ODAM protein expression has been demonstrated in a wide range of normal odontogenic, glandular, and epithelial renewal tissues [10–13] as well as in malignancies including odontogenic tumors, gastric cancer, breast cancer, lung cancer, and melanoma [14–16]. Prior retrospective studies of breast cancer patient biopsies indicated an increase in ODAM expression localized to the cell nucleus associated with advancing disease stage, yet this expression corresponded with improved survival for patients at each stage . A recent study of melanoma patient specimens indicated that nuclear ODAM-expression correlates with sentinel lymph node metastasis in over 70% of cases, indicative of higher stage melanoma at diagnosis and poor prognosis requiring more aggressive therapeutic intervention [2, 19]. These studies have left the role of ODAM in malignancy unclear since, in both breast cancer and melanoma, nuclear ODAM localization corresponds with advancing disease stage yet its influence on disease outcome seemingly differs.
With respect to cellular functions of ODAM, those indicated in ameloblasts are varied, and include an extracellular role at the cell-tooth interface in the junctional epithelium, roles in enamel maturation, and in the response to peridontal disruption [31, 32]. ODAM is secreted [13, 33] yet may also have a role in the cell nucleus regulating matrix metalloproteinase expression via direct chromatin binding . ODAM has thus been suggested to be a matricellular protein exhibiting functions at cellular junctions, in cell signaling, and in direct gene activation . Our previous studies indicated that ectopic ODAM expression in MDA-MB-231 breast cancer cells led to suppression of tumorigenic properties in vitro and in murine tumor models . When the A375 and C8161 human melanoma cell lines were transfected with a gene construct encoding ODAM, their cellular properties were affected in a fashion similar to our studies in MDA-MB-231 cells. Specifically, their growth rate, and migratory ability was decreased and this was associated with increased cell matrix adhesion and morphologic/cytoskeletal rearrangement.
The most significant finding in our studies is the marked suppression of AKT phosphorylation/activation upon ectopic ODAM expression in both melanoma and breast cancer cell lines (Figures 3 and 5). Further, this inhibition of AKT activation was associated with elevated expression levels of PTEN protein, a negative regulator of AKT activation with an essential tumor suppressive role in multiple tissues [35–38]. Dysregulated, active PI3K/AKT/mTOR signaling promotes cell proliferation and survival, and is found in a wide range of tumor types, including melanoma . PTEN expression is frequently absent or decreased in melanoma and many other cancers [40–43], with loss occurring through mutation, deletion, epigenetic silencing, and loss of heterozygocity [44, 45]. The attendant activation of AKT, often in association with ß-catenin stabilization and MAPK activation, serves as a primary driver of growth and metastasis in these tumors .
Knockout mouse studies have demonstrated the tumor suppressive role of PTEN in multiple tissues, and indicate that PTEN function is gene-dosage dependent, as subtle changes in PTEN protein expression level yield significant functional consequences in terms of tumor growth and progression [46, 47]. In each of the melanoma cell lines the increase in PTEN subsequent to ODAM expression was sufficient that AKT activation was profoundly inhibited, and was recovered upon specific silencing of PTEN expression (Figure 4). Accordingly, cell growth and AKT activity were unaffected by ODAM in BT-549 cells that lack PTEN.
As to the mechanism(s) of increased PTEN expression our studies indicate that this corresponds with increased levels of PTEN mRNA in ODAM expressing cells, and likely an increase in de novo protein synthesis (Figure 3). Regulation of PTEN expression is, however, highly complex, mediated at transcription in part by p53 . Further, PTEN protein levels are regulated posttranslationally by ubiquitin-mediated proteasomal degradation elicited by the E3 ubiquitin ligase activities of NEDD4 (neural precursor cell expressed developmentally downregulated protein 4–1), XIAP (X-linked inhibitor of apoptosis protein), and others [49, 50]. PTEN stability and function are further regulated through phosphorylation by casein kinase 2 (CK2), RhoA-associated kinase (RAK), GSK3ß and others [51–53], as well as by direct protein interactions with P-REX2a  and a host of other proteins [45, 55]. Further studies addressing transcriptional regulation of the PTEN gene, PTEN protein stability, and function will be required to fully define the modes of PTEN regulation with respect to ODAM expression and effects on AKT activation.
In a parallel to our observations, overexpression of the matricellular protein SPARC (secreted protein acidic and rich in cysteine) inhibits growth  and migration  of MDA-MB-231 cells, and yields elevated PTEN and growth suppression in neuroblastoma cells . SPARC is the ancestral gene of the SPARCL1 (SPARC-like 1 gene) which is, in turn, the putative progenitor of those in the secretory calcium phosphoprotein (SCPP) gene cluster on human chromosome 4 (at 4q 13.3) which includes ODAM, the α/ß and κ caseins, and FDC-SP (Follicular Dendritic Cell-Secretory Protein) [59, 60]. Matricellular proteins can modulate tumor cell proliferation positively, or negatively, through a variety of mechanisms . SPARC has been reported to function as a tumor suppressor in neuroblastoma, breast, pancreatic, lung and ovarian cancers, yet SPARC is associated with highly aggressive tumor phenotypes in melanomas and gliomas [62–64]. In notable similarity to ODAM action SPARC modulates cell-cell, and cell-matrix interactions, elicits cellular adhesive signaling, and exhibits differential nuclear localization dependent on cellular status [63, 65, 66].
In studies again similar to our observations, overexpression of the Profilin-1 actin-binding protein in MDA-MB-231 cells yields growth suppression and decreased tumorigenicity [67–69]. This is associated with inhibition of AKT activity dependent on elevated PTEN, and with altered cell motility, actin rearrangement, and increased formation of adherens junctions.