CXCR4 expression in papillary thyroid carcinoma: induction by nitric oxide and correlation with lymph node metastasis

<p>Abstract</p> <p>Background</p> <p>Metastasis to regional lymph nodes is a common step in the progression of cancer. Recent evidence suggests that tumor production of CXCR4 promotes lymph node metastasis. Nitric oxide (NO) may also increase metastatic ability in human cancers.</p> <p>Methods</p> <p>Nitrite/nitrate levels and functional CXCR4 expression were assessed in K1 and B-CPAP papillary thyroid carcinoma (PTC) cells after induction and/or inhibition of NO synthesis. CXCR4 expression was also analyzed in primary human PTC. The relationship between nitrotyrosine levels, which are a biomarker for peroxynitrate formation from NO in vivo, CXCR4 expression, and lymph node status was also analyzed.</p> <p>Results</p> <p>Production of nitrite/nitrate and functional CXCR4 expression in both cell lines was increased by treatment with the NO donor DETA NONOate. The NOS inhibitor L-NAME eliminated this increase. Positive CXCR4 immunostaining was observed in 60.7% (34/56) of PTCs. CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis in human PTC.</p> <p>Conclusion</p> <p>Our data indicate that NO stimulates CXCR4 expression in vitro. Formation of the NO biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in human PTC. NO may induce lymph node metastasis via CXCR4 induction in papillary thyroid carcinoma.</p

Neuropilin-2 expression in breast cancer: correlation with lymph node metastasis, poor prognosis, and regulation of CXCR4 expression

<p>Abstract</p> <p>Background</p> <p>Neuropilin-2 (Nrp2) is a receptor for vascular endothelial growth factor-C (VEGF-C), which is a well-known lymphangiogenic factor and plays an important role in lymph node metastasis of various human cancers, including breast cancer. Recently, Nrp2 was shown to play a role in cancer by promoting tumor cell metastasis. CXC chemokine receptor 4 (CXCR4) also promotes tumor metastasis. In the previous studies, we demonstrated that VEGF-C and cytoplasmic CXCR4 expressions were correlated with poorer patient prognosis (BMC Cancer 2008,8:340; Breast Cancer Res Treat 2005, 91:125–132).</p> <p>Methods</p> <p>The relationship between Nrp2 expression and lymph node metastasis, VEGF-C expression, CXCR4 expression, and other established clinicopathological variables (these data were cited in our previous papers), including prognosis, was analyzed in human breast cancer. Effects of neutralizing anti-Nrp2 antibody on CXCR4 expression and chemotaxis were assessed in MDA-MB-231 breast cancer cells.</p> <p>Results</p> <p>Nrp2 expression was observed in 53.1% (60 of 113) of the invasive breast carcinomas. Nrp2 expression was significantly correlated with lymph node metastasis, VEGF-C expression, and cytoplasmic CXCR4 expression. Survival curves determined by the Kaplan-Meier method showed that Nrp2 expression was associated with reduced overall survival. In multivariate analysis, Nrp2 expression emerged as a significant independent predictor for overall survival. Neutralizing anti-Nrp2 antibody blocks cytoplasmic CXCR4 expression and CXCR4-induced migration in MDA-MB-231 cells.</p> <p>Conclusion</p> <p>Nrp2 expression was correlated with lymph node metastasis, VEGF-C expression, and cytoplasmic CXCR4 expression. Nrp2 expression may serve as a significant prognostic factor for long-term survival in breast cancer. Our data also showed a role for Nrp2 in regulating cytoplasmic CXCR4 expression <it>in vitro</it>.</p

Quantitative LC-MS/MS Analysis of Proteins Involved in Metastasis of Breast Cancer.

The purpose of this study was to develop quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of proteins involved in metastasis of breast cancer for diagnosis and determining disease prognosis, as well as to further our understand of metastatic mechanisms. We have previously demonstrated that the protein type XIV collagen may be specifically expressed in metastatic tissues by two dimensional LC-MS/MS. In this study, we developed quantitative LC-MS/MS methods for type XIV collagen. Type XIV collagen was quantified by analyzing 2 peptides generated by digesting type XIV collagen using stable isotope-labeled peptides. The individual concentrations were equivalent between 2 different peptides of type XIV collagen by evaluation of imprecise transitions and using the best transition for the peptide concentration. The results indicated that type XIV collagen is highly expressed in metastatic tissues of patients with massive lymph node involvement compared to non-metastatic tissues. These findings were validated by quantitative real-time RT-PCR. Further studies on type XIV collagen are desired to verify its role as a prognostic factor and diagnosis marker for metastasis

Correlation of concentrations between LC-MS/MS and quantitative real-time RT-PCR for validation of type XIV collagen.

<p>The x-axis represents m-RNA expression of type XIV collagen as determined by quantitative real-time RT-PCR. The y-axis represents protein concentration of type XIV collagen as determined by LC-MS/MS. In the scatter plot, each data point represents protein concentration and m-RNA expression. (A) The protein concentrations are average concentrations of all peptide transitions (614/711, 614/826, and 614/1013) of peptide ITWDPPSSPVK. (A') The protein concentrations are calculated from the best transition (614/711) for peptide ITWDPPSSPVK. (B) The protein concentrations are calculated from the best transition (708/763) for peptide ASAHAITGPPTELITSEVTAR.</p

Concentrations of type XIV collagen determined by quantitative LC-MS/MS analysis with stable isotope-labeled peptides.

<p>*¹: Average retention time; Average retention time of triplicate samples (^#1: Retention time of one sample, ^#2: Average retention time of duplicate samples.-: All samples were below detection limit.).</p><p>*²: Average Conc.; Average intra-assay concentration (n = 3) (^#1: Concentration of one sample, ^#2: Average concentration of duplicate samples, N.D.: Not detected because concentrations of all triplicate samples were not detected, and the concentration was calculated as 0.0.).</p><p>*³: CV of intra-assay; Coefficient of variation of intra-assay reproducibility (n = 3) (N.C.: Not calculated because concentrations of all tripricate samples were not detected.)</p><p>*⁴: Value including interferences</p><p>*⁵: Value including detected samples and non-detected samples</p><p>*⁶: Average conc.; Average concentration from 2 transitions (614/711and 614/826) for peptide 1 and 3 transitions (708/624, 708/763, 708/877) for peptide 2.</p><p>*⁷: Difference of peptide 1; Difference (%) = (Conc. of 614/711-Conc. of 614/826)×100 /Conc. of 614/711, (N.C.: Not calculated because concentration of 614/711 was 0.)</p><p>^#1: Value of 1 sample because other duplicate samples were not detected.</p><p>^#2: Average of duplicate samples because another sample was not detected.</p><p>Concentrations of type XIV collagen determined by quantitative LC-MS/MS analysis with stable isotope-labeled peptides.</p

Type XIV collagen concentrations calculated from the best transitions of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR.

<p>After evaluation of imprecise transitions and using the best transition for the peptide concentration, concentrations of transition 614/711 were used as the concentrations of peptide ITWDPPSSPVK, and concentrations of transition 708/763 were used as the concentrations of peptide ASAHAITGPPTELITSEVTAR.</p

Correlation of type XIV collagen concentrations calculated from the best transitions of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR.

<p>The x-axis represents protein concentration of type XIV collagen calculated from the best transition (708/763) of peptide ASAHAITGPPTELITSEVTAR as determined by LC-MS/MS. The y-axis represents protein concentration of type XIV collagen calculated from the best transition (614/711) of peptide ITWDPPSSPVK as determined by LC-MS/MS. In the scatter plot, each data point represents protein concentration.</p

Average intra-assay concentration of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR of type XIV collagen.

<p>Peptides from each subject (subjects 1–6: non metastasis, subjects 7–18: massive lymph node metastasis) were quantified (n = 3) by LC-MS/MS. The average intra-assay concentration and SD of peptide concentrations from individual proteins are calculated from all transitions (614/711, 614/826 and 614/1013) for peptide ITWDPPSSPVK of Protein 12 (A), 2 transitions (614/711 and 614/826) for peptide ITWDPPSSPVK (A'), and all transitions (708/624, 708/763 and 708/877) for peptide ASAHAITGPPTELITSEVTAR (B) of type XIV collagen. By excluding the low specificity transition 614/1013, the concentration of peptide ITWDPPSSPVK was changed (A) to (A'), which correlated with the concentrations of (B). Significant difference between non metastasis and massive lymph node metastasis in (A') and (B).</p