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S100A7-downregulation inhibits epidermal growth factor-induced signaling in breast cancer cells and blocks osteoclast formation.

Paruchuri V, Prasad A, McHugh K, Bhat HK, Polyak K, Ganju RK - PLoS ONE (2008)

Bottom Line: In addition, reduced phosphorylation of Src at tyrosine 416 and p-SHP2 at tyrosine 542 was observed in these downregulated cell lines.Further studies revealed that S100A7-downregulated cells had reduced angiogenesis in vivo based on matrigel plug assays.Our results also showed decreased tumor-induced osteoclastic resorption in an intra-tibial bone injection model involving SCID mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
S100A7 is a small calcium binding protein, which has been shown to be differentially expressed in psoriatic skin lesions, as well as in squamous cell tumors of the skin, lung and breast. Although its expression has been correlated to HER+ high-grade tumors and to a high risk of progression, the molecular mechanisms of these S100A7-mediated tumorigenic effects are not well known. Here, we showed for the first time that epidermal growth factor (EGF) induces S100A7 expression in both MCF-7 and MDA-MB-468 cell lines. We also observed a decrease in EGF-directed migration in shRNA-downregulated MDA-MB-468 cell lines. Furthermore, our signaling studies revealed that EGF induced simultaneous EGF receptor phosphorylation at Tyr1173 and HER2 phosphorylation at Tyr1248 in S100A7-downregulated cell lines as compared to the vector-transfected controls. In addition, reduced phosphorylation of Src at tyrosine 416 and p-SHP2 at tyrosine 542 was observed in these downregulated cell lines. Further studies revealed that S100A7-downregulated cells had reduced angiogenesis in vivo based on matrigel plug assays. Our results also showed decreased tumor-induced osteoclastic resorption in an intra-tibial bone injection model involving SCID mice. S100A7-downregulated cells had decreased osteoclast number and size as compared to the vector controls, and this decrease was associated with variations in IL-8 expression in in vitro cell cultures. This is a novel report on the role of S100A7 in EGF-induced signaling in breast cancer cells and in osteoclast formation.

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S100A7 knocked-down MDA-MB-468 cells show a decrease in EGF-induced migration and invasion.(A) MDA-MB-468 cells were transfected with vector control or S100A7-shRNA, as described in Materials and Methods. S100A7 expression in these cells was analyzed by Western blotting with anti-S100A7 antibody (upper panel). Equal protein loading in each lane was checked by stripping the blot and probing with β-Actin antibody (lower panel). (B) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to a chemotaxis assay towards EGF (20 ng/ml) using the modified Boyden chamber assay, as described in Materials and Methods. The lower surface of the insert was stained with the Diff-Quik Stain kit and cells were counted in an average of 5 high-power fields (HPF; 20×). Experiments were done in triplicate and repeated three times with similar results. *p<0.05 (C) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to an invasion assay using matrigel-coated transwell plates (BD Biosciences). Chemotaxis towards EGF (20 ng/ml) was then analyzed, as described in Materials and Methods. The cells on the insert were stained with the Diff-Quik Stain kit and total cell numbers were counted (10×). Experiments were done in triplicate and repeated thrice with similar results. *p<0.05.
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pone-0001741-g002: S100A7 knocked-down MDA-MB-468 cells show a decrease in EGF-induced migration and invasion.(A) MDA-MB-468 cells were transfected with vector control or S100A7-shRNA, as described in Materials and Methods. S100A7 expression in these cells was analyzed by Western blotting with anti-S100A7 antibody (upper panel). Equal protein loading in each lane was checked by stripping the blot and probing with β-Actin antibody (lower panel). (B) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to a chemotaxis assay towards EGF (20 ng/ml) using the modified Boyden chamber assay, as described in Materials and Methods. The lower surface of the insert was stained with the Diff-Quik Stain kit and cells were counted in an average of 5 high-power fields (HPF; 20×). Experiments were done in triplicate and repeated three times with similar results. *p<0.05 (C) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to an invasion assay using matrigel-coated transwell plates (BD Biosciences). Chemotaxis towards EGF (20 ng/ml) was then analyzed, as described in Materials and Methods. The cells on the insert were stained with the Diff-Quik Stain kit and total cell numbers were counted (10×). Experiments were done in triplicate and repeated thrice with similar results. *p<0.05.

Mentions: To investigate the involvement of S100A7 in the EGF-mediated effects on migration and invasion, we used stable S100A7-shRNA-downregulated cells (Figure 2A), as previously reported [14]. We observed a reduction in both EGF-induced cell migration on fibronectin-coated plates in Boyden chambers (Figure 2B) and invasive activity on matrigel-coated plates (Figure 2C). The migration of the S100A7-downregulated cells towards EGF was significantly reduced (by 40%) as compared to the vector-transfected controls.


S100A7-downregulation inhibits epidermal growth factor-induced signaling in breast cancer cells and blocks osteoclast formation.

Paruchuri V, Prasad A, McHugh K, Bhat HK, Polyak K, Ganju RK - PLoS ONE (2008)

S100A7 knocked-down MDA-MB-468 cells show a decrease in EGF-induced migration and invasion.(A) MDA-MB-468 cells were transfected with vector control or S100A7-shRNA, as described in Materials and Methods. S100A7 expression in these cells was analyzed by Western blotting with anti-S100A7 antibody (upper panel). Equal protein loading in each lane was checked by stripping the blot and probing with β-Actin antibody (lower panel). (B) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to a chemotaxis assay towards EGF (20 ng/ml) using the modified Boyden chamber assay, as described in Materials and Methods. The lower surface of the insert was stained with the Diff-Quik Stain kit and cells were counted in an average of 5 high-power fields (HPF; 20×). Experiments were done in triplicate and repeated three times with similar results. *p<0.05 (C) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to an invasion assay using matrigel-coated transwell plates (BD Biosciences). Chemotaxis towards EGF (20 ng/ml) was then analyzed, as described in Materials and Methods. The cells on the insert were stained with the Diff-Quik Stain kit and total cell numbers were counted (10×). Experiments were done in triplicate and repeated thrice with similar results. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2254193&req=5

pone-0001741-g002: S100A7 knocked-down MDA-MB-468 cells show a decrease in EGF-induced migration and invasion.(A) MDA-MB-468 cells were transfected with vector control or S100A7-shRNA, as described in Materials and Methods. S100A7 expression in these cells was analyzed by Western blotting with anti-S100A7 antibody (upper panel). Equal protein loading in each lane was checked by stripping the blot and probing with β-Actin antibody (lower panel). (B) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to a chemotaxis assay towards EGF (20 ng/ml) using the modified Boyden chamber assay, as described in Materials and Methods. The lower surface of the insert was stained with the Diff-Quik Stain kit and cells were counted in an average of 5 high-power fields (HPF; 20×). Experiments were done in triplicate and repeated three times with similar results. *p<0.05 (C) Control and S100A7 shRNA-transfected MDA-MB-468 cells were subjected to an invasion assay using matrigel-coated transwell plates (BD Biosciences). Chemotaxis towards EGF (20 ng/ml) was then analyzed, as described in Materials and Methods. The cells on the insert were stained with the Diff-Quik Stain kit and total cell numbers were counted (10×). Experiments were done in triplicate and repeated thrice with similar results. *p<0.05.
Mentions: To investigate the involvement of S100A7 in the EGF-mediated effects on migration and invasion, we used stable S100A7-shRNA-downregulated cells (Figure 2A), as previously reported [14]. We observed a reduction in both EGF-induced cell migration on fibronectin-coated plates in Boyden chambers (Figure 2B) and invasive activity on matrigel-coated plates (Figure 2C). The migration of the S100A7-downregulated cells towards EGF was significantly reduced (by 40%) as compared to the vector-transfected controls.

Bottom Line: In addition, reduced phosphorylation of Src at tyrosine 416 and p-SHP2 at tyrosine 542 was observed in these downregulated cell lines.Further studies revealed that S100A7-downregulated cells had reduced angiogenesis in vivo based on matrigel plug assays.Our results also showed decreased tumor-induced osteoclastic resorption in an intra-tibial bone injection model involving SCID mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
S100A7 is a small calcium binding protein, which has been shown to be differentially expressed in psoriatic skin lesions, as well as in squamous cell tumors of the skin, lung and breast. Although its expression has been correlated to HER+ high-grade tumors and to a high risk of progression, the molecular mechanisms of these S100A7-mediated tumorigenic effects are not well known. Here, we showed for the first time that epidermal growth factor (EGF) induces S100A7 expression in both MCF-7 and MDA-MB-468 cell lines. We also observed a decrease in EGF-directed migration in shRNA-downregulated MDA-MB-468 cell lines. Furthermore, our signaling studies revealed that EGF induced simultaneous EGF receptor phosphorylation at Tyr1173 and HER2 phosphorylation at Tyr1248 in S100A7-downregulated cell lines as compared to the vector-transfected controls. In addition, reduced phosphorylation of Src at tyrosine 416 and p-SHP2 at tyrosine 542 was observed in these downregulated cell lines. Further studies revealed that S100A7-downregulated cells had reduced angiogenesis in vivo based on matrigel plug assays. Our results also showed decreased tumor-induced osteoclastic resorption in an intra-tibial bone injection model involving SCID mice. S100A7-downregulated cells had decreased osteoclast number and size as compared to the vector controls, and this decrease was associated with variations in IL-8 expression in in vitro cell cultures. This is a novel report on the role of S100A7 in EGF-induced signaling in breast cancer cells and in osteoclast formation.

Show MeSH
Related in: MedlinePlus