14-3-3β knockdown inhibits migration and colony formation of esophageal squamous cell carcinoma cells and is associated with weakened p-AKT signaling
Qing-Hua Hu, Man-Qin Zhu, Kuai Chen, Jin-Shi Huang, Qiang Tao, Zhi-Bin Guo
TL;DR
This study shows that reducing 14-3-3β in esophageal cancer cells slows their movement and growth, possibly by weakening the p-AKT signaling pathway.
Contribution
The study reveals a novel link between 14-3-3β and p-AKT signaling in ESCC progression.
Findings
14-3-3β knockdown in KYSE-150 cells reduced migration and colony formation.
Reduced 14-3-3β expression led to decreased p-AKT activity without affecting AKT levels.
No significant changes in apoptosis-related proteins were observed after 14-3-3β knockdown.
Abstract
This study aims to explore the functions and mechanisms underlying the involvement of 14-3-3β in esophageal squamous cell carcinoma (ESCC). Western blot and quantitative real-time PCR (RT-PCR) were utilized to evaluate the levels of 14-3-3β in SHEE, TE-1, and KYSE-150 cells. Following infection with YWHAB-RNAi lentivirus and puromycin screening, the reduction of 14-3-3β expression in KYSE-150 cells was confirmed via western blot and RT-PCR. Subsequently, wound healing, CCK8, and colony-formation assays were performed to assess cell migration and proliferation in KYSE-150 cells with stable low levels of 14-3-3β. Furthermore, western blot analysis was conducted to examine the expression of relevant proteins, elucidating the potential molecular mechanisms underlying 14-3-3β involvement in ESCC progression and metastasis. The expression of 14-3-3β was significantly elevated in KYSE-150…
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Figure 5- —National Natural Science Foundation of China
- —Jiangxi Province Key Laboratory of Child Development
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Taxonomy
Topics14-3-3 protein interactions · Protein Tyrosine Phosphatases · Signaling Pathways in Disease
Introduction
Esophageal cancer ranks among the top 10 deadliest and most common cancers globally, primarily comprising esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) subtypes [1]. ESCC represents approximately 90% of all esophageal cancer cases and is the fourth leading cause of cancer-related mortality in China. Despite considerable progress in diagnosis and treatment, the overall 5-year survival rate remains below 25% for individuals with ESCC [2, 3], primarily due to factors such as local invasion and distant metastasis, which are significant adverse prognostic indicators [4, 5].
The 14-3-3 proteins are a family of intracellular proteins present in all eukaryotic organisms [6]. The 14-3-3 protein family encompasses 7 isoforms—β, ε, ζ, η, θ, γ, and σ—serving as scaffold proteins that interact with a diverse array of molecules. These interactions include transcription factors, signaling molecules, tumor suppressors, cytoskeletal proteins, and apoptotic proteins [7], participating in regulating various biological behaviors. Mounting evidence indicates that 14-3-3β is significantly involved in tumor initiation and advancement, exhibiting elevated expression levels in various cancer types, including gliomas, lung cancer, breast cancer, and squamous cell carcinomas [8, 9]. However, there is limited research on the role and molecular mechanisms of 14-3-3β in the progression and metastasis of esophageal cancer. Nonetheless, a previous study has demonstrated an increase in 14-3-3β expression in ESCC tissues [10], which is suggested to correlate with tumor differentiation. Furthermore, heightened expression of 14-3-3β is noted in highly invasive and metastatic ESCC cell strains [11].
In this study, we explored how 14-3-3β impacts the proliferation and migration of ESCC cells. These findings could offer valuable insights into the molecular underpinnings of ESCC development, potentially informing advancements in diagnosis, treatment, and prognosis for patients with ESCC.
Methods and materials
Reagents and antibodies
The RPMI-1640 medium, fetal bovine serum, and 0.25% EDTA-trypsin were procured from Gibco (USA). Antibiotics including penicillin and streptomycin, as well as BCA protein quantification kits, were obtained from Shanghai Biyuntian Technology Co., Ltd. Phosphate-buffered saline (PBS) powder, skim milk powder, PVDF membrane, and crystal violet were sourced from Beijing Soleibao Technology Co., Ltd. Additionally, the reverse transcription kit was purchased from Promega Corporation, while the quantitative real-time PCR (RT-PCR) kit and CCK-8 kit were obtained from (Beijing) Quanshijin Biotechnology Co., Ltd. Primary antibodies such as BCL2, BAX, GAPDH, and Caspase 9 were acquired from Proteintech, while 14-3-3β was sourced from Abcam, Caspase 3 from Bioss, and AKT and p-AKT from Cell Signaling Technology (CST). Furthermore, lentivirus strains including LV-YWHAB-RNAi (807), LV-YWHAB-RNAi (808), and lentivirus CN207 (as control) were manufactured and synthesized by Jikai Genetics Co., Ltd.
Methods
Cell culture
The TE-1, KYSE-150, and SHEE cells obtained from the Cell Bank of the Chinese Academy of Sciences were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 2% antibiotics (penicillin-streptomycin). The cells were maintained in an incubator at 37 °C with 5% CO_2_. Upon exceeding 90% confluency, the cells were passaged and used for subsequent experiments.
Wound healing assay
The KYSE-150 cells were plated into 6-well culture plates and allowed to proliferate until they achieved over 90% confluence. Subsequently, a sterile pipette tip was used to introduce a uniform vertical scratch through the center of the cell monolayer in each well. After washing the cells with PBS three times, they were cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS). Images were captured under a microscope at 0, 24, and 48 h after scratching. The migration rate of the cells was determined by measuring the width of the scratch area. The formula used for calculating cell migration rate is Cell migration rate = [(scratch width at 0 h - scratch width at 24–48 h)/scratch width at 0 h] × 100% [12].
Colony formation assay
Following trypsinization, the cells were counted, and a total of 2 × 10^2^ cells were seeded into each well of a sterile 6-well plate containing 2 mL of RPMI-1640 medium supplemented with 10% FBS and 2% antibiotics. The cells were then cultured for 2 to 3 weeks at 37 °C with 5% CO_2_, with medium replacements occurring every 3 to 4 days. Upon the colonies reaching approximately 1 mm in diameter, the culture medium was removed, and the cells were washed 3 times with PBS. Subsequently, the cells were fixed with pre-cooled methanol for 15 min, followed by air-drying. After fixation, the cells were stained with crystal violet for 15 min. Excess crystal violet was rinsed off with flowing water, and the cells were air-dried before capturing images. The number of colonies in each group was counted, and the colony formation rate was calculated as (number of colonies/number of cells added at 0 h) × 100%.
CCK8 assay
Cells in the logarithmic growth phase were trypsinized, counted after centrifugation, and then seeded at a density of 2 × 10^3^ cells per well into a 96-well plate, with five replicate wells per group. After cell attachment (approximately 6–8 h), the cells were cultured and assayed at 0, 24, 48, 72, and 96 h. At each designated time point, 10 µL of CCK-8 reagent (Transgen, China) was directly added to each well without replacing the existing culture medium. Following incubation for 3 hours in the incubator, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader (Tecan, Switzerland). Cell viability was expressed as absorbance values. The experiment was independently repeated three times.
Real-time PCR
RNA was extracted from both normal esophageal epithelial cells and ESCC cells using the Trizol method. After measuring and verifying the concentration of the extracted RNA samples, 1 µg of RNA was reverse-transcribed into cDNA. The resulting cDNA was appropriately diluted and used as a template for PCR. The SYBR-Green Real-time PCR Mix, along with primers, template, and water, were mixed according to the manufacturer’s instructions. The relative mRNA expression levels were quantified using the 2^−ΔΔCt^ method, with GAPDH serving as the housekeeping gene.
Western blot
After trypsinization, the cells were washed twice with pre-cooled PBS and then treated with an appropriate amount of protein lysis buffer containing protease inhibitors to halt trypsinization. The cells were lysed on ice for 45 min with mixing at 10-minute intervals. The lysate was centrifuged at 1.2 × 10^4^ rpm (4°C, 15 min), and the supernatant was transferred to a clean EP tube for protein quantification using the BCA method. A total of 30 µg of proteins from each sample was loaded onto SDS-PAGE gel for electrophoresis. The proteins were subsequently transferred to a PVDF membrane and blocked with 5% skim milk for 1 h. After washing three times with TBST for 10 min each, the membrane was incubated with primary antibody overnight at 4°C. The next day, the membrane was washed again with TBST three times, followed by a 1-hour incubation with the secondary antibody at room temperature. Color development was performed after washing the membrane with TBST three times. The results were quantitatively analyzed using Image Lab software, with GAPDH serving as the housekeeping gene [12].
Statistical analysis
Statistical analysis and graph construction were carried out using GraphPad Prism 5.0 software. Experimental measurement data are expressed as mean ± standard deviation x̄±s. The t-test was employed for comparing between two groups, for comparing between multiple groups (e.g., CCK-8 data at different time points), one-way ANOVA was applied, if the overall test was significant, a Tukey’s test was further used for pairwise comparisons. The Shapiro-Wilk test and Levene’s test were used to test for normality and homogeneity of variance. A significance level of P < 0.05 was deemed statistically significant.
Results
Expression of 14-3-3β in the SHEE, TE-1, and KYSE-150 cells
The expression levels of 14-3-3β in the SHEE, TE-1, and KYSE-150 cells were evaluated using western blot and quantitative PCR. The western blot results indicated a significantly higher expression of 14-3-3β in the KYSE-150 cells (1.387 ± 0.114) compared to human esophageal epithelial cells (1.033 ± 0.047) (mean ± SD, n = 3, p < 0.05) (see Fig. 1). However, no significant difference in 14-3-3β expression was noted between the TE-1 (1.083 ± 0353) and SHEE cells (1.033 ± 0.047). The quantitative PCR results were consistent with the WB findings, showing that the expression of 14-3-3β was significantly higher in KYSE-150 cells (1.882 ± 0.148) than in SHEE cells (0.858 ± 0.079) (mean ± SD, n = 2, p < 0.05) (see Fig. 1), while no significant difference was observed between TE-1 (1.105 ± 0.003) and SHEE cells (0.858 ± 0.079). (mean ± SD, n = 2, p > 0.05) (see Fig. 1).
Fig. 114-3-3β expression levels in the SHEE, TE-1 and KYSE-150 cells. A: 14-3-3β protein expression was assessed via western blot analysis. Lane 1: SHEE (normal esophageal epithelial cell line); Lane 2: KYSE-150; Lane 3: TE-1. B: Quantitative analysis based on the results shown in Figure A. C: RT-PCR was employed to measure the mRNA expression of the YWHAB gene. Data are presented as mean ± SD from two independent experiments. *p < 0.05, **p < 0.01 vs. SHEE group
Establishment of a stable 14-3-3β low-expression cell line
The KYSE-150 cells underwent infection with two YWHAB-RNAi viruses (sh1007 and sh1008) along with the CN207 virus (serving as control). After 48 h of infection, red fluorescence confirmed efficient viral activity. Subsequent screening with puromycin revealed approximately 90% of the cultured cells to be positive for red fluorescence on reaching 90% confluence. Cell pellets were collected for quantitative PCR and western blot analyses, demonstrating significantly reduced expression levels of the YWHAB gene (CN207:1.010 ± 0.043; sh1007: 0.166 ± 0.045; sh1008: 0.343 ± 0.061) (mean ± SD, n = 6, p < 0.05) (Fig. 2A and B) and 14-3-3β protein in the RNAi knockdown group (CN207:0.992 ± 0.019; sh1007: 0.609 ± 0.059; sh1008: 0.735 ± 0.059) (mean ± SD, n = 6, p < 0.05) compared to the CN207 control group (Figs. 2C and D). Additionally, both target genes exhibited notably lower expressions in the sh1007 group compared to the sh1008 group, indicating the successful establishment of a KYSE-150 cell line featuring stable and diminished expression of 14-3-3β protein.
Fig. 2. Validation of knockdown efficiency of 14-3-3β expression in KYSE-150 cells. A: Lentiviral infection efficiency was observed under fluorescence microscopy (×100). B: RT-PCR was utilized to detect the down-regulation of the YWHAB gene. C: Western blot analysis was conducted to measure the expression of 14-3-3β protein. Lane 1: CN207 (KYSE-150 cells underwent infection with control viruses CN207); Lane 2: sh1007(KYSE-150 cells underwent infection with YWHAB-RNAi viruses sh1007); Lane 3: sh1008(KYSE-150 cells underwent infection with YWHAB-RNAi viruses sh1008). D: Quantitative analysis based on the results shown in Figure C. Data are presented as mean ± SD from six independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001.The underline below * indicates the comparison between the two groups
Effects of low 14-3-3β expression on cell proliferation, migration, and colony formation of the KYSE-150 cells
The wound healing assay results (Figs. 3A-B) indicated that, after 24 h, cell mobility was significantly reduced in the sh1007 group (63.475% ± 14.086%) of KYSE-150 cells compared to the control group (39.855% ± 5.800%) (mean ± SD, n = 4, P = 0.036, t = 2.686), whereas no notable difference was observed between the sh1008 group (69.915% ± 9.055%) (mean ± SD, n = 4, p > 0.05) and the control. After 48 h, both the sh1007 (49.700% ± 13.829%) and sh1008 groups (75.370% ± 8.956%) exhibited significantly lower cell mobility than the control group (98.863% ± 1.970%) (mean ± SD, n = 4, P < 0.01), with no significant disparity between the sh1007 and sh1008 groups. In the colony formation assay (Figs. 3C-D), the number of colonies in the RNAi knockdown group was markedly reduced compared to the control group (42.10% ± 5.142%), and notably, the sh1007 group (6.40% ± 1.53%) displayed significantly fewer colonies than the sh1008 group (31.40%±6.248%) (mean ± SD, n = 5, P < 0.0001, t = 7.773), consistent with diminished 14-3-3β expression. However, the CCK8 assay results (Fig. 3E) showed no statistically significant difference in cell viability between the RNAi knockdown groups and the control, suggesting that 14-3-3β knockdown inhibited the migration and proliferation of KYSE-150 cells without affecting cell viability.
Fig. 3. Effects of low expression of 14-3-3β on proliferation, migration, and colony formation in the KYSE-150 cells. A: Wound healing assay conducted in KYSE-150 cells. B: Quantitative analysis of the wound healing assay based on Figure A. Data are presented as mean ± SD from four independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001.The underline below * indicates the comparison between the two groups. C: Colony formation assay performed in KYSE-150 cells. D: Quantitative analysis results derived from Figure C. Data are presented as mean ± SD from five independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001.The underline below * indicates the comparison between the two groups E: CCK8 assay results obtained from KYSE-150 cells
Effect of low 14-3-3β expression on the expression of proliferation-related proteins of the KYSE-150 cells
To explore the specific molecular mechanism underlying the influence of 14-3-3β on ESCC proliferation and migration, the western blot assay was employed to analyze proliferation and apoptosis-related proteins. As depicted in Fig. 4, the findings revealed a notable reduction in p-AKT activity (CN207:1.027 ± 0.052; sh1007: 0.424 ± 0.109) in KYSE-150 cells with diminished 14-3-3β expression compared to the control group (mean ± SD, n = 3, P = 0.002, t = 7.036), while total AKT activity remained largely unchanged. Moreover, no significant alterations were observed in the expressions of apoptosis-related proteins, including BCL2, BAX, caspase3, and activated caspase9. The RT-PCR results showed that the mRNA expression of the Cyclin D1, was significantly decreased in the sh1007 group (0.510 ± 0.119, p < 0.01) and sh1008 (0.500 ± 0.76, p < 0.01) compared with the control group (0.983 ± 0.024). However, the gene related to cell invasion (MMP2), apoptosis (FOXO1), cell cycle (P21), and Wnt signaling (β-catenin) no significantly change (Supplementary Fig. 1). These results suggest that the biological effects of 14-3-3β on esophageal squamous cell carcinoma cells may be mediated by PI3K/AKT- Cyclin D1 signaling pathway.
Fig. 4. Effects of low expression of 14-3-3β on apoptosis-related proteins in the KYSE-150 cells. A: Western blot analysis was conducted to assess the expression of apoptosis-related proteins. B: Quantitative analysis was performed based on the results depicted in Figure A. Data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***P < 0.001.The underline below * indicates the comparison between the two groups
Discussion
Esophageal cancer remains a significant health concern in China, with varying incidence rates across regions. ESCC is more commonly observed than EAC [13]. The lack of clear symptoms and reliable early detection methods often lead to the diagnosis of ESCC at intermediate or advanced stages. Although there have been notable advancements in treatment modalities such as surgery, chemotherapy, radiotherapy, and targeted therapies, which have contributed to improved survival rates among patients with esophageal cancer, recurrence rates post-treatment, distant metastases, and limitations in available drugs and therapies following metastasis continue to significantly impact the overall survival rates [14]. Additionally, the molecular mechanisms driving ESCC progression remain poorly understood, highlighting the need for comprehensive investigation into the malignant proliferation and metastasis of ESCC. Identifying biological targets associated with these processes could facilitate early diagnosis, treatment, and prognosis in clinical settings.
The 14-3-3 family consists of highly conserved adaptor proteins found widely across eukaryotic cells. They play crucial roles in regulating various biological processes, including protein subcellular localization, phosphorylation, mitogenesis, cell cycle advancement, and apoptosis. This regulation is achieved through their interaction with specific Ser/Thr phosphorylation motifs present on target proteins [15]. Once bound to the target protein, 14-3-3 proteins exert their influence by modulating various aspects of the target protein’s function, including enzyme activity, stability, cellular localization, and interaction with other proteins [16]. The 14-3-3 protein family is known for its extensive interaction with numerous proteins, a list that continues to grow. Many studies have demonstrated the pivotal role of 14−3-3 proteins in cancer pathogenesis and metastasis. Specifically, aberrant expression of 14-3-3β has been linked to extrahepatic metastasis and poorer survival rates in patients with hepatocellular carcinoma [8, 17]. Moreover, knockdown of 14-3-3β has been shown to curb the proliferation and migration of osteosarcoma cells [6] as well as the proliferation of human glioblastoma cells [18]. Based on findings from previous studies, it is proposed that 14-3-3β may exert crucial functions in the aberrant proliferation of tumor cells, suggesting that novel therapeutic approaches or medications targeting 14-3-3β could hold promise for cancer treatment.
The results of this study demonstrated a noteworthy reduction in the migration rate and cell colony formation of ESCC cells following the downregulation of 14-3-3β. These effects were observed to be associated with the expression levels of 14-3-3β, implying that the suppression of 14-3-3β expression could attenuate the migration and proliferation of ESCC cells.
It is believed that 14-3-3 proteins interact with various signaling molecules, particularly kinases, facilitating their phosphorylation and shielding their phosphorylation sites from dephosphorylation. This process extends their activation duration, suggesting that 14-3-3 proteins have a significant role in serine/threonine kinase-mediated signaling pathways, such as PKC and AKT [19, 20]. The PI3K/AKT signaling pathway has been implicated in the progression of tumors across multiple cancer types, including ESCC [21, 22]. It is regarded as a crucial mutated oncogenic pathway in the development of ESCC [23]. The results of this study revealed that in KYSE-150 cells exhibiting reduced 14-3-3β expression, there was a notable decrease in p-AKT protein levels, while AKT protein expression remained unchanged. AKT serves as a pivotal mediator within the PI3K/AKT pathway, and its activation is closely associated with cancer progression [24, 25]. There is growing evidence indicating that 14-3-3 can influence numerous AKT target proteins, including BAD, TSC2, p27Kip1, YAP, GSK3, PRAS40, and LKB1. This sharing of targets occurs because of the similarity between the recognition motifs of AKT (RxRxxS/T) and 14-3-3 (RSxpS/TxP) [24]. As a result, AKT can create 14-3-3 binding sites on various target proteins. Blocking PI3K/Akt signaling can disrupt the interaction between 14-3-3 proteins and their various targets, thereby reducing their functionality [26].
14-3-3 proteins are involved in tumor progression in various types of cancer. In prostate cancer cells, 14-3-3β binds to BAD, a downstream target of AKT, thereby inhibiting apoptosis [27]. It has also been found in lung cancer that 14-3-3β inhibits BAD by suppressing its phosphorylation and blocking its entry into mitochondria, thereby inhibiting BAD apoptosis [28]; In hepatocellular carcinoma cells, 14-3-3β accumulates in the cytoplasm and leads to the upregulation of EMT factors through the MEK/ERK pathway, promoting the migration and proliferation abilities of cancer cells [29]. Some studies have also shown that 14-3-3β promotes the migration and invasion of human hepatocellular carcinoma cells by regulating the expression of MMP2 and MMP9 through the PI3K/AKT/NF-kB pathway [8]. In breast cancer, 14-3-3β mediates the “dual transformation” of TGF-β from a tumor suppressor factor to a metastasis-promoting factor, which can transform the TGF-β function that inhibits the development of precancerous cells into one that promotes the metastasis of breast cancer cells [30]. Some scholars have also found that 14-3-3β is regulated by IRX5, thereby inhibiting the migration and invasion of breast cancer cells [31].
This study found that knockdown of 14-3-3β significantly inhibited the migration and clone formation ability of KYSE-150 cells, but the cell viability (or short-term proliferation) detected by the CCK-8 method was not significantly affected. This seemingly contradictory result actually reveals that 14-3-3β may preferentially regulate specific programs related to tumor invasiveness and long-term adaptability, rather than fundamental, short-term cell survival and proliferation. Specifically, the colony formation assay assesses the ability of individual cells to continuously proliferate and form colonies over a relatively long period of time (typically 1–2 weeks), a process highly dependent on the cells’ anchor-dependent growth, resistance to lost-free apoptosis, and the ability to cope with initial low-density environmental stress. The CCK-8 experiment mainly reflects the overall metabolic activity or mitochondrial function of cells within a relatively short period (24–96 h) at a higher cell density, and is not sensitive enough to the detection of microenvironmental stress and long-term adaptability. Cyclin D1 is a core protein that drives the cell cycle process, and its expression is positively regulated by AKT signaling. We simultaneously observed a decrease in p-AKT level, down-regulation of Cyclin D1 transcription and impaired clone formation ability in 14-3-3β knockdown cells. These three constitute a coherent causal chain. This strongly indicates that 14-3-3β precisely ensures the adequate expression of Cyclin D1 by maintaining AKT activity, and ultimately promotes the proliferation and long-term survival of ESCC cells. Interestingly, the expression of the migration-related gene MMP2 was not affected, which may explain why there are differences in the sensitivity of cell migration and proliferation phenotypes to 14-3-3β knockdown, and also suggest that the output of this pathway is selective in different biological processes. The high incidence of ESCC is closely related to chronic esophageal inflammation caused by dietary habits (hot food/excessive drinking). This study found that the expression level of 14-3-3β in KYSE-150 (ESCC cells) was significantly higher than that in SHEE (normal esophageal epithelium), and it may respond to the upregulation of inflammatory signals such as IL-6/TNF-α [32]. This mechanism is manifested in liver cancer as 14-3-3η being regulated by HIF-1α to mediate sorafenib resistance [33]. However, it has not been reported in ESCC yet, highlighting disease specificity. Some studies have also found that Porphyromonas gingivalis promotes PD-L1 expression by down-regulating 14-3-3σ and inhibits the anti-tumor immunity of ESCC [34]. Although this study did not involve microorganisms, further research can detect whether 14-3-3β can respond to the inflammatory signals of the oral microbiota. It provides a new direction for the “microbiota-immune-tumor axis” research of ESCC. Based on the fact that baicalein can induce 14-3-3σ to inhibit the proliferation of esophageal adenocarcinoma cells [35], in the future, flavonoids specifically targeting the 14-3-3β ATP-binding domain can be designed to enhance the targeting of ESCC treatment.
Although this study demonstrated that reducing the levels of the 14-3-3β protein significantly inhibited cell migration (as shown in scratch assays) and clone formation, several limitations remain. First, while our loss-of-function (knockdown) experiments clearly established the essential role of 14-3-3β in cell migration, clone formation, and AKT activation, we were unable to successfully develop a stable 14-3-3β overexpression cell model to provide complementary gain-of-function evidence. This limitation restricts our ability to fully assess the phenotypic effects of elevated 14-3-3β expression. The primary cause appears to be technical challenges. Future research should aim to overcome these obstacles, for example by developing an inducible expression system or exploring alternative models to directly validate whether 14-3-3β overexpression is sufficient to enhance migratory and clonogenic capabilities. Such efforts would contribute to a more comprehensive understanding of the oncogenic role of 14−3-3β. Second, another notable limitation was the absence of an evaluation of the impact of 14-3-3β knockdown on cell invasion using Transwell assays, particularly those incorporating matrix gels. While the scratch assay clearly illustrated its inhibitory effect on two-dimensional migration, this does not necessarily reflect a corresponding inhibition of invasion. Invasion involves a more complex process of penetrating the basement membrane, which should be directly assessed in future studies using standard invasion assays, such as Matrigel-coated Transwell systems, once appropriate models are available. Although this study revealed that down-regulation of 14−3-3β expression would inhibit the level of p-AKT, it was not clear whether it interacted directly with AKT or whether it acted by regulating phosphatases such as PP2A. One possibility is the direct action model: 14-3-3β, as a typical phosphorylated serine/threonine-binding protein, directly binds to p-AKT, acting as a ‘molecular chaperone’, stabilizing its conformation and isolating it from phosphatases. Another possibility is the indirect effect model: 14-3-3β affects the phosphorylation homeostasis of AKT by regulating the upstream kinase network or inhibiting the activity of phosphatases (such as PP2A). These two models are not mutually exclusive, but their validation will point to completely different regulatory biology. Therefore, the core of the subsequent work will focus on clarifying the precise model of the interaction between 14-3-3β and AKT. We will verify their direct interaction through Co-IP. The binding dependence was determined by constructing AKT phosphorylation site mutants; And by detecting the changes in phosphatase activity and using small molecule probes, the direct stability and indirect regulatory effects are distinguished. These efforts will eventually map out a complete molecular map from 14-3-3β to AKT activation.
This study focuses on in-depth mechanism analysis of 14-3-3β in the KYSE-150 cell model. Although this is a key first step in clarifying new biological principles, it also has limitations. First, although our loss-of-function (knockdown) experiments clearly demonstrated the necessity of 14-3-3β for cell migration, clone formation and AKT activation, we failed to successfully construct a stable 14-3-3β overexpressing cell model to provide corresponding evidence of functional acquisition. This limits our comprehensive assessment of the impact of elevated 14-3-3β expression levels on cell phenotypes. Future research needs to overcome this technical obstacle, such as developing an inducible expression system or exploring other models to directly verify whether 14-3-3β overexpression is sufficient to drive stronger migration and clone formation capabilities, which will provide a more complete chain of evidence for the carcinogenic function of 14-3-3β. Another significant limitation of this study was the failure to evaluate the effect of 14-3-3β knockdown on cell invasion ability through Transwell assays, particularly the invasion assays containing matrix gels. Although the scratch test clearly demonstrated its inhibitory effect on two-dimensional migration, the inhibition of migration ability is not equivalent to the inhibition of invasion ability. Invasion involves a more complex process of penetrating the basement membrane, which requires direct verification through standard invasion experiments (such as Matrigel-coated Transwell) in the future after obtaining the appropriate model. Notably, due to the tumor heterogeneity of ESCC, the results of this study need to be verified in more ESCC cell lines with different genetic backgrounds and patient-derived organoids. Future research will directly expand on this and evaluate the therapeutic potential of targeting 14-3-3β in animal models, with the ultimate goal of establishing its clinical relevance as a prognostic marker or therapeutic target for ESCC. In addition, all the study findings were based on cell experiments and lacked support from clinical sample analysis and in vivo experiments. The key direction for the future is to collect multi-center clinical coops, analyze the correlation between 14-3-3β expression and TNM stage or patient prognosis (such as survival period, lymph node metastasis) through immunohistochemical analysis, and verify its function in vivo using animal models to promote it as a potential biomarker or therapeutic target. Furthermore, this study still lacks in vivo experimental evidence and failed to explore the influence of the tumor microenvironment. Future research will involve constructing animal models of ESCC transplanted tumors, verifying the function of 14-3-3β in vivo, and focusing on exploring its regulatory role in the tumor microenvironment (such as immune cell infiltration and stromal remodeling), in order to establish a complete regulatory axis from the intrinsic mechanisms of tumor cells to the external microenvironment. These efforts will help establish 14-3-3β as a potential therapeutic target for ESCC with both cell-autonomous and non-cell-autonomous functions.
Conclusions
Our research findings provide concrete evidence highlighting the significant role of 14-3-3β in driving the proliferation and invasion of ESCC. This study lays the groundwork for deeper exploration into the specific molecular mechanisms through which 14-3-3β influences the initiation and advancement of ESCC. Our results suggest that 14-3-3β may promote ESCC cell proliferation and migration by modulating the Akt signaling pathway. Consequently, integrating the assessment of both 14-3-3β and p-Akt could improve the accuracy of prognostic evaluation for patients with ESCC. However, the precise involvement of 14-3-3β in ESCC progression requires further elucidation, emphasizing the need for additional research to clarify the complex relationship between 14-3-3β and ESCC.
Supplementary Information
Supplementary Material 1.
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