The differentiated state of tumor cells in glioblastoma and other cancers affects their growth and response to therapy [11
]. In glioblastoma, expression of CD133 correlates with growth rate and the response to radiation and chemotherapy [37
]. Our findings reveal a role for Numb in establishing the hierarchy of differentiated glioblastoma cell types. A model summarizing the role of Numb in regulating glioblastoma growth and differentiation is shown in Supporting Information Figure S8
We show here that Numb is asymmetrically localized during GSC divisions and regulates the differentiation and growth rate of these cells. High Numb expression increases the number of CD133-hi GSCs while simultaneously decreasing their proliferation. These changes are accompanied by increased expression of radial glia markers such as Pax6 and Sox2. Conversely, low Numb expression leads to the adoption of a highly proliferative CD133-lo/Sox2-lo/Pax6-lo/Tbr2-lo phenotype that is reminiscent of a transit amplifying glial progenitor. Importantly, both Numb-hi/CD133-hi and Numb-lo/CD133-lo cells retain self-renewal capacity. This observation is in agreement with previous reports indicating the existence of a related hierarchy of glioblastoma cells in which both CD133-hi and CD133-lo cells display self-renewal and tumor-initiating capacity [11
In this study, we show that Numb4d7 is upregulated in glioblastoma and increases Notch signaling, AKT activation and GSC growth while preserving asymmetric cell division. Recent studies have reported the existence of Numb isoforms lacking exon 10 that are upregulated in cancer and that are less antagonistic to Notch signaling than previously identified isoforms [42
]. We observed that the frequency and extent of Numb4d7 upregulation are greater in primary human GSCs and in glioblastoma specimens than in glioblastoma cell lines or in nontumor brain specimens, with the ratio of Numb4d7 to Numb4 approaching 1:2 in some tumors. These findings suggest that Numb4d7 opposes the growth-inhibitory effect of Numb4.
Previous studies indicated that Numb regulates Notch degradation by activating the HECT-domain E3 ubiquitin ligase, Itch [3
]. However, another major pathway regulating Notch degradation involves Fbw7, an F-box protein component of the SCF family of ubiquitin ligases [27
]. The gene encoding Fbw7 is a bona fide tumor suppressor gene, and loss of Fbw7 function leads to tumor formation via increased Notch signaling [27
Here, we provide the first evidence that Numb interacts with Fbw7, CUL1, and CAND1, all components of the SCFFbw7 complex. Although Numb4 and Numb4d7 have opposite effects on Notch signaling, they interact similarly with NICD. They differ, however, in their ability to promote SCFFbw7 assembly and to interact with Fbw7 and CAND1, suggesting that these are key regulatory steps in Numb-dependent SCF regulation. Numb4 promotes assembly and activation of the SCFFbw7 complex, and it also increases the association of CAND1 with the SCF complex. Studies suggest that CAND1 not only inhibits SCF assembly but also promotes optimal SCF activity [28
]. Moreover, only a small fraction of CUL1 is bound by CAND1 at any one time [43
], raising the question of how CAND1 can interact with only a small proportion of CUL1 molecules and yet effectively regulate SCF activity. In the case of the SCFFbw7 complex, we propose that Numb4 recruits CAND1 specifically to those SCF complexes where Numb4 is complexed with NICD, Fbw7, and CUL1 (Supporting Information Fig. S6
). After substrate ubiquitination is complete, this association facilitates CAND1-mediated disassembly of the SCF complex, thereby increasing substrate/adaptor recycling. Numb4d7 binds NICD but interacts less effectively with Fbw7 and CAND1. Consequently, it fails to promote SCF assembly or activation. By competing with Numb4 for binding to NICD, Numb4d7 downregulates SCFFbw7 activity and increases NICD levels. Upregulation of Numb4d7 in glioblastoma thus provides a mechanism for increasing cancer stem-like cell diversity while maintaining cancer cell proliferation.
Surprisingly, both Numb4 and Numb4d7 promoted the expression of the CD133-hi/Sox2-hi/Pax6-hi phenotype in GSCs, despite having opposite effects on Notch signaling. Thus, it appears that Numb can affect GSC differentiation through mechanisms that do not require Notch inhibition. Because Numb4 and Numb4d7 are both asymmetrically localized, cosegregation or asymmetric regulation of other cell fate determinants with Numb may explain this phenomenon. Several reports have identified a role for the EGFR in regulating the transition between symmetric and asymmetric division in neural stem cells and in determining the transition between neural stem cells and intermediate progenitors [2
]. The proposed mechanism involves Notch inhibition via EGFR-dependent upregulation of Numb. However, our findings suggest that Numb also maintains neural stem cells by suppressing EGFR expression via endocytic trafficking [5
In leukemia and breast cancer, Numb inhibits growth by stabilizing p53 and inhibiting Notch [6
]. In glioblastoma, Numb knockdown increased GSC proliferation, indicating that the net effect of Numb expression in glioblastoma is to constrain growth. This notion is further supported by our finding of an overall decrease in Numb protein expression in glioblastoma when compared with nontumor brain. This relative decrease in Numb expression may contribute to the preponderance of symmetric divisions observed in GSCs, as has recently been reported for GSCs and for oligodendrogliomas [17
]. Homozygous NUMB deletions and low Numb mRNA expression occurred primarily in a subpopulation of proneural glioblastomas, suggesting that Numb-related defects in asymmetric division play a role in tumorigenesis in this glioblastoma subgroup. Interestingly, a majority of secondary glioblastomas fall within the proneural subclass [30
], and secondary glioblastomas contain fewer CD133-hi GSCs than primary glioblastomas [38
Numb knockdown increased Notch levels and tumor growth, consistent with our finding that the predominant form of Numb (Numb4) in most primary glioblastomas inhibits proliferation. These findings raise questions regarding the functional significance of Numb4d7, which increases Notch and GSC proliferation. Our data suggest that Numb4d7 maintains GSC fate specification while ameliorating the inhibitory effect of Numb4 on tumor growth. Interestingly, a minority of glioblastomas express Numb4 and Numb4d7 in nearly equal amounts (). It will be of interest to determine the net effect of Numb expression on growth in such tumors.