MAP1S Controls Breast Cancer Cell TLR5 Signaling Pathway and Promotes TLR5 Signaling-based Tumor Suppression

<div><p>Targeting TLR5 signaling in breast cancer represents a novel strategy in cancer immunotherapy. However, the underlying mechanism by which TLR5 signaling inhibits cancer cell proliferation and tumor growth has not been elucidated. In this study, we found TLR5 agonist flagellin inhibited the cell state of activation and induced autophagy, and reported that autophagy protein MAP1S regulated the flagellin/TLR5 signaling pathway in breast cancer cells through enhancement of NF-κB activity and cytokine secretion. Remarkably, MAP1S played a critical role in tumor suppression induced by flagellin, and knockdown of MAP1S almost completely abrogated the suppression of tumor growth and migration by flagellin treatment. In addition, elevated expression of MAP1S in response to flagellin feed-back regulated tumor inflammatory microenvironment in the late stages of TLR5 signaling through degradation of MyD88 in autophagy process. These results indicate a mechanism of antitumor activity that involves MAP1S-controlled TLR5 signaling in breast cancer.</p></div

MAP1S links with cancer cell TLR5 signaling pathway.

<p>(A) Whole-cell MALDI-TOF MS spectrum of MCF-7. MCF-7 cells were stimulated with 0.5 µg/ml flagellin for 16 h. Then cells were collected in 10 µl of PBS, and 1 µl suspension was mixed with CHCA matrix and loaded onto MALDI target. (B) MAP1S promotes TLR signaling-induced autophagy. LC3 puncta induced by flagellin in MCF-7 cells were detected by immunofluorescence. Magnification, 63×. (C) MAP1S mRNA expression in untreated, LPS (0.1 µg/ml) or flagellin (0.1 µg/ml) treated MCF-7 cells was detected by quantitative RT-PCR. (D) MCF-7 cells were stimulated by the indicated doses of flagellin for 16 h. MAP1S protein levels were analyzed by western blotting. All data are representative results of 3 independent experiments.</p

Overexpressed MAP1S inhibits tumor cell TLR5 signaling.

<p>(A) MCF-7 cells were transfected with different doses of MAP1S plasmid and NF-κB reporter plasmid and analyzed for luciferase activity induced by flagellin. (B) MCF-7 cells were transfected with AP-1 reporter plasmid, vector control, MyD88 and/or MAP1S and analyzed for luciferase activity. *indicates <i>p</i><0.05, relative to vector control. **indicates <i>p</i><0.001, relative to co-transfection of MyD88 and MAP1S. (C) Left, MCF-7 cells transfected with empty vector or MAP1S expressing plasmid, or wild type MCF-7 cells were treated with 0.5 µg/ml flagellin or vehicle for 24 h. Right, MCF-7/shCtrl and MCF-7/shMAP1S cells were treated with 0.5 µg/ml flagellin or vehicle for 24 h. MyD88 levels were analyzed by western blotting.</p

MAP1S is essential for flagellin to inhibit tumor cell migration.

<p>(A) Left, MCF-7/shCtrl and MCF-7/shMAP1S cells were seeded into the upper chamber of a Transwell insert and treated with 0.5 µg/ml flagellin for 20 h. Cell migration was measured using a Transwell migration assay. Cells were stained with 0.1% crystal violet after nonmigrated cells were scraped on the upper surface of the polycarbonate membrane. Magnification, 200×. Right, quantitative data on cell migration is shown. (B) Left, MAP1S promoted the inhibition of MCF-7 cell migration by flagellin treatment as measured by a scratch wound migration assay. Healing due to cell migration was observed over a period of 6 h and 12 h following scratch wounding. Magnification, 10×. Right, quantitative data from the cell scratch healing assay is shown. The difference in migratory capacity was significant in flagellin-treated MCF-7/shCtrl cells compared with untreated cells and flagellin-treated MCF-7/shMAP1S cells. **indicates <i>p</i><0.001, relative to untreated cells.</p

MAP1S is required for flagellin to inhibit breast cancer cell proliferation.

<p>(A) The cell proliferation of MCF-7/shCtrl and MCF-7/shMAP1S cells was determined using the MTT assay after treatment with 0.5 µg/ml flagellin for the indicated number of days. (B) Four representative 35-mm plates of MCF-7/shCtrl and MCF-7/shMAP1S-2 cells were cultured for 8 days with or without flagellin treatment. Cells were then stained with 1 mg/ml MTT solution for 30 min and images of the colonies that formed were captured with a microscope. The number of colonies (≥100 µm in diameter) was counted using Image-Pro Plus software. **indicates <i>p</i><0.001, relative to untreated cells. (C) MCF-7/shCtrl and MCF-7/shMAP1S-2 cells were stimulated with 0.5 µg/ml flagellin for the indicated number of days. DNA content was assessed by PI staining and flow cytometry. Quantitative data showing different phases of the cell cycle is displayed. (D) MCF-7/shCtrl and MCF-7/shMAP1S-2 cells were treated with 0.5 µg/ml flagellin for the indicated time periods, and Cyclin D1 and p27 level were analyzed by western blotting with specific antibodies. All data are representative results of 3 independent experiments.</p

MAP1S regulates flagellin-induced cytokines and soluble factors in breast cancer cells.

<p>(A) Wild type MCF-7, stable MCF-7/shCtrl and MCF-7/shMAP1S cells were transfected with NF-κB reporter plasmids and analyzed for luciferase activity induced by flagellin. *indicates <i>p</i><0.05, relative to flagellin-treated wild type and shCtrl cells. NC, negative control. (B) MCF-7 cells were stimulated with 0.1 µg/ml flagellin for 6 h for cytokine expression. The mRNA levels were analyzed by quantitative RT-PCR. (C) Wild type MCF-7 cells co-cultured with flagellin-pretreated MCF-7/shCtrl or MCF-7/shMAP1S cells in a co-culture Transwell system. Cell proliferation on the indicated day was detected by the MTT assay. *indicates <i>p</i><0.05, relative to shCtrl cells.</p

The representative N-glycan profiles of membrane proteins from human breast cancer cell line and tissue.

<p>The desialylated N-glycan profiles of membrane proteins from cell lines (n=8) and tumor tissues (n=100). Oligomaltose is used as a sugar mass reference. The number of glucose units (degree of polymerization, DP) in these structures is indicated. N-glycan profiles from RNaseB and serum were used as N-glycan profile controls. RNaseB contained high mannose from M5 to M9. Serum contained various complex N-glycans and the most abundant glycans detected in serum are marked. The vertical axis represents the glycan intensity of the peaks as a percent of the relative fluorescence level. The X-axis represents the retention time of the peaks. The N-glycan structures of the corresponding peaks are shown below the panels. NGA2F is an agalacto core-α-1, 6-fucosylated biantennary glycan; NA2 is a bigalacto biantennary glycan; NA2F is a bigalacto core-α-1, 6-fucosylated biantennary glycan; NA2FB is a bigalacto core-α-1, 6-fucosylated bisecting biantennary glycan; NA3 is tri-antennary; NA3FB is a core-α-1, 6-fucosylated triantennary glycan. The symbols used in the structural formulas are as follows: (○) β-linked N-acetylglucosamine; (●) β-linked galactose; (□) α-linked mannose; (■) β-linked mannose; (▲) α-1, 6-linked fucose.</p

The significantly changed N-glycans of the membrane proteins of human breast cancer cell lines.

<p>(A) The representative N-glycan profiles of membrane proteins from human breast cancer cell lines (n=7). MCF 10A: from breast fibrocystic disease as a non-tumorigenic epithelial cell line; SK-BR-3, MCF-7, Bcap 37, MDA-MB-231, T-47D, Hs 578T and ZR-75-30: cancer cell lines; Serum: from healthy humans. (B–D) The statistically significant difference in N-glycan peaks B1, B2, B4 and B5 between MCF 10A and various cancer cell lines. Peak B2 is significantly increased in cancer cell lines compared with MCF 10A. On the contrary, peaks B1 and B4 are dramatically decreased in cancer cell lines. Particularly, peak B4 is completely lost in Bcap 37, MDA-MB-231 and Hs 578T cell lines. The percentages of each specific peak height in the total peak heights are expressed as mean ± SD. Asterisks indicate statistically significant differences between the various cancer cell lines and the MCF 10A cell line (* <i>p</i> < 0.05, ** <i>p</i> < 0.01). Five major glycan peaks B1-B5 in normal control and breast cancer groups were detected. Experiments were repeated three times.</p

Establishment of the DSA-FACE method for analyzing N-glycan profiling in breast cancer.

<p>(A) Equal amounts (20 μg and 100 μg for cell line and tissue specimens, respectively) of cytoplasmic and membrane proteins (CP and MP) from Bcap 37 cells and tumor tissue were determined by Western blotting using γ-tubulin and Na⁺/K⁺ ATPase-α1 antibody, respectively. Na⁺/K⁺ ATPase-α1 (an integral membrane protein) and γ-tubulin (a core component of the centrosome) were chosen as the cytomembrane and cytoplasm markers, respectively. (B) The representative N-glycan profiles of cytosolic and membrane proteins from cell line (upper panel, n=8) and tissue (lower panel, n=200).</p

The schematic presentation of changes in N-linked glycans in breast cancer.

<p>The increased concentration of NA3FB (peak B5) and decreased concentration of NA2 (peak B1) and NA2FB (peak B4) in breast cancer tissues could be attributed to the increased activity of GnT-V, which competes for substrate with GnT-III and is associated with the consequential elevated level of branched N-glycans.</p