Ferroptosis is a newly-discovered cell death mechanism involved in the progression of various tumors, the role of noncoding RNAs (ncRNAs) in it was relatively less explored. This study identified the low levels of a recently studied long noncoding RNA (lncRNA), A2M-AS1, in pancreatic cancer and suggested its positive correlation with the overall survival time of patients with pancreatic cancer. A2M-AS1 was mainly localized in the cytoplasm, inhibiting the cellular proliferation, migration, and invasion as well as the tumor growth of the pancreatic cancer cells. Moreover, the Erastin-induced ferroptosis increased the expression levels of A2M-AS1. The overexpression of A2M-AS1 promoted ferroptosis in the pancreatic cancer, which was inhibited by the silencing of A2M-AS1. Mechanically, A2M-AS1 could directly interact with the poly (rC) binding protein 3 (PCBP3), which plays an important role in the process of iron metabolism, thereby promoting the ferroptosis in pancreatic cancer. In addition, the A2M-AS1/PCBP3 axis could facilitate the p38 activation and inhibit the phosphorylation of the AKT–mTOR signaling pathway; all these participate in regulating ferroptosis. In conclusion, the regulation of ferroptosis by targeting the A2M-AS1/PCBP3 axis might provide a novel target for the treatment of pancreatic cancer in the future.
A2M-AS1 might be a potential novel therapeutic target for patients with pancreatic cancer in the future.
Pancreatic cancer is a common malignant tumor with a poor prognosis. According to Cancer Statistics 2021, pancreatic cancer is the fourth leading cause of cancer deaths with the lowest 5-year survival rate of about 10% (1). To date, surgical resection remains the only treatment option to cure pancreatic cancer, however, most patients with pancreatic cancer often lose this opportunity due to the early metastasis (2). The advanced pancreatic cancer therapies mainly rely on gemcitabine chemotherapy, but the patients often develop resistance, resulting in unsatisfactory treatment effects. Therefore, finding novel therapeutic options to improve the pancreatic cancer treatment is of utmost importance and significance.
Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs (ncRNAs), having a nucleotide length of >200 bases and no protein-coding potential. Although the studies on lncRNAs are limited, they have a vital role in regulating various cancer types, including hepatic, lung, breast, and colorectal cancers (3–6). However, their role in pancreatic cancer has rarely been explored. Alpha-2-macroglobulin-antisense 1 (A2M-AS1), a recently-studied lncRNA, was reported to be significantly associated with the survival time of patients with pancreatic cancer (7). Meanwhile, A2M-AS1 might lessen the cardiomyocyte injury caused by hypoxia/reoxygenation (8), which are associated with ferroptosis (9, 10). Moreover, A2M-AS1 was found to be related to the ferroptosis-related genes in gastric cancer (11). Ferroptosis is a recently discovered cell death mechanism that is significantly different from traditional cell death processes like necrosis and apoptosis. Numerous studies have proved that ferroptosis is involved in inflammation, nervous system diseases, and the occurrence and development of cancers, including pancreatic cancer (12–15). However, whether A2M-AS1 is involved in the process of ferroptosis in pancreatic cancer remains unknown.
This study investigated the possible role of A2M-AS1 in pancreatic cancer as well as the mechanism involved. A2M-AS1 had low expression in pancreatic cancer and inhibited the proliferation, invasion, metastasis, and tumor growth of the pancreatic cancer cells. Moreover, A2M-AS1 could also promote ferroptosis in the pancreatic cancer cells probably by interacting with poly (rC) binding protein 3 (PCBP3). Together, the results might provide a novel idea for the treatment of pancreatic cancer.
Materials and Methods
Human pancreatic cancer tissue microarray, containing 80 samples of pancreatic cancer tissues and para-cancerous tissues and 20 samples of pancreatic cancer tissues, was purchased from Shanghai Outdo Biotech Company. All the specimens were obtained by surgical resection and diagnosed pathologically as pancreatic cancer. The basic clinicopathologic data of the patients with pancreatic cancer, whose tissue samples were used for the establishment of tissue microarray, are provided in Supplementary Table S1.
The cell lines, including PANC-1 (RRID: CVCL_0480), BxPC-3 (RRID: CVCL_0186), AsPC-1 (RRID: CVCL_0152), and HPDE (RRID: CVCL_4376), were obtained from the Cell Bank of the Chinese Academy of Sciences. Cell line authentication was conducted through STR analysis and cells with a passage number smaller than 20 were used for experiments. The cell cultures were grown in a complete growth medium, containing 10% FBS and 1% penicillin/streptomycin in an incubator with 5% CO2 at 37°C. All the cell cultures were tested regularly every 3 months for mycoplasma contamination. All these reagents were purchased from Gibco.
In situ hybridization
To quantify the expression level of A2M-AS1 in pancreatic cancer tissues, ISH was performed using the double digoxigenin-labeled probes (Outdo Biotech) following the manufacturer's instruction. Briefly, the pancreatic cancer tissues were dried, deparaffinized, and then treated with 5 μg/mL proteinase-K at 37°C. Then, the samples were incubated with the probes at 50°C for 1 hour, followed by incubation with an anti-digoxigenin antibody at 4°C overnight. The probe sequence was 5′-GTTCTTAGTTTATACAATCCATACTCACCTTCTGCCTAAC-3′. The color intensities and scales of the dye were evaluated with scores, ranging from 0 to 3. The scores of 3, 2, 1, and 0 showed strong, medium, weak, and negative results, respectively. Similarly, the scale of dye scores showed 76% to 100%, 51% to 75%, 26% to 50%, and 0% to 25% results, respectively. The scoring was performed independently by three pathologists to obtain the mean scores. The final staining index was calculated by multiplying the two scores.
A2M-AS1 overexpressing lentivirus, PCBP3 overexpressing and silencing lentiviruses and their corresponding negative controls were constructed by the GeneChem Company, whereas the A2M-AS1 silencing lentivirus and its control were constructed by the GenePharma Company. The lentiviral transfection was performed following the instructions of the corresponding manufacturers. PANC-1 or BxPC-3 were selected for 2 weeks using puromycin (2 μg/mL) to obtain the cells stably overexpressing or silencing A2M-AS1. To obtain the cells with stable PCBP3 expression, the PANC-1 and BxPC-3 cells were screened for 4 weeks with 900 or 600 μg/mL G418. The sequences of silencing lentiviral vectors are provided in Supplementary Table S2.
The total RNA extraction from cells was performed with the NucleoZOL Kit (Macherey-Nagel). The extracted RNA was reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and evaluated using the FastStart Universal SYBR Green Master (ROX) Kit (Roche). GAPDH was used as an internal control for evaluating the mRNA expression levels of A2M-AS1 and PCBP3, which were then quantified using the 2−ΔΔCt method. The primer sequences used in this study are listed in Supplementary Table S3.
CCK-8 and EdU assays
The proliferation of cells was evaluated using the CCK-8 assay (DOJINDO) and Cell-Light EdU Apollo567 In Vitro Kit (Ribobio). For the CCK-8 assay, the cells with stable transfection of lentiviruses were seeded in the 96-well plates at a density of 8,000 cells/well and incubated for 96 hours. The medium, containing 10% CCK-8, was added to the pancreatic cancer cells after 0, 24, 48, 72, and 96 hours of inoculation and incubated at 37°C for 4 hours. Then, the absorbance was evaluated at 450 nm using a microplate reader (Nikon). For detecting the condition of cell death after inducing into ferroptosis, the absorbance was detected before Erastin treatment and after 24 hours of intervene via the methods above.
For the EdU assay, the pancreatic cancer cells with stable transfection of lentiviruses were seeded at a density of 105 cells/well in the 96-well plates and fixed with 4% paraformaldehyde after treatment with EdU solution (50 μmol/L) for 5 hours and then permeabilized with 0.5% TritonX-100. Next, the cells were stained with Apollo fluorescent staining solution and Hoechst 33342 and observed under an inverted fluorescence microscope (Olympus BX63) with a 20× objective lens.
Migration and invasion assay
For migration assay, the cells with stable transfection of lentiviruses were seeded in 6-well plates (106 cells/well) and incubated overnight. Scratches were made using a 10-μL micropipette tip perpendicular to the plate bottom and images were taken under the microscope with a 10× objective lens. The healing rates were calculated by measuring the scratch distance after 0 and 24 hours using ImageJ software (version 1.53a; NIH).
For the invasion assay, the cells were added to a 200-μL serum-free medium at a cell density of 105 cells/well and placed in the upper chamber of Transwell (Matrigel was added 2 hours before this). A total of 600 μL complete medium, containing 10% FBS, was added into the lower chamber. After incubating the cells for 24 hours, those on the lower side of the chamber were fixed, stained, and photographed under the microscope with a 20× objective lens. Three visual fields were randomly selected in each chamber, and the migration of cells was quantified using ImageJ software.
RNA pull-down assay and MS
The RNA pull-down assay was performed using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). Briefly, the RNA was firstly bound to the beads and equilibrated with the Protein-RNA Binding Buffer before the addition of protein lysate. The beads were then washed with an appropriate buffer, followed by mixing using a vortex and separating using a magnetic stand. The retrieved proteins were analyzed by the Western blot analysis or resolved using a gradient gel electrophoresis. The extracted proteins were then characterized using a mass spectrometer (AB SCIEX).
RNA immunoprecipitation (RIP) assay
RIP assay was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore), following the manufacturer's instructions. PCBP3 antibody (Abcam) was used for the precipitation of RNA, which was then quantified using RT-qPCR.
Briefly, the PANC-1 and BxPC-3 cells were fixed with 4% formaldehyde and permeabilized with 0.5% TritonX-100. Then, the cells were incubated with an A2M-AS1 probe at 37°C overnight to locate the A2M-AS1 in the cells. The hybridized cells were counterstained with DAPI and observed under the fluorescence microscope with a 40× objective lens.
Western blot analysis
The cell lysis and quantification of proteins were performed as described previously (16). The primary antibodies for AKT, p-AKT, mTOR, p-mTOR, p38, p-p38, and GAPDH and the corresponding anti-rabbit secondary antibodies were purchased from Cell Signaling Technology and used as the previous study (17). The antibodies for PCBP3 and tubulin were purchased from Abcam and Abmart, respectively. GAPDH or tubulin was used as the loading control.
Lipid peroxidation assay
The malondialdehyde (MDA) contents, a natural product of lipid peroxidation, were measured using the Lipid Peroxidation Assay Kit (Beyotime). Pancreatic cancer cells were harvested by washing with cold PBS after reacting with DMSO or Erastin for 24 hours, followed by lysing with RIPA lysis buffer. The protein concentrations were measured by BCA method. Next, 100 μL samples, standard substance, and PBS were added respectively into a 1.5 mL microtube with 200 μL MDA working solution and heated at 100°C for 10 minutes. After immerged in ice water for 10 minutes, these microtubes were centrifuged at 1,000 × g for 10 minutes. Then pipetting 200 μL supernatants to 96-well plate and examine the absorbance of each group at 532 nm by a microplate reader. The final concentration of MDA was calculated according to the standard curve.
An Iron Assay Kit (BJbalb) was used to measure the concentration of Fe in the pancreatic cancer cells. Briefly, after incubated with Erastin or DMSO for 24 hours, pancreatic cancer cells were collected and lysed to collect the supernatant. Then the protein concentration was determined by BCA method. The iron standard working solution, ddH2O and samples (75 μL) were added respectively to the 96-well plate, followed by mixing with Fe assay buffer (200 μL) and chromogenic solution (8.4 μL). After incubating at 37°C for 10 minutes, the absorbance of 96-well plate at 562 nm was examined to calculate the Fe concentrations.
To measure the cellular GSH contents, a GSH Detection Kit (Solarbio) was used, following the manufacturer's instructions. Shortly, after reacting with Erastin or DMSO for 24 hours, cells were acquired, washed with PBS, and lysed with two rounds of freezing and thawing. Then the supernatant was collected by centrifugation at 8,000 × g for 10 minutes and the protein concentrations were evaluated by BCA. Next, adding 100 μL samples or standard substances with various concentrations to the mixture of reagent 2 and 3 in a 96-well plate. After placing for 2 minutes, the absorbance at 412 nm was measured and the contents of GSH in the samples was calculated according to the standard curve.
In vivo experiment
Male BALB/c nude mice (5 weeks old; 16−18 g) were obtained from the Animal Center of Guangxi Medical University and grown under specific pathogen-free conditions. All the animal experiments were approved by the Animal Ethical Committee of Guangxi Medical University. After 1 week of acclimation, a subcutaneous graft tumor model was established. The mice (n = 20) were randomly divided into four groups; Vector group, A2M-AS1 group, Vector + Erastin group, and A2M-AS1 + Erastin group. The Vector and Vector + Erastin groups were injected with 106 cells that stably expressing the lentiviral vector, whereas the other two groups were injected with 106 cells that stably overexpressing A2M-AS1. On the 7th day after inoculation, the Vector + Erastin and A2M-AS1 + Erastin groups were intraperitoneally injected with a 100-μL Erastin solution (40 mg/kg) once every 2 days. The tumor sizes were measured using a pair of Vernier calipers every 4 days, and their tumor volumes were calculated (long diameter × short diameter2 × 0.5). The mice were sacrificed on the 31st day of inoculation and the tumors were completely dissected to measure their sizes and weights.
SPSS 23.0 software was used for the statistical analyses in this study. All the data were expressed as mean ± SD. Chi-square test and t test were used to determine the differences between the two groups. One-way and multiway ANOVA were used to determine the differences among multiple groups. Kaplan–Meier method and log-rank test were used to analyze the survival time and effects of A2M-AS1 expression on the prognosis of patients with pancreatic cancer. P < 0.05 was considered statistically significant.
A2M-AS1 levels were downregulated and correlated with a better prognosis in pancreatic cancer
The tissue microarray included a total of 163 spots (77 paired and 9 single pancreatic cancer tissue samples) in this study after excluding the escaped and empty samples. The ISH result revealed that the expression levels of A2M-AS1 in the pancreatic cancer tissue were significantly lower than the normal adjacent tissue (Fig. 1A–C). The area under the ROC curve was 0.838, suggesting the excellent potential value of A2M-AS1 as a diagnostic marker for pancreatic cancer (Fig. 1D). According to the mean ISH scores of pancreatic cancer tissue (mean value = 0.58), the patients were divided into high and low expression groups. Further analysis revealed that the patients with pancreatic cancer with high A2M-AS1 expression showed an increased survival time (Fig. 1E), suggesting that A2M-AS1 might exert an anti-tumor effect on pancreatic cancer. Moreover, the A2M-AS1 expression showed no obvious association with the clinicopathological characteristics of the patients with pancreatic cancer (Table 1). Therefore, it might serve as an independent biomarker for predicting the prognosis of pancreatic cancer (Supplementary Table S4).
|.||A2M-AS1 level .||.||.|
|Factors .||Low .||High .||Total .||P value .|
|Lymph nodes metastasis|
|.||A2M-AS1 level .||.||.|
|Factors .||Low .||High .||Total .||P value .|
|Lymph nodes metastasis|
A2M-AS1 inhibited the proliferation, migration, and invasion of the pancreatic cancer cells
To explore the localization of A2M-AS1 in pancreatic cancer cells, the FISH assay was performed, showing its localization in both the nucleus and cytoplasm of the pancreatic cancer cells, but mainly in the cytoplasm (Fig. 2A). The expression levels of A2M-AS1 were then compared between the human pancreatic duct epithelial cells (HPDE) and human pancreatic cancer cells, including PANC-1, BxPC-3, and AsPC-1. As compared with HPDE, the expression levels of A2M-AS1 were significantly lower in the pancreatic cancer cells (Fig. 2B). Because AsPC-1 showed a slow growth rate and was difficult to cultivate, which might be due to its relatively poor adhesive ability (18, 19), the PANC-1 and BxPC-3 cell cultures were selected for further experiments.
The A2M-AS1-overexpressing and silencing lentiviral vectors were constructed and transfected into the PANC-1 and BxPC-3 cells for the further investigation of the A2M-AS1 effects on the proliferation, migration, and invasion of the pancreatic cancer cells. A2M-AS1 was successfully overexpressed in the PANC-1 cells and silenced in the BxPC-3 cells (Fig. 2C and D). The impact of A2M-AS1 on the proliferation of pancreatic cancer cells was assessed using CCK-8 and EdU assays. As shown in Fig. 2E to H, the A2M-AS1 overexpression significantly weakened the proliferation of the pancreatic cancer cells, while the silencing of A2M-AS1 enhanced their proliferation. Next, the effects of A2M-AS1 on the invasion and migration of the pancreatic cancer cells were assessed. Similarly, the A2M-AS1 overexpression inhibited the invasion and migration ability of the PANC-1 cells, which were strengthened after the silencing of A2M-AS1 in the BxPC-3 cells (Fig. 2I–L). These results suggested that A2M-AS1 had a crucial role in the progression of pancreatic cancer.
A2M-AS1 promoted ferroptosis of the pancreatic cancer cells
To explore the role of A2M-AS1 in the ferroptosis process of pancreatic cancer cells, the correlation of its expression level with the induction of ferroptosis was investigated. The results showed that the A2M-AS1 expression level increased with the elevation of the Erastin (ferroptosis inducer) concentration (Fig. 3A and B), suggesting that A2M-AS1 may take part in the ferroptosis of the pancreatic cancer cells. Then, the effects of overexpression or silencing of the A2M-AS1 on ferroptosis were explored. As shown in Fig 3C and D, the overexpression of A2M-AS1 significantly decreased the survival rate of pancreatic cancer cells after ferroptosis induction by Erastin, while the silencing of A2M-AS1 resisted the Erastin-induced ferroptosis. Furthermore, the overexpression of A2M-AS1 increased the intracellular concentrations of Fe and MDA and decreased the cellular contents of GSH, while the silencing of A2M-AS1 showed opposite effects (Fig. 3E–J). To further explore the effects of A2M-AS1 on pancreatic cancer in vivo, a xenograft mouse model was constructed. The overexpression of A2M-AS1 dramatically inhibited the growth of pancreatic cancer. The Erastin treatment significantly decreased the tumor volumes and weights, whereas the A2M-AS1 + Erastin group showed the most obvious effects (Fig. 3K–M). These results suggested that A2M-AS1 might promote ferroptosis in the pancreatic cancer cells.
A2M-AS1 affected ferroptosis by interacting with PCBP3
The lncRNAs are known to exhibit biological roles partly through the RNA-interacting proteins. Therefore, an RNA pull-down assay was performed to investigate the interaction of A2M-AS1 with the proteins in regulating ferroptosis. The results showed various differentially-expressed proteins between the A2M-AS1 sense and antisense chain binding proteins (Fig. 4A). The mass spectrometry analysis identified a total of 308 differential proteins, among which, 122 proteins were more likely to be directly bound to A2M-AS1 (−10lgP > 50; Supplementary Table S5). Then, the interactions of these 122 proteins with A2M-AS1 in pancreatic cancer were explored using the GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene=&clicktag=survival). The results showed that the poly (rC) binding protein 3 (PCBP3) expression was strongly correlated with A2M-AS1 (P = 0; Fig. 4B). Moreover, the protein levels of PCBP3 were also positively correlated with the survival time of the patients with pancreatic cancer and were more significant than that of the A2M-AS1 (Fig. 4C). Then, western blot analysis showed that PCBP3 was expressed in the A2M-AS1 sense binding proteins, but absent in the antisense group (Fig. 4D). The RIP assay demonstrated s significant increase in the A2M-AS1 expression in the PCBP3 binding RNA as compared with the control group, suggesting a direct interaction of A2M-AS1 with PCBP3 (Fig. 4E).
Subsequently, the role of PCBP3 in ferroptosis was investigated. The mRNA and protein expression levels of PCBP3 increased significantly after ferroptosis-induction by Erastin in the PANC-1 and BxPC-3 cells (Fig. 4F and G), suggesting the role of PCBP3 in ferroptosis. Next, the effects of altered PCBP3 expression levels on ferroptosis in the pancreatic cancer cells were investigated. The contents of PCBP3 were examined in the PANC-1 and BxPC-3 cells and showed no obvious differences (Fig. 4H). Then the PCBP3 expression was silenced in the PANC-1 and BxPC-3 cells. Interestingly, the silencing of PCBP3 significantly decreased A2M-AS1 expression levels in the pancreatic cancer cells (Fig. 4I and J), further demonstrating their interaction. It was noteworthy that the silencing of PCBP3 significantly decreased the survival rates of the pancreatic cancer cells. On the 7th day of transfection, the percentage of living pancreatic cancer cells was less than 10% (Supplementary Fig. S1). Therefore, the effects of PCBP3 silencing on ferroptosis could not be further investigated.
Hence, the effect of PCBP3 overexpression on ferroptosis was explored. The transfection with PCBP3-overexpressing lentiviral vectors significantly increased the level of PCBP3 (Fig. 4K) and promoted the Erastin-induced ferroptosis in the pancreatic cancer cells (Fig. 4L). Moreover, the transfection also increased the cellular MDA and Fe concentrations (Fig. 4M and N) and decreased the GSH contents in the pancreatic cancer cells (Fig. 4O). Then, a rescue assay was performed and the inhibition of ferroptosis caused by the A2M-AS1 silencing was successfully reversed by overexpressing PCBP3 (Fig. 4P–S). These results revealed that A2M-AS1 might affect ferroptosis by interacting with PCBP3 in the pancreatic cancer cells.
A2M-AS1/PCBP3 modulated ferroptosis by regulating the activation of p38 and AKT/mTOR
Recent studies have shown that p38 phosphorylation can induce ferroptosis (10, 20), whereas the activation of the AKT/mTOR signaling pathway can impede its progression (21, 22). This study investigated the effects of A2M-AS1 on these pathways. As expected, the A2M-AS1 overexpression significantly promoted the activation of p38 and inhibited the stimulation of AKT/mTOR signaling pathways, whereas the A2M-AS1 silencing showed opposite effects (Fig. 5A). The overexpression of PCBP3 also exerted similar effects on the activation of p38 and AKT/mTOR signaling pathways (Fig. 5B). Moreover, the effects of A2M-AS1 silencing on p38 and AKT/mTOR signaling pathways were successfully reversed by the overexpression of PCBP3 (Fig. 5C). These results suggested that the A2M-AS1/PCBP3 axis might modulate ferroptosis by regulating the activation of p38 and AKT/mTOR signaling pathways.
A2M-AS1, a lately studied lncRNA, is located at chr12:p13.31 and has been reported to regulate the development of multiple cancers (23–25). In breast cancer, A2M-AS1 inhibited the action of miR-146b on mucin 19 (MUC19), thereby promoting the metastasis and invasion of the tumor (23, 24). The expression of A2M-AS1 decreased in advanced lung cancer and was positively correlated with the patients’ survival time (25). In pancreatic cancer, the specific role of A2M-AS1 has rarely been studied. This study showed that the expression levels of A2M-AS1 in pancreatic cancer decreased and were positively correlated with the survival time of the patients. Moreover, A2M-AS1 can also be used as a diagnostic and prognostic biomarker for pancreatic cancer. To detect the cellular localization of A2M-AS1, the FISH assay was performed, which showed its localization mainly in the cytoplasm of the pancreatic cancer cells. Then, the A2M-AS1-overexpressing and silencing models were constructed. EdU, CCK8, scratch, Transwell, and tumor formation assays were performed to further explore the effects of A2M-AS1 on the progression of pancreatic cancer. The overexpression of A2M-AS1 inhibited the proliferation, invasion, metastasis, and tumor growth of pancreatic cancer, whereas the A2M-AS1 silencing showed opposite results. These findings suggested the antineoplastic role of A2M-AS1 in pancreatic cancer.
Ferroptosis, first discovered by Dixon in 2012 (26), is a kind of iron-dependent and non-apoptotic programmed cell death. Ferroptosis could affect the progression of various types of cancers, such as liver cancer, gastric cancer, breast cancer, and lung cancer (27–30). However, the existing studies on ferroptosis mainly focused on the regulation of the coding genes (31–34), and only a few studies have investigated the role and mechanism of ncRNAs, such as the lncRNAs. In fact, the ncRNAs play crucial roles in the regulation of ferroptosis. For example, miR-27a-3p promoted the occurrence of ferroptosis by regulating SLC7A11 in non-small cell lung cancer (35). miR-15a-3p facilitated the process of ferroptosis in colorectal cancer by targeting GPX4 (36). Besides, the lncRNA NEAT1 might promote ferroptosis in hepatocellular cancer by regulating the miR-362–3p/MIOX axis (37), whereas the LncRNA BDNF could regulate ferroptosis in gastric cancer through WDR5/FBXW7 axis (38). Moreover, A2M-AS1 has been reported to be associated with the expression levels of ferroptosis-related genes in gastric cancer (11). To investigate whether A2M-AS1 was involved in ferroptosis in pancreatic cancer, its expression in the induction process of ferroptosis was examined. The results showed a significant increase in the levels of A2M-AS1 upon ferroptosis induction by Erastin, indicating that A2M-AS1 might play a role in ferroptosis. Then, the effects of alterations in the A2M-AS1 levels on ferroptosis in pancreatic cancer were studied, which showed that the overexpression of A2M-AS1 could increase cell death as well as cellular concentrations of the ferroptosis-related indicators, such as Fe and MDA. On the other hand, the ROS scavenger GSH largely decreased. The silencing of A2M-AS1 showed opposite effects. Moreover, in the next in vivo experiment, the upregulation of A2M-AS1 was found to improve the therapeutic effects of Erastin on pancreatic cancer. These findings suggested that A2M-AS1 could promote ferroptosis in the pancreatic cancer.
PCBP3 is a member of the PCBP family, which participate in the transport of intracellular iron and act as iron chaperones (39). Iron metabolism is crucial for ferroptosis (40) and PCBP3 regulates the iron balance by binding to ferritin (39). However, the specific role of PCBP3 in ferroptosis remains unknown. In this study, the RNA pull-down and RIP assays were performed, which revealed that PCBP3 directly interacted with A2M-AS1. Moreover, PCBP3 showed a positive correlation with the survival of the patients with pancreatic cancer, which was consistent with the previous study (41). To explore the effects of PCBP3 on ferroptosis, a PCBP3-overexpressing model was constructed and showed a promoting effect on ferroptosis in the pancreatic cancer cells; this was similar to the effects of A2M-AS1 overexpression. To verify the interacting protein of A2M-AS1, a rescue assay was performed. The overexpression of PCBP3 successfully reversed the suppressive effects of A2M-AS1 silencing on ferroptosis, which further demonstrated that A2M-AS1 might promote ferroptosis by interacting with PCBP3. However, this study could not explore the effects of PCBP3 silencing on ferroptosis due to the massive death of the pancreatic cancer cells; this might be due to the possible important biological role of PCBP3 in the pancreatic cancer cells. After silencing PCBP3, the pancreatic cancer cells could not sustain normal metabolism and died. The specific mechanism of this process requires further investigation.
Furthermore, the signaling pathways that the A2M-AS1/PCBP3 axis affected in the action on ferroptosis were studied. Hattori and colleagues discovered that cold stress could induce cell death by activating the p38 signaling pathway (20). Li and colleagues also reported that the inactivation of the p38 signaling pathway could inhibit ferroptosis (10). Besides, the AKT/mTOR signaling pathway was found to protect cells from ferroptosis in the breast cancer cells (22), whereas ferroptosis induction in colorectal cancer cells inhibited this pathway (21). Then, the expression levels of p38 and AKT/mTOR pathway were determined. The overexpression of A2M-AS1 or PCBP3 promoted the activation of p38 and inhibited the phosphorylation of AKT/mTOR, while silencing of A2M-AS1 showed opposite results. Moreover, in the rescue assay, the overexpression of PCBP3 could effectively reverse the effects of A2M-AS1 silencing on the p38 inhibition and AKT/mTOR stimulation. This further proved that the A2M-AS1/PCBP3 axis could affect ferroptosis in pancreatic cancer by regulating the p38 and AKT/mTOR signaling pathways.
In summary, this study revealed that A2M-AS1 could promote ferroptosis in the pancreatic cancer cells by interacting with PCBP3 and regulating the activation of p38 and AKT/mTOR signaling pathways. Therefore, A2M-AS1 might be a potential novel therapeutic target for patients with pancreatic cancer in the future.
No author disclosures were reported.
X. Qiu: Methodology, writing–original draft. Q. Shi: Methodology. X. Zhang: Supervision. X. Shi: Validation. H. Jiang: Data curation, supervision. S. Qin: Supervision, funding acquisition.
This work has been financially supported by the National Natural Science Foundation of China (Grant Nos. 31560257 and 81960439) and the “139” plan for training high-level cadre talents in Guangxi medicine (G201903004).
The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).