The International Journal of Biochemistry & Cell Biology 43 (2011) 682–690 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel Neural cell adhesion molecule potentiates invasion and metastasis of melanoma cells through CAMP-dependent protein kinase and phosphatidylinositol 3-kinase pathways Yu Shi a , Rui Liu a , Si Zhang a , Yin-Yan Xia a , Hai-Jie Yang a,b , Ke Guo b , Qi Zeng b , Zhi-Wei Feng a,∗ a School of Biological Sciences, Nanyang Technological University, #60 Nanyang Drive, Singapore 637551, Singapore b Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore article info Article history: Received 27 November 2010 Received in revised form 18 January 2011 Accepted 19 January 2011 Available online 26 January 2011 Keywords: NCAM Melanoma Metastasis cAMP-dependent protein kinase Phosphatidylinositol 3-kinase abstract Neural cell adhesion molecule (NCAM) has been implicated in tumor metastasis yet its function in melanoma progression remains unclear. Here, we demonstrate that stably silencing NCAM expression in mouse melanoma B16F0 cells perturbs their cellular invasion and metastatic dissemination in vivo. The pro-invasive function of NCAM is exerted via dual mechanisms involving both cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways. Pharmacologic inhibition of PKA and PI3K leads to impaired cellular invasion. In contrast, forced expression of constitutively activated Akt, the major downstream target of PI3K, restores the defective cellular invasiveness of NCAM knock-down (KD) B16F0 cells. Furthermore, attenuation of either PKA or Akt activity in NCAM KD cells is shown to affect their common downstream target, transcription factor cAMP response element binding protein (CREB), which in turn down-regulates mRNA expression of matrix metalloproteinase-2 (MMP-2), thus contributes to impaired cellular invasion and metastasis of melanoma cells. Together, these findings indi- cate that NCAM potentiates cellular invasion and metastasis of melanoma cells through stimulation of PKA and PI3K signaling pathways thus suggesting the potential implication of anti-NCAM strategy in melanoma treatment. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Melanoma arises from the malignant transformation of neural-crest derived pigment-producing cells, melanocytes (Gray- Schopfer et al., 2007). As the leading cause of skin cancer death owing to its highly metastatic nature, melanoma incidence has been increasing at an alarming rate (Gray-Schopfer et al., 2007; Hocker et al., 2008). Though early stage thin lesion of melanoma can be cured by surgical resection, once diagnosed as metastatic phase, it is always fatal, largely refractory to existing therapies with 5-year survival rate of less than 5% (Gray-Schopfer et al., 2007). Therefore, in-depth understanding of the mechanism underlying the complex metastatic process is imperative and would be of great help for novel regime development. On progression to advanced metastatic stage, cancer cells undergo multiple molecular alterations to get equipped to invade neighboring tissues, infiltrate into vascular and lymphatic ves- sels, extravasate and eventually colonize at distant organs (Gupta and Massague, 2006). At molecular level, such changes mainly ∗ Corresponding author. Tel.: +65 65138270; fax: +65 67913856. E-mail address: ZWFeng@ntu.edu.sg (Z.-W. Feng). involve cell adhesion molecules, proteases and proteins regulating actin cytoskeleton reorganization. Ample evidences have demon- strated the pivotal role of cell adhesion molecules belonging to integrin, cadherin or immunoglobulin superfamily in metastatic cascade (McGary et al., 2002). This can be exemplified by neural cell adhesion molecule (NCAM), a membrane bond glycopro- tein belonging to the immunoglobulin superfamily. All NCAM family members come from a single gene via alternative splic- ing. Based on their carboxy-terminal domains, NCAM isoforms can be grouped into three major NCAM classes: the 120 kDa glycosylphosphatidylinositol-linked isoforms and two transmem- brane isoforms of 140 and 180 kDa (Cunningham et al., 1987). Post-translational modification further complicates the struc- ture of NCAM by addition of -2,8-linked sialic acid residues which could alter the functional properties of NCAM protein dur- ing synaptic plasticity and axon pathfinding (Weinhold et al., 2005). NCAM was initially characterized in cells from central nervous system where it modulates intercellular adhesion, neurite out- growth and cell migration. However, studies in various human cancer biopsies as well as mouse tumor models have revealed the importance of NCAM in the progression to tumor malig- nancy (Zecchini and Cavallaro, 2008). For instance, an overall 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.01.016