RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion
Introduction
It is estimated by the NCI SEER that 44,250 American men and 32,000 American women were diagnosed with invasive melanoma of the skin in 2012 and 9180 men and women died of this disease that year. Even with the development of more effective treatments [1], [2], [3], metastatic melanoma is still associated with a poor prognosis [4], [5]. Transcription factors, traditionally considered undruggable, have become the focus of new targeting strategies in melanoma and other cancer types [6], [7]. There is increasing evidence that transcription factors play oncogenic roles in melanoma and this has driven efforts to develop new approaches to target this class of proteins [8].
The RUNX (Runt-related transcription factor) genes comprise a family of three closely related transcription factors, RUNX1, RUNX2 and RUNX3. These genes are defined by a highly conserved 128 amino acid DNA binding/protein–protein interaction domain known as the Runt box [9]. Knock out models have implicated RUNX2 in cartilage and bone development [10], [11]. Some evidence also points to a role for RUNX2 specifically in bone metastasis in advanced breast and prostate cancer. However, the role of RUNX2 in promoting tumor development in hematopoietic lineages and in breast and prostate cancer extends beyond its pro-bone metastatic effects. RUNX2 regulates the expression of genes intimately associated with tumor progression, invasion and metastasis. These genes include osteopontin, bone sialoprotein, collagenases, and FAK/PTK2 [9], [12], [13], [14]. In addition, the pro-angiogenic effects of RUNX2 suggest a major role for this transcription factor in tumor promotion. These effects include endothelial cell proliferation, migration and invasion [15], [16], induction of VEGF expression and physical and functional interactions between RUNX2 and another major pro-angiogenic factor, hypoxia-inducible factor 1-a (HIF1-a) [17], [18].
In one study, two melanoma cell lines showed coexpression of RUNX2 and Bone Sialoprotein, whose expression in vivo correlated with local and regional melanoma spread [19]. Another study indicated that TGFβ, driving metastasis at advanced melanoma stages [20], up regulated RUNX2 expression in the 1205LU melanoma cell line [21]. In addition, the tumor suppressor p14ARF was reported to repress RUNX2 expression in melanoma cell lines. In this study, the authors speculated that increased RUNX2 resulting from p14ARF mutation might contribute to melanoma development [22]. As a first approach to studying the role of RUNX2 in melanoma development, we determined that RUNX2 was overexpressed in melanoma cell lines as compared with primary cultures of melanocytes or an immortalized melanocyte cell line. ShRNA-mediated knock down of RUNX2 in melanoma cell lines negatively affected cell growth and inhibited their migration and invasion in conjunction with a reduction in the levels of the kinase FAK/PTK2 involved in motility and adhesion. The RUNX2 DNA binding inhibitor Cholecalciferol [23] inhibited the activity of the RUNX2-responsive MMP13 promoter, and also decreased melanoma cell growth and their ability to migrate. Furthermore, we addressed the relevance of RUNX2 expression to human melanomagenesis using a melanoma tissue microarray and confirmed overexpression of RUNX2 in melanoma specimens as compared with benign nevi.
Section snippets
Cell lines
WM1552C, WM9, WM1617, WM793, WM278, and 1205LU were kindly provided by Dr. M. Herlyn (Wistar Institute, Philadelphia, PA, USA [24]). These lines were cultured in MCDB153/L-15 (4/1 ratio) medium containing 2% FBS, 5 μg/ml Insulin and 1.7 mM Calcium Chloride. C8161 melanoma cell line was provided by Dr. Mary Hendrix (Children’s Memorial Research Center, Chicago, IL, USA [25] and was grown in D-MEM (Mediatech, 10-013-CV) containing 10% FBS. UACC903 cells were provided by Dr. Jeffrey M. Trent
Overexpression and high activity of RUNX2 in melanoma cell lines
As a first step to address the possible role of RUNX2 in melanoma pathogenesis, RUNX2 mRNA expression was first assessed in two independent primary cultures of human melanocytes and six different human melanoma cell lines using real time PCR. The results showed overexpression of RUNX2 mRNA in the melanoma cell lines as compared with melanocytes (Fig. 1A). By immunoblot, we tested RUNX2 expression in three different primary cultures of melanocytes and in the immortalized melanocyte cell line
Discussion
In the present study, we examined for the first time the role of RUNX2 in melanoma pathogenesis. We demonstrated that RUNX2 mRNA and protein are overexpressed in melanoma cell lines as compared with normal melanocytes. Interestingly, radial growth phase (such as WM35 and WM1552c), vertical growth phase (such as WM793, WM278) and metastases (such as 1205LU, C8161)-derived cell lines had comparable levels of RUNX2, suggesting that RUNX2 is not only involved in the metastatic process, but possibly
Conflict of Interest
No potential conflicts of interest were disclosed.
Acknowledgements
We thank Dr. A. Passaniti and Dr. D. Medina for helpful discussions. We thank Lei Cong and the Tissue Analytical Services at the Rutgers Cancer institute of New Jersey for the immunohistochemical staining of the melanoma tissue microarray slide. We thank Seung-Shick Shin for lysates from nevi and melanoma metastases obtained in an IRB-approved and HIPAA-compliant fashion. We are grateful to Dr.Vasudeva Ginjala for his help on immunofluorescence, to Saurabh Laddha for his help on TCGA analysis
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