A principal function of epithelial cells is adherence to tissue level architecture. At its most basic level, this architecture consists of monolayers of attached cells against a basement membrane. This highly structured organization in epithelial tissues allows them to serve as protective barriers and function to interface with the luminal or outside environment. Certain cellular functions require disengagement from substratum, including functions such as cell migration during development or wound repair, mitotic cell division, and physiologic shedding. Disengagement from substratum requires the activation of autonomous signaling mechanisms not regulated by tissue architecture. The activation of such anchorage-independent programs and the ability to defy tissue-level imposed programs are also important parts of epithelial tumorigenesis. Much of the signaling that underlies the anchorage-dependency and independency of epithelial cells occurs through tyrosine phosphorylation. Here we report that a common signaling pathway that characterizes the anchorage independent growth of epithelial tumor cells is the Src family kinase phosphorylation of their transmembrane substrate Trask.
In untransformed epithelial cells, the phosphorylation of Trask is tightly regulated and Trask phosphorylation is only seen during circumstances of mitotic detachment or physiological shedding such as seen at the apex of the intestinal villi. Trask phosphorylation can be induced in vitro
by forced detachment and growth in suspension. In contrast to the tightly regulated phosphorylation of Trask in untransformed epithelial cells and tissues, we find that the Src phosphorylation of Trask is commonly seen in many epithelial cancers and cancer cell lines. We see Trask phosphorylation in all stages of cancer including pre-invasive cancers such as tubular adenomas of the colon, as well as invasive, and metastatic cancers. As such, the abberant phosphorylation of Trask is not an activity acquired in later stages of invasive or metastatic cancers. It is possible that Trask phosphorylation in pre-invasive cancers identifies those pre-invasive tumors that are at high risk for invasion and dissemination. Future studies may attempt to test that hypothesis, however this may be too simplistic a hypothesis, considering what we know so far about the functions of Trask. It is likely that both the phosphorylation and the dephosphorylation of Trask are important functions in migrating cells and likely also in invading and metastasizing cells and some level of regulation would be optimal for these tumorigenic functions. As such, Trask phosphorylation may be a marker of cellular activities at certain points in time or certain tumor areas. Consistent with this, we see only focal phosphorylation of Trask in many tumors. The spacial focality we seen in the immunostains may merely reflect a snapshot in time captured at the moment of tissue acquisition and fixation, and may be evidence of a wider temporal plasticity in Trask phosphorylation reflecting the dynamic nature of tumor cell adhesion and migration characteristics in vivo
that we are unable to appreciate in these snapshot analyses. The cellular attributes that Trask phosphorylation may be reporting could be disrupted cell-stromal or cell-cell interactions, changes in cytoskeletal signaling, or activation of anoikis resistance. We have evidence that Trask phosphorylation is regulated by cell-cell and cell-matrix engagement (manuscript in submission). Several studies have reported the activation of Src kinases when epithelial cells detach from matrix and its function in averting anoikis when detached (24
). The specific role of Trask/CDCP1 in promoting anoikis resistance was recently demonstrated in a lung cancer cell line (27
The apparent deregulation of Trask in pre-invasive epithelial tumors is interesting but not altogether surprising. Indeed, increased activity of both Src or Yes is an early event in colon neoplasia and seen very frequently in pre-invasive lesions of the colon (28
). The finding of Trask phosphorylation in pre-invasive tumors suggests that some of the molecular and possibly phenotypic attributes of invasive or metastatic epithelial cancers are already present in pre-invasive tumors. Consistent with this, the activation of Src and Yes in pre-invasive tumors of the colon is seen predominantly in tumors at highest risk for progression to invasive cancer (28
). The deregulation of Trask in pre-invasive tumors could have phenotypic consequences, but these would likely be clinically undetectable with little symptomatic consequence. These could include phenotypes such as shedding of tumor cells. However due to the intact nature of the basement membrane in pre-invasive tumors, the shedding would be entirely into the epithelial lumen which would be of little clinical or symptomatic consequence to affected patients. Indeed we do find significant interphase tumor cell shedding in tubular adenomas which we do not see in normal colon tissues (). Trask phosphorylation could also increase the survival of luminally shed cells. Consistent with this, exfoliated tumor cells are detected much more succesfully in the stools of patients with tubular adenomas or colon cancers compared with the stools of patients with inflammatory diseases of the colon or normal colons (31
) and exfoliated breast epithelial cells are detected in nipple aspirates of women with pre-neoplastic lesions of the breast (33
). Although not clinically significant in pre-invasive disease, the phenotypic consequence of deregulated Trask phosphorylation may be much more significant after progression to invasive disease, since the ability to survive and migrate within connective tissues, lymphatics, and the bloodstream can be mediated through Trask phosphorylation. Consistent with this, we do see Trask phosphorylation in tumor emboli ().
When we use the same tumor cells to compare in vitro and in vivo growth, we find that Trask phosphorylation in the in vivo model resembles the anchorage-deprived state of the in vitro model, not the adherent monolayer growth (). This is not surprising, since it is widely recognized that the growth of tumor cell lines in flattened monolayers on tissue culture treated plasticware represents an artificial state with many differences from their growth in vivo.
The mechanisms by which Trask may promote survival in suspended epithelial cells are currently unknown. Trask/CDCP1 was found to bind PKC delta in a phosphorylation-dependent manner through the C2 domain of PKC delta (34
). PKC delta is thought to regulate apoptosis although its role may be complex and both pro-apoptotic and anti-apoptotic functions have been attributed to PKC delta (35
). The functions of PKC delta may be context-dependent. In murine mammary epithelial cells, PKC delta activation promotes the ability to survive and grow in an anchorage-independent manner (36
). Almost surely Trask regulates functions other than survival, since we have previously shown that the forced overexpression and over phosphorylation of Trask retains detached epithelial cells in suspension and prevents them from re-spreading on substrate which suggests that Trask phosphorylation impacts cytoskeletal signaling as well (17
). We have been studying the role of Trask in cytoskeletal signaling and have identified mechanistic links between Trask and components of the actin cytoskeleton, although this evidence is only preliminary at this point.
The fact that the phosphorylation of Trask is entirely dependent on src kinases may suggest its use as an in vivo
marker of the activity of Src kinases. Its phosphorylation in PyMT induced tumors and in many human cancers is consistent with this. However it is unlikely that the level of Trask phosphorylation faithfully reflects the activity of Src kinases. In the tumor cell panel reported here, the level of Trask phosphorylation varies widely and does not parallel the much more uniform activity of Src kinases previously published by ourselves and others (37
). In addition, although epithelial cells undergoing mitosis or detachment show modest increases in Src kinase activity (39
), the abundant phosphorylation of Trask seen in these circumstances greatly exceeds increases in Src kinase activities (26
). In addition, Trask phosphorylation appears to be partly dependent on Trask expression, and its phosphorylation can be induced by its overexpression ((17
) and manuscript in preparation). Clearly, there are many variables other than the steady state activity of Src kinases that regulate the Src-mediated phosphorylation of Trask.
In summary, Trask is a recently identified Src substrate that may be highly relevant to epithelial tumorigenesis. The evidence indicates that Trask is a principal marker of anchorage-independent signaling in epithelial cells and its Src-driven phosphorylation is tightly regulated in epithelial cells. The deregulation of Trask phosphorylation is a common and early event in epithelial tumorigenesis and further mechanistic exploration of its signaling functions should be of high priority.
Statement of translational relevance
Src inhibitors are being studied in patients with epithelial malignancies. This class of anti-tumor agents presents unique challenges in clinical testing and development, principally because the tumorigenic functions and downstream molecular effectors of src, and the expected biological outcomes of src inhibitor therapy are not yet well described. In this paper we present a novel Src substrate that is abberantly phosphorylated in epithelial cancers. Trask phosphorylation is uniquely linked with Src kinases, relatively unique to epithelial tissues, and physiologically relevant to the anchorage independent state in epithelial cells. These attributes make Trask an important new biomarker that may not only be an effector of activated src kinases in epithelial tumors, but may have predictive or pharmacodynamic utility in the clinical development of src inhibitors in epithelial cancers.