<div>Abstract<p>Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF <i>in vivo</i> were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability <i>in vivo</i> than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.</p></div>
Abstract Altered choline phospholipid metabolism in breast cancers provides multiple targets for anticancer therapy. Malignant transformation of breast cancer cells results in a switch from high glycerophosphocholine (GPC) and low phosphocholine (PC) to low GPC and high PC. Glycerophosphocholine phosphodiesterase (E.C. 3.1.4.2; GPC-PDE) catalyzes the degradation of GPC to Cho and glycerol-3-phosphate. The GPC-PDE gene(s) responsible for the low GPC concentration in breast cancer cells have not yet been identified. Glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) is a GPC-PDE that is rapidly inhibited by NaCl and urea (NaCl/urea) in renal cells, and may be a candidate gene for GPC-PDE in breast cancer cells. We chemically inhibited GPC-PDE with NaCl/urea in nonmalignant MCF-12A, and malignant MCF-7 and MDA-MB-231 breast epithelial cell lines. We stably downregulated GDPD5 using short hairpin RNA against GDPD5 (GDPD5-shRNA) delivered by lentiviral transduction in MCF-7 breast cancer cells. Fully relaxed high-resolution 1H magnetic resonance spectroscopy (MRS) of cell extracts was performed on Bruker Avance 500 MR Spectrometer to quantify metabolites. Cell viability/proliferation was measured by WST-1 proliferation assay. Quantitative RT-PCR (qRT-PCR) detected significantly higher GDPD5 mRNA levels compared to the mRNA levels of GDPD1, 2, 3, and 4 in the respective cell line for MCF-12A, MCF-7, and MDA-MB-231 cells. GDPD5 levels were significantly higher in MDA-MB-231 compared to MCF-7 and MCF-12A cells. MRS metabolite quantification demonstrated that exposure of MCF-12A, MCF-7, and MDA-MB-231 cells to NaCl/urea (n=3), as well as transduction with GDPD5-shRNA in MCF-7 cells (n=2), significantly increased GPC and decreased PC, resulting in a decreased [PC]/[GPC] ratio. An increased [PC]/[GPC] ratio is associated with increased malignancy in breast cancer cell lines. We observed a switch from low GPC and high PC to high GPC and low PC following NaCl/urea treatment and following GDPD5-shRNA transduction. GDPD5 inhibition by NaCl/urea significantly decreased cell proliferation/viability in MCF-12A, MCF-7, and MDA-MB-231 cells. Inhibiting or down-regulating GDPD5 altered the choline phospholipid metabolite profile of breast cancer cells toward a less malignant metabolic profile. GDPD5 is at least partially responsible for the decreased GPC levels in breast cancer cells, as indicated by high GDPD5 mRNA and low GPC metabolite levels in MDA-MB-231 cells. Decreased proliferation detected upon GDPD5 inhibition with NaCl/urea further corroborated the importance of GPDP5 in breast cancer. These results indicate that GDPD5 may provide a future target for anticancer therapy. MRS could be used to monitor the GPC increase following downregulation of GDPD5 by RNA interference in such future therapies. This work was supported by NIH R01 CA134695 (to K.G.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 652.
If molecular imaging is to prove clinically useful it will have to surpass current, primarily anatomic techniques in terms of sensitivity and the ability to detect minimal changes in tissue. One of the most important tests for molecular imaging is to determine whether it can image the metastatic potential of tumors. Like all predictive endeavors, the imaging of such "potential" is a daunting task, but one that only molecular imaging--rather than standard, anatomic techniques--is likely to solve. Although difficult, imaging of metastatic potential is also arguably the most important task for molecular imaging of cancer because it is generally the dissemination of malignant tissue, not its prolonged residence in an inopportune site, which kills the patient. Below are examples of uses of molecular imaging of metastases as well as of metastatic potential, the former being a far more developed area of clinical inquiry.
Ranked list of significant probe sets most differentially expressed in MCF-7/Twist primary tumors. Probe sets are ranked by fold change. (XLSX 601Â kb)
