Interleukin 1β-mediated HOXC10 Overexpression Promotes Hepatocellular Carcinoma Metastasis by Upregulating PDPK1 and VASP

Rationale: Metastasis and recurrence are the primary reasons for the high mortality rate of human hepatocellular carcinoma (HCC) patients. However, the exact mechanism underlying HCC metastasis remains unclear. The Homeobox (HOX) family proteins, which are a highly conserved transcription factor superfamily, play important roles in cancer metastasis. Here, we report a novel role of HOXC10, one of the most upregulated HOX genes in human HCC tissues, in promoting HCC metastasis. Methods: The expression of HOXC10 and its functional targets was detected by immunohistochemistry in two independent human HCC cohorts. Luciferase reporter and chromatin immunoprecipitation assays were used to measure the transcriptional regulation of target genes by HOXC10. The effect of HOXC10-mediated invasion and metastasis were analyzed by Transwell assays and by an orthotopic metastasis model. Results: Elevated expression of HOXC10 was positively correlated with the loss of tumor encapsulation and with higher tumor-nodule-metastasis (TNM) stage and poor prognosis in human HCC. Overexpression of HOXC10 promoted HCC metastasis by upregulating metastasis-related genes, including 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and vasodilator-stimulated phosphoprotein (VASP). Knockdown of PDPK1 and VASP inhibited HOXC10-enhanced HCC metastasis, whereas upregulation of PDPK1 and VASP rescued the decreased metastasis induced by HOXC10 knockdown. Interleukin-1β (IL-1β), which is the ligand of IL-1R1, upregulated HOXC10 expression through the c-Jun NH2-terminal kinase (JNK)/c-Jun pathway. HOXC10 knockdown significantly reduced IL-1β-mediated HCC metastasis. Furthermore, Anakinra, a specific antagonist of IL-1R1, inhibited IL-1β-induced HOXC10 upregulation and HCC metastasis. In human HCC tissues, HOXC10 expression was positively correlated with PDPK1, VASP and IL-1R1 expression, and patients with positive coexpression of HOXC10/PDPK1, HOXC10/VASP or IL-1R1/HOXC10 exhibited the poorest prognosis. Conclusions: Upregulated HOXC10 induced by IL-1β promotes HCC metastasis by transactivating PDPK1 and VASP expression. Thus, our study implicates HOXC10 as a prognostic biomarker, and targeting this pathway may be a promising therapeutic option for the clinical prevention of HCC metastasis.

Concentrated lentivirus was transfected into the HCC cells with a multiplicity of infection (MOI) ranging from 20 to 50 in the presence of polybrene (5 μg/ml).
Seventy-two hours after infection, HCC cells were selected for 2 weeks using 2.5 μg/ml puromycin (OriGene). Selected pools of knockdown and over-expressing cells were used for the following experiments.

In vitro invasion and migration assay
For the migration and invasion assay, a 24 well chamber with 8-μm pore filter (Corning corporation, USA) was used. For migration assay, 5×10 5 cells were seeded into the upper chamber in serum-free medium. For invasion assay, 5×10 5 cell were implanted in the top chamber with Matrigel (Corning corporation, USA). After 24-48 hours, the cells were fixed with 95% ethanol and stained with crystal violet. The mean of triplicate assays for each experimental condition was used.

Animal experiment
All animal studies were approved by the Committee on the Use of Live Animals in Teaching and Research, Fourth Military Medical University. Five-weeks-old BALB/C male nude mice were raised in specific pathogen-free conditions in accord with the institutional guidelines for animal care. For in vivo metastasis assay, human luciferase labeled HCC cells (4.0×10 6 ) in the 100 μl of phosphate-buffered saline that were mixed with 100 μl matrigel were injected into the right lobes of livers of the nude mice under anesthesia (10 for each group). The in vivo tumor formation and metastases were monitored using the bioluminescence. For in vivo signal detection, D-luciferin (Perkin-Elmer) at 100 mg/kg was injected intraperitonially into the nude mice. Bioluminescent images were captured using an IVIS 100 Imaging System (Xenogeny). At the 9 weeks, the mice were sacrificed and the livers and lungs were collected and underwent histological examination.

Real-time PCR
Total RNA was extracted using TRIzol Reagent (Invitrogen), and reverse transcription was performed using the Advantage RT-for-PCR Kit (Takara) according to the manufacturer's instructions. For the real-time PCR analysis, aliquots of double-stranded cDNA were amplified using a SYBR Green PCR Kit (Applied Biosystems). The cycling parameters were as follows: 95°C for 15 s, 55-60°C for 15 s, and 72°C for 15 s for 45 cycles. A melting curve analysis was then performed. The Ct was measured during the exponential amplification phase, and the amplification plots were analyzed using SDS 1.9.1 software (Applied Biosystems). For the cell lines, the relative expression levels (defined as the fold change) of the target genes were determined by the following equation: 2 -ΔΔCt (ΔCt = ΔCt target -ΔCt GAPDH , ΔΔCt = ΔCt expressing vector -ΔCt control vector ). The expression level was normalized to the fold change that was detected in the corresponding control cells, which was defined as 1.0.
For the clinical tissue samples, the fold change of the target gene was determined by the following equation: 2 -ΔΔCt (ΔΔCt = ΔCt tumor -ΔCt nontumor ). This value was normalized to the average fold change in the normal liver tissues, which was defined as 1.0. All reactions were performed in duplicate. The primer sequences are listed in Supplementary Table S5.

Luciferase reporter assays
To examine the signaling pathways regulated by HOXC10 and the downstream modulated by HOXC10, the luciferase activity was performed by the Dual Luciferase Assay (Promega, Madison, WI), following to the manufacturer's instructions. Then, the transfected cell was lysed in culture dishes which containing a lysis buffer, and the resulting lysates were centrifuged for 1 minute at maximum speed in an Eppendorf microcentrifuge. Relative luciferase activity (RLA) was analyzed by TD20/20 Luminometer (Turner Biosystems, Sunnyvale, CA), and the transfection efficiencies were normalized according to Rinella activity.