We have found that BMI-1 is highly expressed by ESFT cells and that it promotes anchorage-independent growth and in vivo
tumorigenicity. Importantly, our studies reveal that these tumorigenic properties of BMI-1 are modulated independent of CDKN2A
repression indicating that novel mechanisms of BMI-1 oncogenic activity exist. In fact, in contrast to normal mesenchymal stem cells, altering expression levels of BMI-1 has no significant or consistent impact on CDKN2A
or p16 expression in ESFT. While this may be an artifact of in vitro
culture, it is also possible that non-functional retinoblastoma family proteins prevent BMI-1-mediated repression of CDKN2A
in these cells (36
). Although previous reports have shown that pRB is only rarely mutated in ESFT (25
), recent work suggests that pRB function may be functionally inactivated by EWS-FLI1 itself (40
). Further studies are now required to determine if pRB inactivation contributes to dissociation of BMI-1 from p16 regulation in ESFT.
Although the histogenesis of ESFT remains a mystery, recent studies implicate somatic stem cells as cells of origin (reviewed in (41
)). Given that most somatic stem cells express high-levels of BMI-1 and that expression diminishes during differentiation (42
), it is possible that the high level of BMI-1 expression we observe in ESFT cells is an inherent feature of their cellular origin. Alternatively, expression of the EWS-FLI1 fusion oncogene may be able, in some cell types, to induce BMI-1 as was recently shown in NIH-3T3 cells (43
). Cell type- and differentiation state- appropriate experimental models are now required to test which of these situations exists in the initiation of ESFT. EWS-FLI1-mediated transformation of primary fibroblasts requires inactivation of p16-RB and/or p53 pathways (44
). We speculate that BMI-1-mediated repression of CDKN2A
may confer cellular tolerance to EWS-FLI1 in the ESFT cell of origin and that this epigenetic inactivation of tumor suppressor pathways could explain the relatively low incidence of secondary genetic mutations in primary ESFT (3
). In support of this, we find that BMI-1
levels are, in general, higher among primary tumors than ESFT cell lines () suggesting that mutations in p16 and/or p53 that are more commonly present in cell lines (25
) may at least partially compensate for BMI-1 expression. It has been previously documented that p16 loss in lung tumors correlates with low BMI-1 expression (47
). We are now testing whether there is a relationship between BMI-1 expression and p16 and/or p53 status in primary ESFT and whether differences exist in clinical presentation or outcome between tumors that express high vs.
low levels of BMI-1.
We have shown that altering BMI-1
expression affects the ability of both p16-null and p16-positive cells to form anchorage-independent colonies in vitro
and tumors in vivo.
In corroboration with our findings, several recently published reports have revealed that, in cooperation with other oncogenic lesions such as mutated epidermal growth factor receptor (EGFR) or H-RAS, BMI-1 can transform both CDKN2A
wild-type and CDKN2A
null cells (22
). In addition, BMI-1 knockdown in p16-null DAOY medulloblastoma cells significantly impedes tumor formation in vivo
). Thus, although initial studies of BMI-1 implicated repression of the p16Ink4a
– encoding CDKN2A
locus as the primary mechanism of oncogenic action (11
), more recent data from our lab and others demonstrate a pivotal role for p16-independent mechanisms.
In order to identify potentially novel downstream targets of BMI-1 we performed gene expression profiling of ESFT cells following BMI-1
knockdown and compared BMI-1 responsive genes to those genes similarly affected by BMI-1
knockdown in human medulloblastoma cells (19
). Although a significant subset of genes was commonly regulated by BMI-1 in both tumor types, others were uniquely altered in only one of the model systems. This implies that while some biological pathways are shared among different tumor types, it is likely that at least some downstream effectors of BMI-1 differ among tumors of different cellular origins. Nevertheless, our findings demonstrate that significant commonalities exist. In particular, direct comparison between ESFT and medulloblastoma cells (19
) reveals that alterations in cell adhesion and extracellular remodeling processes are highly over-represented and common to both tumor types. For the current study we have validated that expression of the basement membrane protein nidogen 1 is repressed by BMI-1 in ESFT cells. Nidogen 1 acts as a linker between laminins, collagens and proteoglycans in the extracellular matrix and binds to cell surface integrins (38
). Interestingly, it has recently been reported that NID1
is frequently silenced in colon cancer suggesting that nidogen 1 may have a role as a tumor suppressor gene, preventing invasion and metastasis (48
). In support of this possibility we have found that down-regulation of NID1
promotes adhesion of ESFT cells in vitro
, recapitulating the effects of BMI-1 over-expression. Therefore, we hypothesize that the effect of BMI-1 knockdown on cell adhesion, through modulation of nidogen 1 and/or other adhesion-related proteins, is likely to underlie the delay to in vivo
tumor engraftment that we observe in ESFT cells with reduced levels of BMI-1. Consistent with this hypothesis, delayed engraftment and altered adhesion pathways have also been shown to be a feature of bmi-1
–deficient murine glioma cells (22
). Thus, the cumulative evidence suggests that modulation of adhesion molecules such as nidogen 1 is likely to contribute to the oncogenic function of BMI-1 in ESFT as well as other tumor types.
Finally, polycomb genes, including BMI-1,
play a central role in the repression of differentiation and in the controlled orchestration of normal development (5
). Our finding that developmental pathways are significantly affected by BMI-1
knockdown in ESFT cells suggests that the embryonic function of BMI-1 is being recapitulated in these undifferentiated tumor cells. It is particularly noteworthy that both WNT and NOTCH pathway genes are highly affected by BMI-1 knockdown as both of these developmental pathways have been previously implicated in ESFT growth and tumorigenicity (49
). Intriguingly, our data also show that among the affected developmental processes, BMI-1 loss has its most profound effect on genes that are involved in neural development with BMI-1 knockdown leading to increased expression of neural markers ( and Supplementary Table 1
). These findings corroborate recent documentation of the effects of bmi-1
loss on the phenotype and neural differentiation capacity of murine gliomas (22
). One of the many clinical mysteries surrounding ESFT is the observation that they vary from highly undifferentiated tumors to tumors with obvious neural features. It is tempting to speculate that the phenotype of a particular tumor is either (a) pre-determined by the BMI-1 expression level in the parent cell that originally acquires the EWS-ETS translocation or (b) a consequence of the tumor microenvironment and its downstream effects on BMI-1 expression. More extensive studies are required to evaluate these hypotheses.
In summary, we have shown that BMI-1 functions as an oncogene in ESFT and that it promotes tumorigenicity in a CDKN2A-independent manner, influencing pathways involved in cell adhesion, differentiation and development. Given its central role in the regulation of multiple developmental processes in normal stem cells, we expect that no single gene will be uniquely responsible for the oncogenic effects of BMI-1. Nevertheless, our findings support the hypothesis that regulation of adhesion pathways is central to BMI-1-mediated tumor promotion. Future studies directed at understanding this relationship are likely to proffer attractive and novel targets for therapeutic intervention that may be common to the multiple human cancers that deregulate and over-express BMI-1.