Aspirin regulation of c-myc and cyclinD1 proteins to overcome tamoxifen resistance in estrogen receptor-positive breast cancer cells

Abstract

Tamoxifen is still the most commonly used endocrine therapy drug for estrogen receptor (ER)-positive breast cancer patients and has an excellent outcome, but tamoxifen resistance remains a great impediment to successful treatment. Recent studies have prompted an anti-tumor effect of aspirin. Here, we demonstrated that aspirin not only inhibits the growth of ER-positive breast cancer cell line MCF-7, especially when combined with tamoxifen, but also has a potential function to overcome tamoxifen resistance in MCF-7/TAM. Aspirin combined with tamoxifen can down regulate cyclinD1 and block cell cycle in G0/G1 phase. Besides, tamoxifen alone represses c-myc, progesterone receptor (PR) and cyclinD1 in MCF-7 cell line but not in MCF-7/TAM, while aspirin combined with tamoxifen can inhibit the expression of these proteins in the resistant cell line. When knocking down c-myc in MCF-7/TAM, cells become more sensitive to tamoxifen, cell cycle is blocked as well, indicating that aspirin can regulate c-myc and cyclinD1 proteins to overcome tamoxifen resistance. Our study discovered a novel role of aspirin based on its anti-tumor effect, and put forward some kinds of possible mechanisms of tamoxifen resistance in ER-positive breast cancer cells, providing a new strategy for the treatment of ER-positive breast carcinoma.

Keywords: ER-positive breast cancer; aspirin; c-myc; cyclinD1; tamoxifen resistance.

Figure 1. Detecting the expression of ER α and tamoxifen sensitivity in MCF-7 and MCF-7/TAM cell lines

(A) The expression of ERα was observed using immunofluorescent assays in MCF-7 and MCF-7/TAM cell lines. (B) The survival rate of MCF-7 and MCF-7/TAM cells was tested by MTS Kit after treated with 4-OHT at the indicated concentrations (0–6 μM) (***p < 0.001). (C) MCF-7 and MCF-7/TAM cells were treated with 4-OHT (5 μM) for 72 h, then stained by PI and detected by flow cytometry analysis. (D) Bar chart represented the percentage of G0/G1 phase (***p < 0.001). All the experiments were repeated for at least three times. The results were presented as mean ± SEM.

Figure 2. ASA has an obvious anti-tumor effect on both cell lines and can overcome tamoxifen resistance

(A) After 7-day treatment, with the increasing concentrations of 4-OHT (0 μM, 3 μM and 6 μM), the survival rate of MCF-7 cells decreased and there was an additive inhibitory effect when combined with ASA (2 mM) (***p < 0.001). (B) The difference of survival rate between combined drug group and negative control (4-OHT alone) was observed at the indicated concentration of 4-OHT. There was no significant difference (p > 0.05). (C) After 7-day treatment, with the increasing concentrations of 4-OHT (0 μM, 3 μM and 6 μM), the survival rate of MCF-7/TAM cells decreased when combined with ASA (2 mM) (***p < 0.001), while there was no such phenomenon observed when using 4-OHT alone (p > 0.05). (D) The difference of survival rate between combined drug group and negative control (4-OHT alone) was observed at the indicated concentration of 4-OHT. There was significant difference observed (*p < 0.05 and ***p < 0.001). All the experiments were repeated for at least three times. The results were presented as mean ± SEM.

Figure 3. ASA enhance the anti-tumor effect of 4-OHT through cell cycle arrest

(A) MCF-7 cells were respectively treated with 4-OHT (5 μM), ASA (2 mM), and 4-OHT (5 μM) combined with ASA (2 mM) for 72 h, then detected by flow cytometry analysis. (B) Bar charts represented the percentage of G0/G1 phase in different groups of MCF-7 cells. There was no significant difference observed between ASA group and negative control (p > 0.05), but significant difference was observed among other three groups (**p < 0.01; ***p < 0.001). (C) MCF-7/TAM cells were respectively treated with 4-OHT (5 μM), ASA (2 mM), and 4-OHT (5 μM) combined with ASA (2 mM) for 72 h, then detected by flow cytometry analysis. (D) Bar charts represented the percentage of G0/G1 phase in different groups of MCF-7/TAM cells. There was no significant difference among ASA group, 4-OHT group and negative control (p > 0.05), but when combining 4-OHT with ASA, the percentage of G0/G1 phase increased, which showed significant differences (***p < 0.001). All the experiments were repeated for at least three times. The results were presented as mean ± SEM.

Figure 4. The gene expression of CCND1, MYC and PGR gene was different between MCF-7 and MCF-7/TAM cell line, which changed after treated with 4-OHT combined with ASA

(A) There were differences of the gene expression of CCND1, MYC and PGR gene observed between MCF-7 and MCF-7/TAM cell lines (**p < 0.01, ***p < 0.001). (B) In MCF-7 cell line, the gene expression of CCND1, MYC and PGR was changed in ASA group, 4-OHT group and combination group compared with negative control, significant differences among these treatment groups were observed (*p < 0.05, **p < 0.01 and ***p < 0.001). (C) In MCF-7/TAM cell line, the gene expression of CCND1, MYC and PGR was changed only in ASA group and combination group compared with negative control, significant differences among these treatment groups were observed (*p < 0.05, **p < 0.01 and ***p < 0.001). All the experiments were repeated for at least three times. The results were presented as mean ± SEM.

Figure 5. Western blotting analysis was performed to detect the changes in the protein level before and after treatment with drugs

(A) The cyclinD1 bands of MCF-7 cell line under different drug treatments (4-OHT and ASA separate application or the two drugs combination). (B) Bar charts represented the cyclinD1 level compared with negative control (MCF-7). (C) The cyclinD1 bands of MCF-7/TAM cells under different treatments (4-OHT and ASA separate application or the two drugs combination). (D) Bar charts represented the cyclinD1 level compared with negative control (MCF-7/TAM). (E) The CDK4, CDK6, c-myc, PR bands of MCF-7/TAM cells under different drug treatments (4-OHT and ASA separate application or the two drugs combination). (F–I) Bar charts represented the CDK4, CDK6, c-myc, PR levels compared with negative control (MCF-7/TAM). All the experiments were repeated for at least three times.

Figure 6. MYC gene was knocked down in MCF-7/TAM cell line by shRNA, and cell survival rate and cell cycle was detected after 4-OHT treatment

(A) The transfer efficiency was detected by the percentage of green fluorescent and almost all the cells were infected by lentivirus. (B) The gene expression of MYC in MCF-7/TAM/shMYC was much lower than MCF-7/TAM/U6 (**p < 0.01). (C) The protein bands of c-myc in two cell lines. (D) Bar charts represented c-myc level compared with negative control (MCF-7/TAM/U6). (E) The survival rate of two cell lines under different concentrations of 4-OHT showed an incremental inhibitory effect on proliferation of MCF-7/TAM/shMYC cells but not on MCF-7/TAM/U6 cells (*p < 0.05 and **p < 0.01). (F) Bar charts represented the flow cytometry results about the percentage of G0/G1 phase. The percentage of G0/G1 phase showed a significant increase after 4-OHT treatment in MCF-7/TAM/shMYC cells (*p < 0.05), but not in MCF-7/TAM/U6 cells (p > 0.05). All the experiments were repeated for at least three times. The results were presented as mean ± SEM.

Figure 7. Diagramatic representation of the possible related molecular mechanism of MCF-7/TAM

In the resistant cell line MCF-7/TAM, other mitogens or pathways take place E2-ER complexes’ function, so tamoxifen resistance occur. Aspirin has an effect on inhibiting c-myc and cyclin D1's expression or their upstream regulators’ activity, and partly restores the function of tamoxifen, prompting a potential role in overcoming tamoxifen resistance in ER-positive advanced breast carcinoma.