ASK-ing EMT not to spread cancer

Global communication by the epithelial cell layer is maintained through gap junctional complexes, separated from adjacent tissues by a basal lamina. Mesenchymal cells are loosely organized in a three-dimensional extracellular matrix, comprising connective tissue adjacent to the stroma. Conversion of epithelial cells to mesenhymal cells involves profound phenotypic changes, including loss of cell-cell adhesion, loss of cell polarity, and the acquisition of migratory and invasive properties (1).

The process of epithelial-mesenchymal transition (EMT) is a critical event during embryonic development, required for morphogenetic movements during gastrulation and organ formation, but also a convenient venue for epithelial-derived tumors to become invasive and rapidly metastasize (2, 3). EMT endows cells with migratory and invasive properties, induces stem cell properties, and prevents apoptosis and senescence, thus orchestrating the initiation of metastasis (3, 4). Loss of epithelial-cell markers (E-cadherin, β-catenin) and gain of mesenchymal-cell markers (N-cadherin, vimentin), at the leading edge or invasive front of solid tumors, is associated with progression to metastasis (3). To preserve cellular shape and polarity, the intracellular domains of cadherins connect to the actin cytoskeleton through α-catenin and β-catenin. It is the signaling activities of mesenchymal cells that facilitate migration and survival in an anchorage-independent environment (2, 3). Thus even if there is no histological evidence of cellular intermediates during the transition, activation of mesenchymal genes in epithelial cells is critical for EMT success.

The contribution of a tumor-suppressor gene’s activity to EMT is a fresh determinant in the EMT landscape. In this issue of PNAS, Xie et al. (5) report a unique player in the mechanistic control of EMT, the tumor suppressor gene DAB2IP (also known as ASK1-interacting protein-1), loss of which facilitates EMT leading to metastasis. The documented function of DAB2IP as a scaffold protein in modulating EMT via GSK-3β-catenin recognizes a different “operating forum” for tumor-suppressor action and raises important questions about the dual role of β-catenin in EMT: Not only does it enhance cell-cell adhesion by associating with E-cadherin complexes in adherent junctions, it also functions as a transcriptional coactivator after interacting with TCF/LEF transcription-factor complexes. Indeed, growing evidence suggests that β-catenin-mediated transcription induces Slug or Twist gene expression that further represses E-cadherin, thus contributing to EMT (6, 7). The primary physiological significance of the article by Xie et al. relates to the observation that restoring DAB2IP in metastatic prostate cancer cells reversed EMT, and loss of this tumor suppressor in human cancer cells leads to distant metastatic spread. The findings show that DAB2IP functions as a scaffold protein in regulating EMT by modulating nuclear β-catenin/TCF activity. Prostate epithelial cells can undergo EMT in response to an array of soluble growth factors, including TGFβ1, EGF, IGF1, β2-microglobulin (β2-m), or exposure to a bone microenvironment (8, 9). TGF-β represents a potent EMT inducer in normal development and tumor progression (8), via Smad-dependent and -independent transcriptional pathways. Smad-mediated induction of Snail, Slug, and Twist via high motility group A2 (HMGA2) (7) and Smad-independent phosphorylation of Par6 contribute to dissolution of cell junction complexes (10, 11) (Fig. 1).

Fig. 1.

EMT regulation in prostate cancer cells. Loss of expression of E-cadherin, a key participant in calcium-dependent interactions which form epithelial adherent junctions, is the principal characteristic of EMT. In response to TGFβ1, Smad-mediated EMT proceeds via induction of Snail, Slug, and Twist, and Smad-independent phosphorylation of Par6 contributes to the dissolution of cell junction complexes via Smurf. Snail binds to E-cadherin promoter and represses its transcription during EMT. E-cadherin repressors induce EMT by regulating expression of genes repressing the epithelial character and promoting the mesenchymal state (AR). DAB12P controls epithelial cell movement and survival, stopping EMT. AJ, adherent junctions.

But how can one argue that the propensity of a tumor suppressor to change the state of movement and shape of the cell in its microenvironment leads to its instant acquisition of a therapeutic value in targeting metastastic prostate cancer? The answer lies in the link between EMT and tumor progression to metastasis. In prostate cancer, androgens can suppress E-cadherin expression and induce mesenchymal marker expression. EMT activation may result in increased bone turnover, implicated in bone colonization in metastatic prostate cancer. Alterations in cytoskeleton reorganization and microfilament organization induced by androgens may enable cell migration and metastasis of the escaped prostate tumor cells (12) by providing active movement, assisting cell migration and the dynamics of interaction with adherent molecules in the ECM. The cytoskeletal rearrangements that tumor cells go through during EMT and blood vessel invasion determine the cells’ plasticity and sensitivity to anoikis (apoptosis upon detachment from ECM). Mechanistically, EMT in tumor epithelial cells results from transcriptional reprogramming of abnormal survival signals via growth-factor receptor and kinase signaling regulating apoptosis and survival, such as PI3K, AKT, and mTOR (13, 14).

Is there additional translational importance to these findings, besides the potential value of the tumor suppressor DAB2IP to serve as a prognostic biomarker in prostate cancer? Many critical EMT drivers, such as Snail and Twist, correlate with disease relapse, poor clinical diagnosis, and decreased survival in patients with breast, colorectal, ovarian, or prostate cancer (11, 15, 16), supporting a role for EMT as a predictor of poor outcomes. The significance of EMT during clinical cancer progression has, however, been vigorously debated because of the lack of convincing evidence of EMT in clinical specimens. The skeptics would argue that, in the absence of histological identification of EMT intermediates, it is challenging to distinguish individual mesenchymal cells derived from epithelial tumor cells after EMT from stromal cells or other tumor-associated fibroblasts. Indeed, situated at the interface of tumor and stroma signals, EMT reflects an intricate counterbalance between internal

Restoring DAB2IP in metastatic prostate cancer cells reversed EMT.

growth pressure (by an expanding tumor) and the free edge of the migrating tumor periphery; this “fluid” dynamic of cell movement and polarity can be modified by DAB2IP to reverse metastasis. There is promise in the ability of this uniquely “anointed” tumor suppressor to regulate EMT, functioning not only as a coordinator of cell survival and apoptosis pathways (14), but also offering scaffolding-assistance to stop the metastatic journey of prostate cancer cells at the initiation point (5).

The principal clinical impact of the findings by Xie et al. (5) is the association of the EMT phenotype with therapeutic resistance and invasive metastasis. With the recognition that detection of metastatic lesions at an early stage of the disease or during treatment should increase disease-free survival rates, two questions beg exploration: First, what are the signaling pathways that will allow “smart” therapeutic targeting of tumor-circulating cells undergoing EMT at the initiation of metastasis? Second, do the EMT players serve as unique biomarkers permitting disease categorization based on the cell state of shape and movement? Regarding the former, therapeutic targeting of EMT in metastastic prostate cancer by a proteosome inhibitor suppressing Snail and inducing RKIP (Raf kinase inhibitor protein) via NF-B inhibition (17) has been demonstrated. Given the functional complexity surrounding the contribution of the androgen axis to prostate cancer metastasis, the outcomes of EMT targeting during prostate cancer progression to castration-resistant metastatic disease are unlikely to be functionally transparent. The recently documented inverse relationship between the androgen receptor content and EMT induction implicates the involvement of such a “negative” exchange in the metastatic behavior of prostate cancer cells from recurrent castration-resistant tumors (18). A threshold androgen receptor level may facilitate EMT by promoting β-catenin translocation (19), although DAB21P can reverse EMT and prevent tumor circulating cells from spreading (Fig. 1). The androgen receptor in prostate tumor-associated fibroblasts may participate in the interplay between prostate epithelial and stromal cells to reduce epithelial cell polarity and define an EMT-like signature. A morphologic reflection of transcriptional events dictated by the prostate tumor microenvironment, governs cellular behavior during tumor progression.