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. 2025 Dec 30;31:170. doi: 10.1186/s40001-025-03662-5

DACT1 inhibits cuproptosis and promotes cell malignancy via activation of PI3K/AKT signaling in laryngeal squamous cell carcinoma

Yan Guo 1, Jiarui Zhang 1, Jingchun Ge 1, Liang Li 1, Ming Liu 1, Linli Tian 2,✉
PMCID: PMC12859876  PMID: 41469752

Abstract

Background

Laryngeal cancer has one of the highest mortality rates of all head and neck cancers. DACT1 is a cuproptosis-related gene in laryngeal cancer and serves as a risk factor for patient prognosis. This study aimed to investigate the effects of DACT1 on the malignant behavior and cuproptosis of laryngeal squamous cell carcinoma (LSCC) cells.

Methods

DACT1 expression in LSCC cells was measured using RT-qPCR and western blotting. To establish cuproptosis cell model, TU212 and TU686 cells were incubated with elesclomol (20 nM) and CuCl2 (20 nM) for 24 h. Transfection of shRNA or pcDNA3.1 vectors was performed to interfere DACT1 expression. LY294002 (PI3K inhibitor) and 740Y-P (PI3K agonist) were used to silence and overexpress PI3K signaling in LSCC cells, respectively. A cuproptosis-specific inhibitor, tetrathiomolybdate, was used to suppress cuproptosis in LSCC cells. Cell viability, proliferation, migration, and invasion were assessed using CCK-8 assays, colony formation assays, Transwell migration, and Transwell invasion assays. Copper concentration and reactive oxygen species (ROS) level were also measured. Western blotting was performed to quantify protein levels of cuproptosis-related genes and factors involved in the PI3K/AKT pathway.

Results

DACT1 expression was upregulated in LSCC cells. DACT1 knockdown inhibited LSCC cell proliferation, migration, and invasion. DACT1 depletion enhanced cuproptosis, as evidenced by more pronounced decreases in cell viability, increased intracellular copper concentration and ROS levels, upregulation of HSP70, and downregulation of LIAS. Notably, treatment with the cuproptosis inhibitor tetrathiomolybdate reversed the pro-cuproptosis effects induced by DACT1 silencing. Furthermore, the silencing of DACT1 inactivated the PI3K/AKT signaling, as shown by reduced ratios of p-PI3K/PI3K and p-AKT/AKT. Conversely, DACT1 overexpression activated the PI3K/AKT pathway, an effect that was abolished by LY294002. Moreover, LY294002 reversed the promoting effects of DACT1 on LSCC cell malignancy and its inhibitory effects on cuproptosis. In contrast, activation of the PI3K signaling by 740Y-P reversed the enhancement of cuproptosis caused by DACT1 deficiency.

Conclusion

DACT1 promotes the malignant behavior of LSCC cells and suppresses cuproptosis by activating the PI3K/AKT signaling.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-03662-5.

Keywords: LSCC, DACT1, Cuproptosis, PI3K/AKT, LY294002

Introduction

Laryngeal squamous cell carcinoma (LSCC) is the main pathological subtype of laryngeal carcinoma. In 2020, globally, 184,615 cases of laryngeal cancer were diagnosed, with 99,840 deaths reported [1]. A variety of risk factors include smoking, alcohol consumption, opium use, and infections caused by human papillomavirus and Ebstein-Barr virus [2]. Currently, the primary treatment modalities for laryngeal cancer include surgery, radiotherapy, and chemotherapy [3, 4]. Early-stage LSCC is associated with 5-year survival rate of 70–90%. However, approximately 40% of laryngeal cancer are diagnosed at advanced stages [5], resulting in a significantly reduced 5-year survival rate of only 30% [6]. These statistics indicate that the incidence and mortality rates of LSCC remain concerning. Therefore, there is an urgent need to identify novel diagnostic markers and therapeutic strategies for improving LSCC management.

DACT1 was initially identified as a protein interacting with Dishevelled-1, a key mediator in the Wnt pathway [7, 8]. The role of DACT1 in tumorigenesis appears to vary across different malignancies. It has been reported that DACT1 is overexpressed in colon cancer and squamous cell carcinoma [9, 10], whereas aberrant downregulation of DACT1 is observed in hepatocellular carcinoma, gastrointestinal tumors, and lung cancer [11–13]. Additionally, DACT1 upregulation is related to invasive and metastatic phenotypes of nasopharyngeal carcinoma cells [14]. According to bioinformatics analysis using the UALCAN database (https://ualcan.path.uab.edu/) and Kaplan Merier plotter (https://www.kmplot.com/analysis/index.php?p=home), DACT1 is upregulated in head and neck squamous cell carcinoma (HNSC) samples. Notably, laryngeal cancer is among the most commonly diagnosed malignancies in the head and neck area [15]. Recently, DACT1 has been identified as a cuproptosis-related gene in laryngeal cancer and is considered one of risk factors affecting patient prognosis [16]. However, its effects on malignant behavior and cuproptosis of LSCC cells remain unknown.

Copper, an essential transition metal for cellular enzyme functions, is strongly linked to tumor-related biological behaviors and various signaling pathways [17, 18]. High concentration of copper ions induce lipid peroxidation and cell death [19]. Cuproptosis is a newly identified type of regulated cell death, distinct from other established forms including pyroptosis, ferroptosis, and autophagy [20]. Cuproptosis is triggered when copper binds to lipoylated enzymes within the tricarboxylic acid cycle, resulting in protein aggregation, proteotoxic stress, and eventually cell death [21]. It is marked by an accumulation of excessive copper ions, aberrant clustering of lipoylated proteins within the TCA cycle, depletion of iron-sulfur cluster proteins, and increased expression of HSP70, resulting in proteotoxic stress and oxidative damage [22]. At present, the mechanism by which DACT1 regulates cuproptosis is unclear. A previous study demonstrated that activation of the PI3K/Akt/mTOR pathway reduces the sensitivity of colorectal cancer cells to cuproptosis, thereby promoting malignant cellular process in colorectal cancer [23]. Notably, DACT1 (Dapper1) has been shown to activate the PI3K/Akt pathway and thus regulates glucose and lipid metabolism [24]. Based on this evidence, we propose that DACT1 may activate the PI3K pathway to regulate cuproptosis.

In this study, our goal is to investigate the roles of DACT1 in LSCC progression. We hypothesize that DACT1 is aberrantly expressed in LSCC cells and promotes malignant behavior of LSCC cells. Our findings may provide a novel therapeutic strategy for LSCC treatment.

Methods

Cell culture

Human LSCC cell lines (TU212 and TU686) and normal human bronchial epithelial cells (HBECs) were obtained from EK-Bioscience (Shanghai, China). The cells were cultured in RPMI-1640 medium (Procell, Wuhan, China) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA). These cells were cultured in a humidified incubation with 5% CO2 at 37 °C.

Cell transfection and treatment

To knock down DACT1 expression, specific short hairpin RNA targeting DACT1 (sh-DACT1#1/2) were constructed, with negative control shRNA named sh-NC. For overexpression of DACT1, the pcDNA3.1 vectors containing full sequence of DACT1 (GenePharma) was used, with empty vector as control. These plasmids were purchased from GenePharma (Shanghai, China). The shRNA plasmids (1 μg/well) and pcDNA3.1 vectors (1 μg/well) were transfected into LSCC cells seeded in 24-well plates using Lipofectamine 2000 reagent (Thermo Fisher, Shanghai, China) at a ratio of 3:1 (Lipofectamine: plasmid) for 24 h.

To establish cuproptosis cell model, the transfected TU212 and TU686 cells were incubated with elesclomol (20 nM; MedChem Express, Monmouth Junction, NJ, USA) and CuCl2 (20 nM; Sigma-Aldrich) for 24 h.

To verify the regulatory effect of DACT1 on PI3K/AKT signaling, TU212 and TU686 cells transfected with pcDNA3.1 vectors were then treated with 20 μM LY294002 (a potent PI3K inhibitor, MedChem Express) for 24 h. TU212 and TU686 cells transfected with shRNA were then treated with 20 μM 740Y-P (PI3K agonist, MedChem Express) for 24 h.

A cuproptosis-specific inhibitor (tetrathiomolybdate) was used for suppression of cuproptosis in LSCC cells. In cuproptosis cell model, TU212 and TU686 cells were treated with 20 µM tetrathiomolybdate (HY-128530, Medchem Express) for 24 h.

RT-qPCR

Total RNA was isolated from HBECs, LSCC cells, or shRNA-transfected LSCC cells using TRIzol reagent (Thermo Fisher), followed by reverse transcription into cDNA using a PrimeScript™ RT reagent kit (Takara Biomedical Technology, Beijing, China). Quantitative PCR was conducted using SYBR Green PCR Master Mix (Thermo Scientific) on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The reference gene for normalization was GAPDH. Fold changes were quantified by the 2−△△Ct method. The sequences of primers used in this study were listed as follows: DACT1, forward 5′-AGGAGAAGTTCTTGGAGGAG-3′, reverse 5′-TGAGCTAGGCCGACTGTCTG-3′; GAPDH, forward 5′-GGAGTCAACGGATTTGGT-3′, reverse 5′-GTGATGGGATTTCCATTGAT-3′.

Western blotting

Total protein was isolated from TU212 and TU686 cells using RIPA lysis buffer (Beyotime, Shanghai, China) and quantified via the BCA Protein Assay Kit (Beyotime). Then, total protein (20 μg) was separated by 10–12% SDS-PAGE gel electrophoresis and transferred onto the PVDF membrane. Afterward, the PVDF membrane was blocked in 5% skimmed milk for 1 h and then incubated with specific primary antibodies against DACT1 (#SAB2107759, 1:1000) from Sigma Aldrich (St. Louis, USA), GAPDH (#97,166, 1:1000) from Cell Signaling Technology (Danvers, MA, USA), dihydrolipoamide S-Acetyltransferase (DLAT, #12,362, 1:1000) from Cell Signaling Technology, p-PI3K (ab138364, 1:500, Abcam, UK), PI3K (ab180967, 1:2000, Abcam), p-AKT (ab38449, 1:1000, Abcam), and AKT (ab179463, 1:10,000, Abcam) overnight at 4 °C. Subsequently, the membrane was washed three times with TBST (Beyotime) and incubated with HRP-labeled secondary antibodies for 2 h at room temperature. Finally, the protein bands were illuminated using the enhanced chemiluminescence (Thermo Scientific) and quantified by ImageJ software.

CCK-8 assays

LSCC cell viability was examined using CCK-8 reagent (Beyotime). To determine the effects of DACT1 knockdown/overexpression on LSCC cell viability, TU212 and TU686 cells were seeded onto 96-well plates (1 × 103 cells/well), transfected with shRNA or pcDNA3.1 vectors, and treated with or without LY294002 for 24 h. Next, cells were incubated for 24, 48, or 72 h. At each timepoint, each well was added with 10 μl CCK-8 reagent and cultured for another 2 h. To explore the involvement of DACT1 in cuproptosis, LSCC cells (3 × 103) were seeded onto 96-well plates, transfected with shRNA or pcDNA3.1 vectors, and incubated with elesclomol and CuCl2 for 24 h. Next, cell incubation was performed for 6, 12, 18, or 24 h. Following addition of 10 μl CCK-8 solution at each timepoint for another 2 h of incubation, LSCC cell viability was determined by recording absorbance at 450 nm using a microplate reader (Thermo Fisher).

Colony formation assays

TU212 and TU686 cells were seeded into 6-well plates (1 × 103 cells/well), followed by transfection with shRNA or pcDNA3.1 vectors and treatment of LY294002. After 10 days of incubation under culture conditions, the visible colonies were fixed with 4% paraformaldehyde for 20 min. After fixation, the colonies were stained by 0.1% crystal violet (Beyotime). The number of colonies (containing more than 50 cells) were counted under an inverted light microscope (Olympus, Tokyo, Japan).

Transwell assays

A 24-well transwell chamber with an 8-μm pore polycarbonate membrane (Millipore, Billerica, MA, USA) was used. For cell invasion, the upper chambers were precoated with Matrigel (Corning Incorporated, Corning, NY, USA) diluted with culture medium without fetal bovine serum. For cell migration, the chambers were not precoated with Matrigel. After transfection of shRNA and pcDNA3.1 vectors as well as LY294002 treatment, a total of 2 × 104 cells were added to the upper chambers with 200 μL of serum-free medium. Then, 500 μL of complete medium was added to the bottom chambers. After 24 h, nonmigratory or noninvasive cells in the upper chambers were removed with cotton swabs, and cells migrated or invaded to the lower transwell surfaces were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime) for 3 min. LSCC cells were imaged and counted in five visual fields using an inverted light microscope (Olympus).

Cooper concentration

TU212 and TU686 cells were cultured in 6 cm plates and subjected to cell transfection and treatment for 24 h. Next, cells were collected and disrupted ultrasonically to detect intercellular copper using the Copper Colorimetric Assay Kit (Elabscience, Wuhan, China). The absorbance at 580 nm was determined using a microplate spectrophotometer. A calibration curve was generated by plotting the optical density values against the known copper concentrations of the standard solutions. The concentration of copper ions in the samples was then quantified based on this curve.

ROS detection

TU212 and TU686 cells were exposed to elesclomol and CuCl2, followed by transfection with shRNA or pcDNA3.1 vectors. Next, cells were seeded into 24-well plates (5 × 104 cells/well). Then, serum-free medium containing 10 μM DCFH-DA (Beyotime) was added to each well and incubated at  °C for 1 h in the dark. After washing, a fluorescence microscope (Olympus) was used to capture the images.

Statistical analysis

Data were analyzed by GraphPad Prism software (version 8) and described as the mean ± standard deviation. Student’s t test and one-way or two-way analysis of variance followed by Tukey’s post hoc analysis were used for comparison of statistical disparities. A value of p < 0.05 was considered statistically significant.

Results

Upregulation of DACT1 in LSCC cells

Considering that laryngeal cancer is one of the most frequently occurring malignant tumors in the head and neck region, bioinformatics analysis was performed to analyze DACT1 expression in HNSC tissue samples. As shown in Fig. 1A, the UALCAN database shows the upregulation of DACT1 in HNSC tissues (n = 520) relative to normal samples (n = 44) (p = 1.4 × 10⁻4). RT-qPCR and western blotting confirmed that DACT1 expression was significantly higher in two LSCC cell lines (TU212 and TU686) compared to normal human bronchial epithelial cells (HBEC) (Fig. 1B, C). Given the elevated DACT1 expression in LSCC cells, we effectively knocked down DACT1 expression in LSCC cell lines using sh-DACT1#1/2, as validated by RT-qPCR and western blotting (Fig. 1D, E).

Fig. 1.

Fig. 1

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DACT1 expression is upregulated in LSCC cells. A The UALCAN database (https://ualcan.path.uab.edu/) was employed to analyze DACT1 expression in HNSC samples and matched normal samples. B, C RT-qPCR and Western blotting were carried out to measure mRNA and protein levels of DACT1 in HBECs, TU212, and TU686 cells. ***p < 0.001 versus HBEC group. D, E Relative mRNA expression of DACT1 was measured by RT-qPCR in TU212 and TU686 cells transfected with sh-NC, sh-DACT1#1, and sh-DACT1#2. ***p < 0.001 versus sh-NC group

DACT1 knockdown inhibits malignant behavior of LSCC cells

As shown by CCK-8 and colony formation assays, DACT1 knockdown by sh-DACT1#1/2 significantly reduced optical density (OD) values and the number of colonies formed by TU212 and TU686 cells (Fig. 2A–D). The results demonstrate the suppressive effect of DACT1 knockdown on LSCC cell viability and proliferation. Transwell assays revealed that the proportions of migrated and invaded cells were markedly diminished in the context of DACT1 knockdown (Fig. 2E–G). The findings verify that DACT1 deficiency inhibits LSCC cell migration and invasion.

Fig. 2.

Fig. 2

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DACT1 knockdown inhibits LSCC cell proliferation, migration, and invasion. A, B CCK-8 assays were performed to detect LSCC cell viability in sh-NC, sh-DACT1#1, and sh-DACT1#2 groups. C, D Colony formation assays were conducted to assess LSCC cell proliferation. E–G Transwell assays were carried out to measure the migratory and invasive abilities of TU212 and TU686 cells. scale bar: 200 μm or 100 μm. ***p < 0.001 versus sh-NC group

DACT1 knockdown enhances the sensitivity of LSCC cells to cuproptosis

Given that DACT1 is a cuproptosis-related gene in laryngeal cancer [16], its effects on cuproptosis in LSCC cells were then evaluated. TU212 and TU686 cells were co-cultured with elesclomol and Cucl2, and the morphology is present in Fig. 3A, B. Compared with the control group without any treatment, elesclomol and CuCl2 induced decrease in cell number, and the trend was enhanced by DACT1 depletion (Fig. 3A, B). As suggested by CCK-8 assays, sh-NC, elesclomol, and Cucl2 treatment led to a decline in cell viability compared with the sole sh-NC group, while DACT1 silencing further reduced LSCC cell viability (Fig. 3C, D). In addition, the copper concentration was prominently increased by DACT1 depletion in TU212 and TU686 cells treated with elesclomol and Cucl2 (Fig. 3E, F). Moreover, DACT1 knockdown elevated ROS level in TU212 and TU686 cells without elesclomol and Cucl2 treatment. The ROS level was increased after elesclomol and Cucl2 treatment, and the trend was enhanced by DACT1 knockdown (Fig. 3G–J). The findings indicate that DACT1 knockdown promotes cuproptosis in LSCC cells.

Fig. 3.

Fig. 3

Fig. 3

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DACT1 knockdown enhances the sensitivity of LSCC cells to cuproptosis. TU212 and TU686 cells were transfected with shRNA and incubated with elesclomol and copper for 24 h. A, B The morphology of TU212 and TU686 cells was imaged. scale bar: 100 μm. C, D CCK-8 assays were performed to measure LSCC cell viability. E–F Detection of copper concentration. G–J DCFH-DA method was used to determine intracellular ROS level. scale bar: 100 μm. ***p < 0.001 vs. sh-NC group; ###p < 0.001 vs. sh-NC + Elesclomol + CuCl2 group

DACT1 knockdown enhances the expression of cuproptosis-related genes in LSCC cells

Upregulation of HSP70 and loss of Fe-S cluster proteins such as LIAS are features of cuproptosis [22, 25]. In this study, we found that HSP70 protein level was significantly increased in TU212 and TU686 cells of sh-NC + elesclomol + Cucl2 group relative to the sh-NC group, and the upregulation was enhanced in context of DACT1 deficiency (Fig. 4A–C). In addition, LIAS protein level was decreased in response to elesclomol and Cucl2 treatment, and the silencing of DACT1 further reduced LIAS protein expression (Fig. 4A, D, E). The results indicate the induction of cuproptosis in LSCC cells, and DACT1 depletion enhances cuproptosis.

Fig. 4.

Fig. 4

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DACT1 knockdown regulates cuproptosis-related genes in LSCC cells. A Western blotting was performed to measure protein levels of cuproptosis-related genes (HSP70 and LIAS) in TU212 and TU686 cells of four experimental groups: sh-NC, sh-NC + elesclomol + Cucl2, sh-DACT1#1 + elesclomol + Cucl2, and sh-DACT1#2 + elesclomol + Cucl2 groups. B–E Relative protein levels of HSP70 and LIAS were calculated with normalization to GAPDH. *p < 0.05, **p < 0.01, ***p < 0.001 versus sh-NC group. ##p < 0.01, ###p < 0.001 versus sh-NC + elesclomol + Cucl2 group

Inhibition of cuproptosis using tetrathiomolybdate reverses the promoting effects of DACT1 on cuproptosis

Figure 5A, B reveals that sh-DACT1 significantly reduced the viability of cuproptosis model cells (***p < 0.001), and the suppressive effect was reversed in the sh-DACT1#1 + tetrathiomolybdate group (###p < 0.001). Copper concentration was markedly elevated in cuproptosis model cells of the sh-DACT1#1 group compared to the sh-NC group, while tetrathiomolybdate counteracted the increase in copper level induced by sh-DACT1#1 (Fig. 5C, D). Moreover, upregulation of HSP70 and downregulation of LIAS caused by DACT1 deficiency was reversed by the supplementation of tetrathiomolybdate in cuproptosis model cells (Fig. 5E). The findings further indicate the promoting effect of DACT1 depletion on cuproptosis.

Fig. 5.

Fig. 5

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The promoting effect of sh-DACT1 on cuproptosis was reversed by tetrathiomolybdate (cuproptosis inhibitor). TU212 and TU686 cells exposed to elesclomol and Cucl2 are assigned into sh-NC, sh-DACT1#1, and sh-DACT1#1 + tetrathiomolybdate groups. A, B CCK-8 assays were performed to measure TU212 and TU686 cell viability. C, D Determination of copper concentration. E Western blotting was performed to quantify protein levels of cuproptosis-related genes (HSP70 and LIAS) in LSCC cells, with normalization to GAPDH. ***p < 0.001 versus sh-NC group. ###p < 0.001 versus sh-DACT1#1 group

DACT1 activates PI3K/AKT signaling in LSCC cells

Subsequently, we explored the mechanism related to the induction of cuproptosis mediated by DACT1. As demonstrated by western blotting, DACT1 knockdown reduced ratios of p-PI3K/PI3K and p-AKT/AKT protein levels in LSCC cells (Fig. 6A–C). On the contrary, overexpression of DACT1 increased phosphorylated levels of PI3K and AKT in LSCC cells, and the alterations were reversed by LY294002, (an inhibitor of PI3K/AKT) (Fig. 6D–F). The results validate the promoting effect of DACT1 on the PI3K/AKT signaling in LSCC cells.

Fig. 6.

Fig. 6

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DACT1 activates the PI3K/AKT signaling in LSCC cells. A Western blotting was performed to measure effects of DACT1 depletion on protein levels of PI3K, phosphorylated (p)-PI3K, AKT, and p-AKT. B, C Quantitative analysis of p-PI3K/PI3K and p-AKT/AKT ratios in sh-NC, sh-DACT1#1, and sh-DACT1#2 groups. ***p < 0.001 versus sh-NC group. D The effects of DACT1 overexpression and LY294002 (PI3K inhibitor) on the PI3K/AKT signaling were evaluated by Western blotting. E–F Quantitative analysis of p-PI3K/PI3K and p-AKT/AKT ratios in vector, DACT1, and DACT1 + LY294002 groups. ***p < 0.001 versus vector group; ###p < 0.001 versus DACT1 group

LY294002 reverses the promoting effects of DACT1 on malignant behavior of LSCC cells

DACT1 overexpression led to increases in OD values and the percentage of LSCC cell colonies (Fig. 7A, D, ***p < 0.001). Notably, the promoting effects of DACT1 overexpression on LSCC cell viability and proliferation were counteracted by inhibition of the PI3K pathway using LY294002 (Fig. 7A, D, ###p < 0.001). Moreover, the migration and invasion of LSCC cells were enhanced by DACT1 overexpression, and the changes were inhibited by LY294002 (Fig. 7E, G). The above results demonstrate that the enhancement of DACT1 on LSCC cell behavior was reversed by PI3K inactivation.

Fig. 7.

Fig. 7

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LY294002 reverses the promoting effects of DACT1 on malignant behavior of LSCC cells. A, B CCK-8 assays were performed to measure LSCC cell viability in vector, pcDNA3.1-DACT1 (DACT1), and DACT1 + LY294002 groups. C, D Colony formation assays were conducted to assess LSCC cell proliferation. E–G Transwell assays were performed to measure LSCC cell migration and invasion. scale bar: 100 or 200 μm. ***p < 0.001 versus vector group; ###p < 0.001 versus DACT1 group

LY294002 counteracts the inhibitory effects of DACT1 on cuproptosis of LSCC cells

Compared to the vector group, the vector + elesclomol + Cucl2 group showed a marked reduction in LSCC cell viability (Fig. 8A, B, ***p < 0.001). Under treatment with elesclomol and Cucl2, DACT1 overexpression elevated LSCC cell viability (###p < 0.001), but this pro-survival effect was abolished by LY294002 administration (Fig. 8A, B). As shown in Fig. 8C, D, treatment with elesclomol and Cucl2 led to elevated intracellular copper levels, whereas DACT1 overexpression attenuated copper accumulation. This decrease in copper concentration was substantially reversed by LY294002. Moreover, the induction of cuproptosis using elesclomol and Cucl2 led to an increased ROS level compared to the control vector group, and the alteration was prominently suppressed by DACT1 overexpression (Fig. 8E–H). While LY294002 treatment rescued the decrease in ROS level mediated by DACT1 upregulation (Fig. 8E–H). The findings reveal the inhibitory effect of DACT1 overexpression on cuproptosis in LSCC cells via activation of the PI3K pathway.

Fig. 8.

Fig. 8

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LY294002 counteracts the inhibitory effect of DACT1 on cuproptosis in LSCC. TU212 and TU686 cells were exposed to elesclomol and Cucl2, transfected with pcDNA3.1 vectors, and treated with LY294002. A, B CCK-8 assays were performed to measure cell viability. C, D Detection of copper concentration. E–H DCFH-DA method was used to detect changes in intracellular ROS level. scale bar: 100 μm. ***p < 0.001 versus vector group; ###p < 0.001 versus vector + Elesclomol + CuCl2 group. &&&p < 0.001 versus DACT1 + Elesclomol + CuCl2 group

The regulatory effects of DACT1 on cuproptosis-related genes were counteracted by LY294002

As shown in Fig. 9A–E, treatment with elesclomol and Cucl2 increased HSP70 protein level while decreasing LIAS protein expression. DACT1 overexpression reversed these alterations in cuproptosis-related proteins. Notably, co-treatment with LY294002 restored the elevated HSP70 levels and further reduced LIAS protein expression in LSCC cells (Fig. 9A–E). These findings indicate that DACT1 suppresses cuproptosis through activation of the PI3K signaling pathway.

Fig.9.

Fig.9

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LY294002 counteracts the regulatory effects of DACT1 on cuproptosis-related genes. A Western blot was performed to measure the effects of DACT1 and/or LY294002 on protein levels of cuproptosis-related genes (HSP70 and LIAS). B–E Relative HSP70 and LIAS levels in TU212 and TU686 cells were calculated with normalization to GAPDH. ***p < 0.001 versus vector group. ###p < 0.001 versus vector + Elesclomol + Cucl2 group. &&&p < 0.001 versus DACT1 + Elesclomol + Cucl2 group

Activation of PI3K signaling using 740Y-P reverses the promoting impact of sh-DACT1 on cuproptosis

As shown in Fig. 10A, the silencing of DACT1 lowered ratios of p-PI3K/PI3K and p-AKT/AKT in TU212 and TU686 cells, and the decreases were rescued by 740Y-P (PI3K agonist) treatment. In addition, the silencing of DACT1 reduced the viability of cuproptosis model cells, and the trend was rescued by 740Y-P (Fig. 10B, C). Moreover, the promoting effect of DACT1 depletion on copper concentration was reversed upon 740Y-P treatment (Fig. 10D, E). Furthermore, the silencing of DACT1 increased HSP70 protein level while reducing LIAS protein level in cuproptosis model cells, and the changes were counteracted by 740Y-P treatment (Fig. 10F). The findings indicate that DACT1deficiency promotes cuproptosis through inactivation of the PI3K signaling.

Fig. 10.

Fig. 10

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Activation of the PI3K/Akt signaling using 740Y-P reverses the promoting effect of sh-DACT1 on cuproptosis. A Protein levels of PI3K, phosphor (p)-PI3K, AKT, and p-AKT in TU212 and TU686 cells were quantified using western blotting. Ratios of p-PI3K/PI3K and p-AKT/AKT were quantified. B, C CCK-8 assays were performed to measure the viability of LSCC cells exposure to Elesclomol and Cucl2 in sh-NC, sh-DACT1#1, and sh-DACT1#1 + 740Y-P groups. D, E Effects of DACT1 depletion and PI3K activator on copper concentration were determined. F Western blot analysis was conducted to analyze protein levels of cuproptosis-related genes (HSP70 and LIAS) in sh-NC, sh-DACT1#1, and sh-DACT1#1 + 740Y-P groups. ***p < 0.001 versus sh-NC group. ###p < 0.001 versus sh-DACT1#1 group

Discussion

Although significant advancements have been made in treatment strategies, the prognosis for LSCC remains unsatisfactory. A pressing demand exists for novel molecular markers to facilitate early diagnosis and targeted therapies. DACT1 was initially identified as a Dishevelled-associated antagonist of Wnt/β-catenin and JNK pathways [7, 10]. This gene has been associated with the prognosis of LSCC patients [16]. Our study investigated the roles and mechanisms of DACT1 in LSCC progression and revealed that DACT1 inhibited cuproptosis and promoted cell malignancies through PI3K/AKT pathway activation in LSCC cells.

Proliferation, invasion, and migration represent key hallmarks of cancer [26]. DACT1 has been identified as either an oncogenic or tumor-suppressive gene depending on the context of various tumors. For example, DACT1 promotes cell migration and proliferation in colon cancer [9]. Conversely, it suppresses cell proliferation and migration in cervical [27] and ovarian cancers [28]. In this study, we demonstrated that DACT1 knockdown suppressed the proliferation, migration, and invasion of LSCC cells. The inhibitory effects of DACT1 depletion on these malignant behaviors suggest the tumor-promoting role of DACT1 in LSCC.

Copper, an essential trace element in organisms, participates in various physiological processes, especially tumor growth and metastasis [29]. Aberrant copper concentrations have been detected in laryngeal [30], colorectal [31], endometrial [32], breast [33], and oral cancers [34]. Copper-induced cell death, termed cuproptosis, holds clinical potential for cancer therapy. For example, the administration of elesclomol (a copper ionophore) has shown promise in treating metastatic melanoma, acute myeloid leukemia, ovarian epithelial cancer, fallopian tube cancer, and primary peritoneal cancer [35, 36]. Adding copper to a serum-free medium can enhance the anticancer effect of elesclomol [37]. Furthermore, copper ions can induce ROS production, and excessive ROS generation leads to oxidative stress, cellular damage, and ultimately cell death [38]. Therefore, cuproptosis triggered by excessive copper accumulation represents a novel therapeutic target for anticancer treatment. Elesclomol, a potent copper ionophore, forms a complex with copper ions, inducing mitochondrial oxidative stress and copper-dependent cell death (cuproptosis) [39]. In this study, LSCC cell lines (TU212 and TU686) were treated with a combination of elesclomol and copper to induce cuproptosis [40]. Under these conditions, DACT1 knockdown suppressed TU212 and TU686 cell viability and increased copper concentration and ROS accumulation in LSCC cells, suggesting that DACT1 knockdown promotes cuproptosis in LSCC cells.

Copper is involved in regulating PI3K/AKT signaling. For example, copper directly activates PI3K, thereby inducing AKT activation [41]. Additionally, copper binds to histidine 117 and histidine 203 residues of PDK1, leading to AKT activation [42]. The PI3K/AKT pathway is associated with cell proliferation, survival, gene expression, and protein synthesis [43]. The pathway can reduce the sensitivity to cuproptosis in colorectal and gastric cancers [44, 45]. Upregulation of PI3K and AKT has been observed in LSCC samples [46]. Several molecules that regulate PI3K/AKT signaling, such as BMP2 [47], CH25H [48], and YBX1 [49], have prognostic value for LSCC. DACT1, a cuproptosis-related gene, can activate PI3K/AKT signaling in HepG2 cells [24]. Our study demonstrated that DACT1 knockdown suppressed PI3K and AKT phosphorylation levels in TU212 and TU686 cells. In contrast, overexpression of DACT1 activated the PI3K/Akt signaling. Moreover, the promoting effects of DACT1 on malignant LSCC cell behavior and its suppressive effect on cuproptosis were all reversed by LY294002 (a PI3K/AKT inhibitor). The findings confirmed that DACT1 inhibited cuproptosis and promoted LSCC cell proliferation, migration, and invasion through activation of the PI3K/AKT pathway.

However, this study has several drawbacks. First, the oncogenic role of DACT1 in LSCC was not verified in vivo. Second, clinical samples were not collected in this study, and the association between DACT1 expression and clinicopathological characteristics in patients with LSCC has not been evaluated.

Collectively, this study demonstrates that DACT1 promotes cell proliferation, migration and invasion while inhibiting cuproptosis in LSCC through PI3K/AKT pathway activation. Targeting DACT1 could represent a promising strategy to enhance the efficacy of cuproptosis-induced cancer cell death and improve LSCC treatment outcomes. In future studies, further investigations are required to determine whether DACT1 directly participates in the regulation of cuproptosis.

Supplementary Information

Supplementary material 1 (599.1KB, pptx)

Acknowledgements

Not applicable

Author contributions

Yan Guo was the main designer of this study. Yan Guo, Jiarui Zhang, Jingchun Ge and Linli Tian collected and analyzed the data. Yan Guo, Jiarui Zhang, Liang Li, Ming Liu and Linli Tian drafted the manuscript. All authors read and approved the final manuscript.

Funding

None.

Data availability

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

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Huang J, et al. Updated disease distributions, risk factors, and trends of laryngeal cancer: a global analysis of cancer registries. Int J Surg. 2024;110(2):810–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Liberale C, et al. Updates on larynx cancer: risk factors and oncogenesis. Int J Mol Sci. 2023. 10.3390/ijms241612913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Mannelli G, et al. Conservative treatment for advanced T3–T4 laryngeal cancer: meta-analysis of key oncological outcomes. Eur Arch Otorhinolaryngol. 2018;275(1):27–38. [DOI] [PubMed] [Google Scholar]
  • 4.Djukic V, et al. Laser transoral microsurgery in treatment of early laryngeal carcinoma. Eur Arch Otorhinolaryngol. 2019;276(6):1747–55. [DOI] [PubMed] [Google Scholar]
  • 5.Liu T, et al. Long noncoding RNA NEAT1 functions as an oncogene in human laryngocarcinoma by targeting miR-29a-3p. Eur Rev Med Pharmacol Sci. 2019;23(14):6234–41. [DOI] [PubMed] [Google Scholar]
  • 6.Zhang Q, et al. Evaluation of risk factors for laryngeal squamous cell carcinoma: a single-center retrospective study. Front Oncol. 2021;11:606010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cheyette BN, et al. Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation. Dev Cell. 2002;2(4):449–61. [DOI] [PubMed] [Google Scholar]
  • 8.Zhang L, et al. Dapper 1 antagonizes Wnt signaling by promoting dishevelled degradation. J Biol Chem. 2006;281(13):8607–12. [DOI] [PubMed] [Google Scholar]
  • 9.Yuan G, et al. Oncogenic function of DACT1 in colon cancer through the regulation of β-catenin. PLoS One. 2012;7(3):e34004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Hou J, et al. Cytoplasmic HDPR1 is involved in regional lymph node metastasis and tumor development via beta-catenin accumulation in esophageal squamous cell carcinoma. J Histochem Cytochem. 2011;59(7):711–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Yau TO, et al. HDPR1, a novel inhibitor of the WNT/beta-catenin signaling, is frequently downregulated in hepatocellular carcinoma: involvement of methylation-mediated gene silencing. Oncogene. 2005;24(9):1607–14. [DOI] [PubMed] [Google Scholar]
  • 12.Astolfi A, et al. A molecular portrait of gastrointestinal stromal tumors: an integrative analysis of gene expression profiling and high-resolution genomic copy number. Lab Invest. 2010;90(9):1285–94. [DOI] [PubMed] [Google Scholar]
  • 13.Yang ZQ, et al. Downregulation of HDPR1 is associated with poor prognosis and affects expression levels of p120-catenin and beta-catenin in nonsmall cell lung cancer. Mol Carcinog. 2010;49(5):508–19. [DOI] [PubMed] [Google Scholar]
  • 14.Yang JH, Lin LK, Zhang S. Effects of DACT1 methylation status on invasion and metastasis of nasopharyngeal carcinoma. Biol Res. 2019;52(1):31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cossu AM, et al. Long non-coding RNAs as important biomarkers in laryngeal cancer and other head and neck tumours. Int J Mol Sci. 2019. 10.3390/ijms20143444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Li C, Zhu Y, Shi S. Effective prognostic risk model with cuproptosis-related genes in laryngeal cancer. Braz J Otorhinolaryngol. 2024;90(2):101384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.De Luca A, et al. Copper homeostasis as target of both consolidated and innovative strategies of anti-tumor therapy. J Trace Elem Med Biol. 2019;55:204–13. [DOI] [PubMed] [Google Scholar]
  • 18.Jiang Y, et al. Copper-induced tumor cell death mechanisms and antitumor theragnostic applications of copper complexes. Nanomedicine. 2022;17(5):303–24. [DOI] [PubMed] [Google Scholar]
  • 19.Festa RA, Thiele DJ. Copper: an essential metal in biology. Curr Biol. 2011;21(21):R877-83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Zhang W, et al. Identification of cuproptosis and immune-related gene prognostic signature in lung adenocarcinoma. Front Immunol. 2023;14:1179742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Chen L, Min J, Wang F. Copper homeostasis and cuproptosis in health and disease. Signal Transduct Target Ther. 2022;7(1):378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zhang L, et al. Recent progress of methods for cuproptosis detection. Front Mol Biosci. 2024;11:1460987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Huang T, et al. CEBPB dampens the cuproptosis sensitivity of colorectal cancer cells by facilitating the PI3K/AKT/mTOR signaling pathway. Saudi J Gastroenterol. 2024;30(6):381–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kuang JR, et al. Dapper1 attenuates hepatic gluconeogenesis and lipogenesis by activating PI3K/Akt signaling. Mol Cell Endocrinol. 2017;447:106–15. [DOI] [PubMed] [Google Scholar]
  • 25.Huo S, et al. ATF3/SPI1/SLC31A1 signaling promotes cuproptosis induced by advanced glycosylation end products in diabetic myocardial injury. Int J Mol Sci. 2023. 10.3390/ijms24021667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kuznetsov M, Kolobov A. Investigation of solid tumor progression with account of proliferation/migration dichotomy via Darwinian mathematical model. J Math Biol. 2020;80(3):601–26. [DOI] [PubMed] [Google Scholar]
  • 27.Zeng L, Chen C, Yao C. Histone deacetylation regulated by KDM1A to suppress DACT1 in proliferation and migration of cervical cancer. Anal Cell Pathol. 2021;2021:5555452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Li RN, et al. DACT1 overexpression in type I ovarian cancer inhibits malignant expansion and cis-platinum resistance by modulating canonical Wnt signalling and autophagy. Sci Rep. 2017;7(1):9285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ge EJ, et al. Connecting copper and cancer: from transition metal signalling to metalloplasia. Nat Rev Cancer. 2022;22(2):102–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wojnicka J, et al. The relationship between cancer stage, selected immunological parameters, Epstein-Barr virus infection, and total serum content of iron, zinc, and copper in patients with laryngeal cancer. J Clin Med. 2024. 10.3390/jcm13020511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Squitti R, et al. Serum copper status of patients with colorectal cancer: a systematic review and meta-analysis. J Trace Elem Med Biol. 2024;82:127370. [DOI] [PubMed] [Google Scholar]
  • 32.Atakul T, et al. Serum copper and zinc levels in patients with endometrial cancer. Biol Trace Elem Res. 2020;195(1):46–54. [DOI] [PubMed] [Google Scholar]
  • 33.Szwiec M, et al. Serum levels of copper and zinc and survival in breast cancer patients. Nutrients. 2024. 10.3390/nu16071000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Baharvand M, et al. Serum levels of ferritin, copper, and zinc in patients with oral cancer. Biomed J. 2014;37(5):331–6. [DOI] [PubMed] [Google Scholar]
  • 35.Chen S, et al. Syntheses and antitumor activities of N’1,N’3-dialkyl-N’1,N’3-di-(alkylcarbonothioyl) malonohydrazide: the discovery of elesclomol. Bioorg Med Chem Lett. 2013;23(18):5070–6. [DOI] [PubMed] [Google Scholar]
  • 36.Monk BJ, et al. A phase II evaluation of elesclomol sodium and weekly paclitaxel in the treatment of recurrent or persistent platinum-resistant ovarian, fallopian tube or primary peritoneal cancer: An NRG oncology/gynecologic oncology group study. Gynecol Oncol. 2018;151(3):422–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Nagai M, et al. The oncology drug elesclomol selectively transports copper to the mitochondria to induce oxidative stress in cancer cells. Free Radic Biol Med. 2012;52(10):2142–50. [DOI] [PubMed] [Google Scholar]
  • 38.Lopez J, Ramchandani D, Vahdat L. Copper depletion as a therapeutic strategy in cancer. Met Ions Life Sci. 2019. 10.1515/9783110527872-018. [DOI] [PubMed] [Google Scholar]
  • 39.Zheng P, et al. Elesclomol: a copper ionophore targeting mitochondrial metabolism for cancer therapy. J Exp Clin Cancer Res. 2022;41(1):271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Quan B, et al. LINC02362/hsa-miR-18a-5p/FDX1 axis suppresses proliferation and drives cuproptosis and oxaliplatin sensitivity of hepatocellular carcinoma. Am J Cancer Res. 2023;13(11):5590–609. [PMC free article] [PubMed] [Google Scholar]
  • 41.Liu H, et al. Copper exposure induces hepatic G0/G1 cell-cycle arrest through suppressing the Ras/PI3K/Akt signaling pathway in mice. Ecotoxicol Environ Saf. 2021;222:112518. [DOI] [PubMed] [Google Scholar]
  • 42.Guo J, et al. Copper promotes tumorigenesis by activating the PDK1-AKT oncogenic pathway in a copper transporter 1 dependent manner. Adv Sci. 2021;8(18):e2004303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Jiang N, et al. Role of PI3K/AKT pathway in cancer: the framework of malignant behavior. Mol Biol Rep. 2020;47(6):4587–629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Yi J, et al. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proc Natl Acad Sci U S A. 2020;117(49):31189–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Wang C, et al. Apolipoprotein C-II induces EMT to promote gastric cancer peritoneal metastasis via PI3K/AKT/mTOR pathway. Clin Transl Med. 2021;11(8):e522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Starska K, et al. Fibroblast growth factor receptor 1 and 3 expression is associated with regulatory PI3K/AKT kinase activity, as well as invasion and prognosis, in human laryngeal cancer. Cell Oncol. 2018;41(3):253–68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zhu Q, et al. RUNX1-BMP2 promotes vasculogenic mimicry in laryngeal squamous cell carcinoma via activation of the PI3K-AKT signaling pathway. Cell Commun Signal. 2024;22(1):227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Xiang Z, et al. CH25H promotes autophagy and regulates the malignant progression of laryngeal squamous cell carcinoma through the PI3K-AKT pathway. Cancer Med. 2024;13(20):e70312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Zhao J, Zhang P, Wang X. YBX1 promotes tumor progression via the PI3K/AKT signaling pathway in laryngeal squamous cell carcinoma. Transl Cancer Res. 2021;10(11):4859–69. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (599.1KB, pptx)

Data Availability Statement

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

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