Cancer Letters

Cancer Letters

Volume 348, Issues 1–2, 28 June–1 July 2014, Pages 61-70
Cancer Letters

RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion

https://doi.org/10.1016/j.canlet.2014.03.011Get rights and content

Abstract

In the present study, we investigated the role of the transcription factor RUNX2 in melanomagenesis. We demonstrated that the expression of transcriptionally active RUNX2 was increased in melanoma cell lines as compared with human melanocytes. Using a melanoma tissue microarray, we showed that RUNX2 levels were higher in melanoma cells as compared with nevic melanocytes. RUNX2 knockdown in melanoma cell lines significantly decreased Focal Adhesion Kinase expression, and inhibited their cell growth, migration and invasion ability. Finally, the pro-hormone cholecalciferol reduced RUNX2 transcriptional activity and decreased migration of melanoma cells, further suggesting a role of RUNX2 in melanoma cell migration.

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

References (55)

  • P. Hersey et al.

    A focus on PD-L1 in human melanoma

    Clin. Cancer Res.

    (2013)
  • K.A. Lyseng-Williamson et al.

    Ipilimumab: a guide to its use in advanced melanoma

    Am. J. Clin. Dermatol.

    (2012)
  • P.B. Chapman et al.

    Improved survival with vemurafenib in melanoma with BRAF V600E mutation

    N. Engl. J. Med.

    (2011)
  • G.M. Boyle

    Therapy for metastatic melanoma: an overview and update

    Exp. Rev. Anticancer Ther.

    (2011)
  • R. Siegel et al.

    Cancer treatment and survivorship statistics

    CA Cancer J. Clin.

    (2012)
  • D. Ghosh et al.

    Transcription factor therapeutics: long-shot or lodestone

    Curr. Med. Chem.

    (2005)
  • C. Yan et al.

    Drugging the undruggable: Transcription therapy for cancer

    Biochim. Biophys. Acta.

    (1835)
  • H. Tsao et al.

    Melanoma: from mutations to medicine

    Genes Dev.

    (2012)
  • K. Blyth et al.

    The RUNX genes: gain or loss of function in cancer

    Nat. Rev. Cancer

    (2005)
  • C.W. Chua et al.

    Suppression of androgen-independent prostate cancer cell aggressiveness by FTY720: validating Runx2 as a potential antimetastatic drug screening platform

    Clin. Cancer Res.

    (2009)
  • L. Sun et al.

    Runt-related gene 2 in endothelial cells: inducible expression and specific regulation of cell migration and invasion

    Cancer Res.

    (2001)
  • A.D. Pierce et al.

    Glucose-activated RUNX2 phosphorylation promotes endothelial cell proliferation and an angiogenic phenotype

    J. Cell. Biochem.

    (2012)
  • T.G. Kwon et al.

    Physical and functional interactions between Runx2 and HIF-1alpha induce vascular endothelial growth factor gene expression

    J. Cell. Biochem.

    (2011)
  • M. Riminucci et al.

    Coexpression of bone sialoprotein (BSP) and the pivotal transcriptional regulator of osteogenesis, Cbfa1/Runx2, in malignant melanoma

    Calcif. Tissue Int.

    (2003)
  • A. Lasfar et al.

    Resistance to transforming growth factor beta-mediated tumor suppression in melanoma: are multiple mechanisms in place?

    Carcinogenesis

    (2010)
  • K.S. Mohammad et al.

    TGF-beta-RI kinase inhibitor SD-208 reduces the development and progression of melanoma bone metastases

    Cancer Res.

    (2011)
  • L.M. Packer et al.

    Gene expression profiling in melanoma identifies novel downstream effectors of p14ARF

    Int. J. Cancer

    (2007)
  • Cited by (36)

    • Runt-related transcription factor 2 influences cell adhesion-mediated drug resistance and cell proliferation in B-cell non-Hodgkin's lymphoma and multiple myeloma

      2020, Leukemia Research
      Citation Excerpt :

      In this study, we demonstrated that RUNX2 is expressed in B-NHL and MM cell lines, and that its expression is up-regulated in adherent cells. Previous studies showed that RUNX2 affects migration and invasion by regulating the expression of FAK in melanoma cell lines [24]. In prostate cancer cells, RUNX2 regulates apoptosis by directly controlling the activity of Bcl-2 [7].

    • Transcriptional master regulator analysis in breast cancer genetic networks

      2015, Computational Biology and Chemistry
      Citation Excerpt :

      TFDP3, which was previously commented, is present in 7 out of 10 groups and its dysregulation is linked to evasion of apoptosis. RUNX2 and also FOXJ2 are involved in migration processes (Boregowda et al., 2014; Wang et al., 2014, 2012). HIF3 is involved in the process of angiogenesis (Ando et al., 2013).

    View all citing articles on Scopus
    View full text