and β-catenin signaling, respectively