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ORIGINAL ARTICLE
Year : 2021  |  Volume : 17  |  Issue : 5  |  Page : 1192-1201

Human epidermal growth factor receptor 2 inhibits activating transcription factor 7 to promote breast cancer cell migration by activating histone lysine demethylase 1


1 Department of Breast Surgery, School of Medicine, Women and Children's Hospital, Xiamen University, Xiamen, China
2 Department of General Surgery, The First Affiliated Hospital of Xiamen University, Xinglin Branch, Xiamen, China

Date of Submission23-Apr-2021
Date of Acceptance07-Oct-2021
Date of Web Publication27-Nov-2021

Correspondence Address:
Tao Wang
Department of General Surgery, The First Affiliated Hospital of Xiamen University, Xinglin Branch, Xiamen 361000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.jcrt_649_21

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 > Abstract 


Background: Receptor tyrosine-protein kinase erbB-2 (human epidermal growth factor receptor 2 [HER2])-based therapies can improve the prognosis of HER2-positive breast cancer (BRCA) patients; however, HER2-positive patients with distal metastasis do not gain significant clinical benefit from molecular targeted therapy.
Materials and Methods: A database analysis, immunohistochemistry, and quantitative real-time polymerase chain reaction were used to evaluate the expression of activating transcription factor 7 (ATF7) and its clinical value. A transwell chamber assay was used to assess migration and cell signaling was assessed by immunoblotting.
Results: ATF7 was expressed at a low level in HER2-enriched BRCA specimens compared with normal or HER2-negative specimens, which was corroborated in HER2-positive tissue chips and cultured cells. ATF7 gradually decreased with increased tumor stage and low ATF7 was associated with poor prognosis in HER2-positive BRCA patients. ATF7-upregulation inhibited, whereas ATF7-knockdown promoted migration, activity of matrix metalloproteinase 9 (MMP9), MMP2, and uridylyl phosphate adenosine and plasminogen activator inhibitor-1 (PAI-1) expression in HER2-positive cells. HER2 overexpression markedly reduced ATF7 expression in MCF-10A mammary epithelial cells, along with decreased E-cadherin, and increased N-cadherin and migration, which were abrogated by exogenous ATF7 transfection. Mechanistically, HER2 upregulation mediated the decline of ATF7 and activated histone lysine demethylase 1 (LSD1), followed by elevation of histone H3K9 dimethylation (H3K9me2) and H3K4me2. However, the enhanced effects on LSD1 and H3K9me2, excluding H3K4me2, were abrogated by exogenous ATF7. ATF7 was negatively associated with KDM1A (encoding LSD1 protein) expression.
Conclusions: ATF7 may be a useful diagnostic and prognostic marker for metastatic HER2-positive BRCA. The ATF7/LSD1/H3K9me2 axis may be responsible for metastasis in HER2-positive cells.

Keywords: Activating transcription factor 7, H3K9 dimethylation, histone lysine demethylase 1, human epidermal growth factor receptor 2-positive breast cancer, metastasis


How to cite this article:
Lin J, Mao H, Ji Z, Lin W, Wang T. Human epidermal growth factor receptor 2 inhibits activating transcription factor 7 to promote breast cancer cell migration by activating histone lysine demethylase 1. J Can Res Ther 2021;17:1192-201

How to cite this URL:
Lin J, Mao H, Ji Z, Lin W, Wang T. Human epidermal growth factor receptor 2 inhibits activating transcription factor 7 to promote breast cancer cell migration by activating histone lysine demethylase 1. J Can Res Ther [serial online] 2021 [cited 2022 Jan 17];17:1192-201. Available from: https://www.cancerjournal.net/text.asp?2021/17/5/1192/331311




 > Introduction Top


Breast cancer (BRCA) is recognized as a heterogeneous disease driven by various biologic factors.[1] Human epidermal growth factor receptor 2 (HER2), encoded by the ERBB2 gene, is a key molecular driver for BRCA.[2] High expression of HER2 is observed in approximately 20%–30% of all BRCA cases.[3] Currently, three specific HER2-targeting antibodies, including trastuzumab, pertuzumab, and T-DM1 are widely used for the treatment of HER2-positive BRCA.[4] Although HER2-based therapies have dramatically ameliorated the outcome of early and advanced HER2-positive BRCA, 30%–40% of HER2-positive patients do not benefit from molecular targeted therapy.[5],[6],[7] Therefore, the mechanisms underlying resistance to HER2-based therapies need to be elucidated to improve the prognosis and treatment of HER2-positive BRCA.

The activating transcription factors (ATFs), a class of ATF/cAMP response element-binding proteins, consist of seven members, ATF1 ∼7.[8],[9] Among these, ATF7 is ubiquitously expressed in various tissues, including human tumors.[10],[11] ATF7 shows diverse physiological functions and regulates cell survival.[12] ATF7 knockout, combined with key mutations in ATF2, induces cell death of developing hepatocytes and hematopoietic cells during embryonic development.[13] The elevation of ATF7 in hepatitis B virus-associated hepatocellular carcinoma (HCC) determines the fate of these cancer cells by regulating heat shock protein A member 1B, and subsequently affects liver cancer progression.[14] ATF7 inhibition contributes to the proliferation of gastric cancer.[15] Thus, ATF7 plays a dual role in tumor cell growth. Studies have also demonstrated that the ATF family regulates cancer cell migration through diverse signaling pathways. ATF6 facilitates cell migration by regulating endoplasmic reticulum (ER) stress and MAPK signaling in cervical cancer.[16] ATF1 upregulation leads to the migration and invasion of lung cancer cells.[17] ATF2, which is highly homologous with ATF7, displays oncogenic activities in melanoma and enhances the migration of hepatoma cell lines.[18] However, whether ATF7 affects metastatic events in BRCA cells, particularly in HER2-positive cancer cells, is unclear.

Histone lysine demethylase 1 (LSD1), the first identified histone demethylase, is responsible for removing the methyl group from methylated histone H3 at lysine 4 (H3K4me1/2) and lysine 9 (H3K9me1/2).[19],[20] LSD1 is highly expressed in various tumor types, including BRCA. LSD1 is overexpressed in ER-negative breast tumors and increased expression is associated with disease progression from ductal carcinoma in situ to invasive ductal BRCA.[21] Estrogen signaling induces the activation of LSD1, leading to BRCA cell overproliferation by regulating the degradation of H3K9me2.[22] LSD1 activation promotes invasion and metastasis of BRCA by activating H3K4me2 and H3K9me2 demethylation.[23] ATF7-dependent epigenetic changes cause a decrease in H3K9me2 in testicular germ cells.[24] Furthermore, ATF7-induced tumor cell migration is associated with the LSD1/H3K9me2 signaling pathway.

In the present study, we determined the expression pattern of ATF7 and the diagnostic and prognostic value of ATF7 in HER2-positive BRCA. In addition, the association between HER2 and ATF7 and the role of ATF7 in HER2-mediated cell migration and the underlying epigenetic regulatory mechanism were explored. Our findings provide new insights into the mechanisms by which HER2 promotes cancer cell migration.


 > Materials and Methods Top


Data collection and analysis

Transcriptional expression of ATF7 in human BRCA specimens and normal tissues, association between ATF7 expression status and tumor stage in all BRCA cases, and overall survival according to ATF7 expression status were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) version 2 database.[25] Expression profiles of ATF7 in different BRCA subtype tissues were obtained from the BRCA Gene Expression Miner v4.4 (bc-GenExMiner v4.4). Gene expression profiles of ATF7/ERBB2 (HER2) and the corresponding clinical information associated with HER2-positive BRCA patients were downloaded from the Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/). Association between ATF7 and tumor stage of HER2-positive patients was analyzed by GraphPad Prism 6.0 (San Diego, US, GraphPad Software, Inc.) according to the downloaded database from TCGA. Overall survival of HER2-positive BRCA patients was determined using the online database of Kaplan-Meier Plotter.[26] The associations between ERBB2 expression and ATF7, and between KDM1A expression and ATF7, were assessed by SPSS software using the expression profiles downloaded from TCGA.

Tissue microarray and immunohistochemistry

A tissue microarray containing 96 cases of HER2-negative and 40 cases of HER2-positive BRCA specimens were purchased from OUTDO Biotechnology Co., LTD (Shanghai, China). All experiments were approved by the Ethics Committee of the Women and Children's Hospital. Briefly, tissue slices were dewaxed in xylene, rehydrated in gradient ethanol, and subjected to antigen retrieval using a microwave. After neutralizing endogenous peroxidase using 3% H2O2 and blocking with 10% BSA, the slides were stained with primary antibodies against ATF7 (Abcam, ab231786, UK), LSD1 (Abcam, ab62582, UK), and H3K9m2 (Abcam, ab176882, UK) overnight at 4°C. The next day, the slides were washed three times with tris-buffered saline plus Tween 20 (1xTBST) and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The slides were visualized using DAB (ZL1-9081, ZSGB-BIO, China) and observed under an inverted microscope (IX51, Olympus, Japan). The staining intensity was measured by Image Pro Plus 6.0 software (Rockville, US) and analyzed by three independent pathologists who were blinded to this study. No positive cells were defined as negative (−). The number of positive cells greater than 20%, 20%–50%, and >50% was defined as weakly positive (+), middle positive (++), and strong positive (+++), respectively.

Cell culture

The immortalized human mammary epithelial cell, MCF-10A, luminal A subtype BRCA cell lines MCF-7 and T47D, and HER2-enriched cell lines, SKBR3 and MDA-MB-453, were obtained from the Chinese Academy of Sciences. MCF-10A, SKBR3, and MDA-MB-453 were cultured with RPMI 1640 (SH30809.01, Hyclone, US) medium supplemented with 10% fetal bovine serum (FBS) (SH30070.03, Hyclone, US) in a 37°C humidified incubator with 5% CO2. MCF-7 and T47D cells were cultured with complete Dulbecco's modified Eagle's medium (DMEM; 11320-033, GIBCO, USA) supplemented with 10% FBS.

Cell transfection and measurement

The coding fragment (CDS) of ATF7 was cloned into the pEGFP-C1 plasmid. Human HER2 fragments were subcloned into the Plenti-M3 vector (CH805581, Vigene Biosciences, Jinan, China). Subsequently, the vector plasmid, recombinant ATF7-overexpressed plasmid, and recombinant HER2-overexpressing plasmid were transfected into cells using Turbofect transfection reagent (R0531, Thermo, USA). After 24 h, the cells were harvested for further studies. Knockdown of human ATF7 was done using shRNAs targeting ATF7 (Targeting ATF7 which is a sequence in the CDS), which were synthesized by Guangzhou RiboBio Co., Ltd. To generate ATF7-knockdown cells, cells transfected with ATF7 shRNA were selected in 600 μg/ml G418. For the measurement of LSD1 activity, cells were harvested and lysed, and the lysates were used to monitor the activity of LSD1 using the LSD1 Activity Quantification Assay (Epigentek, P-3079) according to the manufacturer's instructions.

Quantitative real-time polymerase chain reaction

TRIzol reagent (Aidlab, 252250AX) was used to extract total RNA. Then, 1 μg of total RNA was reverse transcribed to cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The polymerase chain reactions (PCRs) were performed using the All-in-One miRNA quantitative real-time PCR (qRT-PCR) Detection Kit (GeneCopoeia). The results were analyzed using a CFX96 instrument (Bio-Rad). The sequences of the primers are presented in [Table 1]. Primers for miR-103a-3p and U6 were purchased from ABM (Vancouver, Canada) (hsa-miR-103a-3p, Cat. # MPH 02063; human U6, Cat. # MPM00001). GAPDH was used as an endogenous control for PCAT18, ATF7, matrix metalloproteinase 9 (MMP9), and MMP2 mRNAs, and universal small nuclear RNA U6 was used as a control for miR-103a-3p. Relative gene expression was calculated by the relative quantification (2− ΔΔ Ct) method.
Table 1: Primer sequences of quantitative real-time polymerase chain reaction

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Western blot analysis

Total protein was isolated from cultured cells using RIPA lysis buffer. Then, 40 μg protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against HER2 (Abcam, ab237715, UK), ATF7 (Abcam, ab231786, UK), E-cadherin (Abcam, ab1416, UK), N-cadherin (Abcam, ab18203, UK), LSD1 (Abcam, ab62582, UK), H3K9m2 (Abcam, ab176882, UK), H3K4me2 (Abcam, ab32356, UK), and β-actin (Sigma, A2228, US). The bands were subsequently visualized using an ECL chemiluminescence reagent kit (Millipore, Billerica, MA, USA) and observed by ChemiDoc MP Imaging System (BIO-RAD).

Transwell assay

A Transwell (PIEP12R48, Millipore, Germany) apparatus was used to evaluate the migration rate of MCF-10A, SKBR3, and MDA-MB-453 cells. Briefly, 800 μL of complete medium was added to the lower chambers. Cells (2 × 105 cells/mL), resuspended in serum-free medium, were added to the upper chamber and allowed to migrate for 12 h. Subsequently, the upper surface of each membrane was cleaned with a cotton swab. Cells attached to the bottom surface were stained with 0.1% crystal violet dye (Sigma) for 20 min and the cells were counted using a DMR inverted microscope (Olympus, IX51).

Statistical analysis

All assays were performed at least three times. The relationship between tumor stage and ATF7 expression was analyzed with GraphPad Prism 6.0 (GraphPad Software, Inc.) using a one-way ANOVA. Data were presented as the mean ± standard deviation. Data between two groups were compared with GraphPad Prism 6.0 using a Student's t-test and data from multiple groups were compared using a one-way ANOVA. Overall survival curves were generated by GEPIA 2 and Kaplan–Meier Plotter. The difference between ATF7 in HER2-negative and HER2-positive BRCA tissues was analyzed by GraphPad Prism 6.0 using Fisher's exact test. The association between ATF7 and/or HER2/LSD1 was determined using SPSS software. Statistically significant differences were defined at P < 0.05.


 > Results Top


Activating transcription factor 7 is minimally expressed in human epidermal growth factor receptor 2-enriched breast cancer

The expression of ATF7 was evaluated in BRCA cases obtained from an online database. As shown in Supplementary [Figure 1] [Figure S1], no difference was observed in ATF7 expression between normal (n = 291) and breast tumor tissues (n = 1085). In contrast, the luminal A subtype (HER2 negative) of BRCA exhibited a significant increase in ATF7 expression, whereas a notable decrease was evident in HER2-enriched and basal-like (triple-negative BRCA [TNBC]) patients [Figure 1]a. ATF7 levels were also significantly increased in luminal A subtype subjects compared with that in HER2-enriched subjects [Figure 1]a. Since HER2 was highly expressed in HER2-enriched subjects and it was not expressed in luminal A subtype subjects, the differential expression of ATF7 in both cancer types was of interest. Combined with the online database and immunohistochemistry data from tissue microarrays, low expression of ATF7 was found in HER2-positive BRCA specimens compared with HER2-negative specimens [Figure 1]b and [Figure 1]c. The results indicated that 28 out of 40 (70%) HER2-positive tumors displayed low expression of ATF7, whereas 37 of 96 (38%) were positive in HER2-negative tumors [P = 0.0065; [Table 2]]. In addition, expression of ATF7 mRNA and protein was increased in MCF-7 and T47D BRCA cells (luminal A subtype) and decreased in SKBR3 and MDA-MB-453 cells (HER2-enriched subtype) compared with MCF-10A normal mammary epithelial cells. This was consistent with our results from the database [Figure 1]d and [Figure 1]e. Protein expression of HER2 in these cell lines was also verified by Western blot (WB) [Figure 1]d. Therefore, ATF7 is expressed at a low level in HER2-enriched BRCA cells.
Figure 1: ATF7 expression in breast cancer. (a) ATF7 expression in different molecular subtypes of breast cancer. *Normal basal-like versus luminal A, HER2-E or basal-like; #Luminal A versus HER2-E, (b) differential expression of ATF7 in HER2-negative and positive breast cancer samples as detected by IHC staining, (c) tissue microarray immunohistochemical staining of ATF7 in HER2-negative and positive breast cancer samples. Brown, ATF7-positive cells. Scale bar, 50 μm, (d) ATF7 and HER2 protein expression in MCF-10A normal mammary epithelial cells, MCF-7, and T47D cells, HER2-enriched SKBR3, and MDA-MB-453 cells as measured by WB analysis. Relative quantitative analysis of ATF7 protein expression is shown on the right, (e) transcriptional expression of ATF7 in MCF-10A, MCF-7, T47D, SKBR3, and MDA-MB-453 cells as measured by qRT-PCR. *HER2-enriched cell lines versus MCF-10A; #Luminal A subtype cells versus MCF-10A; @@ HER2-enriched cell lines versus luminal A subtype cells. **,##,@@P < 0.01. IHC = Immunohistochemistry, ATF7 = Activating transcription factor 7, HER2 = Human epidermal growth factor receptor 2, WB = Western blot, qRT-PCR = Quantitative real-time polymerase chain reaction

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Table 2: Immunohistochemistry staining of activating transcription factor 7 in human epidermal growth factor receptor 2-negative and human epidermal growth factor receptor 2-positive breast cancer tissues

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Activating transcription factor 7 is a potential predictive marker for human epidermal growth factor receptor 2-positive breast cancer patients

Next, the clinical value of ATF7 was explored. In BRCA samples, ATF7 expression status had no association with tumor stage [Figure S2]a. However, ATF7 levels showed a gradually decreased pattern with increased tumor stage in HER2-positive BRCA patients, suggesting a negative association between ATF7 and tumor stage [Figure 2]a. When BRCA patients were divided into two groups according to ATF7 expression, the difference in overall survival was not observed between low ATF7- and high ATF7-expressing groups [Figure S2]b. By contrast, HER2-positive patients with high ATF7 exhibited a favorable clinical outcome compared with low ATF7-expressing patients [Figure 2]b. Thus, ATF7 may be an effective diagnostic and prognostic marker for HER2-positive BRCA patients.

Figure 2: Clinical value of ATF7 in HER2-positive breast cancer patients. (a) HER2-positive breast cancer patients were divided into three groups based on tumor stage (Stage I, II, III, and IV). Subsequently, multiple comparisons between ATF7 expression and tumor stage were conducted using a one-way ANOVA and Tukey's test. *Stage I versus Stage II or Stage IIII; #Stage II versus Stage III; $Stage IV versus Stage III, (b) survival analysis was performed using Kaplan-Meier Plotter in HER2-positive breast cancer patients according to ATF7 expression status (low and high ATF7). *,#,$P < 0.05, **P < 0.01. ATF7 = Activating transcription factor 7, HER2 = Human epidermal growth factor receptor 2

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Activating transcription factor 7 overexpression abolishes human epidermal growth factor receptor2-induced cell migration

The cytological function of ATF7 was investigated in HER2-positive BRCA cells. HER2-overexpression enhanced cell migration and increased the number of migrating MCF-10A cells, which was reversed by exogenous ATF7 transfection [Figure 3]a. In addition, HER2 upregulation increased the expression of MMP9, MMP2, uridylyl phosphate adenosine (uPA), and plasminogen activator inhibitor-1 (PAI-1), which was abolished by ATF7 overexpression [Figure 3]b. Subsequently, the role of ATF7 on cell migration in HER2-enriched cells (SKBR3 and MDA-MB-453 cells) was further confirmed. The efficiency of three candidate shRNAs targeting ATF7 was validated using a WB assay and only shRNA-2 transfection significantly reduced ATF7 expression [Figure S3]a. Therefore, shRNA-2 of ATF7 was selected for subsequent experiments. In ATF7 recombinant plasmid-transfected SKBR3 cells, the number of migrating cells and the expression of MMP9, MMP2, uPA, and PAI-1 significantly decreased; however, ATF7 knockdown markedly restored cell migration and the expression of migration-related genes in SKBR3 cells [Figure 3]c and [Figure 3]d. The efficiency of exogenous recombinant plasmid and ATF7 shRNA in SKBR3 cells was verified [Figure S3]b and [Figure S3]c. Consistently, ATF7 upregulation decreased the number of migrating cells and the expression of MMP9, MMP2, uPA, and PAI-1. In contrast, ATF7 downregulation promoted cell migration and the expression of migration-related genes in MDA-MB-453 cells [Figure 3]e and [Figure 3]f. Therefore, ATF7 regulates BRCA migration in a HER2-dependent manner.
Figure 3: Effect of ATF7 on the migration of MCF-10A, SKBR3, and MDA-MB-453 cells. MCF-10A cells were transfected with HER2-recombinant plasmid alone or with the combination of HER2- and ATF7-recombinant plasmids. Cell migration was determined by Transwell chamber assays. Relative quantification of migrated cells is shown in the right panel. (a) The mRNA expression of MMP9, MMP2, uPA, and PAI-1 as measured by qRT-PCR, (b) SKBR3 cells were transfected with ATF7-recombinant plasmid or ATF7 shRNA. Cell migration was determined by Transwell assays. Relative quantification of migrating cells is shown in the right panel, (c) the mRNA expression of MMP9, MMP2, uPA, and PAI-1 as measured by qRT-PCR, (d) scale bar, 50 μm. MDA-MB-453 cells were transfected with ATF7-recombinant plasmid or ATF7 shRNA. Cell migration was determined by Transwell assays. Relative quantification of migrated cells is shown in the right panel, (e) the mRNA expression of MMP9, MMP2, uPA, and PAI-1 as measured by qRT-PCR, (f) scale bar, 25 μm. *,#P < 0.05, **,##P < 0.01. ATF7 = Activating transcription factor 7, HER2 = Human epidermal growth factor receptor 2, MMP9 = Matrix metalloproteinase 9, uPA = Uridylyl phosphate adenosine, PAI-1 = Plasminogen activator inhibitor-1, qRT-PCR = Quantitative real-time polymerase chain reaction

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Low activating transcription factor 7 is necessary for human epidermal growth factor receptor 2-mediated cell junction damage

In HER2-positive BRCA patients, the expression of ATF7 was negatively associated with ERBB2 levels (encoding HER2) [Figure 4]a. Thus, we suspected that the presence of HER2 affects ATF7 content in BRCA. Once ATF7 was overexpressed in MCF-10A normal mammary epithelial cells, the robust decreased ATF7 was verified, which suggested that HER2 negatively regulated ATF7 expression [Figure 4]b. In addition, HER2 upregulation reduced the level of the cell junction marker, E-cadherin, and increased the level of N-cadherin in MCF-10A cells, which resulted in cell junction damage [Figure 4]b. In contrast, ATF7 overexpression reversed decreased E-cadherin and increased N-cadherin in HER2-overexpressing MCF-10A cells, improving the injury of cell junction [Figure 4]c. The damage of the cell junction was closely associated with cell migration behavior. Consequently, HER2 induced the abnormal expression of cell junction markers which was restored by exogenous ATF7, thus facilitating cell migration.
Figure 4: The association among HER2, ATF7, and cell junction in breast cancer. (a) Gene expression profiles of ATF7 and ERBB2 were obtained from the TCGA database. The association between ATF7 and ERBB2 in HER2-positive samples was determined using SPSS software (n = 79), (b) the HER2 recombinant plasmid was transfected into MCF-10A cells. The protein levels of ATF7, E-cadherin, and N-cadherin were determined by WB, (c) MCF-10A cells were transfected with the HER2-recombinant plasmid alone or with the combination of HER2- and ATF7-recombinant plasmids. Subsequently, the protein levels of ATF7, E-cadherin, and N-cadherin were determined by WB analysis. Relative quantitative analysis of protein expression is shown on the right. R = Pearson's correlation coefficient. ATF7 = Activating transcription factor 7, HER2 = Human epidermal growth factor receptor 2, WB = Western blot, ns = No significance, *,#P < 0.05, **,##P < 0.01

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Activating transcription factor 7 negatively regulates the histone lysine demethylase 1/histone H3K9 dimethylation signaling pathway in human epidermal growth factor receptor 2-overexpressing breast cells

The alteration of LSD1/H3K9me2 signal transduction was observed in HER2-overexpressing MCF-10A breast cells. As shown in [Figure 5]a and [Figure 5]b, HER2-overexpression decreased the expression of ATF7, followed by elevation of LSD1, H3K9me2, and H3K4me2 expression, and the activation of LSD. These changes, excluding H3K4me2, were restored by upregulating ATF7 [Figure 5]a and [Figure 5]b, which suggested that ATF7 was a key determinant in the process of HER2-induced changes in LSD1/H3K9me2 signaling. Furthermore, immunohistochemistry revealed that high expression of LSD1 and H3K9me2 is observed in HER2-positive BRCA specimens compared with HER2-negative specimens [Figure 5]c. A higher number of patients with high LSD1 or H3K9me2 were associated with HER2-positive specimens compared with HER2-negative specimens [[Table 3] and [Table 4]; P = 0.04 and P = 0.0227]. The negative association between ATF7 and KDM1A (LSD1) was also found in HER2-positive samples [Figure 5]d. It is possible that decreased ATF7 in HER2-positive breast cells promotes metastasis by activating the LSD1/H3K9me2 signaling pathway.
Figure 5: Effect of ATF7 on the LSD1/H3K9me2 signaling pathway. (a) MCF-10A cells were transfected with HER2-recombinant plasmid alone or with the combination of HER2- and ATF7-recombinant plasmids. Protein expression of HER2, ATF7, LSD1, H3K9me2, and H3K4me2 was determined by WB analysis. Relative quantitative analysis of ATF7 protein expression is shown on the right, (b) LSD1 activity as measured by the LSD1 Activity Quantification Assay Kits according to the manufacturer's instructions, (c) differential expression of LSD1 and H3K9me2 between HER2-negative and -positive breast cancer samples as determined by IHC staining. Scale bar, 50 μm, (d) gene expression profiles of ATF7 and KDM1A were obtained from the TCGA database. The association between ATF7 and ERBB2 in HER2-positive samples was determined by SPSS software (n = 79). *P < 0.05, **P < 0.01; ns = No significance. ATF7 = Activating transcription factor 7, HER2 = Human epidermal growth factor receptor 2, LSD1 = Histone lysine demethylase 1, H3K9me2 = Histone H3K9 dimethylation, WB = Western blot, IHC = Immunohistochemistry

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Table 3: Immunohistochemistry staining of lysine demethylase 1 in human epidermal growth factor receptor 2-negative and Human epidermal growth factor receptor 2-positive breast cancer tissues

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Table 4: Immunohistochemistry staining of H3K9me2 in human epidermal growth factor receptor 2-negative and human epidermal growth factor receptor 2-positive breast cancer tissues

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 > Discussion Top


As a heterogeneous disease, BRCA is generally classified into four major molecular subtypes: luminal A, luminal B, HER2-positive, and basal-like.[27] Among these subtypes, HER2-enriched patients have a highly invasive tumor with a high recurrence rate, making it the main cause of resistance to HER2-based therapies.[2],[28] In the present study, low expression of ATF7 was demonstrated in HER2-positive BRCA patients. High levels of ATF7 were associated with a good prognosis in the HER2-expressing population. HER2 negatively regulated the expression of ATF7, increased metastasis, was abrogated by exogenous ATF7. Our data provide new strategies for treating drug-resistant HER2-positive BRCA patients through the regulation of ATF7.

ATF4 has been reported to be highly expressed in TNBC patients.[29] Under pathological conditions, a sustained and prolonged expression of ATF3 has been verified in BRCA.[30] Thus, ATF family proteins are closely linked with the progression of BRCA. Although it is was shown that ATF7 is minimally expressed in human gastric cancer,[15] its expression pattern and function in BRCA remains unclear. In the present study, differential expression of ATF7 was not observed in all BRCA specimens. However, in specific molecular subtypes, such as luminal A, HER2-enriched, and TNBC patients, significant changes in ATF7 expression were observed, showing elevation in luminal A patients and a reduction in HER2-enriched and TNBC patients. Luminal A patients lack HER2 expression; however, HER2-enriched patients display high HER2 expression. Therefore, we focused on the contradictory expression pattern of ATF7 in these two subtypes. ATF7 may exert the opposite effect in different subtypes of BRCA. We found that the difference in ATF7 expression in HER2-positive and HER2-negative patients was significant, which we explored in depth; however, the association between ATF7 expression in luminal A and TNBC patients needs to be studied further.

ATF3 is a candidate prognostic biomarker and a potential target for melanoma therapy.[31] ATF4 may also be a valuable prognostic biomarker and therapeutic target, and its inhibition reduces tumor growth, metastases, invasiveness, and epithelial–mesenchymal transition (EMT).[32] ATF5 is decreased in HCC versus matched nontumor hepatic tissue, and low ATF5 expression levels are associated with aggressive tumor behavior and predict a worse clinical outcome in HCC.[33] Although ATF7 may not be a robust diagnostic and prognostic marker for all BRCA patients, its expression gradually decreased with increased tumor stage and high levels of ATF7 are associated with a good clinical outcome in HER2-positive patients. This suggests prognostic value in the subset of HER2-enriched patients. Whether ATF7 possesses diagnostic or prognostic value in luminal A and TNBC patients remains to be determined.

HER2 is an oncogenic factor which promotes cell proliferation, angiogenesis, survival, and metastasis by activating multiple signaling pathways.[34] Among these signaling molecules, HER2 enhances ATF4 expression, followed by upregulation of zinc finger E-box-binding homeobox 1 and reduction of E-cadherin, resulting in cell migration and tumor metastasis in BRCA.[35] However, the expression of ATF7 was negatively associated with HER2 in HER2-positive subjects and HER2 overexpression robustly limited the expression of ATF7. Therefore, HER2 is implicated in the regulation of the ATF family even though they exhibit different roles. ATF5 inhibition decreases colony formation rate, migration, and invasiveness of nasopharyngeal carcinoma cells and sensitizes cancer cells to X-ray irradiation by upregulating the expression of E-cadherin.[36] In contrast, HER2-mediated decreased E-cadherin expression was restored by exogenous ATF7, suggesting that ATF7 is positively associated with E-cadherin in HER2-positive BRCA cells, which is different from that of ATF5. In addition, the decrease in extracellular matrix-related molecules can inhibit EMT and subsequently blocked cell metastasis in numerous cancers. Our data indicate that ATF7 upregulation significantly reduced the expression of extracellular matrix-related molecules in HER2-positive cells, indicating that ATF7-mediated migration inhibition is correlated with extracellular matrix modification. Collectively, ATF7 acts as a downstream suppressor during HER2-mediated metastasis in BRCA cells by affecting cell junctions and EMT.

LSD1 inhibition contributes to anti-tumor effects in luminal BRCA by positively regulating GATA3 and negatively regulating TRIM37.[37] LSD1 knockdown activates the methylation process of H3K4 and H3K9 in BRCA cells to inhibit tumor cell activity; however, LSD1 stability facilitates the demethylation of H3K4me2 and H3K9me2 to accelerate tumor progression.[38],[39] LSD1 inhibitor is considered a potential epigenetic therapeutic strategy for HER2-positive BRCA patients.[40] In the present study, HER2-positive BRCA specimens displayed higher expression of LSD1 and H3K9me2 protein. HER2 overexpression significantly induced the expression of LSD1 along with the activation of H3K4me2 and H3K9me2. Therefore, targeting LSD1-dependent epigenetic changes may contribute to the treatment of HER2-positive BRCA. Interestingly, the binding of ATF7 to transcriptional regulatory regions of several genes for silencing may regulate histone H3K9 dimethylation.[41] In lipopolysaccharide-challenged cells, phosphorylation of ATF7 resulted in the release of ATF7 from chromatin and a decrease in repressive histone H3K9me2 marks.[42] In the present study, exogenous ATF7 prevented HER2-mediated elevation of LSD1 and H3K9me2 expression as well as the activation of LSD1, which indicates that ATF7 negatively regulates the HER2/LSD1/H3K9me2 signaling pathway to inhibit cell migration. However, whether the ATF7-mediated change in H3K9me2 depends on LSD1 has yet to be determined. With respect to H3K4me2, although LSD1 upregulation activated its expression, ATF7 overexpression did not restore its expression. There may be other histone demethylases responsible for its demethylation in HER2-overexpressing breast cells independent of ATF7.


 > conclusion Top


In summary, low expression of ATF7 was observed in HER2-positive BRCA patients and ATF7 is a putative diagnostic and prognostic biomarker for HER2-positive subjects. Low levels of ATF7 were associated with HER2-induced mammary epithelial cell migration. HER2 negatively regulated the expression of ATF7 and elevated ATF7 significantly repressed cell migration in HER2-positive BRCA cells. Moreover, the HER2-mediated decrease of ATF7 re-activated LSD1-induced epigenetic changes in H3K9me2 to promote metastasis in HER2-breast cells. Therefore, our findings provide insight into the regulatory mechanisms of HER2-mediated BRCA cell migration. ATF7-based therapy may represent a promising target strategy for inhibiting metastasis in HER2-positive breast tumors.

Acknowledgments

We thank the support of Gene Expression Profiling Interactive Analysis 2.0 (GEPIA2).

Financial support and sponsorship

This study was supported by the China Postdoctoral Foundation (Grant number 2018M643051).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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