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Statins affect ETS1 -overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency

View Article: PubMed Central - PubMed

ABSTRACT

We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4.

No MeSH data available.


Related in: MedlinePlus

Survival, colony-forming, and cell invasion assays.(A) IC50 values for a panel of BC cell lines treated with simvastatin. Proliferation was assessed 3 days after treating BC cell lines with varying concentrations (0–120 μmol/L) of simvastatin. Dose-response curves were used to calculate the IC50 values. Values are shown are means of three independent experiments. IC50 values less than 20 μmol/L (denoted by the red dashed line) indicate a potential therapeutic effect of simvastatin. White columns, TNBC cells; gray columns, HER-2-positive cells; black columns, hormone receptor-positive cells. (B) Colony-forming assays of TNBC (MDA-MB-231) and ER-α-expressing (MCF 7) cells. Cells were treated with 0.1 μM simvastatin for 3 days and maintained at 37 °C for 14 days. Fresh medium was added after 7 days. Cells were stained with 0.1% crystal violet.
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f2: Survival, colony-forming, and cell invasion assays.(A) IC50 values for a panel of BC cell lines treated with simvastatin. Proliferation was assessed 3 days after treating BC cell lines with varying concentrations (0–120 μmol/L) of simvastatin. Dose-response curves were used to calculate the IC50 values. Values are shown are means of three independent experiments. IC50 values less than 20 μmol/L (denoted by the red dashed line) indicate a potential therapeutic effect of simvastatin. White columns, TNBC cells; gray columns, HER-2-positive cells; black columns, hormone receptor-positive cells. (B) Colony-forming assays of TNBC (MDA-MB-231) and ER-α-expressing (MCF 7) cells. Cells were treated with 0.1 μM simvastatin for 3 days and maintained at 37 °C for 14 days. Fresh medium was added after 7 days. Cells were stained with 0.1% crystal violet.

Mentions: To determine whether statin inhibits cell proliferation, we performed MTT assays. We found that TNBC cells were sensitized to simvastatin compared with non-TNBC cell lines, as indicated by the IC50 values. Six of the 10 TNBC lines tested had an IC50 <20 μmol/L, whereas all non-TNBC cell lines had an IC50 ≥ 20 μmol/L (Fig. 2A). A colony-forming assay was performed by seeding equal numbers of MDA-MB-231 cells and MCF-7 cells in triplicate on soft agar in 6-well tissue culture plates. After treating the cells with 0.1 μM simvastatin, the MDA-MB-231 cells formed fewer colonies than the MCF-7 cells (Fig. 2B). Thus, stimulation with 0.1 μM simvastatin significantly inhibited colony formation in MDA-MB-231 cells (P = 0.002).


Statins affect ETS1 -overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency
Survival, colony-forming, and cell invasion assays.(A) IC50 values for a panel of BC cell lines treated with simvastatin. Proliferation was assessed 3 days after treating BC cell lines with varying concentrations (0–120 μmol/L) of simvastatin. Dose-response curves were used to calculate the IC50 values. Values are shown are means of three independent experiments. IC50 values less than 20 μmol/L (denoted by the red dashed line) indicate a potential therapeutic effect of simvastatin. White columns, TNBC cells; gray columns, HER-2-positive cells; black columns, hormone receptor-positive cells. (B) Colony-forming assays of TNBC (MDA-MB-231) and ER-α-expressing (MCF 7) cells. Cells were treated with 0.1 μM simvastatin for 3 days and maintained at 37 °C for 14 days. Fresh medium was added after 7 days. Cells were stained with 0.1% crystal violet.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015082&req=5

f2: Survival, colony-forming, and cell invasion assays.(A) IC50 values for a panel of BC cell lines treated with simvastatin. Proliferation was assessed 3 days after treating BC cell lines with varying concentrations (0–120 μmol/L) of simvastatin. Dose-response curves were used to calculate the IC50 values. Values are shown are means of three independent experiments. IC50 values less than 20 μmol/L (denoted by the red dashed line) indicate a potential therapeutic effect of simvastatin. White columns, TNBC cells; gray columns, HER-2-positive cells; black columns, hormone receptor-positive cells. (B) Colony-forming assays of TNBC (MDA-MB-231) and ER-α-expressing (MCF 7) cells. Cells were treated with 0.1 μM simvastatin for 3 days and maintained at 37 °C for 14 days. Fresh medium was added after 7 days. Cells were stained with 0.1% crystal violet.
Mentions: To determine whether statin inhibits cell proliferation, we performed MTT assays. We found that TNBC cells were sensitized to simvastatin compared with non-TNBC cell lines, as indicated by the IC50 values. Six of the 10 TNBC lines tested had an IC50 <20 μmol/L, whereas all non-TNBC cell lines had an IC50 ≥ 20 μmol/L (Fig. 2A). A colony-forming assay was performed by seeding equal numbers of MDA-MB-231 cells and MCF-7 cells in triplicate on soft agar in 6-well tissue culture plates. After treating the cells with 0.1 μM simvastatin, the MDA-MB-231 cells formed fewer colonies than the MCF-7 cells (Fig. 2B). Thus, stimulation with 0.1 μM simvastatin significantly inhibited colony formation in MDA-MB-231 cells (P = 0.002).

View Article: PubMed Central - PubMed

ABSTRACT

We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P&thinsp;=&thinsp;&lt;0.001) and DUSP4 mRNA was downregulated (P&thinsp;=&thinsp;&lt;0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P&thinsp;=&thinsp;0.002 in 0.1&thinsp;&mu;M). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4.

No MeSH data available.


Related in: MedlinePlus