<i>BRCA1</i> promoter methylation and immunohistochemical analysis of BRCA1 in early-stage breast tumors.

<p>(A) DNA methylation status of the <i>BRCA1</i> promoter determined by methylation-specific PCR. M-labeled lanes represent PCR products amplified with methylation-specific primers (75 bp). U-labeled lanes indicate the presence of unmethylated genes (82 bp). Patients 1, 2, 3, 4, 6, and 7 show the presence of a PCR product in both reactions, indicating methylation of the <i>BRCA1</i> promoter region. Patients 5 shows unmethylated gene. Molecular weight marker used is a 100-bp ladder. (B) Representative corresponding images of immunohistochemical staining of BRCA1. Scale bars, 100 µm.</p

Multivariate analysis of clinical factors and <i>BRCA1</i> promoter methylation in patients with early-stage breast cancer for overall survival and disease-free survival.

<p>Multivariate analysis of clinical factors and <i>BRCA1</i> promoter methylation in patients with early-stage breast cancer for overall survival and disease-free survival.</p

Inhibition of Chemokine (C-C Motif) Receptor 7 Sialylation Suppresses CCL19-Stimulated Proliferation, Invasion and Anti-Anoikis

<div><p>Chemokine (C-C motif) receptor 7 (CCR7) is involved in lymph-node homing of naive and regulatory T cells and lymphatic metastasis of cancer cells. Sialic acids comprise a group of monosaccharide units that are added to the terminal position of the oligosaccharide chain of glycoproteins by sialyation. Recent studies suggest that aberrant sialylation of receptor proteins contributes to proliferation, motility, and drug resistance of cancer cells. In this study, we addressed whether CCR7 is a sialylated receptor protein and tried to elucidate the effect of sialylation in the regulation of signal transduction and biological function of CCR7. Our results demonstrated that α-2, 3-sialyltransferase which catalyze sialylation reaction <i>in vivo</i> was overexpressed in breast tumor tissues and cell lines. Lectin blot analysis clearly demonstrated that CCR7 receptor was sialyated in breast cancer cells. Chemokine (C-C motif) ligand 19 (CCL19), the cognate ligand for CCR7, induced the activation of extracellular signal-regulated kinase (ERK) and AKT signaling and increased the expression of cell cycle regulatory proteins and proliferation of breast cancer cells. When cells were pre-treated with a sialyltransferase inhibitor AL10 or sialidase, CCL19-induced cell growth was significantly suppressed. CCL19 also increased invasion and prevented anoikis by up-regulating pro-survival proteins Bcl-2 and Bcl-xL. Inhibition of sialylation by AL10 totally abolished these effects. Finally, we showed that AL10 inhibited tumorigenicity of breast cancer in experimental animals. Taken together, we demonstrate for the first time that CCR7 receptor is a sialylated protein and sialylation is important for the paracrine stimulation by its endogenous ligand CCL19. In addition, inhibition of aberrant sialylation of CCR7 suppresses proliferation and invasion and triggers anoikis in breast cancer cells. Targeting of sialylation enzymes may be a novel strategy for breast cancer treatment.</p></div

CCL19-induced anti-anoikis effect was abolished by AL10.

<p>(<b>A</b>) MDA-MB-231 cells were cultured in nonadherent attachment plate in the presence or absence of AL10 or CCL19 (200 ng/mL) for 48 h. Floating cells were collected to examine cell survive by trypan blue exclusion assay and the percentage of dead cells was expressed as Mean±S.E. from three independent assays. *<i>p</i><0.05, when compared to the control group without CCL19 and AL10. (<b>B</b>) Expression of pro-caspase-3, Bcl-2 and Bcl-xL was investigated by Western blot analysis.</p

AL10 inhibited basal and CCL19-induced cyclin D1 expression via a post-transcriptional mechanism.

<p>(<b>A</b>) MDA-MB-231 cells were treated with different combinations of AL10 and CCL19 for 24 or 48 h. Cyclin D1 mRNA level was studied by RT-PCR. (<b>B</b>) MDA-MB-231 cells were pre-treated with LiCl (a GSK-3β inhibitor, 20 mM) for 3 h and then stimulated with CCL19 (200 ng/mL) for 48 h. Cyclin D1 protein level was analyzed by Western blot analysis. (<b>C</b>) MDA-MB-231 cells were treated with different combinations of AL10 and MG132 as described in Materials and Methods. Cyclin D1 protein level was analyzed by Western blot analysis.</p

CCL19 stimulated cell proliferation and cyclin D1 expression.

<p>(<b>A</b>) MDA-MB-231 cells were incubated with CCL19 (200 ng/mL) for 24 or 48 h, MTT assay was performed to investigate cell proliferation, *<i>p</i><0.05, n = 3. (<b>B</b>) MDA-MB-231 cells were pre-treated with AL10 (10 µM) or sialidase (100 or 200 mU) for 3 h and incubated with CCL19 (200 ng/mL) for another 48 h. Protein levels of cyclin A, B1, D1, E, and CDK1 were examined by Western blot analysis.</p

CCL19-induced breast cancer cells invasion was inhibited by AL10.

<p>MDA-MB-231 cells was treated with DMSO or 10 µM of AL10 for 24 h and then harvested for Transwell assay as described in Materials and Methods. Invaded cell number was counted. The number of invaded cells of the control group was defined as 1. *<i>p</i><0.05, when compared to the control group. Representative images of invaded cells were shown.</p

Characteristics of the study sample (N = 504).

<p>BCS = breast conserving surgery, MRM = modified radical mastectomy, TRAM = transverse rectus abdominis myocutaneous flap surgery (mastectomy with reconstruction), ASA = American Society of Anesthesiologists.</p><p>Characteristics of the study sample (N = 504).</p

Inhibition of breast cancer growth by AL10 <i>in vivo</i>.

<p>(<b>A</b>) Highly metastatic murine 4T1-Luc breast cancer cells in which luciferase gene is constitutively expressed were treated with different doses of AL10 for 24 h and the sialylation status was investigated by lectin affinity precipitation and western blotting. (<b>B</b>) 4T1-Luc breast cancer cells (5×10⁵ cells/mice) were orthotopically injected into both mammary gland number four of female BALB/cByJNarl mice. Mice were randomly sorted into two groups. The treatment was initiated with either the vehicle alone (Control) or AL10 (3 mg/kg/day intraperitoneally into each mouse every day). <i>In vivo</i> photon emissions of 4T1-Luc cells in mammary glands were detected and photographed by IVIS 200 image system at day 10. Photon flux reading from mice were averaged and expressed as Mean±S.D. *<i>p</i><0.05, when compared to the control group.</p

Inhibition of CCR7 sialylation attenuated its downstream signaling.

<p>(<b>A</b>) MCF-7 and MDA-MB-231 cells were treated with different dose of AL10 for 24 h and the expression of ST3Gal-I and CCR7 was analyzed by RT-PCR. (<b>B</b>) MCF-7 and MDA-MB-231 cells were pre-treated without or with AL10 for 24 h. A portion of cellular proteins was used to detect the expression of ST3Gal-I and CCR7 by Western blot analysis. Another portion of cellular proteins was incubated with biotinylated M.amurensis lectin II. Streptavidin–agarose beads were added to pull down sialylated proteins and the sialylation status of CCR7 was determined. (<b>C</b>) MDA-MB-231 cells were treated with AL10 (10 µM) for 24 h and then stimulated with CCL19 (200 ng/mL) for 20 min. Total ERK and phospho-ERK were detected by Western blot analysis. (<b>D</b>) MDA-MB-231 cell were pre-treated with different doses of sialidase for 3 h and then stimulated with CCL19 (200 ng/ml) for 20 min. Total ERK and phosphor-ERK were investigated. (<b>E</b>) Cells were treated as described in (C) and total AKT and phospho-AKT were detected by Western blot analysis.</p