Analysis of DEGs

All the procedures of DEGs analysis were performed in R (version 3.6.3) [36]. Data on CRC patients were retrieved from The Cancer Genome Atlas (TCGA) using the TCGAbiolinks package [37]. The GSE39582 dataset [38] was retrieved from the Gene Expression Omnibus (GEO) database. DESeq2 [39] and limma [40] packages were employed to explore DEGs between patients with and without BRAF V600E mutation in TCGA and GSE39582 datasets, respectively. Significantly DEGs with log2 expression fold change (Log2 FC) > 1 or <−1 and adjusted P < 0.05 were sorted based on the adjusted P-value. The common genes from the top-ten upregulated, and top-ten downregulated genes were selected from the two datasets. Multiple linear regression analysis was conducted using the lm function in the R stats package.

Patient samples

Frozen tumor tissues were collected from 44 patients with metastatic CRC who underwent surgery at Zhongshan Hospital Fudan University. There were 7825 patients undergoing surgery for primary tumor of CRC and genetic analysis of BRAF at Zhongshan Hospital Fudan University between June 2015 and December 2018. Among them, formalin-fixed paraffin-embedded (FFPE) tumor tissues of 172 patients with BRAF V600E-mutant CRC were retrospectively collected according to the following inclusion criteria: (1) Patients diagnosed with colorectal adenocarcinoma at Zhongshan Hospital Fudan University between June 2015 and December 2018. (2) Patients who underwent surgery for primary tumor of CRC, and had not received systemic or local anti-tumor treatment before surgery. (3) Patients with BRAF V600E mutation of the primary tumor tissue confirmed via genetic analysis. The exclusion criteria were as follows: (1) Patients with no available FFEP tumor tissue of primary focus. (2) Patients with CRC and other concomitant primary malignant tumors or diagnosed with a hereditary tumor.

Clinical and pathological information was retrieved from the electronic medical records. Postoperative pathological staging for each patient was based on the American Joint Committee on Cancer (AJCC) cancer staging guidelines, 8th edition. The median follow-up time for the retrospective cohort was 32 months. Overall survival (OS) was defined as the time from surgery to death for any reason or the time of the last follow-up.

Immunohistochemistry analysis

Immunohistochemistry (IHC) staining of FFEP slides from patients and xenografts was conducted using an automated system (BenchMark XT, Roche). HPSE antibody (ab85543, Abcam, Cambridge, UK) and p27Kip1 antibody (25614-1-AP, Proteintech, Wuhan, China) at a concentration of 1:100, Ki67 antibody (GB111499, Servicebio, Wuhan, China) 1:200, Cyclin E2 (11935-1-AP, Proteintech) 1:500, Phospho-Akt (Ser473) antibody (4060, CST, MA, USA) 1:50 were used for immunohistochemistry analysis. Two independent pathologists blinded to the study evaluated the staining score of each clinical sample based on the staining extent (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and intensity (0, 0–5%; 1, 6% ~ 25%; 2, 26% ~ 50%; 3, 51% ~ 75%; 4, 75% ~ 100%) of tumor cells. HPSE positive represented more than 5% staining intensity of the tumor cells. Samples with staining extent and intensity scores greater than 1 were denoted as HPSE high, whereas those with scores less than 1 were denoted as HPSE low.

Cell culture

Human embryonic kidney cell line, 293 T and human CRC cell lines, CACO2, COLO320DM, SW480, SW620, COLO205, HT29, and RKO were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). CACO2 and COLO320DM were KRAS/BRAF wild-type cell lines. SW480 and SW620 were KRAS mutant and BRAF wild-type cell lines. COLO205, HT29, and RKO were BRAF V600E-mutant cell lines. 293 T, HT29, and RKO cells were cultured in DMEM/high glucose medium (SH30022.01B, HyClone, Logan, UT, USA) supplemented with 10% FBS (16140071, Gibco, Paisley, UK) and 1% Pen/Strep (16140071, Gibco, Paisley, UK). CACO2 cells were cultured in DMEM/high glucose medium (HyClone) supplemented with 20% FBS (Gibco) and 1% Pen/Strep (Gibco). COLO320DM and COLO205 cells were cultured in RMPI 1640 (HyClone) supplemented with 10% FBS (Gibco) and 1% Pen/Strep (Gibco). 293 T, HT29, RKO, CACO2, COLO320DM, and COLO205 cells were cultured at 37 °C under 5% CO2. SW480 and SW620 cells were cultured in Leibovitz’s L15 medium (11415064, Gibco, Paisley, UK) supplemented with 10% FBS (Gibco) and 1% Pen/Strep (Gibco) at 37 °C without CO2. Short tandem repeat profiling and mycoplasma test were performed for all cell lines to confirm the cell identity and ensure the cells were free from contamination.

Construction of stable transfectants

Plasmids for HPSE silencing were purchased from Genechem (Shanghai, China). Plasmids for CCNE2 overexpression, CDKN1B silencing, and AKT1 overexpression were purchased from Genomeditech (Shanghai, China). Lentivirus was generated using 293 T cells and transduced into cells. Stably transfected cells were selected using puromycin and blasticidin. shRNA targeting sequences for HPSE and CDKN1B were as follows:

shHPSE-1: 5’-TTCCTGAAGGCTGGTGGAGAA-3’,

shHPSE-2: 5’-CTCCGAGAACACTACCAGAAA-3’,

shCDKN1B: 5’-GCAACCGACGATTCTTCTACT-3’.

Colony formation assay

Cells were seeded in six-well plates at 500 cells per well and cultured for 14 days or 1000 cells for 10 days. Colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min. Colonies were then scanned as images and quantified using ImageJ software (Version 1.53a) [41].

Subcutaneous xenograft models

Six-week-old male BALB/c nude mice were purchased from SLAC Laboratory (Shanghai, China). HT29-shHPSE cells and control cells were resuspended in PBS at a density of 1 × 10^7 cells/ml. Cell suspensions (100 μl) were injected subcutaneously into the left axillary area of the nude mice. Twelve mice were randomized into two groups. Mice were monitored daily, and the size of palpable tumors was determined every three days. Tumor volumes were calculated using the formula: π/6 x Length x Width^2. All mice were euthanized when the volume of any of the model mice reached a volume of 1000 mm^3. Tumors were weighed and fixed in 4% paraformaldehyde. FFEP slides of the tumors were prepared for hematoxylin-eosin (HE) and IHC staining. Investigators were blind to the status of the particular mouse during the measurement, dissection, weighing, and photographing of the tumors.

Cell cycle assay

Cells for cell cycle examination were harvested, fixed, and stained with propidium iodide (CCS012, LinkTech, Shanghai, China) according to the manufacturer’s instructions. Flow cytometry was used to explore cell cycle distribution. Results were analyzed using Flowjo software (Version 10.6.2. Ashland, OR: Becton, Dickinson, and Company; 2019).

Heparanase enzyme assay

Heparan degrading enzyme assay kit (MK412, Takara, Shiga, Japan) was used to measure the heparanase enzymatic activity of cells and xenograft tissues following the manufacturer’s instruction.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from frozen CRC tissues and cells using Total RNA Kit I (R6834-02, Omega Bio-Tek, GA, USA) and RNase-Free DNase Set (E1091-02, Omega Bio-Tek, GA, USA) following the manufacturer’s instructions. RNA was reverse transcribed using PrimeScript RT Master Mix (Perfect Real Time) kit (RR036A, Takara, Shiga, Japan). TB Green Premix Ex Taq (Tli RNaseH Plus) kit (RR420A, Takara, Shiga, Japan) was used to perform quantitative PCR. Primers for HPSE and GAPDH were synthesized by Sangon (Shanghai, China) and are listed in Table S4. The 2 (-Delta Delta Ct) method [42] was used for relative quantitative analysis of the expression data using GAPDH as an internal reference gene.

Western blot (WB) analysis

Cells were lysed in SDS lysis buffer (P0013G, Beyotime Biotechnology, Shanghai, China) supplemented with proteinase/phosphatase inhibitor mixture (P1046, Beyotime Biotechnology, Shanghai, China) for protein extraction. BCA kit (P0012, Beyotime Biotechnology, Shanghai, China) was used to determine the total protein concentration. Equal amounts of protein were electrophoresed on 10% SDS–PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 3% BSA solution and incubated with diluted primary antibodies overnight at 4 °C. Further, membranes were washed and incubated with appropriate secondary antibodies for 1 h at room temperature. Protein bands were visualized by an enhanced chemiluminescent method. Antibodies used for WB in this study were as follows: HPSE antibody (ab254254, Abcam), Cell cycle regulation antibody sampler kit (9932, CST), Rb antibody sampler kit (9969, CST), Cyclin E2 antibody (4132, CST), Cyclin D2 antibody (3741, CST), Cyclin D3 antibody (2936, CST), Phospho-Erk1/2 (Thr202/Tyr204) antibody (4370, CST), Erk1/2 antibody (4695, CST), Phospho-Akt (Ser473) antibody (4060, CST), AKT antibody (4691, CST), GAPDH antibody (5174, CST), anti-rabbit IgG, HRP-linked antibody (7074, CST), anti-mouse IgG and HRP-linked antibody (7076, CST). Semi-quantitative analysis of bands was performed using ImageJ software (Version 1.53a) [41].

Statistical analysis

R packages and statistical methods used for each analysis are described in Figure legends. For all experiments, there were at least three independent replicates. Statistical tests were two-sided if applicable. P < 0.05 was considered statistically significant. All statistical analyses and data plotting were performed in RStudio (Version 1.2.1335, R version 3.6.3) [36] and GraphPad Prism software (Version 9.0.0 for Mac OS X, GraphPad Software, San Diego, California USA, www.graphpad.com).