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TFAP2C-mediated transcriptional activation of STEAP3 promotes lung squamous cell carcinoma progression by regulating the β-catenin pathway

Biology Direct volume 19, Article number: 135 (2024) Cite this article

Abstract

Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is associated with the progression of several human malignancies. However, its role in lung squamous cell carcinoma (LUSC) remains unclear. We measured STEAP3 expression in LUSC cell lines and tissues. LUSC cells with stable STEAP3 overexpression and knockdown were obtained through G418 selection. Multiple assays were used to evaluate the malignant phenotypes of LUSC cells and the activation of the β-catenin signaling. The potential transcriptional regulatory factors of STEAP3 were predicted using the JASPAR database, and the correlation between transcription factor AP-2 gamma (TFAP2C) and STEAP3 was analyzed through the GEPIA database. The study evaluated the regulatory relationship between a potential transcription factor and STEAP3 through ChIP and luciferase reporter assays. Additionally, rescue assays were utilized to ascertain whether TFAP2C serves as the upstream regulatory factor of STEAP3, contributing to LUSC progression. Finally, tumor growth and metastasis were evaluated in vivo. STEAP3 expression was notably higher in LUSC, and its overexpression was linked to a poor prognosis. Moreover, STEAP3 overexpression activated the β-catenin pathway, thereby accelerating cell proliferation and metastasis. Conversely, STEAP3 knockdown had an anti-tumor effect in LUSC. Additionally, TFAP2C bound directly to the STEAP3 promoter and positively regulate its expression in LUSC. The anti-tumor effects of TFAP2C knockdown were partially reversed by STEAP3 overexpression. The study indicates that the TFAP2C/STEAP3 axis may be a therapeutic target for LUSC treatment. This enhances our understanding of lung carcinogenesis.

Introduction

Lung cancer represents the second most commonly diagnosed cancer worldwide [1]. The incidence and mortality rates were more favorable in men than in women [1, 2]. Although numerous risk factors have been identified for lung cancer, cigarette smoking remains the leading cause [3]. The incidence and mortality rates of lung cancer have increased in China in recent years [4]. In the United States, the incidence has been declining steadily since 2006 [5]. Accounting for approximately 85% of all cases, non-small cell lung cancer (NSCLC) is the most common subtype [6,7,8]. NSCLC is classified into two major histological subtypes: lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) [9, 10]. Patients with stage I or II NSCLC may currently be treated with surgical resection and adjuvant therapy. Once a patient has been diagnosed with advanced-stage NSCLC, the treatment options shift towards chemotherapy or radiotherapy [11]. Nevertheless, the five-year survival rate remains low due to the limited treatment options available.

Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is a member of the human STEAP family [12]. It is highly expressed in clear cell renal cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, ovarian cancer, and colon cancer [13,14,15,16,17]. The high expression of this gene indicates a poor prognosis [13, 15, 18]. Recent in vitro studies have indicated that STEAP3 promotes colon cancer cell proliferation and migration [16]. Additionally, in hepatocellular carcinoma, overexpression of STEAP3 enhances cell proliferation and stemness [15]; whereas STEAP3 knockdown impairs cell proliferation and tumor growth [15]. Furthermore, the knockdown of STEAP3 exhibits inhibitory effects on glioma cell migration and invasion [18]. However, its function in LUSC remains to be elucidated. Moreover, the specific regulatory mechanisms underlying this role warrant further investigation.

Herein, STEAP3 was highly expressed in LUSC. The malignant phenotypes of LUSC cells were attributed by TFAP2C-mediated transcriptional activation of STEAP3, which regulated the β-catenin pathway.

Materials and methods

Clinical specimens

LUSC and corresponding non-cancerous lung tissues (40 paired samples) were collected from Shengjing Hospital of China Medical University (Shenyang, China). None of the patients received preoperative chemotherapy or radiotherapy. All protocols conformed to the tenets of the Declaration of Helsinki and were approved by the Ethics Committee of Shengjing Hospital, China Medical University. All patients provided written informed consent.

Immunohistochemistry

The tumor tissues underwent dehydration in an ascending alcohol series, clearing in xylene, embedding in paraffin, and sectioning into 5 μm. The sections were then deparaffinized in xylene and rehydrated in a descending alcohol series. Antigen retrieval was performed by heating the sections in a microwave oven for 10 min, followed by immersion in 3% H2O2. The sections were then blocked for 15 min with goat serum. Subsequently, they were exposed to primary antibodies against STEAP3 (#28478-1-AP; Proteintech), PCNA (#ab92552; Abcam, USA), Ki-67 (#ab92742; Abcam), and TFAP2C (#14572-1-AP; Proteintech) at 4℃ overnight. Afterwards, they were incubated for 1 h with HRP-conjugated IgG (#ab205718; Abcam) at 37℃. After development with DAB (Proteintech), the sections were counterstained with hematoxylin, dehydrated, and mounted for analysis. Images were acquired using an Olympus microscope (Japan). Supplemental Table 1 shows the clinical information of patients with LUSC.

Cell culture

NCI-H1703, SK-MES-1, NCI-H520, and NCI-H226 cells were obtained from Procell (Wuhan, China). BEAS-2B cells were purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China) and were cultured in DMEM containing 10% FBS at 37℃, 5% CO2. SK-MES-1 cells were grown in MEM supplemented with 10% FBS at 37℃. NCI-H520, NCI-H226, and NCI-H1703 cells were maintained in RPMI-1640 with 10% FBS at 37℃. RPMI-1640, DMEM, MEM, and FBS were purchased from Gibco (USA).

Quantitative real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen). Reverse transcription was then performed using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Quantitative real-time PCR reactions were then performed using TB Green® Premix Ex Taq™ II FAST qPCR reagent (TaKaRa) on Bio-Rad CFX96 System (USA). GAPDH was used as an internal reference. Supplemental Table 2 lists the primer sequences.

Cell transfection

The shRNAs targeting STEAP3 and TFAP2C (GenePharma, China) were inserted into pLKO.1-neo vector (Addgene, USA). The coding sequence (CDS) regions of the STEAP3 and TFAP2C genes were subcloned into pcDNA-3.1 vector (GenScript). Subsequently, cell transfection was conducted using Lipofectamine 3000 (Invitrogen). The transfected LUSC cells were selected for over four weeks by incubation with G418 (400 µg/mL). Supplemental Table 3 lists the sequences of shRNAs.

Western blot analysis

Total proteins were prepared using a RIPA reagent (CST). Equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes. Following blocking, the membranes were incubated with anti-STEAP3 (#28478-1-AP; Proteintech), anti-Vimentin (#3390; CST, USA), anti-Slug (#9585; CST), anti-c-myc (#10828-1-AP; Proteintech), anti-cyclin D1 (#ab16663; Abcam), anti-TFAP2C (#14572-1-AP; Proteintech), anti-β-catenin (#37447; CST), anti-PCNA (#ab92552; Abcam, USA), and anti-Ki-67 (#ab92742; Abcam) antibodies overnight at 4℃, and then with HRP-conjugated IgG at 37℃ for 1 h (SA00001-1, SA00001-2; Proteintech). An ECL reagent (Abcam) was added to visualize the protein bands.

CCK-8 assay

Cells were inoculated into a 96-well plate and cultured at 37℃. CCK-8 solution (10 µL; Dojindo Laboratories, Japan) was added per well for 2 h. The absorbance was determined at 450 nm.

Colony formation assay

Cells were inoculated into 6-well plates and cultured at 37℃ for 14 days. Cell colonies were fixed in 4% paraformaldehyde (Aladdin, China) for 10 min and stained with 0.5% crystal violet solution (Beyotime). The number of colonies was quantified. Images were taken under an Olympus microscope.

Wound healing assay

Cells were grown in a 6-well plate and cultured until 90% confluence. The culture supernatant was then discarded and a straight scratch was made using a 200 µL sterile pipette tip. The cells in each well were washed three times with PBS to remove non-adherent cells and then serum-free media was added to culture the cells. Images were taken at 0 and 24 h under an inverted microscope (Olympus).

Transwell assays

Cell migration was evaluated using a transwell chamber assay, while cell invasiveness was assessed using Matrigel-precoated transwell chambers. In brief, 600 µL of growth medium with 20% FBS and 100 µL of serum-free cell suspension containing 1 × 104 cells were added to the lower and upper chambers, respectively. After incubation for 24 h at 37℃, the cells were washed, fixed for 30 min with 4% paraformaldehyde, and then stained for 5 min with 0.5% crystal violet. Following PBS washes, the numbers of migrated or invaded cells were counted (DMi8 microscope; Leica, Germany).

ChIP assay

The cells were cross-linked for 10 min with 1% formaldehyde at 37℃ and then incubated for 5 min with glycine at 25℃. Cells were lysed in SDS lysis buffer (Beyotime) containing 1 mM PMSF for 10 min on ice. Genomic DNA was fragmented into 200 to 1000 bp by sonication. ChIP samples were used for the immunoprecipitation with anti-TFAP2C (#ab218107; Abcam) and non-specific IgG (ab172730; Abcam), and 20 µL of the ChIP sample was used as the input. Protein A + G agarose was added to each immunoprecipitation and incubated at 4℃ for 2 h with rotation The immunoprecipitated DNA was then amplified by real-time PCR.

Luciferase reporter assay

293T cells were seeded at 2 × 105 cells/well in 24-well plates. At approximately 80% confluence, the cells were transfected with TFAP2C overexpression plasmid and pGL3-Basic plasmid containing the promoter of the STEAP3 gene. At 24 h post-transfection, luciferase activity was measured using a dual-luciferase reporter assay kit (Promega, USA).

Immunofluorescence assay

The slides were fixed for 15 min using 4% paraformaldehyde, permeabilized for 30 min with 0.1% Triton X-100, and blocked for 15 min with goat serum at 25℃. The slides were then incubated with antibodies against E-cadherin (1:300; #20874-1-AP) and N-cadherin (1:400; #22018-1-AP) at 4℃ overnight, followed by incubation with Cy3-conjugated IgG (1:50; #SA00009-2) for 1 h at 25℃. DAPI (MedChemExpress, USA) was applied to counterstain the nuclei. Immunofluorescence images were collected using an Olympus microscope (Japan). All antibodies were purchased from Proteintech.

In vivo tumor xenograft model

Male BALB/c nude mice, aged 6 weeks and weighing 20 to 24 g, were purchased from Vital River (China) and housed under standard SPF conditions (45–60% humidity; 23–26 °C; 12-hour light/dark cycle) with ad libitum access to standard rodent chow diet. The mice were randomly assigned to four groups and subcutaneously inoculated with 1 × 107 stable transfectants (200 µL per mouse). Tumor long and short diameters were monitored weekly. After 35 days of injection, the mice were euthanized, and the tumors were weighed. Tumor volume was calculated using the following formula: Tumor volume = (long diameter × short diameter2)/2. The protocols adhered to the guidelines of the Guide for the Care and Use of Laboratory Animals: Eighth Edition (NIH) and were approved by the Institutional Animal Ethics Committee of China Medical University.

Metastatic HCC model

Male BALB/c nude mice (6 weeks old, 20 to 24 g) were randomly assigned to four groups. Each mouse was injected with 1 × 106 stable transfectants in 200 µL of cell resuspension via the tail vein. On day 42, tumor metastasis was monitored using an In Vivo Imaging System. The mice were then euthanized, and the lung and liver were collected for analysis.

Hematoxylin and eosin (H&E) staining

Lung and liver tissues were embedded in paraffin and then cut into 4 μm. The slices were deparaffinized, rehydrated, and stained for 5 min with hematoxylin (Beyotime). The slices were then treated with 1% hydrochloric acid for 3 s and stained with eosin (Beyotime) for 3 min. Graded concentrations of ethanol were used to dehydrate the sections. The sections were then exposed to xylene twice each for 10 min each and mounted in neutral balsam. The number of metastatic nodules in the lungs and livers was counted by microscopy (Olympus).

Statistical analysis

Data are presented as mean ± standard deviation. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey post hoc analysis. Comparisons between two groups were analyzed using Student’s t-test. A statistically significant result was considered when P < 0.05.

Results

Elevated STEAP3 expression predicts a poor prognosis in LUSC

The TCGA database demonstrated that STEAP3 expression was elevated in LUSC tissues relative to adjacent non-tumorous tissues (Fig. 1A). Furthermore, the gene level of STEAP3 was upregulated in LUSC tissues (Fig. 1B). Immunohistochemical staining also revealed a notable elevation in STEAP3 expression in LUSC tissues (Fig. 1C), which may provide insight into the potential role of STEAP3 in lung carcinogenesis. To further validate this finding, The expression of STEAP3 was quantified in four LUSC cell lines and normal lung epithelial cells through real-time PCR and western blot analyses. All four LUSC cell lines exhibited higher STEAP3 expression than normal lung epithelial cells. Among the four LUSC cell lines, SK-MES-1 cells exhibited the highest levels of STEAP3 expression, while NCI-H520 cells demonstrated the lowest levels of STEAP3 expression (Fig. 1D, E). Furthermore, Kaplan-Meier survival analyses revealed that LUSC patients with high STEAP3 expression exhibited poorer OS and RFS (Fig. 1F).

Fig. 1
figure 1

STEAP3 is highly expressed in lung squamous cell carcinoma (LUSC) tissues. (A) The mRNA levels of STEAP3 in LUSC and adjacent normal tissues were analyzed using the TCGA database. (B) A total of 40 paired LUSC and adjacent normal lung tissues were collected for mRNA level analysis of STEAP3. GAPDH was employed as the internal control. (C) The protein levels of STEAP3 in LUSC and adjacent normal tissues were analyzed by immunohistochemistry. Scale bar represents 100 μm. (D) The mRNA levels of STEAP3 in four human LUSC cell lines, including NCI-H1703, SK-MES-1, NCI-H520, and NCI-H226, as well as normal bronchial epithelial cells (BEAS-2B), were determined by real-time PCR analysis. GAPDH was employed as the internal control. (E) The protein levels of STEAP3 in four human LUSC cell lines and BEAS-2B were determined by western blot analysis. β-actin was employed as the internal control. (F) The Kaplan-Meier Plotter database was employed to compare overall survival (OS) and recurrence-free survival (RFS) between groups with disparate STEAP3 expression levels. *P < 0.05 and **P < 0.01

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STEAP3 knockdown restrains proliferation and metastasis of LUSC cells

To investigate the potential role of STEAP3, we proceeded to knock down STEAP3 expression in SK-MES-1 cells, which exhibited the highest levels of STEAP3 expression, and to overexpress STEAP3 in NCI-H520 cells, which exhibited the lowest levels of STEAP3 expression. Western blot analysis demonstrated a dramatic downregulation of STEAP3 in SK-MES-1 and upregulation of STEAP3 in NCI-H520 (Fig. 2A). As demonstrated by the CCK-8 assay, STEAP3 knockdown reduced cell proliferation, whereas STEAP3 overexpression promoted cell proliferation (Fig. 2B). Furthermore, the positive regulation of colony formation ability by STEAP3 was confirmed (Fig. 2C). Moreover, following the knockdown of STEAP3, there was an increase in the percentage of cells in the G1 phase, while the percentages of cells in the G2 and S phases were reduced. The overexpression of STEAP3 resulted in a reduction in the proportion of cells in the G1 phase, while simultaneously increasing the percentages of cells in the G2 and S phases (Fig. 2D). Furthermore, STEAP3 knockdown suppressed the migration and invasion of SK-MES-1 cells. STEAP3 overexpression enhanced the migratory and invasive capacities of NCI-H520 cells (Fig. 2E, F). Subsequently, the expression of EMT-associated markers was examined. Notably, STEAP3 knockdown was associated with reduced N-cadherin expression and elevated E-cadherin in SK-MES-1 cells. STEAP3 overexpression reduced E-cadherin expression and resulted in an induction of N-cadherin expression in NCI-H520 cells (Fig. 3A). The knockdown of STEAP3 in SK-MES-1 cells decreased Vimentin and Slug expression, while the overexpression of STEAP3 in NCI-H520 cells led to an increase in their expression (Fig. 3B).

Fig. 2
figure 2

Knockdown of STEAP3 inhibits the proliferation and metastasis of LUSC cells. (A) The SK-MES-1 and NCI-H520 cells were transfected with the recombinant plasmids pLKO.1-sh-STEAP3 and pcDNA-3.1-STEAP3, respectively. Following the selection of stable transfectants, the protein levels of STEAP3 were quantified by western blot analysis. β-actin was employed as the internal control. (B) The assessment of cellular proliferation at various time points was performed using the CCK-8 assay following the knockdown and overexpression of STEAP3. (C) The colony formation assay was employed to evaluate the colony-forming ability of LUSC cells following STEAP3 knockdown and overexpression. (D) Cell cycle distribution was analyzed by flow cytometry. (E) The capacity of the cells to migrate was evaluated using the wound healing assay. Scale bar represents 200 μm. (F) The transwell assay was employed to evaluate the cell migration and invasion ability. Scale bar represents 100 μm. **P < 0.01

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STEAP3 knockdown inhibiting the β-catenin signaling

To ascertain whether β-catenin signaling is implicated in this process, we employed western blot analysis to quantify β-catenin signaling. STEAP3 knockdown impeded β-catenin translocation from the cytoplasm to the nucleus. Conversely, a significant nuclear translocation was observed following STEAP3 overexpression (Fig. 3C). As illustrated in Fig. 3D, STEAP3 knockdown reduced c-myc and cyclin D1 expression levels in SK-MES-1 cells, whereas STEAP3 overexpression led to an increase in their expression levels in NCI-H520 cells.

Fig. 3
figure 3

Knockdown of STEAP3 inhibits the EMT and the β-catenin signaling pathway in LUSC cells. (A) Following the knockdown and overexpression of STEAP3, the cells were subjected to immunofluorescence analysis of the expression levels of E-cadherin and N-cadherin. Scale bar represents 50 μm. (B) Total proteins were extracted from the stable transfectants and then the protein levels of Vimentin and Slug were quantified by western blot analysis. β-actin was employed as the internal control. (C) The protein levels of β-catenin in the cytoplasm and nucleus were quantified by western blot analysis. β-actin was employed as the internal control. (D) The protein levels of c-myc and cyclin D1 were quantified by western blot analysis. β-actin was employed as the internal control. *P < 0.05 and **P < 0.01

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TFAP2C binds directly to the STEAP3 promoter and positively regulates its expression

To gain further insight into the regulatory mechanisms that control STEAP3 expression levels, we set out to identify potential transcription factors that acted as upstream regulators of STEAP3. The GEPIA database demonstrated a significant positive correlation between TFAP2C and STEAP3 expression in LUSC (Fig. 4A). Subsequently, the TCGA database was employed to analyze TFAP2C expression in LUSC and adjacent non-tumorous tissues. TFAP2C expression was significantly higher in LUSC tissues (Fig. 4B). The upregulation of the TFAP2C gene level in LUSC tissues was confirmed by real-time PCR (Fig. 4C). Moreover, our findings revealed a notable positive correlation between TFAP2C and STEAP3 expression in LUSC (R = 0.53, P < 0.01), as evidenced by our analysis of clinical samples (Fig. 4D). Immunohistochemical staining also demonstrated that TFAP2C expression was significantly elevated in LUSC tissues compared to adjacent normal tissues (Fig. 4E). Furthermore, elevated TFAP2C expression was correlated with poor OS in LUSC (Fig. 4F). Subsequently, SK-MES-1 cells were transfected with three specific shRNAs to knock down TFAP2C expression, while NCI-H520 cells were transfected with a TFAP2C overexpression plasmid to overexpress it. All the three shRNAs were found to significantly reduce TFAP2C expression in SK-MES-1 cells, with shRNA-3 exhibiting a stronger knockdown efficiency than the other two shRNAs. Furthermore, TFAP2C was demonstrated to be overexpressed in NCI-H520 cells transfected with a TFAP2C overexpression plasmid (Fig. 4G, H). TFAP2C knockdown lowered STEAP3 expression in SK-MES-1 cells, whereas TFAP2C overexpression led to an increase in STEAP3 expression in NCI-H520 cells (Fig. 4I, J). The experimentally defined transcription factor binding motif of TFAP2C was demonstrated (Fig. 4K). A ChIP assay was then conducted, which demonstrated that STEAP3 enrichment was increased in the anti-TFAP2C group (Fig. 4L). The two potential binding sites with the highest scores, as predicted by the Jaspar database, were selected for validation. TFAP2C overexpression significantly enhanced the luciferase activity of the wild-type STEAP3 promoter. The transcriptional activation was found to be related to binding site 1 (E1), but not the E2 site (Fig. 4M).

Fig. 4
figure 4

The relationship between TFAP2C and STEAP3. (A) The correlation between TFAP2C and STEAP3 was analyzed using the GEPIA database. (B) The mRNA levels of TFAP2C in LUSC and adjacent normal tissues were determined using the TCGA database. (C) A total of 40 paired LUSC and adjacent normal lung tissues were collected for mRNA level analysis of TFAP2C. (D) The correlation between TFAP2C and STEAP3 was analyzed using our own collected clinical samples. (E) The protein levels of TFAP2C in LUSC and adjacent normal tissues were analyzed by immunohistochemistry. Scale bar represents 100 μm. (F) The Kaplan-Meier Plotter database was employed to compare overall survival (OS) between groups with disparate TFAP2C expression levels. (G) The SK-MES-1 cells were transfected with a recombinant plasmid carrying three different shRNAs targeting TFAP2C. The NCI-H520 cells were transfected with a recombinant plasmid carrying full-length CDS region of TFAP2C. The mRNA levels of TFAP2C were examined using real-time PCR. GAPDH was used as the internal control. (H) The protein levels of TFAP2C were quantified by western blot analysis. β-actin was employed as the internal control. (I) Following the knockdown and overexpression of TFAP2C, the cells were subjected to real-time PCR analysis of the mRNA levels of STEAP3. GAPDH was employed as the internal control. (J) The protein levels of STEAP3 were quantified by western blot analysis following the knockdown and overexpression of TFAP2C. β-actin was employed as the internal control. (K) The JASPAR database was used to predict the binding motif of TFAP2C. (L) The binding of transcription factor TFAP2C to the promoter of STEAP3 was examined by ChIP assay. (M) The regulatory relationship between TFAP2C and STEAP3 was analyzed using a dual-luciferase reporter assay. *P < 0.05 and **P < 0.01

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STEAP3 overexpression partially abolishes TFAP2C knockdown-mediated anti-tumor effects

We next determined whether STEAP3 can be regulated by TFAP2C in affecting cell proliferation and metastasis of LUSC cells. TFAP2C knockdown inhibited cell proliferation (Fig. 5A) and caused cell cycle arrest at the G1 phase (Fig. 5B). In addition, the migratory and invasive capabilities were impaired following TFAP2C knockdown (Fig. 5C, D). Moreover, the immunofluorescence assay demonstrated that TFAP2C knockdown significantly reduced N-cadherin and elevated E-cadherin expression (Fig. 5E). The involvement of β-catenin pathway was further validated. Our findings indicated that β-catenin was accumulated in the cytoplasm and its nuclear level was decreased in SK-MES-1 cells following TFAP2C knockdown (Fig. 5F). This was accompanied by a reduction in the expression levels of the downstream target genes, including c-myc and cyclin D1 (Fig. 5G). It is noteworthy that STEAP3 overexpression largely negated the impacts of TFAP2C knockdown on cell proliferation, cell cycle progression, metastasis, and the β-catenin pathway and its downstream target genes (c-myc and cyclin D1) (Fig. 5A–G).

Fig. 5
figure 5

STEAP3 overexpression partially abolishes TFAP2C knockdown-mediated anti-tumor effects. (A) The assessment of cellular proliferation at various time points was performed using the CCK-8 assay following co-transfection. (B) Cell cycle distribution was analyzed by flow cytometry. (C) The capacity of the cells to migrate was evaluated using the wound healing assay. Scale bar represents 200 μm. (D) The transwell assay was employed to evaluate the cell migration and invasion ability. Scale bar represents 100 μm. (E) The expression levels of E-cadherin and N-cadherin were analyzed by immunofluorescence analysis. Scale bar represents 50 μm. (F) The protein levels of β-catenin in the cytoplasm and nucleus were quantified by western blot analysis. β-actin was employed as the internal control. (G) The protein levels of c-myc and cyclin D1 were quantified by western blot analysis. β-actin was employed as the internal control. *P < 0.05 and **P < 0.01

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STEAP3 knockdown slows tumor growth and metastasis

To substantiate its oncogenic role in vivo, mouse xenograft experiments were conducted. Stable transfectants were injected subcutaneously into nude mice and tumor volumes were measured for 35 days. The mice inoculated with transfectants stably knocked down STEAP3 developed smaller tumors, while STEAP3 overexpression contributed to larger tumors than those in the vector group (Fig. 6A). STEAP3 knockdown notably reduced tumor volume and weight, while STEAP3 overexpression increased these parameters (Fig. 6A, B). STEAP3 knockdown significantly decreased STEAP3, PCNA, and Ki-67 expression levels. Conversely, their expression levels were upregulated after STEAP3 overexpression (Fig. 6C, D). Furthermore, STEAP3 knockdown inhibited β-catenin translocation to the nucleus. Conversely, STEAP3 overexpression promoted the its nuclear translocation (Fig. 6E). To determine STEAP3’s role in LUSC metastasis in vivo, nude mice were administered stable transfectants were into to construct metastasis models. In vivo imaging revealed that STEAP3 knockdown suppressed metastasis, whereas STEAP3 overexpression promoted metastasis in nude mice (Fig. 7A). The lungs and livers were excised to evaluate metastasis by H&E staining. The results revealed fewer numbers of metastatic nodules in the lungs and liver following STEAP3 knockdown. The number of metastatic nodules was found to be greater in the mice injected with the STEAP3 overexpression vector than in those injected with an empty vector (Fig. 7B). The knockdown of STEAP3 was observed to result in a notable reduction in the expression levels of STEAP3 in metastatic tumor tissues. Conversely, the overexpression of STEAP3 resulted in increased levels of this protein (Fig. 7C). Additionally, knockdown of STEAP3 resulted in the inhibition of β-catenin nuclear translocation in metastatic tumor tissues. In contrast, STEAP3 overexpression facilitated its translocation to the nucleus (Fig. 7D).

Fig. 6
figure 6

STEAP3 knockdown inhibits tumor growth. (A) The nude mice were administered the stable transfectants via subcutaneous injection. Tumor volumes were measured on a weekly basis from day 7 onwards. (B) On day 35, the tumors were excised and weighed. (C) The expression levels of STEAP3, PCNA, and Ki-67 in tumor tissues were determined by immunohistochemistry. Scale bar represents 50 μm. (D) The protein levels of STEAP3, PCNA, and Ki-67 in tumor tissues were quantified by western blot analysis. β-actin was employed as the internal control. (E) The levels of β-catenin protein in the cytoplasm and nucleus of tumor tissues were quantified by western blot analysis. β-actin was employed as the internal control. **P < 0.01

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Fig. 7
figure 7

STEAP3 knockdown inhibits tumor metastasis. (A) The nude mice were administered stable transfectants to construct metastasis models. In vivo imaging was employed to assess the metastasis of LUSC in nude mice. (B) At the conclusion of the animal experiments, the nude mice were euthanized, and the lungs and livers were obtained from each animal for subsequent analysis. The number of metastatic nodules in the lungs and livers was determined through histological examination using H&E staining. Scale bar represents 250 μm. (C) The protein levels of STEAP3 in metastatic tumor tissues were quantified by western blot analysis. β-actin was employed as the internal control. (D) The levels of β-catenin protein in the cytoplasm and nucleus of metastatic tumor tissues were quantified by western blot analysis. β-actin was employed as the internal control. **P < 0.01

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Discussion

Previous evidence has indicated that high STEAP3 is associated with poor survival in cancer patients, including those with ovarian cancer, hepatocellular carcinoma, and glioma [13, 15, 18]. However, its role in LUSC is currently unclear. STEAP3 was highly expressed in LUSC and its high expression was associated with an unfavorable prognosis. Additionally, the overexpression of STEAP3 activated the β-catenin, promoting LUSC growth and metastasis. The transcriptional activation of STEAP3 by TFAP2C contributes to LUSC progression.

It is reported that STEAP3 is significantly elevated in hepatocellular carcinoma, triple-negative breast cancer, colon cancer, clear cell renal cell carcinoma, and ovarian cancer [13,14,15,16,17]. In hepatocellular carcinoma, STEAP3 has been shown to promote cell proliferation by inducing EGFR nuclear translocation [15]. In colon cancer, STEAP3 triggers a malignant phenotype by affecting histone acetylation [16]. Furthermore, evidence indicates that a reduced expression of STEAP3 results in hepatocellular carcinoma in individuals with a history of cirrhosis [19]. We observed abnormally high expression of STEAP3 in LUSC and its high expression is associated with poor prognosis, suggesting an oncogenic role of STEAP3 in LUSC. Furthermore, overexpression of STEAP3 promoted the proliferation, migration, invasion, and EMT of LUSC cells. Conversely, STEAP3 knockdown was found to have the opposite effects on these malignant phenotypes. Additionally, it was demonstrated that overexpression of STEAP3 accelerated tumor growth and metastasis in vivo, whereas knockdown of STEAP3 inhibited these processes. These results indicate that STEAP3 may contribute to LUSC proliferation and metastasis. Nevertheless, the molecular mechanisms regulating STEAP3 expression during LUSC progression remain poorly understood.

Passer et al. reported that the STEAP3 promoter contained a p53-responsive element. The authors demonstrated that p53-mediated transcriptional activation of STEAP3 in cervical cancer contributes to cell cycle arrest and apoptosis [20]. To gain further insight into the regulatory mechanisms, the JASPAR database was employed to predict the potential transcriptional regulatory factors of STEAP3. The analysis revealed that the promoter region of STEAP3 contained several TFAP2C binding sites. TFAP2C is a transcription factor that controls various biological processes [21]. The aberrant expression of TFAP2C is associated with the malignant development of several cancers [21,22,23,24,25,26]. TFAP2C knockdown inhibited LUSC cell proliferation and metastasis. The findings indicate an oncogenic role of TFAP2C in LUSC, which is consistent with previous studies [26, 27]. Recent studies have demonstrated that TFAP2C accelerates NSCLC progression by influencing the transcription of its target genes [28, 29]. TFAP2C has been demonstrated to elevate TGFBR1 level, which in turn enhances the malignancy and aggressiveness of NSCLC [28]. Moreover, TFAP2C modulates the chemosensitivity of LUAD cells by activating the lncRNA MALAT1 [29]. Nevertheless, the relationship between TFAP2C and STEAP3 is unclear. It was demonstrated that the knockdown of TFAP2C in SK-MES-1 cells reduced STEAP3 expression, while the overexpression of TFAP2C in NCI-H520 cells increased STEAP3 expression. Moreover, The GEPIA database indicated that there was a significant positive correlation between the expression of TFAP2C and STEAP3 in LUSC. The results of our analysis of clinical samples confirmed a notable positive correlation between TFAP2C and STEAP3 expression in LUSC. ChIP and luciferase reporter assays are frequently utilized to explore the regulatory relationship between transcription factors and downstream target genes [30, 31]. Consequently, both functional assays were utilized to verify the regulatory relationship between TFAP2C and STEAP3. ChIP assay demonstrated that TFAP2C bound to the STEAP3 promoter, while dual-luciferase reporter assay confirmed that TFAP2C activated STEAP3 promoter activities. The findings indicate that TFAP2C positively regulates STEAP3 expression in LUSC. To ascertain whether TFAP2C-mediated transcriptional regulation of STEAP3 contributes to the progression of LUSC, rescue experiments were conducted. STEAP3 overexpression attenuated the anti-tumor effects of TFAP2C knockdown on cell proliferation, cell cycle distribution, and metastasis in LUSC. TFAP2C-mediated transcriptional activation of STEAP3 may contribute to the proliferation and metastasis of LUSC.

The β-catenin signaling is essential for maintaining embryonic development and tissue homeostasis [32]. Abnormal activation of this pathway can result in an increased prevalence, poor prognosis, progression of malignancy, and even cancer-related mortality [33]. Overexpression of STEAP3 has been demonstrated to activate the β-catenin pathway, which in turn accelerates CRC development [34]. The study demonstrated that STEAP3 knockdown reduced nuclear β-catenin level, whereas STEAP3 overexpression elevated nuclear β-catenin level in vitro. Moreover, STEAP3 knockdown significantly reduced the relative expression of several Wnt downstream genes, including c-myc and cyclin D1, while STEAP3 overexpression elevated their levels. Similarly, STEAP3 was observed to positively regulate the β-catenin signaling pathway in vivo. These findings indicate that STEAP3 may activate the β-catenin pathway, which in turn accelerates LUSC development. In uterine leiomyomas, TFAP2C upregulation is also associated with the dysregulation of the β-catenin pathway [35]. TFAP2C knockdown inhibited the β-catenin signaling pathway in LUSC. The results of rescue assays revealed that STEAP3 overexpression partially blocked the inhibitory effects of TFAP2C knockdown on the β-catenin pathway in LUSC. The findings suggest that the TFAP2C/STEAP3 axis may facilitate LUSC development by regulating the β-catenin pathway.

In conclusion, STEAP3 was aberrantly expressed in LUSC, and its high expression predicated a poor prognosis. Additionally, STEAP3 knockdown inhibited cell proliferation and metastasis by activating the β-catenin pathway. Moreover, TFAP2C bound directly to STEAP3 promoter, thereby positively regulating its expression in LUSC. The anti-tumor effects of TFAP2C knockdown were partially reversed following STEAP3 overexpression. The findings indicate that TFAP2C-mediated transcriptional activation of STEAP3 contributes to LUSC development by affecting the β-catenin pathway. The TFAP2C/STEAP3 axis may represent a potential therapeutic target for LUSC treatment.

Data availability

No datasets were generated or analysed during the current study.

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  1. Department of Radiology, Shengjing Hospital of China Medical University, No. 36, Sanhao Street, Heping District, Shenyang, 110004, Liaoning, P. R. China

    Tong Sun & Zhiguang Yang

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T.S. performed the experiments and prepared the figures. Z.Y. and T.S. designed the experiments and wrote the manuscript. Z.Y. revised the manuscript. All authors reviewed and approved the final version of the manuscript.

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Correspondence to Zhiguang Yang.

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The study involving human participants was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Shengjing Hospital, China Medical University. All patients provided written informed consent. The animal study was conducted in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals: Eighth Edition (NIH) and was approved by the Institutional Animal Ethics Committee of China Medical University.

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Sun, T., Yang, Z. TFAP2C-mediated transcriptional activation of STEAP3 promotes lung squamous cell carcinoma progression by regulating the β-catenin pathway. Biol Direct 19, 135 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13062-024-00584-w

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