Anticancer peptide MCP-1 induces ferroptosis in liver cancer HCCLM3 cells by targeting FOXM1/ALOXE3 signal pathway

FOXM1 is a crucial oncogenic transcription factor involved in almost all cancer hallmark pathways across all cancer types. Our previous work had found that FOXM1 targeted peptide P201 can strongly inhibit the growth of cancer cells including the liver cancer HCCLM3 cells. In addition, by RNA-seq of HCCLM3 cells treated with MCP-1, an anticancer peptide optimized from P201, ALOXE3, a key feature of ferroptosis was signi�cantly elevated while FOXM1 was down-regulated, we wonder if the cell death of HCCLM3 induced by MCP-1 was associated with ferroptosis. Also, the relationship between FOXM1 and ferroptosis was less understood. Hence, in this study, we explore the effect of MCP-1 on ferroptosis and establish the associations among MCP-1, FOXM1 and ALOXE3 in HCCLM3 cells. The results showed that MCP-1 can signi�cantly induce the elevated expression of ALOXE3, decreased content of GSH, down-regulation of GPX4 expression, increased contents of ROS and total iron in HCCLM3 cells. Also, ferrostatin-1, a speci�c inhibitor for ferroptosis, can reverse the cell death in HCCLM3 cells when co-administrated with MCP-1. TCGA database hepatocellular carcinoma gene expression analysis showed that FOXM1 was negative-related to ALOXE3 and further con�rmed by the results of siRNA knockdown of FOXM1 in HCCLM3 cells. Moreover, the co-expressed genes analysis for FOXM1 and ALOXE3 revealed that many of them were closely involved in the regulation of ferroptosis. Taken together, we discovered and con�rmed the induction of ferroptosis by MCP-1 in liver cancer HCCLM3 cells and primarily established the relationships among MCP-1, FOXM1 and ALOXE3.


Introduction
FOXM1 is a crucial transcription factor belongs to the evolutionarily conserved forkhead box (FOX) superfamily.It has emerged as an important molecule implicated in the initiation, progression, metastasis, angiogenesis and drug resistance of cancers (Bella et al. 2014;Nandi et al. 2018).It is involved in almost all cancer hallmark pathways across all cancer types and proved to be the "Achilles' heel" of cancer (Halasi and Gartel 2013;Raghuwanshi and Gartel 2023).The inhibition of this single transcription factor may induce programmed cell death (PCD) and should be possible to target multiple facets of tumorigenesis (Radhakrishnan and Gartel 2008), suggesting FOXM1 has great potential in anticancer therapy.
Ferroptosis is a term coined by Stockwell and colleagues in 2012 and a newly recognized form of PCD, which is readily mechanistically and morphologically different from other forms of cell death such as apoptosis, necroptosis, and autophagic cell death (Dixon et al. 2012).It is de ned as an iron-dependent, non-apoptotic type of cell death resulted from lipid peroxide accumulation and characterized by the increase of reactive oxygen species (ROS) (Dixon and Pratt 2023;Newton K et al. 2024 ).In ferroptosis, the mitochondrial respiratory chain promotes lipid peroxidation through cytochrome P450 reductase or arachidonate lipoxygenase (ALOX) (Kuang et al. 2020).The mammalian ALOX family consists of six members (ALOXE3, ALOX5, ALOX12, ALOX12B, ALOX15 and ALOX15B), which are responsible for ferroptosis through lipid peroxidation (Li et al. 2020), and that ROS formation catalyzed by the ALOX is a necessary step in ferroptosis (Yang et al. 2016).Accumulating evidence supports the remarkable potential of ferroptosis in cancer treatment and targeted induction of ferroptosis is an effective strategy for the treatment of cancers (Zhao et al. 2022).In addition, pharmacological induction of ferroptosis by bioactive compounds could overcome chemotherapeutic drug resistance (Wang et al. 2023), for example, targeting the STAT3-ferroptosis circuit can promote ferroptosis and restore sensitivity to chemotherapy (Ouyang et al. 2022).Knockdown of FOXM1 can downregulate the expression of ferroptosis-resistant genes and increase malonaldehyde (MDA) and ROS levels in cisplatin-resistant endometrial cancer cells (Peng et al. 2023).Nevertheless, the correlation between ferroptosis and FOXM1 was less reported.In particular, there was no report concerning the association between FOXM1 and ALOXE3.
At present, some anticancer drugs such as cisplatin, lapatinib and sorafenib had been found to induce ferroptosis (Chen et al. 2021).In our previous work, we had found FOXM1 targeted peptide P201, which was obtained from the selection of phage random dodecapeptide library against the DNA binding domain of FOXM1 (FOXM1-DBD), can strongly inhibit the growth of cancer cells including liver cancer cells (Bi et al. 2017;Liu et al. 2017).In addition, by RNA-seq analysis of HCCLM3 cells treated with MCP-1, which was optimized from P201 in our lab, ALOXE3 was found to be signi cantly elevated.We wonder if the cell death of HCCLM3 induced by MCP-1 was associated with ferroptosis in addition to the common PCD.Also, the relationship between FOXM1 and ferroptosis was less understood while they were both involved in drug resistance, the latter is a bottleneck for cancer therapy.Additionally, there was no reports on the study of the correlation between FOXM1 and ALOXE3.Hence, in this work, we rstly con rmed the induction of ferroptosis by MCP-1 in HCCLM3 cells through analyzing the expression or contents of the key features for ferroptosis including ALOXE3, glutathione (GSH), glutathione peroxidase 4 (GPX4), ROS and total iron.Then, ferroptosis inhibitor was used to see if it can reverse the viability of the cells when co-administrated with MCP-1.Later, TCGA hepatocellular carcinoma (HCC) database was used to nd if the expression of FOXM1 and ALOXE3 were correlated to the cancers and lifespans of the patients, in addition, the co-expressed genes for FOXM1 and ALOXE3 in HCC patients were also analyzed.Finally, siRNA knockdown of FOXM1 in HCCLM3 cells was employed to further con rm the correlations between FOXM1 and ALOXE3.

Cell line and culture
The human high metastatic hepatocellular carcinoma HCCLM3 cells (purchased from Bio Biotechnology, Chengdu, P.R. China) were maintained in DMEM high glucose medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and antibiotics (100 U/ml of penicillin and 100 mg/ml of streptomycin, Biosharp, Beijing, P.R. China).The cells were cultured at 37 ℃ in a humidi ed atmosphere of 5% CO 2 .

Peptide design and synthesis
The MCP-1 peptide was composed of three components: a cell-penetrating peptide 9-mer polyarginine (D-enantiomers) at the N-terminus, the dodecapeptide optimized from P201, and a (GS) 2 polypeptide linker between them for exibility.It was chemically synthesized by Shanghai Qiangyao Biol.Com.
(Shanghai, P.R. China).The purity was determined to be greater than 95% by HPLC.In preparation of peptide stock solution, the peptide was dissolved in DMSO at a concentration of 20 µg/µl.It was stored at -80℃ and diluted immediately before use.

qRT-PCR
Cellular total RNA was isolated using Trizol reagent (CWBIO, Jiangsu, P. R. China) according to the manufacturer's instructions.Total RNA was reverse transcribed to cDNA with a reverse transcription kit (Accurate Biology, Hangzhou, P. R. China).qRT-PCR was conducted with a SYBR Green Master Mix Kit (Accurate Biology, Hangzhou, P. R. China) on the StepOne Plus instrument (Thermo, USA).The PCR cycling conditions were 94 ℃ for 3 min, followed by 40 cycles of 94 ℃ for 30 s, 60 ℃ for 20 s and 72 ℃ for 10 s.Each sample was conducted in triplicate.Relative gene expression levels were analyzed using comparative Ct methods where Ct was the cycle threshold number normalized to the internal control of GAPDH.Primers used for qRT-PCR were shown in Table 1.
After washed three times with TBST, the membrane was incubated with peroxidase-conjugated antirabbit secondary antibody (1:8000, Proteintech, Wuhan, P. R. China) for 2 h at room temperature.The membrane was then washed again with TBST three times, immunoreactive bands were visualized by the enhanced chemiluminescence reagent (ECL, Beyotime, Shanghai, P. R. China) and detected by an iBright FL1500 detection system (Thermo Fischer, USA).GAPDH was used as the protein loading control.
Densitometric analysis of each band was measured using ImageJ software for quanti cation.

CCK-8 assay
The cells were seeded at a density of 2×10 3 cells/well into a 96-well plate and incubated at 37℃ with 5% CO 2 overnight.Then, they were co-administrated with various concentrations of ferrostatin-1 (Fer-1) (Adamas, Shanghai, P.R. China) and 15.0 µM MCP-1 for 24 h.After addition of 10 µl CCK-8 solution (Beyotime, Shanghai, P. R. China) to each well, the plate was incubated for another 1 h.The absorbance was measured at 450 nm by using a Synergy H1 microplate reader (Biotek, USA).The percentage of cell viability versus the concentration of Fer-1 and the peptide was then plotted.

GSH determination
The cells were seeded at a density of 3×10 5 cells/well into a 6-well plate and cultured overnight.On the next day, the cells were treated with indicated concentrations of MCP-1 and further incubated for 12 hours.Total protein was extracted and quanti ed.The intracellular GSH content was detected and calculated using a GSH detection kit (Elabscience, Wuhan, P. R. China) according to the manufacturer's instructions.The amount of GSH was expressed as µmol of GSH/l solution.

Detection of iron content
The cells were cultured and treated as that of GSH assay.The total iron content in cells was detected and calculated using a total iron colorimetry test kit (Elabscience, Wuhan, P. R. China) according to the manufacturer's instructions.

ROS assay
The cells were cultured and treated similar to that of GSH assay.For ROS assay, the cultured medium was replaced with serum-free DMEM medium containing DCFH-DA at 1000:1 (v/v) Beyotime, Shanghai, P. R. China) for 20 minutes.The relative level of intracellular uorescence was quanti ed by ow cytometry (BD C6 Plus, Becton Dickinson, USA) and the data were analyzed using FlowJo software.

Bioinformatic analysis
Bioinformatic analysis was performed based on a combination of R software, and web-based public bioinformatics tools.Liver hepatocellular carcinoma (LIHC) data were obtained from The Cancer Genome Atlas (TCGA) (https://gdac.broadinstitute.org/),Kaplan-Meier Plotter (http://kmplot.com/analysis/)and LinkedOmics databases (https://linkedomics.org/login.php)(Vasaikar et al. 2018).RNA-seq data (level 3) for LIHC tissue samples (n = 1109), patients' clinical features (n = 425), and matching adjacent normal tissue samples (n = 50) were obtained from the TCGA dataset (https://portal.gdc.cancer.gov)and integrated using R software (stringr, tidyr, tibble, rstatix, ggprism).The survival curve was generated using the Kaplan-Meier method and compared by log-rank test.For the acquisition of co-expressed genes, LinkedOmics was used to calculate the Pearson's correlation coe cient for each gene expression in the database (the coe cient is between − 1 and 1), then the orders of the positive or negative associated co-expressed genes were obtained.The Gene Set Enrichment Analysis (GSEA) algorithm were used for Gene Ontology biological process (GO-BP) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis and visualization.Enrichment results with FDR < 0.05, or adjusted p < 0.05, were considered statistically signi cant, and simulated for 1000 times.

siRNA transfection
Two siRNAs designed to target human FOXM1 and one non-silencing control siRNA were purchased from GenePharma Com.(Shanghai, P. R. China).For siRNA transfection, the cells were seeded at a density of 5×10 4 cells/well into a 24-well plate and incubated at 37 ℃ with 5% CO 2 overnight.
Transfection was in accordance with the manufacturer's instructions (Polyplus, France).The nal concentrations of siRNAs were 60 pmol/well.After incubation for 24 hours, the transfected cells were harvested for subsequent study.

Statistical analysis
GraphPad Prism 9 was used for all statistical analyses.The data were calculated by three independent experiments and presented as means ± standard deviation (SD), the differences between the two groups were analyzed using Student's t test.The statistical signi cance was denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

MCP-1 induces ferroptosis in HCCLM3 cells
To verify whether MCP-1 can really induce ferroptosis, we rstly con rmed our important nding in our previous transcriptomic sequencing analysis in which the expression of ALOXE3 as the key feature of ferroptosis was signi cantly upregulated by 5.3 times when treated with MCP-1 in HCCLM3 cells.As expected, both the mRNA and protein expression of ALOXE3 in HCCLM3 cells were signi cantly upregulated by 5.7 (p < 0.001) (Fig. 1-a), and 2.9 times (p < 0.001) (Fig. 1-b, c) respectively when treated with 10 µΜ of MCP-1 for 24 hours and analyzed by qRT-PCR and Western blot.They were consistent with our RNA-seq result and the fact that ferroptosis inducers such as the natural small molecule compound talaroconvolutin A can upregulate the expression of ALOXE3 and kill colorectal cancer cells in vitro and in vivo (Xia et al. 2020).Next, the other four important features of ferroptosis including total iron, ROS, GSH and GPX4 were determined.To our current knowledge, abnormal iron metabolism may accelerate the peroxidation of polyunsaturated fatty acids(PUFAs)in cells, it can not only directly catalyze the peroxidation of PUFAs in lipid membrane through the Fenton reaction to produce lipid peroxides but also participate in the synthesis of lipid peroxides as an important cofactor for lipid peroxidase ALOX (Xie et al. 2016;Lei et al. 2022).GSH-GPX4 system is one of the critical ferroptosis defense mechanism, while GPX4 belongs to the GPX protein family and is the only GPX member capable of converting phospholipid hydroperoxides to phospholipid alcohols (Hassannia et al. 2019), they are usually synthesized to resist the lethal accumulation of ROS and reduce its harm to cells.In mitochondria, inactivation of GPX4 weakens the antioxidant capacity of the cell and causes potent ferroptosis (Gan 2021).Therefore, when ferroptosis occurs, the total iron content and ROS will increase while the production of GSH and GPX4 will reduce.In this work, both the intracellular ROS and the total iron content were signi cantly increased while the intracellular GSH and GPX4 were markedly decreased when HCCLM3 cells were treated with 15 µΜ of MCP-1 for 12 hours (p < 0.001, Fig. 2a-d).In addition, the changes of ROS, GSH, GPX4 and total iron were dose dependent.Moreover, Fer-1, a synthetic compound and speci c inhibitor for ferroptosis, was used to see if it can reverse the cell death in HCCLM3 cells when treated with MCP-1.It was reported that Fer-1 can speci cally inhibit ferroptosis but not cell death induced by other apoptosisinducing agents (Dixon et al. 2012;Miotto et al. 2019).As expected, when HCCLM3 cells were coadministrated with Fer-1 and MCP-1, the cell viabilities were signi cantly elevated with the increasing concentration of Fer-1 from 2.5 to 5.0 µM (Fig. 2e), indicating Fer-1 can partly reverse the cell death induced by MCP-1.Collectively, we can conclude that MCP-1 can induce ferroptosis in HCCLM3 cells.

Bioinformatics analysis supported that FOXM1 has close relations to ALOX3 in regulation of LIHC
Since MCP-1 is a FOXM1 targeting peptide which can upregulate ALOXE3 and induce ferroptosis in HCCLM3 cells as convinced above, we proposed there is association between FOXM1 and ALOXE3.In order to con rm this hypothesis, transcriptome data of 374 LIHC samples (including 3 recurrent tumor samples) and 50 paired adjacent tissue controls were collected from TCGA database.Gene expression analysis were rstly performed to compare the differential expressions of FOXM1 and ALOXE3 in the two datasets.The results showed that the expression of FOXM1 in tumor samples was signi cantly higher than that of the controls (P < 0.001) (Fig. 3a-b) as evidenced by many other researches (Barger et al. 2019;Wei et al. 2022).Also, the expression of ALOXE3 in LIHC tissues was signi cantly higher than that of the controls (P < 0.05) while its expression in recurrent tumors was signi cantly lower than that of the primary tumors (P < 0.001) (Fig. 3c-d).This is reasonable, upregulation of ALOXE3 was really reported in HCC and colorectal cancer (Qin et al. 2021; Chen and Li 2022) while markedly down-regulated in human glioblastoma (GBM) (Yang et al. 2021).Next, the impact of the expression of FOXM1 and ALOXE3 on survival of LIHC patients was analyzed by the Kaplan-Meier survival curves (Fig. 4).Expectedly, the patients with higher ALOXE3 expression (P < 0.001) and lower FOXM1 expression (P < 0.001) had higher survival probability and longer life time.Then, the co-expressed genes (Triska et al. 2017; van Dam et al.

2017
) for FOXM1 and ALOXE3 in LIHC patients from LinkedOmics database were analyzed by Pearson's correlation coe cient using R software.The top 50 genes with strong positive or negative correlation to FOXM1 or ALOXE3 were selected to construct the visual heatmap respectively (Fig. 5).Many genes related to the occurrence and development of liver cancer and ferroptosis were involved.For examples of ALOXE3 co-expressed genes, neuronal pentraxin 1 (NPTX1), a downstream target of the AKT pathway, which can inhibit proliferation and promote apoptosis in HCC (Zhao et  Finally, GO and KEGG analysis were used to decipher the involvement of the co-expressed genes including FOXM1 and ALOXE3 in the biological process and metabolic pathways of LIHC (Fig. 6).It was found in GO analysis that both ALOXE3 and FOXM1 were positively involved in the cell cycle G2/M phase transition and negatively involved in cell acetaldehyde metabolism, lipid modi cation, dicarboxylic acid metabolism, drug catabolism, and metabolism of benzene compounds.In KEGG pathway analysis, it was shown that both ALOXE3 and FOXM1 were negatively regulated in fatty acid metabolism and degradation pathway, drug metabolism and valine-leucine-isoleucine degradation.In addition, many terms possibly associated with ferroptosis such as lipid modi cation, lipid catabolic process, lipid homeostasis were enriched in the GO biological process, while fatty acid metabolism, fatty acid degradation, peroxisome were enriched in the pathways of KEGG analysis for ALOXE3.All these results again convinced our primary conclusions that FOXM1 and ALOX3 has close correlations in regulation of LIHC.

FOXM1 knockdown can upregulate the expression of ALOXE3
To further verify the negative correlation between FOXM1 and ALOXE3, siRNA knockdown of FOXM1 was carried to see the expression patterns of FOXM1 and ALOXE3.Two siRNAs (#1 and #2) were designed to knockdown FOXM1 and their effects on the expression of FOXM1 and ALOXE3 were assayed by qRT-PCR and Western blot, respectively.It was shown that the e ciency of knocked down by siFOXM1#1 and #2 for the mRNA expression of FOXM1 was − 19.4% and − 78.0%, (P < 0.001) respectively (Fig. 7-a), while ALOXE3 was upregulated by 3.1 and 2.5 times (P < 0.01) (Fig. 7-b) respectively; Accordingly, the e ciency of knocked down by siFOXM1#1 and #2 for the protein expression of FOXM1 was − 40.5% and − 94.2% (P < 0.001) respectively (Fig. 7-d), while ALOXE3 was upregulated by 1.93 and 1.28 times (P < 0.01) respectively (Fig. 7-e).As FOXM1 transcriptionally regulates the expression of a plethora of genes involved in various cellular processes such as cell cycle, DNA damage response, senescence, apoptosis, migration, invasion, oxidative stress, and drug resistance, many downstream targets such as Cyclin B1, CDC25B, Cyclin A, BCL-2, MMP-2, MMP9, VEGFR were regulated by FOXM1 (Kalathil et al. 2020).The results suggested that FOXM1 is involved in the metabolism of ALOXE3, and ALOXE3 may also be a possible downstream target for FOXM1.It again con rmed our hypothesis that knockdown of FOXM1 can upregulate the expression of ALOXE3, leading to the ferroptosis of the cancer cells.Nevertheless, the relationship between FOXM1 and ALOXE3 needs to be further explored.

Conclusions
In summary, we demonstrated that MCP-1, a FOXM1 targeting peptide optimized from P201, can inhibit the expression of FOXM1 and induce ferroptosis in HCCLM3 cells by upregulating the expression of ALOXE3, total iron content and ROS, while downregulating the content of GSH and expression of GPX4.Meanwhile, it is the rst report putting FOXM1 and ALOXE3 together.It should be noted that since FOXM1 can regulate a large number of target genes involved in various biological processes, the exact regulatory role of FOXM1 in ferroptosis and its association with ALOXE3 are still unclear and needed to be further studied.

Declarations
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