Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

The CSCC tissues were confirmed by histopathological examination and immunostaining for specific markers (Additional file 1: Fig.S1A). Bioinformatics analysis identified 5140 upregulated genes in the CSCC samples, including NOD1 and NOD2 (Additional file 1: Fig.S1B). Preliminary RNA-Seq analysis confirmed that NOD1 and NOD2 were upregulated in the CSCC relative to normal cervix tissues (Fig. 1A), which was further confirmed by RT-PCR (Fig. 1B) and analysis of TCGA data (Fig. 1C). Consistent with this, the NOD1 and NOD2 protein levels were significantly higher in the CSCC tissues compared to the paired adjacent normal cervix tissues (Fig. 1D–F). Furthermore, both NOD1 and NOD2 were overexpressed in embolic tumor cells resulting from lymph-vascular space invasion (LVSI) compared to the primary tumors without LVSI (P < 0.05, Fig. 1G), whereas significantly higher expression of NOD1 was detected in the CSCC tissues of patients with lymph node metastasis (LM) relative to the non-LM samples (P < 0.05, Fig. 1H). Although the tumor stage was not associated with NOD1/NOD2 expression, both were overexpressed in tumors with poorer differentiation (Additional file 1: Fig.S1C and S1D). In clinic practice, LVSI and LM were risk factors for un-promising survival trend. In agreement with the above findings, TCGA data showed that overexpression of NOD1 predicted a worse prognosis in CSCC patients (Fig. 1I, left panel), whereas NOD2 expression level did not show significantly correlation with the overall survival (Fig. 1I, right panel). Interestingly, while the NOD1 and NOD2 mRNA levels showed a positive correlation (Additional file 1: Fig.S1E); the TCGA data showed that both overexpression of NOD1 and NOD2 predicted a worse prognosis trend in CSCC patients but not reached significance. These results maybe according to the small cohort group (Additional file 1: Fig.S1F). Consistent with the patient samples, NOD1/NOD2 mRNA and protein levels were intrinsically expressed in the CSCC cell lines (Fig. 1J) and primary CSCC cells (Fig. 1K). In conclusion, NOD1 may play an important role in the progression of CSCC patients.

The primary cells were purified and identified as previously described (Additional file 2: Fig.S2A). NOD1 and NOD2 were stably upregulated in the primary cells and cell lines with Tri-DAP and MDP stimulation, respectively (Additional file 2: Fig.S2B). In addition, the cell lines were transduced with NOD1/2-overexpressing lentiviruses (Additional file 2: Fig.S2C). Ectopic expression of NOD1 or NOD2 significantly enhanced the colony-forming potential of the CSCC cell lines (Fig. 2A, P < 0.05), which was consistent with their increased viability and proliferation rates in Siha cells (Fig. 2B). On the other hand, the NOD1 and NOD2 ligands only slightly enhanced the proliferative capacity of the CSCC cells (Fig. 2C, D). Consistent with the above, a significantly higher proportion of Siha/LV-NOD1/NOD2 cells were observed in the S phase compared to the Siha/LV-ctrl cells (Fig. 2E, F; P < 0.01). Likewise, the Siha/LV-NOD1 and Siha/LV-NOD2 cells resulted in significantly larger tumors in vivo compared to the control cells (Fig. 2G).

The impact of NOD1 and NOD2 expression on the metastatic potential of CSCC cells was analyzed by in vitro and in vivo assays [28]. NOD1 and NOD2 overexpression significantly increased the extent of wound closure, as well the number of cells that migrated or invaded into the bottom surface of the transwell insert membranes (Fig. 3A–C, Additional file 3: Fig.S3A). The metastatic effect of NOD1/2 was also verified on the primary CSCC cells (Fig. 3D). Furthermore, the number of pulmonary metastatic nodules was markedly higher in the mice injected intravenously with Siha/LV-NOD1/NOD2 cells as opposed to the control Siha cells (Fig. 3E). Although the weight of the tumor-bearing mice was similar across the three groups (Additional file 3: Fig.S3B), the animals harboring Siha/LV-NOD1 or Siha/LV-NOD2 tumors had worse survival rates (Fig. 3F). Taken together, NOD1 and NOD2 significantly promoted CSCC proliferation by accelerating transition into the S phase of the cell cycle, and increased their metastatic potential.

The mechanisms underlying the oncogenic effects of NOD1/NOD2 were further elucidated via pharmacological inhibition with ML-130 as well as siRNA-mediated gene knockdown (Additional file 4: Fig.S4). Inhibition of NOD1 or NOD2 significantly decreased the proliferative capacity (Fig. 4A, B), and the migration and invasion rates of Siha/LV-NOD1 or Siha/LV-NOD2 respectively (Fig. 4C) compared to the vehicle controls. GO and KEGG enrichment analyses further showed that the genes and proteins correlated with the upregulation of NOD1 or NOD2 were involved in cell proliferation, cytokines, and pathways in cancer such as ERK, NF-κB, and IL-8 (Fig. 4D–G). As shown in Fig. 4F, the ERK and NF-κB pathway proteins were also upregulated in the LV-NOD1 and LV-NOD2 cells. The quantity of cytokine in-cell array indicated that IL-6 and IL-8 were upregulated by NOD1 or NOD2 increasing (Fig. 4G upper panels); however, only significant higher IL-8 secretion by Siha/LV-NOD1 or Siha/LV-NOD2 was identified (Fig. 4G, down panels). NOD1/NOD2 increased the secretion of IL-8 but not of IL-6 (Fig. 4G, down panels), which was abrogated by their respective siRNAs (Fig. 5A, left panels) as well as ML-130 (Fig. 5A, right panels) and the NF-κB inhibitor (Fig. 5B). In addition, the proliferation ability of Siha/LV-NOD1 or Siha/LV-NOD2 was significantly inhibited by selective inhibitors of the IL-8 receptor CXCR1/2 (Reparixin), NF-κB (EVP4593), or ERK (SCH772984) (Fig. 5C), and the combination of all three showed a cumulative inhibitory effect (Fig. 5C). Reparixin and EVP4593 also decreased the metastatic potential of Siha/LV-NOD1 and Siha/LV-NOD2 cells (Fig. 5D, E).

The enhanced IL-8 secretion by NOD1/NOD2-overexpressing Siha cells upregulated the adhesion molecule FN1 (Additional file 5: Fig.S5A), and knocking down FN1 inhibited metastasis of Siha/LV-NOD1 and Siha/LV-NOD2 cells (Additional file 5: Fig.S5B). In addition, the elevated FN1 in Siha/LV-NOD1 or Siha/LV-NOD2 cells was downregulated by Reparixin (Additional file 5C). To summarize, the oncogenic effects of NOD1 and NOD2 in CSCC are mediated through multiple pathways; and knocking down either NOD1 or NOD2 in Siha cells downregulated P65/p-P65and ERK/p-ERK significantly (Fig. 5F, G).

Consistent with the in vitro findings, Reparixin or EVP4593 markedly decreased the volume and weight (Fig. 6A, B) of tumors derived from mice with subcutaneous Siha/LV-NOD1 or Siha/LV-NOD2 cells compared to the untreated controls. Reparixin significantly improved the OS of mice bearing Siha/LV-NOD1 tumors subcutaneously (Fig. 6C). In the metastasis models induced by tail vein injection with Siha/LV-NOD1 or Siha/LV-NOD2 cells, Reparixin inhibited the growth of metastatic nodules compared to the placebo controls (Fig. 6D). Finally, Reparixin significantly improved the OS of mice with metastatic Siha/LV-NOD1 nodule xenografts (Fig. 6E). Taken together, the NOD1/ NF-κB/IL-8 axis is a promising therapeutic target in CSCC.

Specimen collection and clinicopathological data review were approved by the Ethics Committee of Cancer Hospital, CAMS (Chinese Academy of Medical Sciences & Peking Union Medical College). This study was performed in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS), and none of the procedures conducted in this study interfered with the treatment plan of the patients. CSCC tissues were collected during surgery or biopsies conducted between Oct 2017 and Dec 2019 at the Cancer Hospital, after obtaining consent from the patients. Totally, fifty-eight CSCC samples and thirty-three normal cervix tissue samples were used for the quantitative real-time PCR (qPCR) assay, and 143 CSCC samples were collected for immunohistochemistry (IHC). Sixteen tumor samples were used for primary cell isolation and subsequent assays.

The NOD1 and NOD2 expression data of CSCC patients was extracted from the Human Protein Atlas (http://​www.​proteinatlas.​org), and the mRNA expression and survival data from The Cancer Genome Atlas (TCGA) databases. The transcriptome sequencing (differential expression genes, DEG) of CSCC and normal cervical tissue (from Cancer Hospital) were identified by BGISEQ platform and analyzed on the DR. TOM network platform of BGI (https://​biosys.​bgi.​com/​#/​report/​login). The sequencing reads which contain low-quality, adaptor-polluted, and high content of unknown base (N) reads should be processed to be removed before downstream analyses. After sequencing data filtering, DEG level was calculated for each sample with RSEM [57]. The target genes were functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and the significant biological processes, cellular components, and molecular functions were identified.

The human CSCC cell lines Siha, CasKi, and C33a were all purchased from Cell Resource Center (Beijing, China). The cell lines were verified by short tandem repeat (STR) sequencing by the Beijing Microread Genetics Company on July 2018. The cells were cultured in DMEM/F12 medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, USA) and 1% penicillin/streptomycin (PS) (Gibco, Thermo Fisher Scientific, USA) at 37 °C under 5% CO₂ (Thermo Technologies, Vancouver, Japan). The cells were treated with Tri-DAP, MDP (InvivoGen, USA), ML-130 (TargetMol, USA), CXCR1/2 inhibitor (Reparixin, Med Chem Express, USA), NF-κB inhibitor (EVP4593, Sellect, USA), or ERK inhibitor (SCH772984, Sellect, USA) as required. Primary CSCC cells were isolated from patient samples as previously described [58]. Briefly, the specimens were minced into 1-mm³ pieces in 6-cm petri dishes, and sequentially digested with 0.05% trypsin containing EDTA (Lonza, Walkersville, MD, USA) and 0.2% type I collagenase (Sigma-Aldrich Corp., St Louis, MO, USA) at 37 °C with constant shaking. FBS was added to terminate the reaction, and the cells were washed and re-suspended in DMEM/F12 complete medium with 5% FBS (Gibco). The primary cells were seeded in a petri dish and cultured for 7–10 days.

Trypsinized primary CSCC cells were re-suspended in MACS (magnetic-activated cell sorting) separation buffer and incubated with anti-EpCAM magnetic microbeads (Miltenyi Biotec Inc, Auburn, CA, USA) according to the manufacturer’s instructions. Then, the EpCAM-positive cells were collected and cultured in high-glucose DMEM medium with FBS (5%). Flow cytometry (FCM) was used to identify the purity of the CSCCs immediately after cell sorting or a short period of culture: Purified primary cells were stained with fluorescent-conjugated antibodies against anti-human EpCAM FITC (BioLegend Inc., San Diego, CA, USA) and anti-human vimentin PE (Miltenyi Biotec Inc, Auburn, CA, USA) on ice. Since vimentin was the cellular marker used, the cells were pretreated with Fixation/Permeabilization reagent (Invitrogen, Carlsbad, CA) according to the protocols recommended by the manufacturer. FCM acquisition was performed using a Beckman coulter-Dxflex flow cytometer. Flow Jo V10 software was used for the data analysis. The purity of the sorted cells reached ∽ 96% purity. After the purification was identified, the cells were collected and cultured for functional examination.

Total RNA was extracted from the cultured cells and frozen tissues using Trizol Reagent (Invitrogen, Carlsbad, CA). The quality and concentration of the RNA were determined using a Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE). The RNA was reverse transcribed to cDNA using a Reverse Transcriptase Kit (Takara, Japan) and amplified by RT-qPCR using Power SYBR Green PCR Master Mix (Life, Applied Biosystems) on the Step One Plus Real-Time PCR System (Life, Applied Biosystems). The target gene expression levels were calculated with the 2^−ΔΔCt method, and each sample was analyzed in triplicates. The primers were synthesized by Sangon Technologies (Shanghai, Corp.) and the sequences were as follows:

The tissue samples were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, cut into 5-μm-thick sections, and coated at 75 °C for 2 h. The sections were deparaffinized using xylene and rehydrated through an ethanol gradient. HE staining was performed as standard protocols. For IHC, the sections were heated in citrate buffer for antigen retrieval, incubated with 2% hydrogen peroxide for quenching endogenous peroxidase, and blocked using 1% goat serum (ZSJQ, Beijing, China). The slices were then incubated overnight with rabbit anti-human vimentin (without diluted, Origene, Beijing, China), mouse anti-human pan-cytokeratin AE1/AE3 (without diluted, Origene, Beijing, China), mouse anti-human Ki67 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human P16 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz, USA), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz, USA) antibodies and the isotype control at 4 °C. After washing with PBST (Phosphate Buffer Solution with Tween-20), the sections were sequentially stained with DAB chromogen (diaminobezidin, ZSJQ, Beijing, China) and hematoxylin (Sigma-Aldrich, USA). NOD1 and NOD2 expression parameters were scored by Image Pro Plus software (USA).

The cells cultured on chamber slides were fixed with cold 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were incubated overnight with rabbit anti-human vimentin (diluted 1:100, Abcam, Cambridge, MA, USA), mouse anti-human AE1/AE3 (diluted 1:100, ZSGB-BIO, Beijing, China), rabbit anti-human CDKN2A/p16INK4a (P16) (diluted 1:200, Abcam), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz) primary antibodies. The slides were then incubated with Alexa Fluor-conjugated secondary antibodies (Abcam) for 1 h at room temperature and counterstained with DAPI (4′,6-diamidino-2-phenylindole, Cat.H3570, Life Technologies). The stained tissues and cells were viewed using Olympus scanner or laser scanning confocal microscope (LSM780; Zeiss), and the staining intensity was evaluated by a pathologist blinded to the samples using image plus software (USA).

Human NOD1 (BC040339.1) and NOD2 (NM022162.2) were respectively cloned into the pLVX-P2A-ZsGreen-T2A-Puro vector at the XhoI and BamHI sites. The lentivirus with pLVX-hNOD1/hNOD2-ZsGreen-Puro was purchased and packaged as per the manufacturer’s description (Likeli Biotec Inc, Beijing, China USA). The CSCC cell lines (Siha, CasKi and C33a) were transduced with the NOD1/NOD2 or empty vector lentiviruses and selected using puromycin. NOD1/2 overexpression was verified by western blotting.

NOD1, NOD2, FN1, and scrambled siRNAs were synthesized by JTS Scientific Company (Beijing, China). The sequences are as follows:

The Siha/LV-NOD1, Siha/LV-NOD2, CasKi/LV-NOD1, and CasKi /LV-NOD2 cell lines were grown till 70% confluency and transfected with the respective siRNAs using Lipofectamine™ 2000 (Invitrogen, Thermo Fisher Scientific). Briefly, siRNA and 1 μl Lipofectamine™ 2000 was respectively diluted in 50 μl Opti-MEM, incubated for 15 min at room temperature, and then mixed. The mixture was incubated for 15 min at room temperature and added to each well. The cells were incubated at 37 °C for 24 h, and the transfection efficacy was tested.

The cultured cells were harvested for cytokine array and western blotting, and the supernatants were collected for ELISA. The cytokine levels were analyzed with the G-Series Human Cytokine Antibody Array 440 as per the manufacturer’s instructions (Ray Biotech Inc. Quantibody service, China). Western blotting was performed using standard protocols after the cells were lysed in RIPA buffer (Sigma, Saint Louis, MO) [59]. The following primary antibodies were used: β-actin (AC-15; 1:2000; Sigma), NOD1 (mouse anti-human, B-4; 1:500; sc-398696, Santa Cruz), NOD2 (mouse anti-human, 2D9; 1:500; sc-56168, Santa Cruz), P65 (rabbit anti-human, D14E12, 1:1000; CST, USA), p-P65 (rabbit anti-human, 93H1, 1:1000; CST, USA), P44/42 MAPK (ERK1/2) (rabbit anti-human, 1:1000; CST, USA), and pP44/42 MAPK (pERK1/2) (Thr202/Tyr204, rabbit anti-human, 1:1000, CST, USA). The IL-8 and IL-6 levels in the supernatants were quantified using LEGEND MAX™ Human IL-8 and Human IL-6 ELISA Kits (Biolegend, USA) according to the manufacturer’s instructions.

Cells were seeded in 6-cm petri dishes at the logarithmic phase of growth, and harvested after 24, 48, 72, 96, and 120 h, respectively. The number of cells was recorded using a cell counter II (Life Corp, USA). The cells were seeded into 96-well plates for the Cell Counting Kit-8 (CCK8) assay (Dojindo Laboratories, Japan), and 10 μl CCK8 solution was added per well at 24, 48, and 72 h of culture. The optical density at 450 nm (OD.450) was measured using a Model 680 Microplate Reader (BIO-RAD, Hercules, CA). Five replicates were tested per sample.

The transfected cells were seeded into 12-well plates and cultured for 24 h. After fixing in cold 4% paraformaldehyde (PFA) for 15 min and permeabilizing with 0.1% Triton X-100 for 15 min, the cells were incubated with EdU (5-Ethynyl-2'-deoxyuridine) (Beyotime Biotechnology, China) for proliferation. The cells were then washed thrice with PBS, counterstained with DAPI (Beyotime Biotechnology, China), and observed under a laser scanning confocal microscope (LSM780; Zeiss). For FCM, the cells were harvested, washed sequentially with citrate buffer and PBS, and incubated with ribonuclease (RNAase) and propidium iodide (PI) (Ref. CYT-PIR-25, Cytognos, Spain) at room temperature for 1 h. The stained cells were acquired in a flow cytometer (Beckman coulter-Dxflex) and analyzed by the Flow Jo V10 software.

The primary cells and cell lines were seeded in six-well plates at the respective densities of 800 cells/well and 500 cells/well. After culturing for 10–14 days, the cells were fixed with cold 4% PFA and stained with crystal violet (Solarbio, Beijing, China).

Siha/LV-Ctrl/NOD1/NOD2 and CasKi/LV-Ctrl/NOD1/NOD2 cells were seeded into 6-well plates at the density of 1 × 10⁶ cells/well in complete DMEM/F12 (10% FBS). After 24 h of culture, the monolayer was scratched using a 10-μl pipette tip, and the wound region was measured at 0, 6, 12, and 24 h under a microscope. The wound healing rate at the different time points was quantified as the width of the wound region relative to the initial width at 0 h.

The different cell lines and primary cells were seeded into transwell chambers (Costar, Cambridge, MA) at the respective densities of 3–5 × 10⁴ cells/well and 0.5–1 × 10⁵ cells/well in 200 μl serum-free DMEM/F12. The lower chambers were filled with 600 μl complete DMEM/F12 (10% FBS). After 20–24 h of incubation, the cells that had migrated through the membrane were fixed and stained, and counted in four randomly chosen fields. The invasive capacity of the cells was similarly analyzed using Matrigel-coated (BD Biosciences, San Jose, CA, USA) transwell membranes.

All animal experiments were approved by the Beijing Municipal Science and Technology Commission, and conducted in accordance with the relevant guidelines. The xenograft model was established by subcutaneously inoculating 8-week-old BALB/c nude mice or 7–8 weeks NOD/SCID mice with 3 × 10⁶ Siha/LV-Ctrl/NOD1/NOD2 cells and bilaterally. Palpable tumors (> 3 mm) appeared 7 days after injection and were measured every 3 days. The mice were euthanized 28–56 days post-inoculation, and the tumors were removed and weighed. The lung metastasis model was established by intravenously injecting 8–9-week-old NOD/SCID mice with 1 × 10⁶ Siha/LV-Ctrl/NOD1/NOD2 cells. Metastatic growth in the lungs was detected by labeling with luciferase or GEP. The mice were sacrificed 56–84 days, and the number of metastatic nodules was counted. For the treatment regimen, the tumor-bearing mice were divided into the placebo control, Reparixin, and EVP-4395 groups, and the tumor growth was monitored as described above.

Statistical analysis was performed using SPSS 19.0 software, and GraphPad Prism 5.0 software was used for plotting graphs. Quantitative variables between two groups were compared by Student’s t test (normal distribution) or Mann-Whitney U test (non-normal distribution), and one-way or two-way ANOVA was used for comparing multiple groups. Pearson χ² test or Fisher’s exact test was used to compare qualitative variables. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. P values of < 0.05 were considered statistically significant.