S100A9 Promotes Resistance to Anti-PD-1 Immunotherapy in Hepatocellular Carcinoma by Degrading PARP1 and Activating the STAT3/PD-L1 Pathway

S100A9 Promotes Resistance to Anti-PD-1 Immunotherapy in Hepatocellular Carcinoma by Degrading PARP1 and Activating the STAT3/PD-L1 Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article S100A9 Promotes Resistance to Anti-PD-1 Immunotherapy in Hepatocellular Carcinoma by Degrading PARP1 and Activating the STAT3/PD-L1 Pathway Xianwei Zhou, Chu Qiao, Xuehui Chu, Haoran Man, Jingxin Liu, Yunzheng Li, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5797937/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1) immunotherapy, have emerged as promising treatments for advanced hepatocellular carcinoma (HCC), significantly improving clinical outcomes. However, resistance to ICIs remains a major challenge, and the underlying mechanisms of this resistance are not yet fully understood. This study aimed to investigate the role of S100 calcium-binding protein A9 (S100A9) in mediating resistance to anti-PD-1 therapy. Approach and Results: We conducted RNA sequencing (RNA-seq) on tumor samples from anti-PD-1 responders and non-responders in HCC patients. Differential expression analysis identified S100A9 as a potential driver gene of resistance to anti-PD-1 therapy. Subcutaneous tumor models and an orthotopic HCC model established via hydrodynamic transfection were utilized to evaluate the impact of S100A9 on the efficacy of PD-1 therapy. Our findings revealed that S100A9 promotes resistance to anti-PD-1 therapy in HCC. Mechanistically, S100A9 directly interacted with PARP1 and induced its degradation via the ubiquitin-proteasome pathway. This process increased STAT3 phosphorylation at Tyr705, thereby enhancing PD-L1 transcription. Notably, treatment with the S100A9 inhibitor Tasquinimod significantly improved the efficacy of anti-PD-1 therapy in HCC. Conclusions Our study reveals that S100A9 facilitates immune evasion in HCC by enhancing PARP1 ubiquitination, STAT3 phosphorylation, and PD-L1 expression. Furthermore, combining S100A9 inhibitors with anti-PD-1 antibodies markedly enhances the therapeutic efficacy of ICIs in HCC. These findings highlight S100A9 as a potential therapeutic target for overcoming resistance to immunotherapy in HCC. Hepatocellular carcinoma Immunotherapy S100 calcium-binding protein A9 Poly (ADP-ribose) polymerase 1 Programmed Death-Ligand 1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Liver cancer is the sixth most common malignant tumor globally and ranks fifth in incidence, constituting the third leading cause of cancer-related mortality worldwide[ 1 , 2 ]. Hepatocellular carcinoma (HCC) is the most prevalent form of liver cancer. Despite considerable advancements in early detection and surgical treatments, the prognosis for HCC remains poor, particularly in advanced stages where patients are ineligible for surgical resection[ 3 , 4 ]. Immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1) immunotherapy, have emerged as promising therapies for advanced HCC, significantly improving treatment outcomes[ 5 , 6 ]. However, the objective response rate (ORR) to PD-1 inhibition remains relatively low, underscoring the need to identify biomarkers that predict response to PD-1/PD-L1 inhibitors. Additionally, resistance to these therapies limits their efficacy in HCC patients, with the underlying mechanisms of resistance still not fully understood[ 7 ]. Key challenges in advancing immunotherapy for HCC include identifying predictive biomarkers for patient selection, overcoming resistance, and developing more effective combination therapies to counteract immune evasion mechanisms[ 8 ]. Biomarkers that predict response to PD-1 inhibitors in HCC are essential for improving treatment outcomes and personalizing therapy[ 9 , 10 ]. Tumor mutational burden (TMB) and microsatellite instability (MSI) are two key biomarkers, with higher TMB and MSI-high status generally correlating with better responses due to enhanced neoantigen presentation[ 11 ]. While PD-L1 expression on tumor cells or immune cells within the tumor microenvironment is commonly used as a biomarker, its predictive value in HCC remains inconsistent[ 12 ]. Tumor-infiltrating lymphocytes (TILs), particularly CD8 + T cells, play a critical role in determining the response to PD-1 inhibitors, with a higher presence of these cells often associated with improved efficacy[ 13 ]. Our previous studies demonstrated that the inflamed class[ 14 ], fatty acid degradation (FAD)-based subtypes[ 15 , 16 ], and the MHC-I-dependent neoantigen presentation pathway were predictive of the response to immunotherapy therapy in HCC[ 17 ]. However, resistance to anti-PD-1 therapy in HCC remains a significant challenge, and the mechanisms underlying this resistance are complex. Investigating these mechanisms is crucial for overcoming resistance and optimizing the clinical application of PD-1 inhibitors in HCC. S100 calcium-binding protein A9 (S100A9) is a prominent member of the S100 protein family. Originally identified as an inflammatory mediator secreted by bone marrow-derived cells in response to cellular damage, infection, or inflammatory stimuli[ 18 ], S100A9 has garnered considerable attention due to its diverse roles. Recent research suggests that S100A9 promotes the initiation and progression of cancer and plays a crucial role in inhibiting tumor immune responses[ 19 , 20 ]. Furthermore, the expression level of S100A9 exhibits a negative correlation with the degree of differentiation in HCC, indicating a poor prognosis for patients with the disease[ 21 , 22 ]. S100A9 activates the inflammatory pathway while concurrently inhibiting immune responses. The mechanisms underlying S100A9's role in HCC remain poorly understood, particularly regarding implications for ICI therapy. Programmed cell death ligand 1 (PD-L1), a crucial immune checkpoint molecule, plays a pivotal role in tumor immune evasion by binding to PD-1 on T cells, inducing immune tolerance, and reducing the cytotoxic activity of tumor-infiltrating T cells, thereby enabling cancer immune escape[ 23 ]. The expression levels of PD-L1 in the tumor microenvironment can predict the efficacy of ICIs on different types of tumors. Therefore, a comprehensive exploration of the regulatory mechanisms governing PD-L1 expression holds promise for revealing new therapeutic targets in cancer immunotherapy[ 24 ]. PD-L1 expression levels in cancer cells can be influenced at the transcriptional and posttranslational levels[ 25 ]. Researchers have demonstrated that STAT3, a pivotal transcription factor, directly enhances PD-L1 expression in human cancer cells by acting on the PD-L1 promoter[ 26 ]. PARP1 (Poly (ADP-ribose) Polymerase 1) is a key member of the poly (ADP-ribose) polymerase family. It primarily recognizes and binds to DNA damage sites, facilitating DNA repair. It plays a crucial role in regulating cell death and inflammatory responses and is involved in key biological processes that maintain chromosomal stability[ 27 ]. Here, we identified that S100A9 promotes resistance to anti-PD-1 immunotherapy in the HCC mice model, consistent with earlier clinical samples. Our findings demonstrated that S100A9 acts as a negative regulator of PARP1 by promoting its ubiquitination and degradation, which enhances STAT3 phosphorylation at Tyr705 and subsequently increases PD-L1 transcription, thereby allowing tumor cells to evade immune system surveillance. Based on these findings, S100A9 might be used as a prognostic marker and a therapeutic target for anti-PD-1 immunotherapy in HCC. MATERIALS AND METHODS Cell culture HCC cell lines MHCC-97H and Huh7 were procured from Wuhan Servicebio Technology Co., Ltd. Cultures were maintained by supplementing the growth medium with 10% fetal bovine serum (FBS) from Wisent Bioproducts China Co., Ltd. The cells were incubated in a humidified atmosphere at 37°C with 5% CO2 to ensure optimal growth conditions. Western blot analysis and antibodies Western blot analysis and antibodies Protein extraction was facilitated by employing RIPA lysis buffer (WB3100, NCM Biotech). The extracted proteins were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Non-specific binding was minimized by pre-incubating the PVDF membranes in a blocking solution of 5% skim milk for 1 hour at ambient temperature. Next, we probed the membranes with specific primary antibodies at 4°C overnight to facilitate antibody-antigen interactions. Following this, the membranes underwent three rounds of washing with Tris-buffered saline with Tween 20 (TBST) to remove unbound antibodies. We then incubated the membranes with horseradish peroxidase-conjugated secondary antibodies for 1 hour to enhance signal detection. Finally, protein expression was visualized by enhanced chemiluminescence (ECL) detection reagents (E412, Vazyme Biotech). We used the following antibodies in Western blotting and immunohistochemistry: Anti-S100A9 (83578-2-RR, 1:1000), Anti-PD-L1 (17952-1-AP, 1:1000), Anti-PARP1 (13371-1-AP, 1:1000), Anti-STAT3 (60199-1-Ig, 1:1000), Anti-β-actin (66009-1-Ig,1:5000), Anti-DYKDDDDK (80010-1-RR,1:5000), Anti-Myc (10828-1-AP, 1:5000), Anti-HA (51064-2-AP, 1:5000) were procured from Wuhan Proteintech BiotechnologyAnti-p-STAT3 (Tyr705) (#9131,1:2000), Anti-Ubiquitin (#3936, 1:5000) were procured from Cell Signaling Technology. Animal studies and In vivo tumor assay In addition, the 5-week-old male C57BL/6 mice used in this study were all purchased from GemPharmatech Co., Ltd. The animals were housed in a pathogen-specific laboratory in an animal research center with strictly controlled environmental conditions, including temperature at 22 ± 2°C, humidity at 60 ± 10%, and a light/dark cycle of 12/12 hours. All animal care procedures and experimental protocols follow the relevant provisions of the Laboratory Animal Care and Use guidelines. The study has also received official approval from the Animal Care and Use Committee of the Laboratory Animal Center of Nanjing Drum Tower Hospital. The stable, transfected Hepa1-6 mouse liver cancer cells (1×10^7 cells) were subcutaneously implanted into male C57BL/6 mice (aged 5–6 weeks, weighing 20–22 grams) to induce tumor growth. When tumors reached a 150–200 mm³ volume, tumor-bearing mice in the S100A9 overexpression and control groups were randomly divided into two subgroups. In the subgroups, tumor-bearing mice were administered intraperitoneal injections of 200 µg anti-PD-1 antibody or IgG every three days, measuring tumor volume. After the experiment, euthanasia was performed through cervical dislocation, followed by a collection of tumors for subsequent immunohistochemical analysis. We used a hydrodynamic tail vein injection model to establish S100A9 overexpression and normal HCC models in situ[ 2 ]. For the in situ HCC models, during week six post-tumor formation, both S100A9 overexpression and normal HCC model groups were randomly divided into two subgroups; each subgroup received either 200 µg of anti-PD-1 antibody or IgG via intraperitoneal injection every three days. At the end of the experiment, mice were euthanized via cervical dislocation, and tumors were harvested for subsequent histological examination. Clinical samples This study rigorously adheres to the fundamental principles outlined in the Declaration of Helsinki. The research team selected a cohort of 41 patients diagnosed with HCC who underwent partial hepatectomy at the Division of Hepatobiliary and Transplantation Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, between January 2019 and December 2021. All participants provided informed consent, allowing the use of tumor tissue for scientific research. The experimental protocol involving human subjects has received formal approval from the Ethics Committee of Nanjing Drum Tower Hospital. Lentivirus Infection Shanghai Genechem Co., Ltd. used the pLVX-AcGFP1-N1 vector to create a lentivirus with a FLAG tag for S100A9 overexpression. This caused the overexpression of the S100A9 protein. This lentivirus was utilized to infect the Hepa1-6 cell line. We used puromycin as a selective agent after infection to enrich cells that successfully integrated the lentiviral genome. Measurement of culture medium supernatant S100A9 The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer's instructions. This kit employs a colorimetric method for detection, quantifying S100A9 in cell culture supernatants at an OD value of 450 nm. Real-Time Fluorescence Quantitative qRT-PCR We extracted total RNA using the Trizol method and then performed reverse transcription. We used a real-time fluorescence quantitative PCR system to do quantitative real-time PCR with ChamQ Universal SYBR qPCR premix, with calculation performed using the 2-ΔΔCt method. The primer sequences used were as follows: β-actin: Forward: 5’-CTACGTCGCCCTGGACTTCGAGC-3’ Reverse 5’-GATGGAGCCGCCGATCCACACGG-3’ S100A9: Forward:5’-AATGGTGGAAGCACAGTTGG-3’ Reverse:5’-TTCCCTTTAGACTTGGTTGG-3’ PARP1: Forward:5’-AAGGCGAATGCCAGCGTTAC-3’ Reverse:5’-GGCACTCTTGGAGACCATGTCA-3’ PD-L1: Forward:5’-GCTGTTGAAGGACCAGCTCT-3′ Reverse:5’-TGGAGGATGTGCCAGAGGTA-3′ Statistical Analysis We subjected all experimental data to rigorous statistical analysis using GraphPad Prism version 9.0 software. The results are depicted as the mean ± standard deviation (SD) to represent the data distribution comprehensively. We used the Student's t-test, to compare two groups. A threshold of p < 0.05 was established to define statistical significance, with more stringent levels of significance indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Nonsignificant differences were denoted by "ns" to distinguish them clearly from statistically significant outcomes. RESULTS S100A9 is highly expressed in anti-PD-1 nonresponders and associated with poor prognosis in HCC. We previously performed RNA sequencing (RNA-seq) on tumor tissue from anti-PD-1 responders and non-responders (Fig. 1 A). To identify potential driver genes associated with immunotherapy resistance, we conducted differential expression analysis comparing tumor tissue with adjacent non-tumor tissue and responders versus non-responders. Genes upregulated in both tumor tissue and non-responders were considered potential driver genes for anti-PD-1 resistance (Fig. 1 B). Using survival and differential expression analyses, we identified three genes—S100A9, PACSIN1, and BNIP3P17—with significantly altered expression levels as potential driver genes (Fig. 1 C). Furthermore, immunohistochemical (IHC) staining of patient tissue samples validated that S100A9 is upregulated in anti-PD-1 non-responders. The survival analysis showed that HCC patients with high S100A9 expression had significantly shorter overall survival (OS) and recurrence-free survival (RFS) compared to those with low expression levels based on both our in-house cohort and several outer cohorts (Fig. 1 E-J). S100A9 inhibits the efficacy of anti-PD-1 therapy in HCC mouse models. To investigate the impact of S100A9 on the efficacy of anti-PD-1 therapy in HCC, we developed subcutaneous and orthotopic HCC mouse models. We first treated mice bearing subcutaneous Hepa1-6 tumors that overexpressed S100A9 or vector with either IgG or anti-PD-1 antibody and monitored tumor growth (Fig. 2 A). The anti-PD-1 therapy significantly inhibited the growth of subcutaneous tumors of control Hepa1-6 cells. In contrast, tumors overexpressing S100A9 exhibited only mild suppression (Fig. 2 B-C). In the orthotopic HCC model, anti-PD-1 therapy significantly reduced the control group's tumor number and liver-to-body weight ratio. However, these reductions were not significant in the S100A9-overexpressing group (Fig. 2 D-F). Subsequently, we performed IHC staining of mice liver tissue, showing that S100A9 suppressed the infiltration of CD4 + and CD8 + T cells into tumor tissue (Fig. 2 G). These results indicated that S100A9 inhibited the efficacy of anti-PD-1 therapy in both subcutaneous and orthotopic HCC models in mice, consistent with findings from clinical samples. S100A9 enhances the transcriptional expression of PD-L1 by activating the STAT3 signaling pathway. PD-L1 was a key predictive biomarker for response to immune checkpoint inhibition via anti-PD-1 therapy in oncology. To investigate whether S100A9's suppression of anti-PD-1 therapy efficacy in HCC was associated with PD-L1, we analyzed the HALLMARK pathways related to S100A9 and PD-L1. Both analyses identified the IL-6/JAK/STAT3 signaling pathway (Fig. 3 A-C). To explore the regulatory relationship between S100A9 and PD-L1, we examined the expression levels of phosphorylated STAT3 and PD-L1 in MHCC-97H cells after S100A9 overexpression. We observed that overexpression of S100A9 in MHCC-97H cells increased STAT3 phosphorylation at Ty705 and significantly upregulated PD-L1 at both mRNA and protein levels (Fig. 3 D-E). Additionally, IHC staining of both patient and mouse tissue samples demonstrated that S100A9 upregulates STAT3 phosphorylation at Ty705 and PD-L1 expression (Fig. 3 F). S100A9 down-regulates PARP1 and promotes STAT3 phosphorylation at Tyr705 and PD-L1 transcription. To elucidate the mechanisms underlying S100A9-mediated STAT3 phosphorylation at Tyr705 and PD-L1 transcription, we overexpressed S100A9 in 293T cells and performed Immunoprecipitation-Mass Spectrometry (IP-MS) to identify interacting proteins, which identified PARP1 as potential Interacting proteins of S100A9 (Fig. 4 A-C). It reported that PARP1 dephosphorylates STAT3 at Tyr705, thereby inhibiting the transcription of PD-L1[ 28 , 29 ]. Thus, we selected PARP1 for further investigation among the identified interactors. Co-immunoprecipitation assays confirmed the interaction between S100A9 and PARP1 in MHCC-97H cells (Fig. 4 D). Immunofluorescence staining showed significant co-localization of S100A9 and PARP1 in Huh7 cells (Fig. 4 E). To further investigate the role of S100A9 in PARP1 regulation, we overexpressed S100A9 in MHCC-97H cells, and assessed PARP1 expression using Western blotting. The results demonstrated a significant downregulation of PARP1 after S100A9 overexpression (Fig. 4 F). Then, we knocked down S100A9 in Huh7 cells and assessed PARP1 expression using Western blotting. The results indicated an upregulation of PARP1 expression after S100A9 knockdown (Fig. 4 G). To identify the specific binding sites and understand the mechanisms underlying this downregulation, we conducted truncation experiments, which revealed that S100A9 interacts with the BRCT domain of PARP1 (Fig. 4 H-I). To further explore the mechanism, we performed protein stability assays in MHCC-97H cells, demonstrating that S100A9 downregulated PARP1 through the ubiquitin-proteasome pathway (Fig. 4 J-M). These results suggested that S100A9 interacted with PARP1 and induced its degradation via the ubiquitin-proteasome pathway, thereby increasing STAT3 phosphorylation at Tyr705 and enhancing PD-L1 transcription. Combining Tasquinimod and anti-PD-1 antibody enhances the efficacy of immune checkpoint inhibitors in HCC mouse model. Tasquinimod, an inhibitor of S100A9, primarily suppressed NF-κB signaling to downregulate S100A9 transcription and enhanced its protein degradation via the ubiquitin-proteasome pathway. The S100A9 inhibitor Tasquinimod has been confirmed to exhibit anti-tumor activity[ 30 , 31 ]. In our study, we established a subcutaneous tumor model using the Hepa1-6 cell line with stable overexpression of S100A9. We then administered either an anti-PD-1 antibody, Tasquinimod, or a combination. Our findings indicate combining Tasquinimod and anti-PD-1 therapy significantly enhances therapeutic efficacy (Fig. 5 A-B). In cellular experiments, applying S100A9 inhibitors led to a significant increase in PARP1 protein levels with rising inhibitor concentrations, while levels of STAT3 phosphorylation at Tyr705 and PD-L1 proteins showed an opposing trend (Fig. 5 C). Furthermore, IHC staining of subcutaneous tumors in mice revealed that Tasquinimod effectively inhibited tumor cell proliferation, enhanced the efficacy of anti-PD-1 therapy, and significantly increased the infiltration of CD4 + and CD8 + T cells into tumor tissues (Fig. 5 D). These results demonstrated that S100A9 inhibitor Tasquinimod can dramatically enhance the efficacy of anti-PD-1 therapy in HCC mouse model through PARP1/STAT3/PD-L1 pathway. In summary, this study demonstrates that S100A9 contributes to resistance against anti-PD-1 therapy in HCC. Mechanistically, S100A9 directly interacts with PARP1 and induces its degradation through the ubiquitin-proteasome pathway. This interaction leads to increased phosphorylation of STAT3 at Tyr705, which subsequently enhances PD-L1 transcription. Notably, treatment with the S100A9 inhibitor Tasquinimod significantly improved the efficacy of anti-PD-1 therapy in HCC (Fig. 6 ). DISCUSSION Despite the promising potential of anti-PD-1 immunotherapy, its effectiveness varies among patients, particularly those with HCC, who often show little to no therapeutic response[ 32 ]. Identifying potential biomarkers and enhancing the efficacy of ICIs in the treatment of HCC is of critical importance. Based on analysis of clinical samples and observations, we identify S100A9 as a crucial factor influencing the efficacy of ICIs in HCC treatment. S100A9 can activate the MAPK signaling pathway, leading to the proliferation and invasion of HCC cells. It can also change reactive oxygen species (ROS) and mitochondrial fission production, worsening the disease after transarterial chemoembolization (TACE)[ 33 , 34 ]. Considering that S100A9 plays a key role in the development and progression of HCC and in its immunotherapy, it may serve as a potential biomarker for evaluating the efficacy of immunotherapy in HCC. Our findings indicate that S100A9 inhibits the effectiveness of anti-PD-1 immunotherapy in HCC by upregulating PD-L1 expression in HCC cells and suppressing anti-tumor immunity. Our study revealed that elevated levels of S100A9 in tumor cells promoted phosphorylation of STAT3 at Tyr705, thereby increasing PD-L1 expression in HCC cells and facilitating tumor immune evasion. Further investigation demonstrated that S100A9 interacted with PARP1, promoting its degradation through ubiquitination, which in turn enhanced STAT3 phosphorylation at Tyr705 and upregulated PD-L1 transcription, ultimately leading to tumor immune escape. Meanwhile, we found that S100A9 inhibitors Tasquinimod could significantly enhance the efficacy of anti-PD-1 therapy in the HCC mouse model. PARP1, an important member of the PARP family, increases the dephosphorylation of STAT3 through poly(ADP-ribosyl)ation, thereby inhibiting PD-L1 transcription[ 29 , 35 ]. Upon DNA damage, PARP1 is recruited to damage sites to aid in DNA repair[ 36 ]. In radiotherapy, PARP1 can bind to TKT, inducing radioresistance by promoting double-strand break repair in HCC[ 37 ]. Notably, recent studies have shown that PARP1 inhibitors can enhance the efficacy of anti-PD-1 therapy in HCC[ 38 , 39 ]. However, our research indicated that S100A9 downregulated PARP1, which might suggest an enhancement of anti-PD-1 therapy effectiveness. Contrary to this expectation, S100A9 inhibited the efficacy of anti-PD-1 therapy due to its strong immunosuppressive effects. HCC cells and monocytes contain S100A9, which they can secrete extracellularly. This creates a positive feedback loop that stimulates the production of more S100A9 in both HCC cells and monocytes. This extracellular S100A9 strongly attracted myeloid-derived suppressor cells (MDSCs) and slowed down immune responses by encouraging the apoptosis of CD4 + and CD8 + T cells[ 20 , 40 ]. Within HCC cells, S100A9 activates the STAT3 signaling pathway and increases PD-L1 expression, aiding in immune evasion. Even though downregulating PARP1 might help the immune system by stopping DNA damage repair and turning on the cGAS/STING pathway in dendritic cells[ 39 ], S100A9 still negatively affects the immune system. In conclusion, although PARP1 downregulation could be beneficial, the negative impacts of S100A9 dominate, reducing the effectiveness of anti-PD-1 therapy. In conclusion, through clinical samples analysis and experimental studies, we have identified the role of S100A9 in treating HCC using ICIs. Given its role in liver cancer, we suggest that S100A9 could serve as a potential biomarker for assessing the efficacy of ICIs in HCC, offering a novel approach to overcoming the resistance of liver cancer to immunotherapy. Abbreviations S100A9, S100 calcium-binding protein A9; PARP1, Poly (ADP-ribose) polymerase 1; PD-L1, Programmed Death-Ligand 1; PD-1, programmed cell death-1; HCC, hepatocellular carcinoma. Declarations Financial Support: This work was supported by the Fundings for Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2021-LCYJ-MS-12, 2023-LCYJ-PY-32), grants from the National Natural Science Foundation of China (No. 82372834, 82173129, and 82103384), grants from Nanjing Special Fund for Science and Technology Development of Health (ZKX21026) and Jiangsu Outstanding Youth Foundation (BK20240119). Data availability statement: Transcriptomic data are available at the Gene Expression Omnibus (GSE202069, GSE14520). Acknowledgements: We are grateful to the patients and investigators who contributed to the publicly available datasets by sharing their data. The authors thank the Biobank of Nanjing Drum Tower Hospital for providing the specimens. Contributors: B.H.L. and D.C.Y. developed the study concept; B.H.L. and D.C.Y. supervised the project. B.H.L. performed the computational analysis; X.W.Z., Q.C., and H.R.M., J.X.L, Y.Z.L., H.L., W.H.W., Y.J.L., and Z.W.L, conducted experiments; X.H.C., X.T.S., and D.C.Y. managed the patients and assessed the clinical response; X.W.Z. and B.H.L. wrote the manuscript with the help of D.C.Y. All authors read and approved the final version of the manuscript. Competing interests: None declared. Ethics approval: The study was approved by the Research Ethics Committee of Drum Tower Hospital, and written informed consent was obtained from each patient. 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PD-L1 and B7-1 Cis-Interaction: New Mechanisms in Immune Checkpoints and Immunotherapies. Trends Mol Med. 2021:207-19. Lin X, Kang K, Chen P, Zeng Z, Li G, Xiong W, Yi M, Xiang B. Regulatory mechanisms of PD-1/PD-L1 in cancers. Mol Cancer. 2024:108. Yi M, Niu M, Xu L, Luo S, Wu K. Regulation of PD-L1 expression in the tumor microenvironment. J Hematol Oncol. 2021:10. Fan Z, Wu C, Chen M, Jiang Y, Wu Y, Mao R, Fan Y. The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation. Acta Pharm Sin B. 2022:1041-53. Chappidi N, Quail T, Doll S, Vogel LT, Aleksandrov R, Felekyan S, Kühnemuth R, Stoynov S, Seidel CAM, Brugués J, Jahnel M, Franzmann TM, Alberti S. PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends. Cell. 2024:945-61.e18. Xiao D, Zeng T, Zhu W, Yu ZZ, Huang W, Yi H, Lu SS, Feng J, Feng XP, Wu D, Wen Q, Zhou JH, Yuan L, Zhuang W, Xiao ZQ. ANXA1 Promotes Tumor Immune Evasion by Binding PARP1 and Upregulating Stat3-Induced Expression of PD-L1 in Multiple Cancers. Cancer Immunol Res. 2023:1367-83. Zhou B, Yan J, Guo L, Zhang B, Liu S, Yu M, Chen Z, Zhang K, Zhang W, Li X, Xu Y, Xiao Y, Zhou J, Fan J, Hung MC, Li H, Ye Q. Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma. Theranostics. 2020:6530-43. Williamson SC, Hartley AE, Heer R. A review of tasquinimod in the treatment of advanced prostate cancer. Drug Des Devel Ther. 2013:167-74. Fan R, Satilmis H, Vandewalle N, Verheye E, De Bruyne E, Menu E, De Beule N, De Becker A, Ates G, Massie A, Kerre T, Törngren M, Eriksson H, Vanderkerken K, Breckpot K, Maes K, De Veirman K. Targeting S100A9 protein affects mTOR-ER stress signaling and increases venetoclax sensitivity in Acute Myeloid Leukemia. Blood Cancer J. 2023:188. Yang X, Yang C, Zhang S, Geng H, Zhu AX, Bernards R, Qin W, Fan J, Wang C, Gao Q. Precision treatment in advanced hepatocellular carcinoma. Cancer Cell. 2024:180-97. Zhong C, Niu Y, Liu W, Yuan Y, Li K, Shi Y, Qiu Z, Li K, Lin Z, Huang Z, Zuo D, Yang Z, Liao Y, Zhang Y, Wang C, Qiu J, He W, Yuan Y, Li B. S100A9 Derived from Chemoembolization-Induced Hypoxia Governs Mitochondrial Function in Hepatocellular Carcinoma Progression. Adv Sci (Weinh). 2022:e2202206. Yan J, Deng M, Kong S, Li T, Lei Z, Zhang L, Zhuang Y, He X, Wang H, Fan H, Guo Y. Transarterial chemoembolization in combination with programmed death-1/programmed cell death-ligand 1 immunotherapy for hepatocellular carcinoma: A mini review. iLIVER. 2022:225-34. Ding L, Chen X, Xu X, Qian Y, Liang G, Yao F, Yao Z, Wu H, Zhang J, He Q, Yang B. PARP1 Suppresses the Transcription of PD-L1 by Poly(ADP-Ribosyl)ating STAT3. Cancer Immunol Res. 2019:136-49. Alemasova EE, Lavrik OI. Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins. Nucleic Acids Res. 2019:3811-27. Geng L, Zhu M, Luo D, Chen H, Li B, Lao Y, An H, Wu Y, Li Y, Xia A, Shi Y, Tong Z, Lu S, Xu D, Wang X, Zhang W, Sun B, Xu Z. TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma. Oncogene. 2024:682-92. Wang C, Tang H, Geng A, Dai B, Zhang H, Sun X, Chen Y, Qiao Z, Zhu H, Yang J, Chen J, He Q, Qin N, Xie J, Tan R, Wan X, Gao S, Jiang Y, Sun FL, Mao Z. Rational combination therapy for hepatocellular carcinoma with PARP1 and DNA-PK inhibitors. Proc Natl Acad Sci U S A. 2020:26356-65. Cao J, Su B, Zhang C, Peng R, Tu D, Deng Q, Jiang G, Jin S, Wang Q, Bai DS. Degradation of PARP1 by MARCHF3 in tumor cells triggers cCAS-STING activation in dendritic cells to regulate antitumor immunity in hepatocellular carcinoma. J Immunother Cancer. 2024. von Wulffen M, Luehrmann V, Robeck S, Russo A, Fischer-Riepe L, van den Bosch M, van Lent P, Loser K, Gabrilovich DI, Hermann S, Roth J, Vogl T. S100A8/A9-alarmin promotes local myeloid-derived suppressor cell activation restricting severe autoimmune arthritis. Cell Rep. 2023:113006. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5797937","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":400497200,"identity":"72129a79-323d-4b53-a315-e796a18a7231","order_by":0,"name":"Xianwei Zhou","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Xianwei","middleName":"","lastName":"Zhou","suffix":""},{"id":400497201,"identity":"5186f915-f625-42da-8380-367e4975dcd7","order_by":1,"name":"Chu Qiao","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chu","middleName":"","lastName":"Qiao","suffix":""},{"id":400497202,"identity":"e339628e-1085-4dc9-a567-5bf52cac6a29","order_by":2,"name":"Xuehui Chu","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Xuehui","middleName":"","lastName":"Chu","suffix":""},{"id":400497203,"identity":"a20c333f-6272-484a-834d-566cae449881","order_by":3,"name":"Haoran Man","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Haoran","middleName":"","lastName":"Man","suffix":""},{"id":400497205,"identity":"a1c2ba5a-3676-4acf-87e1-4c163e6537ae","order_by":4,"name":"Jingxin Liu","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Jingxin","middleName":"","lastName":"Liu","suffix":""},{"id":400497207,"identity":"b8ddfe94-e9b9-4892-af06-ff26b6631b43","order_by":5,"name":"Yunzheng Li","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Yunzheng","middleName":"","lastName":"Li","suffix":""},{"id":400497209,"identity":"756416a1-96cf-4749-94da-086b6239e396","order_by":6,"name":"Huan Li","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Li","suffix":""},{"id":400497211,"identity":"d9fcae28-400d-45b4-a712-1068c4ec8fed","order_by":7,"name":"Xiaodong Shan","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Shan","suffix":""},{"id":400497213,"identity":"d9246a4a-99fb-491b-a0df-a918e0a9483a","order_by":8,"name":"Weihong Wang","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital Clinical College of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Weihong","middleName":"","lastName":"Wang","suffix":""},{"id":400497214,"identity":"2eb2e586-b899-4bb4-9cd0-e5a008ce4d40","order_by":9,"name":"Zaowu Lian","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zaowu","middleName":"","lastName":"Lian","suffix":""},{"id":400497215,"identity":"ad2f8478-0efd-470d-9e88-4c1633868842","order_by":10,"name":"Yanjun Lu","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanjun","middleName":"","lastName":"Lu","suffix":""},{"id":400497216,"identity":"6cd6def7-543c-4351-a3e8-8b3b05f1d1ec","order_by":11,"name":"Decai Yu","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Decai","middleName":"","lastName":"Yu","suffix":""},{"id":400497217,"identity":"21d93ad1-1c82-4937-bbf7-5f2013986661","order_by":12,"name":"Xitai Sun","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Xitai","middleName":"","lastName":"Sun","suffix":""},{"id":400497218,"identity":"6d591333-8238-41fd-942d-3efc6ea2237f","order_by":13,"name":"Binghua Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAp0lEQVRIiWNgGAWjYBACPgkeBgYJBhsefv4GIrWwQbSkyUjOOECKFgaGwzYGDQnEapHuPfbAsu08jwHDAcYPH3OI0SJzLt1Asu02jzlzA7PkzG1EOSzHTAKkxbLhABszLwlazvEYHEggTcsBUrTInDGTkDiXzCM542AzcX7hl+4xk5Yos7Pn528++OEjMVpAgFkCTDE2EKkepPYD8WpHwSgYBaNgJAIAMv8s8r8kuNcAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School","correspondingAuthor":true,"prefix":"","firstName":"Binghua","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-01-09 15:53:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5797937/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5797937/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73788356,"identity":"3a46950c-b995-4b6c-86b5-d3caa202a1b2","added_by":"auto","created_at":"2025-01-14 16:34:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7029066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS100A9 is highly expressed in anti-PD-1 non-responders and associated with poor prognosis in HCC. \u003c/strong\u003e(A) The schematic diagram illustrates the process of RNA sequencing used to identify genes related to anti-PD-1 therapy. (B) Volcano plots display the genes linked to anti-PD-1 therapy, analyzed in responders, non-responders, tumors, and normal tissues. (C) A Venn diagram summarizes the findings related to genes upregulated in non-responders and those associated with overall survival (OS) and relapse-free survival (RFS) from the screening libraries. (D) Representative IHC images demonstrate S100A9 expression in tumor tissue samples from responders and non-responders. (E.G.H.I) Analysis based on the GSE202069, GSE14520, TCGA, and CHCC databases indicates that high expression levels of S100A9 predict poorer outcomes in OS and RFS. (F.H) Furthermore, high expression of S100A9 specifically correlates with worse RFS based on findings from GSE202069 and GSE14520.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5797937/v1/bb90e79395b1d5e892c7f7bd.png"},{"id":73788354,"identity":"af71e54f-150c-4ff4-9fd7-c8f51044dbbd","added_by":"auto","created_at":"2025-01-14 16:34:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10223004,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS100A9 inhibits the efficacy of anti-PD-1 therapy in HCC mouse models. \u003c/strong\u003e(A) Schematic representation of anti-PD-1 resistance treatment strategy in subcutaneous Hepa1-6 tumors. (B) Representative images of subcutaneous Hepa1-6 tumors in each group after treatment (n=7 per group). (C) The growth of subcutaneous Hepa1-6 tumors in C57BL/6 mice treated with IgG or anti-PD-1 antibody that overexpressed S100A9 or normal tumors was measured at designated time points. (n=6 / group, values are mean ± SEM, **p \u0026lt; 0.01, ***p\u0026lt;0.001, NS indicates no significance). (D) Schematic diagram and representative tumor images illustrating the inhibition of anti-PD-1 antibody efficacy by S100A9 in HCC treatment. (E) Number of tumors in each group after anti-PD-1 antibody or IgG treatment. (n=3 / group, values are mean ± SEM, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u0026lt;0.001, NS indicates no significance). (F) The liver-to-body ratio in each group after anti-PD-1 antibody or IgG treatment. (n=3 / group, values are mean ± SEM, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u0026lt;0.001, NS indicates no significance). (G) Representative images of IHC staining of mice tumor samples, including HE, S100A9, CD4, and CD8.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5797937/v1/8f18e5d34f1de3ceb3876519.png"},{"id":73788355,"identity":"874cdf82-c008-408a-a465-7614bc662a7c","added_by":"auto","created_at":"2025-01-14 16:34:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13041957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS100A9 enhances the transcriptional expression of PD-L1 by activating the STAT3 signaling pathway. \u003c/strong\u003e(A) S100A9 and PD-L1 related HALLMARK pathways. (B) Gene set enrichment analysis indicated a significant change in Il-6 Jak Stat3 signaling induced by S100A9. (C) Gene set enrichment analysis indicated a substantial change in Il-6 Jak Stat3 signaling induced by PD-L1. (D) Western blot analysis in MHCC-97H cells showed that S100A9 increased the STAT3 phosphorylation at Tyr705 and PD-L1. (E) q-PCR analysis in MHCC-97H cells showed that S100A9 enhanced the transcription of PD-L1. (F) The representative images of IHC staining for HCC clinical samples and mouse tumor samples include S100A9, STAT3 (Tyr705), and PD-L1.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5797937/v1/9c1cd858f5c05c35679b549c.png"},{"id":73788362,"identity":"3ac7b444-1d94-4837-8a16-064b1f967b5c","added_by":"auto","created_at":"2025-01-14 16:34:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6467888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS100A9 down-regulates PARP1 and promotes STAT3 phosphorylation at Tyr705 and PD-L1 transcription. \u003c/strong\u003e(A) A simplified flowchart illustrates the process of immunoprecipitation combined with mass spectrometry. (B) Coomassie's bright blue staining results showed S100A9 overexpression in 293T cells. (C) LC-MS mass spectrometry identified target proteins, including PARP1, bound to S100A9. (D) Western blotting results from the co-immunoprecipitation experiment in MHCC-97H cells were presented. (E) Immunofluorescence colocalization images demonstrated the interaction between S100A9 and PARP1 in Huh7 cells. (F) Western blotting in MHCC-97H cells indicated that S100A9 downregulated PARP1. (G) Western blotting of S100A9, PARP1, STAT3, p-STAT3 (Tyr705), and PD-L1 was conducted in Huh7 cells. (H) A schematic diagram depicting the functional structure of PARP1 was provided. (I) In 293T cells, the interaction between S100A9 and the functional site of PARP1 was explored, focusing on amino acids 1-1014, 1-779, 1-476, 1-203, and 203-1014 through co-immunoprecipitation. (J) The expression levels of PARP1 were assessed in MHCC-97H cells both before and after treatment with MG132, considering S100A9 overexpression and non-overexpression. (K) The expression status of PARP1 was evaluated in MHCC-97H cells under consistent concentrations of cycloheximide (CHX) and varying treatment durations, with or without S100A9 overexpression. (M-N) Flag-S100A9 and HA-Ub were co-expressed in 293T cells, interacting with MG132, both with and without the presence of MG132. PARP1 was isolated via immunoprecipitation, and its ubiquitin level was detected using an anti-Ub antibody.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5797937/v1/df0554857125dca106015fca.png"},{"id":73788373,"identity":"25f13ee2-4aeb-44d7-982c-e470622dad53","added_by":"auto","created_at":"2025-01-14 16:34:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8241271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe combination of tasquinimod and anti-PD-1 antibody can enhance the efficacy of immune checkpoint inhibitors (ICIs) in treating HCC. \u003c/strong\u003e(A) The schematic diagram illustrated a drug intervention regimen in C57BL/6 mice involving the S100A9 inhibitor Tasquinimod and/or anti-PD-1 antibody. It also included representative images of subcutaneous Hepa1-6 tumors in each treatment group (n=5 per group). (B)The growth of subcutaneous Hepa1-6 tumors was monitored in C57BL/6 mice treated with Tasquinimod and/or anti-PD-1 antibody. Tumor measurements were taken at the indicated time points. (n = 5 per group; values are presented as mean ± SEM; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, NS indicates no significance). (C) The effects of different concentrations of S100A9 inhibitors on S100A9, PARP1, STAT3, p-STAT3 (Tyr705), and PD-L1 levels were assessed in Huh7 cells. (D) Representative images of IHC staining from mouse tumor samples, including HE staining, Ki67, CD4, and CD8, were provided.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5797937/v1/89af88c1fbd5938fe9b7342e.png"},{"id":73788364,"identity":"c8f678d2-7b87-40f3-b56e-687771046a19","added_by":"auto","created_at":"2025-01-14 16:34:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3000325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram summarizing S100A9 Promotes Resistance to Anti-PD-1 Immunotherapy in HCC. \u003c/strong\u003eAn illustration of the proposed working model of S100A9 in HCC (Created by BioRender.com).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5797937/v1/cb8de230f2c45da015adb813.png"},{"id":73883597,"identity":"f029881f-4991-464a-9be6-c6832c57f868","added_by":"auto","created_at":"2025-01-15 14:17:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45562882,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5797937/v1/321037ec-2c31-49d8-94b7-17b9cb2b8fae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"S100A9 Promotes Resistance to Anti-PD-1 Immunotherapy in Hepatocellular Carcinoma by Degrading PARP1 and Activating the STAT3/PD-L1 Pathway","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLiver cancer is the sixth most common malignant tumor globally and ranks fifth in incidence, constituting the third leading cause of cancer-related mortality worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Hepatocellular carcinoma (HCC) is the most prevalent form of liver cancer. Despite considerable advancements in early detection and surgical treatments, the prognosis for HCC remains poor, particularly in advanced stages where patients are ineligible for surgical resection[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1) immunotherapy, have emerged as promising therapies for advanced HCC, significantly improving treatment outcomes[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the objective response rate (ORR) to PD-1 inhibition remains relatively low, underscoring the need to identify biomarkers that predict response to PD-1/PD-L1 inhibitors. Additionally, resistance to these therapies limits their efficacy in HCC patients, with the underlying mechanisms of resistance still not fully understood[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Key challenges in advancing immunotherapy for HCC include identifying predictive biomarkers for patient selection, overcoming resistance, and developing more effective combination therapies to counteract immune evasion mechanisms[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiomarkers that predict response to PD-1 inhibitors in HCC are essential for improving treatment outcomes and personalizing therapy[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Tumor mutational burden (TMB) and microsatellite instability (MSI) are two key biomarkers, with higher TMB and MSI-high status generally correlating with better responses due to enhanced neoantigen presentation[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While PD-L1 expression on tumor cells or immune cells within the tumor microenvironment is commonly used as a biomarker, its predictive value in HCC remains inconsistent[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Tumor-infiltrating lymphocytes (TILs), particularly CD8\u0026thinsp;+\u0026thinsp;T cells, play a critical role in determining the response to PD-1 inhibitors, with a higher presence of these cells often associated with improved efficacy[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our previous studies demonstrated that the inflamed class[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], fatty acid degradation (FAD)-based subtypes[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and the MHC-I-dependent neoantigen presentation pathway were predictive of the response to immunotherapy therapy in HCC[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, resistance to anti-PD-1 therapy in HCC remains a significant challenge, and the mechanisms underlying this resistance are complex. Investigating these mechanisms is crucial for overcoming resistance and optimizing the clinical application of PD-1 inhibitors in HCC.\u003c/p\u003e \u003cp\u003eS100 calcium-binding protein A9 (S100A9) is a prominent member of the S100 protein family. Originally identified as an inflammatory mediator secreted by bone marrow-derived cells in response to cellular damage, infection, or inflammatory stimuli[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], S100A9 has garnered considerable attention due to its diverse roles. Recent research suggests that S100A9 promotes the initiation and progression of cancer and plays a crucial role in inhibiting tumor immune responses[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, the expression level of S100A9 exhibits a negative correlation with the degree of differentiation in HCC, indicating a poor prognosis for patients with the disease[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. S100A9 activates the inflammatory pathway while concurrently inhibiting immune responses. The mechanisms underlying S100A9's role in HCC remain poorly understood, particularly regarding implications for ICI therapy.\u003c/p\u003e \u003cp\u003eProgrammed cell death ligand 1 (PD-L1), a crucial immune checkpoint molecule, plays a pivotal role in tumor immune evasion by binding to PD-1 on T cells, inducing immune tolerance, and reducing the cytotoxic activity of tumor-infiltrating T cells, thereby enabling cancer immune escape[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The expression levels of PD-L1 in the tumor microenvironment can predict the efficacy of ICIs on different types of tumors. Therefore, a comprehensive exploration of the regulatory mechanisms governing PD-L1 expression holds promise for revealing new therapeutic targets in cancer immunotherapy[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. PD-L1 expression levels in cancer cells can be influenced at the transcriptional and posttranslational levels[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Researchers have demonstrated that STAT3, a pivotal transcription factor, directly enhances PD-L1 expression in human cancer cells by acting on the PD-L1 promoter[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. PARP1 (Poly (ADP-ribose) Polymerase 1) is a key member of the poly (ADP-ribose) polymerase family. It primarily recognizes and binds to DNA damage sites, facilitating DNA repair. It plays a crucial role in regulating cell death and inflammatory responses and is involved in key biological processes that maintain chromosomal stability[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we identified that S100A9 promotes resistance to anti-PD-1 immunotherapy in the HCC mice model, consistent with earlier clinical samples. Our findings demonstrated that S100A9 acts as a negative regulator of PARP1 by promoting its ubiquitination and degradation, which enhances STAT3 phosphorylation at Tyr705 and subsequently increases PD-L1 transcription, thereby allowing tumor cells to evade immune system surveillance. Based on these findings, S100A9 might be used as a prognostic marker and a therapeutic target for anti-PD-1 immunotherapy in HCC.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHCC cell lines MHCC-97H and Huh7 were procured from Wuhan Servicebio Technology Co., Ltd. Cultures were maintained by supplementing the growth medium with 10% fetal bovine serum (FBS) from Wisent Bioproducts China Co., Ltd. The cells were incubated in a humidified atmosphere at 37\u0026deg;C with 5% CO2 to ensure optimal growth conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot analysis and antibodies\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot analysis and antibodies\u003c/div\u003e \u003cp\u003eProtein extraction was facilitated by employing RIPA lysis buffer (WB3100, NCM Biotech). The extracted proteins were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Non-specific binding was minimized by pre-incubating the PVDF membranes in a blocking solution of 5% skim milk for 1 hour at ambient temperature. Next, we probed the membranes with specific primary antibodies at 4\u0026deg;C overnight to facilitate antibody-antigen interactions. Following this, the membranes underwent three rounds of washing with Tris-buffered saline with Tween 20 (TBST) to remove unbound antibodies. We then incubated the membranes with horseradish peroxidase-conjugated secondary antibodies for 1 hour to enhance signal detection. Finally, protein expression was visualized by enhanced chemiluminescence (ECL) detection reagents (E412, Vazyme Biotech). We used the following antibodies in Western blotting and immunohistochemistry: Anti-S100A9 (83578-2-RR, 1:1000), Anti-PD-L1 (17952-1-AP, 1:1000), Anti-PARP1 (13371-1-AP, 1:1000), Anti-STAT3 (60199-1-Ig, 1:1000), Anti-β-actin (66009-1-Ig,1:5000), Anti-DYKDDDDK (80010-1-RR,1:5000), Anti-Myc (10828-1-AP, 1:5000), Anti-HA (51064-2-AP, 1:5000) were procured from Wuhan Proteintech BiotechnologyAnti-p-STAT3 (Tyr705) (#9131,1:2000), Anti-Ubiquitin (#3936, 1:5000) were procured from Cell Signaling Technology.\u003c/p\u003e\n\u003ch3\u003eAnimal studies and In vivo tumor assay\u003c/h3\u003e\n\u003cp\u003eIn addition, the 5-week-old male C57BL/6 mice used in this study were all purchased from GemPharmatech Co., Ltd. The animals were housed in a pathogen-specific laboratory in an animal research center with strictly controlled environmental conditions, including temperature at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, humidity at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, and a light/dark cycle of 12/12 hours. All animal care procedures and experimental protocols follow the relevant provisions of the Laboratory Animal Care and Use guidelines. The study has also received official approval from the Animal Care and Use Committee of the Laboratory Animal Center of Nanjing Drum Tower Hospital. The stable, transfected Hepa1-6 mouse liver cancer cells (1\u0026times;10^7 cells) were subcutaneously implanted into male C57BL/6 mice (aged 5\u0026ndash;6 weeks, weighing 20\u0026ndash;22 grams) to induce tumor growth. When tumors reached a 150\u0026ndash;200 mm\u0026sup3; volume, tumor-bearing mice in the S100A9 overexpression and control groups were randomly divided into two subgroups. In the subgroups, tumor-bearing mice were administered intraperitoneal injections of 200 \u0026micro;g anti-PD-1 antibody or IgG every three days, measuring tumor volume. After the experiment, euthanasia was performed through cervical dislocation, followed by a collection of tumors for subsequent immunohistochemical analysis. We used a hydrodynamic tail vein injection model to establish S100A9 overexpression and normal HCC models in situ[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For the in situ HCC models, during week six post-tumor formation, both S100A9 overexpression and normal HCC model groups were randomly divided into two subgroups; each subgroup received either 200 \u0026micro;g of anti-PD-1 antibody or IgG via intraperitoneal injection every three days. At the end of the experiment, mice were euthanized via cervical dislocation, and tumors were harvested for subsequent histological examination.\u003c/p\u003e\n\u003ch3\u003eClinical samples\u003c/h3\u003e\n\u003cp\u003e This study rigorously adheres to the fundamental principles outlined in the Declaration of Helsinki. The research team selected a cohort of 41 patients diagnosed with HCC who underwent partial hepatectomy at the Division of Hepatobiliary and Transplantation Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, between January 2019 and December 2021. All participants provided informed consent, allowing the use of tumor tissue for scientific research. The experimental protocol involving human subjects has received formal approval from the Ethics Committee of Nanjing Drum Tower Hospital.\u003c/p\u003e\n\u003ch3\u003eLentivirus Infection\u003c/h3\u003e\n\u003cp\u003eShanghai Genechem Co., Ltd. used the pLVX-AcGFP1-N1 vector to create a lentivirus with a FLAG tag for S100A9 overexpression. This caused the overexpression of the S100A9 protein. This lentivirus was utilized to infect the Hepa1-6 cell line. We used puromycin as a selective agent after infection to enrich cells that successfully integrated the lentiviral genome.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of culture medium supernatant S100A9\u003c/h2\u003e \u003cp\u003e The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer's instructions. This kit employs a colorimetric method for detection, quantifying S100A9 in cell culture supernatants at an OD value of 450 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReal-Time Fluorescence Quantitative qRT-PCR\u003c/h3\u003e\n\u003cp\u003eWe extracted total RNA using the Trizol method and then performed reverse transcription. We used a real-time fluorescence quantitative PCR system to do quantitative real-time PCR with ChamQ Universal SYBR qPCR premix, with calculation performed using the 2-ΔΔCt method.\u003c/p\u003e \u003cp\u003eThe primer sequences used were as follows:\u003c/p\u003e \u003cp\u003eβ-actin:\u003c/p\u003e \u003cp\u003eForward: 5\u0026rsquo;-CTACGTCGCCCTGGACTTCGAGC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse 5\u0026rsquo;-GATGGAGCCGCCGATCCACACGG-3\u0026rsquo;\u003c/p\u003e\n\u003ch3\u003eS100A9:\u003c/h3\u003e\n\u003cp\u003eForward:5\u0026rsquo;-AATGGTGGAAGCACAGTTGG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse:5\u0026rsquo;-TTCCCTTTAGACTTGGTTGG-3\u0026rsquo;\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePARP1:\u003c/h2\u003e \u003cp\u003eForward:5\u0026rsquo;-AAGGCGAATGCCAGCGTTAC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse:5\u0026rsquo;-GGCACTCTTGGAGACCATGTCA-3\u0026rsquo;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePD-L1:\u003c/h2\u003e \u003cp\u003eForward:5\u0026rsquo;-GCTGTTGAAGGACCAGCTCT-3\u0026prime;\u003c/p\u003e \u003cp\u003eReverse:5\u0026rsquo;-TGGAGGATGTGCCAGAGGTA-3\u0026prime;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eWe subjected all experimental data to rigorous statistical analysis using GraphPad Prism version 9.0 software. The results are depicted as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) to represent the data distribution comprehensively. We used the Student's t-test, to compare two groups. A threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was established to define statistical significance, with more stringent levels of significance indicated as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Nonsignificant differences were denoted by \"ns\" to distinguish them clearly from statistically significant outcomes.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eS100A9 is highly expressed in anti-PD-1 nonresponders and associated with poor prognosis in HCC.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe previously performed RNA sequencing (RNA-seq) on tumor tissue from anti-PD-1 responders and non-responders (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To identify potential driver genes associated with immunotherapy resistance, we conducted differential expression analysis comparing tumor tissue with adjacent non-tumor tissue and responders versus non-responders. Genes upregulated in both tumor tissue and non-responders were considered potential driver genes for anti-PD-1 resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Using survival and differential expression analyses, we identified three genes\u0026mdash;S100A9, PACSIN1, and BNIP3P17\u0026mdash;with significantly altered expression levels as potential driver genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, immunohistochemical (IHC) staining of patient tissue samples validated that S100A9 is upregulated in anti-PD-1 non-responders. The survival analysis showed that HCC patients with high S100A9 expression had significantly shorter overall survival (OS) and recurrence-free survival (RFS) compared to those with low expression levels based on both our in-house cohort and several outer cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eS100A9 inhibits the efficacy of anti-PD-1 therapy in HCC mouse models.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the impact of S100A9 on the efficacy of anti-PD-1 therapy in HCC, we developed subcutaneous and orthotopic HCC mouse models. We first treated mice bearing subcutaneous Hepa1-6 tumors that overexpressed S100A9 or vector with either IgG or anti-PD-1 antibody and monitored tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The anti-PD-1 therapy significantly inhibited the growth of subcutaneous tumors of control Hepa1-6 cells. In contrast, tumors overexpressing S100A9 exhibited only mild suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). In the orthotopic HCC model, anti-PD-1 therapy significantly reduced the control group's tumor number and liver-to-body weight ratio. However, these reductions were not significant in the S100A9-overexpressing group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). Subsequently, we performed IHC staining of mice liver tissue, showing that S100A9 suppressed the infiltration of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells into tumor tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). These results indicated that S100A9 inhibited the efficacy of anti-PD-1 therapy in both subcutaneous and orthotopic HCC models in mice, consistent with findings from clinical samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eS100A9 enhances the transcriptional expression of PD-L1 by activating the STAT3 signaling pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePD-L1 was a key predictive biomarker for response to immune checkpoint inhibition via anti-PD-1 therapy in oncology. To investigate whether S100A9's suppression of anti-PD-1 therapy efficacy in HCC was associated with PD-L1, we analyzed the HALLMARK pathways related to S100A9 and PD-L1. Both analyses identified the IL-6/JAK/STAT3 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). To explore the regulatory relationship between S100A9 and PD-L1, we examined the expression levels of phosphorylated STAT3 and PD-L1 in MHCC-97H cells after S100A9 overexpression. We observed that overexpression of S100A9 in MHCC-97H cells increased STAT3 phosphorylation at Ty705 and significantly upregulated PD-L1 at both mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). Additionally, IHC staining of both patient and mouse tissue samples demonstrated that S100A9 upregulates STAT3 phosphorylation at Ty705 and PD-L1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eS100A9 down-regulates PARP1 and promotes STAT3 phosphorylation at Tyr705 and PD-L1 transcription.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the mechanisms underlying S100A9-mediated STAT3 phosphorylation at Tyr705 and PD-L1 transcription, we overexpressed S100A9 in 293T cells and performed Immunoprecipitation-Mass Spectrometry (IP-MS) to identify interacting proteins, which identified PARP1 as potential Interacting proteins of S100A9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). It reported that PARP1 dephosphorylates STAT3 at Tyr705, thereby inhibiting the transcription of PD-L1[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, we selected PARP1 for further investigation among the identified interactors. Co-immunoprecipitation assays confirmed the interaction between S100A9 and PARP1 in MHCC-97H cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Immunofluorescence staining showed significant co-localization of S100A9 and PARP1 in Huh7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To further investigate the role of S100A9 in PARP1 regulation, we overexpressed S100A9 in MHCC-97H cells, and assessed PARP1 expression using Western blotting. The results demonstrated a significant downregulation of PARP1 after S100A9 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Then, we knocked down S100A9 in Huh7 cells and assessed PARP1 expression using Western blotting. The results indicated an upregulation of PARP1 expression after S100A9 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). To identify the specific binding sites and understand the mechanisms underlying this downregulation, we conducted truncation experiments, which revealed that S100A9 interacts with the BRCT domain of PARP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). To further explore the mechanism, we performed protein stability assays in MHCC-97H cells, demonstrating that S100A9 downregulated PARP1 through the ubiquitin-proteasome pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-M). These results suggested that S100A9 interacted with PARP1 and induced its degradation via the ubiquitin-proteasome pathway, thereby increasing STAT3 phosphorylation at Tyr705 and enhancing PD-L1 transcription.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCombining Tasquinimod and anti-PD-1 antibody enhances the efficacy of immune checkpoint inhibitors in HCC mouse model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTasquinimod, an inhibitor of S100A9, primarily suppressed NF-κB signaling to downregulate S100A9 transcription and enhanced its protein degradation via the ubiquitin-proteasome pathway. The S100A9 inhibitor Tasquinimod has been confirmed to exhibit anti-tumor activity[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In our study, we established a subcutaneous tumor model using the Hepa1-6 cell line with stable overexpression of S100A9. We then administered either an anti-PD-1 antibody, Tasquinimod, or a combination. Our findings indicate combining Tasquinimod and anti-PD-1 therapy significantly enhances therapeutic efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). In cellular experiments, applying S100A9 inhibitors led to a significant increase in PARP1 protein levels with rising inhibitor concentrations, while levels of STAT3 phosphorylation at Tyr705 and PD-L1 proteins showed an opposing trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Furthermore, IHC staining of subcutaneous tumors in mice revealed that Tasquinimod effectively inhibited tumor cell proliferation, enhanced the efficacy of anti-PD-1 therapy, and significantly increased the infiltration of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells into tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results demonstrated that S100A9 inhibitor Tasquinimod can dramatically enhance the efficacy of anti-PD-1 therapy in HCC mouse model through PARP1/STAT3/PD-L1 pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, this study demonstrates that S100A9 contributes to resistance against anti-PD-1 therapy in HCC. Mechanistically, S100A9 directly interacts with PARP1 and induces its degradation through the ubiquitin-proteasome pathway. This interaction leads to increased phosphorylation of STAT3 at Tyr705, which subsequently enhances PD-L1 transcription. Notably, treatment with the S100A9 inhibitor Tasquinimod significantly improved the efficacy of anti-PD-1 therapy in HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDespite the promising potential of anti-PD-1 immunotherapy, its effectiveness varies among patients, particularly those with HCC, who often show little to no therapeutic response[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Identifying potential biomarkers and enhancing the efficacy of ICIs in the treatment of HCC is of critical importance. Based on analysis of clinical samples and observations, we identify S100A9 as a crucial factor influencing the efficacy of ICIs in HCC treatment. S100A9 can activate the MAPK signaling pathway, leading to the proliferation and invasion of HCC cells. It can also change reactive oxygen species (ROS) and mitochondrial fission production, worsening the disease after transarterial chemoembolization (TACE)[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Considering that S100A9 plays a key role in the development and progression of HCC and in its immunotherapy, it may serve as a potential biomarker for evaluating the efficacy of immunotherapy in HCC.\u003c/p\u003e \u003cp\u003eOur findings indicate that S100A9 inhibits the effectiveness of anti-PD-1 immunotherapy in HCC by upregulating PD-L1 expression in HCC cells and suppressing anti-tumor immunity. Our study revealed that elevated levels of S100A9 in tumor cells promoted phosphorylation of STAT3 at Tyr705, thereby increasing PD-L1 expression in HCC cells and facilitating tumor immune evasion. Further investigation demonstrated that S100A9 interacted with PARP1, promoting its degradation through ubiquitination, which in turn enhanced STAT3 phosphorylation at Tyr705 and upregulated PD-L1 transcription, ultimately leading to tumor immune escape. Meanwhile, we found that S100A9 inhibitors Tasquinimod could significantly enhance the efficacy of anti-PD-1 therapy in the HCC mouse model.\u003c/p\u003e \u003cp\u003ePARP1, an important member of the PARP family, increases the dephosphorylation of STAT3 through poly(ADP-ribosyl)ation, thereby inhibiting PD-L1 transcription[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Upon DNA damage, PARP1 is recruited to damage sites to aid in DNA repair[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In radiotherapy, PARP1 can bind to TKT, inducing radioresistance by promoting double-strand break repair in HCC[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably, recent studies have shown that PARP1 inhibitors can enhance the efficacy of anti-PD-1 therapy in HCC[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, our research indicated that S100A9 downregulated PARP1, which might suggest an enhancement of anti-PD-1 therapy effectiveness. Contrary to this expectation, S100A9 inhibited the efficacy of anti-PD-1 therapy due to its strong immunosuppressive effects. HCC cells and monocytes contain S100A9, which they can secrete extracellularly. This creates a positive feedback loop that stimulates the production of more S100A9 in both HCC cells and monocytes. This extracellular S100A9 strongly attracted myeloid-derived suppressor cells (MDSCs) and slowed down immune responses by encouraging the apoptosis of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Within HCC cells, S100A9 activates the STAT3 signaling pathway and increases PD-L1 expression, aiding in immune evasion. Even though downregulating PARP1 might help the immune system by stopping DNA damage repair and turning on the cGAS/STING pathway in dendritic cells[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], S100A9 still negatively affects the immune system. In conclusion, although PARP1 downregulation could be beneficial, the negative impacts of S100A9 dominate, reducing the effectiveness of anti-PD-1 therapy.\u003c/p\u003e \u003cp\u003eIn conclusion, through clinical samples analysis and experimental studies, we have identified the role of S100A9 in treating HCC using ICIs. Given its role in liver cancer, we suggest that S100A9 could serve as a potential biomarker for assessing the efficacy of ICIs in HCC, offering a novel approach to overcoming the resistance of liver cancer to immunotherapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eS100A9, S100 calcium-binding protein A9; PARP1, Poly (ADP-ribose) polymerase 1; PD-L1, Programmed Death-Ligand 1; PD-1, programmed cell death-1; HCC, hepatocellular carcinoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFinancial Support:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work was supported by the Fundings for Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2021-LCYJ-MS-12, 2023-LCYJ-PY-32), grants from the National Natural Science Foundation of China (No. 82372834, 82173129, and 82103384), grants from Nanjing Special Fund for Science and Technology Development of Health (ZKX21026) and Jiangsu Outstanding Youth Foundation (BK20240119).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTranscriptomic data are available at the Gene Expression Omnibus (GSE202069, GSE14520).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWe are grateful to the patients and investigators who contributed to the publicly available datasets by sharing their data. The authors thank the Biobank of Nanjing Drum Tower Hospital for providing the specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributors:\u003c/strong\u003e B.H.L. and D.C.Y. developed the study concept; B.H.L. and D.C.Y. supervised the project. B.H.L. performed the computational analysis; X.W.Z., Q.C., and H.R.M., J.X.L, Y.Z.L., H.L., W.H.W., Y.J.L., and Z.W.L, conducted experiments; X.H.C., X.T.S., and D.C.Y. managed the patients and assessed the clinical response; X.W.Z. and B.H.L. wrote the manuscript with the help of D.C.Y. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e The study was approved by the Research Ethics Committee of Drum Tower Hospital, and written informed consent was obtained from each patient. All animal studies were performed following a protocol approved by the Institutional Ethics Committee for Clinical Research and Animal Trials of Drum Tower Hospital.\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVogel A, Meyer T, Sapisochin G, Salem R, Saborowski A. Hepatocellular carcinoma. Lancet. 2022:1345-62.\u003c/li\u003e\n\u003cli\u003eLlovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, Lencioni R, Koike K, Zucman-Rossi J, Finn RS. Hepatocellular carcinoma. Nat Rev Dis Primers. 2021:6.\u003c/li\u003e\n\u003cli\u003eYang C, Zhang H, Zhang L, Zhu AX, Bernards R, Qin W, Wang C. Evolving therapeutic landscape of advanced hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2023:203-22.\u003c/li\u003e\n\u003cli\u003eSankar K, Gong J, Osipov A, Miles SA, Kosari K, Nissen NN, Hendifar AE, Koltsova EK, Yang JD. Recent advances in the management of hepatocellular carcinoma. 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Cell Rep. 2023:113006.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, Immunotherapy, S100 calcium-binding protein A9, Poly (ADP-ribose) polymerase 1, Programmed Death-Ligand 1","lastPublishedDoi":"10.21203/rs.3.rs-5797937/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5797937/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eImmune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1) immunotherapy, have emerged as promising treatments for advanced hepatocellular carcinoma (HCC), significantly improving clinical outcomes. However, resistance to ICIs remains a major challenge, and the underlying mechanisms of this resistance are not yet fully understood. This study aimed to investigate the role of S100 calcium-binding protein A9 (S100A9) in mediating resistance to anti-PD-1 therapy.\u003c/p\u003e\u003ch2\u003eApproach and Results:\u003c/h2\u003e \u003cp\u003eWe conducted RNA sequencing (RNA-seq) on tumor samples from anti-PD-1 responders and non-responders in HCC patients. Differential expression analysis identified S100A9 as a potential driver gene of resistance to anti-PD-1 therapy. Subcutaneous tumor models and an orthotopic HCC model established via hydrodynamic transfection were utilized to evaluate the impact of S100A9 on the efficacy of PD-1 therapy. Our findings revealed that S100A9 promotes resistance to anti-PD-1 therapy in HCC. Mechanistically, S100A9 directly interacted with PARP1 and induced its degradation via the ubiquitin-proteasome pathway. This process increased STAT3 phosphorylation at Tyr705, thereby enhancing PD-L1 transcription. Notably, treatment with the S100A9 inhibitor Tasquinimod significantly improved the efficacy of anti-PD-1 therapy in HCC.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study reveals that S100A9 facilitates immune evasion in HCC by enhancing PARP1 ubiquitination, STAT3 phosphorylation, and PD-L1 expression. Furthermore, combining S100A9 inhibitors with anti-PD-1 antibodies markedly enhances the therapeutic efficacy of ICIs in HCC. These findings highlight S100A9 as a potential therapeutic target for overcoming resistance to immunotherapy in HCC.\u003c/p\u003e","manuscriptTitle":"S100A9 Promotes Resistance to Anti-PD-1 Immunotherapy in Hepatocellular Carcinoma by Degrading PARP1 and Activating the STAT3/PD-L1 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-14 16:34:10","doi":"10.21203/rs.3.rs-5797937/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cdff8a89-de87-4471-8b19-6ff52b5fdcef","owner":[],"postedDate":"January 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T12:23:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-14 16:34:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5797937","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5797937","identity":"rs-5797937","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}