GLIS Family Zinc Finger 3 Promotes Triple-Negative Breast Cancer Progression by Inducing Cell Proliferation, Migration and Invasion, and Activating the NF-κB Signaling Pathway

Abstract

Triple-negative breast cancer (TNBC) puts a great threat to women’s health. GLIS family zinc finger 3 (GLIS3) belongs to the GLI transcription factor family and acts as a critical factor in cancer progression. Nevertheless, the part of GLIS3 played in TNBC is not known. Immunohistochemical (IHC) staining analysis displayed that GLIS3 was highly expressed in TNBC tissues. The effect of GLIS3 on the malignant phenotype of TNBC was tested in two different cell lines according to GLIS3 regulation. Upregulation of GLIS3 promoted the proliferation, migration, and invasion of TNBC cell lines, whereas the knockdown of GLIS3 suppressed these tumor activities. Inhibition of GLIS3 induced TNBC cell apoptosis. Furthermore, study as immunofluorescence and electrophoretic mobility shift assay confirmed that the nuclear factor-κB (NF-κB) signaling pathway activated by GLIS3 played an important role in TNBC cells’ malignant phenotype. In conclusion, the present work demonstrated that GLIS3 acts as a crucial element in TNBC progression via activating the NF-κB signaling pathway. Accordingly, above mentioned findings indicated that modulation of GLIS3 expression is a potential tactic to interfere with the progression of TNBC.

INTRODUCTION

Breast cancer (BC) as the most common tumor diagnosed in women,¹⁾ is a kind of highly heterogeneous disease and the clinical treatment and prognosis vary greatly between patient to patient.^2,3) It could be divided into several types due to the different subtypes.^4,5) Triple negative breast cancer (TNBC) is a type of BC that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) expression.^6,7) TNBC occupies roughly one-fifth of all BCs and is usually related to the worse clinical outcome.^8–10) Due to the lack of ER, PR and HER2 expression in TNBC, endocrine therapy or molecular targeted therapy is not suitable for it.¹¹⁾ Chemotherapy is currently the main systemic treatment method, but conventional postoperative adjuvant chemo-radiotherapy is not effective and will eventually cause the recurrence of residual lesions.¹²⁾ Consequently, to improve the prognosis of TNBC patients, exploring the molecular mechanisms that affect the occurrence and development of TNBC and formulating new treatment strategies is of great significance.

GLIS3 belongs to the GLI-similar zinc finger protein family and has five C₂H₂-type zinc finger domains.¹³⁾ The characteristic feature of this kind of family is that it exists in two or more conserved C₂H₂ zinc finger domains. It has been proved that GLIS3 acts as both a repressor and activator of transcription and is specifically involved in cell proliferation, differentiation, and development.¹⁴⁾ GLIS3 has been found highly expressed in several cancers. Down-regulation of GLIS3 expression can repress the invasion of melanoma cells cultured in vitro and the migration of melanoma in a chicken transplant model.¹⁵⁾ These studies indicated that enhanced GLIS3 expression promotes tumor cell proliferation, migration, and metastasis. In addition, one clinical study has shown that the relative expression of GLIS3 in human BC tissues was 4 times higher than that in normal tissues, and the highly expression of GLIS3 may have a positive correlation with TNBC.¹⁶⁾ Increased GLIS3 expression has been found in several different cancer cell types, while no links have yet been made between TNBC tumorigenesis and GLIS3 regulation.

Nuclear factor-κB (NF-κB) is a common transcription factor that could mediate the cytoplasmic/nuclear signaling pathways and regulate the expression of cytokines involved in inflammation and immune responses.^17–19) The relationship of the cancer and the NF-κB signaling pathway has been widely studied at before.^20–22) The activation of NF-κB has been proved to be related with the control of apoptotic pathway, cell proliferation, and migration in tumor cells.^23,24) In addition, the abnormal activation of the NF-κB signaling pathway in TNBC leads to the overexpression of downstream signal targets, such as anti-apoptotic genes, which promote tumor progression and chemotherapy resistance.^25,26)

Accordingly, based on these findings, the purpose of this study is to investigate the vital part of GLIS3 plays in TNBC tumor development and whether the NF-κB signaling pathway is involved in GLIS3 regulation.

MATERIALS AND METHODS

Clinical Samples

This study was approved by the Ethics Committee of The Fourth Hospital of Hebei Medical University under code No. 2020KY144. Overall, 40 TNBC specimens, 30 non-TNBC specimens, and 30 normal breast tissues were collected in the Fourth Hospital of Hebei Medical University. All patients involved in this study were aware and provided informed consent.

Cell Cultivate and Transfection

Human normal breast cell line MCF-10A (iCELL, China) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS, Tianhang Biotech, China) and 1% dual antibiotic solution. Carbeniclllin disodium salt and streptomycin sulphate were provided by Solarbio (Beijing, China). TNBC cell lines MDA-MB-436 (iCELL), MDA-MB-231 (iCELL), and MDA-MB-468 (Procell, China) were separately cultured in Leibovitz’s L-15 medium (Procell) supplemented with 10% FBS and 1% dual antibiotic solution. Hs578t (iCELL) was cultured in DMEM medium supplemented with 10% FBS and 1% dual antibiotic solution. All these cells were placed at 37 °C with a 5% CO₂ atmosphere.

Above mentioned cells were provided for testing, and a cell line with relatively low GLIS3 expression (MDA-MB-468) and a cell line with high GLIS3 expression (Hs578t) were selected for the following experiments. For transfection, Opti-MEM (Invitrogen, U.S.A.) was mixed with lipofectamine 3000 (Invitrogen), Opti-MEM was mixed with OE- or small interfering RNA (siRNA)-, respectively. After that, the mixtures were placed together at room temperature for 20 min and then cultured at 37 °C with a 5% CO₂ atmosphere.

Immunohistochemical (IHC) Staining

IHC stains were implemented on 5-µm thick sections of formalin-fixed, paraffin-embedded tissue samples. The slides were first deparaffinized, rehydrated, and followed by antigen retrieval before immunostaining. The tissue sections were then incubated with 0.3% hydrogen peroxide and blocked by 10% normal goat serum (Solarbio). The slides were first incubated with GLIS3 antibody (1 : 100, Biorbyt, U.K.) at 4 °C overnight, and then horseradish peroxidase (HRP) labeled goat anti-rabbit antibody (1 : 500, Thermo Fisher, U.S.A.) added at 37 °C for 1 h. After the DAB substrate solution (Solarbio) was added, hematoxylin (Solarbio) was incubated for counterstaining. All the sections were observed and photographed with a microscope (OLYMPUS, Tokyo, Japan).

Real-Time Quantitative PCR (qRT-PCR)

Total RNA was isolated by using the TRIpure solution (BioTeke, China), incubated for 5 min at 37 °C, and quantified with a spectrophotometer (Nano 2000, Thermo Fisher, U.S.A.). Total RNA was reverse transcribed into cDNA with the help of BeyoRT II M-MLV reverse transcriptase regent (Beyotime, Shanghai, China). The 2 × Taq PCR MasterMix and SYBR Green (Solarbio) regents were used for qRT-PCR analysis and the results were processed by the ExicyclerTM 96 instrument (BIONEER, Korea). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. The primers were kindly provided by GenScript (Nanjing, China): GLIS3 forward 5′-CATCCATCTGCCTGCCTTAA-3′ and GLIS3 reverse 5′-GTCCCAGTCGCTGAACCA-3′. GAPDH forward 5′-GACCTGACCTGCCGTCTAG-3′ and GAPDH reverse 5′-AGGAGTGGGTGTCGCTGT-3′.

Western Blotting

Total protein was extracted with the help of lysis buffer (RIPA buffer, Beyotime). Phenylmethanesulfonylfluoride (PMSF, Solarbio) and protein phosphatase inhibitor (Solarbio) were used in this section. Bicinchoninic acid protein assay kit (Beyotime) was used in this section for protein quantification. After that, 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) gels electrophoresis was used for protein separation and then transferred onto the polyvinylidene difluoride (PVDF) membrane. Bovine serum albumin and 5% non-fat milk powder were prepared for different kinds of antibodies as the blocking buffers. Then, they were immunoblotted with the primary antibody (dilution: 1 : 1000) at 4 °C overnight and then incubated with HRP labeled anti-immunoglobulin G (IgG) (1 : 3000) at 37 °C for 1 h. Antibodies involved in the Western blotting are provided as follows: GLIS3 antibody was purchased from Biorbyt. Proliferating cell nuclear antigen (PCNA), pro/cleaved-caspase-3, Bax, Bcl2, E-cadherin, Vimentin, N-cadherin, p65, and p-p65 antibodies were provided by ABclonal (China). Goat anti-rabbit IgG/HRP and goat-anti mouse IgG/HRP were provided by Solarbio. The housekeeping gene GAPDH antibody was bought from Proteintech (China). After that, the optical density value of the target band was analyzed by a gel image processing system (Gel-Pro-Analyzer software, Beijing, China).

Cell Counting Kit 8 (CCK8) Assay

Cells were seeded in 96-well plates at a density of 3 × 10³ cells/well prior to assay. Each well was incubated with 10 µL Cell Counting Kit-8 solutions (Sigma, U.S.A.) for 1 h. In addition, in some sections, 5 µM BAY11-7082 was added and incubated at 37 °C with a 5% CO₂ atmosphere for 24 h before adding CCK 8 solutions. The absorbance at 450 nm was measured by using a microplate reader (Biotek, U.S.A.).

Cell Cycle Detection

Cell cycle distribution was tested by a cell cycle analysis kit (Beyotime) according to the instruction. Cells were fixed with 70% ethanol for 24 h at 4 °C and then stained for total DNA content with a solution containing 25 µL propidium iodide (PI) and 10 µL RNase A in phosphatebuffer saline (PBS) for 0.5 h at 37 °C in the dark. The result was analyzed with the NovoCyte Flow Cytometer (ACEA, U.S.A.).

Cell Apoptosis

Cell apoptosis was tested by a cell Apoptosis detection kit (KeyGEN BioTECH, China) according to the instruction. The cells were collected by centrifugation; the supernatant was discarded, washed twice with PBS. Binding buffer (500 µL) was added to re-suspend the cells. After adding 5 µL Annexin V-fluorescein isothiocyanate (FITC) and add 5 µL propidium iodide (PI), the mixture was incubated at room temperature for 5–15 min in the dark. The result was analyzed with the NovoCyte Flow Cytometer.

Cell Migration

Cell migration was measured by transwell assay. Cells were washed once with PBS, serum-free medium was added, and the cells were blown off from the culture plate wall and dispersed into single cell suspension. The transwell chamber was placed into a 24-well plate, and 800 µL of culture medium containing 10% FBS was added to the lower chamber. Cell suspension at 200 µL was added into the upper chamber, and the cell number was 3 × 10⁴ cells/well. The plates were incubated in a cell incubator at 37 °C with 5% CO₂ and saturated humidity for 24 h. The chambers were washed twice with PBS, fixed with 4% paraformaldehyde (Aladdin, Chia) for 20 min at room temperature, stained with 0.5% crystal violet (Amresco, U.S.A.) solution for 5 min, and then rinsed with distilled water. The image was captured on an inverted phase-contrast microscope (Olympus).

Cell Invasion

Cell invasion was also measured by transwell assay. The transwell chamber (Corning Costar, U.S.A.) was constructed with the help of Matrigel gel (BD Bioscience, U.S.A.) and serum-free medium. The transwell chamber was taken out and put into a 24-well plate, coated with 40 µL of pre-diluted Matrigel gel on the membrane of the chamber, and placed in the incubator at 37 °C for 2 h. Cells were washed twice with PBS, serum-free medium was added, and the cells were blown off from the culture plate wall and diluted into 1 × 10⁵ cell/mL. The transwell chamber was placed into a 24-well plate, and 800 µL of culture medium containing 10% FBS was added to the lower chamber. Cell suspension at 200 µL was added into the upper chamber, and the cell number was 5 × 10⁴ cells/well. In the last section, BAY11-7082 treated MDA-MB-468 cells were incubated for another 24 h before being tested. The following steps were the same as the cell migration. The image was captured on an inverted phase-contrast microscope.

Immunofluorescence (IF) Staining

Cells were fixed with 4% paraformaldehyde for 15 min and incubated with 0.1% Triton X-100 (Solarbio, Shanghai, China) for 0.5 h at room temperate. The primary antibody (p65 antibody, 1 : 1000, Abclonal, China), Cy3 labeled goat anti-rabbit IgG (1 : 3000, Invitrogen), and 4′,6-diamidino-2-phenylindole (DAPI) were used in this procedure. The images were obverted and collected by using an OLYMPUS-BX53 microscope (OLYMPUS).

Electrophoretic Mobility Shift Assay (EMSA)

In EMSA, the nucleoprotein was extracted and quantified by Nuclear Protein Extraction Kit (Solarbio) and BCA protein assay kit (Beyotime). The DNA binding activity of NF-κB was measured by NF-κB EMSA detection kit (Viagene, China). The regents were added by the following order: 8 µL ddH₂O, 10 µL 10× binding solution, 5 µL nuclear reaction mixtures. All these regents were mixed and incubated for 0.5 h at room temperature. Finally, 0.5 µL biotin-labeled probes was added and incubated for 20 min at room temperature. Then, the samples were electrophoresed and then transferred to the membranes. After that, filter paper was removed. Membranes were cross-linked at 10 cm under UV light for 30 min. ECL luminescent solution was allowed to react for 5 min.

Statistical Analysis

Graphpad software version 8.0 (GraphPad Software Inc. U.S.A.) was used in this work for the statistical analysis. Data between two groups were analyzed using an unpaired t-test while the differences of more than three groups were analyzed by one-way ANOVA. One-way ANOVA followed by Tukey’s, Dunnett’s and Sidak’s multiple comparisons tests were all used in this work. Data were shown as mean ± standard deviation (S.D.). p-Value less than 0.05 was considered as the statistically significant.

RESULTS

GLIS3 Is Overexpressed in TNBC Clinical Tissues and Cell Lines

At first, we explored the expression level of GLIS3 in TNBC clinical samples. In Fig. 1A, we observed the typical IHC images of three kinds of breast tissues. It was clearly to see that GLIS3 was upregulated in non-TNBC tumor tissue when compared with the normal breast tissue, while the TNBC specimen displayed the highest GLIS3 level among them. The clinical information and IHC results of 40 TNBC specimens were summarized in Table 1. The result confirmed that GLIS3 was significantly increased in higher tumor stages and node metastasis patients. Based on the clinical characteristic, we further investigated GLIS3 expression in human normal breast cell line MCF-10A and TNBC cell lines (MDA-MB-436, MDA-MB-468, MDA-MB-231, Hs578t). Results of Western blotting showed a significantly enhanced level of GLIS3 in TNBC cell lines as compared to normal ones (Fig. 1B). In different kinds of TNBC cell lines, GLIS3 was overexpressed in MDA-MB-468 cells and downregulated in Hs578t cells for follow-up studies and the efficiency was convinced by using the qRT-PCR (Fig. 1C) and Western blotting (Fig. 1D). As these result reflected, GLIS3 is overexpressed in TNBC clinical tissues and cell lines.

Fig. 1. GLIS3 Is Overexpressed in Triple-Negative Breast Cancer (TNBC) Clinical Samples and Cell Lines

(A) IHC staining images of human normal breast tissue, non-TNBC tissue, and TNBC tissue. (B) The protein level of GLIS3 in MCF-10A and TNBC cell lines (MDA-MB-436, MDA-MB-468, MDA-MB-231, Hs578t), respectively. MDA-MB-468 and Hs578t cells were transfected with GLIS3-OE and GLIS3-siRNA separately to regulate GLIS3 expression. (C) The mRNA expression level of GLIS3 in GLIS3 overexpressed and suppressed cells (three test repeats). (D) The protein expression level of GLIS3 in GLIS3 overexpressed and suppressed cells. One way-ANOVA followed by Tukey's multiple comparisons test for Figs. 1B–D. Scale bar: 100 µm. Data were expressed as mean ± S.D. and collected from three independent experiments. ns, no significant, * p < 0.05, ** p < 0.01.

Table 1. GLIS3 Expression in Clinical and Pathological Characteristics of 40 TNBC Patients

Characteristics		n
			High	Low	p-Value
Age	<53	15	6	9	0.461
	≥53	25	13	12
Tumor stage	T1	20	5	15	0.025
	T2/T3/T4	20	12	8
Node metastasis	N0	16	4	12	0.020
	N1/N2//N3	24	15	9
TNM	I	9	7	2	0.216
	II/III/IV	31	17	14
Tumor size (cm)	≤2	20	7	13	0.273
	2–5	18	11	7
	>5	2	1	1

GLIS3 Promotes TNBC Cell Proliferation

Giving evidence to the results above mentioned, Hs578t and MDA-MB-468 cells were transfected with GLIS3-siRNA and GLIS3-OE in this work to regulate GLIS3 expression. Afterward, follow-up analysis was introduced to explore how GLIS3 expression influenced TNBC cell progression. In Figs. 2A and B, the CCK-8 results exhibited that overexpressed GLIS3 remarkably improved the viability of cells, while suppressed GLIS3 achieved an opposite result (Fig. 2B). Tumor cells have vigorous proliferation activity, and proliferating cell nuclear antigen (PCNA) can be used as an indicator to evaluate the state of cell proliferation. In Figs. 2C and D, the enhancement of GLIS3 promoted the expression level of PNCA, and suppression of GLIS3 inhibited it. In Fig. 2E, compared with the vector transfected cells, an increased percentage was observed in the S phase and a reduction was observed in the G1 phase in the GLIS3 overexpressed cells. Knockdown of GLIS3 displayed an inhibited proportion in the S phase and an increased cell proportion in the G1 phase when compared with the siNC transfected cells (Fig. 2F).

Fig. 2. GLIS3 Promotes TNBC Cell Proliferation

(A, B) Cell proliferation was evaluated by CCK8 assay (five test repeats). (C, D) The protein expression level of proliferating cell nuclear antigen (PCNA) in GLIS3-regulated cells was tested by Western blotting. (E, F) Cell cycle distribution was determined by flow cytometric assay. Sidak’s multiple comparisons test for Fig. 2A. Unpaired t test for Figs. 2C and E. One way-ANOVA followed by Tukey's multiple comparisons test for Figs. 2B and D. One way-ANOVA followed by Dunnett’s multiple comparisons test for Fig. 2F. Data were expressed as mean ± S.D. and collected from three independent experiments. * p < 0.05, ** p < 0.01.

GLIS3 Decreases TNBC Cell Apoptosis

After confirming the role of GLIS3 in promoting cell proliferation in TNBC, we examined its role in TNBC cell apoptosis by flow cytometry. Compared with the siNC transfected cells, the number of apoptotic cells was increased in GLIS3 suppressed cells (Figs. 3A, B). Based on this, we also tested the level of cleaved-caspase-3, Bax, and Bcl2 in this section to evaluate the effect of GLIS3 on cell apoptosis. The result of the Western blotting showed in Fig. 3C demonstrated that inhibition of GLIS3 increased the level of cleaved-caspase-3 and Bax, repressed the level of Bcl2. The level of pro-caspase-3 was no obviously difference in GLIS3-inhibited and negative control cells. These results suggested that GLIS3 plays an anti-apoptotic role in TNBC.

Fig. 3. GLIS3 Decreases TNBC Cell Apoptosis

(A) Cell apoptosis was measured by flow cytometry. (B) Percentages of TNBC cell apoptosis. (C) The protein expression level of pro-caspase-3, cleaved-caspase-3, Bax, and Bcl2 in TNBC cells. One way-ANOVA followed by Tukey’s multiple comparisons test for Fig. 3B. One way-ANOVA followed by Dunnett’s multiple comparisons test for Fig. 3C. Data were expressed as mean ± S.D. and collected from three independent experiments. * p < 0.05, ** p < 0.01.

GLIS3 Promotes TNBC Cells Migration, Invasion, and EMT

Cell migration and invasion are also an important part of the malignant behavior of tumor cells. Transwell assay was used in this work to detect the migration and invasion ability of TNBC cells. As a result, overexpressed GLIS3 displayed higher migration ability (Figs. 4A, B) and invasive (Figs. 4C, D) cell number than the vector transfected cells. Oppositely, prominently lower migration ability and invasive cell number occurred in GLIS3 downregulated cells when compared to the negative control transfected cells. Based on this, we implied that the high expression of GLIS3 in TNBC may promote the progression of TNBC by enhancing cell migration and invasion ability.

Fig. 4. GLIS3 Promotes TNBC Cell Migration and Invasion

(A, B) The images and quantification results of TNBC cell migration (five test repeats). (C, D) The images and quantification results of TNBC cell invasion (five test repeats). (E, F) The expression level of E-cadherin, N-cadherin and vimentin was tested by Western blotting. Unpaired t test for Figs. 4B (Left), D (Left), and E. One way-ANOVA followed by Dunnett’s multiple comparisons test for Figs. 4B (Right), D (Right), and F. Scale bar: 200 µm. Data were expressed as mean ± S.D. and collected from three independent experiments. ns, no significant, * p < 0.05, ** p < 0.01.

Considering that the cell migration and invasion may relate with the epithelial–mesenchymal transition (EMT) process in cancer cells,^27,28) we measured the expression level of EMT associated markers in Fig. 4E by Western blotting. Upregulated GLIS3 decreased the level of E-cadherin, an epithelial marker, and enhanced the level of N-cadherin and vimentin, the mesenchymal markers. Conversely, downregulated GLIS3 enhanced the level of E-cadherin and showed a repressed level of N-cadherin and vimentin (Fig. 4F). These results indicated that GLIS3 promotes TNBC cells migration, invasion, and EMT.

GLIS3 Activates the NF-κB Signaling Pathway in TNBC Cells

After exploring the effect of GLIS3 on the malignant phenotype of TNBC, we further investigated the potential regulatory mechanism of GLIS3 in TNBC. In previously reported studies, GLIS3 and its family member GLIS1-2 all have been reported were involved in the regulation of NF-κB signaling pathway in different cells and tissues.^29–31) Accordingly, IF staining assay was applied to investigate the effect of GLIS3 up/down-regulation in NF-κB p65 nuclear translocation. As we can see, in vector transfected cells, NF-κB p65 is mainly present in the cytoplasm, while obvious NF-κB p65 staining was shown in the nucleus of GLIS3-overexpressing cells (Fig. 5A), as convinced by the quantification result in Fig. 5C. Conversely, the knockdown of GLIS3 decreased the red fluorescence in the nucleus compared with that of negative control (Figs. 5B, D). In Fig. 5E, p-p65^Ser536 protein expression was enhanced in the GLIS3 upregulated cells, while there was almost no obvious difference of p-65 protein. In the GLIS3 suppressed cells (Fig. 5F), the trend was conversed, the level of p-p65^Ser536 was inhibited when compared with the negative control. In addition, DNA-binding activity may be increased upon activation of NF-κB. By the use of EMSA assay, we found that GLIS3 overexpression increased the intensity of the bands in TNBC cells (Fig. 5G). The opposite result was shown in Fig. 5H, GLIS3 suppression decreased the intensity of the NF-κB-DNA oligonucleotide complex in TNBC cells.

Fig. 5. GLIS3 Activates the NF-κB Signaling Pathway

(A, B) Immunofluorescence staining images of NF-κB p65 nuclear translocation in cells (three test repeats). Red fluorescence: NF-κB p65. Blue fluorescence: DAPI. (C, D) The quantification results of NF-κB p65 nuclear translocation. (E, F) The expression level of p-65 and p-p65^Ser536 in TNBC cells was detected by Western blotting. (G, H) DNA-protein complexes’ binding activity was identified by electrophoretic mobility shift assay (EMSA) analysis. Unpaired t test for Figs. 5C, E, and G. One way-ANOVA followed by Dunnett’s multiple comparisons test for Figs. 5D, F, and H. Scale bar: 100 µm. Data were expressed as mean ± S.D. and collected from three independent experiments. ** p < 0.01.

GLIS3 Affects TNBC Cell Malignant Phenotype via NF-κB Signaling Pathway

To further figure out the impact of the NF-κB signaling pathway in the regulation of GLIS3-mediated TNBC malignant phenotype, we carried out BAY11-7082, an inhibiter of NF-κB, in MDA-MB-468 cells following GLIS3 upregulation. The cell proliferation and invasion were tested in Figs. 6A and B. These results implied that upregulated GLIS3 promoted cell proliferation and invasion, while, the addition of BAY11-7082 alleviated the cell proliferation and invasion ability regulated by GLIS3. These findings indicated that GLIS3 promotes the proliferation and invasion ability of TNBC cells by activating the NF-κB signaling pathway. Figure 6C exhibited the simple scheme of this work. GLIS3 plays a key role in TNBC progression by activating the NF-κB signaling pathway.

Fig. 6. GLIS3 Effects TNBC Cell Malignant Phenotype via Activating the NF-κB Signaling Pathway

After being treated with 5 µM NF-κB inhibitor, BAY11-7082, the cell proliferation (A) and invasion (B) were measured by CCK-8 (five test repeats) and transwell assay (five test repeats). (C) The scheme of GLIS3 affects TNBC progression by activating the NF-κB signaling pathway. One way-ANOVA followed by Tukey’s multiple comparisons test for Figs. 6A and B. Scale bar: 200 µm. Data were expressed as mean ± S.D. and collected from three independent experiments. * p < 0.05.

DISCUSSION

TNBC is a specific and aggressive subtype of BC. It has a poor prognosis value and lacks effective treatments at so far. Therefore, exploring new TNBC treatment strategies is of great clinical significance. Previous studies have confirmed that the NF-κB signaling pathway is associated with the progression of TNBC.^25,26) Some research findings revealed that GLIS3 is highly expressed and may involve in the carcinogenesis and progression of a few cancers.^31–34) Besides that, one previous study revealed that their gene-set enrichment analysis (GSEA) displayed an association of GLIS3 with BC.³⁵⁾ Accordingly, the relationship between GLIS3 and its partners may bring TNBC a new probability in the progression of therapeutic strategies.

In this work, we first analyzed the expression level of GLIS3 in clinical patients with TNBC. IHC images showed that compared with normal tissues, GLIS3 showed high expression in BC patients, especially in TNBC. At the same time, we also used these tissue samples to study the relationship between GLIS3 expression and different grades of TNBC. The results showed that GLIS3 was elevated in TNBC and related with high TNBC stage. We further proved that the GLIS3 expression level in TNBC cell lines was significantly higher than in human normal breast cells by Western blotting. Similarly, Lukashova’s³²⁾ research also demonstrated that overexpression of GLIS3 in cancers with high proliferation indices was related with the poor prognosis value. In addition to this, a study of aggressive mesenchymal melanoma also reported the persistent elevation of GLIS3 expression level in cells.¹⁵⁾ Thereby, the result was similar to the previous studies and indicated a reasonable connection between GLIS3 expression and TNBC progression. Based on the analysis of IHC and Western blotting, these results may suggest that GLIS3 is a crucial element in physiological and pathological cellular processes of tumor.

For further investigation, we tested the role of GLIS3 in TNBC cell proliferation, migration, invasion, and EMT process. Studies have found that PCNA is closely related to DNA synthesis,^36,37) plays an important role in the initiation of cell proliferation, and is a good indicator to reflect the state of cell proliferation.³⁸⁾ We could see from the results that upregulated GLIS3 significantly improved cell viability in MDA-MB-468 cells compared with the vector, while suppression decreased the cell viability, proving that GLIS3 promoted cell proliferation. Kang et al. also implied that GLIS3 acts as a regulator of cell proliferation.³⁹⁾ A similar conclusion was further suggested in this work according to the results that GLIS3 promotes TNBC cell migration and invasion, which was consistent with the previous research.¹⁵⁾ Besides that, enhanced expression level of GLIS3 was shown in this work to reduce the levels of E-cadherin, while downregulation of GLIS3 inhibited the level of N-cadherin and vimentin. In Liu’s study,²⁹⁾ GLIS3 could promote the invasion, migration, and proliferation of glioma cell, which is same as ours. The accurate mechanisms of how GLIS3 improves migration and invasiveness have not been confirmed yet, but some studies indicated that it may be due to the stimulation of EMT.^40,41) EMT is considered as an important process during tumor progression.⁴²⁾ GLIS3 has been considered to interact with the transcriptional co-activator with PDZ-binding motif (TAZ), which was related to the EMT process.^43,44)

In this work, we found that GLIS3 could promote TNBC malignant phenotypes as cell proliferation, migration and invasion. Based on this, we then explored the potential mechanism of GLIS3 on TNBC progression. NF-κB is being considered as the crucial element in the expression of proinflammatory cytokines in cancer cells⁴⁵⁾ as well as takes a great part in tumor growth, metastasis, and inflammatory of different kinds of human cancer.⁴⁶⁾ Some reported studies suggested that there is a strong link between NF-κB signaling pathway and TNBC progression.^47,48) Blocking NF-κB signaling in TNBC cells could reduce tumor outgrowth.⁴⁹⁾ In addition, aberrant activation of the NF-κB pathway has been confirmed could promote TNBC metastasis by regulating epithelial-mesenchymal transition (EMT).^26,47) These results indicated that the activation of NF-κB signaling pathway is very important in affecting malignant phenotypes of TNBC. Importantly, GLIS3 has been proved could promote cell malignant behaviors and NF-κB signaling pathway in glioma.²⁹⁾ Based on these findings, we focused on NF-κB signaling pathway when exploring the potential mechanism of GLIS3 on TNBC progression. In Liu’s research,²⁹⁾ their results indicated that the abrogation of GLIS3 inhibited expression levels of p-p65, which subsequently affected the NF-κB signaling cascade, such as C-Myc and MMP9 in cells, and this trend could be partly counteracted by NF-κB overexpression. Similar with their results, we found that GLIS3 enhanced the levels of p-p65 in TNBC cells and the addition of inhibitor (BAY11-7082) reversed this enhancement. Accordingly, we indicated that GLIS3 may affect the malignant phenotypes of TNBC by activating NF-κB signaling pathway. However, the specific mechanism of how GLIS3 activates this pathway is not unclear at so far. NF-κB signaling pathway could be triggered by a variety of cytokines, growth factors and tyrosine kinases. Moreover, activation of other signaling pathways, such as Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/Akt, are also involved in activation of NF-κB.⁵⁰⁾ GLIS3’s family member GLIS2 and homologs GLI1 both have been shown that could regulate the activity of the NF-κB pathway.^30,31) GLIS2 has been proved was related with the tumor-associated, calcium signal transducer 2, which overexpressed in many cancers and associated with the activation of several kinase pathways, including NF-κB.³¹⁾ Nuclear GLI1 was demonstrated to be closely correlated with nuclear expression of NF-κB p65 in cancer cells.^51,52) However, the specific activation mechanism still needs more experiments to explore.

In summary, the present work confirmed that high expression of GLIS3 probably promoted the performance of proliferation, invasion, and migration in TNBC cells. Additionally, the potential molecular mechanism participated in the malignant phenotypes of TNBC was considered to be related with the activation of the NF-κB signaling pathway.

Acknowledgments

This work was supported by the Natural Science Foundation of Hebei Province (No. H2020206199).

Author Contributions

CHENHAO LI: Conception and design, Collection and assembly of data, Data analysis and interpretation. CUIZHI GENG: Administrative support, Provision of study materials or patients. All authors were involved in Manuscript writing and Final approval of manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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