Introduction

Breast cancer is the most common malignancy following ovarian and cervical cancers in women worldwide(Zou 2004). These years breast cancer is the second leading cause of cancer-related fatalities in women in the United States(Rezaeeyazdi et al. 2022). Early breast cancer detection and therapy have benefited greatly from rapid progress in molecular biology. However, treatment of patients with advanced stage of the disease, especially breast cancer, or extensive invasion and metastasis is often ineffective(Neugut et al. 2014). Mechanisms targeting breast cancer cell metastasis have not yet been fully elucidated. Therefore, it is necessary to identify new molecular biological targets for breast cancer.

Sorting junction proteins (SNXs) are a class of PX domain-containing proteins that play key roles in intracellular protein sorting and protein transport(Teasdale and Collins 2011; Thomas et al. 2014). PX specifically binds to phosphatidyl alcohol groups and affects the activity of related organelles. Based on their structural domain composition, 33 SNXs, including SNX3, SNX4, SNX10, SNX12, SNX22, and SNX-PX-BAR, have been identified in mammals(Gallon and Cullen 2015; Teasdale and Collins 2011; Verges 2007). SNX-PX-BAR has been reported to regulate sorting and transport processes in the body. During evolution, the biochemical role of SNX14 seems to have been largely conserved, and the consequences of its loss-of-function vary from species to species(Verges 2007). SNX14 belongs to the G protein signaling pathway, which is involved in intracellular signal transduction. The onset and progression of cancer are closely correlated with abnormal G protein signaling(Ha et al. 2015). Phosphatidylinositol phosphate-related SNX14 localizes to lysosomes and is a key component of lysosomes. SNX14 is associated with autophagosome accumulation and activation of apoptosis in zebrafish. Mutations in Snx14 cause intracellular lysosome–autophagosome dysfunction syndrome(Akizu et al. 2015). Moreover, overexpression of SNX14 promotes the biosynthesis of lipid droplets, which are nutrient stores used by cells to maintain a stable internal environment(Datta et al. 2019). SNX14 mutations or deletions in human fibroblasts disrupt autophagy; however, its exact function remains unclear(Bryant et al. 2018). Loss of SNX14 can also lead to autophagy(Sait et al. 2022). Therefore, SNX14 may be closely associated with autophagy. Autophagy is crucial for the development, metastasis, and invasion of breast cancer cells(Akkoc et al. 2023).

In this study, we determined the roles and underlying mechanism of SNX14 as well as its effects on autophagy in breast cancer cells. Our findings may aid in the development of effective breast cancer therapies.

Materials and methods

Expression analysis of SNX14 in breast Cancer

Analysis of SNX14 expression in breast cancer tissues (n = 1104) and normal tissues (n = 113) was conducted using the starBase database (https://rnasysu.com/encori/index.php). Immunohistochemical detection results of SNX14 protein in breast tissues (n = 3) and breast cancer tissues (n = 12) were obtained from The Human Protein Atlas database (https://www.proteinatlas.org/), followed by analysis of SNX14 expression in breast cancer tissues. Utilizing a best expression cutoff value of 10.91 from The Human Protein Atlas database, breast cancer patients were stratified into SNX14 low-expression group (n = 702) and SNX14 high-expression group (n = 373), and Kaplan-Meier survival curves were generated.

Cell culture

HEK293T and human breast cancer (MCF7) cells were preserved in our laboratory. HEK293T and MCF7 human breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in a 5% humidified CO2 incubator.

Plasmid construction

Full-length human SNX14 cDNA (NM_001350550.2) was inserted into the pLV-CMV-MCS-PGK puromycin vector to construct a plasmid encoding SNX14 (SNX14-OE). The pLV-CMV-MCS-PGK puro vector was used as a negative control for SNX14-OE. Short hairpin RNA (shRNA) for SNX14 (shSNX14) or a scrambled sequence (as a negative control, shNC) was cloned into PLKO.1-TRC-puro to create a plasmid encoding shSNX14. All primer sequences used in this study are listed Table 1.

Table 1 Sequences of primers used in the paper
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Construction of stable SNX14-knocked down and -overexpressing cell lines

HEK293T cells were co-transfected with 9 µg of plasmids encoding shSNX14 or SNX14-OE in 10-cm plates using Lipofectamine 2000 (Invitrogen) along with the appropriate packaging plasmids (3 µg of pMD2G and 6 µg of pspax2). The lentivirus-containing supernatant was collected after 48 h. After enrichment, the titer was calculated as previously described(Li et al. 2015a). MCF7 cells were transduced with 108 TCID50/mL lentivirus in the presence of 8 µg/mL polypropylene. Puromycin was added at a final concentration of 2.0 µg/mL after replacing the medium with a fresh medium after 48 h. Cells were collected for the determination of SNX14 expression after two weeks.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the cells using TRIzol Reagent (Thermo Fisher Scientific, Inc.) and a Reverse Transcription kit (Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA. RT-qPCR was performed using the SYBR Green PCR kit (TaKaRa, China) on a CFX Connect 96 device (Bio-Rad Laboratories, Inc.). The 2−ΔΔCq approach was used to determine the relative expression(Livak and Schmittgen 2001). All primer sequences are listed in Table 1.

Western blotting

Total cellular protein was extracted using a protein extraction kit (Thermo Fisher Scientific). Total protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Inc.), according to the manufacturer’s instructions. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used for the separation of proteins or cell lysates, and polyvinylidene difluoride membranes were used to transmit the gel, which were blocked with 5% skim milk in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween-20) for 1 h and incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies and detection using an ECL system (Thermo). All antibodies used here are listed in Table 2.

Table 2 Information about antibodies
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

MCF7 cells stably overexpressing SNX14 or shSNX14 were seeded at a density of 10,000 cells/well in a 96-well plate. Briefly, 20 µL of MTT (5 mg/mL) was added to each well after 0, 24, 48, or 72 h, and the cells were incubated at 37 °C for 4 h. After carefully aspirating the supernatant from each well, 150 µL of dimethyl sulfoxide was added to each well, and the cell culture plate was agitated for 10 min to break up the crystals. Data were then recorded after measuring the absorption at 490 nm using a microplate reader (Thermo Fisher Scientific, Inc.). Time was plotted on the x-axis and absorbance was plotted on the y-axis to obtain the cell growth curve.

Electron microscopy analysis

MCF7 cells were collected, centrifuged at 200 × g, and fixed in 0.2 M sodium cacodylate buffer (pH 7.4) containing 2% glutaraldehyde overnight at 4 °C. The samples were subsequently dehydrated and embedded in Epon 812 for ultrathin sectioning post-fixation with cacodylate-buffered 1% osmium tetroxide. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (H-7500; Hitachi, Tokyo, Japan).

Flow cytometry

MCF7 cells (1 × 106) stably overexpressing SNX14 or shSNX14 were placed in a 6-well plate. Following a 24-h culture at 37 °C, adherent and floating cells were separated via centrifugation at 200 × g for 5 min at room temperature, washed with phosphate-buffered saline, and processed to detect apoptosis using an Annexin V-FITC/PI Apoptosis Detection Kit (catalog number: A211-02; Vazyme Biotech Co., Ltd.), according to the manufacturer’s instructions. NovoCyte setup (ACEA Bioscience Inc.) and NovoExpress software 1.4.1 (ACEA Bioscience Inc.) were used to examine the data. The proportion of apoptotic cells (including early and late apoptotic cells) relative to the total number of cells was calculated.

Treatment with inhibitors

MCF7 cells (1 × 106) stably overexpressing SNX14 or shSNX14 were seeded in a 6-well plate overnight. MCF7 cells stably overexpressing SNX14 were treated with 0.5 ng/mL rapamycin, an inhibitor of mechanistic target of rapamycin kinase (mTOR), and those stably overexpressing shSNX14 were treated with 5 mM 3-methyladenine, an inhibitor of phosphoinositide 3-kinase (PI3K). After 24 h, cells were collected for western blotting analysis.

Animal experiments

Briefly, 1 × 106 MCF7 cells stably overexpressing shSNX14 were subcutaneously injected into six-week female BALB/c nude mice procured from the Shanghai Laboratory Animal Research Center. Tumor volumes were measured on days 0, 22, 26, 30, 34, 38, and 42 post-injection. On day 42 post-injection, all mice were euthanized with carbon dioxide at a flow rate of 30–70% of the euthanasia chamber volume. Immunohistochemistry (IHC), hematoxylin and eosin (HE) staining, and western blotting were performed on the obtained tumors. Animal Care and Use Committee of Zhejiang university school of medicine endorsed this study.

HE staining

After fixing in 4% formaldehyde solution and dehydration with ethanol, tumor tissue was embedded in paraffin and cut into sections of 4 μm. After deparaffinization, the sections were stained using a hematoxylin-eosin staining kit (catalog number: G1076; Servicebio) and observed under a light microscope (Nikon).

IHC assay

Tumor sample sections were deparaffinized, rehydrated, and treated with 3% H2O2 before treatment with citrate buffer. The sections were treated with diluted Ki67 antibody (catalog number: 27309-1-AP; Proteintech) for 12 h at 4 °C after blocking with 5% goat serum. After incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (catalog number: SA00001-2; Proteintech), the sections were incubated with DAB for color development. Subsequently, the sections were treated with hematoxylin to stain the nuclei and dehydrated. Finally, clear glue was used to seal the sections, which were observed under an Olympus light microscope (BX51; Olympus Corporation).

Statistical analysis

Data were analyzed using SPSS 17.0, a statistical program. Means and standard deviations were used to express the statistical data. The data were compared across groups using Student’s t-test, and the Shapiro–Wilk test was used to determine if the data were normal. One-way analysis of variance and Tukey’s test were used to determine the significant differences between groups. Differences were considered statistically significant at P < 0.05.

Results

Expression and prognostic implications of SNX14 in breast Cancer

Analysis from the starBase database revealed a significant upregulation of SNX14 expression in breast cancer tissues compared to normal breast tissues (Fig. 1a). Additionally, immunohistochemical examination from The Human Protein Atlas database indicated that SNX14 protein was undetectable in normal breast tissues, whereas in breast cancer tissues, 1/12 of samples showed no SNX14 protein detection, 1/4 exhibited low levels, and 1/3 displayed moderate levels of SNX14 protein (Fig. 1b), indicating an elevation of SNX14 protein in breast cancer tissues relative to normal breast tissues. Kaplan-Meier survival curves from The Human Protein Atlas database further illustrated that breast cancer patients with high SNX14 expression exhibited significantly poorer survival compared to those with low SNX14 expression (Fig. 1c). These findings collectively underscore the overexpression of SNX14 in breast cancer tissues, with higher SNX14 expression correlating with lower survival rates among breast cancer patients.

Fig. 1
figure 1

Expression analysis of SNX14 in breast cancer. (a) Analysis of SNX14 expression levels in breast cancer using starBase (https://rnasysu.com/encori/index.php). (b) Immunohistochemical detection of SNX14 in breast tissue and breast cancer tissue obtained from The Human Protein Atlas. (c) Analysis of the impact of SNX14 expression levels on the survival of breast cancer patients using The Human Protein Atlas (https://www.proteinatlas.org/)

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Identification of stable cells overexpressing SNX14 or shSNX14

Initially, we separately cultured various breast cancer cell lines (MDA-MB-231/436/457/468 and MCF7) along with normal mammary epithelial cells (MCF10A) to observe the expression profile of SNX14. As depicted in Fig. 2a, SNX14 expression levels were notably elevated in breast cancer cells, with the most pronounced increase observed in MDA-MB-436 and MCF7 cells. For ease of experimentation, subsequent studies primarily focused on MCF7 cells. After that, we constructed stable cell lines overexpressing either SNX14 or shSNX14. RT-qPCR revealed a significant increase in SNX14 mRNA levels in cells stably overexpressing SNX14 (Fig. 2b) and a significant decrease in SNX14 mRNA levels in cells stably overexpressing shSNX14 (Fig. 2c). In addition, SNX14 protein levels were significantly increased and decreased in cells stably overexpressing SNX14 and shSNX14, respectively (Fig. 2d and e).

Fig. 2
figure 2

Identification of stable cells overexpressing sorting nexin 14 (SNX14) or short hairpin RNA (shRNA) for SNX14 (shSNX14). (a) Expression of SNX14 in different cell lines. Among them, MCF10A is human normal breast epithelial cells. (b) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of SNX14 mRNA levels in MCF7 cells overexpressing SNX14. (c) RT-qPCR analysis of SNX14 mRNA levels in MCF7 cells overexpressing shSNX14. (d) SNX14 protein expression levels in MCF7 cells overexpressing SNX14 or shSNX14 determined via western blotting. Housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), served as the baseline for all samples. (e) Relative quantification of SNX14 protein levels by calculating the gray scale of western blotting bands

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SNX14 knockdown increases the autophagy and apoptosis and inhibits the survival of MCF7 cells

SNX14 knockdown significantly inhibited the growth of MCF7 cells (Fig. 3a). In contrast, overexpression of SNX14 promoted the proliferation of MCF7 cells compared to the vector group (Fig. 3a). Western blotting revealed that, compared to the control group, the LC3-II/LC3-I ratio and pro-autophagy protein Beclin 1 levels were decreased and p62 protein levels were increased in the SNX14-OE group (Fig. 3b and c). In contrast, the LC3-II/LC3-I ratio and Beclin 1 expression levels were increased and p62 levels were decreased after SNX14 knockdown (Fig. 3b and c). These results suggest that SNX14 has the potential to regulate autophagy in MCF7 cells. Electron microscopy revealed that the SNX14 knockdown group had a much larger number of autophagic lysosomes than the shNC group (Fig. 3d). In summary, SNX14 inhibited autophagy in MCF7 cells. Furthermore, SNX14 knockdown significantly promoted the apoptosis of breast cancer cell MCF7 (P < 0.0001), but its overexpression had no significant effect on the apoptosis of MCF7 cells (Fig. 3e and f).

Fig. 3
figure 3

SNX14 knockdown increases the autophagy and apoptosis and inhibits the survival of MCF7 cells. (a) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of SNX14-overexpressing and knocked-down cells. (b) Beclin 1, p62, TFAR19, and LC3A/B protein levels determined using western blotting in MCF7 cells overexpressing shSNX14 or SNX14. (c) Relative quantification of Beclin 1, p62, TFAR19, and LC3A/B protein levels. (d) Representative image of the electron microscopic analysis of SNX14-overexpressing or knocked-down cells. Magnified areas indicate the autophagosomes. N: cell nucleus; red arrow: mitochondrion; yellow arrow: lysosome; blue arrow: phagocytic vacuole; black arrow: autophagic vacuole; green arrow: autophagic lysosome. (e) Representative image of apoptosis analysis of SNX14-overexpressing or knocked-down cells. (f) Statistical histogram of the apoptosis rate of SNX14-overexpressing or knocked-down cells

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SNX14 may regulate the expression levels of apoptosis-related molecules by activating the mTOR signaling pathway in MCF7 cells

To explore the potential molecular mechanisms by which SNX14 regulates apoptosis in MCF7 cells, we treated SNX14-OE or knockdown cells with rapamycin and 3-methyladenine, respectively. We found that overexpression of SNX14 decreased the protein levels of cleaved caspase 3 and Bcl-2 and decreased the protein levels of BAX (Fig. 4a and b). Rapamycin treatment alleviated the effects of SNX14 overexpression (Fig. 4a and b). Cleaved caspase 3 and Bcl-2 protein levels were decreased and BAX protein levels were increased after SNX14 knockdown in cells (Fig. 4a and b). However, 3-methyladenine treatment exacerbated the effects of SNX14 knockdown (Fig. 4a and b). These data suggest that SNX14 knockdown promotes apoptosis by inhibiting the PI3K signaling pathway.

Fig. 4
figure 4

SNX14 regulates the expression levels of apoptosis-related molecules by activating the mechanistic target of rapamycin kinase (mTOR) signaling pathway in MCF7 cells. (a) Western blotting of SNX14-overexpressing or knocked-down MCF7 cells after rapamycin or 3-methyladenine treatment. (b) Relative quantification of protein by calculating the gray scale of western blotting bands

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SNX14 promotes tumor growth in mice

Subcutaneous tumors formed from MCF7 cells overexpressing shSNX14 in mice were smaller (Fig. 5a), slower in growth (Fig. 5b), and lighter in weight (Fig. 5c) than those formed from MCF7 cells overexpressing shNCs. The ratio of p-AKT/AKT was low and beclin 1 and TFAR19 protein expression levels were increased in the shSNX14 group than in the shNC group (Fig. 5d and e). HE staining revealed increased eosinophilic activity in subcutaneous tumors formed by MCF7 cells with SNX14 knockdown (Fig. 5f). IHC revealed that the shSNX14 group had lower levels of Ki67 than the shNC group (Fig. 5g and h). These findings indicate that SNX14 promotes the growth and proliferation of breast cancer cells via the activation of the AKT signaling pathway.

Fig. 5
figure 5

Silencing of SNX14 inhibits the growth of breast cancer tumors in mice. (a) Images of tumors harvested from shNC and shSNX14 groups. (b) Tumor volumes of shNC and shSNX14 groups. (c) Tumor weights of shNC and shSNX14 groups. (d) Western blotting analysis of the harvested tumors. (e) Relative quantification of protein by calculating the gray scale of western blotting bands. (f) Hematoxylin and eosin (HE) staining images of tumors from shNC and shSNX14 groups. (g) Immunohistochemistry (IHC) images of tumors from shNC and shSNX14 groups. (h) Percentage of Ki-67-positive cells in shNC and shSNX14 groups

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Discussion

Kurten et al. discovered SNXs as a class of proteins containing phagocyte oxidase homology (phoxhomology, PX) in yeast two-hybrid experiments in 1996(Kurten et al. 1996). Currently, 33 members of SNX family have been identified in mammals. Sorting nexin 3 (SNX3) plays a key role in the occurrence and progression of non-small cell lung cancer(Pan et al. 2019). SNX4, another important member of the SNX family, is usually expressed at high levels only in invasive cell lines, indicating its role as a tumor-promoting factor(Leprince et al. 2003). SNX16 is crucial for endocytosis, protein sorting, and cell signal transduction and is strongly associated with the development of various diseases(Wassmer et al. 2009). Zhang et al. reported that the loss of SNX27 inhibits the proliferation of highly aggressive breast cancer MDA-MB-231 cells(Zhang et al. 2019). SNX14 is another important member of SNXs, whose role in cancer development, especially in vivo, remains unknown. Here, we found that the overexpression of SNX14 considerably promoted, whereas its knockdown significantly inhibited the proliferation of breast cancer MCF7 cells. Therefore, variations in the expression levels of SNX14 may considerably and time-dependently control the proliferation of MCF7 human breast cancer cells. We further explored the tumor-promoting effect of SNX14 in vivo by establishing an MCF7 breast cancer tumor-bearing mouse model. Our findings revealed that the suppression of SNX14 expression may drastically slow down the growth of MCF7 breast cancer tumor-bearing mice and decrease the relative expression of Ki67 protein, indicating that the inhibition of proliferation of the tumor cells(Elkablawy et al. 2016; Li et al. 2015b; Pathmanathan and Balleine 2013; Yerushalmi et al. 2010). Ki67 exists only in the active phase of the cell cycle (G1/S/G2/M phase). Given that the expression of Ki67 is closely related to the proliferation and growth of tumor cells, Ki67 is often used as a proliferation marker in routine cancer pathology research(Li et al. 2015b; Pathmanathan and Balleine 2013). Moreover, HE staining revealed that SNX14 knockdown significantly reduced the tumor growth in MCF7 cells.

Autophagy acts as a “double-edged sword” as it is crucial for both carcinogenesis and tumor control(Degenhardt et al. 2006; Gozuacik and Kimchi 2004; Takamura et al. 2011). Autophagy promotes cancer cell survival and metastasis recurrence in breast cancer(Abedin et al. 2007). PI3K/AKT/mTOR pathway is a vital signaling system for regulating autophagy, and mTOR is a major autophagy regulator(Morgensztern and McLeod 2005; Porta et al. 2014). PI3K/AKT controls the action of many inflammatory mediators and activates several intracellular signaling cascades. As a member of the family of heterodimeric lipid kinases, PI3K is activated by various upstream cell surface receptors, phosphorylated by PIP2, and further activated by AKT. AKT regulates downstream effector molecules via phosphorylation cascades and participates in cell proliferation, survival, and other cellular processes(Morgensztern and McLeod 2005). Several malignancies, including breast cancer, are induced by the PI3K/AKT pathway(Paplomata and O’Regan 2014). In this study, we found that, SNX14 overexpression inhibited, whereas SNX14 knockdown promoted autophagy in MCF7 cells. SNX14 overexpression activated the PI3K/AKT signaling pathway. Consequently, we hypothesized that SNX14 overexpression prevents the autophagy of MCF7 cells by stimulating the PI3K/AKT/mTOR signaling pathway. To confirm this, we used the PI3K (3-methyladenine) and mTOR (rapamycin) inhibitors to inhibit the PI3K/AKT/mTOR signaling pathway and found that inhibition of the PI3K/AKT and AKT/mTOR signaling pathways significantly affected SNX14-mediated autophagy. In summary, SNX14 prevents autophagy by activating the PI3K/AKT/mTOR signaling pathway in MCF7 cells.

In conclusion, we determined the role of SNX14 and its underlying molecular mechanism in controlling the development of breast cancer at the cellular, tissue, and molecular levels in this study. In vitro studies revealed that SNX14 controls the emergence and progression of breast cancer by promoting the proliferation and inhibiting the autophagy of MCF7 breast cancer cells. In vivo experiments in nude mice further confirmed that knocking down the expression of SNX14 inhibited the tumorigenicity of MCF7 cells and decreased the growth of tumor cells in tumor tissues in nude mice. Moreover, we found that SNX14 modulates the PI3K/AKT/mTOR signaling pathway to facilitate the autophagy of breast cancer MCF7 cells. Therefore, SNX14 may be a useful therapeutic candidate for the management and treatment of breast cancer.