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Published: 30 October 2023

YTHDC1 as a tumor progression suppressor through modulating FSP1-dependent ferroptosis suppression in lung cancer

Cell Death & Differentiation volume 30, pages 2477–2490 (2023)Cite this article

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Abstract

Ferroptosis is a regulated cell death process initiated by iron-dependent phospholipid peroxidation and is mainly suppressed by GPX4-dependent and FSP1-dependent surveillance mechanisms. However, how the ferroptosis surveillance system is regulated during cancer development remains largely unknown. Here, we report that the YTHDC1-mediated m⁶A epigenetic regulation of FSP1 alleviates the FSP1-dependent ferroptosis suppression that partially contributes to the tumor suppressive role of YTHDC1 in lung cancer progression. YTHDC1 knockdown promoted the lung tumor progression and upregulated FSP1 protein level that resulted in ferroptosis resistance of lung cancer cells. Silencing FSP1 abrogated YTHDC1 knockdown-induced proliferation increase and ferroptosis resistance. Mechanistically, YTHDC1 binding to the m⁶A sites in the FSP1 3’-UTR recruited the alternative polyadenylation regulator CSTF3 to generate a less stable shorter 3’-UTR contained FSP1 mRNA, whereas YTHDC1 downregulation generated the longer 3’-UTR contained FSP1 mRNA that is stabilized by RNA binding protein HuR and thus led to the enhanced FSP1 protein level. Therefore, our findings identify YTHDC1 as a tumor progression suppressor in lung cancer and a ferroptosis regulator through modulating the FSP1 mRNA stability and thus suggest a ferroptosis-related therapeutic option for YTHDC1^high lung cancer.

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Introduction

Ferroptosis is a form of regulated cell death induced by iron-dependent lipid peroxidation [1] and cancer cells have evolved multiple surveillance mechanisms to escape ferroptosis and progress to advanced stages [2]. GPX4 and FSP1 are two major ferroptosis surveillance proteins that detoxify lipid hydroperoxides, either via reduction of phospholipid hydroperoxides (PLOOHs) to corresponding alcohols (PLOHs) or via reduction of ubiquinone to ubiquinol, respectively [3, 4]. FSP1, previously known as a p53-induced target protein [5], suppresses ferroptosis by catalyzing the regeneration of CoQ₁₀ using NAD(P)H in a GPX4-independent manner [6, 7]. Although ferroptosis has been shown to be involved in multiple diseases, little is known about how these two ferroptosis surveillance proteins are regulated during cancer development.

The N6-methyladenosine (m⁶A) modification of RNA is capable of regulating many RNA processes, such as splicing, polyadenylation, nuclear export, and translation, through m⁶A “writers” (methyltransferases), “erasers” (demethylases) and “readers” [8]. Most m⁶A “readers” contain the YTH (YT521-B homology) domain to bind m⁶A modified sites [9, 10] and YTHDC1 is known as the YTH domain-containing nuclear reader and exerts its multifaceted effects to determine the fates of m⁶A-marked mRNA in the nucleus [11]. As a result, YTHDC1 plays an essential role in normal physiological development processes, such as guarding the identity of mouse embryonic stem cells [12, 13] and ensuring the appropriate mouse oocyte development [14]. In addition, YTHDC1 plays different roles in cancers, such as promoting leukemogenesis as an oncogene [15] and predicting the prognosis of endometrial cancer as a biomarker [16]. Interestingly, YTHDC1 is able to form YTHDC1-m⁶A condensates through liquid-liquid phase separation to prevent the degradation of m⁶A-mRNAs that leads to their oncogenic activity in myeloid leukemia [17]. Although YTHDC1 has been reported to mediate the function of several oncogenes or tumor suppressors as an m⁶A reader [18, 19], the specific role and mechanism of YTHDC1 in lung cancer development remains largely elusive.

In this study, we identify YTHDC1 as a tumor progression suppressor in lung cancer and demonstrate that the modulation of the FSP1-dependent ferroptosis activity by YTHDC1 partially contributes to its tumor progression suppressor’s activity.

Results

YTHDC1 is negatively correlated with the progression of lung cancer patients

It has been known that the dysregulation of RNA m⁶A modification by its regulators is critical for cancer initiation and progression [20]. To systematically screen the expression patterns of m⁶A “writers”, “erasers” and “readers” along the tumor progression of lung cancer, we analyzed The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases and found that among the 32 m⁶A modification-related genes, 24 genes were upregulated and 7 genes were downregulated in the lung cancer tissues compared to the normal tissues, and YTHDC1 is one of the downregulated genes (Fig. 1A, Supplementary Fig. S1). Further analysis using TCGA database revealed that 8 of those 24 upregulated genes were continuously upregulated and 3 of those 7 downregulated genes, including YTHDC1, were consistently downregulated in T2-4 tumors compared to T1 tumors (Fig. 1A, Supplementary Fig. S2). Interestingly, PCIF1 and YTHDC1 were still downregulated in N2-3 tumor tissues with lymph node spread compared with N0-1 tumor tissues (Fig. 1A, Supplementary Fig. S3), and YTHDC1 is the only one gene that was gradually decreased with the progression of lung cancer from stage I to the advanced stage III-IV (Fig. 1A, Supplementary Fig. S4). Therefore, these analyses revealed that YTHDC1 was the only m⁶A modification regulator that was persistently decreased during the initiation and progression of lung cancer (Fig. 1A), suggesting a potential tumor suppressive role for YTHDC1 in lung cancer progression.

**Fig. 1: YTHDC1 is negatively correlated with the progression of lung cancer.**

To further assess the clinical relevance of YTHDC1 in lung cancer patients, we performed a meta-analysis to assess the association of YTHDC1 expression with outcomes of lung cancer patients using the Kaplan–Meier Plotter (KMplot) program. Using median gene expression values as the cutoff, we observed low expression of YTHDC1 was significantly associated with worse overall survival in patients with lung cancer (Fig. 1B). This association remains significant by a multivariate Cox proportional-hazards regression analysis after adjustment for tumor stage, gender, and smoking history (Fig. 1C). Taken together, these data suggest that YTHDC1 may function as a tumor progression suppressor in lung cancer.

YTHDC1 downregulation promotes lung cancer progression

To verify the findings obtained from those bioinformatics and clinical analyses, we examined YTHDC1 protein levels in 95 lung tumor tissues (Supplementary Table S1) by immunohistochemical staining and divided these tumor samples into YTHDC1^low (immunostaining score ≤ 75) and YTHDC1^high (immunostaining score > 75) groups according to its nuclear staining (Fig. 2A). Kaplan–Meier analysis indicated that low level of nuclear YTHDC1 was significantly associated with worse outcome in patients with lung cancer (Fig. 2B). Although it is known as a nuclear protein, YTHDC1 was detected in both cell nucleus and cytoplasm in the lung tumor tissues. Based on the subcellular localization of YTHDC1 protein, we divided tumor samples into two groups: predominant nuclear localization (nuclear immunostaining score > cytoplasmic immunostaining score) and predominant cytoplasmic localization (nuclear immunostaining score ≤ cytoplasmic immunostaining score) (Fig. 2C). Kaplan–Meier analysis showed that predominant cytoplasm localization group had a worse prognosis of overall survival in lung cancer patients (Fig. 2D), suggesting that less nuclear YTHDC1 level is correlated with progression of lung cancer as well.

**Fig. 2: YTHDC1 suppresses lung cancer progression.**

To directly evaluate the potential tumor progression suppressor activity of YTHDC1 in lung cancer, we established YTHDC1-overexpressing stable cell line by introducing Flag-tagged YTHDC1 into H460 cells (Fig. 2E), a lung cancer cell line with relatively low YTHDC1 protein levels compared to other cell lines (Supplementary Fig. S5A). Overexpression of YTHDC1 significantly decreased the proliferation and colony formation of H460 cells (Fig. 2F, G), suggesting a tumor suppressive activity of YTHDC1 in lung cancer cells. To further confirm these results, we stably knocked down YTHDC1 in A549 and H1299 cells by two independent YTHDC1 shRNAs (Fig. 2H, Supplementary Fig. S5B). YTHDC1 knockdown significantly promoted the proliferation of A549 and H1299 cells in vitro (Fig. 2I) as well as significantly increased the growth of A549 xenograft tumors and their size in vivo (Fig. 2J–L, Supplementary Fig. S5C). Taken together, the clinical and functional data indicate that YTHDC1 has a tumor suppressive role in lung cancer progression.

YTHDC1 downregulation increases the ferroptosis resistance

To explore the potential mechanisms that YTHDC1 suppressed lung cancer progression, we conducted an RNA-seq for control and stable YTHDC1-knockdown A549 cells and found 291 differentially expressed genes (DEGs), including 151 upregulated genes and 140 downregulated genes (q < 0.05) (Supplementary Fig. S5D, Supplementary Table S2). KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis of these DEGs revealed ferroptosis as one of the most affected cellular processes in YTHDC1-knockdown cells (Fig. 3A), and the altered genes (including TP53, CP and SLC40A1) in the ferroptosis category were validated by qRT-PCR in the YTHDC1 knockdown cells (Supplementary Fig. S5E). By using gene set enrichment analysis (GSEA), we found a correlation between YTHDC1 and ferroptosis activity (Fig. 3B), suggesting that YTHDC1-knockdown might lead to ferroptosis suppression in lung cancer. Although KEGG pathway analysis indicated that apoptosis was one of the affected cellular processes as well, the caspase-3 protein (a key mediator and a well-established cellular marker of apoptosis) level was not altered in YTHDC1-knockdown cells (Supplementary Fig. S5F).

**Fig. 3: YTHDC1 promotes ferroptosis activity.**

To validate the association between YTHDC1 and ferroptosis activity, we knocked down YTHDC1 in A549 and H1299 cells and found that more colonies were formed in YTHDC1 knockdown cells with RSL3 treatment (Fig. 3C, D). Furthermore, both A549 and H1299 cells with YTHDC1 knockdown were more resistant to RSL3-induced ferroptosis (Fig. 3E, F; Supplementary Fig. S6A). The similar resistant results were found for both A549 and H1299 cells with YTHDC1 knockdown treated with other ferroptosis inducers, including Erastin (Fig. 3G, H), FIN56 (Supplementary Fig. S6B), and FINO₂ (Supplementary Fig. S6C). As ferroptosis can be indicated by enhanced PTGS2 expression and increased ROS level [21], we found that knocking down YTHDC1 down-regulated PTGS2 mRNA expression (Fig. 3I) and decreased the cellular ROS level upon the RSL3 treatment (Fig. 3J). Furthermore, ferroptosis is characterized morphologically by smaller mitochondria with condensed mitochondrial membrane densities, reduced or vanished mitochondria crista and ruptured outer mitochondrial membrane [22]. As shown in Fig. 3K, RSL3 treatment induced the typical morphological alterations of mitochondria, indicative of ferroptosis occurrence, whereas YTHDC1 knockdown alleviated these RSL3-induced mitochondrial changes in A549 cells.

We further directly evaluated the lipid peroxidation by using BODIPY-C11, a well-known lipid peroxidation marker. Since our shRNA-YTHDC1 vectors contained GFP fluorescence, which interfered with the detection of lipid peroxidation with BODIPY-C11, we established the YTHDC1 knockout A549 cells using CRISPR/Cas9 (Supplementary Fig. S6D). Although we didn’t select the single clone with undetectable YTHDC1 protein level, our pooled population A549 cells with YTHDC1 knockout showed a decreased lipid peroxidation level upon the RSL3 treatment and more resistant to RSL3-induced ferroptosis indicated by an increased IC50 value (Supplementary Fig. S6E, F). Therefore, YTHDC1 downregulation leads to ferroptotic resistance in lung cancer cells.

YTHDC1 regulates FSP1 at the post-transcriptional level in an m⁶A-dependent manner

GPX4 and FSP1 are two independent regulators to suppress ferroptosis [23]. Interestingly, both A549 and H1299 cells with YTHDC1 knockdown showed more resistant to RSL3-induced ferroptosis (Fig. 3E, F), but more sensitive to FSP1 inhibitor iFSP1 (Fig. 4A), suggesting that YTHDC1 knockdown may differentially regulate GPX4 and FSP1 levels in lung cancer. Indeed, YTHDC1 knockdown did not simultaneously upregulate the GPX4 and FSP1 mRNA expression in A549 and H1299 cells (Fig. 4B), instead markedly upregulated the FSP1 protein levels (Fig. 4C). A negative correlation between the transcription levels of YTHDC1 and FSP1 protein levels was also identified when examining cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) (Supplementary Fig. S7A). In line with these findings, we also found that overexpression of Flag-tagged YTHDC1 decreased the FSP1 protein level in H460 cells (Supplementary Fig. S7B). Taken together, these data suggest that YTHDC1 negatively regulates FSP1 protein levels.

We next explored the underlying mechanism how YTHDC1 regulates FSP1. The mRNA levels of FSP1 were marginally increased in YTHDC1 knockdown cells (Fig. 4B), suggesting that YTHDC1 regulated the protein level of FSP1 was not at the transcriptional level. Given that YTHDC1 is an m⁶A reader, we analyzed m⁶A sites in FSP1 mRNA by SRAMP prediction program and found most of the m⁶A sites were located in the 3’-untranslated region (UTR) of FSP1 mRNA (Fig. 4D). Actually, methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP)-qPCR assays demonstrated that FSP1 mRNA was modified by m⁶A in both A549 and H1299 cells and FSP1 mRNA was recognized and bound by YTHDC1 (Fig. 4E). By using siRNAs to knock down METTL3 (an m⁶A writer) to decrease the total m⁶A methylation level, we found that the mRNA level of FSP1 was not changed, but the FSP1 protein level was decreased in A549 cells (Fig. 4F, G). Additionally, METTL3 knockdown partially abrogated the YTHDC1 knockdown caused upregulation of FSP1 protein (Fig. 4H). All these data suggest that YTHDC1 suppresses FSP1 at the post-transcriptional level in an m⁶A-dependent manner.

YTHDC1 has previously been implicated in regulating gene expression at the post-transcriptional level through mediating the nuclear export or the decay of m⁶A methylated mRNAs [24,25,26]. First, we found that knockdown of YTHDC1 slightly increased the nuclear fraction of FSP1 mRNA in A549 cells (Fig. 4I), and did not disturb the nuclear fraction and cytoplasmic fraction of FSP1 mRNA in H1299 cells (Fig. 4J), ruling out the possibility that YTHDC1 regulates FSP1 through the mRNA nuclear export. We then treated cells with Actinomycin D, an inhibitor of RNA polymerase II, to block new RNA synthesis and found that YTHDC1 knockdown enhanced the stability of FSP1 mRNA in both A549 and H1299 cells (Fig. 4K, L), suggesting that YTHDC1 regulates the stability of FSP1 mRNA through m⁶A-mediated RNA decay processing.

YTHDC1 knockdown produces a longer 3’-UTR-containing FSP1 mRNA through alternative polyadenylation

Two poly(A) sites are identified in the 3’-UTR of FSP1 mRNA in addition to the m⁶A sites (Fig. 4D), raising a possibility that YTHDC1 may regulate the RNA decay of FSP1 mRNA through alternative polyadenylation (APA). To determine which poly(A) site of FSP1 mRNA YTHDC1 uses, we designed two paired qRT-PCR primers that specifically recognized the longer 3’-UTR of FSP1 mRNA to monitor the distal poly(A) site usage of FSP1 mRNA (Fig. 4D). Knockdown of YTHDC1 significantly increased the levels of the longer 3’-UTR-containing FSP1 mRNA in A549 and H1299 cells (Fig. 5A), suggesting that YTHDC1 knockdown promoted the distal poly(A) site usage to produce a longer 3’-UTR-containing FSP1 mRNA isoform. To further investigate how YTHDC1 regulates the 3′-UTR processing, we use the STRING database (version: 11.5) to search the top 10 proteins that are predicted to physically interact with YTHDC1 and found that seven out of ten YTHDC1 binding proteins are the components of the cleavage and polyadenylation (CPA) complex [27], which has been shown to play a critical role in APA formation and regulation (Supplementary Fig. S8A). Most of the seven YTHDC1 binding CPA factors, including CSTF3, CSTF1, CPSF1, CPSF2, and FIP1L1, are reported to use the proximal poly(A) sites that result in shorter 3′-UTR-containing mRNA [28]. We showed that YTHDC1 was able to co-immunoprecipitate with CSTF3, the core CPA complex factor, in both A549 and H1299 cells (Fig. 5B). Immunofluorescent staining further confirmed that Flag-tagged YTHDC1 co-stained with CSTF3 in A549 cells (Fig. 5C). These data suggest that YTHDC1 interacts with CPA complex to promote the proximal poly(A) site usage.

**Fig. 5: YTHDC1 knockdown regulates the alternative polyadenylation and increases the long isoform of *FSP1* mRNA, which can be bound and stabilized by HuR protein.**

HuR stabilizes the longer 3’-UTR-containing FSP1 mRNA to enhance FSP1 protein level

Longer 3′-UTR usually leads to the decreased stability of mRNA due to harboring miRNA regulatory sites [29]. However, some longer 3′-UTRs that harbor RNA regulatory elements (such as AU-rich elements) are able to increase the mRNA stability due to the binding of RNA binding proteins (RBPs) to these regulatory elements. By using ENCORI database, we identified 87 RBPs that bind to FSP1 mRNA (Supplementary Table S3). Gene Ontology (GO) analysis showed that the 87 RBPs were significantly enriched in mRNA 3′-UTR binding and regulating 3′-UTR-mediated mRNA stability. Among the GO terms, four proteins (HuR, HNRNPC, TARDBP and RBM10) were enriched in 3′-UTR-mediated mRNA stabilization (Supplementary Fig. S8B), which may increase the stability of FSP1 mRNA. We then validated the binding effect of FSP1 mRNA 3’-UTR (FSP1 mRNA region: 1430-3137) to the HuR, HNRNPC, TARDBP, and RBM10 proteins using catRAPID omics v2.1, and found that HuR protein showed the highest ranking of 3′-UTR binding effect (Fig. 5D). Previous studies have demonstrated that HuR was capable of stabilizing the target 3′-UTR-containing mRNA through AREs (AU-rich elements) [30]. The RIP assay confirmed that HuR was able to bind to FSP1 mRNA in A549 cells (Fig. 5E), while knocking down HuR with siRNAs decreased stability of FSP1 mRNA that also resulted in the decreased FSP1 protein level (Fig. 5F, G). Functionally, A549 cells with HuR knockdown were more sensitive to RSL3-induced ferroptosis (Fig. 5H), suggesting that HuR mediates the stabilization of FSP1 mRNA and ferroptosis resistance caused by YTHDC1 knockdown.

We further mapped the binding regions of HuR to the FSP1 mRNA. The in vitro RNA pull-down assays showed that HuR mainly interacted with the 3′-UTR of FSP1 mRNA (Fig. 5I), which is further confirmed by the decreased binding of mutant 3′-UTR of FSP1 mRNA to HuR proteins, wherein the three potential HuR binding sites ATTTA were mutated into AGGGA in the 3′-UTR of FSP1 mRNA (Fig. 5J). Two luciferase reporter plasmids containing 3′-UTR of FSP1 mRNA (wild type: ATTTA; mutant type: AGGGA) were constructed and transfected into A549 cells. As expected, the luciferase activity was significantly reduced with the knockdown of HuR for wild type luciferase reporter but not for the mutant luciferase reporter (Fig. 5K). The RIP assay further showed that knockdown of YTHDC1 increased the binding of HuR to FSP1 mRNA (Fig. 5L). Taken together, these results demonstrate that YTHDC1 knockdown promotes the stability of FSP1 mRNA with a longer 3′-UTR through APA, and HuR subsequently binds to the longer 3′-UTR of FSP1 mRNA to stabilize FSP1 mRNA and enhance its protein level.

YTHDC1 provides a ferroptotic vulnerability for lung cancer treatment

To test whether the upregulation of FSP1 mediates the tumor suppressor activity of YTHDC1 in lung cancer cells, we first examined the cell viability of YTHDC1-overexpressing H460 cells treated with ferroptosis inhibitors (Fer-1). Interestingly, the inhibiting effect of YTHDC1 on cell growth was partially restored by treating cells with Fer-1 (Supplementary Fig. S9A), whereas Fer-1 treatment did not significantly increase cell viability for vector control H460 cells and A549 cells (Supplementary Fig. S9A, B), suggesting that YTHDC1 overexpression suppresses cancer cell growth partially through ferroptosis. In parallel with this, we used siRNA to knock down FSP1 (Supplementary Fig. S9C) and found that downregulating FSP1 did decrease the proliferation of YTHDC1-knockdown A549 cells (Fig. 6A). To directly determine whether YTHDC1 modulates ferroptosis activity through FSP1. We knocked down FSP1 in A549 cells and found that FSP1 knockdown abrogated the RSL3-induced ferroptosis resistance in YTHDC1-knockdown cells (Fig. 6B), indicating that the YTHDC1-knockdown caused RSL3 resistance was mediated at least partially by the enhanced FSP1-dependent ferroptosis suppression.

The effects of YTHDC1 on ferroptosis may suggest a ferroptotic vulnerability for YTHDC1^high lung cancer. To test that, we first examined the YTHDC1 and FSP1 levels in four lung cancer cell lines (H1299, H1975, H460, and A549). We found that H460 cell showed the highest FSP1 level and lowest YTHDC1 level, while H1299 cell showed the lowest FSP1 level and highest YTHDC1 level (Supplementary Fig. S9D). The IC50 assay showed that H1299 cells with YTHDC1^high genotype were more sensitive to RSL3-induced ferroptosis than H460 cells with YTHDC1^low genotype (Fig. 6C), suggesting that YTHDC1^high lung cancer might be more susceptible for ferroptosis inducers. In line with these findings, overexpression of YTHDC1 in H460 cells increased their susceptibility to ferroptosis (Fig. 6D). To further test the effects of YTHDC1 on ferroptosis in tumor tissues, we examined the levels of 4-HNE (byproducts of lipid peroxidation) in the same 95 lung tumor tissues as above using immunohistochemical staining. We found that levels of 4-HNE in YTHDC1^high lung tumor tissues were higher than that in YTHDC1^low lung tumor tissues (Fig. 6E, F). Taken together, these data suggested that YTHDC1 increased the ferroptotic vulnerability in lung cancer.

Discussion

In summary, we demonstrate in this study that YTHDC1 functions as a tumor progression suppressor in lung cancer through regulating FSP1-mediated ferroptosis suppression. Mechanistically, YTHDC1 binds to the 3′-UTR of FSP1 mRNA that recruits APA processing factor CSTF3 to generate unstable FSP1 mRNA isoform with shorter 3′-UTR; downregulation of YTHDC1 decreases the recruitment of CSTF3 to generate the FSP1 mRNA isoform with longer 3′-UTR, which is subsequently stabilized by HuR to increase its protein level (Fig. 7). This mechanism may explain why YTHDC1^low lung cancer patients have a poor prognosis whereas YTHDC1^high lung cancer may have a therapeutic vulnerability for ferroptosis treatment.

**Fig. 7: A working model shows how YTHDC1 induces ferroptosis by regulating FSP1 in lung cancer.**

YTHDC1 is identified as the mainly nuclear m⁶A reader to control the fate of m⁶A modified transcripts in different stages of the mRNA life cycle in the nucleus, such as mRNA splicing, transcription and nuclear export [11]. Particularly, it has been recently proposed that the formation of YTHDC1 nuclear condensates by liquid-liquid phase separation is a general mechanism to underlie its diverse functions in the nucleus [17, 31]. The tumor suppressor gene PTEN and oncogene MYC have been reported to be regulated by YTHDC1 [17, 32], indicating a differential role of YTHDC1 in cancer development. Our study here demonstrated that YTHDC1 is able to suppress lung cancer progression, at least partially, through regulating FSP1-mediated ferroptosis suppression, suggesting a new tumor suppressive role of YTHDC1 in lung cancer progression. Whether YTHDC1 functions as an authentic tumor suppressor in lung tumorigenesis, however, needs to be further investigated.

Ferroptosis is a non-apoptotic form of regulated cell death that can be triggered by phospholipid peroxidation of polyunsaturated fatty acids (PUFAs) and involved in multiple diseases such as neurodegeneration and cancer [33, 34]. Cancer cells are usually more metabolically active and tend to produce more ROS and PUFAs that make them more prone to ferroptosis [23]. However, cancer cells have evolved a variety of mechanisms to suppress ferroptosis at the genetic and epigenetic levels. GPX4 and FSP1 are the two important evolved surveillance mechanisms to protect cancer cells from ferroptosis [23]. We found that knockdown of YTHDC1 in lung cancer cells did not alter the GPX4 level, but remarkably promoted the protein level of FSP1 in an m⁶A dependent manner, which may provide new therapeutic targets for cancers [35, 36]. A recent study reported that FSP1 was a transcriptional target of NRF2 and mediated ferroptosis-resistance in KEAP1 deficient lung cancer cells [37]. A549 and H460 cells, two cell lines used in our study, have been reported to have mutant KEAP1 and thus exhibited high NRF2 activity and FSP1 expression. Our results showed that A549 and H460 cells harbored higher FSP1 protein levels and were more resistant to RSL3 than H1299 cells which contains wild type KEAP1. Interestingly, in addition to the regulation of FSP1 mRNA at the transcriptional level by NRF2, our study further demonstrated that FSP1 mRNA was regulated at the post-transcriptional level by YTHDC1 as well, which suggests another layer of FSP1 regulation.

APA is a widely-used gene regulation mechanism for many cellular functions [38] and dysregulation of APA contributes to the development of various diseases, including cancer [39]. A most recent study showed that APA events with more longer isoforms are found in Ythdc1-deficient oocytes [14]. In consistent with these results, we identified that YTHDC1 is able to regulate the stability of FSP1 mRNA through APA and knockdown of YTHDC1 increased the abundance of FSP1 mRNA isoforms with longer 3′-UTR. Usually longer 3′-UTR containing mRNA tends to have a decreased stability due to the miRNA regulatory sites in the mRNA 3′-UTR region [29, 40]. However, several mRNAs with longer 3’-UTRs have increased mRNA stability due to the AREs in the 3′-UTR, which can recruit HuR protein and stabilize the target longer 3′-UTR isoforms through ARE-mediated stabilization [29, 41]. Our study showed that the longer 3′-UTR of FSP1 mRNA harbored ARE sites and can be stabilized by the HuR binding, therefore increasing the FSP1 protein levels in YTHDC1 knockdown cells. Loss of YTHDC1 has been shown to alter 3′-UTR length for more than 800 genes in oocytes and most of them are favoring the longer 3′-UTR [14], however, the detailed biological outcomes and mechanisms are still elusive. Our findings that AREs-mediated HuR binding and stabilizing the longer 3′-UTR containing mRNA in YTHDC1 knockdown cells may provide a mechanism to explain how YTHDC1 regulates the RNA processes.

A reasonable hypothesis has been proposed that ferroptosis is a natural tumor suppressive mechanism contributing to the antitumor activity of the tumor suppressors [42]. In support of this hypothesis, several tumor suppressors, such as p53, BAP1 and fumarate hydratase (FH), exert their tumor suppressor’s functions through ferroptosis [42,43,44]. Especially, the ferroptosis-promoting effect of p53 alone has been implied to be sufficient to suppress tumorigenesis in vivo [45]. Tumor cells do not undergo spontaneous ferroptosis unless the ferroptosis defense systems are damaged by GPX4 or FSP1 dysregulation [46]. Our results that the suppression of tumor cell growth by overexpression of YTHDC1 can be partially abrogated by ferroptosis inhibitor Fer-1 suggest that YTHDC1 regulates tumor progression at least partially through ferroptosis. We have further shown that YTHDC1-mediated FSP1 protein regulation may alter the FSP1-dependent ferroptosis surveillance system and, therefore, contributes to the tumor suppressor’s activity of YTHDC1 in the lung cancer progression. However, whether there are other pathways (such as p53) and to what degree they are involved in YTHDC1’s tumor-suppressive activity as well as which endogenous ferroptosis inducers that YTHDC1-FSP1 signaling axis responds warrant further investigation.

Materials and methods

Cell lines, animals, and reagents

The lung cancer cell lines A549, H1299, H1975, and H460 were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China), and cultured in RPMI-1640 (Gibco, Life Technology, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco). All the cells were maintained at 37 °C with 5% CO₂. The male BALB/c-nude mice were purchased from the Vital River (Charles River China, Beijing, China). RSL3, Erastin, iFSP1, Fer-1 and Actinomycin D were purchased from MCE (New Jersey, USA). FIN56 and FINO₂ were purchased from Selleck (Texas, USA).

Tissue microarray (TMA) and immunohistochemical analysis

Tissue microarrays contained 98 lung cancer tissues (3 tissues were excluded from the analysis due to low tumor-cell content) were obtained from Shanghai Outdo Biotech Company (Shanghai, China) with the agreement of the patients with an informed consent form. IHC staining was performed using rabbit polyclonal antibody against YTHDC1 (Abcam, ab122340) or mouse monoclonal antibody against 4-HNE (Abcam, ab48506). Immunostaining was scored based on the extent of positive cell staining (0–100%) and the staining intensity (0, no staining; 1, slight staining; 2, moderate staining; and 3, strong staining). The final immunostaining score with a potential range of 0–300 was calculated as follows: Immunostaining score = [extent of positive cell staining (0–100%) × staining intensity (0–3)] × 100.

RNA extraction and qRT-PCR analysis

The total RNA was isolated using the Eastep Super Total RNA Extraction Kit (Promega, Shanghai, China), and the nuclear and cytoplasmic RNA of cells were isolated using PARIS™ Kit (Invitrogen, NY, USA) following the manufacturer’s instructions. A total of 1000 ng RNA was used as a template for cDNA synthesis using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time PCR was performed using the SYBR Premix Ex Taq (TaKaRa). Results were normalized to the housekeeping gene GAPDH and relative gene expression levels were calculated using 2^^−(ΔΔCt) method. Primers sequences were provided in Supplementary Table S4.

Plasmid construction and cell transfection

For YTHDC1 knockdown, shRNA and corresponding control lentiviruses were synthesized by GeneChem (Shanghai, China), and infected cells were selected using puromycin (2 μg/ml; Biosharp, Anhui, China). For YTHDC1 knockout, CRISPR/Cas9 and corresponding control lentiviruses were synthesized by GeneChem. For knockdown of FSP1, METTL3, and HuR, siRNAs targeting FSP1, METTL3 or HuR were purchased from the GenePharma (Shanghai, China). The siRNAs were transfected into cells with Lipofectamine 2000 (Invitrogen). To construct a plasmid expressing YTHDC1, the coding sequence of human YTHDC1 was synthesized and cloned into the GV492 vector (GeneChem). The lentivirus containing Flag-tagged YTHDC1 gene were transfected into cells and were screened under 2 μg/ml puromycin selection. YTHDC1 shRNA, YTHDC1 sgRNA, METTL3 siRNA, FSP1 siRNA, and HuR siRNA target sequences were shown in Supplementary Table S4.

Cell viability assay in vitro

The cell viability was detected using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay, or CellTiter-Lumi™ Luminescent Cell Viability Assay Kit (Beyotime, Shanghai, China) based on quantification of adenosine triphosphate (ATP), which represented the number of active cells.

Colony formation assays

Cells were seeded in 6-well plates and media were changed every 3 days. Colonies were fixed with methanol and stained with 0.1% crystal violet (Beyotime) for 15 min. The visible colonies were then counted. All of the experiments were performed in triplicate.

Animal experiments in vivo

For subcutaneous xenograft model, ten 5 weeks old male BALB/c nude mice were randomly divided into two groups equally. Stable transfected A549 cells (5.0 × 10⁶), suspended in 200 μL of 50% Matrigel (Corning, New York, USA), were injected subcutaneously into the flanks of the nude mice. The tumor volumes were measured and were estimated by using the formula: V = 0.5 × D × d² (V, volume; D, longitudinal diameter; d, latitudinal diameter). The GFP fluorescent images of subcutaneous tumor were photographed and analyzed with Bruker Molecular Imaging Software (USA). All experimental animal procedures were approved by the Experimental Animal Welfare and Ethics, Zhongnan Hospital of Wuhan University.

Cellular ROS and lipid peroxidation measurement

For cellular ROS detection, cells (1 × 10⁵) were grown in 6-well plates overnight and were then treated with RSL3 (800 nM) for 24 h, followed by the addition of DHE (KeyGEN BioTECH, Jiangsu, China) for 60 min. Cells were harvested and washed with PBS and then were subjected to flow cytometry to measure the levels of cellular ROS. For cellular lipid peroxidation detection, cells (1 × 10⁵) were grown in 6-well plates overnight and were then treated with RSL3 (1 μM) for 24 h, followed by the addition of BODIPY 581/591 C11 (8 μM, ABclonal, Wuhan, China) for 60 min at 37 °C in the dark. Cells were harvested and washed with PBS and then were subjected to flow cytometry using the 488 nm and 561 nm laser for excitation. The BODIPY 581/591 C11 value was calculated as the ratio of the green fluorescence (which indicates oxidized probe) to total (green + red, which indicates total reduced plus oxidized probe) fluorescence.

RNA stability and Actinomycin D treatment

Cells (3.0 × 10⁵) were seeded into 6-well plates overnight and were then treated with Actinomycin D (5 μg/mL), and then were harvested for RNA purification at 0, 2, 4, 6, 8, or 10 h. FSP1 mRNA levels were subsequently measured by qRT-PCR to test whether YTHDC1 or HuR could regulate the stability of FSP1 mRNA.

Transcriptome sequencing and analysis

The transcriptome sequencing was conducted at Shenzhen BGI. In brief, the total RNA was extracted from A549 cells and then the RNA was subjected to sequencing with DNBSEQ platform in the company of BGI (Shenzhen, China). The data were analyzed and visualized with the Dr. Tom System (BGI).

Luciferase reporter assay

To detect the HuR binding sites in the 3′-UTR of FSP1 mRNA, the wild type (ATTTA) or mutant type (AGGGA) sequences of 3′-UTR of FSP1 mRNA were inserted into the pmirGLO luciferase vectors. 5 × 10³ A549 cells were seeded into 96-well plates in triplicate for each group. Cells were co-transfected with pmirGLO-FSP1-wild type 3′-UTR (or pmirGLO-FSP1- mutant type 3′-UTR) and siRNAs targeting HuR. Firefly and Renilla luciferase activities were measured at 48 h after transfection using the Dual-Luciferase Assay System (Beyotime) following the manufacturer’s instructions. The relative luciferase activity was calculated using firefly/renilla luciferase activity.

Western blot and co-immunoprecipitation

Western blot (WB) and co-immunoprecipitation (Co-IP) were performed as described previously [47]. The antibodies used in this study were rabbit polyclonal antibody against YTHDC1 (1:1000; Abcam), rabbit monoclonal to GPX4 (1:1000; Abcam), rabbit monoclonal to HuR (1:1000; Abcam), rabbit monoclonal to GAPDH (1:1000; Abcam), mouse monoclonal to FSP1 (1:1000; Santa Cruz, USA), rabbit polyclonal to CSTF3 (1:1000; ABclonal), and mouse monoclonal to Caspase-3 (1:1000; Santa).

RNA immunoprecipitation assay and methylated RNA immunoprecipitation (MeRIP) assay

RNA immunoprecipitation (RIP) assay was performed using EZ-Magna RIP^TM RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) following the manufacturer’s protocol. In briefly, cells were lysed using the RIP lysis buffer. Immunoprecipitation were conducted with magnetic beads coupled with anti-HuR, anti-YTHDC1, or IgG. The enrichment of FSP1 mRNA in immunoprecipitated RNAs was assessed by qRT-PCR assay. The methylated RNA immunoprecipitation (MeRIP) was performed to determine the m⁶A levels in FSP1 mRNA as described previously [48]. In briefly, 5 μg anti-m⁶A antibody (ABclonal) or IgG were bound to magnetic beads (Magna RIP Kit, Millipore). A total of 50 μg RNA from the cells was immunoprecipitated with anti-m⁶A antibody in RIP immunoprecipitation buffer (Magna RIP Kit, Millipore) overnight at 4 °C. After treating with proteinase K, RNA was extracted with an RNeasy Mini Kit (Qiagen). The enrichment of FSP1 in m⁶A immunoprecipitated RNAs was assessed by qRT-PCR assay.

RNA pull-down

Biotin-labeled FSP1 mRNA was obtained through in vitro transcription from vector pcDNA3.1-FSP1 mRNA 1-1429 and pcDNA3.1-FSP1 mRNA 1430-3137 using Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche). After treating with RNase-free DNase I (Promega), the biotin-labeled RNA was purified with an RNeasy Mini Kit (Qiagen, Germany) and then added into the A549 cell lysates. After incubating for 1 h, the biotin-labeled RNA was immunoprecipitated with Dynabeads^TM M-270 Streptavidin (Invitrogen). Proteins were then eluted from the dynabeads and detected by WB.

Immunofluorescence cell staining and transmission electron microscopy

A549 cells transfected with Flag-tagged YTHDC1 fusion vector were grown on a sterile confocal petri dish (Biosharp), fixed in methanol for 15 min, permeabilized by 0.1% Triton X-100, and then blocked with 1% BSA for 30 min. Mouse anti-Flag (Sigma) and rabbit polyclonal to CSTF3 (ABclonal; A11665) were incubated overnight at 4 °C. After washing for three times, the cells were probed with Alexa-Fluor-555-conjugated goat anti-rabbit-IgG (Abcam) and Alexa-Fluor-647-conjugated goat anti-mouse-IgG (Abcam) for 1 h at room temperature, followed by nuclear counterstaining with DAPI (Beyotime). Cells were observed with a confocal fluorescence microscopy (Leica, Germany). For detecting the morphological changes of ferroptosis, cells were observed in a transmission electron microscopy (TEM) by Hitachi (HT7700 Exalens).

Bioinformatics analysis

To assess the clinical significance of YTHDC1 in lung cancer, the lung cancer RNA-seq data and clinical data were downloaded from the GTEx Portal (version V8) database and the Cancer Genome Atlas (TCGA) (version 2019-7-20). We compared the gene expression levels among patients subgrouped by tumor size, lymph node spread, and TNM stage. To evaluate the prognostic value of YTHDC1, Kaplan–Meier curves were generated using the Kaplan–Meier Plotter (KMplot) program (www.kmplot.com, version 2022). Four probes (228556_at, 212455_at, 214814_at and 240459_at) targeting YTHDC1 were used. The patients were classified into two groups using median gene expression value as the cutoff.

Statistical analysis

Statistical analyses were performed using the SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Results were analyzed using the Wilcoxon rank-sum test, Student’s t-test, Kruskal–Wallis test or repeated measures analysis of variance. A two-sided P-value less than 0.05 was taken as statistically significant.

Data availability

All data needed to evaluate the conclusions are present in the paper and the supplementary files.

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Funding

This work was supported by the National Natural Science Foundation of China (No. 82002447 to SY; 82173131 to HH), the Outstanding Young and Middle-aged Talents Training Program of Zhongnan Hospital of Wuhan University (Grant No. ZNYQ2022003 to SY), the Science and Technology Innovation Commission of Shenzhen Municipal Government Grants (JCYJ20210324104007022 to HH), Guangdong provincial fundings: 2021KTSCX108 (to XH), and the Program of Excellent Doctoral (Postdoctoral) of Zhongnan Hospital of Wuhan University (No. ZNYB2020028 to SY).

Author information

These authors contributed equally: Shuai Yuan, Shu Xi, Hong Weng.
These authors jointly supervised this work: Yafei Li, Xian-Tao Zeng, Hailiang Hu.

Authors and Affiliations

Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
Shuai Yuan, Shu Xi, Hong Weng, Meng-Meng Guo, Jin-Hui Zhang, Zhi-Ping Yu, Meng-Yang Liu, Dao-Jing Ming & Xian-Tao Zeng
Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Shenzhen, China
Haozhe Zhang, Zengzhen Xing, Rajiv Kumar Sah, Yi Zhou, Xin Hong & Hailiang Hu
Department of Urology, The Second Affiliated Hospital of Nanchang University, Nanchang, China
Zhaojun Yu & Tao Zeng
Cancer Center, Faculty of Health Sciences, MoE Frontier Science Center for Precision Oncology, University of Macau, Taipa, Macau, SAR, China
Gang Li
Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen, China
Xin Hong & Hailiang Hu
Department of Epidemiology, College of Preventive Medicine, Army Medical University, Chongqing, China
Yafei Li

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Shuai Yuan
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Contributions

HH led the study by designing, interpreting results, obtaining the funding and reviewing the manuscript. SY, SX, and HW participated in performing the experiments, results interpretation, manuscript writing. MMG, JHZ, ZPY, HZ, ZY, ZX, DJM, RKS, YZ, and TZ participated in performing the experiments and collecting the data. MYL performed the bioinformatics analysis and participated in results interpretation. GL and XH participated in the manuscript discussion. YL and XTZ participated in study design, results interpretation and reviewing the manuscript.

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Correspondence to Yafei Li, Xian-Tao Zeng or Hailiang Hu.

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Yuan, S., Xi, S., Weng, H. et al. YTHDC1 as a tumor progression suppressor through modulating FSP1-dependent ferroptosis suppression in lung cancer. Cell Death Differ 30, 2477–2490 (2023). https://doi.org/10.1038/s41418-023-01234-w

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Received: 09 January 2023
Revised: 08 October 2023
Accepted: 17 October 2023
Published: 30 October 2023
Issue Date: December 2023
DOI: https://doi.org/10.1038/s41418-023-01234-w

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