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Introduction

Breast cancer (BRCA) is the most commonly diagnosed malignancy among women worldwide (1). Approximately 2.1 million patients with BRCA were newly diagnosed, and >0.62 million of those patients succumbed to the disease in 2018 (2). Based on the absence or presence of human epidermal growth factor (HER)2, estrogen receptors (ERs) or progesterone receptors (PRs), BRCA has been divided into three subtypes, namely the ER+/HER2−, HER+ and ER−/PR−/HER2− subtypes (3). Although the majority of patients are diagnosed at the non-metastatic and curable stage, the remaining women with advanced-stage BRCA are confronted with a dire predicament, as there are no effective therapies available. Therefore, identifying novel molecular biomarkers or essential contributors for BRCA may aid in the development of effective therapies against this malignancy.

The histidine triad (HIT) protein superfamily is classified into at least three subgroups, containing histidine triad nucleotide-binding protein (HINT), fragile histidine triad diadenosine triphosphatase and galactose-1-phosphate uridylyltransferase (4). Increasing evidence has indicated that the HINT subfamily function as tumor suppressors in various types of cancer. The depletion of HINT1 in mice has been shown to promote the development of N-nitrosomethylbenzylamine-induced squamous tumors in the forestomach and 7,12-dimethylbenz[a]anthracene-induced BRCA (5,6). Long-term observations have revealed that HINT1-knockout mice exhibit a higher incidence of spontaneous tumors compared with wild-type mice (6). The decreased expression of HINT1 has been found in gastric cancer and hepatocellular carcinoma (HCC) (7,8). Similar to HINT1, HINT2 has also been identified as a tumor suppressor in various types of cancer. HINT2 expression has been found to be downregulated in HCC and colorectal cancer (9,10). The decreased expression of HINT2 has been shown to promote the proliferation, migration and tumorigenesis of cancer cells (9,11). Nevertheless, the significance of HINT3 in cancer, including in BRCA, remains unclear.

In the present study, the clinical relevance of HINT3 and its potential role in BRCA were investigated. In vitro studies using cell lines and in vivo experiments using a nude mouse model were performed to investigate the functions of HINT3 in BRCA growth. A potential role of HINT3 in regulating AKT/mammalian target of rapamycin (mTOR) signaling was also explored using reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and dual-luciferase activity assays. Taken together, the results of the present study demonstrate that HINT3 functions as a tumor suppressor, and inhibits the activation of the AKT/mTOR signaling cascade in BRCA.

Materials and methods

The cancer genome atlas (TCGA) database analysis

The clinical significance of HINT3 in BRCA was analyzed using the UALCAN website (http://ualcan.path.uab.edu), which provided in-depth analyses of TCGA (http://cancergenome.nih.gov) for 31 different cancer subtypes (12). First, 1,097 cancerous and 114 normal tissues were used to analyze HINT3 expression in BRCA and normal tissues. Secondly, 114 normal, 183 stage I, 615 stage II, 247 stage III and 20 stage IV BRCA tissues were used to analyze the correlation between HINT3 expression and the BRCA stage. Thirdly, 114 normal, 516 N0 (no regional lymph node metastasis), 362 N1 (metastases in 1–3 axillary lymph nodes), 120 N2 (metastases in 4–9 axillary lymph nodes) and 77 N3 (metastases in ≥10 axillary lymph nodes) BRCA tissues were used to analyze the association between HINT3 expression and nodal metastasis status. Fourthly, 114 normal tissues, 566 luminal, 37 HER2-positive and 116 triple-negative cancer tissues from TCGA database were used to analyze the expression of HINT3 and BRCA subclasses. Finally, Spearman's correlation analysis was used to investigate the correlation between HINT3 and PTEN in patients with BRCA from TCGA database.

Human BRCA samples

Human BRCA samples were collected from 14 patients prior to their receiving any treatment intervention at the General Hospital of Ningxia Medical University (Yinchuan, China) between January, 2019 to September, 2020. The patient characteristics are presented in Table I. Written informed consent was obtained from all the patients. The present study was approved by the Ethics Committee of General Hospital of Ningxia Medical University (no. 2022-68). The ethics approval was obtained in January, 2019.

Table I. Association of HINT3 expression with the clinicopathological characteristics of 14 patients with breast cancer.

Table I.

Association of HINT3 expression with the clinicopathological characteristics of 14 patients with breast cancer.

	HINT3 expression
Characteristic	High	Low	P-value
Age, years
<60	5	5	NS
≥60	2	2
Stage I/II/III
I	3	3	NS
II/III	4	4
Ki-67 status
Positive	6	2	NS
Negative	1	5
Estrogen receptor status
Positive	6	2	NS
Negative	1	5
Progesterone receptor status
Positive	6	2	NS
Negative	1	5
Triple-negative
Yes	1	5	NS
No	6	2

[i] For the analysis of the association between the HINT3 expression levels and clinical features, Fisher's exact test was used to analyze the categorical variables. The median expression level was used as the cut-off. The low expression of HINT3 in 14 patients was classified as values below the 50th percentile. A high HINT3 expression in 14 patients was classified as values at or above the 50th percentile. P<0.05 was considered to indicate a statistically significant difference. HINT3, histidine triad nucleotide-binding 3; NS, not significant.

Cells and cell culture

The human. The MCF-7 and MDA-MB-231 cells were cultured in HyClone® medium (Cytiva), the MDA-MB-436 and MDA-MB-468 cells were cultured in L15 medium (Gibco; Thermo Fisher Scientific, Inc.), and the MCF10A cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), which was supplemented with 10% Gibco® fetal bovine serum (Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin solution (HyClone; Cytiva). The cell culture was maintained at 37°C in an atmosphere of 5% CO2.

Lentivirus-mediated HINT3 knockdown and overexpression

A lentivirus vector (GeneChem, Inc.) was used to knock down HINT3 in the MCF-7 and MDA-MB-231 cells due to the expression of HINT3. This system comprised three vectors: pGCSIL-GFP (inserted with the targeted shRNA), pHelper1.0 (gag/pol) and Helper2.0 (VSVG). The sequences of the shRNAs were as follows: Negative control, 5′-TTCTCCGAACGTGTCACGT-3′; shHINT3-1, 5′-GCGAGAATGAGGACCTAATTT-3′; and shHINT3-2, 5′-GAGTCAATTCCTATTGGTTTA-3′. After cloning the shRNA into the pGCSIL-GFP vectors, 15 µg of pGCSIL-GFP-shHINT3-1/2 vectors were co-transfected with 5 µg of pHelper1.0 and 5 µg of Helper2.0 into 293FT cells (cat. no. R7007, Thermo Fisher Scientific, Inc.) using Invitrogen™ Lipofectamine™ 2000 reagent (Thermo Fisher Scientific, Inc.). To overexpress HINT3, the coding sequence of HINT3 was synthesized and inserted into the pCDH-EGFP lentivirus vectors (GeneChem, Inc.). The lentivirus was packaged by co-transfecting 10 µg of pCDH-empty or 10 µg of pCDH-HINT3, 10 µg of PSPAX2 and 10 µg of PDM2G in 293FT cells using Lipofectamine 2000 for 72 h. Viral supernatants were harvested at 48 and 72 h following transfection, and then filtered and centrifuged at 72,000 × g/min for 2 h at 4°C for subsequent infection. The cells were infected with virus at a multiplicity of infection of 3 for 24 h. The knockdown and overexpression efficiency was subsequently assessed using western blot analysis.

Western blot analysis

Total proteins were extracted from the shCtrl, shHINT3-1 and shHINT3-2, Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells by lysing them in lysis buffer (Beyotime Institute of Biotechnology). BCA protein assay, using a BCA kit provided by the Beyotime Institute of Biotechnology, was performed to detect the amounts of protein. The proteins were loaded on to 10% SDS-PAGE gels for separation, and subsequently transferred onto PVDF membranes (ISEQ00010, MilliporeSigma). Subsequently, the membranes were blocked with 5% non-fat milk for 60 min at room temperature and incubated with the following primary antibodies at 4°C overnight: Anti-HINT3 (1:800; cat. no. ab121960, Abcam), anti-phosphatase and tensin homolog (PTEN; 1:1,000; cat. no. CST9188), phosphorylated (p)-AKT (1:1,000; cat. no. CST13038), AKT (1:1,000; cat. no. CST4691), p-S6 (1:1,000; cat. no. CST2215) and S6 (1:1,000; cat. no. CST2217) (all from Cell Signaling Technology, Inc.), mTOR (1:1,000, cat. no. ab32028, Abcam) and p-mTOR (1:1,000, cat. no. ab109268, Abcam). The loading control antibodies used were: GAPDH (1:1,000, sc-32233; Santa Cruz Biotechnology, Inc.) and β-actin (1:3,000; cat. no. 66009-1-Ig, ProteinTech Group, Inc.). The secondary antibodies, HRP conjugates (1:6,000; cat. nos. SA00001-1 and SA00001-2) were from ProteinTech Group, Inc. The membranes were then washed with TBST containing 1% Tween-20 three times and incubated with the secondary antibodies for 2 h at room temperature. After washing with PBS for three times, the membranes were subjected enhanced chemiluminescence using an ECL kit (Promega Corporation). ImageJ software (V1.8.0, National Institutes of Health) was performed to analyze the intensity of target proteins.

RT-qPCR analysis

The shCtrl, shHINT3-1 and shHINT3-2, Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells were lysed using Invitrogen® TRIzol™ reagent (Thermo Fisher Scientific, Inc.). Total RNA was extracted using an Ultrapure RNA kit from CoWin BioAciences (cat. no. CW0581), following the manufacturer's protocol. After reverse transcribing the RNA into cDNA using an RT-PCR kit (K1002S, Promega Corporation), the qPCR reaction was performed on a Bio-Rad Laboratories CFX96 machine with SYBR™-Green PCR Master Mix (Thermo Fisher Scientific, Inc.). The sequences of the qPCR primers were as follows: HINT3 forward, 5′-CTGGTTGAGAACATGGTAACT-3′ and reverse, 5′-TGATCAGCTGTGATAAACCAAT-3′; PTEN forward, 5′-TTTGAAGACCATAACCCACCAC-3′, and reverse, 5′-ATTACACCAGTTCGTCCCTTTC-3′; and GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′, and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. GAPDH served as the internal control. The thermocycling conditions were as follows: An initial denaturation at 94°C for 10 min, followed by 45 of cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The 2−ΔΔCq method was performed for quantification (13).

Cell Counting Kit-8 (CCK-8) assay

Equal numbers (3,000 cells per well) of shCtrl, shHINT3-1, shHINT3-2, Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells were seeded into 96-well plates, which contained 200 µl HyClone culture medium (HyClone; Cytiva) with 10% FBS (cat. no. 26010074, Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin solution (cat. no. 10378016, Gibco; Thermo Fisher Scientific, Inc.). After 1, 2, 3 and 4 days, 20 µl CCK-8 reagent (Beyotime Institute of Biotechnology) were added to each well, and the cells were incubated at 37°C for 3 h. The optical density (OD) was detected at 450 nm using a SPECTROstar® Nano microplate reader (BMG LABTECH).

Colony formation assay

Equal numbers of shCtrl, shHINT3-1, shHINT3-2 (500 cells per well for HINT3 knockdown), Ctrl and HINT3-overexpressing (2,000 cells per well for HINT3 overexpression) MCF-7 and MDA-MB-231 cells were seeded into six-well plates. Following culture for 10 days, the colonies were fixed with methanol for 10 min at room temperature and stained using Giemsa solution (48900; MilliporeSigma) for 20 min at room temperature. Images of the colonies were captured using a camera (D810A; Nikon Corporation) and the numbers of the colonies were counted using Photoshop CS5 (Adobe Systems, Inc.).

Transwell assay

Filter migration chambers (8.0 µm; Corning, Inc.) were used to detect the migration of the shCtrl, shHINT3-1 and shHINT3-2-transfected MCF-7 and MDA-MB-231 cells. In brief, 60,000 cells were seeded onto the upper surface of the chambers. The lower chamber was filled with 200 µl HyClone culture medium (HyClone; Cytiva) containing 10% FBS (cat. no. 26010074, Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin solution (cat. no. 10378016, Gibco; Thermo Fisher Scientific, Inc.). Following incubation of the chambers at 37°C for 24 h, the cells on the upper surface were removed using cotton tips. The cells on the lower surface were fixed with methanol for 10 min at room temperature and stained using crystal violet (cat. no. 94448, MilliporeSigma) for 20 min at room temperature. The stained cells were photographed using a light microscope (Ei-BI-1000X, Nikon Corporation). The magnification was ×20.

Cell apoptosis detection

Cell apoptosis was assessed using an Invitrogen® PI/Annexin V-APC assay kit (cat. no. V35133, Thermo Fisher Scientific, Inc.). First, the cells were collected from six-well plates using EDTA-free trypsin (Corning, Inc.). Subsequently, cell apoptosis was performed according to the manufacturer's protocol. Briefly, the cells were incubated with Annexin-V and PI at room temperature for 20 min in the dark. The samples were analyzed using a FACScan (BD Biosciences) flow cytometer.

Dual luciferase activity reporter assay

The promoter sequence of PTEN was inserted into the pGL3.basic reporter vector (cat. no. E1751, Promega Corporation), whereas the coding sequence of HINT3 was inserted into pCDNA3.1 vectors. The MCF-7 cells were transfected with 0.1 µg pGL3.basic, TK and shRNAs or pCDNA3.1 vectors using Lipofectamine® 2000(Thermo Fisher Scientific, Inc.). Following transfection for 48 h, the firefly and Renilla luciferase activities were measured using the dual luciferase reporter kit (cat. No. E1910, Promega Corporation), following the manufacturer's protocol.

5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay

EdU incorporation assay was performed to determine DNA synthesis. Briefly, the cells were incubated with 50 µM EdU (Guangzhou RiboBio Co., Ltd.) for 24 h. The cells were then collected and centrifuged at 300 × g for 5 min at room temperature. Red fluorescent reactive dye (Thermo Fisher Scientific, Inc.) was added to the cells for 30 min at room temperature in the dark, followed by washing with PBS with 1% BSA and fixing with 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 15 min at room temperature. The fixation solution was then removed, and the cells were resuspended in saponin-based permeabilization buffer (MilliporeSigma) and incubated for 15 min at room temperature. Subsequently, the cells were analyzed under a fluorescence microscope (Olympus Corporation).

Immunohistochemistry (IHC)

A total of 24 paired breast cancer tissues and adjacent normal tissues were collected without any intervention. The paraffin-embedded tissues (4-µm-thick) derived from the patients were deparaffinized, dehydrated and treated with 3% hydrogen peroxide for 10 min to neutralize endogenous peroxidase activity. Antigen retrieval was performed by immersing the slides in 10 mM of boiling citrate buffer (cat. no. ab64214, Abcam) (pH 6) for 22 min using a microwave oven and then cooling with cold Tris-buffered saline (TBS, pH 7.4) for 20 min. The sections were pre-incubated in 0.1 M Tris-HCl buffer (cat. no. 26-575, Moltox) (pH 7.5) containing 3% BSA at room temperature, and then in TBS containing 0.02% biotin for 15 min to reduce nonspecific staining. The following antibodies were used to assess HINT3 and Ki-67 expression: Anti-HINT3 (1:100; cat. no. ab121960, Abcam) and anti-KI-67 (1:100, IR626, clone MIB-1; Dako; Agilent Technologies, Inc.) at 4°C for 30 min. After washing with PBS three times, the immunoreactions were visualized using the rabbit/mouse EnVision-HRP and DAB + kit (Dako; Agilent Technologies, Inc.). IHC was performed to analyze the expression of HINT3. HINT3 expression was examined using signal intensity as follows: 0 (negative), 1 (low), 2 (moderate) and 3 (high) using a Nikon microscope (ECLIPSE E200, Nikon Corporation).

In vivo xenograft assay

Female Balb/c nude mice (weight, 14–16 g, 6–8 weeks) were purchased from the Animal Center of General Hospital of Ningxia Medical University, and were fed a standard diet and water in a specific pathogen-free room. The temperature was maintained at 25°C and the room was lit with a 12-h light/12-h dark cycle. A total of 10 mice were randomly divided into two groups (n=5 in each group). A total of 1×107 MDA-MB-231 cells and a total of 107 MCF-7 cells were subcutaneously implanted into the right armpit of 5-week-old female Balb/c nude mice, as previously described (14–18). The volume of the xenograft tumors was calculated using the following formula: v=ab2/2, where ‘a’ represents the long diameter and ‘b’ is the short diameter. Animal health and behavior were monitored at least twice a week. None of the mice died during the experiment. All the animals were euthanized when the tumor volume reached approximately 1,500 mm3. The mice implanted with MDA-MB-231 cells were euthanized using CO2 (a flow rate of 50% chamber volume/min) after 45 days, whereas those implanted with the MCF-7 cells were euthanized after 48 days. The death of the mice was confirmed when the mice exhibited loss of breathing, and no response to stimuli. The animal experiments were approved by the Ethics Committee of General Hospital of Ningxia Medical University (no. 2022-68, which was the same ethics approval document as that for the human experiments), and were performed according to the animal guidelines of the General Hospital of Ningxia Medical University. The ethics approval for the study was obtained in January, 2019.

Statistical analysis

Statistical analyses were performed using GraphPad prism 8 software (GraphPad Software, Inc.). The data are presented as the mean ± standard error of mean (SEM). An unpaired Student's t-test was used for analyzing differences between two groups, and one-way ANOVA followed by Tukey's post hoc test for multiple group comparisons. The χ2 test or Fisher's exact test were used to analyze categorical variables. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of HINT3 is reduced in BRCA tissues, and is inversely associated with tumor stage

TCGA database contains a large amount of information on the transcript abundance of various genes in a variety of cancer types. In the present study, to evaluate the clinical relevance of HINT3 in BRCA, the mRNA expression levels of HINT3 were analyzed using TCGA database. The analysis revealed that HINT3 expression was decreased in BRCA tissues compared with normal tissues (Fig. 1A). Subsequently, normal and BRCA tissues were collected, and subjected to RT-qPCR in order to assess the HINT3 mRNA level. The results obtained demonstrated that the mRNA level of HINT3 was evidently decreased in BRCA tissues (Fig. 1B and C). Since BRCA is a malignancy with a rapid rate of disease progression and a high potential for metastasis, HINT3 expression in BRCA tissues was subsequently analyzed according to different cancer stages and the metastasis status. Based on the samples analyzed, HINT3 expression was significantly decreased in breast cancer tissues, compared with adjacent normal tissues (Fig. 1D and Table II). In addition, it was found that the low expression of HINT3 was associated with Ki-67 positive staining, although this was not associated with the cancer stage of the patients (Table I). Based on TCGA database, the median expression levels of HINT3 in normal tissues, and in cancer tissues of stages I–IV were 31.635, 26.23, 25.56, 26.527 and 22.656, respectively. Compared with the normal tissues, HINT3 expression was found at lower levels in the cancer tissues. The expression of HINT3 was lowest in cancer tissues of stage IV, although compared with the other stages, this difference was not found to be significant (Fig. 1E). In addition, the median expression level of HINT3 in normal and cancer tissues of N0, N1, N2 and N3 (see the Materials and methods section for a fuller explanation of these terms) was 31.635, 25.716, 25.738, 27.973 and 25.045, respectively. Compared with the normal tissues, HINT3 expression was lower in cancer tissues with a varied metastatic status. Nevertheless, the P-values were >0.05 among cancer tissues with N2 metastatic status and normal tissues (Fig. 1F); thus, these results were not statistically significant. Finally, the median expression level of HINT3 in normal tissues, and in luminal, HER2-positive and triple-negative cancer tissues was 31.635, 26.597, 20.385 and 26.338, respectively. Although HINT3 was decreased in cancer tissues compared with normal tissues, no significant differences were observed among the subgroups (Fig. 1G). In addition, the expression of HINT3 was examined in several BRCA cell lines, and it was found that its expression was decreased in MCF7, MDA-MB-231 and MDA-MB-436 cell lines, and increased in MCF10A and MDA-MB468 cell lines (Fig. 1H). Therefore, HINT3 expression was clearly shown to be reduced in BRCA tissues, although the change in levels was not significantly associated with either the stage of cancer or the metastatic status of the patients. Taken together, these findings indicate that HINT3 may participate in the development of BRCA.

Figure 1. HINT3 expression is decreased in BRCA tissues. (A) The transcript of HINT3 was analyzed in BRCA tissues (n=1,097; median, 25.962) and normal tissues (n=114; median, 31.635) from TCGA database (***P<0.001). (B) The mRNA expression of HINT3 was examined using RT-qPCR in BRCA tissues and paired normal tissues. Statistical significance was analyzed using the Mann-Whitney test (*P<0.05). (C) HINT3 mRNA expression was determined using RT-qPCR in BRCA tissues and normal tissues. Statistical significance was analyzed using the Mann-Whitney test (*P<0.05). (D) The expression of HINT3 was analyzed in BRCA tissues using immunohistochemistry. (E) The expression of HINT3 was analyzed in BRCA tissues of different individual stages from TCGA database. The median expression levels of HINT3 in normal tissues, and stage I, stage II, stage III and stage IV cancer tissues were found to be 31.635, 26.23, 25.56, 26.527 and 22.656, respectively; *P<0.05. (F) The expression of HINT3 was analyzed in BRCA tissues with a different metastatic status from TCGA database. The median expression of HINT3 in normal tissues, N0, N1, N2 and N3 cancer tissues was found to be 31.635, 25.716, 25.738 and 27.9733, respectively (**P<0.01, ***P<0.001). (G) The expression of HINT3 was analyzed in BRCA tissues based on BRCA subclasses from TCGA database. The median expression of HINT3 in normal tissues, luminal, HER2-positive and triple-negative cancer tissues was 31.635, 26.597, 20.385 and 26.338, respectively (*P<0.05). (H) The expression of HINT3 in different breast cancer cell lines was examined using western blot analysis. BRCA, breast cancer; TCGA, The Cancer Genome Atlas; HINT3, histidine triad nucleotide-binding 3; RT-qPCR, reverse transcription-quantitative PCR.

Table II. The expression of HINT3 in normal and breast cancer tissues determined using immunohistochemistry.

Table II.

The expression of HINT3 in normal and breast cancer tissues determined using immunohistochemistry.

HINT3 expression status	Tumor tissue	Adjacent normal tissue	χ2 test value	P-value
High expression	3	10	7.04	0.008
Low expression	11	4
Total	14	14

[i] HINT3, histidine triad nucleotide-binding 3.

HINT3 inhibits the proliferation and colony formation of BRCA cells

To explore the role of HINT3 in BRCA cells, the lentivirus-mediated overexpression of HINT3 in BRCA cells was first performed. Since HINT3 expression was found to be decreased in BRCA cells, including MCF-7 and MDA-MB-231 cells, these cell lines were selected for further investigations. The results of western blot analysis revealed that HINT3 was overexpressed in both BRCA cell lines transfected with overexpression vector (i.e., MCF-7 and MDA-MB-231 cells) (Fig. 2A). Subsequently, the effects of HINT3 on BRCA cell proliferation were examined using CCK-8 and colony formation assays. HINT3 overexpression was found to inhibit the proliferation of both the MCF-7 and MDA-MB-231 cells (Fig. 2B). The overexpression of HINT3 also led to the suppression of the colony formation ability of both cell lines (Fig. 2C). Subsequently, the knockdown efficiency of HINT3 was assessed using western blot analysis of these cells. The results revealed that the protein levels of HINT3 were significantly reduced in the shHINT3-1- and shHINT3-2-transfected MCF-7 and MDA-MB-231 cells (Fig. 2D). The decreased levels of HINT3 clearly promoted the proliferation of both MCF-7 and MDA-MB-231 cells (Fig. 2E). Consistently, the knockdown of HINT3 enhanced the proliferation and colony-forming ability of the MCF-7 and MDA-MB-231 cells (Fig. 2F and G). Taken together, these results suggest that HINT3 functions as a tumor suppressor protein in BRCA.

Figure 2. HINT3 suppresses the proliferation of BRCA cells. (A) Western blot analysis of HINT3 in Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells. GAPDH served as the internal control (***P<0.001). (B) Cell proliferation was determined using CCK-8 assay (*P<0.05 and **P<0.01, vs. control). (C) Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells were subjected to colony formation assay (colony images are presented on the left panel, and the quantification of the results is shown on the right panel (*P<0.05, vs. control). (D) Western blot analysis of HINT3 in shCtrl, shHINT3-1 and shHINT3-2 MCF-7 and MDA-MB-231 cells. GAPDH served as the internal control. (E) Cell proliferation was determined using CCK-8 assay (*P<0.05 and **P<0.01, vs. control). (F and G) shCtrl, shHINT3-1 and shHINT3-2 MCF-7 and MDA-MB-231cells were subjected to colony formation assay; (F) colony images, and (G) the quantification of the same results (*P<0.05 and **P<0.01, vs. control). BRCA, breast cancer; HINT3, histidine triad nucleotide-binding 3; Ctrl, control.

HINT3 reduces EdU incorporation and promotes the apoptosis of BRCA cells

Proliferative cells require increased DNA synthesis, and EdU incorporation is an assay that enables the analysis of DNA synthesis to be made (19). Thus, in the present study, DNA synthesis was then examined using EdU staining in BRCA cells. Compared with the EdU staining density in the shCtrl MCF-7 and MDA-MB-231 cells, the positive signal of EdU was markedly increased in the shHINT3-1 and shHINT3-2 cells (Fig. 3A). By contrast, the incorporation of EdU was suppressed by the overexpression of HINT3 in the MCF-7 and MDA-MB-231 cells (Fig. 3B). Subsequently, the present study examined whether HINT3 regulates the apoptosis of BRCA cells. These experiments revealed that HINT3 knockdown significantly reduced the apoptosis of the MCF-7 and MDA-MB-231 cells (Fig. 3C). On the other hand, HINT3 overexpression significantly promoted the apoptosis of both cell lines (Fig. 3D). Taken together, these experiments confirmed that HINT3 suppressed DNA synthesis, and promoted the apoptosis of BRCA cells.

Figure 3. HINT3 inhibits the EdU incorporation of BRCA cells. (A) EdU staining was performed in shCtrl, shHINT3-1 and shHINT3-2 MCF-7 and MDA-MB-231 cells. Staining images are presented on the left panel, whereas the quantification of the same results is presented on the right panel (*P<0.05 and **P<0.01, vs. control). (B) EdU staining was performed in Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells. Again, staining of the images is presented on the left panel, and the quantification of the same results is presented on the right panel (**P<0.01, vs. control). (C) Cell apoptosis was examined using PI/Annexin V-APC staining in shCtrl, shHINT3-1 and shHINT3-2 MCF-7 and MDA-MB-231 cells. Cell apoptosis images are presented on the left panel, and the quantification of the results is presented on the right panel (**P<0.01, vs. control). (D) Cell apoptosis was examined using by PI/Annexin V-APC staining in the Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells The cell apoptosis images are presented on the left, and the quantification of the results is presented on the right panel (**P<0.01, vs. control). BRCA, breast cancer; HINT3, histidine triad nucleotide-binding 3; Ctrl, control; EdU, 5-ethynyl-2′-deoxyuridine.

HINT3 suppresses the migration of MCF-7 and MDA-MB-231 cells

BRCA is a malignancy with a high potential for metastasis. Subsequently, the role of HINT3 in BRCA metastasis was investigated using Transwell assay. The results obtained revealed that HINT3 knockdown increased the migration of MCF-7 cells, whereas the ectopic expression of HINT3 suppressed the migratory capacity of the cells (Fig. 4A and B). Similar results were obtained in HINT3-silenced and -overexpressing MDA-MB-231 cells (Fig. 4C and D).

Figure 4. HINT3 suppresses the migration of BRCA cells. (A) Transwell analysis of migration was performed in shCtrl, shHINT3-1 and shHINT3-2 MCF-7 cells. (B) Transwell analysis of migration was performed in Ctrl and HINT3-overexpressing MCF-7 cells. (C) Transwell analysis of migration was performed in shCtrl, shHINT3-1 and shHINT3-2 MDA-MB-231 cells. (D) Transwell analysis of migration was performed in Ctrl and HINT3-overexpressing MDA-MB-231 cells. (A-D) Representative images are presented on the left panels, and the quantification of the same results is presented on the right panels (**P<0.01, vs. control). BRCA, breast cancer; HINT3, histidine triad nucleotide-binding 3; Ctrl, control.

HINT3 suppresses PTEN/AKT/mTOR signaling in BRCA cells

Subsequently, the molecular changes in MCF-7 and MDA-MB-231 cells that were associated with the knockdown or overexpression of HINT3 were explored. It was found that HINT3 knockdown and overexpression led to the down- and upregulation of PTEN at the mRNA level, respectively (Fig. 5A). Spearman's correlation analysis in patients with BRCA from TCGA database revealed that HINT3 inversely correlated with PTEN in BRCA cancer samples (Fig. 5B). HINT3 is a nuclear protein that may participate in regulating the transcriptional activity of downstream genes (20) (PMID: 17870088). Subsequently, in the present study, luciferase activity reporter assay was performed to examine whether HINT3 regulates the transcriptional activity of PTEN. The results obtained revealed that HINT3 promoted the transcriptional activity of PTEN in the MCF-7 and MDA-MB-231 cells (Fig. 5C). The results of western blot analysis also revealed that PTEN protein expression was decreased following the silencing of HINT3, and was increased following the ectopic expression of HINT3 (Figs. 5D and S1). Consistently, it was found that the phosphorylation, but not the protein expression, of AKT, mTOR and S6 were enhanced and suppressed by HINT3 knockdown and overexpression, respectively in the MCF-7 and MDA-MB-231 cells (Figs. 5D and S1). Taken together, these experimental findings suggested that PTEN/AKT signaling may be involved in the tumor-suppressive role of HINT3 in BRCA.

Figure 5. HINT3 inhibits AKT signaling in BRCA cells. (A) RT-qPCR analysis of PTEN in shCtrl, shHINT3-1 and shHINT3-2, and in Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells (**P<0.01, vs. control). (B) The correlation analysis between PTEN and HINT3 expression in TCGA database is shown. (C) Dual luciferase activity was examined in MCF-7 and MDA-MB-231 cells transfected with HINT3-overexpressing vectors or shHINT3-1, pGL3.Basic and TK vectors (*P<0.05, vs. control). (D) shCtrl, shHINT3-1 and shHINT3-2, Ctrl and HINT3-overexpressing MCF-7 and MDA-MB-231 cells were subjected to western blot analysis to assess the protein levels of PTEN, p-AKT, AKT, p-mTOR, mTOR, p-S6, S6 and β-actin. β-actin served as the internal control. The quantification of proteins is presented in Fig. S1. PTEN, phosphatase and tensin homolog; BRCA, breast cancer; HINT3, histidine triad nucleotide-binding 3; mTOR, mammalian target of rapamycin; Ctrl, control; p-, phosphorylated.

HINT3 inhibits AKT activity and tumor xenograft growth

To explore the functions of HINT3 in vivo, MDA-MB-231 and MCF-7 cells infected with Ctrl or HINT3-overexpressing lentivirus were subcutaneously implanted into the right armpits of 5-week-old female nude mice. The mice were sacrificed either on day 45 (for the MDA-MB-21 cells) or day 48 (for the MCF-7 cells), as detailed in the Materials and methods section. In the present study, the maximum tumor volume obtained was measured. These results revealed that HINT3 overexpression inhibited the tumorigenesis and progression of MDA-MB-231 cells (Fig. 6A and B). Consistent results were observed in the MCF-7 cells with the overexpression of HINT3 (Fig. 6C and D). It was also found that HINT3 overexpression suppressed AKT and S6 phosphorylation in the tumors obtained following the implantation of the MDA-MB-231 and MCF-7 cells (Figs. 6E and F, and S2). Taken together, these results suggest that HINT3 can block AKT activity and the tumor growth of BRCA cells in nude mice.

Figure 6. HINT3 overexpression suppresses AKT activity and the tumorigenesis of MDA-MB-231 and MCF-7 cells. (A and B) MDA-MB-231 cells expressing Ctrl or HINT3-overexpressing lentivirus were subcutaneously implanted into the right armpits of 4-week-old female nude mice. The mice were sacrificed at 45 days after implantation. (A) Images of the tumors are shown. (B) The tumor growth curve and tumor weights are shown (*P<0.05 and **P<0.01, vs. control). (C and D) MCF-7 cells expressing Ctrl or HINT3-overexpressing lentivirus were subcutaneously implanted into the right armpits of 5-weeks old female nude mice. The mice were sacrificed by the 48th day after implantation. (C) Images of the tumors are shown. (D) The tumor growth curve and tumor weights are shown (**P<0.01, vs. control). (E and F) Western blot analysis of HINT3, p-AKT, AKT, p-S6, S6 and β-actin in the tumor tissues. The quantification of proteins is presented in Fig. S2. β-actin served as the internal control. HINT3, histidine triad nucleotide-binding 3; Ctrl, control; p-, phosphorylated.

Discussion

Unlike HINT1 and HINT2, the role of HINT3 has yet to be fully elucidated in cancer development. In the present study, it was demonstrated that HINT3 functioned as a tumor suppressor in BRCA. In vitro, HINT3 knockdown promoted the proliferation, colony growth, EdU incorporation and migration of BRCA cells. By contrast, opposite results were obtained in HINT3-overexpressing BRCA cells. In vivo, the tumorigenesis of MDA-MB-231 and MCF-7 cells was suppressed via the ectopic expression of HINT3.

In total, ~10% of patients with BRCA exhibit inherited genetic variants, and among these, mutations of BRCA1 and BRCA2, which are involved in DNA repair, play crucial roles in BRCA development (21–24). Furthermore, the most commonly mutated genes or amplified genes, such as TP53, PIK3CA, MYC, PTEN, CCND1, ERBB2, FGFR1 and GATA3, play critical roles in the progression of BRCA (25). These genetic alterations provide possible avenues for therapeutic intervention in patients with BRCA. One representative drug is poly(ADP-ribose) polymerase (PARP) inhibitor, which is used to treat patients with BRCA who have BRCA mutations (26). However, the effectiveness of this treatment remains unsatisfactory, and therefore the use of PARP inhibitor is limited. It is thus necessary to identify novel drug targets to cure this malignancy. The HINT family contains 3 family members, including HINT1, HINT2 and HINT3. A previous study demonstrated that HINT1 displayed tumor-suppressive properties and regulated tumorigenesis signaling pathways, and may thus function as a diagnostic target in tumors (27). Another member, HINT2 has been reported to be downregulated in hepatocellular carcinoma tissues from patients with a poor prognosis (28). To date, at least to the best of our knowledge, no studies have investigated the role of HINT3 in tumor formation or progression. In the present study, it was found that HINT3 functioned as a tumor suppressor in BRCA. HINT3 expression was found to be decreased in BRCA tissues. Functional assays revealed that the ectopic expression of HINT3 suppressed the proliferative and migratory capacity of the MCF-7 and MDA-MB-231 cells. DNA synthesis, which is the hallmark of cell cycle progression, was also suppressed by HINT3. Moreover, cell apoptosis was promoted by HINT3 overexpression, and was suppressed by HINT3 knockdown. In vivo, HINT3-overexpressing MDA-MB-231 and MCF-7 cells developed xenograft tumors at a slower rate compared with Ctrl MDA-MB-231 and MCF-7 cells. Taken together, these findings indicated that HINT3 exhibited tumor-suppressive functions in BRCA cells; thus, it may be suitable as a diagnostic target for breast cancer.

PTEN is a well-known tumor suppressor (29). The inactivation of PTEN leads to the enhanced phosphorylation and activity of AKT. The activation of AKT contributes to cancer development via the regulation of distinct downstream targets, including glycogen synthase kinase 3β, FOXO, p21 and caspase 9 (30). The AKT/mTOR pathway is involved in the regulation of cell growth, differentiation, motility and survival, processes which are usually enhanced in various types of cancer, such as colorectal cancer, ovarian and cervical cancer; the inhibitor of mTOR signaling, suppresses colorectal cancer development, and thus, it has been considered as a potential target for cancer therapy (31,32).

The absence of PTEN, or mutations of PTEN are commonly observed in BRCA (33–35). However, the upstream regulator of PTEN is not yet well known in BRCA. In the present study, it was found that HINT3 negatively regulated the mRNA and protein expression of PTEN. HINT3 knockdown and overexpression up- and downregulated the phosphorylation level of AKT, respectively. Furthermore, the results of dual luciferase activity reporter assay demonstrated that HINT3 promoted the transcriptional activity of PTEN gene in BRCA cells. Of note, HINT3 overexpression also suppressed the activity of AKT in BRCA tumors, suggesting that the HINT3-induced inactivation of AKT is involved in the tumorigenesis of BRCA. Collectively, these results suggest that HINT3 suppresses the development of BRCA, at least partly by inactivating the AKT signaling pathway. Accordingly, owing to the key roles of PTEN and the AKT/mTOR pathway, HINT3 may play crucial roles in other diseases, such as human immunodeficiency virus and coronavirus disease 2019, as well as in autoimmune diseases, such as multiple sclerosis and systemic lupus erythematosus (36–39).

However, the present study had several limitations which should be mentioned. The molecular mechanisms through which HINT3 downregulation may operate in BRCA remain unknown. Whether HINT3 downregulation is associated with promoter hypermethylation, or whether it is regulated by other factors at the transcriptional level in BRCA requires further investigation. Moreover, the association between HINT3 and the stage/metastasis and status/molecular subtype of patients with BRCA was found not to be significant, as shown from TCGA database. Two reasons may account for these observations: i) The patients originated from different countries; or ii) the numbers of patients with BRCA differed when comparing the different groups. In addition, the small sample size limited the analysis of HINT3 in the clinicopathological characteristics of these patients. In the future, a larger number of patients with BRCA in China need to be examined in order to analyze the association between HINT3 expression and the stage/metastasis and status/molecular subtype of the patients. Additionally, HINT3-knockout mice need to be established to explore the functional roles of HINT3 in vivo. Furthermore, clinical analyses will be required in the future. This will be crucial in terms of evaluating the diagnostic value of HINT3 in BRCA.

In the future, the authors aim to explore the role of HINT3 knockdown in the development of BRCA cell tumorigenesis, in which drugs, including AKT inhibitors, will be applied for rescue experiments. Moreover, the molecular mechanisms, as well as the clinical application of HINT3 also need to be investigated in the future.

In conclusion, the present study demonstrated that HINT3 expression was decreased in BRCA tissues. HINT3 knockdown contributed to the development of BRCA via the activation of the PTEN/AKT signaling pathway. The present study supports the role of HINT3 as a novel tumor suppressor in BRCA cells.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was financially supported by the National Natural Science Foundation of China (grant no. 81760482) and the Natural Science Foundation of Ningxia (grant nos. NZ17138, 2019AAC03214, 2018AAC03162, 2019AAC03232 and 2018AAC03165). The funding provided contributed to the procurement of the experiment materials used in the present study.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

YL, BL and JL designed the study. YL, BL, HL, DC, YG and YW conducted the experiments and analyzed the results. wrote and revised the manuscript. All authors have read and approved the final manuscript. JL and YL confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of General Hospital of Ningxia Medical University (no. 2022-68). Written informed consents were collected from all patients. The animal experiments were approved by the Ethics Committee of General Hospital of Ningxia Medical University (no. 2022-68), and were performed according to the animal guidelines of General Hospital of Ningxia Medical University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References