Interleukin-7 Contributes to the Invasiveness of Prostate Cancer Cells by Promoting Epithelial–Mesenchymal Transition

Precise mechanisms underlying interleukin-7 (IL-7)-mediated tumor invasion remain unclear. Thus, we investigated the role of IL-7 in tumor invasiveness using metastatic prostate cancer PC-3 cell line derivatives, and assessed the potential of IL-7 as a clinical target using a Janus kinase (JAK) inhibitor and an IL-7-blocking antibody. We found that IL-7 stimulated wound-healing migration and invasion of PC-3 cells, increased phosphorylation of signal transducer and activator of transcription 5, Akt, and extracellular signal-regulated kinase. On the other hand, a JAK inhibitor and an IL-7-blocking antibody decreased the invasiveness of PC-3 cells. IL-7 increased tumor sphere formation and expression of epithelial–mesenchymal transition (EMT) markers. Importantly, lentiviral delivery of IL-7Rα to PC-3 cells significantly increased bone metastasis in an experimental murine metastasis model compared to controls. The gene expression profile of human prostate cancer cells from The Cancer Genome Atlas revealed that EMT pathways are strongly associated with prostate cancers that highly express both IL-7 and IL-7Rα. Collectively, these data suggest that IL-7 and/or IL-7Rα are promising targets of inhibiting tumor metastasis.

(100 ng/mL)-stimulated PC-3 cells were treated with M25 at the indicated concentrations for 30 min and then subjected to immunoblot analysis of STAT5 phosphorylation. (B) PC-3 cells were allowed to invade through matrigel (250 µg/mL) for 24 h after treatment with IL-7 (10 ng/mL) in the presence of M25 or isotype control Abs (Iso) (100 µg/mL). Invading cells were stained with crystal violet (left panel), counted using ImageJ software, and plotted as a graph (right panel).

In vitro cell growth assay
Cells were seeded into 96-well plates at a density of 1×10 3 /well in 100 µL and treated with IL-7 (10 ng/mL) for 24 h. MTT assay was performed at 4 h before the end of the incubation; 20 µL MTT solution (5 mg/mL in PBS) were added to each well, and the cells were incubated at 37°C for 4 h. The medium was removed, and 150 µL DMSO were added to each well. The plate was gently rotated on an orbital shaker for 10 min to dissolve the precipitate completely. The absorbance was measured at 560 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

In vivo tumor growth
NOD/SCID/IL-2Rγ-null (NSG) mice were purchased from the Jackson Laboratory and maintained at the animal facility of Seoul National University College of Medicine. Experiments involving mice were approved by the Institutional Animal Care and Use Committee of Seoul National University (authorization no. SNU140716-1-1).
To determine in vivo tumor growth, 6-8-week-old male NSG mice were injected subcutaneously with 5×10 5 cells in 100 µL PBS using a 29-gauge insulin needle. Tumor volume was measured in vivo using an external caliper from 1 week after implantation of the tumor cells.

RT-PCR
For semi-quantitative RT-PCR, total RNA was extracted using TRIzol® (Life Technologies) from cells stimulated with or without IL-7 (10 ng/mL) for the indicated times. cDNA was synthesized from the extracted RNA using the Transcript First Strand cDNA Synthesis Kit (Roche Applied Science). PCR was performed using the AccuPower® PCR PreMix (Bioneer) (see Table S1 for primer sequences) and a conventional PCR System (Bioneer). The amplified PCR product was resolved on an agarose gel stained with ethidium bromide. All samples were first adjusted to contain equal input amounts of GAPDH cDNA for semi-quantitative PCR.

Gelatin zymography
To determine the amounts of MMP2 and MMP9 secreted into the culture medium, zymography was used for semi-quantitative analysis of gelatinase levels.

In vitro Stimulation of androgen receptor blocker
Androgen receptor (AR)-positive human prostate cancer cell lines VCaP and LNCaP-LN3 were obtained from the Korean Cell Line Bank and grown in RPMI complete medium. To examine the IL-7Ra expression in response to an AR blocker, cells were stimulated in the presence of bicalutamide (Sigma-Aldrich) or PBS control for the indicated times. The expression of IL-7Ra was analyzed by quantitative RT-PCR (Applied Biosystems) and flow cytometry (BD Biosciences) by staining cells with Phycoerythrin-conjugated anti-human IL-7Rα Abs (BD Biosciences). The relative gene expression levels of IL7RA were normalized to GAPDH.