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Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death.

Choi HJ, Lui A, Ogony J, Jan R, Sims PJ, Lewis-Wambi J - Breast Cancer Res. (2015)

Bottom Line: Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death.Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells.Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Estrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance.

Methods: Real-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression.

Results: We found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells.

Conclusion: Overall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.

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Related in: MedlinePlus

IFITM1 knockdown decreases migration and invasion in AI-resistant MCF-7:5C breast cancer cells. (A) MCF-7:5C cells were transfected with control siRNA (siCon) or IFITM1 siRNA (siIFITM1) for 24 hours and knockdown of IFITM1 protein expression was confirmed by Western blot (right panel) and real-time PCR analyses (left panel). Standard deviations are shown. **P <0.01 versus siCon. (B and C) The effect of IFITM1 knockdown on cell migration (B) and invasion (C) was assessed by transwell migration assay and matrigel invasion assay. Cells that invaded through the Matrigel-coated transwells were fixed, stained, visualized by light microscopy and photographed. Quantitation of the Transwell assay is also shown (B, right panel). Ten random fields were counted per insert at 20X. **P <0.01. (D) Effect of IFITM1 knockdown on cell viability in resistant MCF-7:5C cells. Cells were transfected with siCon or siIFITM1 for 24 hours, then DNA content of cells was analyzed using flow cytometry as described in the Methods section. The arrow is sub G1 phase apoptosis. MTT assay (bottom panel) was also performed in the IFITM1-knockdown cells at 24 hours. All the illustrated data are expressed as mean values of three independent experiments. Standard deviations are shown. AI, aromatase inhibitor; IFITM1, interferon induced transmembrane protein1; MTT, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide.
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Fig6: IFITM1 knockdown decreases migration and invasion in AI-resistant MCF-7:5C breast cancer cells. (A) MCF-7:5C cells were transfected with control siRNA (siCon) or IFITM1 siRNA (siIFITM1) for 24 hours and knockdown of IFITM1 protein expression was confirmed by Western blot (right panel) and real-time PCR analyses (left panel). Standard deviations are shown. **P <0.01 versus siCon. (B and C) The effect of IFITM1 knockdown on cell migration (B) and invasion (C) was assessed by transwell migration assay and matrigel invasion assay. Cells that invaded through the Matrigel-coated transwells were fixed, stained, visualized by light microscopy and photographed. Quantitation of the Transwell assay is also shown (B, right panel). Ten random fields were counted per insert at 20X. **P <0.01. (D) Effect of IFITM1 knockdown on cell viability in resistant MCF-7:5C cells. Cells were transfected with siCon or siIFITM1 for 24 hours, then DNA content of cells was analyzed using flow cytometry as described in the Methods section. The arrow is sub G1 phase apoptosis. MTT assay (bottom panel) was also performed in the IFITM1-knockdown cells at 24 hours. All the illustrated data are expressed as mean values of three independent experiments. Standard deviations are shown. AI, aromatase inhibitor; IFITM1, interferon induced transmembrane protein1; MTT, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide.

Mentions: There is evidence that IFITM1 overexpression induces tumor resistance to natural killer (NK) cells in gastric tumor cells and it facilitates migration and invasion of gastric cancer cells [29]. In addition, overexpression of IFITM1 has been shown to promote head and neck tumor invasion by mediating the expression of matrix metalloproteinases 12 and 13 [32]. To investigate the role of IFITM1 in breast cancer progression, we examined the influence of IFITM1 knockdown on migration and invasion of AI-resistant MCF-7:5C breast cancer cells. Western blot and real-time PCR analysis confirmed that IFITM1 protein and mRNA expression suppressed by siRNA in MCF-7:5C cells compared with siCon-transfected cells (Figure 6A). Silencing of IFITM1 markedly reduced the migratory ability (Figure 6B) and invasion capacity (Figure 6C) of AI-resistant MCF-7:5C cells. The cell migration and invasion counted from 10 randomly selected areas per well at 24 hours showed that siRNA knockdown of IFITM1 inhibited migration by 54% (Figure 6B, bar graph) and invasion by approximately 78% (Figure 6C) compared with siCon-transfected cells. To confirm that the inhibitory effect of IFITM1 knockdown on migration and invasion was not due to cell death we measured apoptosis (via flow cytometry) and cell viability in IFITM1-knockdown MCF-7:5C cells at the same time point (24 hours) the migration and invasion assays were performed. As shown in Figure 6D, IFITM1 knockdown did not induce cell death (top panel) or reduce cell viability (bottom panel) at 24 hours; hence, its inhibitory effect on migration and invasion at 24 hours is not due to cell death. However, we should note that knockdown of IFITM1 does cause significant cell death at 72 hours; hence, migration and invasion would be inhibited at the later time points due to cell death. This result suggests that overexpression of IFITM1 enhances the ability of AI-resistant MCF-7:5C cells to migrate and invade and its suppression has the opposite effect.Figure 6


Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death.

Choi HJ, Lui A, Ogony J, Jan R, Sims PJ, Lewis-Wambi J - Breast Cancer Res. (2015)

IFITM1 knockdown decreases migration and invasion in AI-resistant MCF-7:5C breast cancer cells. (A) MCF-7:5C cells were transfected with control siRNA (siCon) or IFITM1 siRNA (siIFITM1) for 24 hours and knockdown of IFITM1 protein expression was confirmed by Western blot (right panel) and real-time PCR analyses (left panel). Standard deviations are shown. **P <0.01 versus siCon. (B and C) The effect of IFITM1 knockdown on cell migration (B) and invasion (C) was assessed by transwell migration assay and matrigel invasion assay. Cells that invaded through the Matrigel-coated transwells were fixed, stained, visualized by light microscopy and photographed. Quantitation of the Transwell assay is also shown (B, right panel). Ten random fields were counted per insert at 20X. **P <0.01. (D) Effect of IFITM1 knockdown on cell viability in resistant MCF-7:5C cells. Cells were transfected with siCon or siIFITM1 for 24 hours, then DNA content of cells was analyzed using flow cytometry as described in the Methods section. The arrow is sub G1 phase apoptosis. MTT assay (bottom panel) was also performed in the IFITM1-knockdown cells at 24 hours. All the illustrated data are expressed as mean values of three independent experiments. Standard deviations are shown. AI, aromatase inhibitor; IFITM1, interferon induced transmembrane protein1; MTT, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide.
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Related In: Results  -  Collection

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Fig6: IFITM1 knockdown decreases migration and invasion in AI-resistant MCF-7:5C breast cancer cells. (A) MCF-7:5C cells were transfected with control siRNA (siCon) or IFITM1 siRNA (siIFITM1) for 24 hours and knockdown of IFITM1 protein expression was confirmed by Western blot (right panel) and real-time PCR analyses (left panel). Standard deviations are shown. **P <0.01 versus siCon. (B and C) The effect of IFITM1 knockdown on cell migration (B) and invasion (C) was assessed by transwell migration assay and matrigel invasion assay. Cells that invaded through the Matrigel-coated transwells were fixed, stained, visualized by light microscopy and photographed. Quantitation of the Transwell assay is also shown (B, right panel). Ten random fields were counted per insert at 20X. **P <0.01. (D) Effect of IFITM1 knockdown on cell viability in resistant MCF-7:5C cells. Cells were transfected with siCon or siIFITM1 for 24 hours, then DNA content of cells was analyzed using flow cytometry as described in the Methods section. The arrow is sub G1 phase apoptosis. MTT assay (bottom panel) was also performed in the IFITM1-knockdown cells at 24 hours. All the illustrated data are expressed as mean values of three independent experiments. Standard deviations are shown. AI, aromatase inhibitor; IFITM1, interferon induced transmembrane protein1; MTT, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide.
Mentions: There is evidence that IFITM1 overexpression induces tumor resistance to natural killer (NK) cells in gastric tumor cells and it facilitates migration and invasion of gastric cancer cells [29]. In addition, overexpression of IFITM1 has been shown to promote head and neck tumor invasion by mediating the expression of matrix metalloproteinases 12 and 13 [32]. To investigate the role of IFITM1 in breast cancer progression, we examined the influence of IFITM1 knockdown on migration and invasion of AI-resistant MCF-7:5C breast cancer cells. Western blot and real-time PCR analysis confirmed that IFITM1 protein and mRNA expression suppressed by siRNA in MCF-7:5C cells compared with siCon-transfected cells (Figure 6A). Silencing of IFITM1 markedly reduced the migratory ability (Figure 6B) and invasion capacity (Figure 6C) of AI-resistant MCF-7:5C cells. The cell migration and invasion counted from 10 randomly selected areas per well at 24 hours showed that siRNA knockdown of IFITM1 inhibited migration by 54% (Figure 6B, bar graph) and invasion by approximately 78% (Figure 6C) compared with siCon-transfected cells. To confirm that the inhibitory effect of IFITM1 knockdown on migration and invasion was not due to cell death we measured apoptosis (via flow cytometry) and cell viability in IFITM1-knockdown MCF-7:5C cells at the same time point (24 hours) the migration and invasion assays were performed. As shown in Figure 6D, IFITM1 knockdown did not induce cell death (top panel) or reduce cell viability (bottom panel) at 24 hours; hence, its inhibitory effect on migration and invasion at 24 hours is not due to cell death. However, we should note that knockdown of IFITM1 does cause significant cell death at 72 hours; hence, migration and invasion would be inhibited at the later time points due to cell death. This result suggests that overexpression of IFITM1 enhances the ability of AI-resistant MCF-7:5C cells to migrate and invade and its suppression has the opposite effect.Figure 6

Bottom Line: Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death.Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells.Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Estrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance.

Methods: Real-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression.

Results: We found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells.

Conclusion: Overall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.

Show MeSH
Related in: MedlinePlus