Integrin α4 Enhances Metastasis and May Be Associated with Poor Prognosis in MYCN-low Neuroblastoma

Abstract

High-risk neuroblastoma is associated with an overall survival rate of 30-50%. Neuroblastoma-expressed cell adhesion receptors of the integrin family impact cell adhesion, migration, proliferation and survival. Integrin α4 is essential for neural crest cell motility during development, is highly expressed on leukocytes, and is critical for transendothelial migration. Thus, cancer cells that express this receptor may exhibit increased metastatic potential. We show that α4 expression in human and murine neuroblastoma cell lines selectively enhances in vitro interaction with the alternatively spliced connecting segment 1 of fibronectin, as well as vascular cell adhesion molecule-1 and increases migration. Integrin α4 expression enhanced experimental metastasis in a syngeneic tumor model, reconstituting a pattern of organ involvement similar to that seen in patients. Accordingly, antagonism of integrin α4 blocked metastasis, suggesting adhesive function of the integrin is required. However, adhesive function was not sufficient, as mutants of integrin α4 that conserved the matrix-adhesive and promigratory function in vitro were compromised in their metastatic capacity in vivo. Clinically, integrin α4 is more frequently expressed in non-MYNC amplified tumors, and is selectively associated with poor prognosis in this subset of disease. These results reveal an unexpected role for integrin α4 in neuroblastoma dissemination and identify α4 as a potential prognostic indicator and therapeutic target.

Fig 1. Integrin α4 expression may be associated with worse prognosis in MYCN^low NB.

Twenty-eight human primary neuroblastoma samples were stained for integrin α4 (HRP). Serial sections of α4 positive samples were stained for CD45. (A) Example of NB patient samples with tumor cell and leukocyte α4 staining (N13) or leukocyte α4 only (N73) Black arrows indicate α4-positive tumor cells (CD45^-). (B) Integrin α4 status of human NB samples. (C) For the 28 samples analyzed, patient status was stratified by MYCN amplification and integrin α4 expression then grouped according to their last known disease status. (D) Kaplan-Meier curve of relapse-free survival for non-MYCN amplified patients with high or low α4 gene expression (raw p = 4.4 x 10^–5; bonf. p = 3.8 x 10^–3). Analysis was performed using the Seeger dataset from the R2: Genomic analysis and visualization platform.

Fig 2. Integrin α4 promotes human NB cell adhesion and migration.

(A) Map of the cytoplasmic domain of the chimeric integrin α4-gfp fusion protein. (B) Flow cytometry analysis of NB5 cells stably expressing a full-length integrin α4-GFP fusion construct (shaded peak) or an eGFP control vector (open peak) (α4 antibody; P1H4). (C) Attachment to 5 ug/ml GST-CS1 FN (p<0.00001) or plasma FN (30 minutes). (D) Transwell haptotaxis to 5 ug/ml GST-CS1 FN for 3 hours (p<0.001). (E) Haptotaxis to GST-CS1 FN of C1300 eGFP and α4-GFP cells pre-treated with 10 ug/ml of antibodies against integrins α4 (P1H4) (p<0.01), β1 (P4C10) (p<0.05) or β5 (P1F6). (F) Scratch wound healing on 5 ug/ml pFN. (G) Quantification of wound closure in E (p<0.001). (H) Proliferation (by live cell count) over 6 days. Values for wound healing migration are means ± s.d. (n = 6) for a representative experiment (of three).

Fig 3. Integrin α4 promotes mouse NB cell adhesion and migration.

(A) Flow cytometry analysis of C1300 cells stably expressing a full-length integrin α4-GFP fusion construct (shaded peak) or an eGFP control vector (open peak) (α4 antibody; P1H4). (B) Adhesion of cells that were untreated (p<0.001) or pre-treated with an anti-α4 antibody (P1H4) to 2 ug/ml recombinant mouse (rm) VCAM-1 after 30 minutes (p<0.001). (C) Haptotaxis to 2 ug/ml rm-VCAM-1 after 3 hours (p<0.01). (D) Proliferation (by live cell count) over 6 days.

Fig 4. Integrin α4 does not drive NB tumor growth.

(A) Soft agar colony growth of C1300 eGFP or α4-gfp cells after 4 days. (B) C1300 eGFP or α4-GFP cells were injected subcutaneously (SQ) into 8-week-old A/J mice (n = 7). Tumors were harvested and weighed 11 days later. Each filled circle represents one mouse. Gray lines denote the means of each group. (C) C1300 eGFP or α4-GFP cells were injected into the adrenal gland (orthotopic site) of 8-week-old A/J mice (n = 10 eGFP, 8 α4-GFP). After 2 weeks, tissues were harvested and examined for macroscopic tumors.

Fig 5. Integrin α4 promotes NB metastasis.

(A) C1300 egfp or α4-gfp cells were injected into the tail vein of 8-week-old A/J mice. After 18–24 days, tissues were harvested and examined for macroscopic lesions. (B) The number of macroscopic metastatic liver lesions was assessed by counting after Bouin’s staining (n = 10, p<0.05). Gray lines denote the means of each group. (C) Images of Bouin’s, hematoxylin and eosin, or human integrin α4 stained tissues.

Fig 6. The α4 cytoplasmic tail is dispensible for NB adhesion and migration in vitro.

(A) Sequences of full-length and truncated α4 cytoplasmic domains. (B) Flow cytometry analysis of C1300 cells stably expressing full-length or truncated α4 and α4-GFP fusion protein (α4 antibody; P1H4) (shaded peaks) compared to secondary only control (open peaks). C1300 cell adhesion (C) and haptotaxis (D) to 2 ug/ml rmVCAM-1. (C) GFP-fusion restores C1300 cell adhesion to wildtype levels (Δcyto vs. Δcyto-GFP, p<0.01). (E) C1300 cell proliferation over 5 days. (F) C1300 colony formation in soft agar after 4 days. Values for proliferation are means ± s.d. (n = 3) of a representative experiment (of two). Values for the soft agar growth assay are the means ± s.d. (n = 6) of a representative experiment.

Fig 7. The cytoplasmic tail is important for α4-mediated NB metastasis.

(A) Number of metastatic liver lesions in mice receiving tail vein injection of C1300 α4-gfp cells pre-treated with 10 ug/ml anti-α4 antibody (P1H4) or control IgG. Tissues were harvested and examined for metastases after 21 days (n = 4, p<0.05). 1 x 10⁶ C1300 full-length or truncated α4 cells were injected into the tail vein of 8-week-old A/J mice (n = 8 α4-GFP, 6 Δcyto-GFP, 4 Δcyto). Tissues were harvested after 21 days. (B) The number of macroscopic metastatic lesions in tissues harvested. (C) Number of macroscopic liver lesions (p<0.01). Gray lines denote the mean of each group (A, C).