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. 2013 Jan 3;32(1):97-105.
doi: 10.1038/onc.2012.24. Epub 2012 Feb 13.

CXCL13 activation of c-Myc induces RANK ligand expression in stromal/preosteoblast cells in the oral squamous cell carcinoma tumor-bone microenvironment

Y Sambandam  1 , K Sundaram, A Liu, K L Kirkwood, W L Ries, S V Reddy
Affiliations

CXCL13 activation of c-Myc induces RANK ligand expression in stromal/preosteoblast cells in the oral squamous cell carcinoma tumor-bone microenvironment

Y Sambandam et al. Oncogene. .

Abstract

CXC chemokine ligand-13 (CXCL13) has been implicated in oral squamous cell carcinoma (OSCC) tumor progression and osteolysis. The tumor necrosis factor family member RANKL (receptor activator of NF-κB ligand), a critical bone resorbing osteoclastogenic factor, has an important role in cancer invasion of bone/osteolysis. Here, we show high-level expression of CXCL13 in primary human OSCC tumor specimens; however, human bone marrow-derived stromal (SAKA-T) and murine preosteoblast (MC3T3-E1) cells produce at very low level. Recombinant CXCL13 (0-15 ng/ml) dose dependently induced CXCR5 expression in SAKA-T and MC3T3-E1 cells. Conditioned media obtained from OSCC cell lines increased the RANKL expression and an antibody against the CXCL13 specific receptor, CXCR5 markedly decreased RANKL expression in these cells. Furthermore, CXCL13 increased hRANKL-Luc promoter activity. Superarray screening identified c-Myc and NFATc3 transcription factors upregulated in CXCL13-stimulated SAKA-T cells. Immunohistochemical analysis of OSCC tumors that developed in athymic mice demonstrated RANKL and NFATc3 expression in tumor and osteoblast cells, however, showed p-c-Myc expression specific to osteoblastic cells at the tumor-bone interface. We further identified NFATc3 expression, but not c-Myc activation in primary human OSCC tumor specimens compared with adjacent normal tissue. Also, CXCL13 significantly increased p-ERK1/2 in SAKA-T and MC3T3-E1 cells. siRNA suppression of c-Myc expression markedly decreased CXCL13-induced RANKL and NFATc3 expression in preosteoblast cells. Chromatin-immuno precipitation assay confirmed p-c-Myc binding to the hRANKL promoter region. In summary, c-Myc activation through CXCL13-CXCR5 signaling axis stimulates RANKL expression in stromal/preosteoblast cells. Thus, our results implicate CXCL13 as a potential therapeutic target to prevent OSCC invasion of bone/osteolysis.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest with this work.

Figures

Figure 1
Figure 1
CXCL13 expression in OSCC, stromal/preosteoblast cells. (A) Representative sections showing immunohistochemical staining for CXCL13 expression in OSCC tumor specimens and control adjacent normal tissues from human subjects. (B) CXCL13 levels in serum free conditioned media (CM) obtained from SCC-14a, human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast cells (MC3T3-E1) as measured by ELISA. (C) CXCL13 stimulation of CXCR5 receptor expression in bone marrow stromal/preosteoblast cells. SAKA-T and MC3T3-E1 cells were treated with recombinant CXCL13 (15 ng/ml) for 0–6 h and the total cell lysates were analyzed for CXCR5 expression by Western blot. β-actin expression served as control. Data represent three replicate studies (*p<0.05).
Figure 2
Figure 2
CXCL13 stimulation of RANKL expression in stromal/preosteoblast cells. (A-i) Real time RT-PCR analysis for RANKL mRNA expression in MC3T3-E1 preosteoblast cells stimulated with CM (0–30%) obtained from different OSCC cell lines (SCC-1, SCC-11B and SCC-14a). (A-ii) SCC-14a cell CM stimulates RANKL expression in stromal/preosteoblast cells. Human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast cells (MC3T3-E1) were stimulated with SCC-14a CM (20%) or recombinant CXCL13 (15 ng/ml) in the presence and absence of anti-CXCR5 antibody (10 μg/ml) for 6 h. The total cell lysates were subjected to Western blot analysis for RANKL expression. (B) CXCL13 stimulation of RANKL mRNA expression in SAKA-T and MC3T3-E1 cells. Cells were stimulated with CXCL13 (15 ng/ml) and total RNA isolated from these cells were analyzed for RANKL mRNA expression by Real-time RT-PCR. The relative mRNA expression was normalized with respect to GAPDH amplification. The values are expressed as mean ± SD (*p<0.05). (C) CXCL13 dose-dependent stimulation of RANKL expression in SAKA-T, MC3T3-E1 and mouse bone marrow derived primary stromal cells. Cells were treated with different concentrations (0–15 ng/ml) of CXCL13 for 6 h and total cell lysates were analyzed for RANKL expression by Western blot. β-actin expression serves as control. Data shown are representative of three replicate studies.
Figure 3
Figure 3
CXCL13 transcriptional control of RANKL expression in human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. (A) SAKA-T and (B) MC3T3-E1 cells were transiently transfected with hRANKL-luc P#3 plasmid by lipofectamine and cultured with CXCL13 (0–15 ng/ml) for 6 h. Cells mock-transfected with empty vector (EV) served as control. Total cell lysates prepared were assayed for luciferase activity. (C) MC3T3-E1 cells were treated with different concentrations of recombinant CXCL13 (0–20 ng/ml) for 6 h. Total cell lysates obtained were analyzed for c-Myc, NFATc3 and RANKL expression by Western blot. (D) CXCL13 activation of c-Myc in SAKA-T and MC3T3-E1 cells. Cells were treated with CXCL13 (15 ng/ml) for 0–60 min and total cell lysates obtained were analyzed for p-c-Myc by Western blot. β-actin expression served as control. Data represent three replicate studies and mean ± SD (*p<0.05).
Figure 4
Figure 4
RANKL, p-c-Myc, NFATc3 expression in OSCC tumor-bone microenvironment. (A) OSCC tumor progression in athymic mice. NCr-nu/nu athymic mice were subcutaneously injected with SCC-11B cells (7×106) with or without stable RANKL-Luc reporter gene construct in PBS over calvaria. Tumors were allowed to grow for five weeks and imaged the calvarial region of mice facing up towards the CCD (charge-coupled device) camera using IVIS imaging system at 20 min after luciferin (150 mg/kg, i.p). (B) Immuno-histochemical analysis of RANKL, NFATc3, p-c-Myc expression and alkaline phosphatase (ALP) activity at the tumor-bone interface in athymic mice. (C) Immunohistochemical analysis of p-c-Myc and NFATc3 expression in primary human OSCC tumor specimens and adjacent normal tissues. Data shown are representative of three replicate studies.
Figure 5
Figure 5
CXCL13 activation of c-Myc expression induces RANKL expression. (A) CXCL13 activation of ERK1/2 in human bone marrow stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. Cells were stimulated with CXCL13 (15 ng/ml) for different time points (0–60 min) and total cell lysates were subjected to Western blot analysis for ERK1/2 and pERK1/2 expression. (B) siRNA suppression of NFATc3 inhibits RANKL but not c-Myc expression and (C) siRNA suppression of c-Myc inhibits RANKL and NFATc3 expression in MC3T3-E1 cells. Cells were transiently transfected with double stranded siRNA against c-Myc or NFATc3 using oligofectamine. After 48 h, cells were treated with CXCL13 (15 ng/ml) for 6 h and total cell lysates were analyzed by Western blot for RANKL, c-Myc and NFATc3 expression. β-actin expression serves as control. Data shown are representative of three independent experiments.
Figure 6
Figure 6
CXCL13 induces c-Myc activation in human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. (A) MC3T3-E1 cells were stimulated with CXCL13 (15 ng/ml) for 6 h and c-Myc nuclear translocation was analyzed by confocal microscopy. (B) ChIP assay for c-Myc binding to hRANKL promoter region. SAKA-T cells were stimulated with and without CXCL13 (15 ng/ml) for 6 h and ChIP assay for c-Myc binding to hRANKL promoter was performed as described in methods. (C) Quantitative real-time PCR analysis of chromatin immune complexes for p-c-Myc binding to RANKL promoter region. The DNA amplification was normalized with respect to input. Data represent triplicate studies and mean ± SD (*p<0.05).
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References

    1. Mao L, Hong WK, Papadimitrakopoulou VA. Focus on head and neck cancer. Cancer Cell. 2004;5:311–316. - PubMed
    1. Funk GF, Karnell LH, Robinson RA, Zhen WK, Trask DK, Hoffman HT. Presentation, treatment, and outcome of oral cavity cancer: a National Cancer Data Base report. Head Neck. 2002;24:165–180. - PubMed
    1. Choi S, Myers JN. Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy. J Dent Res. 2008;87:14–32. - PubMed
    1. Nomura T, Shibahara T, Katakura A, Matsubara S, Takano N. Establishment of a murine model of bone invasion by oral squamous cell carcinoma. Oral Oncol. 2007;43:257–262. - PubMed
    1. Mishra A, Bharti AC, Varghese P, Saluja D, Das BC. Differential expression and activation of NF-kappaB family proteins during oral carcinogenesis: Role of high risk human papillomavirus infection. Int J Cancer. 2006;119:2840–2850. - PubMed

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