P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells

Abstract

Background/aim: Interferon-induced trans-membrane protein 1 (IFITM1) is known to be involved in breast cancer progression. We aimed to investigate its role in estrogen receptor (ER)-positive breast cancer cells with wild-type p53 and tamoxifen-resistant breast cancer cells.

Materials and methods: The ER-positive breast cancer cell lines, MCF-7 with wild-type p53 and T47D with mutant p53, were used. We established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen.

Results: IFITM1 inhibition in MCF-7 cells significantly decreased cell growth and migration. MCF-7 cells with suppression of IFITM1 using siRNA or ruxolitinib showed reduced cell viability after tamoxifen treatment compared with that in the control MCF-7 cells. Unexpectedly, mRNA and protein levels of IFITM1 were decreased in TamR cells compared with those in MCF-7 cells. TamR cells with suppression of IFITM1 using siRNA or ruxolitinib showed no change in cell viability after treatment with tamoxifen. P53 knockdown using siRNA reduced the mRNA levels of IRF9 and increased mRNA and protein levels of SOCS3 in MCF-7 cells, suggesting that loss or mutation of p53 can affect the induction of IFITM1 via the JAK/STAT signaling pathway in breast cancer. Furthermore, MCF-7 cells with p53 knockdown using siRNA showed no decrease in cell viability after tamoxifen treatment or IFITM1 inhibition, indicating that p53 status may be important for cell death after tamoxifen treatment or IFITM1 inhibition.

Conclusion: IFITM1 inhibition may enhance the sensitivity to tamoxifen based on p53-dependent enhancement of IFN signaling in wild-type p53, ER-positive breast cancer cells.

Keywords: Breast cancer; P53; interferon-induced transmembrane protein 1; tamoxifen.

Figure 1

IFITM1 inhibition suppressed cell proliferation and migration in MCF-7 cells. (A) The box plot downloaded from FireBrowse Gene Expression Viewer (Broad Institute of MIT and Harvard; http://firebrowse.org) indicated that IFITM1 expression in tumors was relatively higher than that in normal control tissues in breast cancer. (B-E) siControl (CTL) and siIFITM1 were transfected into MCF-7 cells. After siRNA knockdown, the protein (B) and mRNA (C) levels of p53 and IFITM1 were determined using western blotting and RT-PCR. IFITM1 knockdown significantly decreased cell growth (D) and migration (E) compared to those in the control. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (C and E) and one-way ANOVA with Turkey’s post hoc test (D). *p<0.1, **p<0.01 and ***p<0.0001.

Figure 2

IFITM1 inhibition enhanced sensitivity to tamoxifen in MCF-7 cells. (A) After transfection of siControl (CTL) and siIFITM1 in MCF-7 cells, they were treated with 9 μM tamoxifen for 48 h, and then cell viability was measured using the WST-8 assay. RT-PCR (B) and western blotting (C) were used to measure the IFITM1 expression in MCF-7 cells after treatment with 20 μM ruxolitinib for 48 h. (D) Cell viability of MCF-7 cells was measured after treatment with tamoxifen (9 μM) and ruxolitinib (20 μM) for 48 h. (E) Cellular apoptosis in MCF-7 cells was evaluated using flow cytometry after treatment with tamoxifen (9 μM) and ruxolitinib (20 μM) for 48 h. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (A and B) and one-way ANOVA with Turkey’s post hoc test (D and E). *p<0.1, **p<0.01 and ***p<0.0001.

Figure 3

TamR cells showed decreased IFITM1 and p53 expression. (A) For colony formation assay, MCF-7 and TamR cells were kept for 10 days after treatment with tamoxifen and then stained using crystal violet and imaged. (B) The viability of MCF-7 cells decreased after treatment with tamoxifen (3 and 9 μM) for 72 h, whereas that of TamR cells did not change. (C) The protein expression levels of p53, IFITM1, ERα, STAT1, and phosphorylated STAT1 were determined using western blotting in MCF-7 and TamR cells. (D) The mRNA levels of p53 and IFITM1 were measured using RT-PCR in MCF-7 and TamR cells. Western blotting (E) and RT-PCR (F) were used to measure p53 and IFITM1 expression in MCF-7 cells after treatment with tamoxifen (3 and 9 μM) for 48 h. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (B and D) and one-way ANOVA with Turkey’s post hoc test (F). *p<0.01, ** p<0.001 and ***p<0.0001.

Figure 4

IFITM1 inhibition did not affect cell survival in TamR cells. (A) After transfection of siControl (CTL) and siIFITM1 in TamR cells, cells were treated with 9 μM tamoxifen for 48 h, and then cell viability was measured using the WST-8 assay. (B) Cell viability of TamR cells was measured after treatment with tamoxifen (9 μM) and ruxolitinib (20 μM) for 48 h. (C) Cellular apoptosis in TamR cells was evaluated using flow cytometry after treatment with tamoxifen (9 μM) and ruxolitinib (20 μM) for 48 h. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (A) and one-way ANOVA with Turkey’s post hoc test (B and C). *p<0.01, **p<0.001 and ***p<0.0001.

Figure 5

P53 status can affect IFITM1 expression and cell death after tamoxifen treatment or IFITM1 inhibition in MCF-7 cells. (A) After transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 cells, the mRNA levels of p53, IFITM1, and IRF9 were measured using RT-PCR. (B) The protein expression levels of p53, IFITM1, SOCS1, and SOCS3 were measured using western blotting 48 h after the transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 cells. The mRNA levels of SOCS1 and SOCS3 were measured after transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 (C) and TamR cells (D). (E) Decreased protein and mRNA levels of IFITM1 were observed in T47D cells with mutant p53 compared with those in MCF-7 cells. (F) IFITM1 and IRF9 mRNA levels were decreased and SOCS3 mRNA levels were increased in breast cancer with mutant p53 compared with those in breast cancer with wild-type p53. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (A, C, D, and E). *p<0.05, **p<0.005 and ***p<0.0005.

Figure 5

P53 status can affect IFITM1 expression and cell death after tamoxifen treatment or IFITM1 inhibition in MCF-7 cells. (A) After transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 cells, the mRNA levels of p53, IFITM1, and IRF9 were measured using RT-PCR. (B) The protein expression levels of p53, IFITM1, SOCS1, and SOCS3 were measured using western blotting 48 h after the transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 cells. The mRNA levels of SOCS1 and SOCS3 were measured after transfection of siControl (CTL), sip53, and siIFITM1 in MCF-7 (C) and TamR cells (D). (E) Decreased protein and mRNA levels of IFITM1 were observed in T47D cells with mutant p53 compared with those in MCF-7 cells. (F) IFITM1 and IRF9 mRNA levels were decreased and SOCS3 mRNA levels were increased in breast cancer with mutant p53 compared with those in breast cancer with wild-type p53. Data are expressed as mean±standard deviation (SD). p-Values were calculated using unpaired t-test (A, C, D, and E). *p<0.05, **p<0.005 and ***p<0.0005.

Figure 6

P53 status is important for cell death after tamoxifen treatment or IFITM1 inhibition. The protein expression levels of p53 and IFITM1 were measured after simultaneous inhibition of IFITM1 and p53 using siRNA transfection (A), and cell viability was measured using the WST-8 assay after treatment with tamoxifen (9 μM) for 48 h (B). Data are expressed as mean±standard deviation (SD). p-Values were calculated using one-way ANOVA with Turkey’s post hoc test. *p<0.05, **p<0.005 and ***p<0.0005.