Ets2 has different functions in different cell types and tumors. Previously we have shown that Ets2 supports mammary tumors through a stromal effect [6
] that was identified as fibroblasts [27
]. However, in intestine an Ets2 copy-number dependent repression of Apcmin
induced tumors was found in the DS1 region of the mouse ortholog of human chromosome 21 [4
]. Recently a different region of Hsa21 that does not include Ets2 was implicated in inhibiting tumor angiogenesis of transplantable tumors in a second mouse model of Down’s syndrome [5
]. Our results indicate that Ets2 acts within intestinal epithelial cells to repress chemical carcinogen induced colon tumors and this activity is likely associated with restricting intestinal stem cells. Thus Ets2 may be additive with other mouse orthologs and human genes located on Hsa21 to repress tumors. However, this Ets2 contribution may be specific to intestinal tumors driven by the Wnt pathway. Thus multiple genes of Hsa21 may contribute to resistance to cancer via different tumor specific mechanisms. A role for Ets2 within intestinal stem cells was anticipated from the identification of Ets2 as a target of the Wnt pathway [1
] and its regulation by Ascl2 in intestinal stem cells [3
]. However, in contrast to the conditional inactivation of Wnt targets Ascl2 and Myc, inactivation of Ets2 within the intestinal epithelium leads to increased crypt cell proliferation and increased tumor sensitivity [3
] rather than the loss of Myc or Ascl2 deficient crypts. Ets2 acts to moderate the proliferation response of the Wnt pathway within the intestine like Lgr5, a stem cell marker and Wnt target [30
]. The potential interaction of Ets2 within the Wnt pathway in altering colon stem cell fate is shown in .
We deduce that Ets2 regulates colonic stem cell self renewal because of the over representation of uniformly Ets2 deficient crypts. Competition between intestinal stem cells results in monoclonal crypt conversion, even between equally competitive cells [31
]. The identification of crypts of Ets2+/+
, V-Cre and Ets2flox/+
, V-Cre that uniformly express the R26R reporter gene are examples of normal monoclonal conversion [34
]. The competitive advantage of Ets2 deficient stem cells may be reflected by the increased number or activity of proliferative cells at the bottoms of colon crypts. While the epithelium autonomous function of Ets2 revealed by Villin-Cre might be elicited through cells adjacent to the stem cells [33
], the over representation of Ets2 deficient tumor cells within tumors suggests a tumor cell autonomous function. Direct proof of a stem cell autonomous function must necessarily await quantitation of isolated colonic stem cells in the presence and absence of active Ets2.
Stem cell colonization and crypt fission appear closely related. However, the regulation of crypt fission has not been studied extensively. APC mutations that drive adenoma formation from a stem cell, results in increased crypt fission perhaps even in the absence of increased epithelial cell proliferation [23
]. The re-emergence of intestinal crypts that express Myc or Ascl2 after the conditional inactivation of either gene reveals a connection between competitive advantage of stem cells and increased crypt fission [3
]. Fission has been implicated as a mechanism for spread of mutations in the intestine of both mice and humans [36
]. To date alterations in genes that affect intestinal stem cells (APC, Myc, Ascl2), also affect crypt fission. The possible expansion of Ets2 deficient stem cells within individual crypts and by crypt fission may result in an expanded population of colonic stem cells. One consequence of a larger population of Ets2 deficient colonic stem cells number would be an expected increase in AOM induced tumor sensitivity because the intestinal stem cells are the cell of origin of intestinal tumorigenesis in mice [9
]. This prediction was supported by the AOM/DSS experiments with Ets2flox/flox
, V-Cre and the Ets2A72/A72
The activation of Ets2 through the phosphorylation of threonine 72 is important for its tumor repressive activity. This may reflect the dual roles of Ras/Erk activation in senescence of normal cells and its oncogenic role in cancer. For intestinal stem cells Ets2 may integrate the increased activity of the Wnt pathway and growth factor signaling normally associated with mitotic activity. Ets2 contributes to the regulation of multiple genes that may influence intestinal stem cell self renewal or differentiation. Cdx2 expression is decreased within the colons of Ets2 deficient mice rescued from placental insufficiency [10
]. Cdx2 regulates early intestinal morphogenesis as well as early embryonic axis development [38
] and suppresses tumorigenesis in the distal colon in response to AOM injections [39
]. MMP9 expression is decreased in some but not all expressing tissues of Ets2 deficient mice [13
] and is important for differentiation towards the absorptive lineage. MMP-9 deficiency may lead to Notch1 inhibition and subsequent accumulation of goblet cells [40
]. MMP9 also acts as a tumor suppressor in colitis associated cancer [41
]. Finally Ets2 has been suggested to be important in restricting the self renewal of primary fibroblasts by regulating p16 [42
]. Ets2 regulation of p16 may be restricted by degradation of Ets2 by the Cdh1 component of the anaphase promoting complex during the cell cycle [43
]. Confirmation of involvement of any or all of these Ets2 targets will be most definitively evaluated in isolated Ets2 deficient colonic stem cells.
In summary our results indicate that while Ets2 is a Wnt pathway target gene within intestinal stem cells, its loss provides a competitive advantage for intestinal stem cells to colonize crypts, increase basal crypt cell proliferation and increases crypt fission. Ets2 loss may increase the number or sensitivity of colon stem cells for tumor initiation.