PADI3 plays an antitumor role via the Hsp90/CKS1 pathway in colon cancer

BALB/c nude mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility. The study was approved by the Ethics Committee of Shandong Province Qianfoshan Hospital, Jinan, China (Approval number: S0087).

The patients information including gender, age (20 < adults < 60 years old), pathological stage are clear and available, primary colon cancer tissues diagnosed by pathology, and there is available corresponding adjacent tissue as control.

All solid colon cancer tissues and adjacent tissues used in this study were obtained from patient surgeries at the Shandong Provincial Qianfoshan Hospital in Jinan, Shandong, P. R. China. Tumor diagnosis was performed via histological methods, and pathological categorization was performed according to the World Health Organization (WHO) classification system.

The following primary antibodies used in this study were commercially obtained: PADI3 (Abcam, ab172959), GAPDH (Abcam, ab181603), His-tag (CST, 12698S), Flag (CST, 14793S), red fluorescent protein (RFP) (Abcam, ab28664), CKS1 (ThermoFisher, 36-6800), CDK1 (ThermoFisher, 33-1800), Hsp90 (CST, 4874S), and p27^Kip1 (CST, 3686S). The following secondary antibodies were used in this study: goat anti-rabbit (Affinity, s0001), goat anti-mouse (Affinity, s0002) and donkey anti-goat (Abcam, ab6881).

Total RNA of tissues or cells was extracted using Trizol reagent (Life Technologies, USA). The extracted RNA was reverse-transcribed into first-strand cDNA in a final volume of 10 µL using an RNA PCR Kit (Toyobo, Japan). The reverse-transcribed first-strand cDNA was used as the template for real-time PCR with the forward primer and the reverse primer. The conditions of real-time PCR were as follows: 10 s at 95 °C; 45 cycles of 5 s at 60 °C and 10 s at 72 °C; and 30 s at 65 °C. This experiment was performed in triplicate. Real-time PCR was performed using a 10 µL total volume that contained 1 µL of cDNA, 2 µL of ddH₂O, 5 µL of SYBR Green Real-time PCR Master Mix (Toyobo, Japan), 1 µL of forward primer and 1 µL of reverse primer. GAPDH was used for quantity and quality control using the forward primer of human GAPDH-QF and the reverse primer of human GAPDH-QR. The primer sequences in detail used in this study are shown in Additional file 1: Table S1. Data were analyzed using the formula R = 2^{−[ΔCt sample − ΔCt control]}, where R is the relative expression level, ΔCt sample is the difference between the Ct of the gene and the average GAPDH in the experiment sample, and ΔCt control is the difference between the Ct of the gene and the average GAPDH in the control sample.

One hundred micrograms of tissue were homogenized in 500 μL of cell lysis buffer (KeyGEN BioTECH, China) and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected as the total protein, and the protein concentrations were determined using the Bradford Protein Assay Kit (Beyotime, China). About 30 μg total proteins for each sample was loaded and separated by 12.5% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a 0.22 μm aperture polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The PVDF membrane was then incubated with blocking buffer (5% skim milk powder dissolved in TBS) for about 1 h at room temperature. After washed thrice with TBST (10 mM Tris–HCl, pH of 7.5, 150 mM NaCl, and 0.05% Tween-20), the PVDF membrane was then incubated with primary antibody (1:1000 in Primary Antibody Dilution Buffer (Beyotime, China)) for 16 h or overnight at 4 °C. After washed thrice with TBST, the PVDF membrane was then incubated with HRP-conjugated secondary antibody (1:5000 in blocking buffer) for about 1 h at room temperature. The PVDF membrane was then washed thrice with TBST, and the acquisition of enhanced chemiluminescence (ECL) images was carried out with the Typhoon Trio System (GE Healthcare, USA).

The PADI3 sequence was obtained from GenBank (https://​www.​ncbi.​nlm.​nih.​gov/​genbank/​). Sequence analysis was performed using BLASTX software (http://​www.​ncbi.​nlm.​nih.​gov/​). Gene translation and protein prediction were performed using ExPASy (http://​www.​au.​expasy.​org/​).

Cell proliferation assays were performed using a Cell Counting Kit-8 (Dojindo, Japan). The recombinant plasmids were transfected into SW620 or HCT116 cells using PolyJet™ DNA In Vitro Transfection Reagent (SignaGen, USA) according to the manufacturer’s instructions. After culturing for 12 h, the cell culture medium was discarded, and 100 μL of fresh complete culture medium (containing 10% FBS and a 1% penicillin/streptomycin solution) was added and separately cultured for another 12 h, 24 h or 48 h at 37 °C with 5% CO₂. Then, the CCK-8 solution (Dojindo, Japan) at a concentration of 10 μL/well was added into the cell culture medium, and culturing was continued for another 2 h. The absorbance was measured with a spectrophotometer at 450 nm (SpectraMax 190, Molecular Device, USA). Graphs showing cell growth were generated from the average values of five wells in each group, and data were obtained from 3 independent experiments.

The full coding sequence of CKS1 was inserted into Ubi-CKS1-3FLAG-CBh-gcGFP-IRES-Puro lentiviral vectors containing the GFP reporter gene (Genechem, Shanghai, China). Recombinant Ubi-CKS1-3FLAG-CBh-gcGFP-IRES-Puro viruses were transfected into HCT116 cells and screened with 2 μg/mL puromycin. Ubi-MCS-3FLAG-CBh-gcGFP-IRES-Puro was transfected into HCT116 cells as the control group. Twelve BALB/c nude mice at 6 weeks old (Vital River, China) were randomly divided into two groups to establish the tumor-bearing mouse model. Two hundred microliters of CKS1-expressing HCT116 cells (10⁷/mL in phosphate-buffered saline (PBS)) were injected into the dorsal flank of each mouse in the experimental group, and 200 µL of GFP-expressing HCT116 cells (10⁷/mL in phosphate-buffered saline (PBS)) were injected into the dorsal flank of each mouse in the control group. Tumors were dissected at 42 days after cell injection; the size and weight of the tumors were measured by routine methods. This experiment was repeated three times independently.

To fully study the function of CKS1 in colon cancer, the expression profile of it was examined using western blot and qRT-PCR in colon cancer tissues and their corresponding adjacent tissues which were obtained from 12 different patients. Results showed that there was only a little expression of CKS1 in the adjacent tissues. However, a high expression level of CKS1 was detected in the corresponding colon cancer tissues both in translational level (Fig. 1a, b) and in transcriptional level (Fig. 1c). This finding suggests that CKS1 mainly expressed in colon cancer tissues and may play an important role in the tumorigenesis of colon cancer.

To investigate the role of CKS1 in colon cancer, CKS1 was transfected into HCT116 cells and SW620 cells to study the proliferation ratio and colony formation activity. RFP was transfected into HCT116 cells and SW620 cells, separately, as the controls. The results showed that in both the HCT116 cells and SW620 cells, CKS1-overexpressing cells had a higher cell proliferation activity (Fig. 2a, b) and colony formation ability (Fig. 2c, d) than the control groups. These results indicate that CKS1 may take part in tumorigenesis of colon cancer via promoting cell proliferation and colony formation in vitro.

To further study the function of CKS1 in colon cancer, lentiviral-coated CKS1 was transfected into HCT116 cells, and this CKS1-overexpressing HCT116 cells were injected into 6-week-old BALB/c nude mice, whereas GFP-overexpressing HCT116 cells was used to inject 6-week-old BALB/c nude mice as the control group. Results showed that lentiviral-coated CKS1 was transfected into HCT116 cells successfully (Fig. 3a), and this CKS1-expressing HCT116 cells can significantly promote tumor growth in vivo (Fig. 3b, c), these larger tumor tissues obtained from experiment group has a high expression level of CKS1 than tissues obtained from control group (Fig. 3d). This finding indicates that high level of CKS1 can promote colon cancer tumor growth in vivo.

Earlier reports have shown that the Hsp90 inhibitor 17-AAG can suppress CKS1, CDK1 expression, promote p27^kip1 expression and lead to G1-phase arrest in colon cancer cells. Our previous study found that PADI3 can inhibit cell proliferation via inducing G1-phase arrest. According to that, we speculate that PADI3 may play its anti-tumor role via affect Hsp90, CKS1, CDK1 and p27^kip1 expression in colon cancer.

To verify these results, we detected the expression pattern of PADI3, Hsp90, CKS1, CDK1 and p27^kip1 in colon cancer tissues and their corresponding adjacent tissues, results showed that both PADI3 and p27^kip1 had lower expression level in colon cancer tissues than in the corresponding adjacent tissues. In contrast, Hsp90, CKS1 and CDK1 had higher expression level in colon cancer tissues than in the corresponding adjacent tissues (Fig. 4a, b). Further study found that 17-AAG can suppress CKS1 and CDK1 expression and promote p27kip1 expression in HCT116 cells, which agree with the earlier study (Fig. 4c, d).

To determine whether PADI3 has a similar function as 17-AAG, we transfected HCT116 cells with PADI3-overexpressing plasmids and measured the expression levels of Hsp90, CDK1, CKS1 and p27^kip1. Results showed that PADI3 functions similar to 17-AAG, which can inhibit Hsp90, CDK1, CKS1 expression and promote p27^kip1 expression (Fig. 4e, f) These results indicate that PADI3 may be a promising inhibitor of both Hsp90 and CKS1.

To verify the function of PADI3 in CKS1-induced tumorigenesis in vitro, cell proliferation and colony formation activity were measured. The results showed that the transfection of the PADI3 overexpression plasmid into lentivirus-transfected CKS1-stably expressed HCT116 cells blocked CKS1 expression-induced colony formation (Fig. 5a, b)and cell proliferation (Fig. 5c), which is similar to the function of 17-AAG. These results indicate that PADI3 can block CKS1 overexpression induced colon cancer cell colony formation and proliferation ability in vitro.

To further study the molecular mechanism of PADI3 in regulating CKS1, we transfected HCT116 cells with a lentivirus-coated PADI3-expressing plasmid and screened using puromycin (2 μg/mL) to establish a PADI3-stably expressing HCT116 cell line. The Hsp90-expressing plasmid was then transfected into the PADI3-stably expressing HCT116 cells. Results showed that overexpression of Hsp90 can practically recover the PADI3-affected CKS1, CDK1 and p27^kip1 expression both in translational level (Fig. 6a) and in transcriptional level (Fig. 6b). CCK-8 analysis was also performed to verify the results, which showed that the overexpression of Hsp90 in PADI3-stably expressing HCT116 cells can overcome PADI3-suppressed cell proliferation (Fig. 6c).

CKS1 is essential for cell proliferation via regulating the cell cycle transition from the G1 to the S phase [24], plays an important role in tumorigenesis as an oncogene, and previous research suggests that inhibiting CKS1 expression may be an excellent strategy for cancer therapy [7, 25]. An increasing number of studies have found that CKS1 is highly expressed in various malignant tumors. However, the function and molecular mechanism of CKS1 in colon cancer is still unclear. In this study, we found that CKS1 has a higher expression level in colon cancer tissues than in their corresponding adjacent tissues. In vitro, the overexpression of CKS1 can promote cell proliferation and colony formation in both SW620 cells and HCT116 cells. In vivo, CKS1-overexpressing HCT116 cells injected into BALB/c nude mice led to larger tumors than those in the controls, and these results agree with the results of previous studies which showed that CKS1 contributes to tumorigenesis in colon cancer [26, 27]. The suppression of CKS1 expression may be a beneficial strategy for colon cancer therapy.

Hsp90 is well known as an oncogene, and a high expression level of Hsp90 is associated with decreased survival and poor prognosis in various cancers [28‐30]. Inhibiting Hsp90 expression has been considered to be a promising antitumor treatment [31‐33]. Previous studies found that inhibitors of Hsp90 can block cell proliferation, but the molecular mechanism is still unclear. In this study, we found that 17-AAG, an Hsp90 inhibitor, can suppress CKS1 and CDK1 expression and promote p27^kip1 expression. CKS1, CDK1, and p27^kip1 are essential for cell cycle control and cell proliferation [34, 35]. Thus, inhibit Hsp90 expression can downregulate CKS1 expression and lead to the suppression of cell proliferation in colon cancer cells.

The discovery of Hsp90 and CKS1 inhibitors with low toxicity and high efficiency is essential for colon cancer therapy. Our previous study showed that PADI3 plays an antitumor role in colon cancer by suppressing cell proliferation and cell colony formation, but the molecular mechanism is still unclear. In this study, we found that PADI3 exerts its antitumor activity by suppressing Hsp90 and CKS1 expression, and Hsp90 is essential for PADI3 to suppress CKS1 expression. Although the key functional domain and sites of PADI3 in this process require further exploration in future studies.