HDAC Inhibitor Regulates the Tumor Immune Microenvironment via Pyroptosis in Triple Negative Breast Cancer

doi:10.21203/rs.3.rs-3815673/v1

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Research Article

HDAC Inhibitor Regulates the Tumor Immune Microenvironment via Pyroptosis in Triple Negative Breast Cancer

https://doi.org/10.21203/rs.3.rs-3815673/v1

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published 11 Jun, 2024

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Objective

Pyroptosis, an inflammatory form of cell death, promotes the release of immunogenic substances and stimulates immune cell recruitment, a process which could turn cold tumours into hot ones. Thus, instigating pyroptosis in triple-negative breast cancer (TNBC) serves as a viable method for restoring anti-tumor immunity.

Methods

We analyzed the effects of Histone Deacetylase Inhibitors (HDACi) on TNBC cells using the CCK8 and colony formation assay. Apoptosis and lactate dehydrogenase (LDH) release assays were utilized to determine the form of cell death. The pyroptotic executor was validated by qRT-PCR and Western Blot. Transcriptome was analyzed to investigate pyroptosis-inducing mechanisms. A subcutaneously transplanted tumor model was generated in BALB/c mice to evaluate infiltration of immune cells.

Results

HDACi significantly diminished cell proliferation, and pyroptotic “balloon”-like cells became apparent. HDACi led to an intra and extracellular material exchange, signified by the release of LDH and the uptake of propidium iodide. Amongst the gasdermin family, TNBC cells expressed maximum quantities of GSDME, and expression of GSDMA, GSDMB and GSDME were augmented post HDACi treatment. Pyroptosis was instigated via the activation of the caspase 3-GSDME pathway with the potential mechanisms being cell cycle arrest and altered intracellular REDOX balance due to aberrant glutathione metabolism. In vivo experiments demonstrated that HDACi can activate pyroptosis, limit tumor growth, and escalate CD8 + lymphocyte and CD11b + cell infiltration along with an increased presence of granzyme B in tumors.

Conclusion

HDACi can instigate pyroptosis in TNBC, promoting infiltration of immune cells and consequently intensifying the efficacy of anticancer immunity.

TNBC

pyroptosis

HDACi

anti-tumor immunity

Pyroptosis, a highly inflammatory mode of necrotic cell death initially identified in immune cells, is triggered when gasdermin D (GSDMD) protein is cleaved at its N-terminal and C-terminal domains upon the activation of caspase 1 (Casp1)[1–3]. This cleavage deactivates GSDMD-C's inhibitory effect on the GSDMD-N domain. Consequently, GSDMD-N accumulates in cellular membranes, building pores that allow intracellular and extracellular material exchange. This induces cellular swelling, giving the cells a balloon-like appearance[4–6]. Concurrently, the cells release their contents, including cytokines, recruiting more immune cells to amplify the immune response against the invading pathogens[7–9]. Unlike apoptosis, pyroptosis sparks a potent immune reaction to these pathogens. The gasdermin (GSDM) family of proteins is highly conserved, and human gasdermins can be categorized into 6 distinct groups: GSDMA, GSDMB, GSDMC, GSDMD, GSDME/DFNA5, and DFNB59/Pejvakin. Mice harbour ten types of gasdermins, including three GSDMA homologs (GSDMA1-3), four GSDMC homologs (GSDMC1-4), as well as GSDMD, GSDME and DFNB59[4, 10]. Subsequent research revealed that the majority of GSDM members can trigger pyroptosis under specific conditions[11–15]. During tumor cell pyroptosis, the release of the cell components, especially the immunogenic substances, recruits more immune cells to infiltrate the tumor tissue, converting the 'cold tumor' into a 'hot tumor', thereby enhancing the immune response within that tumor and facilitating tumor suppression.

In immune cells, pyroptosis necessitates inflammasome assembly to activate caspase 1-gasdermin D (Casp1-GSDMD), initiating the process[3]. However, pyroptosis in tumor cells does not rely on inflammasome. By directly overexpressing the gasdermin A N-terminal (GSDMA-N) structural element in tumor cells, pyroptosis can be successfully initiated. Remarkably, pyroptosis in just 10% of tumor cells can lead to total eradication of breast graft tumors[16]. Gasdermin B (GSDMB) presents in high levels in digestive tract epithelial tumor cells. The GSDMB can be cleaved by granzyme A, secreted by tumor-infiltrating natural killer (NK) cells and cytotoxic T lymphocytes (CTL). This pore-forming functionality of GSDMB N-terminal aids tumor clearance in mice[11]. Tumor cell pyroptosis can also be induced through the activation of caspase 8-gasdermin C (Casp8-GSDMC) by the metabolite alpha-ketoglutaric acid and the TNF-α signal[12, 13]. Regarding GSDME-dependent pyroptosis, evident in breast cancer[17], oral cancer[18], lung cancer[19], melanoma[20], and gastrointestinal tumors[21], several drugs can activate caspase 3 (Casp3), cleave GSDME, and release the GSDME N-terminal to oligomerize into the cell membrane. Similarly, GSDME can be cleaved by granzyme B (GzmB) released by NK cells and T lymphocytes at the same action site as caspase 3. This also induces pyroptosis, playing an anticancer role that can either suppress cancers or trigger cytokine release syndrome[14, 15]. GSDME-dependent pyroptosis is commonplace in tumor cells. Still, GSDME expression is inhibited by DNA methylation, but deleting this methylation can enhance its expression and boost pyroptosis[22].

Breast cancer is the leading cause of cancer death among women globally. Classifications based on estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) overexpression statuses yield Luminal A, Luminal B, HER2 overexpressing, and triple-negative/basal-like breast cancers (TNBC)[23]. TNBC is hard to treat with hormone or targeted drugs due to absent ER, PR, and HER2 expression. For these cases, chemotherapy remains the primary treatment, despite its high hematologic toxicity[24]. The adoption of immunotherapy for TNBC is underway, but monotherapies underwhelm[25]. TNBC's immunosuppressive environment limits the success of standalone chemotherapy or tumor immunotherapy[26, 27]. Given this, finding novel therapeutic targets for TNBC and reconfiguring the tumor immune microenvironment is of high significance. Inducing pyroptotic death in cancer cells to trigger immunogenic substance release redefines 'cold tumors' as 'hot', augmenting immune efficacy as a promising anti-cancer strategy. This principle of inducing pyroptotic death in breast cancer cells suggests a strategy for restoring anti-tumor immunity.

In our prior research[28], we observed that the use of histone deacetylase inhibitors (HDACi) induces pyroptosis in breast cancer cells. This finding hints at the possibility that epigenetic modifications can influence the pyroptotic process. The current study corroborates this observation, specifically demonstrating that HDACi regulates the expression of gasdermins, and results in pyroptotic cell death. When HDACi was applied to TNBC cells, noticeable morphological changes were observed, including 'balloon'-like cell swelling. This alteration was accompanied by an escalation in cell death and concurrent lactate dehydrogenase (LDH) release, a well-known marker of pyroptosis. Moreover, following HDACi treatment in mice, growth inhibition of grafted tumors was evident, simultaneously enhancing the infiltration of immune cells in these tumors. These observations signify an improved tumor immune microenvironment amongst the treated group. Importantly, our research indicates that high expression of gasdermins is insufficient to induce pyroptosis. GSDMs require activation under specific conditions, to release functional structure domains with pore-forming capabilities, thus initiating the process of pyroptosis.

Cell Culture and Drug Treatment

Triple-negative breast cancer cell lines MDA-MB-231, BT-549 (human) and 4T1 (mouse) were procured from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The mouse TNBC cell line EO771 was a kind gift from Professor Lei Ping, Tongji Medical College, Huazhong University of Science and Technology. The human renal epithelial cell line HEK293T was obtained from the Shanghai Institute. MDA-MB-231, EO771, and HEK293T cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum while BT-549 and 4T1 were maintained in RPMI-1640 medium fortified with 10% fetal bovine serum. HDACIs, namely Entinostat (MS275, Cat# HY-12163) and Vorinostat (SAHA, Cat# HY-10221), were procured from MedChemExpress (MCE, Shanghai, China). The drug concentration for TNBC cell treatment was based on the detected half-maximal inhibitory concentration (IC50 value). Cells were harvested for subsequent experimental procedures after 48 hours of HDACi treatment.

Cell Proliferation-Toxicity Assay and Colony Formation Assay

Cell proliferation-toxicity was assessed using the Cell Counting Kit-8 (CCK-8, Cat# HY-K0301, MCE, Shanghai, China). TNBC cells were cultured in 96-well plates at a density of 2000 cells/100µL per well. Cells were treated with HDACi in concentrations ranging from 0µM to 128µM for 24 hours, 48 hours, and 72 hours. After the treatment period, 10µL of CCK-8 solution was added to each well, plates incubated for an hour, and the absorbance at 450nm was subsequently measured. Cell viability curves and IC50 values were plotted and calculated using GraphPad Prism 8.

In the colony formation assay, 1000 cells were suspended in 2mL medium per well in a six-well plate and cultured for 14 days. After fixation with 4% paraformaldehyde, cells were stained with crystal violet. The number of cell colonies was quantified using Image J software.

Annexin V-FITC/PI Apoptosis Assay

The Annexin V-FITC/PI Double Stain Apoptosis Detection kit (Cat# KGA108) was purchased from KeyGEN Bio TECH (Jiangsu, China). TNBC cells were cultured in six-well plates and treated with HDACi for 48 hours. Cells were harvested, stained with Annexin V-FITC and Propidium Iodide (PI) after suspension in binding buffer, and incubated in the dark for 15 minutes. Apoptosis detection was performed via flow cytometry and Flowjo_V10 software was used for data analysis.

LDH Release Assay

The Cytotoxicity LDH Assay Kit-WST (Cat# CK12, DOJINDO LABORATORIES, Shanghai, China) facilitates the measurement of cellular damage by gauging the activity of lactate dehydrogenase (LDH) present in the cytoplasm and released into the medium. This LDH release occurs whenever cell membrane damage transpires, thus making LDH a stable and reliable indicator of cell mortality and injury. For this procedure, TNBC cells were first placed in 96-well plates for a day, and thereafter incubated in fresh medium which contained either HDACi or PBS for 48 hours. Subsequently, cell lysis buffer, amounting to 10% of the original medium volume, was added to the sample well showing maximum enzyme activity, and incubated at 37℃ for half an hour. Following this, a total of 100µL working solution was added to each well and, after mixing, was incubated in the absence of light for an identical time span. As a final step, 50µL of stop solution was introduced into every well, with immediate absorbance measurement taken at 490 nm. The calculation for determining the LDH release rate follows this formula: (drug-treated sample - PBS-treated control sample) / (maximum enzyme activity sample - PBS-treated control sample) × 100%.

Quantitative Real-Time PCR

Total RNA was extracted utilizing the SteadyPure Universal RNA Extraction Kit (Cat# AG21017, Accurate Biology, Hunan, China). The resulting RNA was then reverse-transcribed into cDNA using the Evo M-MLV RT Premix for qPCR (Cat# AG11706, Accurate Biology, Hunan, China). The qRT-PCR reaction setup was based on the SYBR Green Pro Taq HS Premix (Rox Plus) (Cat# AG11718, Accurate Biology, Hunan, China), into which 2µL of cDNA was introduced into a 20µL system. A two-step PCR procedure was adhered to. The preliminary stage entailed predenaturation at 95℃ for a duration of 30 seconds. Following this was the amplification and extension stage, conducted over 40 cycles, with denaturation, annealing/elongation temperatures stipulated at 95°C and 60°C respectively. The specific primer sequences are outlined in the supplementary table 1.

Western Blot Assay

Collected cells were treated with RIPA lysis buffer (Cat# G2002, Servicebio, Wuhan, China) containing a protease inhibitor cocktail (Cat# G2006, Servicebio, Wuhan, China), and this was lysed on ice for 30 minutes to extract intracellular proteins. The electrophoresis procedure employed the AGE Gel Fast Preparation Kit (10%) (Cat# PG112, Epizyme Biotech, Shanghai, China), Tris-glycine SDS-PAGE SWE High-resolution fast electrophoresis buffer (Cat# G2081, Servicebio, Wuhan, China), and the Ice-free fast transfer buffer (Cat# G2028, Servicebio, Wuhan, China). Operating at a constant pressure of 80V initially, protein samples were equally loaded before shifting to a 200V constant pressure for electrophoresis. Depending on the molecular weight of the target protein, the protein within the gel was transferred onto a PVDF membrane, employing a transfer program set at 400mA constant current for 25 minutes. Post transfer, the protein-free rapid sealing solution (Cat# G2052, Servicebio, Wuhan, China) was used to seal the membrane at room temperature for 15 minutes. The membrane was then subjected to incubation with primary antibodies that targeted specific bands at 4℃ overnight. Following this, species-specific secondary antibodies were used to incubate the membranes at room temperature for an hour. The usage of the antibodies listed in the supplementary table 2.

Construction of Plasmids and Stable Cell Lines

The study utilized a pLKO.1-TRC cloning vector (Cat# 10878, Addgene) as an shRNA vector, which was cleaved by AgeⅠ and EcoRⅠ enzymes. The vector was then conjugated with the insert fragment shRNA oligos using T4 ligase to produce the target plasmid. This target plasmid was packaged alongside the psPAX and pMD2.G packaging plasmids at a ratio of 4:3:1 in HEK293T cells, creating lentivirus. Centrifugation at 30,000g for four hours was employed to collect the virus in the supernatant. Subsequently, the MDA-MB-231 and 4T1 cell lines were developed into stable cell lines for GSDMEsh and GSDMAsh. Following lentivirus transfection, puromycin was used to screen transfected positive cells, resulting in stable transmissible cell lines. The effectiveness of the knockdown was gauged with qRT-PCR, and it revealed a ~ 75% reduction in mRNA expression. Sigma website (https://www.sigmaaldrich.cn/) was utilized for filtering shRNA sequences. Refer to the supplementary table 3 for specific sequences.

Immunohistochemistry (IHC) Test

The procedure was undertaken in accordance with the SABC Immunohistochemical Staining Kit's directions (Cat# SA1028, Bosterbio, Wuhan, China). The paraffin sections were heated at 60℃ for two hours for dewaxing. Following dewaxing, the tissue sections were rehydrated using an eco-friendly dewaxing solution and ethanol with a gradient concentration. To deactivate endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxide at room temperature for 15 minutes. Antigens underwent acid repair or alkaline repair based on their properties. The tissue sections were cooled to room temperature after antigen repair. The tissues were treated with 5% BSA sealing solution for 30 minutes at 37℃. Properly diluted primary antibody was added and left to incubate at 4℃ overnight. Post the primary antibody wash with PBS, biotin-labeled anti-rabbit IgG was added and kept at 37℃ for 60 minutes. After which SABC was added and left at 37℃ for 30 minutes. The color was developed by DAB, while the reaction time was monitored under a microscope. Hematoxylin re-dyed the tissue sections for a duration of 0.5-2min. Following the dehydration and transparency process of the tissue sections, neutral gum was used to seal the slices. The immunohistochemical primary antibodies were documented in the supplementary table 2.

Slides were scanned using the SQS-40 slide scan system (Teksqray, Shenzhen, China). 4 to 6 40× images were randomly chosen from each tissue section from the scanned images. The Image J software was utilized to convert the immunohistochemical images into 8-bit grayscale images, with the grayscale value being converted into optical density (OD) value for data acquisition. Integrated optical density (IOD) was used to analyze the protein expression from the derived data.

In Vivo Anti-tumor Therapy

A subcutaneous transplanted tumor model was established in BALB/c mice using 4T1 cells. BALB/c mice, sourced from Gempharmatech in Jiangsu, China, were quarantined and then housed in SPF-sanitized animal facilities. After a week-long acclimatization in these facilities, each mouse was inoculated with 5×10^5 4T1 cells to induce tumor development. Tumor growth was monitored by measuring both its longest and shortest diameters, used to compute the tumor volume. When the tumors reached a diameter of 5mm, the mice were randomly distributed into a treatment group and a control group. Mice in the treatment group received intraperitoneal injections of 100µL SAHA (50mg/kg), while those in the control group got 100µL solvent injections, both administered the subsequent day. The experiment was concluded after three weeks, with the mice euthanized in accordance with animal ethics. Tumor samples were subsequently collected, cleaned with sterile PBS, and subsequently divided into three parts. One part was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. The second part was deep-frozen at -80℃, while the last part was frozen with RNAlater to prevent RNA degradation. The experimental procedures were approved by the Animal Ethics Committee of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology (TJH-202207028) and carried out by trained personnel utilizing appropriate animal-handling practices in the SPF animal room.

Statistical Analyses

All data underwent statistical analysis in Graphpad Prism 8.0, complemented with data visualization via Afinity Designer software. Image J software facilitated the quantification of cell clones and the transition of the immunohistochemical images into 8-bit grayscale images. Flow cytometry results were analyzed with Flowjo_V10 software. The expression of gasdermin genes in human breast cancer was scrutinized using UALCAN website (https://ualcan.path.uab.edu/index.html, query date: September 10, 2023). The correlation between gene expression and immune cell infiltration was obtained at the SangerBox website (http://sangerbox.com/home.html, query date: October 2, 2023). Transcriptome sequencing and analysis were executed on the DNBSEQ-T7 sequencing platform by YingziGene Technology (Wuhan, China). Unpaired T-tests compared the differences between two peer groups with normally distributed variables. ANOVA (parametric method) and the Kruskal-Wallis test (non-parametric method) allowed for the comparison of mean values across several groups. All conducted tests were bilateral, with p-values less than 0.05 considered statistically significant.

Profile of Gasdermin Family Members in Breast Cancer

Given that pyroptosis is executed by gasdermins, we explored the expression profiles of GSDMA-E in breast cancer patients using the TCGA database (UALCAN website). We observed the highest expression of GSDMD per million transcripts in breast cancer while GSDMA and GSDMC demonstrated the lowest expression. GSDMB and GSDME demonstrated medium expression levels (Fig. 1-A). Comparative analysis with normal breast samples revealed upregulation of GSDMD and downregulation of GSDMB and GSDME in tumor samples (Fig. 1-A). In breast cancer subtypes (Fig. 1-B), the expression of GSDMA and GSDMB correlated with HER2-positive breast cancer. A differential expression of GSDMC, GSDMD, and GSDME was noted in TNBC and normal samples. By employing the ESTIMATE approach, we assessed the correlation between GSDM gene expression and immune cell infiltration in breast cancer. This evaluation revealed a strong association between the expression of GSDMA, GSDMC, and GSDME with immune infiltration (Fig. 1-C).

HDACi-Induced Pyroptosis and Apoptosis in TNBC Cells

Our prior research indicated that HDACi could prompt pyroptosis in TNBC cells. Consequently, we administered two different HDACi, SAHA and MS275, to human TNBC cells (MDA-MB-231 and BT-549) and murine TNBC cells (4T1 and EO771). According to the cell viability curve (Fig. 2-A, B) and half-maximal inhibitory concentration (IC50, Table 1), the proliferative suppression of HDACi on TNBC cells was not significant in the initial 24 hours but became substantial at the 48- and 72-hour timepoints. Diverse cell lines displayed differential responses to HDACi treatment. Specifically, MDA-MB-231 and BT-549 were sensitively inhibited by SAHA, with a 48-hour IC50 of about 5µM, while EO771 displayed resistance to both SAHA and MS275, with a correspondingly higher IC50 value (48-hour IC50 > 10µM). After treating TNBC cells with the 48-hour IC50 of MS275 and SAHA, all four cell lines exhibited a "balloon"-like morphology under phase-contrast microscopy, with 4T1 demonstrating the most pronounced pyroptotic form (Fig. 2-C, D). Since both HDACi could induce pyroptosis in TNBC cells, and considering the superior performance of SAHA, we primarily employed SAHA for succeeding experiments. Following treatment of MDA-MB-231 and 4T1 with SAHA, a marked reduction in cell colony formation became evident (Fig. 2-E), asserting the inhibitory impact of SAHA on TNBC cell proliferation.

Table 1

IC50 values of TNBC cells at different time post-HDACi treatment
Cell line	Time	IC50-SAHA(µM)	IC50-MS275(µM)
MDA-MB-231	24h	56.37	191.6
	48h	5.354	25.91
	72h	2.328	7.048
BT-549	24h	24.84	105.5
	48h	5.521	4.757
	72h	2.249	1.897
4T1	24h	23.47	75.18
	48h	7.242	34.98
	72h	3.716	17.29
EO771	24h	126.8	230.6
	48h	13.78	78.67
	72h	6.33	9.357

A distinguishing feature of pyroptosis vis-a-vis apoptosis is the formation of pores in the cell membrane. These pores facilitate the interchange of intracellular and extracellular substances, such as propidium iodide (PI) dye entering the cell and lactate dehydrogenase (LDH) escaping the cell. Upon SAHA administration, we noted a surge in Annexin V-FITC and PI double-positive cells in four cell lines following the increasing concentrations (Fig. 3-A). Especially in FITC + 4T1 and BT-549, PI + cells accounted for the majority. While in MDA-MB-231 and EO771, FITC + PI + double-positive cells were also present, but the proportion of FITC + PI- cells (generally considered to be the early stage of apoptosis) dominated. This suggested that pyroptosis and apoptosis coexist in TNBC cells. Concurrently, enhanced LDH release was observed in the cell supernatant following SAHA treatment (Fig. 3-B). Combining cell morphology, double-positive cell proportions, and LDH release, the results substantially validated HDACi induction of pyroptosis in TNBC cells.

Activation of the Caspase 3-Gasdermin E Pathway Leading to Pyroptosis

It remains uncertain which GSDM protein (GSDMA-E) would induce pyroptosis in TNBC. To resolve this, further exploration of the executors was undertaken. We evaluated the mRNA expression of GSDM family members in human TNBC cell lines MDA-MB-231, BT-549, and mouse TNBC cell lines 4T1, EO771 (Fig. 4-A). Analysis revealed in MDA-MB-231, BT-549 and 4T1, the highest expression of GSDME followed by GSDMD, while GSDMA and GSDMC showed limited expression, and EO771 showed higher expression of GSDMD. As an epigenetic modifier, HDACi also possess functional capacities of managing gene expression. Hence, we observed the mRNA expression of GSDM post-SAHA treatment and noted that SAHA could manipulate expression in TNBC cell lines (Fig. 4-A). In MDA-MB-231, SAHA promoted the expression of GSDMB and GSDME, with a ~ 3-fold increment in GSDMB and a ~ 1.5-fold increment in GSDME. In BT-549, SAHA increased the expression of GSDME nearly 3-fold. In 4T1 and EO771, while GSDME expression change was not as dramatic as in MDA-MB-231 and BT-549, GSDMA expression was considerably increased, almost matching GSDME expression levels. And GSDMD expression in EO771 was down-regulated post-SAHA treatment.

Accordingly, we conducted a further analysis of GSDME protein in TNBC cells (Fig. 4-B). Upon increasing SAHA concentration, the bands corresponding to the GSDME N-terminal intensified. Usually, activated caspase 3 cleaves GSDME at specific sites, resulting in the GSDME-C terminal and the GSDME-N terminal. The inactive forerunners of caspase 3 are functionalized upon cleavage into active structures of molecular weights 17kDa and 12kDa. With SAHA concentration elevation, it was noticed that the expression of activated caspase 3 also increased. Preliminary results suggested that the caspase 3-GSDME pathway was activated during SAHA-induced pyroptosis of TNBC cells.

To further authenticate the role of GSDME protein in such execution, we constructed GSDME knockdown cell lines in MDA-MB-231 and 4T1 using shRNA lentiviruses. After GSDME knock-down, SAHA-treated cells released less LDH as compared to control cell lines. Moreover, microscopic observations revealed fewer dying cells, especially in the "balloon" form (Fig. 4-C, D). This suggested that reduced expression of GSDME mitigated the degree of pyroptosis. However, these cells also showed lower densities than the control group, and cells manifesting apoptotic morphology were still observed, indicating that SAHA not only induced cell pyroptosis but also apoptosis.

HDACi Regulation of GSDM Family Member

It was surprising to note that HDACi induced substantial pyroptosis in TNBC cells via caspase 3-GSDME. Treating TNBC cells with HDACi promoted the expression and activation of GSDME. HDACi also significantly increased the expression of GSDMA. Thereby, further exploration was warranted to assess if GSDMA played a role in the pyroptotic process of 4T1. Our results demonstrated a significant increase in GSDMA protein levels, but no bands were found at the GSDMA N-terminal location (Fig. 5-A). We also created stable knockdown of GSDMA in 4T1 cells. Even after achieving approximately 80% knock-down efficiency of GSDMA (Fig. 5-B), LDH release level was not mitigated (Fig. 5-C). The function of GSDMA on the pyroptosis in 4T1 was not significant, which may relate to the non-activation of the GSDMA N-terminal.

To investigate the activation conditions of the caspase 3-GSDME pathway, we performed transcriptome sequencing on 4T1 cells, where the most noticeable pyroptotic phenomenon was observed. HDACi treatment produced 5,243 differentially expressed genes, including 2,573 up-regulated genes and 2,670 down-regulated genes (Fig. 5-D). The Gene Ontology (GO) enrichment pathway suggested that HDACi may induce cell cycle arrest (Fig. 5-E). Key genes in cell cycle regulation were examined, which revealed diminished cyclin-Cdk expression at cell cycle checkpoints, including Ccnd1-Cdk6/Cdk4, Ccna2-Cdk2, and Ccnb1-Cdk1 (Supplementary Fig. 1-A). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway displayed significantly enriched metabolic pathways, with glutathione metabolism demonstrating the highest rich factor (Fig. 5-F). Glutathione metabolism participates in the intracellular REDOX reaction equilibrium[29, 30]. Thus, this prompted further analysis of the expression of genes that produce reactive oxygen species (ROS) and antioxidant systems (Fig. 5-G), which revealed an imbalance in gene expression that might lead to intracellular oxidative stress post-HDACi treatment.

HDACi-Induced Tumor Pyroptosis and Improved Immune Infiltration

As we have earlier authenticated HDACi's induction of pyroptosis in TNBC cells via activating the caspase 3-GSDME pathway, next, we endeavored to explore the effects of HDACi on the tumor immune microenvironment in mice. Upon subcutaneous tumor formation in BALB/c mice (Fig. 6-A), SAHA was administred via intraperitoneal injection at a concentration of 50mg/kg. Tumor gross view (Fig. 6-B) revealed significant inhibition of breast tumor growth and relatively small-sized and lightweight tumors than the control group (Fig. 6-C). There was no significant difference in the body weight curve of the control and administration group, bolstering the claim that SAHA had minimal impact on normal mouse growth (Fig. 6-D). Subsequent analysis showed delayed tumor growth in SAHA-treated mice compared to the control group starting around one week after treatment commencement, and the gap widened further with continued administration (Fig. 6-E). Post-harvesting the mouse tumors, western blot and immunohistochemical tests were performed to detect the executor of pyroptosis. The expression of GSDME N-terminal structure and cleaved caspase 3 was more intense in the administration group than the control (Fig. 6-F).

Following pyroptotic death, tumor cells transitioned from being non-immunogenic to immunogenic, eliciting an anti-tumor immune response (Fig. 6-G). Tumor cells produce signaling molecules such as calreticulin, which is displayed on the cell surface, and also secrete high mobility group protein 1 (HMGB1). In SAHA-treated mouse tumors, HMGB1 and calreticulin expression were elevated, particularly HMGB1, whose expression in the control group was minimal. Pyroptosis of tumor cells triggers both innate and specific immune response. We examined the myeloid marker CD11b (including macrophages and dendritic cells), anti-tumor effector cells (CD8 + T cells) and effector molecule granzyme B (GZMB). The results revealed enhanced infiltration of CD8 + T cells and CD11b + cells in the tumor in the administration group than the control, with increased expression of GZMB, which has a direct tumor-killing effect. We also examined the expression of cytokines in tumor tissues and found that the mRNA expressions of Ifn-g, Ccl5 and Cxcl10, which are related to the chemotaxis and activity of CD8 + T cells, were upregulated, while the mRNA expression of the immunosuppressive molecule Pd-l1 was decreased (Supplementary Fig. 1-B). In conclusion, HDACi can induce tumor pyroptosis and stimulate the anti-tumor immune response.

This study's results indicate that HDACi can inhibit the proliferation of TNBC cells and induce pyroptosis, a type of programmed cell death. Within 48 hours of treatment with HDACi, TNBC cells visibly developed a "balloon" shape, exhibited an internal release of lactate dehydrogenase (LDH) and allowed the penetration of propidium iodide (PI) dye. It is noteworthy that the extent of pyroptosis varied among different TNBC cell types, with the 4T1 cells demonstrating the most prominent pyroptosis phenotype. TNBC cells primarily expressed gasdermin E (GSDME) from the gasdermin (GSDM) family, and the implementation of pyroptosis was driven by the caspase 3-GSDME pathway. Knocking down GSDME alleviated pyroptosis to a certain degree. And the pyroptotic death of the tumor cells was immunogenic. This immunogenicity contributed to higher expression levels of HMGB1 and calreticulin, which in turn promoted the infiltration of CD11b + cells and CD8 + T cells into the tumor. This led to an enhanced expression of the granzyme B (GZMB), a potent tumoricidal molecule, thereby advancing the immunity against the tumor.

HDACi, as a coordinator of histone acetylation modification, can regulate gene expression via epigenetic changes. Although previous studies reported DNA methylation inhibiting GSDME expression in tumor cells, demethylation with decitabine restored its expression[31, 32]. Yet, only decitabine did not cause pyroptosis. In contrast, the use of chemotherapy agents beyond decitabine resulted in a visible pyroptosis effect on the tumor cells[22]. HDACi treatment caused differing degrees of increase in the expression of GSDMA, GSDMB and GSDME. Nevertheless, despite GSDMA expression level being almost identical to GSDME, no activation of GSDMA was detected, proven by the unaltered level of LDH release after the GSDMA knockdown. This indicates that GSDMA was not activated in TNBC cells, and only activated GSDM proteins can carry out pyroptotic activities.

Following 48 hours of HDACi treatment, the pathways regulating cell cycle and glutathione metabolism were enriched. This suggests that cell cycle arrest and oxidative stress were significant for GSDME activation. The process of oxidative stress triggering pyroptosis through the caspase 3-GSDME pathway has been observed in various tumor types. Glutathione (GSH), as the most abundant antioxidant, primarily ensures cell redox equilibrium[29, 30]. This intracellular redox balance is maintained by the interaction between reactive oxygen species (ROS) and antioxidants[33, 34]. Studies have shown that Tom20, a protein on the mitochondrial outer membrane, senses ROS signals and recruits Bax to form a mitochondrial pore. As a result, cytochrome c is released into the cytoplasm, activating caspase 3, which cleaves GSDME and induces melanoma cell pyroptosis[35]. In breast cancer cases, the chemotherapy drug Doxorubicin (DOX) caused ROS accumulation within cells, stimulating cell death via the JNK phosphorylation and caspase 3-GSDME pathways[36]. However, adding the antioxidant GSH to ROS-stimulated TNBC cells prevented pyroptosis[37]. This suggests that oxidative stress and antioxidants play a crucial role in regulating cell death. On one side, HDACi treatment enhances GSDME expression in TNBC cells, while on the other, it may disrupt redox equilibrium, creating an environment that activates the caspase 3-GSDME pathway which in turn led to cell pyroptosis.

This study, however, had its limitations. Firstly, we only revealed that HDACi constructs a platform for TNBC cell pyroptosis by activating the caspase 3-GSDME pathway, with the underlying mechanisms being cell cycle arrest and oxidative stress, neglecting further verification processes. Additionally, we focused solely on understanding HDACi's role in activating GSDME and inducing pyroptosis in TNBC cells, overlooking the impact HDACi potentially has on the histone acetylation modifications and consequent gene expression. Consequently, this led to findings that were preliminary at best, indicating that HDACi heightened the expression of GSDME, enabling the caspase 3-GSDME pathway to induce pyroptosis in TNBC cells, restrained breast tumor growth, and encouraged immunity against the tumor.

Competing Interests

The authors have no competing interests to declare that are relevant to the content of this article.

Acknowledgements

We thank all members of the Thyroid and Breast Surgery Laboratory as well as various colleagues in the Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

This work was supported by the Key Research and Development Program of Hubei Province (Grant No. 2022BCA007), the Scientific Research Staring Foundation for the Returned Overseas Chinese Scholars, Tongji Hospital (Grant No. 2022hgry010) and the Knowledge Innovation Program of Wuhan-Shuguang Project (Grant No.2023020201020490).

Author Contribution

T. Xu contributed to the study’s conception and design. Y. Du and X. Li supervised the study. X. Yang and X. Cui performed the experiment. X. Yang and X. Cui contributed equally to the study. G. Wang and Y. Wu provided the study materials and realized in vivo experiments. X. Yang analyzed, interpreted the data and wrote the manuscript. All authors read and approved its final version.

Data availability

Authors confirm that the data supporting the results of this study are available within the article. The transcriptome sequencing data analysed during the current study are available from the corresponding author on reasonable request.

Contributions

Ethics declarations

Ethical approval

The animal experiments conducted in this study have been reviewed and approved by the Animal Ethics Committee of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology (TJH-202207028). We aimed to minimize the suffering and the number of animals. The mice were kept on a 12-hour light/dark cycle with access to food and water ad libitum.

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No competing interests reported.

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published 11 Jun, 2024

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HDAC Inhibitor Regulates the Tumor Immune Microenvironment via Pyroptosis in Triple Negative Breast Cancer

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Abstract

Objective

Methods

Results

Conclusion

Figures

Introduction

Methods

Cell Culture and Drug Treatment

Cell Proliferation-Toxicity Assay and Colony Formation Assay

Annexin V-FITC/PI Apoptosis Assay

LDH Release Assay

Quantitative Real-Time PCR

Western Blot Assay

Construction of Plasmids and Stable Cell Lines

Immunohistochemistry (IHC) Test

In Vivo Anti-tumor Therapy

Statistical Analyses

Results

Profile of Gasdermin Family Members in Breast Cancer

HDACi-Induced Pyroptosis and Apoptosis in TNBC Cells

Activation of the Caspase 3-Gasdermin E Pathway Leading to Pyroptosis

HDACi Regulation of GSDM Family Member

HDACi-Induced Tumor Pyroptosis and Improved Immune Infiltration

Discussion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

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