PFKFB3 attenuates cisplatin-induced ferroptosis in gastric cancer via dephosphorylation of SLC7A11

doi:10.21203/rs.3.rs-3432510/v1

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PFKFB3 attenuates cisplatin-induced ferroptosis in gastric cancer via dephosphorylation of SLC7A11

https://doi.org/10.21203/rs.3.rs-3432510/v1

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6-phosphofructo-2-kinase (PFKFB3) is an isoenzyme of the PFKFB family, of which numerous studies have revealed the involvement in tumorigenesis and malignant behaviors in a non-glycolysis-dependent manner. Based on our findings of PFKFB3 in trastuzumab resistance, interestingly, we have found that PFKFB3 significantly attenuated cisplatin cytotoxicity both in vivo and in vitro. We demonstrated that overexpression of PFKFB3 markedly inhibited Erastin- and cisplatin-induced ferroptotic cell death. We further showed that Cystine/glutamate antiporter (xCT) interacts with the phosphatase domain of PFKFB3 and can be dephosphorylated at serine 26 (S26) by PFKFB3. The dephosphorylation of S26 greatly enhances xCT transporter activity, is critical for the production of GSH, and inhibits cisplatin-induced ferroptosis. Notably, erastin reversed PFKFB3-mediated resistance to cisplatinboth in vivo and in vitro. Collectively, our findings open a door to uncover how PFKFB3 promotes cisplatin resistance and may provide a potential target for gastric cancer treatment.

Health sciences/Biomarkers/Predictive markers

Biological sciences/Cancer/Gastrointestinal cancer/Gastric cancer

PFKFB3

gastric cancer

ferroptosis

cisplatin

Gastric cancer is responsible for more than one million new cases in 2020 and approximately 769,000 deaths. It remains one of the most prevalent cancers worldwide, ranking fifth for cancer incidence and fourth for mortality globally. [1] Effective and curative treatment for gastric cancer requires surgical or endoscopic resection with lymphadenectomy to an optimal extent. Meanwhile, preoperative or postoperative chemotherapy is important to achieve better surgical outcomes and is now the standard for managing patients with locally advanced gastric cancer in most countries.[2] Chemotherapy regimens for gastric cancer include ECF (epirubicin, cisplatin, and 5-fluorouracil), ECX (epirubicin, 5-fluorouracil, and capecitabine), and FLOT (5-fluorouracil, folinic acid, oxaliplatin, and docetaxel). However, cisplatin resistance has been a major problem as it compromises the efficacy of chemotherapy.[2]

Cisplatin, one of the best and first metal-based chemotherapeutic drugs, has been used for the treatment of various human solid cancers, including ovarian, testicular, bladder, head and neck, lung, cervical cancer, melanoma, lymphoma, etc.[3, 4] The mechanisms of its anticancer activity are associated with oxidative stress, DNA lesions, DNA repair, and cell cycle arrest. However, resistance to cisplatin is a significant and serious drawback that attenuates its efficacy and limits its clinical application [3–5]. Multiple pharmacokinetic, cellular, and molecular mechanisms underlying cisplatin resistance have been reported, including changes in the expression or modification of cell membrane transporters, endocytosis reduction, epigenetic modulation, microRNA involvement, microenvironment changes, accelerated DNA repair, and defects in signal transduction pathways that elicit apoptosis. [4, 6] However, an effective synergy that can reverse cisplatin resistance and enhance the anticancer activity of cisplatin remains to be explored.

Ferroptosis is a type of regulated cell death that can be triggered by, for instance, Erastin and RSL3, defined as iron-dependent cell death that occurs as lipid-based reactive oxygen species accumulate under conditions of impaired glutathione (GSH)-dependent lipid peroxide repair systems, with molecular mechanisms that are distinct from other forms of regulated cell death.[7, 8] Ferroptosis, as reported, is involved in the initiation and progression of various diseases such as neurodegeneration, ischemia-reperfusion injury, and cancer.[9] Emerging evidence has shown that ferroptosis activation has great potential for the treatment of cancers. The cystine/glutamate antiporter (xCT) and glutathione peroxidase (GPX4) are two critical molecules related to ferroptosis. xCT is a transmembrane transporter that transports cystine in exchange for glutamate, which is essential for GSH-dependent lipid peroxide repair systems and counteracts ferroptosis. GSH depletion by blocking cystine uptake resulting from targeting xCT renders cancer cells sensitive to chemotherapy and radiotherapy.[10–12]

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is a bifunctional enzyme that has both kinase and phosphatase activities, catalyzing both the synthesis and degradation of fructose-2,6-biphosphate. Numerous studies have revealed the involvement of PFKFB3 in tumorigenesis and malignancy via its interference with cancer metabolism.[13, 14] Our previous research found that PFKFB3 mediates trastuzumab resistance in gastric cancer by regulating vascular normalization in the tumor microenvironment through its kinase activity.[15] Hence, therapies targeting PFKFB3 or its downstream or upstream pathways may have great potential in cancer treatment.[16, 17] Although pharmaceutical options that serve the purpose are springing up, effective ones are still lacking.

In the present study, we found that PFKFB3 not only promotes gastric cancer malignancy, but also mediates cisplatin resistance by inhibiting ferroptosis. Our study also revealed that PFKFB3 dephosphorylates SLC7A11, the main functioning subunit of xCT, at serine 26, which has a ferroptosis-inhibiting effect. Furthermore, PFKFB3-induced cisplatin resistance, by inhibiting ferroptosis through SLC7A11 serine 26 (S26) dephosphorylation, can be reversed by Erastin, an agonist of ferroptosis, both in vitro and in vivo. These findings may provide potential targets for cancer treatment. (Fig. 6F)

PFKFB3 is up-regulated in GC tissues and correlates with the malignant phenotype of GC cells and cisplatin resistance

We conducted IHC staining of PFKFB3 and found an up-regulation of PFKFB3 staining intensity in gastric cancer tissues compared to that in normal tissues (Fig. 1A). Analysis of TCGA and GEO database yielded similar results (Fig. 1B and Fig.S1A, B). Survival analysis of TCGA and GSE22377 cohorts also revealed that a high level of PFKFB3 was significantly associated with reduced overall survival of patients with GC (Fig. 1C and Fig.S1C, D). These findings confirmed the clinical value of PFKFB3, which is consistent with the findings of previous studies.[15, 18, 19] To further evaluate the effect of PFKFB3 on gastric cancer cells, CCK8 assay and colony formation were performed to detect the proliferation of GC cells. The results showed that overexpression of PFKFB3 significantly increased the viability of MGC-803 and SGC-7901 cells, whereas knockdown of PFKFB3 decreased the viability of SNU216 cells (Fig. 1D, E and Fig.S1L). The colony formation assay showed consistent results with increased colony number and enlarged colony size induced by PFKFB3 overexpression, as well as fewer and smaller colonies resulting from PFKFB3 knockdown (Fig. 1F, G and Fig.S1E). However, in the scratch assay, no significant difference in wound healing was observed between the two groups of cells (Fig.S1F-H).

Based on our findings regarding PFKFB3 in trastuzumab resistance, we tested the effect of PFKFB3 on the sensitivity of gastric cancer cells to chemotherapy. Interestingly, the results showed that PFKFB3 significantly attenuated cisplatin cytotoxicity in vitro (Fig. 3A-D). A subcutaneous xenograft tumor model was established to further study whether PFKFB3 exhibits pro-tumor activity and reduces the efficacy of cisplatin in vivo. We found that tumors inoculated with PFKFB3-overexpressing GC cells grew faster and larger, whereas those inoculated with PFKFB3 knockdown showed the opposite effect. Tumors inoculated with PFKFB3-overexpressing GC cells were less responsive to cisplatin treatment (Fig. 1H, I and Fig.S1M). Gene sets enrichment analysis of the TCGA transcriptome profiles showed that the activation of cisplatin resistance signaling pathway were significantly different between PFKFB3-high and PFKFB3-low groups (Fig.S1I-K). These results confirmed the distinct pro-tumor effects of PFKFB3 and that PFKFB3 induces cisplatin resistance.

PFKFB3 expression is associated with the resistance to Erastin-induced ferroptosis

To determine the mechanism underlying PFKFB3’s pro-tumor activity, GSEA was used to elucidate the biological pathways associated with PFKFB3 expression in GC. The results showed that PFKFB3 was correlated with the ferroptosis signaling pathway (Fig. 2A). Then, to validate how PFKFB3 acts on ferroptosis, PI staining and CCK8 were performed and it turned out that PFKFB3 overexpression led to resistance to Erastin-induced ferroptotic cell death, similar to the recovery effect of ferrostatin-1 (Fig. 2B, C and Fig.S2A, B). Erastin treatment significantly induced cellular GSH depletion, MDA production, and ROS accumulation, which could be partly prevented by PFKFB3 overexpression or ferrostatin-1 (Fig. 2D-G). On the other hand, knockdown of PFKFB3 further enhanced the cytotoxicity of Erastin and decreased GSH levels, while further increasing MDA production and intracellular ROS levels, and these effects could be reversed by Ferrostatin-1 (Fig.S2A-F). Hence, a conclusion can be made that PFKFB3 expression is associated with the resistance to Erastin-induced ferroptosis.

As a well-known inducer of ferroptosis, Erastin blocks SLC7A11-mediated cysteine transport into the cells to initiate ferroptosis. Therefore, we measured the expression of SLC7A11 in GC cells with PFKFB3 overexpression or knockdown by qPCR and western blotting. However, no significant change in SLC7A11 expression were observed (Fig. 2H-J). Similarly, overexpression or knockdown of SLC7A11 had no significant effect on the expression of PFKFB3 (Fig.S2G-I).

PFKFB3 mediates cisplatin resistance of gastric cancer cells via its inhibition effect on cisplatin-induced ferroptosis

As previously mentioned, ferroptosis-inhibiting PFKFB3 is a critical inducer of cisplatin resistance in gastric cancer. Our experiments verified this assumption, demonstrating that overexpression of PFKFB3 significantly attenuated cisplatin cytotoxicity, whereas knockdown of PFKFB3 promoted cisplatin-induced cell death (Fig. 3A-D and Fig.S3A, B).

As ferroptosis is reported to be one of the crucial mechanisms for the effect of cisplatin[20], a chemotherapeutic drug of clinical importance, which was also confirmed in our experiments with down-regulated intracellular GSH and Cys levels and up-regulated MDA and lipid ROS levels in cisplatin-treated gastric cancer cells (Fig. 3E-H and Fig.S3D), we assumed that PFKFB3 induces cisplatin resistance via its inhibitory effect on cisplatin-induced ferroptosis. MGC-803 and SGC-7901 cells stably overexpressing PFKFB3 were treated with cisplatin, and intracellular GSH, MDA, Cys, and ROS levels were tested. Compared to the control group, GSH (Fig. 3M, N) and Cys (Fig. S3E, F) levels increased, whereas MDA (Fig. 3I, J) and ROS (Fig. 3Q, R) levels significantly decreased. Transmission electron microscopic images of PFKFB3 overexpressed MGC-803 gastric cancer cells also revealed less atrophic, less dense, and larger mitochondria(Fig.S3C). Similarly, we treated NCI-N87 and SNU-216 cell lines with PFKFB3 knockdown and cisplatin, and the results showed down-regulated GSH (Fig. 3O, P) and Cys (Fig.S3G, H) levels and up-regulated MDA (Fig. 3K, L) and ROS (Fig. 3S, T) levels. Therefore, PFKFB3 induces cisplatin resistance through its inhibitory effect on cisplatin-induced ferroptosis.

PFKFB3 regulates SLC7A11 serine 26 dephosphorylation

As mentioned above, PFKFB3 and SLC7A11 did not affect each other’s expression. Since kinase and phosphatase activities are the key components of PFKFB3 function, phosphoproteomics was performed using gastric cancer cells with PFKFB3 overexpression or knockdown. The results showed that PFKFB3 was closely associated with the dephosphorylation of SLC7A11 at serine 26 (Fig. 4A).

To verify whether PFKFB3 has a direct effect on SLC7A11, we first used immunofluorescence to determine if they colocalized in cells, and the result was in line with our speculation (Fig. 4G). We performed a series of co-immunoprecipitation experiments to further explore their direct interactions. First, we pulled down PFKFB3 in HEK-293T cell lysates and detected SLC7A11 in the co-immunoprecipitated proteins with PFKFB3 (Fig. 4B). Next, we pulled down SLC7A11 and analyzed the co-precipitated complexes, in which we observed the presence of PFKFB3 and a significantly down-regulated phosphorylation level of SLC7A11 in the cell extracts from PFKFB3-overexpressing HEK-293T cells compared to the empty vector (Fig. 4C). Next, to identify the critical domain of PFKFB3 for its effect on SLC7A11, we constructed HEK-293T cells overexpressing PFKFB3 (full-length)-GFP (hereafter referred to as PFKFB3(FL)-GFP), PFKFB3(1-245)-GFP, and PFKFB3(246–520)-GFP (Fig. 4D). We then pulled down SLC7A11, detected the presence of PFKFB3 using an anti-GFP antibody, and analyzed the phosphorylation levels of SLC7A11 in the extracts from the aforementioned cells. The results demonstrated that only PFKFB3(FL)-GFP and PFKFB3(246–520)-GFP could be co-precipitated with SLC7A11 (Fig. 4F), and only in these two cell lysates could the down-regulated phosphorylation level of SLC7A11 be detected (Fig. 4E). In conclusion, PFKFB3 directly dephosphorylates SLC7A11 at serine 26 through its 246–520 region.

PFKFB3 inhibits cisplatin-induced ferroptosis via SLC7A11 S26 dephosphorylation

Based on the previously reported significance of SLC7A11 in ferroptosis, we speculated that PFKFB3 inhibited cisplatin-induced ferroptosis by dephosphorylating SLC7A11 at serine 26 (S26). Therefore, we constructed SLC7A11 mutants with activated or inactivated serine 26, that is, by replacing serine with glutamic acid and alanine, respectively (later referred to as S26A and S26E, respectively) (Fig. 5A and Fig.S4A, B). In cells stably overexpressing PFKFB3 and co-expressing S26E mutants, the up-regulation of cisplatin-induced GSH levels by PFKFB3 and the down-regulation of ROS and MDA levels were reversed compared with the SLC7A11 wild type (hereafter referred to as WT) (Fig. 5C-E, J). The cytotoxicity of cisplatin attenuated by PFKFB3 was also enhanced (Fig. 5B and Fig.S4E). Cell lines expressing S26A with PFKFB3 knockdown were also constructed, in which S26A up-regulated the decreased GSH level while downregulating ROS and MDA levels and cell death compared with WT (Fig. 5F-I, K and Fig.S4F). Hence, PFKFB3 inhibits cisplatin-induced ferroptosis via SLC7A11 S26 dephosphorylation, which activates SLC7A11 and induces cisplatin resistance in gastric cancer cells.

Erastin rescues PFKFB3-induced cisplatin resistance of gastric cancer

Since Erastin can induce ferroptosis by inhibiting system \({xc}^{-}\),[21] we think it may block the activation of SLC7A11 by PFKFB3 and reverse this type of cisplatin resistance. The results of the in vitro and in vivo studies are consistent with this inference. When co-administered with Erastin, cisplatin regained its cytotoxicity and efficacy in inducing ferroptosis in GC cells overexpressing PFKFB3, as indicated by the CCK-8 assay and PI staining (Fig. 6A, B). This enhancement has also been observed in vivo. In the subcutaneous xenograft tumor model, when mice were treated with both cisplatin and Erastin, the tumors were smaller and grew more slowly than those treated with cisplatin alone. When additionally treated with Erastin, the tumors formed by PFKFB3-overexpressing GC cells were almost at the same volume as those formed by control cells. (Fig. 6C-E) Thus, the regulatory axis -- the inhibition of cisplatin-induced ferroptosis and the induction of cisplatin resistance by PFKFB3 through the phosphorylation modulation of SLC7A11 -- was identified.

In the present study, we demonstrated an unprecedented pathway that induces cisplatin resistance in gastric cancer, in which PFKFB3 dephosphorylates SLC7A11 at serine 26 and consequently inhibits cisplatin-induced ferroptosis, thereby attenuating cisplatin cytotoxicity.

The sensitivity of cancer cells to cisplatin is modulated in many ways, as each mechanism of cisplatin cytotoxicity also provides cells with a target for modification in self-defense. For instance, cisplatin binds to DNA and causes damage; in response, resistant cells develop an enhanced NER system, accelerating DNA repair to quickly remove lesions.[13] Recently, increasing evidence has revealed that cisplatin can induce ferroptosis, an important tumor-suppressing mechanism, in cancer cells.[20] However, in several types of cisplatin-resistant cancer cells, reduction of ferroptosis levels regulated by various pathways has been detected, and inducing ferroptosis or targeting ferroptosis-inhibiting pathways upon administration of cisplatin renders cancer cells more sensitive to cisplatin.[10, 20, 22–24]. In this study, we found that PFKFB3 not only exhibited tumor-promoting activity, but also inhibited cisplatin-induced ferroptosis, and thus induced cisplatin resistance by the detection of cell viability and GSH, MDA, and ROS levels under cisplatin treatment.

PFKFB3 is a bifunctional enzyme that catalyzes the synthesis and degradation of fructose-2,6-biphosphate. Compared to the other three isoenzymes (PFKFB1, 2, and 4), it had the highest kinase: phosphatase activity ratio, yielding a high glycolysis rate.[13] According to previous studies as well as ours, PFKFB3 is up-regulated in many human cancers or cells in the cancer microenvironment because of its glycolytic activity and intense involvement in cancer metabolism, shifting from restricted to high glucose uptake and increased glycolysis to meet the demand for biosynthesis for rapid and uncontrolled proliferation.[15, 25, 26] It has also been reported as a considerable inducer of cisplatin resistance in some other cancers, and this type of cisplatin resistance can be reversed by targeting PFKFB3 (for example, using 3PO, a PFKFB3 inhibitor), knocking PFKFB3 down, or targeting the upstream or downstream of PFKFB3.[15, 27] Herein, we propose a novel mechanism by which PFKFB3 induces cisplatin resistance in addition to the acknowledged mechanisms, such as inhibition of cisplatin-induced apoptosis or enhanced glycolysis; that is, PFKFB3 inhibits cisplatin-induced ferroptosis via dephosphorylation of SLC7A11 at serine 26.

SLC7A11, one of the key molecules involved in ferroptosis, is the main functional subunit of xCT, a cystine/glutamate antiporter that exports intracellular glutamate and imports extracellular cystine at a 1:1 ratio. In this process, cystine serves as both an antioxidant and precursor of many other antioxidants, directly affecting ferroptosis.[28] Many factors have been found to regulate ferroptosis through modulating the activity or expression, or even both, of SLC7A11 [10, 29], in which we found PFKFB3 also plays a significant part. A common and important regulator of SLC7A11 is the modulation of its phosphorylation level, which governs the activation of SLC7A11 and controls its transport activity. Previous studies have reported a few potential phosphorylation sites in SLC7A11, including S26, S51, S261, S481, T45, and T364, of which S26 has been proven to be critical and significant.[30] It has been reported previously and further confirmed in our study that phosphorylation-resistant mutation of S26 increases, while mutation of SLC7A11, in which S26 is constantly phosphorylated, fails to sustain cystine uptake. A study demonstrated that mTORC2, an intrinsic negative regulator of SLC7A11, inhibits the activity of SLC7A11 by phosphorylating it at S26.[30] We found in our study, we found that PFKFB3 directly dephosphorylates SLC7A11 at serine 26, and in this way, activates SLC7A11, augments intracellular cystine, buffers ROS, diminishes ferroptosis, and consequently attenuates cisplatin efficacy, resulting in cisplatin resistance. This is unanticipated because, in the present study, the effects of PFKFB3 on tumorigenesis, tumor progression, and aggressiveness rely on its activity in glycolysis and PPP, where it usually modulates the phosphorylation level of carbohydrates, resulting in carbohydrate metabolism reprogramming.[13, 25–27] However, we found that PFKFB3 altered metabolism not through its glycolytic activity, but through its kinase activity, which activated an amino acid transporting protein to alter intracellular amino acid metabolism.

Having clarified the mechanism underlying PFKFB3-induced cisplatin resistance, we sought to reverse this by targeting this pathway. A small molecule compound named Erastin, which targets the voltage-dependent anion channel (VDAC), p53, the cystine-glutamate transport receptor (system \({xc}^{-}\)), and others, can induce ferroptosis, showing great potential in cancer treatment. It has been shown that Erastin can enhance the sensitivity of several cancer cells to chemotherapy and radiotherapy.[21] In our study, we applied Erastin to ferroptosis-inhibited and cisplatin-resistant systems induced by PFKFB3 and found that Erastin could significantly promote ferroptosis, and gastric cancer cells regained sensitivity to cisplatin under Erastin treatment, both in vitro and in vivo. This provides further evidence supporting the clinical application of Erastin as a chemosensitizing agent.

In conclusion, our study not only revealed that PFKFB3 could directly dephosphorylate SLC7A11 to promote its activity in countering ferroptosis but also identified the PFKFB3/SLC7A11 axis as a major culprit responsible for cisplatin resistance in gastric cancer. Fortunately, we found that this effect can be reversed by Erastin. With the increasing number of studies, the prospect of sensitization of cisplatin in cisplatin-resistant gastric cancer and the clinical application of Erastin in chemotherapy has become apparent.

Patient tissue specimens

Primary tumor tissues and adjacent normal tissues from 18 patients with gastric cancer were obtained from Nanfang Hospital, Southern Medical University. Ethics approval and prior patient consent were obtained from the Institutional Research Ethics Committee to use the clinical specimens for research purposes. The study conformed to the principles set out in the Declaration of Helsinki.

Cell lines

The human gastric cancer cell lines SGC-7901, MGC-803, SNU-216, and NCI-807 were obtained from the American Type Culture Collection (ATCC). Cell lines were authenticated by short tandem repeat (STR) fingerprinting. Cells propagated in the recommended medium. All cells were maintained in 5% CO2 at 37°C.

Immunohistochemistry (IHC)

In summary, the sections were dewaxed in xylene, gradient ethanol, and PBS and boiled in sodium citrate buffer for antigen retrieval. Endogenous peroxidase blockers (ZSGB-BIO, Shanghai, China) were used to inactivate endogenous peroxidase, followed by 10% goat serum (ZSGB-BIO, Shanghai, China) to block nonspecific binding sites for antibodies. Then, the sections were incubated with primary antibodies against xCT (1:100, Affinity) and PFKFB3 (1:250, Proteintech) at 4 ℃ overnight. On the second day, the sections were incubated with anti-rabbit secondary antibodies (ZSGB-BIO, Shanghai, China) and stained using a DAB kit (Beyotime, Shanghai, China). Finally, the staining intensity of all sections was observed, scored, and recorded under a microscope by two investigators in a double-blind manner.

Cell viability determination

The cells were seeded in 96-well plate at 5000 cells/well after cell counting. To track proliferation, cell numbers were measured using the CCK-8 assay (Yeason, Shanghai, China) at 0, 24, 48, 72, 96, and 120 h after adherence. To detect cell viability after treatment with different agents, cells were treated with the indicated drugs after adherence, and cell numbers were measured by the CCK-8 assay (Yeason, Shanghai, China) 24 h after drug treatment.

The CCK-8 assay was performed using the following procedure: First, CCK-8 (Yeason, Shanghai, China) was diluted with DEME. The medium was then replaced by the diluted CCK-8 solution, and the system was incubated in a cell culture environment for 2 h. Finally, absorbance at a wavelength of 570 nm was measured using a BioTek® 800™ TS Absorbance Reader.

Colony formation assay

Cells were seeded in 6-well plates at 1000 cells/well after cell counting and cultured in a cell culture environment for 10 days. Each well was gently washed with PBS, followed by incubation with 1 mL 4% paraformaldehyde for 15 min for fixation. Next, the colonies were stained with 1 mL of 5% crystal violet solution for 10–20 minutes. The plates were washed with running water and dried. Finally, the colonies were photographed macroscopically and microscopically.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted using the RNA-Quick Purification Kit (ESscience, Shanghai), and reverse transcription was performed using the Hifair® III 1st Strand cDNA Synthesis SuperMix (Yeasen, Shanghai, China) following the manufacturer’s instructions. Real-time PCR was conducted using Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) in QuantStudio 5. Each reaction was performed in duplicates. Fixed threshold settings were used to determine the cycle threshold (CT) data and the mean CT was determined from duplicate PCRs. A comparative CT method was used to compare each group with the control group. All mRNA levels were normalized to those of β-actin. The relative gene expression normalized to that of the control was measured using Eq. 2-ΔCT, in which ΔCT = CT gene – CT control.

Western blotting

PFKFB3, xCT, GPX4, and xCT phosphorylation levels were assessed by western blotting. Cells were collected and lysed using RIPA lysis buffer (Fdbio Science, Zhejiang, China) containing 1% protease inhibitor and 1% phosphatase inhibitors (Beyotime, Shanghai, China). Then, lysates were centrifuged at 12000r/min at 4 ℃ for 30 min and the supernatants were collected. Protein concentrations were quantified using BCA Protein Assay Reagent (Beyotime, Shanghai, China), and the protein was denatured by boiling in Loading Buffer(Fdbio Science, Zhejiang, China) at 100 ℃ for 10 min. Equal amounts of proteins(30–50 µg) were loaded and separated on a polyacrylamide gel(One-Step PAGE Gel Fast Preparation Kit (10%), Vazyme) and transferred to PVDF membranes(Merck, Germany). The PVDF membranes were then blocked with PBS-5% fat-free milk at room temperature for 1 h and then incubated with primary antibodies against xCT(1:500, Affinity), PFKFB3(1:500, Proteintech), GFP(1:500, Proteintech), and GPX4(1:500, Affinity) at 4 ℃ overnight. The next day, the PVDF membranes were incubated with secondary antibodies(1:5000, Abbkine) at room temperature for 1 h. Finally, the protein quantity was detected using Tanon 5200 Multi.

Detection of intracellular lipid-ROS level

The detection of intracellular lipid-ROS level was performed following instructions. Cells were seeded in 60 mm culture dishes. At the time of detection, cells were collected in 1.5 mL EP tubes and washed twice with PBS. The cells were then incubated with the ROS probe 2-7-dichlorodihydrofluorescein diacetate(10 µM) in the dark in a cell culturing environment for 30 min with constant shaking at an interval of 5 min. The cells were then washed twice with PBS and resuspended. The fluorescence that labels the ROS level was determined using Beckman CytoFLEX.

Animal experiments

BALB/C nude mice (6-week-old) were obtained from the Laboratory Animal Unit, Southern Medical University, China, and kept in a specialized animal facility with access to food and water. Cells were inoculated subcutaneously, and the volume of the tumors was measured daily. By the time the tumor volume reached 100–300 mm², the mice were treated with the indicated drugs, and the tumor volume was tracked. After two weeks, the mice were euthanized by spinal cord transection, and tumor weight were measured. All experimental procedures were approved by the Institutional Review Board of the Nanfang Hospital of Southern Medical University and were performed following the Declaration of Helsinki.

Statistical analyses

All statistical analyses were performed using GraphPad Prisms 8 (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as the mean of three independent samples ± standard deviation of the mean (SD). Unless otherwise stated, the variance between groups being statistically compared was similar. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

AUTHOR CONTRIBUTIONS

Methodology: ZY Lian, K Yang, CY Sui; Formal analysis: ZK He, JN Wu, ZY Yan; Investigation: PH Zhang, XX Yao; Writing—original draft preparation: JL Shi, ZK He, ZY Lian; Writing—review and editing: HJ Deng, GX Li, J Yu.

Disclosure of Potential Conflicts of Interest

The authors declare no potential conflicts of interest.

FUNDING

This work was supported by grants from the National Natural Science Foundation of China (No.82003289), the Guangdong Provincial Key Laboratory of Precision Medicine for Gastrointestinal Cancer (2020B121201004), the Natural Science Foundation of Guangdong Province (2021A1515011721, 2022A1515010657, 2022A1515012629, 2022A1515111182), the Presidential Foundation of Nanfang Hospital, Southern Medical University (2021B006).

DATA AVAILABILITY

All relevant data are available to any researcher from the authors for non-commercial purposes without breaching participant confidentiality.

COMPETING INTERESTS

There are no competing financial interests in relation to the work described.

Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians 2021; 71: 209-249.
Smyth EC, Nilsson M, Grabsch HI, van Grieken NC, Lordick F. Gastric cancer. Lancet (London, England) 2020; 396: 635-648.
Ghosh S. Cisplatin: The first metal based anticancer drug. Bioorganic chemistry 2019; 88: 102925.
Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O et al. Molecular mechanisms of cisplatin resistance. Oncogene 2012; 31: 1869-1883.
Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. European journal of pharmacology 2014; 740: 364-378.
Shen DW, Pouliot LM, Hall MD, Gottesman MM. Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes. Pharmacological reviews 2012; 64: 706-721.
Xu T, Ding W, Ji X, Ao X, Liu Y, Yu W et al. Molecular mechanisms of ferroptosis and its role in cancer therapy. Journal of cellular and molecular medicine 2019; 23: 4900-4912.
Mou Y, Wang J, Wu J, He D, Zhang C, Duan C et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancer. Journal of hematology & oncology 2019; 12: 34.
Hirschhorn T, Stockwell BR. The development of the concept of ferroptosis. Free radical biology & medicine 2019; 133: 130-143.
Roh JL, Kim EH, Jang HJ, Park JY, Shin D. Induction of ferroptotic cell death for overcoming cisplatin resistance of head and neck cancer. Cancer letters 2016; 381: 96-103.
Wang Z, Ding Y, Wang X, Lu S, Wang C, He C et al. Pseudolaric acid B triggers ferroptosis in glioma cells via activation of Nox4 and inhibition of xCT. Cancer letters 2018; 428: 21-33.
Magri J, Gasparetto A, Conti L, Calautti E, Cossu C, Ruiu R et al. Tumor-Associated Antigen xCT and Mutant-p53 as Molecular Targets for New Combinatorial Antitumor Strategies. Cells 2021; 10.
Shi L, Pan H, Liu Z, Xie J, Han W. Roles of PFKFB3 in cancer. Signal transduction and targeted therapy 2017; 2: 17044.
Kotowski K, Rosik J, Machaj F, Supplitt S, Wiczew D, Jabłońska K et al. Role of PFKFB3 and PFKFB4 in Cancer: Genetic Basis, Impact on Disease Development/Progression, and Potential as Therapeutic Targets. Cancers 2021; 13.
Yao X, He Z, Qin C, Zhang P, Sui C, Deng X et al. Inhibition of PFKFB3 in HER2-positive gastric cancer improves sensitivity to trastuzumab by inducing tumour vessel normalisation. British journal of cancer 2022; 127: 811-823.
Wang Y, Qu C, Liu T, Wang C. PFKFB3 inhibitors as potential anticancer agents: Mechanisms of action, current developments, and structure-activity relationships. European journal of medicinal chemistry 2020; 203: 112612.
Bartrons R, Rodríguez-García A, Simon-Molas H, Castaño E, Manzano A, Navarro-Sabaté À. The potential utility of PFKFB3 as a therapeutic target. Expert opinion on therapeutic targets 2018; 22: 659-674.
Peng F, Li Q, Sun JY, Luo Y, Chen M, Bao Y. PFKFB3 is involved in breast cancer proliferation, migration, invasion and angiogenesis. International journal of oncology 2018; 52: 945-954.
Jiang YX, Siu MKY, Wang JJ, Leung THY, Chan DW, Cheung ANY et al. PFKFB3 Regulates Chemoresistance, Metastasis and Stemness via IAP Proteins and the NF-κB Signaling Pathway in Ovarian Cancer. Frontiers in oncology 2022; 12: 748403.
Guo J, Xu B, Han Q, Zhou H, Xia Y, Gong C et al. Ferroptosis: A Novel Anti-tumor Action for Cisplatin. Cancer research and treatment 2018; 50: 445-460.
Zhao Y, Li Y, Zhang R, Wang F, Wang T, Jiao Y. The Role of Erastin in Ferroptosis and Its Prospects in Cancer Therapy. OncoTargets and therapy 2020; 13: 5429-5441.
Fu D, Wang C, Yu L, Yu R. Induction of ferroptosis by ATF3 elevation alleviates cisplatin resistance in gastric cancer by restraining Nrf2/Keap1/xCT signaling. Cellular & molecular biology letters 2021; 26: 26.
Li H, Yang P, Wang J, Zhang J, Ma Q, Jiang Y et al. HLF regulates ferroptosis, development and chemoresistance of triple-negative breast cancer by activating tumor cell-macrophage crosstalk. Journal of hematology & oncology 2022; 15: 2.
Bi G, Liang J, Zhao M, Zhang H, Jin X, Lu T et al. miR-6077 promotes cisplatin/pemetrexed resistance in lung adenocarcinoma via CDKN1A/cell cycle arrest and KEAP1/ferroptosis pathways. Molecular therapy Nucleic acids 2022; 28: 366-386.
Cantelmo AR, Conradi LC, Brajic A, Goveia J, Kalucka J, Pircher A et al. Inhibition of the Glycolytic Activator PFKFB3 in Endothelium Induces Tumor Vessel Normalization, Impairs Metastasis, and Improves Chemotherapy. Cancer cell 2016; 30: 968-985.
Xintaropoulou C, Ward C, Wise A, Queckborner S, Turnbull A, Michie CO et al. Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment. BMC cancer 2018; 18: 636.
Li FL, Liu JP, Bao RX, Yan G, Feng X, Xu YP et al. Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis. Nature communications 2018; 9: 508.
Koppula P, Zhuang L, Gan B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein & cell 2021; 12: 599-620.
Kim EH, Shin D, Lee J, Jung AR, Roh JL. CISD2 inhibition overcomes resistance to sulfasalazine-induced ferroptotic cell death in head and neck cancer. Cancer letters 2018; 432: 180-190.
Gu Y, Albuquerque CP, Braas D, Zhang W, Villa GR, Bi J et al. mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT. Molecular cell 2017; 67: 128-138.e127.

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PFKFB3 attenuates cisplatin-induced ferroptosis in gastric cancer via dephosphorylation of SLC7A11

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Abstract

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INTRODUCTION

RESULT

PFKFB3 expression is associated with the resistance to Erastin-induced ferroptosis

PFKFB3 mediates cisplatin resistance of gastric cancer cells via its inhibition effect on cisplatin-induced ferroptosis

PFKFB3 regulates SLC7A11 serine 26 dephosphorylation

PFKFB3 inhibits cisplatin-induced ferroptosis via SLC7A11 S26 dephosphorylation

Erastin rescues PFKFB3-induced cisplatin resistance of gastric cancer

DISCUSSION

MATERIALS AND METHODS

Patient tissue specimens

Cell lines

Immunohistochemistry (IHC)

Cell viability determination

Colony formation assay

RNA extraction and quantitative real-time PCR (qRT-PCR)

Western blotting

Detection of intracellular lipid-ROS level

Animal experiments

Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

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