Cellular transformation in mammals is a complex and often multigenic process. Therefore, identifying and testing the genetic and epigenetic bases of particular malignancies has been challenging. In recent years, various studies have identified the tumor suppressor PTEN as a target in multiple tumor types and disease models. Studies of malignancies driven by loss-of-function PTEN
mutations and gain-of-function mutations in PI3K subunit genes have established the relevance of the PI3K pathway in promoting cancer (Yuan and Cantley, 2008
). Surprisingly, a tumor suppressor role for INPP5D
, the gene encoding SHIP, which also regulates PI3K signaling, has not been demonstrated (Rohrschneider et al., 2000
). In this paper, we provide the first evidence that SHIP acts as a tumor suppressor and that it acts in concert with PTEN to suppress B cell transformation.
Elevated PI3K signaling has recently been noted in selected B cell malignancies, namely MCL and DLBCL (Rudelius et al., 2006
; Uddin et al., 2006
). MCL patients are highly responsive to rapamycin treatment to inactivate mTOR activity (Witzig and Kaufmann, 2006
), and Akt activity is enhanced in many MCL lines that are dependent on PI3K for cell cycle progression and survival (Rudelius et al., 2006
). Similarly, bPTEN/SHIP−/−
lymphoma cells are responsive to PI3K inhibitors and rapamycin (unpublished data). Although the incidence of mutations in PTEN
in human cancers is second only to that of TP53
(Stiles et al., 2004
; Cully et al., 2006
), we previously found that its loss in B cells does not cause transformation (Anzelon et al., 2003
). In the case of DLBCL, reduced PTEN expression or predicted oncogenic PIK3CA
mutations occurred in 37 and 8% of cases, respectively. Both alterations were associated with poor overall survival (Abubaker et al., 2007
). Consistent with these findings, PTEN expression was reduced but still present in primary MCL cases exhibiting elevated Akt activity (Rudelius et al., 2006
), raising the possibility that SHIP expression or function may also be affected.
has recently been identified as a miR-155 target, causing reduced SHIP expression in a subset of DLBCL cases (Pedersen et al., 2009
). This finding was confirmed in a miR-155 transgenic B lymphoma model (Costinean et al., 2009
) and also reported in macrophages responding to inflammatory stimuli (O’Connell et al., 2009
). Intriguingly, these findings suggest that epigenetic down-regulation of SHIP expression by inflammation-induced miR-155 expression may promote transformation of B cells with impaired PTEN function.
PTEN regulates PI(3,4,5)P3
levels in all resting and stimulated cells. SHIP, in contrast, is expressed only in hematopoietic cells and depends on membrane recruitment via phosphotyrosine residues or SH3-based interactions for its activation (Leslie et al., 2008
). In particular, upon recruitment to ITIM residues after coengagement with FcγRIIB, SHIP mediates an inhibitory effect on BCR signaling. Thus, in the absence of PTEN, BCR-induced PI(3,4,5)P3
production likely remains dampened by SHIP. Accordingly, we postulate that disease in bPTEN/SHIP−/−
mice is promoted, at least in part, by tonic and/or antigen-mediated BCR signaling, which becomes lymphomagenic in the absence of both phosphatases. Indeed, BCR recognition of microbial or auto-antigens is thought to promote B-NHLs including MZL, CLL, and FL (Damle et al., 1999
; Guyomard et al., 2003
; Hervé et al., 2005
). Clonal bPTEN/SHIP−/−
lymphoma populations support a role for antigen-mediated selection in lymphomagenesis. Moreover, heightened serum IgM and IgM deposits in bPTEN/SHIP−/−
heart and kidneys (unpublished data) are consistent with poly- or autoreactive antigen receptor signaling that may help drive lymphoma expansion in this animal model. Interestingly, however, bPTEN/SHIP−/−
lymphoma cells, which do not spontaneously proliferate, are also quite hypoproliferative in response to BCR stimulation in ex vivo cultures. It is possible that this reflects a refractory or tolerized state, perhaps caused by sustained antigen receptor ligation or tonic signaling. However, biochemistry studies indicate that Akt and other downstream effectors of the PI3K pathway are hyperactivated both basally and after various stimuli, including ligation of the antigen receptor. Thus, it is clear that B cells lacking PTEN and SHIP are not completely refractory to BCR ligation and that activation of PI3K effectors are enhanced after this stimulus. This observation, taken with the collective results presented here, suggests that despite the hypoproliferative phenotype observed ex vivo, bPTEN/SHIP−/−
lymphoma progression is driven, at least partly, by signals through the antigen receptor but likely requires the contribution of additional receptor-mediated signals.
In this regard, BAFF, which promotes B cell survival and supports BCR-dependent clonal expansion of normal B cells, is an attractive candidate. BAFF activates B cells to enter G1
phase of the cell cycle marked by up-regulation of cyclin D, but cell cycle progression is halted before S phase without p27 down-regulation and cyclin E up-regulation (Huang et al., 2004
). Notably, here we found that BAFF alone stimulated proliferation of bPTEN/SHIP−/−
B cells and that the combination of BAFF and anti-IgM stimuli synergistically enhanced proliferation. Indeed, in addition to its role in dampening BCR signaling, SHIP has been linked to suppression of BAFF-induced signals and acts in a BCR-dependent fashion to suppress signaling via other receptors such as CXCR4 (Brauweiler et al., 2007
; Crowley et al., 2009
). Thus, it is possible that in the absence of PTEN, SHIP prevents transformation by regulating PIP3
levels induced by the BCR and other mitogenic signals and that in the absence of both phosphatases, the combination of unchecked signals via multiple receptors promotes disease.
A role for BAFF in B cell malignancies has been suggested. Transgenic expression of either BAFF (when combined with TNF deficiency) or the related molecule APRIL results in B cell neoplasia in mice (Mackay et al., 1999
; Planelles et al., 2004
). Studies in humans implicate BAFF and/or APRIL in mature B cell lymphomas, multiple myeloma, B cell chronic lymphocytic leukemia, and Waldenstrom’s macroglobulinemia (Shivakumar and Ansell, 2006
; Tangye et al., 2006
). We did not find evidence of autocrine BAFF production by bPTEN/SHIP−/−
cells. Moreover, serum BAFF levels were not reduced, even in mice bearing a large tumor load (unpublished data), confirming that BAFF is not limiting in vivo. bPTEN/SHIP−/−
B cells acquire the ability to proliferate to BAFF, which typically only promotes survival. Although BAFF is absolutely required for late maturation of WT B cells, transfer of bPTEN/SHIP−/−
B cells into BAFF−/−
recipients showed that its presence is not a strict requirement for B lymphoma progression. Nonetheless, lymphoma progression appeared to be less aggressive in BAFF−/−
than in TCR-β/δ−/−
recipients, suggesting that acute depletion of BAFF could deliver a therapeutic benefit in reducing tumor burden.
In summary, the bPTEN/SHIP−/− model presented in this paper provides the first evidence that SHIP is a tumor suppressor. As such, this model will be useful for elucidating the specific roles of PTEN and SHIP in B cell neoplasia, as well as providing a novel platform for interrogating and therapeutically manipulating the PI3K pathway in cancer. In a broader sense, the bPTEN/SHIP−/− mice also provide a system with which to investigate B cell–intrinsic (e.g., the BCR) and microenvironmental (e.g., BAFF/APRIL) factors that promote lymphoma induction and progression.