Inhibition of SUV39H1 reduces tumor angiogenesis via Notch1 in oral squamous cell carcinoma

Statement of ethics

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (protocol code WDRY2021-KS019 and date of approval) for studies involving humans. The animal study protocol was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (protocol code ZN2022085 and date of approval).

Patients

OSCC and matched paracancer tissues were diagnosed by two independent pathologists in the Department of Pathology of Zhongnan Hospital of Wuhan University in accordance with the 2006 WHO grading system. This study included 80 OSCCs and 40 paired paracancer tissues. Please refer to Table 1 for the general information of the patient.

Reagents, antibodies, cell lines and cultures

N-(N-(3, 5-difluorophenyl-l-propyl))-S-phenylglycine t-butyl ester (DAPT, gamma-secretase inhibitor, inhibits the cleavage of Notch1) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Chaetocin (SUV39H1 inhibitors; catalog No. S8068) from Selleck China (Shanghai, China). Antibody SUV39H1 (catalog number: AV32470) was purchased from Sigma-Aldrich, Notch1 (catalog No. 3608), CD31 (catalog No. 3528), and VEGF (catalog No. 50661) from Cell Signaling Technology (Danvers, MA, USA). Antibody SUV39H1 (catalog number: 28074-1-AP) was purchased from Proteintech (WuHan, Hubei province, China). The Human OSCC cell line (Cal27) was purchased from ATCC. Cal27 cells were cultured in DMEM medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) in a humid atmosphere of 37 °C, 95% air, and 5% carbon dioxide.

Immunohistochemistry

Simply, slices are dewaxed in xylene, rehydrated in graded ethanol and double distilled water, and then heat-induced antigen extraction. After the primary antibody (SUV39H1 1:200, Notch1 1:100, CD31 1:200, Ki67 1:2,000) was incubated at 4 °C overnight, the secondary antibody was incubated at room temperature for 1 h. Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the density determination. CD31 labeled microvessels, counting capillaries and microvessels in tissue sections. A single brown endothelial cell or cluster of endothelial cells was counted as one vessel, but vessels with thick muscle layer and lumen area larger than 8 red blood cell diameter were not counted. The counting method was to first scan the whole section with a 10*10 low-power lens to determine the field of view with the densest microvessels, and then count all the stained microvessels within the field of view with a high-power lens. The average of the three field counts was taken as the number of microvessels in the cuticle. The tissue sections were scored according to the staining degree (0–3 corresponding to negative staining, light brown, dark brown) and positive range (1–4 corresponding to 0–25%, 26–50%, 51–75%, 76–100%) under the optical microscope. The two scores are added together and compared.

Animal experiments

The in vivo study was approved and supervised by the Animal Care and Use Committee of Wuhan University, and was conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Female BALB/c nude mice without thymus (18–20 g; 6–8 weeks of age) were obtained from Wuhan SLAC Laboratory Animal Co., Ltd. (Wuhan, China). At the Laboratory Animal Center of Wuhan University, animals are kept in pressurized ventilation cages, according to the institution’s regulations (five animals per cage). The mice were placed in a suitable sterile cage with a filter lid in the Laboratory Animal Center of Wuhan University and fed and watered at will. Mice were maintained on a 12-h light/dark cycle. When the cell growth was exponential, CAL27 cells (4 × 10⁶, 0.2 ml serum-free medium) were injected subcutaneously into the flank of mice.

The mice were randomly divided into two groups (five animals per group), give Chaetocin (0.25 mg/kg, intraperitoneally (i.p.), once a day; n = 5) or normal saline (control, 100 ul i.p once a day; n = 5) 3 weeks of infusion. Tumor growth was determined by measuring tumor size three times a week. The tumor volume was determined by the formula (width² × length)/2. The mice were euthanized by cervical dislocation method at a specified time point or maximum diameter of tumor more than 2 cm. The tumors were collected for immunohistochemical analysis. The raw data are provided in the Supplemental Material.

RNA interference experiment

Briefly, CAL 27 cells were seeded in 6 cm petri dishes and grew to 80%. SUV39H1 siRNA was were purchased from Shanghai Genechem Co., LTD (Shanghai, China) and transfected with Hiperfect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Western blot confirmed that at a specific time (36 h), the knockout efficiency of SUV39H1 protein was reduced by at least 90%. SUV39H1 siRNA forward 5′-CGUGGAUUGUCUCAGGGAATT-3′; Downstream 5′-UUCCCU GAGACAAUCCACGTG-3′.

Conditioned medium, transwell assay and tube formation

The treated CAL27 cells (SUV39H1 siRNA or/and DAPT) were washed twice with phosphate buffer (PBS). Endothelial basal medium (EBM; Lonza, Walkersville, MD, USA) with serum removed was continued to grow for 24 h. The cleared supernatant was collected as conditioned medium (CM) and stored at −80 °C.

HUVECs cells were suspended in six-well culture plates, and grown to 90% confluence. The Transwell Boyden chamber (Corning Life Sciences, Corning, NY, USA) was used to measure migration of endothelial cells. Starved HUVECs cells (5 × 10⁴ cells/well) incubated in 100 μl of ECM were placed in the upper wells, whereas CM, as a chemo-attractant, was added to the lower wells. Then, cells were incubated for 12 h at 37 °C. HUVECs on the upper surface were carefully scrubbed off using a cotton swab, and cells adhering to the lower membrane were fixed with 4% formaldehyde, stained by crystal violet, and observed under microscope. The rat tail glue was configured first: rat tail glue was composed in the ratio of A:B:C = 9:1:1. Liquid A was 0.02N acetic acid plus 50× Collagen solution (0.02N acetic acid 7.8 μl + Collagen 130 μl + H₂O 6.3 ml), and liquid B was 10 × Eagle’s medium solution. C is a 2% sodium carbonate solution. Rat tail gum solution was added to 24-well plate, and the volume of 500 μl/well was added. The solution was set at 37 °C for 1 h, and after solidified, it was washed gently with PBS. HUVEC was inoculated with 5 × 10⁴ cells per well. After several hours of culture, the cells were adherent to the wall. Ordinary medium was replaced with conditioned medium collected after treatment. This should be stopped after 24 h. The supernatant was removed and discarded with gum then gently washed three times with PBS. Then, 4% paraformaldehyde should be used to fix for 10 min; wash with water and then gently wash with PBS. The culture plates were placed under an inverted microscope, and six fields of view were randomly selected to take photos to observe and analyze the number of tubes.

Tumor genome atlas (TCGA) analysis

Data on target gene transcription in different cancer patients were obtained from the GEPIA database (GEPIA http://gepia.cancer-pku.cn/).

Western blot assay

Briefly, CAL27 cells were treated with the indicated concentrations in DMEM containing 2% FBS for 24 or 36 h. Then the cells were lysed, and the total protein was separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). The immunoblots were cultured overnight at 4 °C with the corresponding primary antibodies in blocking solution, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Pierce Chemical, Rockford, IL, USA). Then, the blots were developed with a West Pico ECL kit.

Statistical analysis

Statistical analysis was performed using Graphpad Prism software (GraphPad, Inc., La Jolla, CA, USA), and the data were expressed as mean ± mean standard error (SEM). Two-way ANOVA analysis was used for analyzing differences between animal treatment results. One-way analysis of variance (ANOVA) and student t test were used to analyze the differences with the control group and among each group. The relationship between SUV39H1, Notch1 and CD31 expression was analyzed statistically by Pearson with two tails.

Correlation between SUV39H1, Notch1, and MVD expression in OSCC

We studied the correlation between SUV39H1, Notch1 and CD31 protein expression by immunohistochemical staining. As showed in Fig. 2A, SUV39H1 was mainly expressed in the nucleus. Notch1 is mainly expressed in the cell membrane, and CD31 is present in cell membranes, cell junctions, and blood vessels. Tissue scores of SUV39H1, Notch1 and MVD were calculated, and then correlation analysis was performed for each of the two proteins selected. Our results showed a significant positive correlation between SUV39H1 and tissue scores of Notch1 and MVD (Figs. 2B and 2C). In addition, Notch1 expression in OSCC was positively correlated with MVD (Fig. 2D). We investigated the correlation between NOTCH1 and CD31 expression in head and neck squamous cell carcinoma in the GEPIA database and found that the expression of NOTCH1 was positively correlated with that of CD31 (Fig. S1). It is worth noting that the correlation between SUV39H1 and NOTCH1 and MVD is weak, which may indicate that SUV39H1 may not be directly involved in tumor angiogenesis in OSCC. This needs to be further clarified in future studies.

Chaetocin attenuates OSCC xenograft tumor growth in nude mice

We used Chaetocin treatment ectopic xenograft tumor derived from CAL27 cells to further determine SUV39H1 function in OSCC. Chaetocin is used to inhibit SUV39H1 function. As shown in Figs. 3A–3D, compared with the control group, chaetocin obviously inhibited tumor growth. Treatment doses of Chaetocin has no obvious toxicity in mice (Fig. 3E). These results suggest that Chaetocin may effectively inhibit tumor growth in OSCC.

Chaetocin inhibit tumor angiogenesis in OSCC

After the animal experiment, we collected the transplanted tumor tissue. Immunohistochemical study showed that in the groups treated Chaetocin, SUV39H1, Notch1, CD31, Ki67 staining intensity is lower than the control group (Fig. 4A). We calculated the SUV39H1, Notch1, MVD group score. In OSCC, SUV39H1, Notch1, MVD, Ki67 was significantly lower after Chaetocin treatment (Fig. 4B).

SUV39H1 affects tumor angiogenesis by regulating Notch1

We constructed SUV39H1siRNA and examined its inhibitory effect on SUV39H1 expression (Fig. 5A). As shown in Fig. 5B, SUV39H1siRNA reduced HUVEC migration and tube formion capacity compared with control medium. Similar results were obtained for DAPT treatment. SUV39H1siRNA as well as DAPT treatment further reduced HUVEC migration and tube formion ability (Figs. 5C and 5D). SUV39H1siRNA decreased the expression of Notch1 and VEGF. DAPT decreased the expression of VEGF, but had little effect on the expression of SUV39H1. After SUV39H1siRNA and DAPT were treated simultaneously, the expressions of Notch1 and VEGF were further decreased (Fig. 5E). As shown in Fig. 5F, after treatment with Chaetocin and DAPT, the gray scale of SUV39H1, NOTCH1, and VEGF strips was lower than that after Chaetocin or DAPT alone treatment. The results of Chaetocin treatment were similar to those of SUV39H1 siRNA treatment.