Introduction

RACK1 belongs to the tryptophan-aspartate repeat (WD-40) protein family. It is a scaffold protein with several protein binding sites because its protein molecule comprises seven WD-40 motifs that collectively form a spatial structure resembling a seven-bladed propelleris [1]. Previous studies have identified that RACK1 is abnormally expressed in a variety of malignancies, including lung, breast, gastric, and oral squamous cell carcinoma [2,3,4,5]. Our recent study also confirmed that RACK1 is highly expressed in cervical cancer tissue. Thereafter, from the studies of cervical cancer, we have further discovered that by inhibiting miR-1275, RACK1 facilitates the expression and intracellular signaling of Galectin-1, causing cervical cancer to invade and undergo epithelial-mesenchymal transition. Additionally, the O-GlcNAc modification of RACK1 enhances its regulation of tumor-associated lymphatic vessel formation and cervical lymph node metastasis through Galectin-1 [2].

With the characteristics of a scaffold protein, RACK1 can participate in the biological processes such as intracellular signal transduction and microRNA maturation, thus regulating various biological functions of cells, such as growth, differentiation and migration. Multiple studies have shown that the expression of RACK1 is related to non-small cell lung cancer, breast cancer, esophageal squamous cell carcinoma and other tumors.

Cervical cancer has the fourth highest incidence and mortality rates among women worldwide, and the highest among female reproductive system tumors in China [6, 7]. Radiation surgery, radiotherapy, or chemotherapy can be used to treat patients with early or locally advanced cervical cancer [8]. However, a large number of cervical cancer patients are already at the progressive stage when they are diagnosed. The treatment options for such patients are limited, and usually, palliative treatment is the only option to prolong patient survival and maintain quality of life [9]. Nevertheless, the prognosis remains poor. Few studies have revealed a connection between RACK1 and the growth of cervical cancer cells. The specific regulatory mechanism has not been elucidated. As a result, more research into RACK1's molecular regulatory mechanisms in cervical cancer is required.

A newly discovered form of cell death, ferroptosis, is characterized by the iron-dependent accumulation of lipid peroxides [10]. The cellular morphological characteristics of ferroptosis are cell shrinkage and increased density of mitochondrial membrane, which are distinct from the other cell death processes like apoptosis, autophagy, and necrosis [11]. Ferroptosis can be influenced by several factors, among which SLC7A11, GPX4 and ACSL4 are key factors associated with ferroptosis [12, 13]. The amino acid transporter protein System Xc-, which is found on the cell membrane, is made up of the SLC7A11 and SLC3A2 subunits [14]. Extracellular cystine and intracellular glutamate are transported into and out of a cell by System Xc- antiporter. Upon cellular uptake, a reduction of cystine to cysteine results in glutathione synthesis. Furtherly, glutathione peroxidase 4 (GPX4) converts the reduced glutathione into an oxidized form while converting the lipid peroxides into non-toxic lipid alcohols, thereby inhibiting the onset of ferroptosis [15, 16]. Although SLC7A11 and GPX4 are closely related through GSH, their expression is not necessarily relevant in all cases [17]. ACSL4 catalyzes the acylation of lysophospholipids, facilitating the incorporation of polyunsaturated fatty acids into phospholipids and ferroptosis [18]. The presence of regulatory effect of RACK1 on ferroptosis in cervical cancer cells needs to be further investigated.

Abnormal protein glycosylation is closely related to tumor formation, progression, metastasis, and treatment [19]. Abnormal fucosylation is a type of glycosylation that has the closest relationship with malignant tumors [20]. Fucosylation is a modification process of attaching uridine diphosphate-fucose (GDP-fucose) to its receptor molecule that is catalyzed by fucosyltransferases (FUTs). Humans have 13 types of FUTs that catalyzeα(1,2)-, α(1,3/4)-, α(1,6)-, and O-fucosylations [21]. The α(1,2)- and α(1,3/4)-fucosylations occur at or near the end of N-glycan or O-glycan chains whereas O-fucosylation directly attaches GDP-fucose to protein Ser/Thr residues. On the other hand, at the starting point of the N-glycan chain, GDP-fucose is attached to N-acetylglucosamine residues by α(1,6)-fucosylation. Therefore, it is also known as core fucosylation [22]. The core fucosylation of AFP in hepatocellular carcinoma has become a highly sensitive and specific diagnostic indicator for liver cancer [23]. It is widely used in the clinical laboratory testing. FUT8 is the only glycosyltransferase that catalyzes core fucosylation. Numerous studies have reported significant upregulation of FUT8 expression in a variety of malignant tumors, such as breast cancer, colorectal cancer, prostate cancer, and hepatocellular carcinomas, which is closely related to the tumor characteristics and patient prognosis [24]. Nevertheless, the effects of the following two components, including the regulatory mechanism of RACK1 on FUT8 expression and the changes in core fucosylation on ferroptosis in cervical cancer need further exploration.

In summary, we are the first to find that upregulation of RACK1 expression enhanced FUT8 expression, which in turn affected the core fucosylation of cystine transporter protein SLC7A11. Elevated levels of core fucosylation enhanced the stability and expression of SLC7A11 protein, inhibiting ferroptosis of cervical cancer cells.

Material and method

Cells and reagents

The human cervical cancer cell lines HeLa and SiHa were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai,China), and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS,Gibco,USA) and 1% penicillin–streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Z-VAD-FMK (HY-16658B) and MG-132 (HY-13259) were purchased from MedChemExpress (Shanghai, China). Necrosulfonamide (S8251), Deferoxamine (S5724) and 3-Methyladenine (S2767), Erastin(S7242) were purchased from Selleck Chemical (Shanghai, China).

Colony formation assay

Seeded 1,000 cells/well in 6-well plates for the colony formation assay. After 5, 9, 14 days of incubation with appropriate media, colonies were fixed for 30 min with 4% paraformaldehyde and stained for 15 min with 0.5% Crystal Violet (Sigma). PBS was used to rinse the cells after removing the crystal violet. The colonies were allowed to dry in ambient air at room temperature. Colonies for each cell line were counted using ImageJ software.

Cell viability assay

The cell growth of SiHa and HeLa were measured by the Cell Counting Kit-8 (CCK-8,SolarBio,China). Cells were seeded into a 96-well plate at 3,000 cells/well. After treatments and incubation, CCK-8 reagent(10 μl) was added into each well and incubated for 2 h accordance with the manufacturer's instructions. The absorbance was measured at 450 nm.

BrdU incorporation assay

Transfected cells were seeded in 96-well plates at a density of 1 × 103 cells per well. Upon adherence, the cells were incubated at 37 °C for 24 h in a CO2 incubator after the addition of bromodeoxyuridine (BrdU) diluent according to the manufacturer's instructions. BrdU-positive cells were measured by flow cytometry.

Assessment of lipid peroxidation

Cells were seeded in six-well plates 1 × 105 cells/well and treated with ferroptosis inducers on the following day. After 24 h, cells were exposed to 1 mM Liperfluo (DojinDo, Japan) for 30 min at 37 °C and then harvested. The cells were re-suspended in 1 × PBS. The data were collected and analyzed using a flow cytometer and analyzed using FlowJo v10.8 software.

Assessment of ROS

Intracellular ROS was measured using a ROS Assay Kit (DojinDo, Japan) following the manufacturer's instructions. To begin, cells were seeded onto 6-well plates and treated for 30 min at 37 °C with the prepared 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA) solution. The cells were washed twice with HBSS before being digested with trypsin. Cell precipitates were collected by centrifugation and resuspended in HBSS. Finally, ROS levels were analyzed using a flow cytometer.

Assessment of malondialdehyde (MDA)

The MDA level was measured using the MDA Assay Kit (Dojindo, Japan) according to the manufacturer’s instructions. In short, the MDA in cells reacted with thiobarbituric acid (TBA) to form a colored MDA-TBA adduct, and the mixture was incubated for 15 min at 95 °C followed by ice-cooling for 10 min. The absorbance at 532 nm was measured.

Immunoprecipitation and western blot

The cells were lysed by an immunoprecipitation buffer (IP buffer) containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 15 mM MgCl2, and 5 mM EDTA, for the immunoprecipitation assay. After centrifugation, the supernatant was incubated overnight with Flag beads or indicator antibodies and protein A/G beads at 4 °C. Beads were rinsed thoroughly with immunoprecipitation lysis buffer before being boiled and centrifuged. For whole cell extracts, cells were lysed with RIPA buffer(Thermo,USA) separated by SDS–PAGE and transferred onto PVDF membranes. After blocking, membranes were treated with primary antibody overnight at 4 °C. The second day, membranes were incubated with horseradish peroxidase-linked secondary antibody at room temperature for 1 h. Visualize protein bands using ECL substrates (Thermo, USA).

RNA extraction, cDNA synthesis, and real-time polymerase chain reaction (PCR)

According to the manufacturer’s instructions, total RNA was extracted from the harvested cells using TRIzol Reagent (Invitrogen, USA), and cDNA was synthesized from 2 μg of total RNA using the PrimeScript RT reagent Kit (Takara, Japan). Real-time PCR was performed on the ABI StepOne Plus system (Applied Biosystems, USA) using the SYBR Green PCR Master Mix (Applied Biosystems, USA). The primers used to amplify the specifific genes were obtained from Sangon Biotech (China).

Label and capture nascent RNA

In accordance with the manufacturer's instructions, the Click-iT Nascent RNA Capture Kit (Invitrogen, USA) was performed to capture newly synthesized RNA. In brief, after incubating the cell culture for 4 h with 0.5 mM 5-ethyluridine (EU), total RNA tagged with EU was extracted using TRIzol reagent (Invitrogen, USA). The EU-labeled RNA was then biotinylated by biotin azide and finally, the biotinylated nascent RNA was pulled down using streptavidin magnetic beads.

miRNA analysis and transfections

TaqManTM MicroRNA Assays (Thermo, USA) were used to evaluate miRNA expression. RNU6B served as the endogenous control. Real-time PCR was performed by TaqMan® Universal PCR Master Mix II without UNG according to the manufacturer’s instructions. Hsa-miR-1275 mimics were purchased from Thermo Fisher Scientific, and transfected with Lipofectamine 3000 (Life Technologies, USA).

Plasmids

Previous research described the creation of a RACK1 knockdown cell line (shRACK1) [25]. Human RACK1 expression plasmids have previously been described [26]. Transfection experiments of plasmids applied Lipofectamine 3000 reagents(Life Technologies, USA) according to the manufacturer’s instructions.

Statistical analysis

All data are displayed as mean ± SD and were sourced from at least three independent replicates. The statistical differences were analyzed using Student’s t-test for two experimental groups and Analysis of variance (ANOVA) for three or more groups. P < 0.05 was considered statistically significant. Statistical analyses were carried out using Prism 10.0 (GraphPad) and SPSS 22.0 software (IBM, USA).

Results

RACK1 knockdown induced ferroptosis in cervical cancer cells

To investigate the effect of RACK1 on the growth of cervical cancer cells, two cervical cancer cell lines HeLa and SiHa with RACK1 knockdown were constructed. Colony formation assays indicated that RACK1 knockdown decreased cellular viability on days 5, 9, and 14 with the growth of tumor cells (Fig. 1A). To further analyze cell proliferation, BrdU incorporation assays were also performed on the corresponding days 5, 9, and 14. Results showed that proliferative capacity of both tumor cells were not affected by RACK1 down-regulation (Fig. 1B). Cervical cancer cells with RACK1 knockdown were treated with different kinds of death inhibitors, including necrosulfonamide, a necrosis inhibitor; Z-VAD-FMK, an apoptosis inhibitor; 3-methyladenine, an autophagy inhibitor; and deferoxamine (DFO), an iron-binding agent. Among them, DFO is a specific ferroptosis inhibitor that functions by chelating intracellular iron, thus preventing the iron-dependent lipid peroxidation characteristic of ferroptosis [27]. The results showed that only DFO significantly reversed the inhibitory effect of RACK1 silencing on cell viability, while the other inhibitors exhibited faint effects, suggesting that knockdown of RACK1 may lead to ferroptosis in cervical cancer cells (Fig. 1C). Additionally, we examined the effects of RACK1 on the biochemical processes of ferroptosis via accumulation of lipid peroxidation, reactive oxygen species (ROS) and malondialdehyde (MDA). Results showed that lipid peroxidation, ROS levels, and MDA were markedly increased in RACK1-downregulated cervical cancer cells (Fig. 1D, E, F). Together, these results suggest that RACK1 restricts ferroptosis in cervical cancer cells.

Fig. 1
figure 1

RACK1 knockdown induced ferroptosis in cervical cancer cells. A Colony formation assay of HeLa and SiHa cells with or without RACK1 knockdown, monitored on days 5, 9, and 14, to assess its influence on cell viability. B BrdU incorporation assay of HeLa and SiHa cells with or without RACK1 knockdown, monitored on days 5, 9, and 14, followed by anti-BrdU staining and FACS analysis. C HeLa and SiHa cells of RACK1 knockdown were treated with or without Z-VAD-FMK (20 μM), necrosulfonamide (1 μM), 3-Methyladenine (5 mM) or Deferoxamine (20 μM) for 48 h followed by cell viability assay. D The relative lipid peroxidation levels were assayed in HeLa and SiHa cells with or without RACK1 knockdown. E The relative ROS levels were assayed in HeLa and SiHa cells with or without RACK1 knockdown. F The relative MDA levels were assayed in HeLa and SiHa cells with or without RACK1 knockdown. **, P < 0.01, ***, P < 0.001, NS, no significance

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RACK1 promoted SLC7A11 protein expression in cervical cancer cells

To investigate the regulatory mechanism of RACK1 on ferroptosis in cervical cancer cells, we further analyzed the effect of RACK1 on the key regulator of ferroptosis. Western blot confirmed that knockdown of RACK1 had no effect on the expression of GPX4 and ACSL4, but downregulated the protein level of SLC7A11 (Fig. 2A). This suggested that while RACK1 regulates SLC7A11, GPX4 expression may be regulated by other pathways that are not regulated by RACK1. Similarly, ACSL4 expression may not be directly affected by RACK1, but rather by the entire cellular lipid composition and environment. Meanwhile, real-time PCR showed that there was no significant difference in the mRNA level of SLC7A11 when RACK1 was knocked down (Fig. 2B), suggesting the regulation of RACK1 on SLC7A11 expression might occur at translational or post-translational levels. Moreover, rescue of SLC7A11 expression could remarkably abolish the accumulation of lipid peroxidation, the inhibition of cell viability, and the increases in ROS and MDA levels induced by RACK1 knockdown (Fig. 2C, D, E, and F). Together, these data imply that RACK1 stimulates the expression of SLC7A11 and inhibits ferroptosis phenotype in cervical cancer.

Fig. 2
figure 2

RACK1 promoted SLC7A11 protein expression in cervical cancer cells. A Western blot of RACK1-knockdown HeLa and SiHa cell lysates to detect ferroptosis related proteins. B Effect of RACK1 knockdown on SLC7A11 mRNA level by real-time PCR. C, D, E, F The relative lipid peroxidation (C), cell viability (D), relative ROS (E), and relative MDA levels (F) were detected in HeLa and SiHa of RACK1 knockdown with or without exogenous SLC7A11 expression. **, P < 0.01, ***, P < 0.001, NS, no significance

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RACK1 enhanced the core fucosylation and protein stability of SLC7A11 in cervical cancer cells

To investigate the molecular mechanism underlying SLC7A11 expression regulation by RACK1, cycloheximide (CHX) pulse chase analysis was conducted. CHX is a potent inhibitor of protein synthesis, enabling the monitoring of degradation of existing proteins over time [28, 29]. Results showed that a significantly faster rate of protein degradation was observed for SLC7A11 in RACK1 knockdown cells (Fig. 3A), suggesting that RACK1 plays a key role in maintaining the stability of SLC7A11 protein. To investigate the pathway through which RACK1 affected the protein degradation of SLC7A11, we added the proteasome pathway inhibitor MG-132 and the lysosomal pathway inhibitor chloroquine to the cervical cancer cells [28, 29], respectively, followed by western blotting. Results demonstrated that MG-132 instead of chloroquine significantly reversed the RACK1 knockdown induced down-regulatory effect on SLC7A11 (Fig. 3B).

Fig. 3
figure 3

RACK1 enhanced the core fucosylation and protein stability of SLC7A11 in cervical cancer cells. A HeLa and SiHa cells of RACK1 knockdown were applied to cycloheximide chase analysis for SLC7A11 at indicated time points. B HeLa and SiHa cells with or without RACK1 knockdown were treated with 50 μM MG132 for 4 h or chroloquine for 12 h followed by SLC7A11 detection. C Endogenous SLC7A11 were immunoprecipitated from HeLa cells for lectin blotting. D SLC7A11 protein was detected in HeLa and SiHa of RACK1 knockdown with or without exogenous FUT8 expression

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To understand whether post-translational glycosylation was involved in RACK1-mediated SLC7A11 stabilization, SLC7A11 protein was immunoprecipitated and was further analyzed for multiple kinds of glycan structures through lectin blot. Lectins are a class of proteins with antigenic and agglutinating activities that specifically recognize complex carbohydrate structures in glycoproteins and glycolipids [30]. Among these, core fucosylation promotes the addition of α(1,6)-fucose to the innermost GlcNAc residue of N-glycans, and Lens Culinaris Agglutinin (LCA) has a recognized high affinity and specificity for core fucosylation [24, 31]. The staining of LCA, which recognizes the core fucose subunit, was decreased upon RACK1 downregulation (Fig. 3C), indicating a lower presence of fucose residues available for binding by LCA, which directly reflects the diminished core fucosylation on SLC7A11. As FUT8 is the only glycosyltransferase that catalyze synthesize of core fucosylation, FUT8 expression plasmids were transfected in tumor cells to explore whether core fucosylation was involved in SLC7A11 expression mediated by RACK1. Results showed that rescue of FUT8 abrogated the inhibition of shRACK1 on SLC7A11 protein expression (Fig. 3D). These results demonstrate RACK1 enhances protein stability and expression of SLC7A11 by promoting core fucosylation.

RACK1 regulated FUT8 and core fucosylation via miR-1275

Further studies were done to explore the regulatory mechanism of RACK1 on core fucosylation of SLC7A11. Real-time PCR analysis validated that RACK1 promoted the mRNA levels of FUT8 in both of cervical cancer cells (Fig. 4A). The regulation of mRNA levels includes both transcriptional and post-transcriptional levels, and detection of newly synthesized mRNA of FUT8 using the Click-iT Nascent RNA Capture Kit and real-time PCR showed that alteration of RACK1 expression induced no significant difference in nascent FUT8 RNA levels (Fig. 4A). This suggests that the regulation of FUT8 by RACK1 might occur at the post-transcriptional level rather than the transcriptional level. Since previous studies identified a role for RACK1 in regulating miRNA expression [32], we next investigated whether RACK1 could regulate FUT8 mRNA levels at the post-transcriptional stage through certain miRNAs. In previous research, we found that miR-1275 was regulated by RACK1 in cervical cancer during screening of four miRNA online databases and real-time PCR assay [2]. In this study, using miRWalk2.0 software, seven potential miRNAs, including miR-1275, were predicted to target the 3’-UTR of FUT8 mRNA with high probability (Fig. 4B). Further sequence mapping confirmed complementarity between FUT8 3’-UTR and miR-1275 (Fig. 4C). Additionally, transfection of miR-1275 mimics significantly abolished up-regulation of FUT8 induced by RACK1 overexpression (Fig. 4D and E). Moreover, transfection of miR-1275 mimics also significantly reduced the cell viability induced by RACK1 overexpression upon the treatment of erastin, a ferroptosis inducer (Fig. 4F). These results indicate that regulation of RACK1 on FUT8 expression is mediated by miR-1275.

Fig. 4
figure 4

RACK1 regulated FUT8 and core fucosylation via miR-1275. A Effect of RACK1 overexpression on FUT8 mRNA and newly synthesized FUT8 RNA levels by real-time PCR. B Venn diagram of overlapping predicted miRNAs from miRWalk2.0 including miRWalk, Starbase, and Targetscan databases. C Computer prediction of conserved binding sites within the 3’-UTR of FUT8 mRNA for the miR-1275. D, E FUT8 mRNA (D) and protein (E) were detected in HeLa and SiHa expressing RACK1 with or without miR-1275 mimics transfection. F HeLa and SiHa cells were transfected as indicated, and treated with increasing dose of Erastin for 24 h, followed by CCK-8 assay. *, P < 0.05, **, P < 0.01, ***, P < 0.001, NS, no significance

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Discussion

RACK1 is a typical scaffold protein, which is involved in various biological processes regulating tumorigenesis [1]. With the characteristics of a scaffold protein, RACK1 can participate in the biological processes such as intracellular signal transduction and microRNA maturation, thus regulating various biological functions of cells, such as growth, differentiation and migration [2]. Multiple studies have shown that the expression of RACK1 is related to non-small cell lung cancer, breast cancer, esophageal squamous cell carcinoma and other tumors [3, 4]. Our previous research also demonstrated that RACK1 promotes carcinogenesis and chemoresistance in hepatocellular carcinoma [25, 26], along with that RACK1 promotes cervical cancer cell invasion and EMT, as well as lymphangiogenesis and lymph node metastases in a galectin-1-dependent manner [2]. Ferroptosis is a novel regulatory and programmed cell death driven by iron dependent phospholipid peroxidation. It has exhibited promising efficacy in various anticancer treatments, such as radiotherapy, certain chemotherapy and immunotherapy [12]. However, research on the mechanism of ferroptosis in cervical cancer is still limited. Here, we report that RACK1 may promote the expression of FUT8 in cervical cancer cells by lowering the levels of miR-1275 and enhance the stability of SLC7A11 protein induced by core-fucosylation, thus inhibiting ferroptosis in cervical cancer cells.

During our experiments, we discovered that the viability of cervical cancer cell clones was significantly reduced after knockdown of RACK1 (Fig. 1A), suggesting that RACK1 plays a crucial role in promoting cell growth. However, the results of the BrdU incorporation assay (Fig. 1B) showed that RACK1 had no significant effect on cell proliferation. Since colony formation reflected the long-term survival and reproduction of cells, whereas the BrdU assay measures short-term DNA synthesis [33], one possibility is that RACK1 may not alter cell proliferation but affect other factors related to cell survival, such as resistance to cell death mechanisms, which was verified later that RACK1 facilitated ferroptosis resistance via SLC7A11 up-regulation. In addition, as a multifunctional scaffolding protein, RACK1 may affect cellular homeostasis, including interactions with the microenvironment, which may support cancer cell survival and resistance to death. This area, although not well understood, warrants further investigation.

In this study, we found that the increased expression of FUT8 was associated with the tumorigenic effect of RACK1 in cervical cancer cells. Despite the fact that RACK1's scaffold characteristic determines its interaction with multiple transcription factors and regulation of gene transcription [34], our results showed no changes in nascent mRNA level of FUT8 in cervical cancer cells overexpressing RACK1 (Fig. 4B), indicating that RACK1 may regulate FUT8 at a post-transcriptional stage rather than at a transcriptional level. In addition, RACK1 has been proposed to exhibit multiple functions in miRNA transcription, miRNA maturation, and miRNA loading into miRISCs [35,36,37]. Recently, we revealed that RACK1 can modulated a number of miRNAs in gastric cancer cells, with miRNA-302c being implicated in RACK1-mediated suppression of interleukin-8, demonstrating that RACK1 might modulate mRNA expression through specific miRNAs [32]. Our previous research showed that miRNA-1275 was negatively regulated by RACK1 in cervical cancer cells [2], and we further confirmed that miRNA-1275 can target FUT8 mRNA 3'-UTR(Fig. 4C). Although FUT8 mRNA was previously identified to be a target of miR-122 and miR-34a, which have been shown to cause a drop in FUT8 levels as well as impact core fucosylation of secreted proteins in hepatocarcinoma cells [38]. As a result, the mechanism of regulating FUT8 at the transcriptional level varies among different tumors.

As mentioned above, ferroptosis is driven by iron dependent phospholipid peroxidation. The antioxidant defense system that cells have developed can shield them from increased ROS production and oxidative stress, the most important of which is that reduced glutathione (GSH) reduces lipid peroxide with GPX4 (Glutathione peroxidase 4), and intracellular GSH synthesis relies on the extracellular intake of cysteine [39, 40]. The cystine/glutamate transporter system (also known as the Xc- system) on the cell membrane consists of the light chain subunit SLC7A11 (also known as xCT) and the heavy chain subunit SLC3A2, which are covalently linked by disulfide bonds. The primary functional component, SLC7A11, mediates the exchange of intracellular glutamine and extracellular cystine, and then cystine is converted into cysteine as the rate-limiting precursor for GSH biosynthesis [17, 41]. Based on a series of studies, high expression of SLC7A11 has been observed in various human malignancies tumors and is associated with poor survival [42,43,44]. The increased SLC7A11 can accelerate the uptake of cystine and the synthesis of GSH, thus protecting cancer cells from oxidative stress and ferroptosis. Consistent, the decreased SLC7A11 can result in the depletion of GSH and trigger ferroptosis, indicating that SLC7A11 acts as a key factor in modulating ferroptosis [17]. Exploring the precise mechanism of SLC7A11 regulation can help identify new therapeutic targets for inducing ferroptosis in cancer treatment.

As illustrated recently, SLC7A11 can be transcriptionally regulated by by ATF4 [41], NRF2 [45], ATF3 and mutant p53 [46]. Notablely, ATF4 and NRF2 interact with each other on the SLC7A11 promoter, and synergistically regulate the transcription of SLC7A11 under stress conditions [45]. Another interesting topic in SLC7A11 research is to reveal the critical role of epigenetic regulation of SLC7A11 transcription in governing ferroptosis. Earlier studies identified a dynamic equilibrium between H2A ubiquitination and deubiquitination by PRC1 and BAP1, which might be important for suppressing the expression of SLC7A11 [47]. Wang et al. linked H2Bub-mediated epigenetic mechanisms to transcriptional activation of SLC7A11 and ferroptosis repression [48]. It was shown that overexpression of KDM3B, a H3K9 demethylase, decreases H3K9 methylation and upregulates SLC7A11 expression, leading to enhanced resistance to ferroptosis [49]. As another example, ARID1A, binds on the SLC7A11 promoter and facilitates transcriptional activation of SLC7A11 through chromatin remodelling [50].

Although many studies have demonstrated the importance of transcriptional regulation by transcription factors and epigenetic regulators of SLC7A11 in ferroptosis, it is still unclear how the stability of SLC7A11 is regulated in human cancers. Interestingly, a recent study showed that overexpression of the cancer stem cell marker CD44 in human gastrointestinal tumor cells can enhance the stability of SLC7A1 by promoting the interaction between SLC7A11 and OTUB1. CD44, as a positive regulatory factor for SLC7A1, can promote the recruitment of OTUB1, leading to a decrease in the ubiquitination degree of SLC7A11, an extension of protein half-life, and stable protein levels, thereby reducing the susceptibility of cancer cells to oxidative stress and ferroptosis [51]. Our studies showed significant change of core-fucosylation for SLC7A11 in state of RACK1 enhancement, which was not reported before (Fig. 3C). SLC7A11 is the only membrane protein that maintains cellular redox homeostasis by uptake of cysteine [17], and this subcellular localization and structural property allows it to be modified by glycosylation, which may explain why SLC7A11, rather than other ferroptosis regulators, is specifically affected by RACK1.

Aberrant glycosylation is a prevalent characteristic observed in cancer cells and can affect tumour invasion, metastasis and immune evasion. A pivotal modification in tumor glycosylation is core fucosylation, a process exclusively mediated by fucosyltransferase 8 (FUT8), which facilitates the addition of α1,6-fucose to the innermost GlcNAc residue of N-glycans [24]. Core fucosylation encoded by FUT8 has been demonstrated to be upregulated in various cancers, such as lung, breast, liver, and pancreatic [31, 52,53,54]. In hepatocellular carcinoma (HCC), AFP is a gold standard biomarker for the diagnosis, but the specificity is relatively low. Increased fucosylation is a promising marker for monitoring the progression from chronic liver disease to HCC, and levels of FUT8 have been shown to be increased on the cell surface and in the serum s