Wnt/β-Catenin Signaling Regulates CXCR4 Expression and [68Ga] Pentixafor Internalization in Neuroendocrine Tumor Cells

Abstract

Loss of Somatostatin Receptor 2 (SSTR2) expression and rising CXC Chemokine Receptor Type 4 (CXCR4) expression are associated with dedifferentiation in neuroendocrine tumors (NET). In NET, CXCR4 expression is associated with enhanced metastatic and invasive potential and worse prognosis but might be a theragnostic target. Likewise, activation of Wnt/β-catenin signaling may promote a more aggressive phenotype in NET. We hypothesized an interaction of the Wnt/β-catenin pathway with CXCR4 expression and function in NET. The NET cell lines BON-1, QGP-1, and MS-18 were exposed to Wnt inhibitors (5-aza-CdR, quercetin, and niclosamide) or the Wnt activator LiCl. The expressions of Wnt pathway genes and of CXCR4 were studied by qRT-PCR, Western blot, and immunohistochemistry. The effects of Wnt modulators on uptake of the CXCR4 ligand [⁶⁸Ga] Pentixafor were measured. The Wnt activator LiCl induced upregulation of CXCR4 and Wnt target gene expression. Treatment with the Wnt inhibitors had opposite effects. LiCl significantly increased [⁶⁸Ga] Pentixafor uptake, while treatment with Wnt inhibitors decreased radiopeptide uptake. Wnt pathway modulation influences CXCR4 expression and function in NET cell lines. Wnt modulation might be a tool to enhance the efficacy of CXCR4-directed therapies in NET or to inhibit CXCR4-dependent proliferative signaling. The underlying mechanisms for the interaction of the Wnt pathway with CXCR4 expression and function have yet to be clarified.

Keywords: BON-1; CXCR4; MS-18; NET; QGP-1; Wnt; [68Ga] Pentixafor; neuroendocrine tumor; β-catenin.

Figure 1

mRNA expression of ß-Catenin, TCF7, MYC, and CCDN1 (Cyclin D1) after treatment with (a) 5-aza CdR; (b) quercetin; (c) niclosamide; and (d) LiCl in BON-1, QGP-1, and MS-18 cells (logarithmic scale). The data are shown as percentages of untreated control cells. The expression levels were normalized to housekeeping gene GAPDH (mean ± SD; n = 6 experimental repeats; * p < 0.05, ** p < 0.01).

Figure 2

mRNA expression of CXCR4 after treatment with Wnt modulators in BON-1, QGP-1, and MS-18 cells (logarithmic scale). The data are shown as percentages of untreated control cells. The expression levels were normalized to housekeeping gene GAPDH (mean ± SD; n = 6 experimental repeats; * p < 0.05, ** p < 0.01).

Figure 3

Immunoblot staining of CXCR4 in BON-1, QGP-1, and MS-18 either untreated (control) or after incubation with Wnt modulators. ß-Actin is exposed as the loading control.

Figure 4

Immunohistochemistry staining of CXCR4 in BON-1, QGP-1, and MS-18 cells either before (control) or after incubation with Wnt modulators. The bar in the images represents 75 μm. The percentage of CXCR4-positive cells after immunohistochemical staining in BON-1, QGP-1, and MS-18 after Wnt modulator treatment is shown in Supplementary Materials Figure S2.

Figure 5

Specific uptake of radiolabeled [Ga68] Pentixafor in BON-1, QGP-1, and MS-18 cells after incubation with Wnt modulators. The data are presented as percentages of untreated controls (mean ± SD; n = 3 experimental repeats; * p < 0.05, ** p < 0.01, and *** p < 0.001).