DHA inhibits Gremlin-1-induced epithelial-to-mesenchymal transition via ERK suppression in human breast cancer cells

Abstract

Docosahexaenoic acid (DHA) is an omega-3 fatty acid abundant in fish oils. It is known to have an inhibitory effect on various diseases such as inflammation, diabetes, and cancer. Epithelial-to-mesenchymal transition (EMT) is a process that epithelial cells gain migratory property to become mesenchymal cells involved in wound healing, organ fibrosis, and cancer progression. Gremlin-1 (GREM1) is a bone morphogenetic protein antagonist known to play a role in EMT. However, the role of GREM1 in the induction of EMT in human breast cancer cells and the effect of DHA on GREM1-induced EMT remain unclear. Establishment of GREM1 knockdown cell lines was performed using lentiviral shRNAs. Expression of EMT markers was determined by qRT-PCR and Western blotting. Effect of GREM1 and/or DHA on cell migration was investigated using wound healing assay. The level of GREM1 expression in human breast cancer tissues was determined by Oncomine database mining. GREM1 induced the expression of genes including N-cadherin, vimentin, and Slug. GREM1 promoted the migration of human breast cancer cells. GREM1 enhanced the expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and the ERK activation was involved in EMT. Interestingly, DHA reduced the expression of GREM1. DHA also inhibited the expression of mesenchymal cell-associated genes and cell migration induced by GREM1. Furthermore, DHA suppressed the expression of p-ERK induced by GREM1. These results indicate that GREM1-ERK axis plays a role in EMT in human breast cancer cells and DHA is a putative compound that can inhibit EMT by inhibiting GREM1 signal transduction.

Keywords: Breast cancer; DHA; EMT; GREM1; extracellular signal-regulated kinases.

Figure 1. DHA inhibits TGF-β-induced EMT in human breast cancer cells

Cells were starved overnight and stimulated with TGF-β (10 ng/ml) for an additional 48 h. RNA was analyzed by qPCR analysis (A). Cells were starved overnight and pretreated with TGF-β (10 ng/ml) for 24 h and then incubated with DHA (25 μM) for another 24 h. RNA was collected and analyzed by qPCR analysis (B) and proteins were performed by Western blot analysis (C). MDA-MB-453 cells were seeded in 6-well plates and wounded by 1 ml pipette tip. The cells were incubated with TGF-β (10 ng/ml) or DHA (25 μM) for 48 h, separately or in combination (D and E). *, P<0.05; **, P<0.01; ***, P<0.001, and NS=non-significant.

Figure 2. GREM1 is overexpressed in human breast cancer tissues

Cells were starved overnight and stimulated with TGF-β (10 ng/ml) for an additional 48 h. RNA was analyzed by qPCR analysis (A) and protein lysates were subjected to immunoblot analysis (B). Oncomine microarray database was used to analyze GREM1 mRNA expression in breast cancer versus normal breast tissues. Four datasets were included in the meta-analysis (C); **, P< 0.01.

Figure 3. GREM1 increases EMT in human breast cancer cells

Cells were starved overnight and stimulated with GREM1 (10 and 50 ng/ml) for an additional 48 h. RNA was analyzed by qPCR analysis (A). Cells were starved overnight and stimulated with GREM1 (50 ng/ml) for an additional 48 h. Protein lysates were subjected to immunoblot analysis (B). Each cell line was established by using lentiviral shRNA system. RNA was collected and indicated genes were quantitated by qPCR analysis (C). MDA-MB-453-shCtrl or MDA-MB-453-shGREM1 cells were seeded into 6-well plates and wounded with 1 ml pipette tip. Each cell line was incubated with vehicle or TGF-β (10 ng/ml) for 48 h. The wound closure was monitored by photography at the indicated time (D and E). Cells were starved overnight and treated with GREM1 (50 ng/ml) for 30 min. Protein lysates were subjected to immunoblot analysis (F). Cells were starved overnight and co-treated with GREM1 (50 ng/ml) and U0126 (10 μM) for 30 min (pERK and ERK) or 48 h (N-cadherin, vimentin, and Slug). Protein lysates were subjected to immunoblot analysis (G). *, P<0.05; **, P<0.01; ***, P<0.001, and NS = non-significant.

Figure 4. DHA inhibits GREM1 expression in human breast cancer cells

Cells were starved overnight and stimulated with DHA (25 μM) for 24 h. RNA was collected and analyzed by qPCR analysis (A) and proteins were subjected to Western blot analysis (B). Cells were starved overnight and pretreated with TGF-β (10 ng/ml) for 24 h and then incubated with DHA (25 μM) for another 24 h. RNA was collected and analyzed by qPCR analysis (C) and proteins were subjected to Western blot analysis (D). **, P<0.01 and ***, P<0.001.

Figure 5. DHA inhibits GREM1-induced EMT

Cells were starved overnight and pretreated with GREM1 (50 ng/ml) for 24 h and then incubated with DHA (25 μM) for another 24 h. RNA was collected and analyzed by qPCR analysis (A) and proteins were performed by Western blot analysis (B). MDA-MB-453 cells were seeded in 6-well plates and wounded by 1 ml pipette tip. The cells were incubated with GREM1 (50 ng/ml) or DHA (25 μM) for 48 h, separately or in combination (C and D). Cells were starved overnight and co-treated with GREM1 (50 ng/ml) and DHA (25 μM) for 30 min. Protein lysates were subjected to immunoblot analysis (E). *, P<0.05; **, P<0.01; ***, P<0.001, and NS=non-significant.