Involvement of CXCR4 chemokine receptor in metastastic HER2-positive esophageal cancer

Abstract

A functional linkage of the structurally unrelated receptors HER2 and CXCR4 has been suggested for breast cancer but has not been evaluated for esophageal carcinoma. The inhibition of HER2 leads to a reduction of primary tumor growth and metastases in an orthotopic model of esophageal carcinoma. The chemokine receptor CXCR4 has been implicated in metastatic dissemination of various tumors and correlates with poor survival in esophageal carcinoma. The aim of this study was to investigate a correlation between the expression levels of HER2 and CXCR4 and to evaluate the involvement of CXCR4-expression in HER2-positive esophageal carcinoma. The effects of HER2-inhibition with trastuzumab and of CXCR4-inhibition with AMD3100 on primary tumor growth, metastatic homing, and receptor expression were evaluated in vitro and in an orthotopic model of metastatic esophageal carcinoma using MRI for imaging. The clinical relevance of HER2- and CXCR4-expression was examined in esophageal carcinoma patients. A significant correlation of HER2- and CXCR4-expression in primary tumor and metastases exists in the orthotopic model. Trastuzumab and AMD3100 treatment led to a significant reduction of primary tumor growth, metastases and micrometastases. HER2-expression was significantly elevated under AMD3100 treatment in the primary tumor and particularly in the metastases. The positive correlation between HER2- and CXCR4-expression was validated in esophageal cancer patients. The correlation of CXCR4- and HER2-expression and the elevation of HER2-expression and reduction of metastases through CXCR4-inhibition suggest a possible functional linkage and a role in tumor dissemination in HER2-positive esophageal carcinoma.

Figure 1

A Effect of trastuzumab and ADM3100 treatment on proliferation (%) of OE19 cells compared to control in the lactate-dehydrogenase assay. Receptor inhibition leads to reduced proliferation of cells. It shows a significant reduction of cell proliferation under HER2- and CXCR4-receptor inhibition after treatment with trastuzumab (p = 0.005) as well as with AMD3100 (p = 0.02) compared to the untreated control. B Microscopic evaluation shows dose-dependent effect of SDF-1α-stimulated cell migration on OE19 cells. C A relevant effect of SDF-1α on cell migration is observed at 250 ng/ml compared to unstimulated cells (control).

Figure 2

A Significant differences in tumor weights between control and trastuzumab-treated groups (p<0.00), control and combination trastuzumab/AMD3100-treated groups (p<0.00), trastuzumab and AMD3100-treated groups (p = 0.04), and AMD and combination trastuzumab/AMD3100-treated groups (p = 0.02). Although the effect of AMD3100 on the primary tumor weight was not as relevant as the effect of trastuzumab, a potent effect was achieved by AMD3100 treatment alone, compared to the untreated group. B MRI-based tumor volumetry confirmed the results of tumor weight. The tumor weights at time of autopsy correlated significantly with the volumetric measure by MRI (correlation coefficient: 0.837, p<0.01). C Micrometastases in liver and lung after treatment with AMD3100 and trastuzumab, were analysed by real-time PCR according to the level of human gapdh. AMD3100 and trastuzumab-treated mice showed with a mean delta-ct-value of −2 and −3 strong reductions in lung metastasis of 75 to nearly 100%. Additionally the trastuzumab-treated mice had a strong reduction in liver metastasis represented by a mean delta-ct-value of −3. The AMD3100/trastuzumab combination group had a reduced rate of lung (delta-ct −2), and liver (delta-ct −3) metastasis. D Disseminated tumor cells were detected by cytokeratin and HER2 immunhistochemical staining. Figure 3B shows a bone marrow sample. Human cell with a strong positivity for HER2 is detectable (red). * Due to space limitations, AMD3100 was abbreviated to AMD in Figures 2a and c .

Figure 3

A CXCR4-expression of OE19 cells determined by fluorescence immunostaining (IgG1-control) B Confirmation of Her2-amplification determined by fluorescence in situ hybridization (red: Her2-gene loci, green reference CENT-17-loci) C CXCR4 and HER-2 mRNA-expression analysis of esophageal cancer cell line OE19 compared to MDA-MB-231 and SKBr-3 cell lines and null control (nc). D CXCR4 and HER2 expression level analysis determined by immunostaining in primary tumor, liver, lung and lymph node. Representative images are shown from the tissues of an untreated animal (magnification ×100). E Intensity of HER2- and CXCR4-expression was scored in primary tumor and metastases. Positivity-scores of primary tumor and respective metastases were matched to evaluate the occurrence of and correlation of primary tumor expression and that of its respective metastases between the therapeutic groups. Trastuzumab treatment led to an absence of metastases and thus could not be included. * Due to space limitations, AMD3100 was abbreviated to AMD in Figure 3e .