Activation of VCAM-1 and its associated molecule CD44 leads to increased malignant potential of breast cancer cells

Abstract

VCAM-1 (CD106), a transmembrane glycoprotein, was first reported to play an important role in leukocyte adhesion, leukocyte transendothelial migration and cell activation by binding to integrin VLA-1 (α4β1). In the present study, we observed that VCAM-1 expression can be induced in many breast cancer epithelial cells by cytokine stimulation in vitro and its up-regulation directly correlated with advanced clinical breast cancer stage. We found that VCAM-1 over-expression in the NMuMG breast epithelial cells controls the epithelial and mesenchymal transition (EMT) program to increase cell motility rates and promote chemoresistance to doxorubicin and cisplatin in vitro. Conversely, in the established MDAMB231 metastatic breast cancer cell line, we confirmed that knockdown of endogenous VCAM-1 expression reduced cell proliferation and inhibited TGFβ1 or IL-6 mediated cell migration, and increased chemosensitivity. Furthermore, we demonstrated that knockdown of endogenous VCAM-1 expression in MDAMB231 cells reduced tumor formation in a SCID xenograft mouse model. Signaling studies showed that VCAM-1 physically associates with CD44 and enhances CD44 and ABCG2 expression. Our findings uncover the possible mechanism of VCAM-1 activation facilitating breast cancer progression, and suggest that targeting VCAM-1 is an attractive strategy for therapeutic intervention.

Figure 1.

(A) RT-qPCR analysis of VCAM-1 expression in breast tumor tissues (tumor stage 1 + 2 AB and 3) and noncancerous controls; (B) a,b, Representative IHC staining of VCAM-1 in normal breast tissues; c–f, VCAM-1 expression in human breast tumor tissues. The magnifications are indicated.

Figure 2.

(A) RT-qPCR analysis for VCAM-1 mRNA expression in a, NMuMG and b, MDAMB231 cells after treatment with control (1× PBS), TGFβ (5 ng/mL), TNFα (10 ng/mL), or IL-6 (1 ng/mL) overnight. Columns, mean of triplicate samples; bars, SE. * p < 0.01; (B) VCAM-1 protein expression in a, NMuMG and b, MDAMB231 cells by western blotting; (C) IF staining for VCAM-1 expression in MDAMB231 cells with or without cytokine treatments. DNA was visualized with DAPI staining.

Figure 3.

(A) Western blot VCAM-1 protein expression in NMuMG cells transfected with the pBabe-eGFP or the pBabe-VCAM-1 plasmid. β-actin results indicate similar sample loads; (B) a, Transwell migration analysis of NMuMG cells (control and VCAM-1 over-expression) towards 10% FBS + insulin. Photo images are representative fields of the migration of VCAM-1 over-expressing cells and the control group after crystal violet dye staining; b, The cell numbers of migrated NMuMG (eGFP control) and VCAM-1 NMuMG cells from the upper insert to the bottom surface of the lower well under a light micropore (100×) after overnight incubation; c, Migrated NMuMG VCAM-1 cells in the lower wells showed high clonogenic ability after 2 weeks of incubation; (C) EMT-related protein expression in eGFP control and VCAM-1 over-expressing NMuMG cells was determined by Western blot analysis.

Figure 4.

(A) a, Western blot analysis of MDAMB231 VCAM-1 siRNA cells showed reduced VCAM-1 expression after IL-6 treatment compared with control cells; b, VCAM-1 siRNA knockdown blocks TGF-β1 or IL6-induced MDAMB231 cell migration and colony formation. The lower chamber was filled with 2 mL regular medium (normal) or contained TGFβ1 (5 ng/mL) or IL-6 (1 ng/mL) as a chemoattractant; c, Representative photos showed the influence of VCAM-1 siRNA on the number of colony-forming MDAMB231 cells as evaluated by clonogenic assay; (B) The inhibition of migration and colony formation by VCAM-1 knockdown was confirmed by shRNA knockdown stable clones. a,b, similar results confirmed the influence of VCAM-1 shRNA knockdown on the migratory ability and number of colony-forming MDAMB231 cells. Columns, mean of triplicate samples; bars, SE; p-value was determined by student’s t-test (* p < 0.01).

Figure 5.

(A) The level of VCAM-1 protein expression in stable transfection clones was determined by western blotting; (B) Tumors were established using MDAMB231 eGFP and VCAM-1 shRNA knockdown clones implanted by s.c. injection and analyzed after eight weeks. Western blot analysis was used to confirm VCAM-1 knockdown efficacy in xenograft tumors (upper insert pictures); (C) Tumor weights were measured after autopsy and tumor volumes were calculated using the formula 4/3πr₁²r₂ (r₁ < r₂) as described in Materials and Methods. Mean ± SE (n = 6). * p < 0.01.

Figure 6.

(A) VCAM-1 over-expression contributes to increased chemoresistance in a, NMuMG and b, MDAMB231 cells by MTT analysis. Samples were analyzed after 48 h of drug treatment, * p < 0.01; (B) Increased chemoresistance markers and VCAM-1 expression in NMuMG cells after long-term exposure to low-dose doxorubicin. Relative mRNA levels are expressed as arbitrary units normalized to the data from controls, * p < 0.01; (C) VCAM-1 regulates migration and chemoresistance-related genes in NMuMG cells as determined by RT-qPCR, * p < 0.01; (D) Over-expression of VCAM-1 increases CD44 and ABCG2 protein levels, and VCAM-1 physically interacts with CD44 molecules in control and VCAM-1-over-expressing NMuMG cells as determined by western blot and immunoprecipitation analyses.