NMR metabolomics of MTLn3E breast cancer cells identifies a role for CXCR4 in lipid and choline regulation.

The alpha chemokine receptor CXCR4 is up-regulated in certain types of breast cancer. Truncation of the C-terminus of this receptor alters cell morphology and increases invasiveness and metastatic potential. Here, to better understand the effects of CXCR4 expression and truncation in breast cancer cells, we have used high resolution magic angle spinning (HR-MAS) NMR studies of rat breast carcinoma MtLn3E cells to characterise the metabolite complement of cells heterologously expressing human CXCR4 or its C-terminal truncation mutant, Δ34-CXCR4. Notable reductions in choline levels were detected when either cells expressing wild-type CXCR4 or Δ34-CXCR4 were compared with cells containing an empty expression vector. Cells expressing CXCR4-Δ34 had reduced lipid content when compared with either the wild-type CXCR4 expressing cells or those containing the empty expression vector. Taken together, our results show that distinct effects on the metabolite complement can be linked to either CXCR4 expression or CXCR4 regulation. The metabolite markers for these two effects identified in the present study can, in turn, be used to further investigate the role of CXCR4 in metastasis. Keywords: NMR metabolomics, High resolution magic angle spinning (HR-MAS), CXCR4, breast cancer cells Introduction The 352 amino acid CXC chemokine receptor type 4 (CXCR4) selectively binds the CXC chemokine stromal cell-derived factor-1 (SDF-1) also known as CXCL12. This G-protein coupled receptor (GPCR) is implicated in a variety of human diseases; it serves as a coreceptor for entry of T-tropic HIV viruses that target CD4-positive T-cells,1,2 was found to be expressed in >20 types of human cancers3,4 and has been linked to enhanced cancer cell motility and metastasis. In particular, an important role for CXCR4 has been demonstrated in breast cancer metastasis where signalling through CXCR4 leads to actin polymerization, formation of pseudopodia, chemotaxis, and increased metastasis in cancer cells.5 The C-terminus of CXCR4 is crucial to this activity and expression of a C-terminal truncation mutant in MCF-7 cells was shown to lead to altered cell morphology, higher growth rate, and increased cell motility, when compared with wild type CXCR4 or vector transduced cells.6 Recent results also show that the truncation of the C-terminus of CXCR4 leads to increased invasive potential of mammary carcinoma cells in vitro in a MatriGel invasion assay and increased metastatic potential in vivo in a murine model of spontaneous breast cancer metastasis.7 This behaviour is likely to be linked to the substantially reduced receptor internalization and hence degradation observed in cells expressing the truncated CXCR4.7 Although CXCR4 truncation mutations have been found to play an important role in Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome,8,9 as yet, no such deletions have been observed in cancer. Nevertheless, different CXCR4 mutations have been reported in a number of cancers and are implicated in promoting CXCR4 persistence at the cell surface and/or conferring migratory advances to the cell lines established from these cancers.10-12 Magnetic resonance spectroscopy (MRS) has been shown to have potential in providing information about tumour phenotypes in human samples in vivo. For instance, it has been used to distinguish tumour tissue from non-lactating human breast tissue13-15 and for the evaluation of human breast lesions. Furthermore, the composite total choline resonance in proton spectra was used to distinguish malign from benign tissue17,18 and for monitoring the success of neoadjuvant chemotherapy in locally advanced human breast cancer19. In order to gain insight into the metabolic changes associated with increased CXCR4 expression or truncation of its C-terminus, we have characterized the changes in the small molecule complement of a breast cancer cell model designed to distinguish CXCR4-induced changes and truncation-induced alterations. Specifically, we applied high resolution magic angle spinning (HR-MAS) NMR techniques to lyophilised and rehydrated rat mammary adenocarcinoma MTLn3E cells, either transduced with an empty vector or stably expressing either wildtype or C-terminally truncated CXCR4.