USP29 and SMURF1 orchestrate FSP1-mediated ferroptosis suppression to facilitate chemoresistance in gastric cancer

Domain: neuroscience
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
Gastric cancer (GC) ranks as the third leading cause of cancer-related mortality. Chemoresistance poses a major obstacle to successful treatment for GC patients. Here, we demonstrate that ferroptosis suppressor protein 1 (FSP1) enables chemoresistance in GC by inhibiting ferroptosis, resulting in diminished chemotherapy response and reduced patient survival rate. USP29 and SMURF1 are subsequently discovered as the deubiquitinase and E3 ligase of FSP1, respectively, orchestrating the ubiquitination of FSP1 to modulate ferroptosis and chemoresistance in GC. In the ubiquitination process, SMURF1 is observed to interact with and polyubiquitinate FSP1 within K63/K193 sites. Notably, pharmacological inhibition of FSP1 by iFSP1 is shown to synergize with chemotherapeutic agents across chemoresistant cellular and mouse models. Our findings thus underscore the pivotal role of the USP29 and SMURF1 in regulating GC chemoresistance via FSP1-mediated ferroptosis suppression. FSP1 inhibition may represent a promising therapeutic avenue to overcome chemoresistance in GC.

Introduction
Gastric cancer is the fifth most diagnosed malignancy globally and stands out as a leading cause of cancer-related mortality1,2. Diagnosis typically occurs at advanced stages, precluding surgical intervention and contributing to a bleak prognosis3. Chemotherapy remains a cornerstone of gastric cancer treatment. Nevertheless, the emergence of chemoresistance has a marked impact on therapeutic efficacy, potentially resulting in treatment failure4,5. Consequently, there is an imperative need to investigate the precise mechanisms underpinning drug resistance and develop clinical strategies to address this challenge.

Cell survival and cell death are intricately controlled by a myriad of signals and molecules6,7. Regulated cell death (RCD) represents an orchestrated process that holds major implications across various facets of mammalian biology, encompassing development, homeostasis, and pathogenesis8–10. In recent years, multiple forms of RCD have emerged as critical players in human health and disease. Among these, ferroptosis stands out as a distinctive mode of regulated cell demise instigated by iron-driven lipid peroxidation11,12. Ferroptosis ensues when perturbations in intracellular iron levels precipitate an excessive buildup of deleterious lipid reactive oxygen species (ROS), overwhelming the cell’s antioxidant defenses and culminating in membranous structural damage13,14. Noteworthy efforts have elucidated numerous proteins and pathways governing the intricate regulation of ferroptosis.

Ferroptosis suppressor protein 1 (FSP1) has emerged as a regulator of ferroptosis inhibition15,16. Operating in concert with the glutathione-GPX4 axis, FSP1 functions as a nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent coenzyme Q (CoQ) oxidoreductase. Within the FSP1-NADPH pathway, CoQ10 catalyzes the direct synthesis of lipid-soluble antioxidants and sequesters lipid peroxyl radicals to mitigate lipid peroxidation within various biological membranes. Recent investigations have elucidated the roles of post-translational modifications (PTMs) in the regulation of ferroptosis, encompassing phosphorylation, acetylation, ubiquitination, methylation, and SUMOylation17–20. Ubiquitination entails the addition of ubiquitin moieties to lysine residues of target proteins, dictating their degradation and turnover dynamics21. Notably, recent studies have underscored the crucial regulatory functions of ubiquitination in the context of ferroptosis. Specifically, the OTU deubiquitinase ubiquitin aldehyde binding 1 (OTUB1) has been shown to interact directly with SLC7A11, stabilizing it through deubiquitination22. Additionally, research by Yang et al. unveiled the induction of Nedd4 by erastin, leading to increased ubiquitination and degradation of VDAC2/323. Nonetheless, the precise mechanisms by which ubiquitination and deubiquitination modulate FSP1 stabilization remain largely unexplored.

In this study, our findings show that FSP1 enhances chemoresistance and correlates with inferior survival outcomes in GC patients. Notably, depletion of FSP1 promotes chemosensitivity through the induction of ferroptosis. USP29 and SMURF1 orchestrate the protein homeostasis of FSP1 and thereby modulating ferroptosis and chemoresistance in GC. Importantly, the inhibitor of FSP1, iFSP1, exhibits synergistic effects with chemotherapy, effectively reversing chemoresistance. Therefore, we propose that FSP1 could serve as a predictive biomarker for chemoresistance and adverse survival prognosis in GC, and targeting FSP1 may represent a promising strategy for overcoming chemoresistance.

Results
FSP1 expression serves as an indicator for unfavorable chemotherapy response and survival in GC
To investigate the key genes contributing to chemoresistance in cancer, cisplatin treatment data in Genomics of Drug Sensitivity in Cancer (GDSC) (https://www.cancerrxgene.org/) were analyzed using weighted correlation network analysis (WGCNA)24. Initially, sample clustering was performed to identify outliers, revealing that all cancer cell lines were clustered together (Supplementary Fig. 1a). A soft threshold of beta = 18 was selected to construct a scale-free network (Supplementary Fig. 1b). Subsequently, 7 modules were delineated by grouping genes with similar expression patterns (Supplementary Fig. 1c). Notably, the turquoise module exhibited the strongest positive correlation with cisplatin resistance (Supplementary Fig. 1d), establishing it as a pivotal module for further investigation. Module membership and gene significance related to cisplatin resistance within the turquoise module are illustrated in Supplementary Fig. 1e. Interestingly, we found that the ferroptosis pathway was enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Supplementary Fig. 1f). Furthermore, the gene expression profiles of cells sensitive or resistant to cisplatin were analyzed. Setting the cut-off criteria as fold change > 2 and P < 0.01, we identified 156 genes with expression changes (94 genes were upregulated and 62 genes were downregulated) in the cisplatin-resistant group compared with cisplatin sensitive group (Supplementary Fig. 1g). Next, we took the intersection of genes in the turquoise module, 94 upregulated genes and genes in the ferroptosis pathway and obtained only one gene, FSP1 (Supplementary Fig. 1h), highlighting FSP1 as a potential key gene driving cisplatin resistance in cancer cells through the ferroptosis pathway. Indeed, we identified a positive correlation between FSP1 expression levels and chemotherapeutics IC values in the GDSC dataset (Supplementary Fig. 1i). Notably, this relationship was particularly pronounced in GC cells (Supplementary Fig. 1j), prompting our focus on GC for further investigation.

Although surgery is the main curative approach for resectable GC, the prognosis of patients remains unfavorable post-curative resection. Preoperative chemotherapy offers potential advantages like reducing the primary tumor size to enhance the chances and effectiveness of curative resection, as well as eradicating potential micro-metastases to prevent or lower tumor recurrence and spread, thus enhancing the management of tumor-related symptoms2. Nevertheless, ~15% of GC patients who receive preoperative chemotherapy face the risk of tumor progression3. Thus, accurately predicting patients’ response to preoperative chemotherapy remains particularly crucial.

To validate the findings from WGCNA and explore the role of FSP1 in chemotherapy response and outcomes in GC, we conducted a prospective clinical cohort study. A total of 164 GC patients scheduled with preoperative neoadjuvant therapy were enrolled in this study. The expression levels of FSP1 were evaluated in preoperative biopsy samples from the GC cohort, and no substantial differences in clinical characteristics were observed between the FSP1 low and high expression groups (Supplementary Table 1). Remarkably, we found that patients with low FSP1 exhibited favorable response, whereas those with elevated FSP1 expression demonstrated poor responses to preoperative neoadjuvant therapy (Fig. 1a–d and Supplementary Table 1). Subsequently, we performed immunohistochemistry (IHC) staining of 4-HNE to assess cell ferroptosis in GC specimens after preoperative neoadjuvant therapy (Fig. 1e). The results showed that FSP1 expression negatively correlates with 4-HNE levels (Fig. 1f). Furthermore, patients with high FSP1 expression show poorer survival in long-term follow-up (Fig. 1g, h). Collectively, these findings suggest that FSP1 correlates with chemoresistance and indicates poor patient outcomes in GC.

FSP1 renders chemoresistance in GC via repressing ferroptosis
To explore the causal relationship and underlying mechanism, we utilized two gastric cancer cell lines that acquired resistance to cisplatin for subsequent study (Supplementary Fig. 2a, b). The initial results revealed elevated expression of FSP1, but unchanged expression of other ferroptosis factors, and decreased level of lipid peroxidation induced by cisplatin in chemoresistance cell lines (Fig. 2a, b and Supplementary Fig. 2c–e). We then used two independent single guide RNAs (sgRNAs) to knockout FSP1 in AGS-R and MGC-803-R cells (Fig. 2c and Supplementary Fig. 2f), the results showed that FSP1 depletion markedly enhances the level of cisplatin-induced lipid peroxidation and vulnerability to cisplatin, without affecting DNA damage repair (Fig. 2d, e and Supplementary Fig. 2g–j). Reintroduction of wild-type (WT) FSP1, but not impaired myristoylation and plasma-membrane localization mutant (G2A), or unfunctional CoQ oxidoreductase mutant (E156A), could rescue cisplatin-induced lipid peroxidation and sensitivity to cisplatin contributed by FSP1 deficiency (Fig. 2f–h and Supplementary Fig. 2k–m). Furthermore, only Liproxstatin-1 (Lip-1, a ferroptosis inhibitor), but not Z-VAD-FMK (a pan-caspase inhibitor), 3-Methyladenine (3-MA, an autophagy inhibitor), or necrostatin 2 racemate (Nec-1s, a necroptosis inhibitor) could restore cisplatin susceptibility in FSP1-KO cells (Fig. 2i and Supplementary Fig. 2n).

The encouraging in vitro results prompted us to explore the impact of FSP1 on the chemoresistance of GC in mouse xenograft models. In vivo growth curves showed that the FSP1 KO tumors exhibited a modest reduction in size compared to tumors derived from parental cells prior to cisplatin treatment, but FSP1 KO was strikingly sensitive to cisplatin treatment (Fig. 2j and Supplementary Fig. 2o). Consistent results were observed upon the sacrifice of the mice and subsequent calculation of tumor weight (Fig. 2k and Supplementary Fig. 2p). Moreover, ferroptosis inhibitor of liproxstatin-1 (Lip-1) effectively reversed the heightened cisplatin sensitivity induced by FSP1 depletion in the in vivo setting (Fig. 2j, k and Supplementary Fig. 2o, p). Taken together, these data demonstrated that FSP1 facilitates gastric cancer chemoresistance in vitro and in vivo by suppressing ferroptosis.

USP29 binds and stabilizes FSP1
Due to the notable elevated protein levels, but not mRNA expression, of FSP1 in cisplatin-resistant cells compared to sensitive cells (Fig. 2a and Supplementary Figs. 2c and 3a), we investigated its stability in these cells. The results revealed a notable increase in FSP1 stability in resistant cells, accompanied by reduced ubiquitination levels (Fig. 3a, b and Supplementary Fig. 3b, c). Next, we set out to investigate the potential deubiquitinases (DUBs) associated with FSP1 by overexpressing a series of Flag-tagged DUBs in HEK293T cells and conducting co-immunoprecipitation (Co-IP) assays. As shown in Supplementary Fig. 3d, DUB3, USP11, USP29, USP33, CYLD, and USP43 exhibited observable interactions with FSP1 among the tested DUBs.

To further elucidate the roles of these six DUBs in modulating ferroptosis through their interactions with FSP1, we individually silenced them in HT1080 cells (Supplementary Fig. 3e) and evaluated cell viability following treatment with RSL3 or erastin. The results demonstrated that knockdown of USP29 most substantially sensitized cells to both RSL3 and erastin (Supplementary Fig. 3f, g). We further validated that silencing USP29 with short hairpin RNAs (shRNAs) enhanced ferroptosis in HT1080 induced by RSL3 or erastin (Supplementary Fig. 3h–j). Consequently, our study focuses on USP29 for an initial detailed investigation based on these compelling findings.

To further confirm the interaction between USP29 and FSP1, endogenous co-immunoprecipitation (Co-IP) assays were conducted, demonstrating that reciprocal immunoprecipitation of endogenous USP29 (or FSP1) effectively precipitated FSP1 (or USP29) (Fig. 3c, d). The binding regions between USP29 and FSP1 were delineated through a series of truncation experiments involving USP29 (Fig. 3e). Notably, the enzymatic domain (UCH domain) of USP29 was identified as crucial for mediating the USP29-FSP1 interaction (Fig. 3f). Consistently, knockdown of USP29 using two distinct USP29-specific shRNAs resulted in a marked reduction in FSP1 protein abundance, without affecting its mRNA levels or the expression of other ferroptosis factors (Fig. 3g and Supplementary Fig. 4a, b), implying a post-transcriptional regulatory role of USP29 on FSP1 expression. Moreover, the diminished FSP1 levels in USP29-depleted cells could be restored by treatment with the proteasome inhibitor MG132 (Fig. 3h), indicating that USP29 regulates FSP1 levels via proteasomal degradation.

Furthermore, reintroduction of shRNA-resistant WT USP29 into USP29 knockdown cells successfully restored FSP1 protein levels (Fig. 3i). To further address whether USP29 regulates FSP1 protein stability, cells were treated with cycloheximide (CHX) to determine the half-life of FSP1. The results revealed a notable decrease in FSP1 stability upon USP29 depletion, which could be rescued by ectopic expression of USP29 in the depleted cells (Fig. 3j, k). Collectively, these findings substantiate the role of USP29 in regulating FSP1 protein abundance and stability in a proteasome-dependent manner.

To further elucidate the mechanism by which USP29 mediates FSP1 stabilization, we investigated whether USP29 deubiquitinates FSP1 in cellular contexts. Our results showed that knockdown of USP29 led to a notable increase in polyubiquitination of FSP1 within cells (Fig. 3l). Conversely, ectopic expression of WT USP29, but not the catalytically inactive (CI) mutant USP29 (substitution of cysteine 294 to serine, C294S), resulted in a marked decrease in the polyubiquitination levels of FSP1 (Fig. 3m). To further probe the direct deubiquitination activity of USP29 towards FSP1, in vitro deubiquitination assay were conducted. The results showed that recombinant WT USP29, but not the USP29 CI mutant, demonstrated the ability to deubiquitinate FSP1 in vitro (Fig. 3n).

It is widely recognized that ubiquitin can form diverse types of ubiquitin chains through various lysine residues (Ub-K6, Ub-K11, Ub-K27, Ub-K29, Ub-K33, Ub-K48, and Ub-K63)25. Typically, ubiquitination via K48 of the ubiquitin chain marks proteins for proteasomal degradation, while K63-linked ubiquitination often modulates signaling pathways. To further address whether USP29 specifically deubiquitinates FSP1 on the K48-linked polyubiquitin chains, ubiquitination assays were performed using either K48/K63-intact or K48R/K63R ubiquitin variants. Expectedly, our results revealed extensive ubiquitination of FSP1 primarily through K48 linkages (Fig. 3o). Subsequently, we examined the impact of USP29 depletion on these linkages (Ub-K48) and observed that the loss of USP29 led to increased K48-linked polyubiquitination of FSP1 (Fig. 3p). Overall, these data strongly demonstrate that USP29 deubiquitinates FSP1 K48-linked polyubiquitin chains both in vitro and in vivo.

USP29 enables GC chemoresistance through FSP1-mediated ferroptosis inhibition
To investigate the involvement of USP29-FSP1 signaling in GC chemoresistance, we initially assessed the protein expression levels of USP29 and FSP1 in various cancer cell lines. The results revealed a strong positive correlation between USP29 and FSP1 expression across the majority of these cell lines (Supplementary Fig. 4c, d) as well as within the GC specimens (Supplementary Fig. 4e, f). Moreover, the elevated expression of USP29 also suggests inferior survival in the GC cohort (Supplementary Fig. 4g, h). Next, we knocked out USP29 in AGS-R and MGC-803-R cells (Fig. 4a and Supplementary Fig. 5a), the results showed that USP29 depletion markedly increases the level of cisplatin induced lipid peroxidation and susceptibility to cisplatin (Fig. 4b, c and Supplementary Fig. 5b, c). The heightened cisplatin induced lipid peroxidation and sensitivity to cisplatin observed with USP29 depletion was reversed upon re-introduction of WT USP29, but not the USP29 CI mutant (Fig. 4d–f and Supplementary Fig. 5d–f). Given prior findings indicating that USP29 plays a role in stabilizing AURKB to promote tumor growth in GC26, potentially contributing to drug resistance, we investigated the impact of AURKB on chemosensitivity in the context of USP29 depletion. The results showed that overexpression of AURKB in USP29 knockout cells did not rescue cell viability following cisplatin exposure (Supplementary Fig. 5g–j), suggesting that chemoresistance lead by USP29 is independent of AURKB. On the other hand, re-expressing WT FSP1, but the mutant of G2A or E156A, was able to restore ferroptosis and cisplatin sensitivity contributed by USP29 loss (Fig. 4g–i and Supplementary Fig. 5k–m). Furthermore, only ferroptosis inhibitor could reverse cisplatin susceptibility in USP29-null cells (Fig. 4j and Supplementary Fig. 5n). Consistent results were also observed in mouse models (Fig. 4k, l and Supplementary Fig. 5o, p). Collectively, these data suggest that USP29 promotes chemoresistance in GC through FSP1-medicated ferroptosis suppression.

SMURF1 enables FSP1 degradation and chemosensitivity of GC
After elucidating the function of the USP29-FSP1 axis in GC chemoresistance, we sought to identify the specific E3 ubiquitin ligase responsible for this process. Utilizing the UbiBrowser website tool (http://ubibrowser.bio-it.cn/ubibrowser/), we conducted predictive analysis to determine the E3 ubiquitin ligase involved in the degradation of FSP1. Subsequently, the top seven E3 ligases identified through the prediction were selected as potential candidates for further investigation (Fig. 5a). Through Co-IP assays, we confirmed a robust interaction between FSP1 and SMURF1, while no substantial interactions were observed with the other candidate ligases or the paralog SMURF2 (Fig. 5b and Supplementary Fig. 6a, b). Reciprocal immunoprecipitation experiments involving SMURF1 and FSP1 antibodies indicated mutual binding between the two proteins (Fig. 5c, d), suggesting an interaction between FSP1 and SMURF1 in cells. Notably, the WW domain of SMURF1 was identified to be crucial for mediating the SMURF1-FSP1 interaction (Fig. 5e). Silencing SMURF1 increases FSP1 while SMURF1 overexpression decreases FSP1 protein expression level (Fig. 5f, g), without affecting its mRNA level (Supplementary Fig. 6c, d). Additionally, a negative correlation between SMURF1 expression and FSP1 levels was observed in the GC cells and specimens (Supplementary Fig. 6e–g) and high expression of SMURF1 indicates better survival in the GC cohort (Supplementary Fig. 6h, i). Notably, following SMURF1 knockdown, the half-life of FSP1 substantially increased (Supplementary Fig. 6j, k), an effect that could be reversed upon re-expression of WT SMURF1 but not the catalytically inactive mutant (C725A) lacking ubiquitin ligase activity (Fig. 5h, i). Through ubiquitination assays, we found that depletion of SMURF1 led to a marked reduction in the ubiquitination of FSP1, which could be rescued by reintroducing WT SMURF1 but not by the C725A mutant of SMURF1 (Fig. 5j, k). In vitro ubiquitination assay also showed that SMURF1 WT, but not C725A mutant, can ubiquitinate FSP1 (Fig. 5l). Collectively, these findings provide compelling evidence that SMURF1 functions as the E3 ligase responsible for directly ubiquitylating and destabilizing FSP1 protein.

Subsequently, we investigated the role of SMURF1 in GC chemoresistance. Ectopic expression of SMURF1 resulted in a substantial enhancement of ferroptosis and sensitivity to cisplatin in drug-resistant GC cells, with susceptibility being rescued only by WT FSP1 or ferroptosis inhibitor treatment (Fig. 5m–p and Supplementary Fig. 6l–o). Consistent conclusions were drawn from in vivo experiments (Fig. 5q, r and Supplementary Fig. 6p, q). Taken together, our findings demonstrate that SMURF1 enhances chemosensitivity in GC by promoting the degradation and inhibition of FSP1-mediated ferroptosis.

SMURF1 regulates GC chemoresistance by destabilizing FSP1 through specific ubiquitination sites
To further understand the molecular basis of how FSP1 is ubiquitinated, we utilized UbiBrowser website tool again to predict the specific lysine sites within FSP1 targeted by the SMURF1 E3 ligase. Predictions identified three potential ubiquitination sites on FSP1 (K63, K193, and K225). Subsequently, we generated mutants for these lysine residues individually (KR) and in combination. Ubiquitination assays revealed that the single-site mutant (K225R) had minimal impact on overall ubiquitination, while single-site mutants (K63R and K193R) or two-site mutants, K63R/K225R (2KR3) and K193R/K225R (2KR2) sharply reduced FSP1 ubiquitination levels (Fig. 6a). Furthermore, the two-site mutant K63R/K193R (2KR1) or three-site mutant K63R/K193R/K225R (3KR) nearly abolished FSP1 ubiquitination (Fig. 6a). Consistently, overexpression of SMURF1 led to the degradation of WT FSP1 protein but not the K63R/K193R mutant FSP1 protein in a dose-dependent manner (Fig. 6b). Moreover, the half-life of K63R/K193R mutant FSP1 protein was notably prolonged compared to WT FSP1 (Fig. 6c, d). These findings collectively suggest that SMURF1 mediates the ubiquitination of FSP1 at the K63 and K193 sites.

We proceeded to explore the functional implications of ubiquitination on FSP1 at these specific sites. By overexpressing SMURF1 in AGS-R and MGC-803-R cells and reintroducing either WT or the K63R/K193R mutant FSP1 (Fig. 6e and Supplementary Fig. 7a), we observed that the increased cisplatin-induced lipid peroxidation and sensitivity to cisplatin induced by SMURF1 overexpression were reversed upon re-expression of WT FSP1 (Fig. 6f, g and Supplementary Fig. 7b, c). In addition, reconstitution with the mutant lacking ubiquitination (K63R/K193R) led to a further reduction in cisplatin-induced lipid peroxidation and cell death (Fig. 6f, g and Supplementary Fig. 7b, c). These findings unequivocally demonstrate that SMURF1 regulates the chemosensitivity of GC by promoting FSP1 degradation through ubiquitination at the K63 and K193 sites.

Targeting FSP1 reverses GC chemoresistance
Our data have convincingly illustrated that FSP1 plays a central role in mediating chemoresistance in GC. We next asked whether iFSP1, the first reported inhibitor for FSP1 that is specific for the human enzyme, in combination with chemotherapeutic agents could effectively eradicate tumor cells and reverse chemoresistance. Initially, we observed that iFSP1 synergistically enhanced the effectiveness of cisplatin or 5-FU in both GC cell lines and mouse models (Supplementary Fig. 8a–g). Moreover, in chemoresistant cells, iFSP1 markedly augmented the anti-tumor efficacy of cisplatin in both in vitro and in vivo settings, consequently improving overall survival rates (Fig. 7a–d and Supplementary Fig. 8h–k). Furthermore, we established six cases of GC patient-derived organoids (PDO) to examine the combination efficacy of FSP1 inhibition and chemotherapeutic agents (Fig. 7e, f and Supplementary Table 2). Expectedly, iFSP1 markedly enhanced the sensitivity of PDO to cisplatin or 5-FU (Fig. 7g, h and Supplementary Fig. 8l, m). We also obtained a fresh GC tumor specimen exhibiting high FSP1 expression (Fig. 7i) from a patient who had undergone a second surgical resection due to rapid disease progression following chemotherapy, indicating resistant to chemotherapy (Supplementary Table 2). We successfully established patient-derived xenograft (PDX) models, and the results showed that a combination of iFSP1 and cisplatin worked effectively in killing tumor cells and prolonging survival (Fig. 7j–l). We next examined the body weight and TUNEL staining of the liver and kidneys in mouse experiments to assess any side effects or potential toxicity for a combination of iFSP1 and chemotherapeutic agents. The results indicated that combo drug treatment was well-tolerated, as evidenced by the absence of any notable effects on mouse body weight, as well as a lack of substantial systemic toxicity observed in TUNEL-stained sections of the liver and kidneys (Supplementary Fig. 9a–h). Collectively, these findings from both in vitro and in vivo studies highlight the promising therapeutic potential of iFSP1 in conjunction with chemotherapy for overcoming chemoresistance in GC.

Methods
Study approval
All mouse experiments were approved by the Institutional Animal Care and Use Committee of Mayo Clinic. Collection of the biopsy samples was performed according to the relevant ethical standards and was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. All registered patients provided informed consent.

Cell culture and plasmids
Cell lines used in this study: HEK-293T (ATCC #CRL-3216), AGS (ATCC #CRL-1739), HT-1080 (ATCC #CCL-121), MGC-803 (Ubigene #YC-C081), SGC-7901 (Ubigene #YC-C055), BGC-823 (Beyotime #C6123). All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2. The culture medium composition for each cell line was consistent with previously described42. Cell line identities were authenticated by the Medical Genome Facility (MGF) at Mayo Clinic (Rochester, MN) using short tandem repeat (STR) profiling. Mycoplasma contamination was routinely monitored every 6 months using the EZ-PCR Mycoplasma Test Kit (Biological Industries). No cell line was used for more than 20 passages after thawing from frozen stocks.

WT USP29 was purchased from Addgene (Plasmid #22599) and subcloned into PLVX3 lentiviral. WT FSP1 was purchased from Horizon (Clone Id: 3506309) and subcloned into PLVX6 lentiviral. WT SMURF1 was purchased from Addgene (Plasmid #11752). WT His-Ub (Plasmid #107392) and WT HA-Ub (Plasmid #17608) were purchased from addgene. All the mutant of FSP1, USP29, HA-Ub, and SMURF1 were generated by site-directed mutagenesis according to standard protocol.

Antibodies and reagents
Antibodies used in this study are described in the Supplementary Table 3. Cycloheximide (CHX) and MG132 were purchased from Selleck. RSL3, erastin, liproxstatin-1, Z-VAD-FMK, 3-Methyladenine, necrostatin 2 racemate, IgG agarose, anti-Flag agarose and anti-HA agarose were purchased from Sigma Aldrich.

Lentivirus production and infection
Short hairpin RNAs (shRNAs) used in this study are listed in the Supplementary Table 4. TransIT-X2 (MIRUS Bio) was used to co-transfect the shRNA vector, the envelope plasmid pMD2.G, and the packaging plasmid psPAX2 into HEK293T cells. The medium was changed at 16 h post transfection and harvested at 48 h after transfection. The medium was filtered through a 0.45 μm syringe filter and supplemented with 8 μg/mL polybrene for infection. The infected cells were selected by 4 μg/mL puromycin.

Generation of CRISPR–Cas9 KO cell lines
To delete the USP29 or FSP1 gene in AGS-R and MGC-803-R cell lines, we designed two independent guide RNAs (gRNAs) using the CRISPR design tool (https://www.genscript.com) for each gene. The gRNA sequences were as follows: USP29 #1: TGTCGAGGTTCATCTTCAAG; and USP29 #2: ATTGAACTCTACGAATGTAT; FSP1 #1: CCTCTTTGCCCGAGTACTTG; and FSP1 #2: GCTCAACAAGTCAGAACTAA. The gRNA sequences were cloned into pSpCas9(BB)-2A-GFP (PX458) all-in-one plasmid (no. 48138, Addgene) according to the standard protocol. The gRNA/Cas9 expression constructs were co-transfected into AGS-R or MGC-803-R cells. At 24 h after transfection, the cells were enriched by fluorescent-based sorting using a FACSAria SORP (BD Biosciences) and transferred into 96-well plates at ~1 cell per well. Single cells were grown for ~6 weeks and colonies were screened for KOs by genomic sequence and western blotting.

Western blot and immunoprecipitation
We prepared cell lysates, performed immunoprecipitation, and immunoblotting as previously described43. Briefly, cells were lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). Whole-cell lysates were centrifuged at 12,000 × g for 15 min. Supernatant containing proteins incubated with 2 μg of antibody and protein A or protein G sepharose beads overnight at 4°C. The immunocomplexes were then washed with NETN buffer and separated by SDS-PAGE. Immunoblotting was performed following standard procedures.

Protein stability assay
To examine the protein turnover, cycloheximide (CHX, 0.1 mg/ml) was added to cell culture medium and cells were harvested at the indicated time points. Cells were lysed and subjected to western blot. Then, we quantified of the protein levels relative to β-Actin by ImageJ.

Denaturing Ni-NTA pull-down
Cells were harvested and lysed in urea buffer (8 M Urea, 0.1 M NaH2PO4, 300 mM NaCl, 0.01 M Tris (pH 8.0)). Lysates were then sonicated and incubated with Ni-NTA agarose beads for 1–2 h at room temperature. Beads were washed 5 times with urea buffer. The immunocomplexes were boiled in 1× Laemmli buffer and subjected to Western blot.

Deubiquitination in vivo and in vitro
The in vivo and in vitro deubiquitination assays were performed as described previously44,45. Briefly, after transfection indicated vectors and treated with MG132, the cells were lysed in the SDS buffer (62.5 mM Tris-HCl (PH 6.8), 2% SDS, 10% glycerol) containing 20 mM NEM and 1 mM iodoacetamide and boiled for 15 min. Then diluted 10 times with NETN buffer containing 20 mM NEM and 1 mM iodoacetamide. The lysates were immunoprecipitated with the HA beads overnight at 4°C. After NETN buffer washing, the immunocomplexes were subjected to Western blot. For the in vitro deubiquitination assay, ubiquitinated FSP1 protein was incubated with purified Flag-USP29 WT and USP29 CI mutant in deubiquitination buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol) for 4 h at room temperature.

In vitro ubiquitination assay
For in vitro ubiquitination, ubiquitination kit (Enzo lifescience) was used and obtained following manufacturer’s instructions. Briefly, purified GST-FSP1, His-SMURF1 WT and C725A were incubated with 100 nM E1 (UBE1), 1 μM E2 (UbcH7), 5 mM Mg-ATP, 2.5 μM Ub or recombinant Ub variant and 1× ligase reaction buffer at 37°C for 45 min in 25 μl of reaction mixture, boiled in sample buffer and subjected to Western blot analysis.

Immunofluorescence staining
Cells cultured on coverslips were treated with cisplatin for 24 h. And then washed with PBS, pre-extracted with a solution containing 20 mM HEPES pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose, and 0.5% NP-40 for 2 min on ice, incubated in 3% paraformaldehyde for 15 min, and permeabilized in 0.25% Triton X-100 solution for 5 min at room temperature. Subsequently, samples were incubated overnight at 4°C with the primary antibody, followed by three times washes and a 1-h incubation with the secondary antibody at room temperature. DAPI staining was performed to visualize nuclear DNA. Finally, coverslips were mounted onto glass slides with an anti-fade solution and observed using a Nikon Eclipse 80i fluorescence microscope.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the cells using Trizol reagent and reversely transcribed into cDNA by utilizing the Takara RR037A kit. The SYBR (Takara, RR820A) and CFX96 Real-Time System (Bio-Rad, USA) were used to conduct the real-time PCR according to manufacturer’s instruction. Expression of β-Actin gene was used as an inner control. Primer sequences used in qRT-PCR are listed in Supplementary Table 5.

Colony formation assays
Cells (500 per well) were seeded in triplicate in 6-well plates. 24 h later, cells were treated with indicated reagents, and cultured for 10–14 days at 37°C. After the colony formed, washed the colonies with PBS and stained with the Coomassie buffer and counted. Lip-1 50 nM, Z-VAD 10 μM, 3-MA 10 mM and Nec-1s 1 μM were used in the colony formation assays.

MTS cell proliferation assay
Cell proliferation was evaluated using the MTS assay (Promega, USA) according to manufacturer’s instruction. Briefly, cells were seeded in 96-well plates (2000 cells per well) and treated as the indicated. The culture medium was replaced with 100 μL fresh medium containing 10 μL of the MTS solution for each well of the plate. After incubating at 37°C incubator for 1 h, cell growth was measured in a microplate reader with absorbance at 490 nm.

C11-BODIPY staining
The indicated cells are seed in six-well plates with or without cisplatin treatment. 24 h later, the cells were stained with 5 μM C11-BODPY 581/591 (SIGMA ALDRICH, D-3861) at 37°C for 30 min prior to trypsinization. After trypsinization and resuspension in phosphate-buffered saline (PBS), flow cytometry was employed to analyze the fluorescence emission peak from 590 to 510 nm. The gating strategy for all flow cytometry analyzes is shown in Supplementary Fig. 10.

Culture of human gastric cancer organoids
Gastric cancer tissues were obtained after surgery and minced in the laboratory. After being washed 6 times by DPBS supplemented with 1% of penicillin-streptomycin, the tumor samples were minced into pieces and then digested by tumor tissue digestion solution (bioGenous, K601003) for 15 min at 37°C. DPBS plus 1% penicillin-streptomycin was added to stop digestion. Samples were then filtered through 100-μm cell strainers, followed by 1800 rpm, centrifugation for 5 min. The centrifugated cells were mixed with Matrigel (BD Pharmingen) and seeded in 24-well-plates. After the Matrigel containing tumor cells was fixed at 37°C for 10 min, the culturing medium was added into wells. Gastric cancer organoids derived from patients (PDOs) were cultured in gastric cancer organoid kit (bioGenous, K2179-GC), and incubated at 37°C with 5% CO2.

Organoid viability assay
Approximately 5000 tumor organoids were cultured in 10 µl of Matrigel per well in a 96-well plate. Following 2–4 days of culture, the specified reagents were diluted to various concentrations and introduced into the tumor organoid medium for an additional 3–6 days. At the end of the experiments, CCK8 (Beyotime, C0040) solution at a ratio of 1:100 was applied to each well and allowed to incubate for 60 min at 37°C and 5% CO2. The absorbance at 450 nm wavelength was measured using a Multiskan Sky with Touch Screen (Thermo Scientific).

GC organoid live/dead staining
PDOs were cultured in a single confocal microscope Petri dish with the specified treatment for 72 h. Following this, the culture medium was removed, and the samples in the dish were rinsed twice with PBS. Subsequently, a staining solution was prepared by adding 1 μl of calcein-AM (1 μM) and 1 μl of EthD-1 (5 μM) to 0.3 ml of PBS. The GC organoids were then stained with calcein-AM and EthD-1 for 20 min at room temperature in dark. Next, the samples in the dish underwent counter-staining with 4, 6-diamidino-2-phenylindole (Sigma) and were subjected to confocal microscopic analysis. Images were captured using a Zeiss LSM900 equipped with Z-stack technology. Image analysis was performed using the ZEN-blue (v. 2.0) software package. Quantification of live and dead cells was carried out by measuring the total cell area of each dye using ImageJ (v. 1.8.0).

Xenograft mouse model
BALB/c female/male mice in the age of 4–6 weeks old were utilized in this study. Indicated cells (2 × 10^6 cells per mouse) were injected subcutaneously into the left posterior flanks of the mice. Tumor volume were monitored by measurements of tumor length (L), and width (W) (tumor volume = L × W × W × 1/2). When tumors reached a volume of ~100 mm^3, mice were randomly allocated into indicated groups. Mouse were treated with cisplatin (8 mg/kg) or 5-FU (10 mg/kg) by intraperitoneal injections 5 times per week, or iFSP1 (10 mg/kg) by daily intraperitoneal injections. For liproxstatin-1 rescue experiment, liproxstatin-1 (10 mg/kg) was intraperitoneally injected once every 3 days for 6 times. At the end of the study, mice were sacrificed and then removed the tumors, weighed and processed. The maximal tumor size has not exceeded the range permitted by the ethics committee (tumor size ≤ 2000 mm^3).

Patient-derived xenograft
The PDX experiment was conducted in strict accordance with the ethical guidelines and was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Fresh tumor specimens were obtained from a patient with recurrent gastric cancer who was undergoing surgical resection following chemotherapy. Immediately after collection, the tissues were placed in cold sterile physiological saline supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution to maintain cell viability. Specimens were minced into 1–2 mm^3 fragments using sterile scalpels and washed three times with cold saline to remove excess blood and debris. Minced tumor pieces were then mixed 1:1 with reduced growth factor Matrigel (BD Biosciences, Inc) and injected subcutaneously into 6–8-week-old Balb/c nu/nu mice using an 18-gauge needle. Successful engraftment was defined as sustained tumor growth beyond 100 mm^3 for at least 4 weeks. Subsequent passages were performed in a similar manner in Balb/c nu/nu mice. Only F3 or higher-generation models were utilized for experimental studies to ensure genetic stability. The administration of group allocation and treatment followed the same protocol as the xenograft mouse model.

Immunohistochemistry (IHC)
IHC assays were performed as previously described with the indicated antibodies46. The expression scores were assessed based on the German semi-quantitative scoring system according to the staining intensity and the staining region as previously described. The final staining score ≥ 4 was identified as high expression; otherwise, it was identified as low expression. The Pearson correlation coefficient and χ^2 test were used for statistical analysis of the correlation of IHC expression.

Statistical analysis
All data in bar and line graphs are presented as mean ± SD of at least three independent experiments. All the Western blotting data were repeated independently three times with similar results. Differences between two groups were analyzed using unpaired Student’s t test, 1-way analysis of variance (ANOVA), or 2-way ANOVA. All findings were considered significant at a P value threshold of 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript Zheming Wu, Xinyi Tu et al. propose that Ferroptosis Suppressor Protein 1 (FSP1) contributes to\nchemotherapy resistance in patients with gastric cancer by inhibiting ferroptotic cell death. Furthermore, they characterized\nUSP29 and SMURF1 as regulators of cellular FSP1 abundance, with USP29 being a deubiquitinating enzyme that protects\nFSP1 from proteasomal degradation, and SMURF1 being the corresponding E3 ligase ubiquitinating FSP1 and therefore,\nreducing FSP1 protein levels. Finally, they present treatment with small molecule iFSP1 as a promising strategy to inhibit\ntumor growth in patients with gastric cancer.\nThis manuscript shows a detailed and well-performed study that not only uncovers a novel ferroptosis-regulating axis\nbetween an E3 ligase and a DUB, but also proposes a druggable target for gastric cancer therapy. Since the development of\nclinically approved ferroptosis-related drugs is still ongoing and hampered by drug resistance of tumors, these findings could\nbe clinically relevant in the future.\nThe presented data seems to be profound, since all findings are concluded from experiments containing several cancer cell\nlines, patient data and sufficient independent replicates.\nThe bioinformatic analysis performed in Suppl. Figure S1 is outside of the scope of our expertise, so we cannot comment on\nthis. However, the results gained from this analysis were validated clearly with biochemical experiments and seem to be\nplausible and explained well in the background of ferroptosis and ubiquitin signaling.\nWe propose the following points of manuscript improvement:\nMajor:\n- Treatment with iFSP1 (Figure 7): Since there are not many in vivo studies done with iFSP1 yet, do you report any side\neffects or potential toxicity? Moreover, is it possible that cells develop a resistance against iFSP1 over time, for example by\nupregulating USP29 and/or downregulating SMURF1? It would be interesting to see expression levels after a long-time\ntreatment with iFSP1.\n- How are the GPX4 level in these gastric cancer cells? Are they generally low or reduced after cisplatin/iFSP1 treatment?\nFundamentally, GPX4 alone should be able to inhibit ferroptosis even in the absence of FSP1 or with FSP1 inhibition.\nMinor:\n- In the abstract, the abbreviation FSP1 is explained as “ferroptosis suppression factor” instead of “ferroptosis suppressor\nprotein 1”\n- When screening for the FSP1-associated DUB, how were the selected DUBs chosen (Supp. Figure S3D)? Could there be\neven another DUB regulating FSP1?\n- How was SMURF1 predicted as E3 ligase by the tool UbiBrowser? When searching for FSP1, I only find MARCHF5 and\nMARCHF6, have you tested these too?\n- It has been shown that iFSP1 can only target human FSP1, but not murine FSP1 (PMID: 37080964, PMID: 37957306) –\nthis should be mentioned (or potentially discussed) in case readers want to use non-xenograft models involving mouse cells.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "Lou and colleagues’ manuscript offers insights into how USP29 and SMURF1 regulate the stability of FSP1, highlighting a\nconnection between ferroptosis evasion and chemoresistance in gastric cancer (GC). The findings imply that targeting FSP1\ncould potentially help in overcoming acquired drug resistance. This study is particularly timely and significant for the fields of\ncell death and cancer biology. It illustrates the role of USP29 and SMURF1 in maintaining FSP1 stability, facilitating\nferroptosis evasion and chemoresistance in gastric cancer cells. Moreover, the research underscores the importance of the\ninterplay between ubiquitin-mediated proteostasis and cell death pathways. By addressing the following points, the study\ncould enhance its rigor and clarity, elevating its value for specialized researchers as well as those in broader clinical and\ncell biology disciplines.\nSeveral comments and suggestions are provided to clarify and strengthen the manuscript.\n1. In Figure 1, including more detailed explanations about the connection between diseased tissue and surgical outcomes\nwould assist readers with diverse backgrounds in grasping the importance of preoperative biopsies and the data on\nneoadjuvant therapy. This context would strengthen the study’s rationale.\n2. The authors focus on two GC cell lines (AGS and MGC-803) that developed resistance to cisplatin (AGS-R, MGC-803-R).\nHowever, it's unclear whether other GC cell lines can also acquire resistance or if these two were the only ones examined.\nProviding clarity on why these specific lines were chosen, along with information on the evaluation of any additional lines,\nwould enhance confidence in the findings' generalizability. These cells would also benefit from a more comprehensive\ncharacterization, for example by performing RNAseq of the naïve and resistant pair.\n3. In lines 130–131, the manuscript notes that cisplatin-resistant lines exhibit both “elevated expression of FSP1 and\nincreased levels of lipid peroxidation.” However, this logic should be reconciled with the overall model, which posits that\nFSP1 typically suppresses lipid peroxidation. A more explicit explanation connecting these observations to the proposed\nmechanism (where higher FSP1 generally reduces lipid peroxidation) would reduce potential confusion.\n4. The manuscript discusses a screen of various deubiquitinating enzymes (DUBs). It would be beneficial to elaborate on\nthe criteria or logic behind the selection of the tested DUBs. It would also strengthen the findings to show that the increased\nsensitivity to ferroptosis in DUB (USP29) knockout cells truly stems from changes in FSP1 levels. For instance, measuring\nshifts in known USP29 or SMURF1 substrates later in the text, as well as examining other ferroptosis regulators in these\nknockout cells, could bolster the conclusion that the observed effects are FSP1-dependent.\n5. The manuscript posits that the USP29 catalytic (UCH) domain mediates the binding to FSP1; however, it's unclear how\nthis interaction does not impede the catalytic function itself. Additional mechanistic discussion would elucidate how USP29\ncan bind FSP1 through its enzymatic domain without negatively affecting enzymatic activity.\nFor Figure 3, the legend should clarify what the green region or domain signifies to help readers understand the schematic\nbetter.\n6. Please clarify how the authors differentiate between increased protein stability and reduced protein translation as the\nmain reasons for altered FSP1 levels. Including data on FSP1 mRNA levels (noted in some figures) or ribosome profiling\ncould strengthen the argument that the changes are due to protein degradation rather than gene expression variations.\n7. A fundamental inquiry is whether depleting FSP1 or USP29 in cisplatin-sensitive parental cells (instead of only in\ncisplatin-resistant cells) would have a more pronounced effect on cisplatin sensitivity. Clarifying whether FSP1 mediates\nchemoresistance specifically in “FSP1-high” cells or also impacts “FSP1-low” cells would contribute to understanding its\nbroader relevance.\n8. In lines 258–259, the phrase “Silence or overexpression of SMURF1 revealed a negative regulatory effect on FSP1\nprotein expression levels” should be revised for clarity. If shSMURF1 increases FSP1 while SMURF1 overexpression\ndecreases FSP1, this should be rewritten to avoid confusion and ensure alignment with the data.\n9. In the half-life experiments (Figure 6), a SMURF1 band appears at the shSmurf EV 0h time point but is absent in other\npanels. Please provide clarification as to why SMURF1 is visible there and not in the others; it may be an artifact or a\nmissing element in the figure panels.\n10. The manuscript frequently uses the term “significant,” but the accompanying statistical analyses are not consistently\nmentioned. Please ensure that all assertions of significance are backed by transparent statistical methods and that relevant\np-values or statistical tests are included.\n11. While the manuscript illustrates the opposing roles of USP29 and SMURF1 in regulating FSP1, it should be noted that\nthey do not necessarily function as part of a singular, linear complex. Thus, characterizing it as the “USP29-SMURF1 axis”\ncould oversimplify the complexities of the interplay between ubiquitination and\ndeubiquitination. Providing a more nuanced explanation that acknowledges the\nindependent modulation of FSP1 by each enzyme would more accurately portray the\nintricacies of the ubiquitin-proteasome system.\n12. In Figure 8, replace the term “lipide peroxide” with “lipid peroxidation” or an equivalent standard term to ensure clarity\nand consistency with the ferroptosis literature.\n13. The impact of the iFSP1 in vivo is rather surprising given that the stability of iFSP1 in plasma is about 20´.\nImportantly here, would be to study the response of the xenograft proficient and deficient in FSP1 upon treatment with\ncisplatin in order to properly validate the role of FSP1 in vivo.", "reviewer_4": "In this manuscript, the Lou lab reports that the USP29-SMURF1 axis regulates the stability of FSP1, shedding light on the\nrole of FSP1-mediated ferroptosis inhibition in chemotherapy resistance in gastric cancer. Additionally, the authors identify\ntwo enzymes for the first time, USP29 as the deubiquitinase of FSP1 and SMURF1 as the E3 ligase of FSP1. Finally, the\nauthors demonstrate that FSP1 inhibitors have a synergistic effect with chemotherapy drugs, suggesting that FSP1 could act\nas a novel target for overcoming chemotherapy resistance in the treatment of gastric cancer. I have listed my concern as\nbelow.\nMajor Points:\n1. Causal Relationship between Ferroptosis and Chemotherapy Resistance. The authors should provide clear evidence to\nestablish the causal relationship between ferroptosis and chemotherapy resistance in gastric cancer (GC). Are other\nferroptosis-inhibiting factors, such as GPX4 and FDFT1, also involved in this process? The authors should perform\nadditional analysis to confirm if the chemotherapy resistance phenotype can be rescued by GPX4-KO or FSP1-KO.\n2. Mechanism of FSP1-Mediated Chemotherapy Resistance. The authors claim that high expression of FSP1 leads to\nchemotherapy resistance. Does FSP1 promote DNA damage repair or deactivate the chemotherapy drugs? Whether\nchemotherapy resistance is due to enhanced ferroptosis? Do other mechanisms, such as DNA repair, contribute to the\nobserved resistance? The authors should evaluate the effect of FSP1 overexpression on DNA damage response (DDR) and\napoptosis in GC cells.\n3. USP29-SMURF1-FSP1 Axis and Chemotherapy Resistance. Based on the existing data, it is reasonable to speculate\nthat under chemotherapy conditions, the FSP1 deubiquitination process regulated by USP29 might outweigh the FSP1\ndegradation mediated by SMURF1. However, the authors did not provide more evidence to support this hypothesis.\nMoreover, the functional connection between the USP29-SMURF1-FSP1 axis and chemotherapy resistance in GC is not\nsufficiently established.\n4. Generalizability of Chemotherapy Sensitivity Assays. One of the main concerns is that the chemotherapy sensitivity\nassays, both in vitro and in vivo, appear overly simplistic. Many of these experiments were conducted using AGS and MGC-\n803 cell lines. What is the functional role of the USP29-SMURF1-FSP1 axis in patient-derived xenograft (PDX) and patient-\nderived organoid (PDO) models? Additionally, the study relies solely on cisplatin for the functional studies throughout the\nmanuscript. To strengthen the conclusions, the authors should validate their findings using other chemotherapy drugs.\n5. Ubiquitination Experiments to Confirm SMURF1’s Role. The authors need to conduct in vitro ubiquitination assays to\nconfirm that SMURF1 functions as an E3 ligase for FSP1. Moreover, the authors should investigate which specific structural\ndomain of SMURF1 mediates its E3 ligase activity. Identifying this domain would provide further insight into the mechanism\nby which SMURF1 regulates FSP1 stability.\nMinor Points:\n1. Figure 3. Many GFP and His bands are overexposed. The quality of these Western blot images should be improved.\n2. Figures 3L-3N. The negative control should be clearly labeled and addressed."}, "author_response": "Point-by-point response to the reviewers’ comments.\nWe sincerely express our gratitude to the reviewers for their thoughtful assessment of\nour work. Their comments were well received, and the resulting changes have\nimproved manuscript quality.\nOur responses to their comments/questions are in blue. The response to Reviewer 1\nstarts on Page 1, Reviewer 2 on Page 7, Reviewer 3 on page 8, and Reviewer 4 on\npage 22.\nReviewer #1:\nIn this manuscript Zheming Wu, Xinyi Tu et al. propose that Ferroptosis Suppressor\nProtein 1 (FSP1) contributes to chemotherapy resistance in patients with gastric\ncancer by inhibiting ferroptotic cell death. Furthermore, they characterized USP29\nand SMURF1 as regulators of cellular FSP1 abundance, with USP29 being a\ndeubiquitinating enzyme that protects FSP1 from proteasomal degradation, and\nSMURF1 being the corresponding E3 ligase ubiquitinating FSP1 and therefore,\nreducing FSP1 protein levels. Finally, they present treatment with small molecule\niFSP1 as a promising strategy to inhibit tumor growth in patients with gastric cancer.\nThis manuscript shows a detailed and well-performed study that not only uncovers a\nnovel ferroptosis-regulating axis between an E3 ligase and a DUB, but also proposes\na druggable target for gastric cancer therapy. Since the development of clinically\napproved ferroptosis-related drugs is still ongoing and hampered by drug resistance of\ntumors, these findings could be clinically relevant in the future.\nThe presented data seems to be profound, since all findings are concluded from\nexperiments containing several cancer cell lines, patient data and sufficient\nindependent replicates.\nThe bioinformatic analysis performed in Suppl. Figure S1 is outside of the scope of\nour expertise, so we cannot comment on this. However, the results gained from this\nanalysis were validated clearly with biochemical experiments and seem to be\nplausible and explained well in the background of ferroptosis and ubiquitin\nsignaling.\nthe suggestions provided, which helped us to craft an improved version of our\nmanuscript.\nWe propose the following points of manuscript improvement:\nMajor:\n- Treatment with iFSP1 (Figure 7): Since there are not many in vivo studies done with\niFSP1 yet, do you report any side effects or potential toxicity? Moreover, is it possible\nthat cells develop a resistance against iFSP1 over time, for example by upregulating\nUSP29 and/or downregulating SMURF1? It would be interesting to see expression\nlevels after a long-time treatment with iFSP1.\nResponse: Thank you for highlighting this important point. Here, we show the mouse\nbody weight and TUNEL staining of the liver and kidneys in mouse experiments to\nassess any side effects or potential toxicity for a combination of iFSP1 and\nchemotherapeutic agents. The results indicated that combo drug treatment is well-\ntolerated, as evidenced by the absence of any notable effects on mouse body weight,\nas well as a lack of substantial systemic toxicity observed in TUNEL-stained sections\nof the liver and kidneys (Supplementary Fig. S9a-h).\nTo test whether cells develop a resistance against iFSP1 over time, we continuously\ntreated cells with iFPS1 (0.5 M) for more than 1 month and examined their\nsensitivity to iFSP1. The MTS assay result showed that cells subjected to long-term\ntreatment of iFSP1 exhibited similar sensitivity to iFSP1 compared to parental cells\n(Fig. i). Moreover, western blots showed moderate decreased FSP1 level, which is\nconsistent with Wu et al.’s study1, while no marked change of USP29 and SMURF1\nin cells after iFSP1 long period treatment (Fig. j). All the results suggested that cells\ndo not develop a resistance against iFSP1 over time.\n2\ns9a-h body weight and quantification of TUNEL-positive cells in liver and kidney of\nBGC-823 (s9a, s9b), AGS-R (s9c, s9d), MGC-803-R (s9e, s9f), and PDX (s9g, s9h)\nxenograft tumor models with the indicated treatment. i Sensitivity of parental cells\nand cells after iFSP1 long time treatment to iFSP1. j FSP1, USP29 and SMURF1\nexpression levels in parental cells and cells after iFSP1 long time treatment.\nReference\n1. Wu YC, et al. Targeting of FSP1 regulates iron homeostasis in drug-tolerant\npersister head and neck cancer cells via lipid-metabolism-driven ferroptosis.\nAging (Albany NY). 2024; 16(1): 627-647.\n- How are the GPX4 level in these gastric cancer cells? Are they generally low or\nreduced after cisplatin/iFSP1 treatment? Fundamentally, GPX4 alone should be able\nto inhibit ferroptosis even in the absence of FSP1 or with FSP1 inhibition.\nResponse: We agree with this point that GPX4, or other ferroptosis factors are able to\ninhibit ferroptosis even in the absence of FSP1 or with FSP1 inhibition, therefore\n3\npotentially function in chemoresistance. To address this concern, we examined GPX4,\nACSL4, FTH1, and FDFT1 levels besides FSP1 in cisplatin-sensitive and resistant\ncells. The results showed that only FSP1 expression levels increased in resistant cells,\nsuggesting that FSP1, rather than other ferroptosis factors play a crucial role in GC\nchemoresistance in our experimental models.\nExpression levels of ferroptosis factors in AGS and MGC-803 cisplatin sensitive and\nresistant cells.\nMinor:\n- In the abstract, the abbreviation FSP1 is explained as “ferroptosis suppression factor”\ninstead of “ferroptosis suppressor protein 1”\nResponse: Thank you for your careful review. We have corrected the term of FSP1 to\n“ferroptosis suppressor protein 1” in our revised manuscript.\n- When screening for the FSP1-associated DUB, how were the selected DUBs chosen\n(Supp. Figure S3D)? Could there be even another DUB regulating FSP1?\nResponse: Thank you for pointing out this issue. To identify potential DUBs that can\ndeubiquitinate and stabilize FSP1, we conducted a screening with a panel of\ndeubiquitinases available in our lab. We did identify several DUBs that interact with\nFSP1; among them, USP29 is the most significant DUB regulating FSP1 and\nferroptosis. We believe that USP29 plays a predominant role in the stabilization of\nFSP1 in our experimental models. However, we cannot exclude the possibility that\nthere exists another DUB for FSP1 in other experimental models and conditions.\n4\n- How was SMURF1 predicted as E3 ligase by the tool UbiBrowser? When searching\nfor FSP1, I only find MARCHF5 and MARCHF6, have you tested these too?\nResponse: Thank you for your insightful comment. Actually, there are two versions of\nUbiBrowser tool, http://ubibrowser.bio-it.cn/ubibrowser/ (version 1) (Fig. a) and\nhttp://ubibrowser.bio-it.cn/ubibrowser_v3/home/index/ (version 2) (Fig. b),\nrespectively. When we conducted our research, only version 1 was available. Here we\nalso examined the interaction of MARCHF5 and MARCHF6 with FSP1. The results\nshowed a quite weak binding between MARCHF5 or MARCHF6 with FSP1 compared\nto SMURF1 (Fig. c).\na, b Predicted E3 ligases of FSP1 (AIFM2) in UbiBrowser version 1 (a) and\nUbiBrowser version 2 (b). c The indicated Flag-tagged vectors were transfected into\nHEK293T cells. 48 h later, cells were lysed and immunoprecipitated with Flag beads.\nThe immunoprecipitates were then blotted with the indicated antibodies.\n- It has been shown that iFSP1 can only target human FSP1, but not murine FSP1\n(PMID: 37080964, PMID: 37957306) – this should be mentioned (or potentially\ndiscussed) in case readers want to use non-xenograft models involving mouse cells.\nResponse: Thank you for your valuable suggestion. We mentioned that iFSP1 can only\ntarget human FSP1 in our revised manuscript.\n5\n“We next asked whether iFSP1, the first reported inhibitor for FSP1 that is specific for\nthe human enzyme, in combination with chemotherapy could effectively eradicate\ntumor cells and reverse chemoresistance.”\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: We sincerely thank the reviewer for the careful and thorough evaluation of\nour manuscript.\nLou and colleagues’ manuscript offers insights into how USP29 and SMURF1\nregulate the stability of FSP1, highlighting a connection between ferroptosis evasion\nand chemoresistance in gastric cancer (GC). The findings imply that targeting FSP1\ncould potentially help in overcoming acquired drug resistance. This study is\nparticularly timely and significant for the fields of cell death and cancer biology. It\nillustrates the role of USP29 and SMURF1 in maintaining FSP1 stability, facilitating\nferroptosis evasion and chemoresistance in gastric cancer cells. Moreover, the\nresearch underscores the importance of the interplay between ubiquitin-mediated\nproteostasis and cell death pathways. By addressing the following points, the study\ncould enhance its rigor and clarity, elevating its value for specialized researchers as\nwell as those in broader clinical and cell biology disciplines.\nSeveral comments and suggestions are provided to clarify and strengthen the\nmanuscript.\nResponse: We greatly appreciate the reviewer for recognition of novelty and\nsignificance of our manuscript, and for the thoughtful comments and constructive\nsuggestions, which helped to improve the quality of our work. We have carried out\nthe experiments that the reviewer suggested and revised the manuscript accordingly.\n1. In Figure 1, including more detailed explanations about the connection between\ndiseased tissue and surgical outcomes would assist readers with diverse backgrounds\nin grasping the importance of preoperative biopsies and the data on neoadjuvant\ntherapy. This context would strengthen the study’s rationale.\nResponse: We appreciate the reviewer's insightful suggestion. In our revised\nmanuscript, we incorporated the background regarding the importance of preoperative\nbiopsies in predicting the effectiveness of neoadjuvant therapy for GC.\n“Although surgery is the main curative approach for resectable GC, the prognosis of\npatients remains unfavorable post curative resection. Preoperative chemotherapy\noffers potential advantages like reducing the primary tumor size to enhance the\nchances and effectiveness of curative resection, as well as eradicating potential\n8\nmicrometastases to prevent or lower tumor recurrence and spread, thus enhancing the\nmanagement of tumor-related symptoms1. Nevertheless, approximately 15% of GC\npatients who receive preoperative chemotherapy face the risk of tumor progression2.\nThus, accurately predicting patients' response to preoperative chemotherapy remains\nparticularly crucial.”\nReferences\n1. Smyth EC, et al. Gastric cancer. Lancet 396, 635-648 (2020).\n2. Li GZ, et al. Surgical Management of Gastric Cancer: A Review. JAMA Surg. 157,\n446-454 (2022).\n2. The authors focus on two GC cell lines (AGS and MGC-803) that developed\nresistance to cisplatin (AGS-R, MGC-803-R). However, it's unclear whether other GC\ncell lines can also acquire resistance or if these two were the only ones examined.\nProviding clarity on why these specific lines were chosen, along with information on\nthe evaluation of any additional lines, would enhance confidence in the findings'\ngeneralizability. These cells would also benefit from a more comprehensive\ncharacterization, for example by performing RNAseq of the naïve and resistant pair.\nResponse: Thank you for your insightful comments. In our research, we focused on\nchemoresistance in gastric cancer (GC), with AGS and MGC-803 cell lines being the\nmost frequently utilized cells for investigating chemoresistance in GC1-5. Drawing\nfrom our robust data obtained from the two cell lines, they suffice to substantiate our\nfindings, despite the potential for other cell lines to develop resistance as well.\nFurthermore, to boost confidence in the generalizability of our findings, we\nestablished six cases of GC patient-derived organoids (PDO) to examine the\ncombination efficacy of FSP1 inhibition and chemotherapeutic agents (Fig. 7e, f).\nExpectedly, iFSP1 markedly enhances the sensitivity of PDO to cisplatin or 5-FU\n(Fig. 7g, h and Supplementary Fig. 8l, m). We also obtained a fresh GC tumor\nspecimen exhibiting high FSP1 expression (Fig. 7i) from a patient who had undergone\na second surgical resection due to rapid disease progression following chemotherapy,\n9\nindicating resistant to chemotherapy. We successfully established patient-derived\nxenograft (PDX) models, and the results showed that a combination of iFSP1 and\ncisplatin worked effectively in killing tumor cells and prolonging survival (Fig. 7j-l).\nCollectively, these PDO and PDX data provide additional support for our conclusions.\n7e Representative bright-field images of the 6 cases of cultured gastric cancer patient-\nderived organoids (PDO). 7f Immunoblot showing FSP1 expression levels in the 6\nGC PDO. 7g, s8l Organoid viability of PDO #4 with the indicated treatment. 7h, s8m\nRepresentative micrographs (left) and quantitation (right) of PDO #4 with the\nindicated treatment. Organoids were stained with Calcein-AM (live cells, green) and\nEthD-1 (dead cells, red) for 30 min and then fluorescent signals were captured. 7i\nthe patient-derived tumor xenograft (PDX) models. PDX cells were injected\nsubcutaneously. When tumors reached approximately 100 mm3, mice were randomly\nassigned to the indicated treatment (n = 5 for each group). 7k Quantification of the\nweight of the tumors in different groups of the PDX models. 7l Survival curves of the\nPDX models (n = 8 for each group).\nReferences\n1. Mo D, et al. Human Helicase RECQL4 Drives Cisplatin Resistance in Gastric\nCancer by Activating an AKT-YB1-MDR1 Signaling Pathway. Cancer Res.\n2016; 76(10): 3057-3066.\n2. Peng L, et al. circCUL2 regulates gastric cancer malignant transformation and\ncisplatin resistance by modulating autophagy activation via miR-142-3p/ROCK2.\nMol Cancer. 2020; 19(1): 156.\n3. Mora-Lagos B, et al. Functional and transcriptomic characterization of cisplatin-\nresistant AGS and MKN-28 gastric cancer cell lines. PLoS One. 2020; 15(1):\ne0228331.\n4. Wu Q, et al. Downregulation of LncRNA CCAT1 Enhances Chemosensitivity in\nCisplatin-Resistant Gastric Cancer Cells. Drug Dev Res. 2025; 86(1): e70048.\n5. Huang D, et al. Cisplatin resistance in gastric cancer cells is associated with\nHER2 upregulation-induced epithelial-mesenchymal transition. Sci Rep. 2016; 6:\n20502.\n3. In lines 130-131, the manuscript notes that cisplatin-resistant lines exhibit both\n“elevated expression of FSP1 and increased levels of lipid peroxidation.” However,\nthis logic should be reconciled with the overall model, which posits that FSP1\ntypically suppresses lipid peroxidation. A more explicit explanation connecting these\nobservations to the proposed mechanism (where higher FSP1 generally reduces lipid\nperoxidation) would reduce potential confusion.\nbe “elevated expression of FSP1 and decreased levels of lipid peroxidation.” as our\nresults shown in Fig. 2a, b and Supplementary Fig. S2c, d. We have corrected this\nmistake in our revised manuscript.\n4. The manuscript discusses a screen of various deubiquitinating enzymes (DUBs). It\nwould be beneficial to elaborate on the criteria or logic behind the selection of the\ntested DUBs. It would also strengthen the findings to show that the increased\nsensitivity to ferroptosis in DUB (USP29) knockout cells truly stems from changes in\nFSP1 levels. For instance, measuring shifts in known USP29 or SMURF1 substrates\nlater in the text, as well as examining other ferroptosis regulators in these knockout\ncells, could bolster the conclusion that the observed effects are FSP1-dependent.\nResponse: We appreciate the reviewer's insightful suggestions. To identify potential\nDUBs that can deubiquitinate and stabilize FSP1, we conducted a screening with a\npanel of deubiquitinases available in our lab and revealed that USP29 is the most\nsignificant DUB regulating FSP1 and ferroptosis. Knockdown of USP29 using two\ndistinct USP29-specific shRNAs resulted in a marked reduction in FSP1 protein\nabundance (Fig. 3g), without affecting other ferroptosis factors (Supplementary Fig.\nS4b). Furthermore, the heightened cisplatin induced lipid peroxidation and sensitivity\nto cisplatin observed with USP29 depletion was reversed upon re-introduction of WT\nFSP1 (Fig. 4g-i and supplementary Fig. s5k-m). Given prior findings indicating that\nUSP29 plays a role in stabilizing AURKB to promote tumor growth in GC1,\npotentially contributing to drug resistance, we investigated the impact of AURKB on\nchemosensitivity in the context of USP29 depletion. The results showed that\noverexpression of AURKB in USP29 knockout cells did not rescue cell viability\nfollowing cisplatin exposure (Supplementary Fig. s5g-j), suggesting that\nchemoresistance lead by USP29 is independent of AURKB. Collectively, these data\nsuggest that USP29 promotes chemoresistance in GC through FSP1-medicated\nferroptosis suppression.\n12\n3g, s4b FSP1 (3g) and other ferroptosis factors (s4b) protein levels after transduced\nwith lentivirus carrying control or two independent USP29 shRNAs. 4g, s5k\nImmunoblots of FSP1 and USP29 in AGS-R (4g) and MGC-803 (s5k) cells stably\nexpressing control sgRNA, USP29 sgRNA #2, or USP29 sgRNA #2 reintroduced\nwith the indicated FSP1 vectors. 4h, s5l Cells from (4g/s5k) or were treated with or\nwithout cisplatin (2M) for 24 h, and then BODIPY C11 levels were detected. 4i,\ns5m Cells from (4g/s5k) were exposed to the indicated dose of cisplatin for 2 weeks,\ncolony formation assay was then performed to detect the cell survival. s5g-j\nImmunoblots of USP29 and AURKB, and colony formation assay treated with\n13\ncisplatin for AGS-R (s5g, h) and MGC-803-R (s5i, j) cells stably expressing control\nsgRNA, USP29 sgRNA #2, or USP29 sgRNA #2 reintroduced with AURKB vector.\nReference\n1. Tu, et al. USP29 activation mediated by FUBP1 promotes AURKB stability and\noncogenic functions in gastric cancer. Cancer Cell Int 24, 33 (2024).\n5. The manuscript posits that the USP29 catalytic (UCH) domain mediates the\nbinding to FSP1; however, it's unclear how this interaction does not impede the\ncatalytic function itself. Additional mechanistic discussion would elucidate how\nUSP29 can bind FSP1 through its enzymatic domain without negatively affecting\nenzymatic activity.\nFor Figure 3, the legend should clarify what the green region or domain signifies to\nhelp readers understand the schematic better.\nResponse: Thank you for your constructive comments and suggestions. Since the\ncysteine 294 within USP29 catalytic (UCH) domain is crucial for its enzyme activity,\nwe constructed another vector of USP29 (300-600 aa) and performed Co-IP with\nFSP1. The results showed that USP29 (300-600 aa) can strongly bind with FSP1 (Fig.\na), suggesting that the interaction between USP29 and FSP1 does not impede the\ncatalytic function of USP29. Furthermore, deubiquitinases (DUBs) have evolved their\ncatalytic domains to serve a dual purpose: to recognize and bind substrates (typically\nubiquitin or ubiquitin-conjugated proteins) and to catalyze the cleavage of the\nisopeptide bond. This dual functionality is not counterproductive; rather, it is central\nto efficient catalysis. Here’s why substrate binding does not negatively affect the\nreaction.\nIn revised Fig. 3 legend, we included the meaning of green labels as suggested.\n“The green triangles indicate the band of the specific USP29 truncates.”\n14\na The indicated Flag-tagged USP29 were transfected into HEK293T cells. 48 h later,\ncells were lysed and immunoprecipitated with Flag beads. The immunoprecipitates\nwere then blotted with the indicated antibodies.\n6. Please clarify how the authors differentiate between increased protein stability and\nreduced protein translation as the main reasons for altered FSP1 levels. Including data\non FSP1 mRNA levels (noted in some figures) or ribosome profiling could strengthen\nthe argument that the changes are due to protein degradation rather than gene\nexpression variations.\nResponse: Thank you for pointing out this issue. Knockdown of USP29 using two\ndistinct USP29-specific shRNAs resulted in a marked reduction in FSP1 protein\nabundance, without affecting FSP1 mRNA levels (Fig. 3g and Supplementary Fig.\ns4a), implying a post-transcriptional regulatory role of USP29 on FSP1 expression.\nMoreover, the diminished FSP1 levels in USP29-depleted cells could be restored by\ntreatment with the proteasome inhibitor MG132 (Fig. 3h), indicating that USP29\nregulates FSP1 levels via proteasomal degradation. Furthermore, FSP1 stability (by\ntreating cells with CHX, a protein translation inhibitor) sharply decreased upon\nUSP29 depletion, which could be rescued by ectopic expression of USP29 in the\ndepleted cells (Fig. 3j, k). Collectively, these findings substantiate the role of USP29\nin regulating FSP1 protein abundance and stability in a proteasome-dependent\nmanner.\n15\n3g FSP1 protein level after transduced with lentivirus carrying control or two\nindependent USP29 shRNAs. s4a Relative USP29 or FSP1 mRNA level after\ntransduced with lentivirus carrying control or two independent USP29 shRNAs. 3h\nImmunoblots of FSP1 and USP29 in HEK293T cells stably expressing control or\nUSP29 shRNAs treated with vehicle or MG132. 3j, k Cells stably expressing control\nshRNA, USP29 shRNA and USP29 shRNA together with shRNA-resistant USP29\nwere treated with cycloheximide (CHX) (50 g/mL), and harvested at the indicated\ntime points. Immunoblots of FSP1 and USP29 (3j) and the quantification of the FSP1\nlevel relative to -Actin (3k) were shown.\n7. A fundamental inquiry is whether depleting FSP1 or USP29 in cisplatin-sensitive\nparental cells (instead of only in cisplatin-resistant cells) would have a more\npronounced effect on cisplatin sensitivity. Clarifying whether FSP1 mediates\nchemoresistance specifically in “FSP1-high” cells or also impacts “FSP1-low” cells\nwould contribute to understanding its broader relevance.\nResponse: Thank you for your insightful comment. To enhance confidence in our\nfindings' generalizability, we utilized another two GC cell lines sensitive to cisplatin.\nThe in vitro and in vivo data revealed that inhibition of FSP1 has a more pronounced\neffect on chemosensitivity (Supplementary Fig. s8a-g), suggesting that iFSP1 can be\nbroadly used to sensitize cells to chemotherapy.\n16\ns8a-d The indicated cells were exposed to vehicle, cisplatin (20 nM), 5-FU (20 nM),\niFSP1 (10 nM) or their combination for 2 weeks, colony formation assay was then\nperformed to detect the cell survival. s8e Growth curves of the BGC-823 xenograft\ntumor models. BGC-823 cells were injected subcutaneously. When tumors reached\napproximately 100 mm3, mice were randomly assigned to the indicated treatment (n =\n5 for each group). s8f Quantification of the weight of the tumors in different groups of\nthe xenograft tumor models. s8g Survival curves of the BGC-823 xenograft tumor\nmodels (n = 8 for each group).\n8. In lines 258-259, the phrase “Silence or overexpression of SMURF1 revealed a\nnegative regulatory effect on FSP1 protein expression levels” should be revised for\nclarity. If shSMURF1 increases FSP1 while SMURF1 overexpression decreases\nFSP1, this should be rewritten to avoid confusion and ensure alignment with the data.\nResponse: Thank you for your careful review. To avoid confusion, we rewritten the\nsentence to “silencing SMURF1 increases FSP1 while SMURF1 overexpression\ndecreases FSP1 protein expression level” as suggested.\n9. In the half-life experiments (Figure 6), a SMURF1 band appears at the shSmurf1\nEV 0h time point but is absent in other panels. Please provide clarification as to why\nelement in the figure panels.\nResponse: Thank you for pointing out this issue. According to the description you\nprovided, we infer that you should refer to Fig 5h. The first lane of Fig. 5h indicates\nknocking down SMURF1 and without CHX treatment. In the first lane, a weak band\nof SMURF1 is observed due to the knockdown efficiency of SMURF1 shRNA does\nnot reach 100%, while SMURF1 expression in the subsequent three lanes is not\nvisible as a result of the treatment with CHX leading to the degradation of SMURF1.\nSimilar results were also observed in Supplementary Fig. 6j, k.\n5h, i Cells stably expressing SMURF1 shRNA reintroduced with empty vector, WT,\nor C725A vectors of SMURF1 were treated with CHX (50 g/mL), and harvested at\nthe indicated time points. Immunoblots of FSP1 and SMURF1 (h) and the\nquantification of the FSP1 level relative to -Actin (i) were shown. s6j, k Cells stably\nexpressing control or SMURF1 shRNAs were treated with CHX (50 g/mL), and\nharvested at the indicated time points. Immunoblots of FSP1 and SMURF1 (s6j) and\nthe quantification of the FSP1 level relative to -Actin (s6k) were shown.\n10. The manuscript frequently uses the term “significant,” but the accompanying\nstatistical analyses are not consistently mentioned. Please ensure that all assertions of\nsignificance are backed by transparent statistical methods and that relevant p-values\nor statistical tests are included.\nof significance are limited specifically to statistical significance. In our revised\nmanuscript, we substituted other instances to 'substantial', 'marked', or a similar\nsynonym.\n11. While the manuscript illustrates the opposing roles of USP29 and SMURF1 in\nregulating FSP1, it should be noted that they do not necessarily function as part of a\nsingular, linear complex. Thus, characterizing it as the “USP29-SMURF1 axis”\ncould oversimplify the complexities of the interplay between ubiquitination and\ndeubiquitination. Providing a more nuanced explanation that acknowledges the\nindependent modulation of FSP1 by each enzyme would more accurately portray the\nintricacies of the ubiquitin-proteasome system.\nResponse: Thank you for your insightful comment. In order to prevent\noversimplification of the intricate interactions between USP29 and SMURF1 in\nmodulating FSP1 and its mediated ferroptosis, we revised the title to “USP29 and\nSMURF1 orchestrate FSP1-mediated ferroptosis to facilitate chemoresistance in\ngastric cancer”. And also revised “USP29-SMURF1 axis” accordingly in manuscript.\n12. In Figure 8, replace the term “lipid peroxide” with “lipid peroxidation” or an\nequivalent standard term to ensure clarity and consistency with the ferroptosis\nliterature.\nResponse: Thank you for your careful review. Here we replaced the term “lipid\nperoxide” with “lipid peroxidation” in Fig. 8 as suggested.\n19\n13. The impact of the iFSP1 in vivo is rather surprising given that the stability of\niFSP1 in plasma is about 20´. Importantly here, would be to study the response of the\nxenograft proficient and deficient in FSP1 upon treatment with cisplatin in order to\nproperly validate the role of FSP1 in vivo.\nResponse: Thank you for raising this important point. To validate the role of FSP1 in\ncisplatin sensitivity in vivo, we knocked out FSP1 in AGS-R and MGC-803-R cells\nand conducted animal experiments. In vivo growth curves showed that the FSP1 KO\ntumors exhibited a modest reduction in size compared to tumors derived from\nparental cells prior to cisplatin treatment, but FSP1 KO was strikingly sensitive to\ncisplatin treatment (Fig. 2j and Supplementary Fig. s2o). Consistent results were\nobserved upon sacrifice of the mice and subsequent calculation of tumor weight (Fig.\n2k and Supplementary Fig. S2p). Moreover, the ferroptosis inhibitor of liproxstatin-1\n(Lip-1) effectively reversed the cisplatin response induced by FSP1 depletion in the in\nvivo setting (Fig. 2j, k and Supplementary Fig. S2o, p). Taken together, these data\ndemonstrated that FSP1 facilitates gastric cancer chemoresistance in vivo by\nsuppressing ferroptosis. Moreover, several studies have also indicated that iFSP1\ndemonstrates good efficacy in animal experiments1,2. Additionally, Nakamura et al.'s\nNature study3 reported that icFSP1, another FSP1 inhibitor, works effectively in their\nin vivo experiments despite displaying comparable stability to iFSP1.\n20\n2j, s2o Growth curves of the AGS-R (2j) and MGC-803-R (s2o) xenograft tumor\nmodels. Cells stably expressing control or FSP1 sgRNA were injected\nsubcutaneously. When tumors reached approximately 100 mm3, mice were randomly\nassigned to the indicated treatment (n = 5 for each group). 2k, s2p Quantification of\nthe weight of the tumors in different groups of the xenograft tumor models.\nReferences\n1. Wu YC, et al. Targeting of FSP1 regulates iron homeostasis in drug-tolerant\npersister head and neck cancer cells via lipid-metabolism-driven ferroptosis.\nAging (Albany NY). 2024; 16(1): 627-647.\n2. Cheu JW, et al. Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor\nFerroptosis and Anti-tumor Immune Responses in Liver Cancer. Cell Mol\nGastroenterol Hepatol. 2023; 16(1): 133-159.\n3. Nakamura T, et al. Phase separation of FSP1 promotes ferroptosis. Nature. 2023;\n619(7969): 371-377.\nIn this manuscript, the Lou lab reports that the USP29-SMURF1 axis regulates the\nstability of FSP1, shedding light on the role of FSP1-mediated ferroptosis inhibition\nin chemotherapy resistance in gastric cancer. Additionally, the authors identify two\nenzymes for the first time, USP29 as the deubiquitinase of FSP1 and SMURF1 as the\nE3 ligase of FSP1. Finally, the authors demonstrate that FSP1 inhibitors have a\nsynergistic effect with chemotherapy drugs, suggesting that FSP1 could act as a novel\ntarget for overcoming chemotherapy resistance in the treatment of gastric cancer. I\nhave listed my concern as below.\nResponse: We thank the reviewer for the appreciative words on our study and for the\nsuggestions provided, which helped us to produce an improved version of our\nmanuscript.\nMajor Points:\n1. Causal Relationship between Ferroptosis and Chemotherapy Resistance. The\nauthors should provide clear evidence to establish the causal relationship between\nferroptosis and chemotherapy resistance in gastric cancer (GC). Are other ferroptosis-\ninhibiting factors, such as GPX4 and FDFT1, also involved in this process? The\nauthors should perform additional analysis to confirm if the chemotherapy resistance\nphenotype can be rescued by GPX4-KO or FSP1-KO.\nResponse: We thank the reviewer for the insightful comments. To test whether other\nferroptosis factors function in GC chemoresistance, we examined GPX4, ACSL4,\nFTH1, and FDFT1 levels besides to FSP1 in cisplatin sensitive and resistant cells. The\nresults showed that only FSP1 expression levels increased in resistant cells (Fig. 2a\nand Supplementary Fig. s2c,d), suggesting that FSP1, rather than other ferroptosis\nfactors play a crucial role in chemoresistance. Furthermore, FSP1 depletion markedly\nenhances the level of cisplatin induced lipid peroxidation and vulnerability to cisplatin\n(Fig. 2c-e and Supplementary Fig. s2f-h). Reintroduction of wild-type (WT) FSP1,\nbut not impaired myristoylation and plasma-membrane localization mutant (G2A),\nor unfunctional CoQ oxidoreductase mutant (E156A), could rescue cisplatin induced\n22\nlipid peroxidation and sensitivity to cisplatin contributed by FSP1 deficiency (Fig. 2f-\nh and Supplementary Fig. s2k-m). All these data demonstrated that FSP1, not other\nferroptosis factors, facilitates gastric cancer chemoresistance by suppressing\nferroptosis.\n2a, s2c, s2d Expression levels of the indicated ferroptosis factors in cisplatin sensitive\n(S) and resistant (R) cells. 2c, s2f Immunoblot of FSP1 in AGS-R (2c) and MGC-803\n(s2f) cells stably expressing control or two independent FSP1 sgRNAs. 2d, s2g Cells\nfrom (2c/s2f) were treated with or without cisplatin (2M) for 24 h, and then\nBODIPY C11 levels were detected. 2e, s2h Cells from (2c/s2f) were exposed to the\nindicated dose of cisplatin for 2 weeks, colony formation assay was then performed to\ndetect the cell survival. 2f, s2k Immunoblot of FSP1 in AGS-R (2f) and MGC-803\n23\n(s2k) cells stably expressing control sgRNA, FSP1 sgRNA #1, or FSP1 sgRNA #1\nreintroduced with the indicated FSP1 vectors. 2g, s2l Cells from (2f/s2k) were treated\nwith or without cisplatin (2M) for 24 h, and then BODIPY C11 levels were\ndetected. 2h, s2m Cells from (2f/s2k) were exposed to the indicated dose of cisplatin\nfor 2 weeks, colony formation assay was then performed to detect the cell survival.\n2. Mechanism of FSP1-Mediated Chemotherapy Resistance. The authors claim that\nhigh expression of FSP1 leads to chemotherapy resistance. Does FSP1 promote DNA\ndamage repair or deactivate the chemotherapy drugs? Whether chemotherapy\nresistance is due to enhanced ferroptosis? Do other mechanisms, such as DNA repair,\ncontribute to the observed resistance? The authors should evaluate the effect of FSP1\noverexpression on DNA damage response (DDR) and apoptosis in GC cells.\nResponse: Thank you for your valuable suggestion. We examined DNA damage-\ninduced by cisplatin after depletion of FSP1. The results showed unchanged -H2AX\nfoci formation in FSP1 KO cells following cisplatin treatment (Supplementary Fig.\ns2i, j). Furthermore, only Liproxstatin-1 (Lip-1, a ferroptosis inhibitor), but not Z-\nVAD-FMK (a pan-caspase inhibitor), 3-Methyladenine (3-MA, an autophagy\ninhibitor), or necrostatin 2 racemate (Nec-1s, a necroptosis inhibitor) could restore\ncisplatin susceptibility in FSP1-KO cells (Fig. 2i and Supplementary Fig. s2n).\nCollectively, these data suggested that FSP1 promotes chemoresistance in GC by\nmodulating ferroptosis, rather than through DNA damage repair or other cell death\npathways.\n24\ns2i, j Representative micrographs (left) and quantitation (right) for -H2AX foci\nformation in the indicated cells after cisplatin treatment. 2i, s2n Control or FSP1-\nknockout AGS-R (2i) or MGC-803-R (s2n) cells were exposed to the indicated dose of\ncisplatin with or without the indicated reagents for 2 weeks, colony formation assay\nwas then performed to detect the cell survival.\n3. USP29-SMURF1-FSP1 Axis and Chemotherapy Resistance. Based on the existing\ndata, it is reasonable to speculate that under chemotherapy conditions, the FSP1\ndeubiquitination process regulated by USP29 might outweigh the FSP1 degradation\nmediated by SMURF1. However, the authors did not provide more evidence to\nsupport this hypothesis. Moreover, the functional connection between the USP29-SMURF1-FSP1 axis and chemotherapy resistance in GC is not\nsufficiently established.\nResponse: We appreciate the reviewer raising this question. By investigating the\nstability of FSP1 in cisplatin sensitive and resistant cells, results revealed a notable\nincrease in FSP1 stability in resistant cells (Fig. 3a and Supplementary Fig. s3b),\naccompanied by reduced ubiquitination levels (Fig. 3b and Supplementary Fig. s3c),\nsuggesting that the deubiquitinase USP29 mediated deubiquitination outweighs the E3\nligase SMURF1 mediated ubiquitination for FSP1 protein. In terms of the functional\n25\nstudy of the USP29-SMURF1-FSP1 axis in GC chemoresistance, re-expressing WT\nFSP1 in USP29 depletion cells or SMURF1 overexpression cells was able to restore\nferroptosis and cisplatin sensitivity (Fig. 4g-i, 5m-o and Supplementary Fig. s5k-m,\ns6l-n), indicating that USP29 and SMURF1 coordinately regulate chemoresistance in\ngastric cancer by modulating FSP1-mediated ferroptosis.\n26\n3a, s3b Cisplatin sensitive or resistant AGS (3a) or MGC-803 (s3b) cells were treated\nwith 50 g/mL cycloheximide (CHX) for indicated time points and then FSP1 levels\nwere detected with western blot (left panel). FSP1 band intensity was quantified by\nImage J, which was normalized to -Actin and then compared to the t = 0 time point\n(right panel). 3b, s3c Ubiquitinated FSP1 was detected by anti-Ub antibody in the\nindicated cells treated with MG132 (50 M) for 4 h before harvested. 4g, s5k\nImmunoblots of FSP1 and USP29 in AGS-R (4g) or MGC-803-R (s5k) cells stably\nexpressing control sgRNA, USP29 sgRNA #2, or USP29 sgRNA #2 reintroduced\nwith the indicated FSP1 vectors. 4h, s5l Cells from (4g/s5k) were treated with or\nwithout cisplatin (2M) for 24 h, and then BODIPY C11 levels were detected. 4i,\ns5m Cells from (4g/s5k) were exposed to the indicated dose of cisplatin for 2 weeks,\ncolony formation assay was then performed to detect the cell survival. 5m, s6l\nImmunoblots of Flag and FSP1 in AGS-R (5m) or MGC-803-R (s6l) cells stably\nexpressing empty vector or Flag-SMURF1 reintroduced with empty vector or the\nindicated FSP1 vectors. 5n, s6m Cells from (5m/s6l) were treated with or without\ncisplatin (2M) for 24 h, and then BODIPY C11 levels were detected. 5o, s6n Cells\nfrom (5m/s6l) were exposed to the indicated dose of cisplatin for 2 weeks, colony\nformation assay was then performed to detect the cell survival.\n4. Generalizability of Chemotherapy Sensitivity Assays. One of the main concerns is\nthat the chemotherapy sensitivity assays, both in vitro and in vivo, appear overly\nsimplistic. Many of these experiments were conducted using AGS and MGC-803 cell\nlines. What is the functional role of the USP29-SMURF1-FSP1 axis in patient-\nderived xenograft (PDX) and patient-derived organoid (PDO) models? Additionally,\nthe study relies solely on cisplatin for the functional studies throughout the\nmanuscript. To strengthen the conclusions, the authors should validate their findings\nusing other chemotherapy drugs.\nResponse: We appreciate the reviewer's insightful suggestions. To enhance\nconfidence in our findings' generalizability, we utilized another two GC cell lines and\ntreated with cisplatin and 5-FU. The in vitro and in vivo data revealed that inhibition\n27\nof FSP1 synergizes with both chemotherapy drugs of cisplatin and 5-FU\n(Supplementary Fig. s8a-g). Furthermore, to boost confidence in the generalizability\nof our findings, we established six cases of GC patient-derived organoids (PDO) to\nexamine the combination efficacy of FSP1 inhibition and chemotherapeutic agents\n(Fig. 7e, f). Expectedly, iFSP1 markedly enhances the sensitivity of PDO to cisplatin\nor 5-FU (Fig. 7g, h and Supplementary Fig. 8l, m). We also obtained a fresh GC\ntumor specimen exhibiting high FSP1 expression (Fig. 7i) from a patient who had\nundergone a second surgical resection due to rapid disease progression following\nchemotherapy. We successfully established patient-derived xenograft (PDX) models,\nand the results showed that a combination of iFSP1 and cisplatin worked effectively\nin killing tumor cells and prolonging survival (Fig. 7j-l). Collectively, these PDO and\nPDX data provide additional support for our conclusions.\ns8a-d The indicated cells were exposed to vehicle, cisplatin (20 nM), 5-FU (20 nM),\niFSP1 (10 nM) or their combination for 2 weeks, colony formation assay was then\nperformed to detect the cell survival. s8e Growth curves of the BGC-823 xenograft\ntumor models. BGC-823 cells were injected subcutaneously. When tumors reached\napproximately 100 mm3, mice were randomly assigned to the indicated treatment (n =\n5 for each group). s8f Quantification of the weight of the tumors in different groups of\nthe xenograft tumor models. s8g Survival curves of the BGC-823 xenograft tumor\nmodels (n = 8 for each group).\n28\n7e Representative bright-field images of the 6 cases of cultured gastric cancer patient-\nderived organoids (PDO). 7f Immunoblot showing FSP1 expression levels in the 6\nGC PDO. 7g, s8l Organoid viability of PDO #4 with the indicated treatment. 7h, s8m\nRepresentative micrographs (left) and quantitation (right) of PDO #4 with the\nindicated treatment. Organoids were stained with Calcein-AM (live cells, green) and\nEthD-1 (dead cells, red) for 30 min and then fluorescent signals were captured. 7i\nIHC staining of FSP1 in the GC patient. Scale bars are indicated. 7j Growth curves of\nthe patient-derived tumor xenograft (PDX) models. PDX cells were injected\nsubcutaneously. When tumors reached approximately 100 mm3, mice were randomly\nassigned to the indicated treatment (n = 5 for each group). 7k Quantification of the\nweight of the tumors in different groups of the PDX models. 7l Survival curves of the\nPDX models (n = 8 for each group).\n5. Ubiquitination Experiments to Confirm SMURF1’s Role. The authors need to\n29\nconduct in vitro ubiquitination assays to confirm that SMURF1 functions as an E3\nligase for FSP1. Moreover, the authors should investigate which specific structural\ndomain of SMURF1 mediates its E3 ligase activity. Identifying this domain would\nprovide further insight into the mechanism by which SMURF1 regulates FSP1\nstability.\nResponse: Thank you for your valuable suggestions. The binding regions between\nSMURF1 and FSP1 were delineated through a series of truncation experiments\ninvolving SMURF1. Notably, the WW domain of SMURF1 was identified as crucial\nfor mediating the SMURF1-FSP1 interaction (Fig. 5e). Furthermore, we performed in\nvitro ubiquitination assay using purified FSP1 and SMURF1 proteins and confirmed\nthat SMURF1 can ubiquitinate FSP1, while mutation of the C725 in the HECT\ndomain abolished the E3 ligase activity (Fig. 5l).\n5e Full length and different fragments of Flag-tagged SMURF1 were transfected into\nHEK293T cells. 48 h later, cells were lysed and immunoprecipitated with Flag beads.\nThe immunoprecipitates were then blotted with the indicated antibodies. 5l In vitro\nubiquitination of FSP1 by SMURF1. Purified GFP-FSP1, His-SMURF1 WT and\nC725A proteins were subjected to in vitro ubiquitination assay in the presence or\nabsence of E1 and E2.\n1. Figure 3. Many GFP and His bands are overexposed. The quality of these Western\nblot images should be improved.\n2. Figures 3L-3N. The negative control should be clearly labeled and addressed.\nResponse: Thank you for careful review of our manuscript. We repeated the\nexperiments in Fig 3l-p and showed bands without overexposure. Furthermore, we\nuniformed the negative control with empty vector (EV) in Fig. 3m, n.\n3l Cells stably expressing control or USP29 shRNAs were transfected with His-Ub\nand GFP-FSP1, and then were treated with MG132 for 4 h before harvested.\nCovalently modified proteins purified on Ni-NTA beads under denatured conditions.\nUbiquitinated FSP1 was detected by anti-GFP antibody. 3m HEK293T cells\ntransfected with the indicated vectors were treated with MG132 for 4 h before\nharvested. Covalently modified proteins purified on Ni-NTA beads under denatured\nconditions. Ubiquitinated FSP1 was detected by anti-GFP antibody. 3n\nDeubiquitination of FSP1 in vitro by USP29. Ubiquitinated FSP1 was incubated with\npurified GST fusion USP29 WT or USP29 CI in vitro and then blotted with indicated\nantibodies. 3o FSP1 expression vectors and indicated HA tag ubiquitin were\ntransfected into USP29 knockdown cells. Cell lysates were boiled and\nimmunoprecipitated with HA beads and immunoblotted as indicated. 3p HEK293T\ncells transfected with indicated vectors were treated with MG132 for 4 h before\n31\nharvested. Covalently modified proteins purified on HA beads under undenatured\nconditions. Ubiquitinated FSP1 was detected by anti-GFP antibody.\n32"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The author have done a great effort to address the points raised.\nWe still have one major concern:\nWhy is ferroptosis induced by iFSP1 in vivo in the presence of full GPX4 capacity? This is very puzzling.\nIn 7 b/c and j/k, the effect of iFSP1 is very low, while the combination with cisplatin is much higher. How do the authors rule\nout other forms of cell death (e.g. apoptosis) in vivo?\nFurthermore, in 2j the combination of sgFSP1+cisplatin is only rescued by 50% with Lip-1. How about the other 50%? Other\ncell death modality?\nThis ALL needs clarification (experimental and discussion)… in its current form, it is not convincing that it is only ferroptosis\nthat is induced.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "The authors have thoroughly addresses the points raised during the revision. I have nothing to add at this stage.", "reviewer_4": "The authors have comprehensively addressed my previous review comments by providing a clearer framework for\nelucidating the mechanism of FSP1-mediated chemotherapy resistance. One remaining question merits further\ninvestigation.\nCould the authors examine the protein levels of FSP1/ USP29/SMURF1 and ubiquitination status of FSP1 in untreated and\ncisplatin-treated AGS cells, as well as other GC cell line? Should cisplatin treatment induce alterations in these protein\nlevels or the enzymatic activities of USP29/SMURF1, it would be valuable to explore the underlying regulatory mechanisms."}, "author_response": "Point-by-point response to the reviewers’ comments.\nWe sincerely thank the reviewers for their thoughtful evaluation of our revision work.\nTheir additional comments were highly appreciated. Our replies to their comments are\nhighlighted in blue.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe author have done a great effort to address the points raised.\nWe still have one major concern:\nWhy is ferroptosis induced by iFSP1 in vivo in the presence of full GPX4 capacity?\nThis is very puzzling.\nResponse: Thank you for diligently reviewing our manuscript and for bringing this\nmatter to our attention. There are three reasons that may potentially address the\nconcern you raised:\n(1) As illustrated in the figure below, the level of GPX4 expression in gastric cancer\n(GC) is comparatively low among various types of cancer, indicating that GC\nexhibits a reduced reliance on GPX4. Furthermore, Doll S, et al. revealed that\nelevated FSP1 expression is associated with a diminished reliance on GPX4 in a\npanel of 559 cancer cell lines through an analysis of the cancer dependency map.1\nIn our study, we have shown that cisplatin-resistant cells exhibited markedly\nhigher levels of FSP1 compared with parental cells, suggesting a substantial\nreduction in GPX4 dependency within these cells.\nExpression levels of GPX4 for various cancer types in TCGA.\n(2) Emerging evidence have demonstrated that DNA damage-inducing agents, such as\nirradiation and PARP inhibitors, can induce ferroptosis independently of GPX4\nlevel change.2-6\n(3) Other studies have also demonstrated that FSP1 inhibition alone can inhibit tumor\ngrowth by inducing ferroptosis in vitro and in vivo.7-9\nReference\n1. Doll S, Freitas FP, Shah R, et al. FSP1 is a glutathione-independent ferroptosis\nsuppressor. Nature. 2019; 575(7784): 693-698.\n2. Lang X, Green MD, Wang W, et al. Radiotherapy and Immunotherapy Promote\nTumoral Lipid Oxidation and Ferroptosis via Synergistic Repression of SLC7A11.\nCancer Discov. 2019; 9(12): 1673-1685.\n3. Ye LF, Chaudhary KR, Zandkarimi F, et al. Radiation-Induced Lipid Peroxidation\nTriggers Ferroptosis and Synergizes with Ferroptosis Inducers. ACS Chem Biol.\n2020; 15(2): 469-484.\n4. Hong T, Lei G, Chen X, et al. PARP inhibition promotes ferroptosis via repressing\nSLC7A11 and synergizes with ferroptosis inducers in BRCA-proficient ovarian\ncancer. Redox Biol. 2021; 42: 101928.\n5. Shen D, Luo J, Chen L, et al. PARPi treatment enhances radiotherapy-induced\nferroptosis and antitumor immune responses via the cGAS signaling pathway in\ncolorectal cancer. Cancer Lett. 2022; 550: 215919.\n6. Chen PH, Tseng WH, Chi JT. The Intersection of DNA Damage Response and\nFerroptosis-A Rationale for Combination Therapeutics. Biology (Basel). 2020;\n9(8): 187-198.\n7. Nakamura T, Hipp C, Santos Dias Mourão A, et al. Phase separation of FSP1\npromotes ferroptosis. Nature. 2023; 619(7969): 371-377.\n8. Wu YC, Huang CS, Hsieh MS, et al. Targeting of FSP1 regulates iron homeostasis\nin drug-tolerant persister head and neck cancer cells via lipid-metabolism-driven\nferroptosis. Aging (Albany NY). 2024; 16(1): 627-647.\n9. Cheu JW, Lee D, Li Q, et al. Ferroptosis Suppressor Protein 1 Inhibition Promotes\nTumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer. Cell Mol\nGastroenterol Hepatol. 2023; 16(1):133-159.\nIn 7 b/c and j/k, the effect of iFSP1 is very low, while the combination with cisplatin\nis much higher. How do the authors rule out other forms of cell death (e.g. apoptosis)\nin vivo?\nFurthermore, in 2j the combination of sgFSP1+cisplatin is only rescued by 50% with\nLip-1. How about the other 50%? Other cell death modality?\nThis ALL needs clarification (experimental and discussion)… in its current form, it is\nnot convincing that it is only ferroptosis that is induced.\nResponse: We appreciate the reviewer for raising this question. As mentioned above,\nemerging evidence indicates that DNA-damaging agents can trigger ferroptosis. This\nfinding may help explain why the efficacy of iFSP1 alone is minimal compared to its\ncombination with cisplatin. In order to exclude the possibility of other types of cell\ndeath in FSP1 inhibition-induced cisplatin susceptibility, we initially exposed FSP1-\nnull cells to inhibitors targeting alternative forms of cell death. The results showed\nthat FSP1 deficiency-induced cisplatin susceptibility was only rescued by ferroptosis\ninhibitor, rather than by inhibitors targeting other forms of cell death, thus confirming\nits specificity for ferroptosis (Fig. 2i and Supplementary Fig. s2n).\n2i, s2n Control or FSP1-KO AGS-R (2i) and MGC-803-R (s2n) cells were exposed to\nthe indicated dose of cisplatin with or without the indicated reagents for 2 weeks,\ncolony formation assay was then performed to detect the cell survival.\nFor in vivo liproxstatin-1 rescue experiment in our study, liproxstatin-1 (10 mg/kg)\nwas intraperitoneally injected once every 3 days for 6 times. The dosage and\nfrequency of liproxstatin-1 administration in our study were significantly lower\ncompared to other research studies1-4. Furthermore, due to the intricate nature of\nmouse models, it has been demonstrated in other studies that complete rescue of\nmouse growth inhibition induced by ferroptosis is not always achieved with\nliproxstatin-1.5-8 Combined with our in vitro rescue data, we hypothesized that the\n50% rescue rate was attributed to the lower dosage of liproxstatin-1 administration\nand the complexity of mouse models rather than other mechanisms of cell death. We\nhave included specific information regarding the administration of liproxstatin-1 in\nmice in the Method of revised manuscript.\nReference\n1. Kim R, Hashimoto A, Markosyan N, et al. Ferroptosis of tumour neutrophils\ncauses immune suppression in cancer. Nature. 2022; 612(7939): 338-346.\n2. Wang W, Green M, Choi JE, et al. CD8+ T cells regulate tumour ferroptosis\nduring cancer immunotherapy. Nature. 2019; 569(7755): 270-274.\n3. Zou Y, Palte MJ, Deik AA, et al. A GPX4-dependent cancer cell state underlies\nthe clear-cell morphology and confers sensitivity to ferroptosis. Nat Commun.\n2019; 10(1): 1617.\n4. Wu M, Zhang X, Zhang W, et al. Cancer stem cell regulated phenotypic plasticity\nprotects metastasized cancer cells from ferroptosis. Nat Commun. 2022; 13(1):\n1371.\n5. Lang X, Green MD, Wang W, et al. Radiotherapy and Immunotherapy Promote\nTumoral Lipid Oxidation and Ferroptosis via Synergistic Repression of SLC7A11.\nCancer Discov. 2019; 9(12): 1673-1685.\n6. Zhang T, Sun L, Hao Y, et al. ENO1 suppresses cancer cell ferroptosis by\ndegrading the mRNA of iron regulatory protein 1. Nat Cancer. 2022; 3(1): 75-89.\n7. Mao C, Lei G, Horbath A, et al. Unraveling ETC complex I function in\nferroptosis reveals a potential ferroptosis-inducing therapeutic strategy for LKB1-\ndeficient cancers. Mol Cell. 2024; 84(10): 1964-1979.e6.\n8. Lei G, Zhang Y, Koppula P, et al. The role of ferroptosis in ionizing radiation-\ninduced cell death and tumor suppression. Cell Res. 2020; 30(2): 146-162.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nReviewer #3 (Remarks to the Author):\nThe authors have thoroughly addresses the points raised during the revision. I have\nnothing to add at this stage.\nWe would like to thank the reviewer for the support and constructive feedback.\nReviewer #4 (Remarks to the Author):\nThe authors have comprehensively addressed my previous review comments by\nproviding a clearer framework for elucidating the mechanism of FSP1-mediated\nchemotherapy resistance. One remaining question merits further investigation.\nCould the authors examine the protein levels of FSP1/USP29/SMURF1 and\nubiquitination status of FSP1 in untreated and cisplatin-treated AGS cells, as well as\nother GC cell line? Should cisplatin treatment induce alterations in these protein\nlevels or the enzymatic activities of USP29/SMURF1, it would be valuable to explore\nthe underlying regulatory mechanisms.\nResponse: We sincerely thank the reviewer for the careful and thorough evaluation of\nour manuscript, and for the thoughtful comments. We treated AGS and MGC-803\ncells with cisplatin (2 M) for 48 hours and examined the protein levels of\nFSP1/USP29/SMURF1 and ubiquitination status of FSP1. The results showed no\nchanges in the protein levels of FSP1/USP29/SMURF1 or the ubiquitination status of\nFSP1 (Figure a,b) after acute treatment of cisplatin. We speculate that the increased\nexpression of FSP1 in cisplatin-resistant cells is a result of natural selection during\nprolonged cisplatin treatment. In our study, we focus more narrowly on the role of\nFSP1 in cisplatin-resistant cells, demonstrating that it is a potential target in\ncombination therapies to overcome cisplatin resistance.\na AGS and MGC-803 cells were treated with or without cisplatin (2 M) for 48 hours\nand FSP1/USP29/SMURF1 protein levels were detected with western blot. b AGS and\nMGC-803 cells transfected with indicated vectors were treated with or without MG132\nfor 4 h before harvested. Covalently modified proteins purified on GFP beads under\nundenatured conditions. Ubiquitinated FSP1 was detected by anti-HA antibody."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Thanks to the authors for the explanation.\nI still don't fully believe this only ferroptosis -- more some mixed-effect. However, the study is good and can be published.\nI would strongly suggest that the authors discuss some of these points as this would make the study more accessible to\nreaders.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThanks to the authors for the explanation.\nI still don't fully believe this only ferroptosis -- more some mixed-effect. However,\nthe study is good and can be published.\nI would strongly suggest that the authors discuss some of these points as this would\nmake the study more accessible to readers.\nResponse: We appreciate the reviewer for careful and thorough evaluation of our\nmanuscript, and for the valuable suggestion. We added discussion about the\npossibility of mixed cell death effect in the first paragraph of Discussion section in\nour revised manuscript and marked with yellow background.\n“Chemoresistance in GC can arise from a dynamic cell death response, where cancer\ncells switch between different cell death pathways, including apoptosis, autophagy,\nnecrosis, and ferroptosis. This shift enables GC cells evade the effects of\nchemotherapy, thereby diminishing the efficacy of treatment. While the possibility\nthat mixed cell death contributing to chemoresistance in GC does exist, our findings\nsuggest that FSP1-mediated ferroptosis may play a key role in chemoresistance of GC\nvia WGCNA.”\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nWe thank the reviewer for evaluating our revised manuscript."}]

Decision letter