Low-dose SAHA enhances CD8+T cell-mediated antitumor immunity by upregulating MHC I expression in non-small cell lung cancers

doi:10.21203/rs.3.rs-3585533/v1

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Low-dose SAHA enhances CD8+T cell-mediated antitumor immunity by upregulating MHC I expression in non-small cell lung cancers

https://doi.org/10.21203/rs.3.rs-3585533/v1

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Non-small cell lung cancers (NSCLCs) is a highly aggressive lung cancer with poor responses to traditional therapies like surgery, radiotherapy, and chemotherapy. While immunotherapy has become an effective approach treating multiple types of cancer, solid tumors frequently exhibit immune escape through various mechanisms, including downregulation of MHC I expression. Whether upregulating MHC I expression can improve the immunotherapeutic effect for NSCLCs remains unexplored. Suberoylanilide hydroxamic acid (SAHA) is a potent histone deacetylase (HDAC) inhibitor, which has been applied clinically to treat lymphoma, but high-dose of SAHA kills tumor cells and normal cells without preference. Here, we show that low-dose SAHA enhances CD8 + T cell-mediated antitumor immunity via upregulating MHC I expression in NSCLC cells. We also provide evidence that high levels of MHC I induced by SAHA promoted the activation, proliferation, and cytotoxicity of specific CD8⁺T cells in mouse models. Mechanistically, low-dose SAHA increases the levels of H3K9ac and H3K27ac in the promoters of STAT1, Smad2 and Smad3 genes in NSCLC cells by inhibiting HDAC activity, resulting in elevated expression levels of STAT1, Smad2 and Smad3. The nuclear translocation of phosphorylated STAT1 and Smad2/3 upregulates the expression of MHC I in NSCLC cells remarkably. Thus, we uncovered a key mechanism of SAHA-mediated enhanced antitumor immunity, providing insights into a novel immunotherapy strategy for NSCLC.

NSCLC

SAHA

MHC I

STAT1

Smad2/3

antitumor immunity

Lung cancer continues to be a major health problem associated with high morbidity and mortality[1–3]. More than 85% of lung cancers are non-small cell lung cancers (NSCLCs) [4]. The traditional treatment for NSCLC includes surgery, radiotherapy, and chemotherapy which have limited curative effects and obvious side effects[5, 6]. While traditional lung cancer treatment has reached a bottleneck, immunotherapy has gained considerable attention[7–13]. Importantly, enhancing antitumor immunity by targeting the immune system is the key strategy of treatment[14–16]. However, several studies have demonstrated that tumor cells play an important role in regulating host immune responses and have various ways to escape immune surveillance and attacks[17–20]. During the tumor immune response, major histocompatibility complex class I (MHC I) combined with antigen peptides is indispensable for specific CD8 + T cell activation and proliferation[18, 21]. Nevertheless, one of the principal mechanisms of immune escape by solid tumors is the downregulation of MHC I, resulting in a weakened antitumor effect[22–25]. Thus, increasing the expression of MHC I on the surface of tumor cells is one of the most critical aspects for improving the immunotherapeutic effect for NSCLC.

Suberoylanilide hydroxamic acid (SAHA) is a classic chemotherapeutic drug, that is a potent histone deacetylase (HDAC) inhibitor. According to previous studies, HDAC inhibitors can enhance T cell-mediated antitumor immunity and exert multiple cytotoxic actions in cancer cells, including inhibiting tumor proliferation, invasion, metastasis, and differentiation[26–30]. To take advantage of these biological effects, SAHA has been reported to be clinically applied to treat lymphoma that continues to deteriorate or relapse. Despite the common use of SAHA to kill tumor cells, it has been demonstrated that SAHA at low doses participates in immune regulation, such as promoting MHC I expression. However, the immunotherapeutic effect of SAHA in NSCLC remains elusive.

STAT1 in the cytoplasm can be phosphorylated and forms heterodimers upon stimulation by extracellular signals, and translocation of p-STAT1 from the cytoplasm into the nucleus has been considered a key regulator of tumor immune surveillance[31–33]. In addition, STAT1, Smad2 and Smad3 can be phosphorylated and translocated into the nucleus upon stimulation to control the transcription of immune regulatory genes[34, 35]. However, the molecular mechanism by which low-dose SAHA exerts antitumor effects in NSCLC is poorly understood, and whether STAT1 and Smad2/3 are involved in SAHA-mediated immune regulation is unclear.

In the present study, we demonstrated that low-dose SAHA can upregulate the expression of MHC I on the surface of NSCLC cells, thereby enhancing specific CD8 + T cell activation and proliferation. Moreover, we provide evidence that SAHA can activate the STAT1 and Smad2/3 signaling pathways to achieve a strong immunotherapeutic outcome for NSCLC.

Chemicals and reagents

Suberoylanilide hydroxamic acid (SAHA) and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Sigma-Aldrich. Lipofectamine 2000 was purchased from Invitrogen. siRNAs targeting human STAT1, Smad2, and Smad3 were purchased from GenePharma (Shanghai, China), and mouse STAT1, Smad2, and Smad3 were purchased from Gene Biosystems(Anhui, China). For the antibodies used in western blot analysis, antibodies against human and mouse p-STAT1, p-Smad2/3, and STAT1 were purchased from Cell Signaling Technology, antibodies against human and mouse Smad2, Smad3, β-actin, GAPDH, and mouse MHC I were purchased from Proteintech (Wuhan, China), and antibodies against human MHC I were obtained from Huaan (Hangzhou, China). All antibodies used for flow cytometry were purchased from BioLegend. All antibodies used for chromatin immunoprecipitation were purchased from Cell Signaling Technology. For the antibodies used in immunofluorescence staining, anti-STAT1 and anti-Smad2 were purchased from Proteintech (Wuhan, China) and anti-Smad3 was purchased from ImmunoWay Biotechnology. The STAT1 and Smad2/3 inhibitors, nifuroxazide and ITD-1, were purchased from Selleck. IFN-γ and TNF-α ELISA kits were purchased from Dakewe (Beijing, China). The Apoptosis Detection Kit was purchased from BD Biosciences. The HDAC Activity Direct Assay Kit was purchased from Epigentek.

Animals and cell lines

Male C57BL/6 mice (6–8 weeks old) were purchased from the Shanghai SLAC Laboratory Animal Center (Shanghai, China) and male BALB/C-nude mice (6–8 weeks old) were purchased from Vital River. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of USTC. A549 and NCI-H520 human lung cancer cell lines were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The mouse Lewis lung cancer cell line was purchased from Fuheng Biology (Shanghai, China). A549 and Lewis cells were cultured in DMEM and NCI-H520 cells were cultured in RPMI1640 culture medium. All cell lines were cultured in an incubator at 37°C in a humidified atmosphere with 5% CO₂.

Western blot analysis

Proteins from tumor cells were extracted with RIPA buffer and the concentrations of the lysates were determined using the BCA protein assay kit (Thermo Scientific). Samples were loaded and separated on a 10% gel by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) and probed with specific primary antibodies at 4°C overnight. Then, the membranes were incubated with peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally, the protein bands were detected using an ECL western blot detection kit.

Quantitative real-time PCR (qPCR)

Total RNA of the cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. qPCR was performed using SYBR Master Mix (Takara Bio) on an ABI 7500 system. The mRNA levels were normalized to that of GAPDH. The primer sequences are listed in Table S1.

Chromatin immunoprecipitation-qPCR

Chromatin immunoprecipitation was conducted according to the manufacturer’s protocols (Cell Signaling Technology). Briefly, the tumor cells were crosslinked, and chromatin was sheared and extracted. Immunoprecipitation was performed using anti-p-Smad2, anti-H3K9ac and anti-H3K27ac antibodies. The sequences of primers used for ChIP‒qPCR are listed in Table S2.

Gene silencing experiments

siRNAs targeting human or mouse STAT1, Smad2, and Smad3 were transfected into tumor cells using Lipofectamine 2000 according to the manufacturer’s protocol. The expression of the indicated genes was verified by qPCR. The siRNA sequences are listed in Table S3.

Flow cytometric analysis

The surface staining antibodies used were as follows: anti-human HLA-A, -B, and -C (W6/32), anti-human CD80 (2D10), anti-human CD86 (IT2.2), anti-mouse H-2Kb/H-2Db (28-8-6), anti-mouse CD80 (16-10A1), anti-mouse CD86 (GL-1), and anti-mouse CD8a (53 − 6.7). For intracellular cytokine staining, tumor-infiltrating lymphocytes were isolated and cultured for 4 h in the presence of Cell Stimulation Cocktail with Brefeldin A (BioLegend). The lymphocytes were treated with anti-mouse IFN-γ (XMG1.2) following the cytoplasmic protein staining protocol (BioLegend), and flow cytometric analysis was performed by flow cytometer (BD Calibur).

Immunofluorescence staining

Tumor cells seeded in the confocal dish were fixed with 15% formaldehyde for 15 min at room temperature and then permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% BSA for 1 h, the cells were incubated with the primary antibodies overnight at 4°C, followed by the fluorescent secondary antibodies for 1 h at room temperature. Finally, the nuclei were stained with DAPI (1 µg/ml). The fluorescent cells were observed by confocal microscopy (ZEISS LSM880).

Immunization of mice

Lewis cells were subjected to three cycles of rapid freezing at -80°C and thawing at 37°C to prepare tumor cell lysates, followed by centrifugation at 2,000 × g for 10 min to remove cellular debris. Emulsions containing equal volumes of Lewis cell lysate and complete Freund’s adjuvant (CFA, Sigma‒Aldrich) or incomplete Freund’s adjuvant (IFA, Sigma‒Aldrich) were prepared by vortexing the suspensions for 30 min until a stable oil-in-water emulsion was achieved. C57BL/6 mice were immunized by subcutaneous injection of 100 µg of Lewis cell lysate in CFA on Day 0 and boosted with 100 µg of Lewis cell lysate in IFA on Days 7 and 14. Splenic CD8⁺T cell were isolated by magnetic sorting (Miltenyi Biotec) and cocultured with SAHA-pretreated Lewis cells. The proliferation of CFSE-labeled T cells and apoptosis of Lewis cells were detected.

Animal experiments in vivo

In the Lewis tumor model, C57BL/6 mice or nude mice were challenged subcutaneously with 5×10⁵ Lewis cells. Five days after tumor cell inoculation, mice were treated intratumorally with SAHA or intraperitoneally with nifuroxazide and ITD-1 every other day. Palpable tumors were measured by calipers, and the tumor volume was calculated according to the following formula: volume = (length×width²)/2, where length and width are measured in millimeters.

Isolation of tumor-infiltrating lymphocytes (TILs)

Lewis cell-derived tumor tissues were removed and digested with collagenase Ⅳ (10 mg/mL), hyaluronidase (100 µg/mL), and DNA enzyme (20 µg/mL) for 2 h at 37°C, and homogenized with frosted slides. After lysis of red blood cells, lymphocytes were separated using the mouse lymphocyte separation solution (Dakewe, China).

Database search

The CISTROME DB database (http://cistrome.org/db) was used to extract the binding abundance of H3K9ac and H3K27ac modifications in the promoter regions of STAT1, Smad2, and Smad3 and the data was visualized in the UCSC database (http://genome.ucsc.edu). We used the JASPAR database to predict the binding sites of Smad2 to the promoter regions of HLA-A, HLA-B, and HLA-C genes.

Statistical analysis

Two-tailed unpaired t-test was performed to evaluate the differences between any two groups. One-way ANOVA was performed to assess the difference in mean between groups. The data are expressed as the mean ± SD of three independent experiments. GraphPad Prism software 9.3 and SPSS 19.0 were used to analyze the data. Differences were considered as statistically significant when p < 0.05.

Low-dose SAHA upregulates the expression of MHC I in NSCLC cell lines

HDACIs have been reported to be associated with the repression of MHC I in various tumors[36, 37]. Therefore, we postulated that SAHA, a classic HDAC inhibitor, might upregulate the expression of MHC I in NSCLC. First, a CCK8 assay was employed to determine the cytotoxicity of SAHA in A549 and NCI-H520 (hereafter, H520) cells, which are two typical human NSCLC cell lines. We found that cell viability was inhibited in a dose-dependent manner, especially SAHA at higher concentrations, which significantly decreased the survival rate of A549 and H520 cells (Fig. S1A). To identify the immune regulatory effect of SAHA in NSCLC cells, when the cell viability was approximately 85%, 3 µM of SAHA was added to A549 and H520 cells (Fig. S1A). Surprisingly, qPCR results showed that the expression levels of HLA-A, HLA-B, and HLA-C were clearly elevated after 24 h (Fig. 1A, B). In parallel, the protein levels of MHC I in SAHA-treated A549 and H520 cells were increased as well, as analyzed by western blotting and flow cytometry at 24 h and 48 h (Fig. 1C-F). The same results were also observed in Lewis cells, a typical mouse NSCLC cell line (Fig. 1G, H). At the same time, 3 µM of SAHA inhibited the activity of total HDAC (Fig. S1B). Based on these results, SAHA was used at a concentration of 3 µM in the subsequent experiments. In addition to HLA-A, HLA-B, and HLA-C, the gene encoding MHC I molecules (Β2m) was upregulated in SAHA-treated cells, as were genes directing peptide transporters (Tap1 and Tap2) (Fig. 1I-K). Moreover, the expression of costimulatory molecules such as CD80 and CD86 was upregulated dramatically with SAHA treatment (Fig. S1C-E). Taken together, the above results suggest that low-dose SAHA upregulates the expression of both MHC I and related molecules, such as peptide transporters and costimulatory molecules, in NSCLC cell lines without affecting cell viability, thereby indicating the potential of SAHA in immune regulation.

SAHA promotes STAT1 and Smad2/3 phosphorylation and nuclear translocation to increase MHC I expression in NSCLC cell lines

Next, we investigated the molecular mechanism by which SAHA upregulated MHC I expression. According to previous literature, STAT1 is a key regulator of MHC I and MHC II expression[36–40], and HDACI treatment can activate Smad2/3 to enter the nucleus and regulate the expression of downstream genes[41]. Here, we treated both human and mouse NSCLC cells with SAHA for 24 h and 48 h. As expected, the total protein levels of STAT1, Smad2 and Smad3 were increased significantly (Figs. 2A, B and S2A). Since the activation of STAT1 and Smad2/3 is characterized by phosphorylation, H520, A549 and Lewis cells were treated with SAHA at different time points. As shown in Figs. 2C, D and S2B, SAHA could rapidly phosphorylate STAT1 and Smad2/3 at 10 min in NCI-H520 and A549 cells and 60 min in Lewis cells. To further study whether SAHA promoted the translocation of cytoplasmic p-STAT1 and p-Smad2/3 to the nucleus, cell fractionation experiments and western blotting were performed to analyze SAHA-treated NSCLC cells. The addition of SAHA led to an increase in the expression of p-STAT1 and p-Smad2/3 in the nuclei of H520, A549 and Lewis cells (Figs. 3A, B and S2C). Simultaneously, the immunofluorescent analysis further confirmed the nuclear accumulation of STAT1, Smad2 and Smad3 with SAHA treatment for 24 h and 48 h (Fig. 3C-F). These findings imply that SAHA activates NSCLC cells by STAT1 and Smad2/3 phosphorylation and their nuclear translocation.

Then, we wondered whether phosphorylated STAT1 and Smad2/3 could influence MHC I expression after nuclear translocation. Previous studies have demonstrated that STAT1 can act as a transcription factor targeting the promoter regions of MHC I genes to regulate their expression[38]. In the case of Smad2/3, the JASPAR website showed that there were multiple binding sites of Smad2 at the promoter regions of HLA-A, B, and C genes. Therefore, chromatin immunoprecipitation (ChIP)-qPCR was conducted and further confirmed that p-Smad2 indeed bound to the promoters of HLA-A, HLA-B, and HLA-C, and the binding of p-Smad2 could be promoted significantly by SAHA (Fig. 3G, H). Smad2 and Smad3 translocate into the nucleus together once activated [39], and our above data demonstrate that p-Smad2 is the binding site of activated Smad2/3. Taken together, our results indicate that the activation of STAT1 and Smad2/3 by SAHA can increase MHC I expression by binding to the promoters of the HLA-A, B, and C genes.

SAHA-induced MHC I upregulation is blocked by STAT1 and Smad2/3 knockdown

To confirm the role of STAT1 and Smad2/3 in SAHA-induced MHC I upregulation, the expression of STAT1, Smad2 and Smad3 in NCI-H520 and A549 cells was silenced by siRNA. The protein levels of MHC I were obviously decreased upon STAT1, Smad2 and Smad3 knockdown, even though the cells were treated with SAHA for 48 h (Figs. 4A-C and S3A-C). Consistent with these results, silencing of STAT1, Smad2 and Smad3 in Lewis cells was executed (Fig. S3D-F) and markedly reduced SAHA-mediated MHC I upregulation, as shown by both western blotting and flow cytometry (Fig. 4D-I). In summary, the above data reveal that SAHA upregulates the expression of MHC I in NSCLC cells by activating the STAT1 and Smad2/3 pathways.

SAHA drastically boosts acetylation of the promoter regions of STAT1, Smad2 and Smad3

The above data showed that the expression of STAT1, Smad2 and Smad3 in NSCLC cells was increased after SAHA treatment, which made us proceed to investigate how SAHA influenced these signaling molecules. It was reported that HDACIs could enhance the expression of targeted molecules by promoting the acetylation of genes [40]. Interestingly, we discovered H3K9ac and H3K27ac modifications in the promoter regions of STAT1, Smad2 and Smad3 in A549 via analysis of the CISTROME DB and UCSC databases (Fig. 5A). To verify this finding, ChIP‒qPCR was carried out to test the histone acetylation. As expected, H3K9 and H3K27 were acetylated in the promoter regions of STAT1, Smad2 and Smad3 in H520 and Lewis cells, which were enhanced dramatically with SAHA treatment (Fig. 5B-G). Collectively, these observations indicate that SAHA triggers upregulation of STAT1, Smad2 and Smad3 by boosting acetylation of the promoter regions of the corresponding genes.

SAHA remarkably enhances antitumor immunity in a mouse model

Next, we sought to validate the therapeutic effect of SAHA in tumor-bearing mice. To this end, C57BL/6 mice were inoculated subcutaneously with Lewis cells, followed by intratumoral SAHA injection every other day. As shown in Fig. 6A, compared to the control group, SAHA significantly inhibited tumor growth, indicating that SAHA could generate an antitumor effect. In addition, the same experiment was conducted in T cell-deficient nude mice, and we found that SAHA failed to exert an antitumor effect (Fig. 6B), suggesting that SAHA may elicit potent antitumor T cell immunity. To further clarify the immunomodulatory effect of SAHA on T cells, tumor-infiltrating lymphocytes (TILs) were isolated from Lewis cell-derived tumors after intratumoral SAHA injection for five consecutive days. Flow cytometric analysis showed that the percentage of CD8 + T cells in TILs was increased significantly (Fig. 6C). More importantly, SAHA induced a high frequency of IFN-γ + CD8 + T cells in TILs (Fig. 6D). In addition, IFN-γ levels were strikingly upregulated, while TNF-α levels were moderately upregulated in TILs, as demonstrated by ELISA (Fig. S4A, B). All these results reveal the activation and proliferation of tumor-infiltrating CD8 + T cells in the presence of SAHA in vivo.

To further evaluate the influence of SAHA on Lewis-specific CD8 + T cells in vitro, splenic CD8 + T cells purified from mice, immunized with Lewis cell lysates emulsified in Freund’s adjuvant, were cocultured with Lewis cells pretreated with SAHA for 48 h. As a result, SAHA-treated Lewis cells had a greater ability to stimulate T cell proliferation (Fig. 6E) and a higher rate of early and late apoptosis (Fig. 6F). Together, the above data indicate that SAHA can remarkably enhance CD8 + T cell-mediated antitumor immunity by upregulating MHC I expression in Lewis cells.

Inhibition of STAT1 and Smad2/3 represses SAHA-mediated antitumor immunity

We then investigated the potential significance of STAT1 and Smad2/3 blockade in SAHA-mediated antitumor immunity. Here, STAT1 and Smad2/3 inhibitors (nifuroxazide and ITD- 1) were used to block the signaling pathway in SAHA-treated mice. As shown in Fig. 7A, SAHA failed to generate a therapeutic effect against Lewis cell-derived tumors when STAT1 and Smad2/3 inhibitors were injected intraperitoneally. Notably, the increase in the percentage of CD8 + T cells and IFN-γ-secreting CD8 + T cells in Lewis cell-derived tumor-infiltrating lymphocytes was also blocked by the inhibitors (Fig. 7B, C), revealing that STAT1 and Smad2/3 are involved in the immunomodulatory effect of SAHA. Moreover, the ability of SAHA-treated Lewis cells to stimulate T cell proliferation was strikingly repressed by silencing STAT1, Smad2, and Smad3 (Fig. 7D). Consistently, when STAT1, Smad2, and Smad3 were knocked down, coculture of specific CD8 + T cells and SAHA-treated Lewis cells did not lead to an increase in the rate of apoptosis (Fig. S4C). Altogether, these results suggest that SAHA-mediated antitumor immunity is regulated through the STAT1 and Smad2/3 pathways.

In recent years, HDAC inhibitors have been increasingly applied as therapeutic agents in the treatment of a variety of cancers and other diseases [41–43]. SAHA as a potent HDAC inhibitor has shown remarkable performance in the treatment of cutaneous T-cell lymphomas since its introduction to the market [44], but its performance in NSCLC is not clear. In the current study, SAHA was applied at low doses to treat NSCLC cells without affecting cell viability. We discovered that SAHA increased the levels of H3K9ac and H3K27ac in the promoters of the STAT1, Smad2 and Smad3 genes in NSCLC cells by inhibiting HDAC activity, thereby driving an increase in their expression levels. Subsequently, STAT1 and Smad2/3 were phosphorylated and translocated from the cytosol into the nucleus upon SAHA stimulation, resulting in a remarkable increase in the expression of MHC I on the surface of both human and mouse NSCLC cells. More importantly, we provided evidence that high levels of MHC I induced by SAHA promoted the activation, proliferation, and cytotoxicity of specific CD8 + T cells, thereby enhancing the antitumor immunotherapeutic effect of low-dose SAHA (Fig. 8). To our knowledge, this is the first report to reveal a novel immunotherapeutic property of SAHA for NSCLC.

Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality and therefore is a great threat to the health and life of the population[45–47]. Approximately 85% of lung cancers are NSCLC, most of which are already in advanced stages at the time of diagnosis[2, 48, 49]. Surgery, radiotherapy, and chemotherapy constitute the traditional clinical treatment for NSCLC, which has great limitations and impacts the quality of life of patients. In addition, due to immune deficiency or damage caused by radiotherapy and chemotherapy, residual tumor cells develop further which is characterized by proliferation, invasion, and metastasis. Thus, it is necessary to explore effective therapeutic approaches with fewer side effects for NSCLC to meet critical medical needs. In recent years, tumor immune escape has attracted a great deal of attention and, treatment targeting the immune system is taking the stage.

Tumor immune escape is an important strategy for tumor survival and harbors a variety of mechanisms. Nevertheless, one of the principal mechanisms is the downregulation of MHC I, which has been observed in approximately 40%-90% of human tumors and is usually associated with poor prognosis[17, 25, 50]. More importantly, the MHC I-antigen peptide complex plays a pivotal role in stimulating CD8 + T cell immunity[51], while costimulatory molecules such as CD80 and CD86 provide the second signal for T cell activation. In general, the downregulation of MHC I in tumor cells is often accompanied by low levels of CD80 and CD86, so tumor-specific CD8 + T cells cannot be activated completely[52]. Therefore, increasing the expression of MHC I and costimulatory molecules in NSCLC cells will improve the antitumor immune response to some extent.

The significant finding of this study is the identification that low-dose SAHA can kill NSCLC cells through immune regulation rather than direct induction of tumor cell death. Previous studies have confirmed that activated STAT1 enters the nucleus to regulate the expression of human MHC genes[53], and translocation of phosphorylated Smad2/3 into the nucleus is associated with the process of immune regulation[54]. On this basis, we found that in NSCLC cells, MHC I upregulation and the proliferation and killing ability of specific CD8 + T cells could be repressed by STAT1 and Smad2/3 blockade, implying the synergistic effects of STAT1 and Smad2/3 in SAHA-induced immune regulation.

In summary, the data presented in this study clearly show that MHC I expression in NSCLC cells can be upregulated by low-dose SAHA, uncovering a key mechanism of SAHA-mediated enhanced antitumor immunity, thus opening the door to a novel immunotherapeutic strategy for NSCLC with potential applications.

DATA AVAILABILITY

All source data are available from the corresponding author upon reasonable request.

ACKNOWLEDGEMENTS

This research study was supported by the National Natural Science Foundation of China (81972006) to BW. This work was also supported by the National Natural Science Foundation of China (82103307), Anhui Provincial Natural Science Foundation (2108085QH350), Scientific Research Project of Anhui Provincial Higher Education Institutions (2022AH051254) and the Fundamental Research Funds for the Central Universities (WK9110000074) to WD.

AUTHOR CONTRIBUTIONS

BW: conception and design. WD: conception and design, animal experiments, data analysis and interpretation, and manuscript writing. HW: conception and design, data analysis and interpretation. BH, YC, RY, YH, YK, DL, PL: performed the experiments. SZ, CW, PW, XZ: analyzed and interpreted the data. JX, SY, DC: completed critical revisions and proofread the manuscript. All authors reviewed the manuscript and approved the final manuscript.

COMPETING INTERESTS

The authors declare no competing interests.

Oudkerk M, Liu S, Heuvelmans MA, Walter JE, Field JK. Lung cancer LDCT screening and mortality reduction - evidence, pitfalls and future perspectives. Nat Rev Clin Oncol. 2020; 18(3): 135-151.
Arbour K, Riely G. Systemic Therapy for Locally Advanced and Metastatic Non-Small Cell Lung Cancer: A Review. JAMA. 2019;322:764-774.
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA-A CANCER JOURNAL FOR CLINICIANS. 2021;71:209-249.
Herbst RS, Morgensztern D, Boshoff C. The biology and management of non-small cell lung cancer. Nature. 2018;553:446-454.
Di Maio M, Gridelli C, Gallo C, Shepherd F, Piantedosi F, Cigolari S, et al. Chemotherapy-induced neutropenia and treatment efficacy in advanced non-small-cell lung cancer: a pooled analysis of three randomised trials. The Lancet Oncology. 2005;6:669-677.
De Ruysscher D, Faivre-Finn C, Nackaerts K, Jordan K, Arends J, Douillard J, et al. Recommendation for supportive care in patients receiving concurrent chemotherapy and radiotherapy for lung cancer. Annals of oncology : official journal of the European Society for Medical Oncology. 2020;31:41-49.
Billan S, Kaidar-Person O, Gil Z. Treatment after progression in the era of immunotherapy. The Lancet Oncology. 2020;21:e463-e476.
Camidge D, Doebele R, Kerr K. Comparing and contrasting predictive biomarkers for immunotherapy and targeted therapy of NSCLC. Nature reviews Clinical oncology. 2019;16:341-355.
Dammeijer F, Lievense L, Veerman G, Hoogsteden H, Hegmans J, Arends L, et al. Efficacy of Tumor Vaccines and Cellular Immunotherapies in Non-Small-Cell Lung Cancer: A Systematic Review and Meta-Analysis. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2016;34:3204-3212.
Tartour E, Zitvogel L. Lung cancer: potential targets for immunotherapy. The Lancet Respiratory medicine. 2013;1:551-563.
Osmani L, Askin F, Gabrielson E, Li Q. Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): Moving from targeted therapy to immunotherapy. Seminars in Cancer Biology. 2018;52:103-109.
Carlisle J, Steuer C, Owonikoko T, Saba N. An update on the immune landscape in lung and head and neck cancers. CA: a cancer journal for clinicians. 2020;70:505-517.
Massafra M, Passalacqua MI, Gebbia V, Macri P, Lazzari C, Gregorc V, et al. Immunotherapeutic Advances for NSCLC. Biologics-Targets & Therapy. 2021;15:399-417.
Cannone G, Comacchio GM, Pasello G, Faccioli E, Schiavon M, Dell'Amore A, et al. Precision Surgery in NSCLC. Cancers. 2023; 15(5): 1571.
French J, Bible K, Spitzweg C, Haugen B, Ryder M. Leveraging the immune system to treat advanced thyroid cancers. The lancet Diabetes & endocrinology. 2017;5:469-481.
Tubin S, Khan MK, Gupta S, Jeremic B. Biology of NSCLC: Interplay between Cancer Cells, Radiation and Tumor Immune Microenvironment. Cancers. 2021; 13(4): 775.
Burr M, Sparbier C, Chan K, Chan Y, Kersbergen A, Lam E, et al. An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer. Cancer cell. 2019;36:385-401.e388.
Chew G, Campbell A, De Neef E, Sutliff N, Shadle S, Tapscott S, et al. DUX4 Suppresses MHC Class I to Promote Cancer Immune Evasion and Resistance to Checkpoint Blockade. Developmental cell. 2019;50:658-671.e657.
McGranahan N, Rosenthal R, Hiley C, Rowan A, Watkins T, Wilson G, et al. Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution. Cell. 2017;171:1259-1271.e1211.
Schölzel K, Schildberg F, Welz M, Börner C, Geiger S, Kurts C, et al. Transfer of MHC-class-I molecules among liver sinusoidal cells facilitates hepatic immune surveillance. Journal of hepatology. 2014;61:600-608.
Dersh D, Hollý J, Yewdell J. A few good peptides: MHC class I-based cancer immunosurveillance and immunoevasion. Nature reviews Immunology. 2020; 21(2): 116-128.
Sari G, Rock K. Tumor immune evasion through loss of MHC class-I antigen presentation. Current Opinion in Immunology. 2023; 83: 102329.
Jongsma MLM, Neefjes J, Spaapen RM. Playing hide and seek: Tumor cells in control of MHC class I antigen presentation. Molecular Immunology. 2021;136:36-44.
Shklovskaya E, Rizos H. MHC Class I Deficiency in Solid Tumors and Therapeutic Strategies to Overcome It. International Journal of Molecular Sciences. 2021; 22(13): 6741.
Dersh D, Yewdell J. Immune MAL2-practice: breast cancer immunoevasion via MHC class I degradation. The Journal of clinical investigation. 2021; 131(1).
Qu K, Zaba L, Satpathy A, Giresi P, Li R, Jin Y, et al. Chromatin Accessibility Landscape of Cutaneous T Cell Lymphoma and Dynamic Response to HDAC Inhibitors. Cancer cell. 2017;32:27-41.e24.
Faião-Flores F, Emmons M, Durante M, Kinose F, Saha B, Fang B, et al. In VivoHDAC Inhibition Enhances the Efficacy of MEK Inhibitor Therapy in Uveal Melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2019;25:5686-5701.
Bose P, Dai Y, Grant S. Histone deacetylase inhibitor (HDACI) mechanisms of action: emerging insights. Pharmacology & therapeutics. 2014;143:323-336.
Wang J, Hasan F, Frey A, Li H, Park J, Pan K, et al. Histone Deacetylase Inhibitors and IL21 Cooperate to Reprogram Human Effector CD8 T Cells to Memory T Cells. Cancer immunology research. 2020;8:794-805.
Akimova T, Beier U, Liu Y, Wang L, Hancock W. Histone/protein deacetylases and T-cell immune responses. Blood. 2012;119:2443-2451.
Meissl K, Amenitsch L, Witalisz-Siepracka A, Klein K, Lassnig C, Puga A, et al. STAT1 Isoforms Differentially Regulate NK Cell Maturation and Anti-tumor Activity. Frontiers in Immunology. 2020; 11: 2189.
Putz E, Gotthardt D, Hoermann G, Csiszar A, Wirth S, Berger A, et al. CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance. Cell reports. 2013;4:437-444.
Li X, Pan KF, Vieth M, Gerhard M, Li WQ, Mejias-Luque R. JAK-STAT1 Signaling Pathway Is an Early Response to Helicobacter pylori Infection and Contributes to Immune Escape and Gastric Carcinogenesis. International Journal of Molecular Sciences. 2022; 23(8): 4147.
Park B, Freeman Z, Ghasemzadeh A, Chattergoon M, Rutebemberwa A, Steigner J, et al. TGFβ1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer. Cancer discovery. 2016;6:1366-1381.
Kashiwagi I, Morita R, Schichita T, Komai K, Saeki K, Matsumoto M, et al. Smad2 and Smad3 Inversely Regulate TGF-β Autoinduction in Clostridium butyricum-Activated Dendritic Cells. Immunity. 2015;43:65-79.
Cornel AM, Dunnebach E, Hofman DA, Das S, Sengupta S, van den Ham F, et al. Epigenetic modulation of neuroblastoma enhances T cell and NK cell immunogenicity by inducing a tumor-cell lineage switch. Journal for Immunotherapy of Cancer. 2022; 10(12).
Holay N, Giri U, Miljanic M, Soleimani M, Shaikh O, Capasso A, et al. Unleashing the potential of HDACi to augment immunotherapy for the treatment of colorectal cancer. Cancer Research. 2019; 79(13_Supplement): 1088-1088.
Darnell JE, Jr., Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415-1421.
Ervin EH, French R, Chang CH, Pauklin S. Inside the stemness engine: Mechanistic links between deregulated transcription factors and stemness in cancer. Seminars in cancer biology. 2022;87:48-83.
Liang T, Wang FL, Elhassan RM, Cheng YM, Tang XL, Chen WA, et al. Targeting histone deacetylases for cancer therapy: Trends and challenges. Acta Pharmaceutica Sinica B. 2023;13:2425-2463.
Schelker C, Nowak-Sliwinska P, Borchard G. HDACIs and TKIs combinations and their liposomal delivery for cancer treatment. Journal of Controlled Release. 2023;358:59-77.
Xiang XS, Li PC, Wang WQ, Liu L. Histone deacetylases: A novel class of therapeutic targets for pancreatic cancer. Biochimica Et Biophysica Acta-Reviews on Cancer. 2022; 1877(1): 188676.
Ferreira JP, Pitt B, Zannad F. Histone deacetylase inhibitors for cardiovascular conditions and healthy longevity. Lancet Healthy Longevity. 2021;2:E371-E379.
Marks PA, Breslow R. Dimethyl sulfoxide to vorinostat: Development of this histone deacetylase inhibitor as an anticancer drug. Nature Biotechnology. 2007;25:84-90.
Bray F, Ferlay J, Soerjomataram I, Siegel R, Torre L, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2018;68:394-424.
Gridelli C, Rossi A, Carbone DP, Guarize J, Karachaliou N, Mok T, et al. Non-small-cell lung cancer. Nat Rev Dis Primers. 2015;1:15009.
Siegel R, Miller K, Jemal A. Cancer statistics, 2020. CA: a cancer journal for clinicians. 2020;70:7-30.
Socinski M, Obasaju C, Gandara D, Hirsch F, Bonomi P, Bunn P, et al. Clinicopathologic Features of Advanced Squamous NSCLC. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2016;11:1411-1422.
Ettinger D, Wood D, Akerley W, Bazhenova L, Borghaei H, Camidge D, et al. Non-Small Cell Lung Cancer, Version 6.2015. Journal of the National Comprehensive Cancer Network : JNCCN. 2015;13:515-524.
Gettinger S, Choi J, Hastings K, Truini A, Datar I, Sowell R, et al. Impaired HLA Class I Antigen Processing and Presentation as a Mechanism of Acquired Resistance to Immune Checkpoint Inhibitors in Lung Cancer. Cancer discovery. 2017;7:1420-1435.
Leone P, Shin E, Perosa F, Vacca A, Dammacco F, Racanelli V. MHC class I antigen processing and presenting machinery: organization, function, and defects in tumor cells. Journal of the National Cancer Institute. 2013;105:1172-1187.
Majzner R, Mackall C. Tumor Antigen Escape from CAR T-cell Therapy. Cancer discovery. 2018;8:1219-1226.
Simpson J, Al-Attar A, Watson N, Scholefield J, Ilyas M, Durrant L. Intratumoral T cell infiltration, MHC class I and STAT1 as biomarkers of good prognosis in colorectal cancer. Gut. 2010;59:926-933.
Xu W, Liu H, Liu Z, Wang H, Zhang F, Wang H, et al. Histone deacetylase inhibitors upregulate Snail via Smad2/3 phosphorylation and stabilization of Snail to promote metastasis of hepatoma cells. Cancer letters. 2018;420:1-13.

There is NO conflict of interest to disclose.

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Low-dose SAHA enhances CD8+T cell-mediated antitumor immunity by upregulating MHC I expression in non-small cell lung cancers

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Abstract

Figures

INTRODUCTION

Materials and methods

Chemicals and reagents

Animals and cell lines

Western blot analysis

Quantitative real-time PCR (qPCR)

Chromatin immunoprecipitation-qPCR

Gene silencing experiments

Flow cytometric analysis

Immunofluorescence staining

Immunization of mice

Isolation of tumor-infiltrating lymphocytes (TILs)

Database search

Statistical analysis

RESULTS

Low-dose SAHA upregulates the expression of MHC I in NSCLC cell lines

SAHA-induced MHC I upregulation is blocked by STAT1 and Smad2/3 knockdown

SAHA drastically boosts acetylation of the promoter regions of STAT1, Smad2 and Smad3

SAHA remarkably enhances antitumor immunity in a mouse model

Inhibition of STAT1 and Smad2/3 represses SAHA-mediated antitumor immunity

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

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