The tumor cells were cultured as previously described (Zhu et al., 2021). This research employed human breast cancer cells (T47D, MCF-7, BT-549, MDA-MB-231), glioma cell line (T98G), and human mammary epithelial cells (MCF-10A) sourced from the Chinese Academy of Sciences, Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were cultured in RPMI 1640 medium (Gibco, United States) or Dulbecco’s modified Eagle’s medium (DMEM, Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, United States).

PACAP38 and PACAP6-38 were purchased from MedChemExpress (MCE, United States). PACAP38 was solubilized in sterile water to reach 1 mg/mL, aliquoted, and kept at −80°C for further utilization. DNA Methylation Inhibitors (5-Azacytidine, 5Aza), Histone Acetylation Inhibitors (HATi, Anacardic acid, ANA), and Histone Deacetylation Inhibitors (HDACi, Vorinostat, SAHA) were purchased from Selleck Chemicals (Selleck, United States).

Protein expression was assessed by Western blotting. Cells were lysed using RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor-supplied for protein extraction. The BCA protein assay kit (Beyotime, China) was utilized to quantify proteins. Protein was loaded into 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in equivalent amounts for electrophoresis, followed by being transferred to PVDF membranes (Millipore, United States). After transfer, the PVDF membranes was first blocked with 5% no-fat milk for 1 h, followed by incubated with primary antibodies overnight at 4°C. At last, they were further incubated with HRP-conjugated secondary antibodies at room temperature (RT) for 1 h. The primary antibodies listed below were employed: anti-SOX6 (Proteintech, United States), anti-β-catenin (CST, United States), anti-LEF1 (CST, United States), anti-TCF1/TCF7 (CST, United States), anti-MMP7 (CST, United States), anti-cJUN (CST, United States), anti-c-myc (CST, United States), anti-CD44 (CST, United States), anti-Tubulin (CST, United States), anti-GAPDH (CST, UUnited States). The Acetyl-Histone Antibody Sampler Kit (CST, United States) was used to evaluate the acetylation states of histones H2A, H2B, H3, and H4. The dilution ratios for all primary antibodies utilized ranged from 1:500 to 1:1000. Secondary antibodies goat anti-rabbit-HRP (CST, United States) and goat anti-mouse-HRP (CST, United States), both used at 1:3000. ECL system (GE Healthcare, Little Chalfont, United Kingdom) was used for visualizing signals.

The Cell Counting Kit-8 (CCK8) and colony formation assay were used to measure cell proliferation. The CCK8 test was conducted using CCK8 assay kits (Dojindo, Japan). Briefly, 96-well plates were seeded with 2 × 10³ cells, and the absorbance at 450 nm was recorded.

For colony formation assay, cells were plated in 6-well plates and subjected to several treatments based on their assigned group. Following an additional 10 days of cultivation, cells were then fixed in 4% paraformaldehyde and stained with 0.5% crystal violet for further visualizing colony formation.

For Edu assay, cells were plated in 24-well plates, EdU solvent was added to the medium for the cells to ingest and staining and assaying were performed 2 h later.

Flow cytometry was used for the analysis of the cell cycle change. The detailed operational procedures are elucidated in our previous article (Li et al., 2019).

Experiments and procedures were carried out with the consent of the Animal Ethic Committee of Shanghai Ruijin Hospital (the Ethical Clearance number is SYXK 018-0027). 5 × 10⁶ T47D cells diluted with 200 µL phosphate-buffered saline (PBS) were injected subcutaneously in the proximal right hind limb of female nude BALB/c mice aged 5–6 weeks old.

When the longest diameter of the tumors in tumor-bearing mice reached approximately 5–6 mm, a total of four cohorts of five mice each were utilized: the control group (Ctrl), PACAP38 group (PACAP38), irradiation group (IR), and the combined irradiation and PACAP38 treatment group (PACAP38+IR). A dose of 10 Gy X-ray irradiation was applied to the tumor sites of the mice in the IR and PACAP38+IR groups. The mice in PACAP38, PACAP38+IR groups were administered intraperitoneal injection of PACAP38 (10 µg/100 µL) diluted in 0.9% normal saline solution administration for three consecutive days (the day before irradiation, the day of irradiation, the day following irradiation). Mice in the control group and the irradiation group were given equal volumes of 0.9% saline solution. Tumor sizes in all groups were recorded and subsequently monitored every other day from the day of IR. The formula for calculating tumor volume was 0.5 × length × width². When the maximum tumor volume of approximately 1,000 mm³ in the mice was observed, euthanasia was performed, and tumor specimens were dissected, photographed, and fixed to enable further analysis.

A medical linear accelerator (Varian Trilogy, FL, United States) was used for irradiation with an X-ray beam energy of 6 MV and a dose rate of 300 cGy/min. The external beam irradiation treatment plan and positioning strategy are detailed in our previous study (Li et al., 2019). For in vivo irradiation of tumors, nude mice bearing a xenograft of breast cancer cells were intraperitoneally administered pentobarbital 0.2% at a dose of 60 mg/kg for anesthesia. Subsequently, X-rays were locally delivered to the xenograft site.

Following a 12-h treatment of T47D cells with RPMI 1640 complete medium containing 100 nM PACAP38 or an equivalent volume of sterile water for control, cellular total RNA was extracted. The RNA was then further enriched with polyA + by Dynabeads Oligo (dT) magnetic beads. The polyA + RNA was prepared as the input material for library preparations using a NEBNext^® Ultra™ II mRNA Library Prep Kit for Illumina^® (NEB, United States). Following this HaploX Medical Laboratory Co. Ltd. China carried out paired-end sequencing on an Illumina PE150, with the abundance of gene expression being displayed using fragments per Kilobase of Transcript per Million mapped reads (FPKM).

Three pairs of SOX6-siRNAs (si-SOX6 1#, si-SOX6 2#, si-SOX6 3#) and their negative control siRNAs (si-NC) sequences were designed and manufactured by Genomeditech (Shanghai, China). The siRNA sequences are shown in Supplementary Table S1. For more on siRNA knockdown operations, please see our prior publication (Su et al., 2020). Western blotting was used to determine the knock-down efficiency. The pair of siRNAs with the highest knock-down efficiency was used for subsequent experiments.

Lentiviral vectors were amplified using DH5α, and HEK293T cells were used for lentiviral packaging. Plasmids were transfected into cells with Polyethylenimine (PEI). The culture supernatant were collected at 48 and 72 h post-transfection, centrifuged, filtered, and concentrated, then infect the target cells, as previously described (Zhu et al., 2021).

For cell IHC analysis, cells were cultured in 6-well plates for 48 h. Then fix the cells on ice with pre-cooled acetone for 15 min. 0.5% Triton X-100 solution was used for immersing cells for 15 min in RT and were incubated with 3% H2O2 for 15 min and blocked for 1 h with 2% BSA in RT. Next, the cells were successively incubated with primary antibodies for SOX6 (1:300, Proteintech, United States), secondary antibody (Abcam, United States), and tertiary antibody (SAB complex) for 12 h, 30 min, and 30 min respectively at 37°C. Under the condition of light-avoidance, the cells were observed under the microscope to react with DAB to brown color for about 5 min. Then after counterstaining with hematoxylin for 1 min, Dehydration was carried out using graded alcohols, and the clearing was carried out with xylene. The rate of positive protein expression in cells was immediately examined using a microscope (Olympus BX51).

Cells were cultured in 6-well plates for 48 h before the IF experiment (Zhu et al., 2021). The detailed experimental procedures can be found in our previous study. The primary antibody dilutions SOX6 (1:300, Proteintech, United States) were used. The fluorescence intensity was evaluated using fluorescence microscopy (Olympus BX51) and quantified by ImageJ.

The tissue microarray, comprising 131 invasive breast cancer tissue specimens sourced from patients, was obtained from Shanghai Outdo Biotech. The development and application of this tissue microarray product received ethical approval from the Ethics Committee of Shanghai Outdo Biotech.

Statistical analyses were conducted utilizing GraphPad Prism9 (GraphPad Software). Data were presented as means with standard deviation (SD). The comparison between the two groups employed the unpaired Student’s t-test, while multiple group comparisons were assessed through one‐way analysis of variance (ANOVA). Survival analysis was performed using the Kaplan-Meier method, and significance in survival discrepancies was determined by log-rank tests. A stringent significance threshold was employed in this study, and a two-tailed test with a p-value below 0.05 was considered statistically significant. Graphs were generated using GraphPad Prism9 software for comprehensive data visualization.

Based on the application concentration range of PACAP38 in previous research, we first explored how PACAP38 treatment affected the growth activity of cancer cells across a concentration range from 0.1 nM to 200 nM by CCK8 assays (Maugeri et al., 2016; Hajdú et al., 2021; Bensalma et al., 2019). The results indicated a substantial suppression of glioma and breast cancer cells (T98G, T47D, BT-549) proliferation by PACAP38 at the dose of 0.1 nM–200 nM (Figure 1A). What’s more, the dose-dependent property of this inhibitory activity is most pronounced within the range of 10–100 nM. We chose an effective dose of 100 nM in the subsequent cellular experiments in vitro.

We further explored the impact of PACAP38 (100 nM), applied independently and in combination with irradiation, on glioma and breast cancer cells proliferation ability by CCK8 assay and Edu assay, the colony formation capability by colony formation assay, and cell cycle by flow cytometry. The results indicated that PACAP38, in synergy with irradiation, reduced the proliferation activity (Figures 1B, C) and colony formation capability (Figure 1D) of the glioma and breast cancer cells more effective than PACAP38 was applied independently. When PACAP receptors antagonist PACAP6-38 (1 μM) was applied simultaneously with PACAP38, the suppression of colony formation capability of PACAP38 on these cancer cells was attenuated (Figure 1D). Flow experiments showed that PACAP38 increased the proportion of cells arrested in the G2-M phase that was blocked, and a higher proportion of cells were arrested in the G2-M phase when PACAP38 was co-irradiated, whereas PACAP6-38 reversed this effect of PACAP38 (Supplementary Figure S1).

To study the impact of PACAP38 on tumor growth in vivo, we established a xenograft model by subcutaneously implanting T47D cells into mice. We observed substantial tumor suppression in mice that received PACAP38, irradiation, and PACAP38 combined with irradiation treatment (Figures 2A, B). Furthermore, the combination of PACAP38 and irradiation suppressed tumor proliferation in vivo more effective than administered with PACAP38 or irradiation alone (Figures 2A,B). The percentage of Ki-67-positive cells in dissected tumor specimens was utilized to measure the proliferation index. The IHC analyses indicated a decreased Ki-67 percentage in the PACAP38 group compared to the Ctrl group, and in the IR + PACAP38 group compared to the IR group (Figure 2C).

To further investigate how PACAP38 suppress the growth of tumors, we conducted RNA-sequencing (RNA-seq) analysis to investigate transcriptional divergence between T47D cells treated with or without PACAP38. The transcriptional variances between the two groups revealed 174 significant differentially expressed genes (DEGs, cutoff point: |log2 fold change| > 1; p-value <0.05), of which 137 genes exhibited upregulation and 37 genes demonstrated downregulation in the PACAP38-treated group (Figure 3A). The information on all differentially expressed genes is presented in Supplementary Table S2. Three of these DEGs belong to the transcription factor family, with two genes (SOX6, ZNF98) upregulated and GFI1 downregulated after PACAP38 treatment.

We further validated that the level of SOX6 protein was also upregulated in PACAP38-treated T98G, T47D, BT-549 cells by western blotting (Figures 3B, C). Moreover, IF analysis showed that PACAP38 treatment enhanced the localization of the SOX6 protein in nuclear, where serving as the site for transcription factors to regulate downstream gene expression (Figures 3D, E).Immunohistochemical analysis from the preceding animal experiments indicates that PACAP38 treatment enhances SOX6 expression in tumor tissues across different groups (Figure 3F).

Pan-cancer analysis via Kaplan-Meier Plotter datasets (http://kmplot.com/) based on TCGA, GEO, and EGA databases also indicated higher SOX6 mRNA expression in adjacent cancer tissue (normal) compared with the corresponding cancerous tissues (tumor) (Figure 4A). The SOX6 mRNA levels comparison between human breast cancer tissue and normal tissue by means of the GEPIA platform (http://gepia.cancer-pku.cn/), which is based on the TCGA and GTEx databases indicated that the levels of SOX6 expression were elevated in normal tissues (N) than in tumor tissues (T) (Figure 4B). Next, SOX6 protein levels were detected in a normal human breast epithelial cell line (MCF-10A) and several human breast cancer cell lines (T47D, BT-549, MCF-7, MDA-MB-231). The findings revealed a higher expression of SOX6 protein in normal breast epithelial cells contrast with breast cancer cell lines (Figures 4C, D).

Employing the breast cancer tissue microarray from Shanghai Outdo Biotech, a correlation analysis was conducted on the immunohistochemical expression of SOX6 in relation to clinical prognosis. The results demonstrate that high expression levels of SOX6 are indicative of improved overall survival (OS) and relapse-free survival (RFS) outcomes (Figures 4E–G). The characteristics of the clinical and pathological information of the patients involved in the chip and the correlation between the clinical and pathological characteristics of the patients and SOX6 expression were showed in the Supplementary Material (Supplementary Table S3). The results showed that there was no significant correlation between SOX6 expression and age, pathological grade, tumour size, lymph node metastasis, clinical stage and molecular type. Kaplan-Meier (KM) survival analysis of breast cancer patients via Kaplan-Meier Plotter datasets based on the TCGA, GEO, and EGA RNA databases revealed that patients with higher SOX6 mRNA levels owned an improved OS and RFS than those with lower SOX6 mRNA levels, regardless of the breast cancer subtype (Figures 4H, I). KM analysis of the GEPIA platform based on the TCGA database showed that those with higher levels of SOX6 mRNA among glioma patients had better OS and RFS than those with lower levels of SOX6 mRNA (Figures 4J, K). These results supported the SOX6 gene’s tumor-suppressive role in glioma and breast carcinoma. Based on this research, we reason that PACAP38 may suppress cancer proliferation via upregulating SOX6 expression.

To elucidate the role of SOX6 in PACAP38-mediated tumor suppression, we employed siRNA-mediated knockdown and lentiviral overexpression of SOX6 in tumor cell lines in T47D and T98G cells. The knockdown (Figures 5A, B) and overexpression (Figures 5C, D) efficiency were assessed by Western blot assays. Three different pairs of siRNAs sequences were used, and si-SOX6 2# was chosen as the most effective pair for further work. Colony formation assays indicated that knockdown of SOX6 could reverse the suppression of PACAP38 on T47D and T98G cells proliferation (Figures 5E, F), which indicated that the capacity of PACAP38 to suppress cancer cells proliferation is dependent upon upregulating SOX6. Additionally, the reduced proliferative capacity of tumor cells following SOX6 overexpression further validates its tumor-suppressive function.

Next, we investigated the mechanisms through which PACAP38 regulates SOX6 gene expression. Epigenetic modifications are currently recognized as significant factors in the regulation of tumor pathology. We examined whether PACAP38 influences the epigenetic modifications of tumor cells at both the pre-transcriptional and post-transcriptional levels. Our findings revealed that the addition of the DNA methylation inhibitor (5-Aza) did not affect SOX6 expression when used alongside PACAP38 treatment (Supplementary Figure S2). However, the application of a HAT inhibitor (ANA) in conjunction with PACAP38 reduced the upregulation of SOX6 expression (Figure 6A). These results suggest that PACAP38 may regulate SOX6 expression through histone acetylation. We further assessed changes in histone acetylation levels following PACAP38 treatment. The results demonstrated that PACAP38 upregulated multiple types of histone acetylation including H2B, H3, and H4, with the most pronounced effects observed on histones H3 and H4 (Figure 6B). Collectively, these findings indicate that PACAP38 likely regulates SOX6 gene expression via histone acetylation.

We further explored the tumor suppression mechanisms operating downstream of the PACAP38/SOX6 axis in cancer cells. Our previous experiments indicated that PACAP38 exerted its antineoplastic effects by upregulating SOX6 expression. Wnt/β-catenin is a common downstream target pathway of SOX6. We speculated that PACAP38 may suppress Wnt/β-catenin signaling by upregulating SOX6 expression. Thus, we performed western blot assays in T47D and BT-549 cells to elucidate the mechanism responsible for the anti-cancer effects mediated by the PACAP38/SOX6 axis. Hereon, our research revealed that PACAP38 downregulated the crucial effector (β-catenin), nuclear transcription factors (LEF1, TCF1/TCF7), and downstream target genes (MMP7, cJUN, c-myc and CD44) associated with the Wnt/β-catenin signaling in cancer cells. However, when PACAP38 was applied concurrently with the knockdown of the SOX6 gene in cancer cells, the inhibitory effects of PACAP38 on proteins associated with the Wnt/β-catenin pathway were reversed (Figures 6C,D).