Our understanding of the roles that proteins play in hematological malignancies is further advanced than our understanding in solid tumors. Germline mutations are widely used to study both hematological malignancies and solid tumors; however, in hematological malignancies our ability to isolate stem cells from mice, genetically manipulate these ex vivo, and reintroduce them into syngenic animals allows us to determine the role of each protein or pathway in a natural setting. Our understanding of the pathways impacting solid tumor development is commensurately poorer because the cell of origin of many solid tumors remains a mystery and an allograft may not recapitulate the environment in which tumors evolve.
The cki family of proteins was originally identified by their ability to bind to G1 cdks, ultimately inhibiting kinase activity and preventing progression through the G1-S transition. However, we have begun to appreciate that this is only one biochemical activity, and growth suppression is only one role that these proteins have. For example, p21 and p27 play distinct roles in the growth and differentiation of OPC (Casaccia-Bonnefil et al, 1997
; Zezula et al, 2001
; Doetsch et al, 2002
). These proteins are also useful prognostic markers in ODG, albeit in a reciprocal fashion with high p27-staining indices associating with good prognosis, and high p21-staining indices associating with poor prognosis (Cavalla et al, 1999
; Miettinen et al, 2001
). In our studies we found that p21 facilitates the development of PDGF-induced ODG in mice. Thus, p21 makes a contribution to tumor progression. It is ‘oncogenic.'
How common are tumor-promoting activities? While there is an abundance of examples where cki are growth suppressive, there are a smaller number in which an ‘oncogenic' role is consistent with the data. The biochemical activities of cki might reflect the specific cell types or conditions in which they are studied; thus, ‘oncogenic' activity might be restricted to certain cell types or carcinogenic insults. In a Pten/Nkx3.1-deficient prostate model (Gao et al, 2004
), an MMTV-erbB2/neu mammary model (Muraoka et al, 2002
), and an MMTV-Wnt1 mammary model (Jones et al, 1999
), the complete absence of p21 or p27 reduces tumor development, suggesting that at least some level of p21 or p27 might be required for tumor progression under these conditions. In these three studies the cki was nuclear. A more recent study in a p27ck(−) knock-in animal model suggested a cdk-independent function promoting stem cell expansion and tumor development, and p27ck(−) protein was both nuclear and cytoplasmic (Besson et al, 2007
). In addition to ODG, there are suggestions for p21 ‘oncogenicity' in other human cancers as well, including prostate (Aaltomaa et al, 1999
; Baretton et al, 1999
; Omar et al, 2001
), cervical (Bae et al, 2001
; Cheung et al, 2001
), breast (Ceccarelli et al, 2001
), squamous cell carcinoma (Sarbia et al, 1998
), and tall-cell and well-differentiated papillary thyroid cancer (RG, BS and AK, unpublished data). Consequently, a growth- or tumor-promoting role is not unusual, but our understanding of it at the molecular and cellular levels is largely based on inferences drawn from subcellular localization and evaluation of the affect of protein levels on proliferation and apoptosis. Genetic evidence validating such notions has been elusive.
What biochemical activities of p21 and p27 might be important for ‘oncogenicity'? cki are found in multiple protein complexes, sometimes operating in distinct subcellular locations (Coqueret, 2003
; Denicourt and Dowdy, 2004
; Child and Mann, 2006
). These features might account for their ‘oncogenic' role (McAllister et al, 2003
; Denicourt and Dowdy, 2004
; Wu et al, 2006
). Some of these interactions occur when the cki are in the cytosol. In neuronal cells and mouse embryo fibroblasts, cytoplasmic p27 interacts with rhoA to affect cell migration (Besson et al, 2004
; Nguyen et al, 2006
). Cytoplasmic p27 can also interact with grb2 (Moeller et al, 2003
). Reducing cytosolic p27 inhibits cancer cell motility and tumorigenicity by affecting rho and akt signaling pathways (Wu et al, 2006
). Binding of cytosolic p21 to procaspase 3 (Suzuki et al, 1998
; Dotto, 2000
; Glaser et al, 2001
; Weiss, 2003
) or ask1 (Asada et al, 1999
; Zhan et al, 2007
) can desensitize tumor cells to apoptotic stimuli. Conversely, nuclear roles should be considered. As mentioned previously, nuclear p21 and p27 facilitate tumor development in the Pten/Nkx3.1, MMTV-Wnt1, and MMTV-erbB2/neu models. Nuclear cki can promote the accumulation of cyclin D–cdk4 (Cheng et al, 1999
; Weiss et al, 2000
), and p21 can interact with a surfeit of transcription factors and chromatin remodeling proteins (Dotto, 2000
; Gartel, 2006a
). However, establishing that a particular interaction is responsible, in situ
, in a developing tumor is a considerable challenge. Furthermore, given the cornucopia of possible interactions, it is unlikely that a single mechanism explains its role in all tumors.
In the studies presented here we have shown that p21 accumulates in the nucleus of ODG tumor cells and in glial cells stimulated by PDGF signaling. We have shown that this is associated with the accumulation of nuclear cyclin D1 and formation of cyclin D–cdk4 complexes, and increased proliferation and reduced apoptosis. Most importantly, by using somatic cell engineering, we established that p21 acts cell autonomously to promote tumor development, and this depends on the Cy element. Through this element, p21 interacts with cyclin–cdk complexes, and interacts with components of the receptor trafficking and endosome sorting machinery. Nevertheless, the status of p21 had no effect on the accumulation of PDGF receptors at the cell surface, and we were able to bypass the effect of p21 deficiency by enforcing accumulation of functional cyclin D1. Mutants of cyclin D1 that fail to accumulate in the nucleus but bind cdk4, or that accumulate in the nucleus but fail to bind cdk4 were both unable to support tumor development. All together, this suggests that p21 promotes ODG by stabilizing cyclin D1–cdk4 in the nucleus. Although this mechanism has been suggested before, specifically for p27 in the Pten/Nkx3.1 and MMTV-erbB2 models, and for p21 in the MMTV-Wnt1 model, this is the first time that a genetic proof has been used to assess the veracity of this model.
Nevertheless, our approach to identify protein domains will also benefit from further biochemical refinement. For example, it was surprising that the ability of the p21Cy2 and p21Cy1Cy2 mutants were comparable, albeit there was a ‘cy-dose' dependency to the onset of morbidity. We expected that the p21Cy2 mutant, with an intact Cy1 element, would support tumor development, just like Np21. The fact that it does not suggests that its interactions with other proteins in the cell could affect its availability to associate with cyclin D–cdk4, which is unaffected in vitro (data not shown). Additionally, overexpression of the mutants from a heterologous promoter might allow ‘weak' alleles to have functional affect.
In the absence of a genetic analysis, suggestions based on knowing where a cki accumulates and the effect of its absence on proliferation and apoptosis might be incorrect. For example, it is difficult to reconcile the suggestion that p21 supports cyclin D–cdk4 accumulation in the MMTV-Wnt1 model, when Yu et al (2001)
later demonstrated that cyclin D1 was not required in this model. Ultimately, identifying the correct mechanism is critical for providing insight into how to modulate p21 levels for therapeutic gain.