CXCL12-mediated HOXB5 overexpression facilitates Colorectal Cancer metastasis through transactivating CXCR4 and ITGB3

Background: Metastasis is the major reason for the high mortality of colorectal cancer (CRC). However, the molecular mechanism underlying CRC metastasis remains unclear. Here, we report a novel role of homeobox B5 (HOXB5), a member of the HOX family, in promoting CRC metastasis. Method: The expression of HOXB5 and its target genes were examined by immunohistochemistry in human CRC. Chromatin immunoprecipitation and luciferase reporter assays were performed to measure the transcriptional regulation of target genes by HOXB5. The metastatic capacities of CRC cells were evaluated by in vivo lung and liver metastatic models. Results: The elevated expression of HOXB5 was positively correlated with distant metastasis, higher AJCC stage, and poor prognosis in CRC patients. HOXB5 expression was an independent and significant risk factor for the recurrence and survival in CRC patients. Overexpression of HOXB5 promoted CRC metastasis by transactivating metastatic related genes, C-X-C motif chemokine receptor 4 (CXCR4) and integrin subunit beta 3 (ITGB3). C-X-C motif chemokine ligand 12 (CXCL12), which is the ligand of CXCR4, upregulated HOXB5 expression through the extracellular regulated protein kinase (ERK)/ETS proto-oncogene 1, transcription factor (ETS1) pathway. The knockdown of HOXB5 decreased CXCL12-enhanced CRC metastasis. Furthermore, AMD3100, a specific CXCR4 inhibitor, significantly suppressed HOXB5-mediated CRC metastasis. HOXB5 expression was positively correlated with CXCR4 and ITGB3 expression in human CRC tissues, and patients with positive co-expression of HOXB5/CXCR4, or HOXB5/ITGB3 exhibited the worst prognosis. Conclusion: Our study implicates HOXB5 as a prognostic biomarker in CRC, and defines a CXCL12-HOXB5-CXCR4 positive feedback loop that plays an important role in promoting CRC metastasis.

ITGB3 and CXCL12 genes were constructed in PLKO.1-TRC (Addgene) and designated as LV-HOXB5, LV-CXCR4, LV-ITGB3 and LV-CXCL12. An empty vector was used as the negative control and was designated as LV-control. The lentivirus and cell infection were produced according to the pLKO.1 lentiviral vector protocol recommended by Addgene. Briefly, the lentiviral plasmid and packaging plasmids pMD2. G and psPAX2 (Addgene plasmid #12259 and #12260) were transfected into HEK-293T cells with transfection reagent (Lipofectamine®3000, Thermo Fisher Scientific) and OPTI-MEM media (Invitrogen, Waltham, MA, USA).
The lentiviruses were harvested twice on days 4 and 5. Viruses were filtered with a 0.45-μm filter and stored at -80 °C. Lentiviral infection of target cells was performed in cell culture media with 5 μg/ml polybrene (Sigma H9268). Seventy-two hours after infection, cells were selected for 2 weeks using 2.5 μg/ml puromycin (OriGene).
Selected pools of cells were used for the following experiments.

Transient transfection
A total of 1×10 5 serum-starved cells were plated in each well of a 24-well plate and allowed to attach for 12-24 hours. Then, a mixture of Lipofectamine 3000 (Invitrogen, USA) containing 0.02 μg of the pRL-TK plasmids, 0.18 μg of the promoter reporter plasmids and 0.6 μg of the expression vector plasmids was used to cotransfect cells for 5 hours based on the manufacturer's instructions. The cells were then washed and incubated with 1% FBS-supplemented fresh medium for 48 hours.

Luciferase reporter assay
The Dual Luciferase Assay (Promega, USA) was used to quantify luciferase activity following the manufacturer's instructions. Transfected cells were subjected to cell lysis in a culture dish with lysis buffer. Subsequent lysates were transferred to an Eppendorf microcentrifuge before being centrifuged for 1 minute at maximum speed.
The efficiency of transfection was normalized to Renilla activity, and relative luciferase activity was quantified with a Modulus TM TD20/20 Luminometer (Turner Biosystems, USA).

In vitro migration and invasion assays
The invasive and migratory capabilities of each cell line were assessed with an 8-µm pore, 24-well Transwell plate (Corning, USA). For invasion assays, chamber inserts were first coated with 60 μL of Matrigel (Corning, 200 mg/mL) and left to dry overnight under sterile conditions. The next day, the uppermost chamber was plated at a cell density of 1×10 5 . For cell migration assays, the upper chamber, which was lined with a noncoated membrane, was plated with cells at a density of 5×10 4 . Each assay was repeated thrice, and three different inserts were used to obtain a mean cell number in five fields per membrane.

Western blot analyses
Proteins from lysed cells were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with 5% milk in TBST (120 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20) for 1 hour at room temperature. Blots were incubated with a specific antibody overnight at 4 °C.
Western blotting of β-actin on the same membrane was used as a loading control. The membranes were incubated with primary antibodies overnight at 4 °C. The membranes were then washed with PBS 3 times and incubated with an HRP-conjugated secondary antibody. Proteins were visualized using a Immobilon TM Western Chemiluminescent HRP substrate(Millipore, USA).
The primary antibodies used are listed below.

Antibodies
Source Concentration  Table S4 lists all sequences of all primers used.

CCK-8 assay
For the CCK-8 assay, cells were seeded into 96-well plates at a density of 1000 cells in 100 μl of complete medium per well. At each time point, the original medium was replaced with CCK-8 solution (TransDetect Cell Counting Kit, Transgene, Beijing, China) and complete medium mixed at a 1:9 ratio, and the cells were then incubated at 37 °C for 2 h. The absorbance of each sample was recorded at 450 nm using a microplate reader (Tecan Group, Ltd, Zürich, Switzerland) and each sample was measured three times.

Colony formation assay
Transfected cells (1000 per well) were cultured in 6-well plates. After 14 days of culture, the cells formed stable colonies. The cell colonies were fixed with 4% paraformaldehyde for 10 minutes and then stained with a crystal violet solution for 10 minutes. Colonies containing more than 50 cells were counted and each group included three replicates.

In vivo tumor growth in the xenograft model
Six-week-old BALB/C nude mice were cared for and maintained based on our institution's protocols for ethical animal care. The Committee on the Use of Live

Animals in Teaching and Research (CULATR) of the Fourth Military Medical
University approved all animal experiments. For the in vivo growth assay, suspended treated cells were subcutaneously injected into the flank of each mouse (6 mice per group, 1 × 10 6 cells in 150 μl of PBS per mouse). The mice were weighed and the tumor size was measured using vernier calipers. The tumor volume was calculated using the following equation: V (mm 3 ) = 0.5 × L (mm) × W 2 (mm 2 ). After 4 weeks, all mice were sacrificed. Then, tumor weight measured.

Quantification and Statistical Analysis
The quantitative data were compared between groups using the Student's t-test.
Categorical data were analyzed using the Fisher's exact test. The cumulative recurrence and survival rates were determined using the Kaplan-Meier method and log-rank test. The Cox proportional hazards model was used to determine the independent factors that influence survival and recurrence based on the variables that had been selected from the univariate analyses. A value of p < 0.05 was considered to be significant. All the analyses were performed using the SPSS software (version 19.0). (I) Schematic overview of experimental processing was shown. AMD3100 or vehicle (3 mg/kg) was administered intraperitoneally to the nude mice every two days from day 8 after implantation to day 42.

References
All the data are shown as the mean ± s.d. * P < 0.05 ** P ˂ 0.01