E6/E7-P53-POU2F1-CTHRC1 axis promotes cervical cancer metastasis and activates Wnt/PCP pathway

Cervical cancer is an infectious cancer and the most common gynecologic cancer worldwide. E6/E7, the early genes of the high-risk mucosal human papillomavirus type, play key roles in the carcinogenic process of cervical cancer. However, little was known about its roles in modulating tumor microenvironment, particular extracellular matrix (ECM). In this study, we found that E6/E7 could regulate multiple ECM proteins, especially collagen triple helix repeat containing 1 (CTHRC1). CTHRC1 is highly expressed in cervical cancer tissue and serum and closely correlated with clinicopathological parameters. CTHRC1 promotes cervical cancer cell migration and invasion in vitro and metastasis in vivo. E6/E7 regulates the expression of CTHRC1 in cervical cancer by E6/E7-p53-POU2F1 (POU class 2 homeobox 1) axis. Futhermore, CTHRC1 activates Wnt/PCP signaling pathway. Take together, E6/E7-p53-POU2F1-CTHRC1 axis promotes cervical cancer cell invasion and metastasis and may act as a potential therapeutic target for interventions against cervical cancer invasion and metastasis.

maintained at 37 °C in an incubator with a 5% CO2 atmosphere.

Whole-genome microarray construction
A whole-genome microarray of silencing E6/E7-group and control group in Caski and Ms751 cell lines were done in Shanghai BoHao Biotechnology Co. Ltd (Hao Biotechnology Co, Shanghai, China). according to manufacturer's protocol. In brief, 1.65μg Cy3-labeled cRNA from two biological replicates was hybridized to using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US). After 17 hours, slides were washed in staining dishes

Immunohistochemical staining
Paraffin embedded tissue samples were cut into 4-μm-thick sections slides, then were stained with primary antibodies for CTHRC1 antibody (mouse monoclonal antibody, 1:100 dilution, HuaAn Biotechnology, Hangzhou, China) overnight at 4°C, All the steps of the immunohistochemical staining were performed according to standard procedures as described. After treatment with diaminobenzidine and counterstaining with hematoxylin, all the sections were observed and photographed with a microscope (Axio Imager: Carl Zeiss). Scoring was conducted according to the ratio and intensity of positive-staining cells: 0-5% scored 0; 6-35% scored 1; 36-70% scored 2; more than 70% scored 3.
The final score of CTHRC1 expression was designated as low or high expression group as follows: low expression: score 0-1; high expression: score 2-3. All the scores ofCTHRC1 expression were done in a blinded manner and determined independently by two senior pathologists.

Cell Viability Assay
Cell viability was detected using a standard Cell CountingKit-8 assay. Control and sh-CTHRC1, Lenti-CTHRC1 cervical cancer cells were seeded into 96-well plates (100μl per well) at a density of 2000 cells per well with 100μl of complete culture medium. Each group contains five wells. 10μl Cell Counting Kit-8 (CCK-8, WST-8, Dojindo, Japan) solution was added to each well after 0 h, 24 h, 48 h, 72 h and 96 h, respectively. In viable cells, CCK8 was metabolized to produce a colorimetric dye that is detected at 450 nm using a microplate reader. The experiment was repeated three times, the optical density was measured using microplate reader at a wavelength of 450nm.

In Vitro Migration and Invasion Assays
For the transwell migration assay, 20000 Control and sh-CTHRC1, Lenti-vector and Lenti-CTHRC1 cervical cancer cells were placed on the top chamber of each insert with the noncoated membrane (Millicell). Cells were trypsinized and resuspended in medium and 700-900μl medium supplemented with 10% fetal bovine serum and Siha cells were added rCTHRC1 protein followed gradient doses of 0 nM, 10 nM, 1000 nM respectively were injected into the lower chamber. After 24 hours for Cevical cancer cells in the migration assays, any cells remaining in the top chambers or on the upper membrane of the inserts were carefully removed. After fixation and staining in a dye solution containing 0.1% crystal violet and 20% methanol, cells adhering to the lower membrane of the inserts were counted and imaged through an IX71 inverted microscope (Olympus Corp. Tokyo, Japan). We carried out invasion assay by adding 100μl matrigel (BD Bioscience, Franklin Lakes, NJ) into top chamber of transwell and placed 8×10^4 Control and sh-CTHRC1,Lenti-CTHRC1 cervical cancer cells onto the matrigel. 48 hours later, the transwell for invasion was ceased and staining.

In vivo tumor xenograft model
Six-week-old female nude (nu/nu) mice (SLAC, Shanghai, China) were injected subcutaneously in the right flank with the stable single cell clones of Siha cells at 5×10 6 infected with Lenti-CTHRC1 or Lenti -Vector in 100μl serum-free medium for each nude mouse, Each group contained 6 mice, the tumor weights were measured and recorded. After 6 weeks, mice were sacrificed, and their tumors were dissected, fixed with phosphate-buffered neutral formalin and prepared for standard histologic examination. Mice were manipulated and housed according to protocols approved by the East China Normal University Animal Care Commission.

In vivo pulmonary metastasis model
The stable single cell clones of Siha cells at 1.5 × 10^6 infected with Lenti-CTHRC1 or Lenti -Vector, respectively, were suspended in100 μ l serum-free DMEM/matrigel (1:1) for each nude mouse. These cells were injected intravenously into nude mice (6 in each group, 6 weeks female BALB/c-nu/nu).
After 4 weeks, mice were sacrificed, and their lungs were dissected, fixed with phosphate-buffered neutral formalin and prepared for standard histological examination. Mice were manipulated and housed according to protocols approved by the East China Normal University Animal Care Commission. All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health.

Transcriptional reporter gene assay
Siha and Hela cells were seeded in 96-well plates and transfected with mixture of 100 ng TCF/catenin reporter plasmid (WNT/β-catenin signaling), or 100 ng ATF2 reporter plasmid (WNT/PCP signaling). After 48 hours of incubation, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega, Madison, WI) from the cell lysates.

qChIP-PCR Analysis
Caski cells were subjected to ChIP with the Pierce Agarose ChIP Kit (Thermo).
Briefly, cells were treated with 37% formaldehyde to crosslink proteins, and terminated with 0.125M glycine. After being performed with sonication, chromatinprotein complexes were immunoprecipitated with 5 mg of anti-POU2F1 antibodies (NB100-91899, Novus), TP53 (SAB1306667, SIGMA) antibodies or 1 mg of mouse IgG. Real-time PCR was performed to amplify the regions of interest or internal negative control regions. The primers used for these studies are listed in Supplementary