Research ArticleArticles
Open Access

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

DER SHENG SUN, JUNG-SOOK YOON, YONG-SEOK KIM and HYE SUNG WON
Cancer Genomics & Proteomics September 2024, 21 (5) 511-522; DOI: https://doi.org/10.21873/cgp.20468

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.

Key Words:

Breast cancer is one of the most common cancers in women and a highly heterogeneous disease. It is classified as hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-positive, and triple-negative breast cancer, according to the expression of estrogen receptor (ER), progesterone receptor, and HER2 (1). HR-positive breast cancer is the most common subtype, for which endocrine therapy is used as an important treatment option besides cytotoxic chemotherapy. Numerous preclinical and clinical studies have been conducted to overcome endocrine resistance and improve the efficacy of endocrine therapy (2, 3).

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is an important signaling cascade for various cytokines and growth factors, including interferons (IFN) and interleukins. Multiple studies revealed that JAK/STAT pathway dysregulation was associated with various cancers and autoimmune diseases (4-6). The JAK/STAT pathway plays a key role in the transcriptional activation of interferon-stimulated genes (ISGs), which contribute to multiple cellular and immune responses in various ways. Briefly, IFN activates JAK and tyrosine kinase, leading to phosphorylation of STAT1 and STAT2 and dimerization. These STAT dimmers bind to IFN response factor 9 (IRF9) to form IFN-stimulated gene factor 3, which translocates into the nucleus and binds to IFN-stimulated response elements to induce the expression of ISGs (4, 7).

The interferon-induced transmembrane (IFITM) proteins are members of the ISG family, which are up-regulated by both type I and type II IFNs. These proteins are important for immunity and anti-viral response by preventing viruses from traversing the lipid bilayer of the cell and accessing the cytoplasm (8). Recent studies reported that IFITM proteins were up-regulated in several types of cancers and associated with tumorigenesis, including cell proliferation, invasion, angiogenesis, and drug resistance (9, 10).

The tumor suppressor p53, encoded by the TP53 gene, is critical for regulating DNA repair and cell division. Loss of p53 function not only impairs its antitumor activity but also leads to the gain of novel oncogenic functions (11). TP53 is the most frequently mutated gene in human cancers. TP53 mutation, which usually results in loss of function of the p53 protein, is found in approximately 12%-32% of luminal breast cancer subtypes (12). Moreover, p53 status is associated with the prognosis and affects the response to treatment, including endocrine therapy for breast cancer (11, 13-15). With remarkable advances in immuno-oncology, recent studies are focused on the emerging roles of p53 in immune regulation, including its interaction with interferon signaling (13, 16, 17).

This study aimed to investigate the potential of IFITM1 as a druggable target in MCF-7 cells, which are an ER-positive breast cancer cell line with wild-type p53, and tamoxifen-resistant breast cancer (TamR) cells. Our findings suggest that p53 enhanced IFN signaling in MCF-7 cells and p53 status may significantly impact growth inhibition by targeting IFITM1 in HR-positive breast cancer.

Materials and Methods

Cell culture. The ER-positive luminal breast cancer cell lines, MCF-7 and T47D, were obtained from the American Type Culture Collection and Korean Cell Line Bank, respectively. MCF-7 cells have a wild-type p53 with a functional p53 protein, whereas T47D cells have a mutated p53 with a loss of p53 function (18). MCF-7-derived TamR cells were established by long-term culture of MCF-7 cells with 4-hydroxytamoxifen (4-OH TAM, #54965-24-1, Selleckchem, Houston, TX, USA) in an estrogen-free medium as previously described (19). MCF-7, T47D, and TamR cells were cultured in RPMI 1640 medium (#LM011-03, Welgene, Republic of Korea) and phenol-red-free RPMI 1640 medium (#LM011-02, Welgene, Republic of Korea) containing 10% fetal bovine serum and 1% penicillin/streptomycin. For TamR cell culture, 3 μM of 4-OH-TAM was added in the culture medium. Cells were cultured at 37°C in a humidified incubator containing 5% CO2.

Reagents and antibodies. The drugs 4-OH-TAM (#54965-24-1) and ruxolitinib (#S1378) were purchased from Selleckchem. Ruxolitinib is an orally available selective JAK1/2 inhibitor approved by the Food and Drug Administration for use in myelofibrosis, polycythemia vera, and graft-versus-host disease (4). Tamoxifen and ruxolitinib were each dissolved in DMSO at a stock concentration of 10 mM. Anti-p53 (#sc-126), anti-IFITM1 (#sc-374026), anti-ERα (#sc-71064), anti-SOCS1 (#sc-518028), anti-SOCS3 (#sc-51699), and anti-β-actin (#sc-47778) antibodies were purchased from Santa Cruz Technology Inc. (Dallas, TX, USA). Anti-STAT1 (#9172) and anti-pSTAT1 (Tyr701) (#9167) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Small interfering RNA (siRNA) transfection. MCF-7 and TamR were transiently transfected with p53 siRNA (#sc-29435, Santa Cruz), IFITM1 siRNA (#sc-44549, Santa Cruz), and negative control siRNA (#sc-37007, Santa Cruz) using the Lipofectamine® RNAiMAX reagent (#13778-075, Thermo Fisher Scientific, Waltham, MA, USA). A total of 2.5×105 and 1×104 cells were seeded per well in 6- and 96-well plates, respectively. Each siRNA was used at a concentration of 20 nM, and cells were incubated for 72 h after transfection. The rest of the steps were performed according to the manufacturer’s manual. The efficiency of knockdown was determined using real-time polymerase chain reaction (RT-PCR) and western blotting.

Real-time polymerase chain reaction. Total RNA was extracted using an RNeasy mini kit (#74104, Qiagen, Hilden, Germany). cDNA synthesis was performed using 1 μg total RNA and RevertAid First Strand cDNA Synthesis kit (#K1622, Thermo Fisher Scientific) according to the manufacturer’s manual. RT-PCR was conducted using iTaq™ Universal SYBR® Green Supermix (#172-5121, Bio-Rad, Hercules, CA, USA) and Bio-Rad CFX96 real-time system (Bio-Rad) with specific primers for p53 (F: 5′-GTTCCGAGAGCTGAATGAGG-3′, R: 5′-TCTGAGTCAGGCC CTTCTGT-3′), IFITM1 (F: 5′-TCCACCGTGATCAACATCCA-3′, R: 5′-AATCAGGGCCCAGATGTTCA-3′), IRF9 (F: 5′-CCACCGA AGTTCCAGGTAACAC-3′, R: 5′-AGTCTGCTCCAGCAAGTA TCGG-3′), SOCS1 (F: 5′-TTCGCCCTTAGCGTGAAGATGG-3′, R 5′-TAGTGCTCCAGCAGCTCGAAGA-3′), SOCS3 (F: 5′-CATCTCTGTCGGAAGACCGTCA-3′, R 5′-GCATCGTACTGGT CCAGGAACT-3′), and β-actin (F: 5′-CCTGGCACCCAGCA CAAT-3′, R 5′-GCCGATCCACACGGAGTA-3′). Relative mRNA expression levels were normalized using β-actin.

Western blot analysis. Protein was obtained from the harvested cells using RIPA Lysis and Extraction Buffer (#89900, Thermo Fisher Scientific) with Halt™ Protease and Phosphatase Inhibitor Cocktail (#78440, Thermo Fisher Scientific). Proteins on acrylamide gel were transferred to polyvinylidene difluoride membrane (#IPVH00010, Merck Millipore, Burlington, MA, USA). The membranes were incubated with 5% non-fat dried milk for 1 h at room temperature and then incubated with diluted primary antibodies at 4°C overnight. Protein signals were detected using Western Blotting Luminol Reagent (#sc-2048, Santa Cruz).

Cell viability assay. A total 1×104 cells were seeded per well in a 96-well plate. Cell viability was evaluated in triplicate using Cell Counting Kit 8 (WST-8; #ab228554, Abcam, Cambridge, MA, USA) according to the manufacturer’s manual. The absorbance was measured at 460 nm using SpectraMax® i3x (Molecular Devices, San Jose, CA, USA).

Migration assay. A total of 1×105 cells were seeded on the upper chamber of a transwell (#3464, Corning, Kennebunk, ME, USA) in 200 μl of serum-free media. Then, 750 μl of culture media was added to the lower chamber of the transwell. The membrane of the upper chamber was fixed using cold methanol and stained using 0.2% crystal violet solution (#V5265, Sigma, St. Louis, MO, USA) after 24 h. The stained membranes were imaged using an optical microscope.

Colony-forming assay. A total of 5×103 MCF-7 and TamR cells were seeded per well in a 6-well plate. Cells were treated with or without tamoxifen and grown for 10 days with fresh media replacement every 5 days. The colonies were stained using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide solution (#ab211091, Abcam) and scanned as a whole plate.

Flow cytometry analysis of apoptosis. A total of 2.5×105 MCF-7 and TamR cells were seeded per well in a 6-well plate. Cells were treated with or without 9 μM tamoxifen and 20 μM ruxolitinib for 72 h. Cells were harvested using trypsin-ethylenediamine tetraacetic acid, and the supernatant was included. For analysis of apoptosis, cells were stained according to the manual of Annexin V-FITC Apoptosis Staining/Detection Kit (#ab14085, Abcam) and detected using FACSCanto™ II Flow Cytometer (BD Biosciences, San Jose, CA, USA). The results were obtained using BD FACSDiva Software (BD Biosciences).

Analysis using a public database. The FireBrowse gene expression viewer (Broad Institute of MIT and Harvard; http://www.firebrowse.org/) is used to visualize expression data collected from various whole genome RNA-seq studies. The Cancer Genome Atlas (TCGA) RNA-seq data of IFITM1 in different types of cancers was downloaded using FireBrowse. The cBioPortal database (http://www.cbioportal.org/) is used to explore and visualize genomic data of patients with cancer from various sources, including TCGA. We used cBioPortal to analyze the mRNA expression levels according to p53 status.

Statistical analysis. The data are presented as mean±standard deviation (SD). Statistical analysis was based on data obtained from at least two or three independent experiments performed in triplicate. The differences between two groups were calculated using unpaired t-test. Alternatively, more than two groups were compared using ANOVA with Tukey’s post hoc test. p<0.05 was considered statistically significant.

Results

IFITM1 inhibition suppressed cell proliferation and enhanced sensitivity to tamoxifen in MCF-7 cells. Using the FireBrowse portal, we found that IFITM1 expression was up-regulated in breast cancer compared to matched normal tissue (Figure 1A). This finding indicates that IFITM1 may have oncogenic activities in breast cancer. To evaluate the effect of IFITM1 on cell survival and migration, we used siRNA to knockdown IFITM1 expression in MCF-7 cells (Figure 1B and C). We found that IFITM1 inhibition using siRNA significantly decreased cell growth and migration compared with those in control siRNA-treated MCF-7 cells, indicating that IFITM1 has oncogenic potential in wild-type p53, ER-positive MCF-7 cells (Figure 1D and E). The cell viability assay indicated that MCF-7 cells with IFITM1 inhibition using siRNA showed more reduced cell viability after tamoxifen treatment than that in control siRNA-treated cells (Figure 2A). Ruxolitinib, which is a selective, oral inhibitor of JAK1 and 2, was used to inhibit IFITM1, and its use effectively suppressed the mRNA and protein levels of IFITM1 (Figure 2B and C). Cell viability was significantly reduced after treatment with ruxolitinib and tamoxifen compared with tamoxifen alone (p<0.0001; Figure 2D). Annexin V-propidium iodide staining showed that using ruxolitinib with tamoxifen increased the total number of dead cells from 14.0% (tamoxifen only) to 19.7% (tamoxifen + ruxolitinib; Figure 2E). This finding indicates that IFITM1 inhibition enhanced the sensitivity to tamoxifen in MCF-7 cells.

Figure 1.
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.
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.

TamR cells showed decreased IFITM1 expression and IFITM1 inhibition did not affect cell proliferation. We evaluated whether IFITM1 played the same role in TamR cells. To confirm the resistance to tamoxifen in TamR cells, we performed cell viability and colony-forming assays of MCF-7 and TamR cells after treatment with tamoxifen. Colony formation was inhibited by tamoxifen treatment in MCF-7 cells but not in TamR cells (Figure 3A). Cell viability was significantly decreased in MCF-7 cells in a dose-dependent manner, whereas no change was observed in the cell viability of TamR cells (p<0.001 and <0.0001 for 3 and 9 μM, respectively; Figure 3B). Unexpectedly, protein and mRNA levels of IFITM1 were decreased in TamR cells compared with those in MCF-7 cells. We found that protein and mRNA levels of p53 were also decreased in TamR cells compared with those in MCF-7 cells (Figure 3C and D). We evaluated changes in protein and mRNA levels of IFITM1 and p53 after tamoxifen treatment in MCF-7 cells to validate this observation. We found that IFITM1 and p53 expression were decreased after tamoxifen exposure in MCF-7 cells, similar to that in TamR cells (Figure 3E and F). TamR cells with the inhibition of IFITM1 using siRNA showed no change in cell viability after treatment with tamoxifen, unlike that in MCF-7 cells (Figure 4A). Ruxolitinib treatment also did not reduce cell viability and increase apoptosis in TamR cells, suggesting that IFITM1 inhibition does not affect cell survival in TamR cells (Figure 4B and C).

Figure 3.
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.
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.

P53 status can affect IFITM1 expression in MCF-7 cells. Because the expressions of both p53 and IFITM1 were observed to decrease in TamR cells, we evaluated their interactions. We found that p53 knockdown using siRNA decreased IFITM1 expression in MCF-7 cells, whereas IFITM1 inhibition using siRNA did not affect or slightly increased p53 expression. Therefore, we investigated the effects of p53 knockdown on other molecules related to the induction of ISGs, including IRF9 and suppressors of cytokine signaling (SOCS). IRF9 is a key transcriptional factor in the JAK/STAT signaling pathway, leading to the expression of hundreds of ISGs (4). SOCS, especially SOCS1 and SOCS3, are negative feedback regulators in the JAK/STAT signaling pathway and prevent continuous activation by switching off the signaling cascade (20). P53 knockdown using siRNA reduced mRNA levels of IRF9 and increased mRNA and protein levels of SOCS3 in MCF-7 cells (Figure 5A-C). We also found increased mRNA levels of SOCS3 in TamR cells (Figure 5D). T47D cells, which express inactive mutant p53 protein, showed decreased mRNA and protein levels of IFITM1 compared with those in MCF-7 cells (Figure 5E). We verified the relationship between p53 and IFITM1 using cBioPortal containing TCGA data from 825 breast cancer cases. IFITM1 and IRF9 mRNA levels were decreased, and SOCS3 mRNA levels were slightly increased in breast cancer cases with TP53 mutations compared with breast cancer cases without TP53 mutations (Figure 5F). Taken together, these findings suggest that loss or mutation of p53 can affect the induction of IFITM1 via the JAK/STAT signaling pathway in breast cancer.

Figure 5.
Figure 5.
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.

P53 status is important for cell death after tamoxifen treatment or IFITM1 inhibition. MCF-7 cells showed increased tamoxifen sensitivity after IFITM1 inhibition, whereas TamR cells showed decreased expression of both IFITM1 and p53 and were unaffected by IFITM1 inhibition. Therefore, we evaluated whether p53 knockdown affects cell death by IFITM1 inhibition in MCF-7 cells. MCF-7 cells with p53 knockdown using siRNA showed no decrease in cell viability after tamoxifen treatment or IFITM1 inhibition (Figure 6A and B). This indicates that p53 status may be important for cell death by tamoxifen or IFITM1 inhibition.

Figure 6.
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.

Discussion

IFITM1, which is mainly located in the plasma membrane, is important for immunity, anti-viral activity, and cellular functions, such as adhesion and proliferation through interactions with other membrane proteins (8). It has been shown to play different roles in various types of cancers: its up-regulation and down-regulation occur depending on the cancer type, and the exact role in tumorigenesis is complex in each cancer (9, 10, 21). Although IFITM1 is mainly overexpressed and has pro-oncogenic functions in many types of cancers, the precise regulation of its expression and molecular mechanisms of oncogenic processes have not been completely elucidated.

In previous studies on breast cancer, IFITM1 overexpression enhanced the aggressiveness of triple-negative breast cancer cells (22) and aromatase inhibitor-resistant breast cancer cells (23), suggesting that targeting IFITM1 may benefit patients with these diseases. Fu et al. reported seven inflammatory-related genes associated with the prognosis of breast cancer through analysis of TCGA data. IFITM1 was identified as one of those prognostic inflammatory-related genes, and up-regulated IFITM1 expression was associated with poor survival outcomes in breast cancer patients (24). In the present study, we investigated the role of IFITM1 in wild-type p53, ER-positive MCF-7 breast cancer cells and MCF-7-derived TamR cells. We found that IFITM1 inhibition decreased cell proliferation and migration in MCF-7 cells, and a combination of tamoxifen and ruxolitinib enhanced cell death in MCF-7 cells. Considering this oncogenic potential of IFITM1, we expected that IFITM1 expression would be increased in TamR cells, but we found the opposite result: TamR cells showed decreased IFITM1 expression. In addition, IFITM1 inhibition did not induce a decrease in cell viability of TamR cells. Regarding the mechanism of decreased IFITM1 expression in TamR cells, we noted that p53 expression was decreased along with that of IFITM1. This similar pattern, in which the expressions of p53 and IFITM1 decreased, was also observed even after treating MCF-7 cells with tamoxifen. According to these findings, we confirmed that IRF9 was down-regulated and SOCS3 was up-regulated by p53 knockdown, suggesting that p53 might regulate the expression of ISGs, such as IFITM1, through the control of positive and negative regulators in the JAK/STAT signaling pathway. These findings were consistent with previous reports on a mechanistic link between p53 and ISG induction. Muñoz-Fontela et al. reported a significant function of p53 in the activation of the IFN pathway. They found that p53 enhanced IFN signaling by the direct transcriptional induction of IRF9 (25). In addition to IRF9, p53 can activate the expression of several immune response genes, including chemokine ligand 2, IFN regulatory factor 5, immune-stimulated gene 15, and Toll-like receptor 3. Yan et al. and Zhu et al. demonstrated the role of p53 in promoting IFN signaling. They showed that p53-knockout mice were more susceptible to the influenza A virus and had significantly changed the expression of genes associated with IFN signaling (26). In another study, the up-regulation of ISGs in p53-deficient cells was attenuated after exposure to influenza A virus and IFN, suggesting that p53 plays an essential role in enhancing IFN signaling against viral infection (27). Recent evidence indicated that p53 is involved in crucial roles of tumor immunology and the regulation of immune response by directly activating key regulators of the immune signaling pathway, regulating the production of several cytokines and chemokines, and regulating immune checkpoint programmed cell death ligand 1 expression (16, 17, 28). Therefore, the role of p53 is expected to become more important in the era of immuno-oncology.

Our findings indicate the possibility of IFITM1 being a druggable target in wild-type p53, HR-positive breast cancer, but not in tamoxifen-resistant breast cancer. P53-dependent up-regulation of IFITM1 expression may contribute to tumor aggressiveness in MCF-7 cells, and IFITM1 inhibition showed enhanced sensitivity to tamoxifen. In addition, p53 expression was decreased in MCF-7 cells after tamoxifen treatment and in TamR cells. Notably, p53 knockdown eliminated the effect of cell death induced by IFITM1 inhibition in MCF-7 cells. These findings suggest that functionally active p53 may be an important factor involved in growth inhibition after tamoxifen treatment or IFITM1 inhibition in HR-positive breast cancer.

Several studies have shown that loss of functional p53 is correlated with poor prognosis and resistance to endocrine therapy. Fernandez-Cuesta et al. reported that p53-mutated cells were more resistant to the cytotoxic effects of tamoxifen than p53-wild-type cells (29). Lu et al. reported that the reactivation of p53 using an MDM2 inhibitor might be an effective therapeutic strategy for the treatment of endocrine-resistant breast cancer retaining wild-type p53 (30). Berns et al. reported that TP53 mutations were significantly associated with a poor response to tamoxifen, with an odds ratio of 0.22 in patients with advanced breast cancer (15). Consistent with these reports, our findings suggest a functional link between the p53 status and response to certain oncological therapeutic modalities in wild-type p53, HR-positive breast cancer.

Several preclinical studies showed the anticancer effect of ruxolitinib in breast cancer cell lines under various conditions (10, 22, 23). Despite the favorable efficacies of ruxolitinib in preclinical studies, early-phase studies using ruxolitinib failed to show clinically meaningful responses in metastatic breast cancer. Makhlin et al. examined the activity of ruxolitinib in combination with endocrine therapy in the endocrine-resistant metastatic setting (31). In a phase II single-arm trial, combining ruxolitinib with exemestane in patients with HR-positive metastatic breast cancer after progression on non-steroidal aromatase inhibitors, had a clinical benefit rate of 24% in the overall study population, but the pre-specified response criteria were not met (31). These findings highlight the need for predictive biomarkers to select patients for whom drugs targeting JAK/STAT signaling may be more effective in breast cancer, and further investigation is needed on the role of p53 with regards to that.

Regarding SOCS3 negatively regulating JAK/STAT signaling, increasing evidence shows that SOCS3 affects tumor cell growth, metastasis, and chemotherapy resistance through various signaling pathways and immune molecules (5, 20, 32). Consistent with our findings on SOCS3 in TamR cells, Cheng et al. reported gene expression pattern changes in response to tamoxifen resistance by analyzing gene expression profiles from the Gene Expression Omnibus database (33). They showed that SOCS3 was up-regulated in the tamoxifen-resistant MCF-7 cells compared with wild-type MCF-7 cells and 16 genes, including SOCS3, were significantly associated with relapse-free survival in patients with HR-positive breast cancer who had undergone endocrine therapies.

Conclusion

Our findings suggest that IFITM1 inhibition may enhance the sensitivity to tamoxifen based on p53-dependent enhancement of IFN signaling in wild-type p53, HR-positive breast cancer cells. This anticancer effect by IFITM1 inhibition may be abolished in tumors with an altered p53 status and after the development of tamoxifen resistance. Therefore, the strategy of early combining drugs targeting IFITM1 with endocrine therapy may be a better treatment option for patients with HR-positive breast cancer retaining functional p53. Further investigation is warranted to understand the interplay between IFN and p53 signaling pathway.

Footnotes

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Authors’ Contributions

    All Authors have read and approved the final manuscript and made the following contributions: HSW: Conceptualization, funding acquisition, writing – review and editing. YK: Formal analysis, supervision, writing – review and editing. JY: Data curation, formal analysis, methodology. DSS: Visualization, writing – original draft.

  • Funding

    This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (Ministry of Science and ICT) (No. 2021R1C1C101045, H.S.W) and by The Catholic University of Korea, Uijeongbu St. Mary’s Hospital Clinical Research Laboratory Foundation made in the program year of 2023 (H.S.W). The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

  • Received May 13, 2024.
  • Revision received June 20, 2024.
  • Accepted July 3, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells
DER SHENG SUN, JUNG-SOOK YOON, YONG-SEOK KIM, HYE SUNG WON
Cancer Genomics & Proteomics Sep 2024, 21 (5) 511-522; DOI: 10.21873/cgp.20468
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