Mucin 5AC Promotes Breast Cancer Brain Metastasis through cMET/CD44v6

Abstract

Purpose: Breast cancer brain metastasis remains a significant clinical problem. Mucins have been implicated in metastasis; however, whether they are also involved in breast cancer brain metastasis remains unknown. We queried databases of patients with brain metastasis and found mucin 5AC (MUC5AC) to be upregulated and therefore sought to define the role of MUC5AC in breast cancer brain metastasis.

Experimental design: In silico dataset analysis, RNA-sequence profiling of patient samples and cell lines, analysis of patient serum samples, and in vitro/in vivo knockdown experiments were performed to determine the function of MUC5AC in breast cancer brain metastasis. Coimmunoprecipitation was used to unravel the interactions that can be therapeutically targeted.

Results: Global in silico transcriptomic analysis showed that MUC5AC is significantly higher in patients with breast cancer brain metastasis. Analysis of archived breast cancer brain metastasis tissue further revealed significantly higher expression of MUC5AC in all breast cancer subtypes, and high MUC5AC expression predicted poor survival in HER2+ breast cancer brain metastasis. We validated these observations in breast cancer brain metastatic cell lines and tissue samples. Interestingly, elevated levels of MUC5AC were detected in the sera of patients with breast cancer brain metastasis. MUC5AC silencing in breast cancer brain metastatic cells reduced their migration and adhesion in vitro and in brain metastasis in the intracardiac injection mouse model. We found high expression of cMET and CD44v6 in breast cancer brain metastasis, which increased MUC5AC expression via hepatocyte growth factor signaling. In addition, MUC5AC interacts with cMET and CD44v6, suggesting that MUC5AC promotes breast cancer brain metastasis via the cMET/CD44v6 axis. Inhibition of the MUC5AC/cMET/CD44v6 axis with the blood-brain barrier-permeable cMET inhibitor bozitinib (PLB1001) effectively inhibits breast cancer brain metastasis.

Conclusions: Our study establishes that the MUC5AC/cMET/CD44v6 axis is critical for breast cancer brain metastasis, and blocking this axis will be a novel therapeutic approach for breast cancer brain metastasis.

Figure 1.. MUC5AC expression in breast cancer brain metastasis:

A. Comparison of mucin expression between BC and brain metastasis samples, using online available Gene Expression Omnibus (GEO) public datasets (GSE184869). B. Volcano plot showing the differential expression of different genes in BC and BCBrM cell lines (MDA-231BR vs MDA-231). The blue dots show downregulated and red dots show upregulated genes. C. Representative IHC of normal breast, invasive ductal carcinoma (IDC), invasive ductal carcinoma with HER2⁺, lymph node metastasis and breast cancer brain metastasis TMA slides for MUC5AC and D. H-score quantification of MUC5AC expression in different cohorts of breast cancer (BC) patients. E. Patient serum data shows the expression of MUC5AC in healthy control and different cohorts of BC with and without CNS metastasis. F. MUC5AC expression in different metastatic cell lines derived from lung, brain, and bone. Data presented as mean ± SD from experiments done in triplicate: * (p≤0.05), ** (p≤0.01), *** (p≤0.001), **** (p≤0.0001). Scale bar: 100μm.

Figure 2.. MUC5AC expression in HER2⁺ BC brain metastasis and its impact on prognosis.

A. Aperio Digital Imaging was utilized for reading the MUC5AC expression in BCBrM tissues and, strong staining scores in the tumor regions were considered as positive. B and C. Graphs show % MUC5AC positive tumor area for a cohort of BCBrM (n=50), HER2⁺ (n=26) and TNBC (n=24) subtypes. Data represented mean with 95% IC. D. Percent survival for BCBrM patients (n=47) based on tumor MUC5AC expression levels (long-rank test (P=0.0779)). E. Percent survival for HER2⁺ BM patients (n=23) based on tumor MUC5AC expression levels (long-rank test (P=0.0251)). F. Percent survival for TNBC BM patients (n=24) based on tumor MUC5AC expression levels (long-rank test (P=0.9544)). G. Fluorescence microscopy images showed the expression of MUC5AC in human HER2⁺ and TNBC BM-PDXs. H. Representative Western blot shows MUC5AC expression in human BC (parent), BCBrM and HER2 overexpressing BR cells.

Figure 3.. MUC5AC silencing in BCBrM cells reduces brain metastasis in vitro and in vivo models.

A. Western blot showing knockdown of MUC5AC in the MDA-231BR HER2 cells. Representative images (B) and quantification (C) of trans-endothelial cell migration of MDA-231BR HER2 shSCR and shMUC5AC cells. Representative fluorescence microscopy images of brain slices (organotypic culture) (D) and quantification (E) showing GFP⁺ MDA-231BR shSCR and shMUC5AC invasion into the brain tissues. Representative images (F) and quantification (G) of adhesion assay in control (shSCR) and shMUC5AC MDA-231BR HER2 cells using fluorescence and brightfield microscopy, respectively. H. IVIS images of nude mice intracardially injected with shSCR or shMUC5AC MDA-231BR HER2 cells (1×10⁵ in 100μl PBS) expressing luciferase at day 0 and day 58 end of the experiment with ex-vivo images of the brain showing brain metastasis. Athymic nude mice were injected with luciferin (150 mg/kg in sterile PBS) 5min before euthanasia; following IVIS imaging and collection of the brain with metastatic lesions. I. Quantified luminescence ratio on day 58, MDA 231BR HER2⁺ shSCR vs shMUC5AC (*, P < 0.05). J. H&E staining of the brain tissue samples obtained after injecting mouse with shSCR and shMUC5AC MDA-231BR HER2 cells. Data presented as mean ± SD from experiments done in triplicate; * (p≤0.05), ** (p≤0.01), *** (p≤0.001), **** (p≤0.0001). Scale bar: 100μm.

Figure 4.. MUC5AC and associated signaling.

A. Western blot of cMET in MDA-231P, MDA-231BR, MDA-231BR HER2. B. Representative IHC image showing the expression of cMET in human BM-PDXs. C. Western blot images show CD44v6 in MDA-231P, MDA-231BR, MDA-231BR HER2. D. Representative IHC image showing the expression of CD44v6 in human BM-PDXs. E and F. Representative fluorescence microscopy images showing co-localization of cMET and MUC5AC (E), CD44v6 and MUC5AC (F) in BM-PDXs. G and H. Immunoprecipitation of 5AC (MUC5AC) and probed for cMET and CD44v6 shows specific interaction of 5AC (MUC5AC) with cMET and CD44v6, respectively. I and J. Immunoprecipitation of cMET and probed for CD44v6 (I) and immunoprecipitation of CD44v6 and probed for cMET (J) to show interaction between cMET and CD44v6. Scale bar: 100μm.

Figure 5.. PLB1001 inhibits cMET and MUC5AC expression.

A. Cell viability profile of PLB1001 for MDA-231BR HER2 and HCC1954BR cell lines. B-D. Western blots data showing p-cMET, cMET, CD44v6, and MUC5AC expression following PLB1001 treatment (10 μM and 20μM as indicated) in HCC1954BR cells (B) and MDA-231BR HER2 (C and D). E. Representative fluorescence microscopy images of MUC5AC (red) and p-cMET (green) expression in MDA-231BR HER2 cells upon PLB1001 treatment (20μM). Representative images (F) and quantification (G) of wound healing assay of vehicle and PLB1001 treated MDA-231BR HER2 cells. Representative images (H) and quantification (I) of trans-endothelial cell migration assay of vehicle and PLB1001 treated MDA-231BR HER2 cells. J. Western blot images for MUC5AC in MDA-231BR HER2 and HCC1954BR treated with indicated HGF concentration with or without PLB1001 (20μM) for 48h. Data presented as mean ± SD from experiments done in triplicate; *** (p≤0.001), **** (p≤0.0001).

Figure 6.. Targeting MUC5AC-cMET axis in-vivo in brain metastatic PDXs.

A. Schematic diagram and timeline of procedure to determine the effect of PLB1001 in BrM PDXs using orthotopic and intracardiac models. A piece of G5–3 GFP⁺ PDX tumor (HER2⁺, MUC5AC⁺) was implanted either in the 4th mammary fat pad (MFP) of 20 NSG female mice (14–18 weeks old) or injected intracardially in 20 NSG female mice (11–13 weeks old), with estrogen supplementation (E2-pellet, 1mg). Treatment was initiated when the tumor reached a volume of about 100 mm³ in the MFP model. Treatments vehicle (DMSO, n=10) vs. PLB1001 (50mg/kg, n=10) were administered by oral gavage in 200μl of normal saline solution from Monday to Friday for 4 weeks at the same time (1pm), tumor volumes were measured daily. One week after finishing the treatments, the animals were euthanized with an overdose of Ketamine/Xylazine and perfused with PBS/4% PFA. Brains, livers, and lungs ex-vivo were analyzed under the microscope to find GFP⁺ metastases, and the tumors were dissected and weighed. Animals with ulcerated tumors or tumor volumes greater than 2cm³ were euthanized according to our animal protocol. In intracardiac model, female NSG mice were randomized into two groups: Vehicle (DMSO) and PLB1001 (50mg/kg), treatments were administered by oral gavage as described above. Two weeks after last treatment, mice were euthanized. Brain was harvested and H&E was performed. Created in BioRender. Khan, P. (2024) https://BioRender.com/c23y938. B. Tumor volume (mm³) of G5–3 GFP⁺ PDX (HER2⁺, MUC5CA⁺) primary tumors in NSG female mice treated with either Vehicle (DMSO, n=10) or PLB1001 (50 mg/kg, n=10) for 4 weeks. C. Tumor weight of G5–3 GFP⁺ PDX (HER2⁺, MUC5CA⁺) primary tumors at the endpoint. D. Representative image of ex-vivo G5–3 GFP⁺ PDX (HER2⁺, MUC5CA⁺) primary tumors under a stereomicroscope at the endpoint, one week after the 4-week treatment period in NSG female mice. Representative H&E images (E) and percentage (F) of mice showing leptomeningeal and brain metastasis in G5–3 PDX treated with either vehicle or PLB1001 (50mg/kg). G. Schematic representation of the role of MUC5AC/CD44v6/cMET axis in the BCBrM process. Created in BioRender. Khan, P. (2024) https://BioRender.com/w65d274. Blue arrow indicates the time point where the differences between treatments start to become significant (p<0.05). Data were analyzed by 2-way ANOVA with uncorrected Fisher’s LSD and are presented as mean ± SEM. Data were analyzed using an unpaired t-test and are presented as mean ± SEM. Scale bar: 100μm.