Growth factor-stimulated RTK signaling, particularly aberrant EGF receptor activation, is gaining much attention in both cancer and CSC biology. Multiple TKIs and blocking monoclonal antibodies are already in clinical use for various cancers such as lung, head/neck, breast, colorectal, and others [10–12,42
], with many second-generation EGFR inhibitors also in clinical trials [43,44
]. In brain tumors, EGFR is overexpressed in up to 50% of GBMs [45
] with the EGFR variant III (EGFRvIII) mutation (confers constitutive EGF signaling) present in up to 40% of EGFR-amplified GBMs [5,46
]. Despite the seemingly critical role of EGFR signaling in GBM, initial clinical trials have only shown modest improvements in a small percentage of patients [13–16
]. These initial clinical results demonstrate the importance of understanding GBM resistance mechanisms to EGFR and other molecular inhibitors toward developing more effective treatments.
Within GBM, the CSC subpopulation likely harbors intrinsic or rapidly acquired resistance to EGFR inhibitors as for other chemotherapeutics [26,27,47
]. In this study, we removed exogenous mitogens present in GBM CSC culture medium as a means of investigating GBM CSC responses to disruption of EGFR signaling. We identified activation of compensatory ERBB family members, specifically ERBB2 and ERBB3, in GBM CSCs deprived of exogenous EGF, suggesting blockade of multiple ERBB family members is required for successful GBM CSC eradication. This was shown through addition of lapatinib, a dual TKI directed at EGFR and ERBB2, to effectively prevent GBM CSC colony formation in all tested independent GBM CSC lines.
These results of EGFR blockade within GBM CSCs are in line with and extend recently reported results [25,28,29
]. Soeda et al. [28
] tested multiple growth factors for their ability to promote sphere formation in three independent brain tumor lines and found that EGF stimulation was required for sphere formation. In contrast, Kelly et al. [25
] successfully generated CSC cultures from 8 of 11 GBM samples in the absence of exogenous mitogens (-EGF/-bFGF). Although timing of the studies was different, as Soeda et al. [28
] tested sphere formation after 7 days and Kelly et al. [25
] established ongoing cultures directly from patient specimens, these studies suggest variable GBM CSC growth factor requirements between samples. Our results for three established GBM CSC lines correlates with the findings of Kelly et al. [25
]; when cultured without exogenous mitogens (EGF and bFGF), GBM CSCs continued to self-renew and maintained multipotency and tumor initiation capability. The comparison controls of normal NSCs did not form spheres or survive without exogenous growth factors, in agreement with Kelly et al. [25
], so growth factor-free survival seems to be a CSC oncogenic property. In this study, we demonstrate that continued GBM CSC propagation after EGF removal is at least partially due to activation of ERBB family signaling. Our data along with the reported studies suggest that EGFR signaling is required for GBM CSC propagation, but the CSCs can rapidly activate related RTKs to maintain downstream signaling pathways for survival.
Although not investigated in this study, it will be important to determine the drivers of the compensatory ERBB signaling found in GBM CSCs after EGF deprivation. Genetically, the constitutively active EGFRvIII mutation was not identified in any of the GBM CSC lines (), and we have additionally demonstrated that the main EGFR kinase domain abnormalities found in lung cancer are not present in the tested GBM CSC lines (data not shown). Autocrine or paracrine stimulation of EGFR through ligands such as EGF or amphiregulin, among others, may also confer survival and proliferative cues to GBM CSCs independent of exogenous EGF. These types of signaling may explain the small but significant effect of cetuximab at inhibiting GBM CSC colony formation in medium without exogenous mitogens (); however, lapatinib treatment resulted in greater inhibition of colony formation, suggesting that autocrine or paracrine EGFR stimulation is only partially responsible for GBM CSC exogenous mitogen independence. Finally, multiple reports have now identified neuregulin ligands as potential drivers in ERBB-dependent cancers such as head and neck, colon, and lung [42,48
]. Neuregulin ligands activate ERBB family members ERBB3 or ERBB4 that dimerize with other family members as well as activating their own downstream signaling [49,50
], and future work will need to determine the role of neuregulins in GBM CSC growth and resistance to EGFR inhibitors.
The role of ERBB family signaling in resistance to molecular inhibitors is rapidly becoming appreciated in many forms of cancer [34,42,51–53
]. In glioma, the role of ERBB2 and ERBB3 signaling in progression and resistance remains largely unknown. Some GBM cell lines express ERBB2, including A172 and U251MG, and have been shown to be sensitive either to ERBB2-inhibiting antibodies or lapatinib [36,54
]. Detection of ERBB2 in GBM and grade 3 gliomas has also been reported, with low-ERBB2 expression correlated with a better prognosis [55
]. Early clinical trials have demonstrated reduced pEGFR and pAKT after 7 days of lapatinib treatment in six of nine patients tested; however, completion of the trial with reported survival is still forthcoming [38
Lastly, a caveat of the current study relates to lapatinib dosing. In addition to specific EGFR- and ERBB2-targeted inhibition, other “off-target” lapatinib activities have been reported—including inhibition of glutathione S
-transferase P1 (GSTP1) [41
], inhibition of nuclear translocation of EGFR and ERBB2 [56
], and inhibition of sterol regulatory element-binding protein-1 (SREBP-1) to block fatty acid synthesis [38
]. Future studies will investigate the contribution (if any) of these “nonspecific” lapatinib effects in targeting GBM CSC proliferation.
In summary, this study provides an explanation for previous confounding reports showing the importance and necessity of aberrant EGFR activation in GBMs, yet failure of EGFR-targeted inhibition therapy in GBM clinical trials. We show that a subset of GBM cells, the GBM CSCs, exhibit therapeutic resistance and proliferate despite EGF deprivation or cetuximab-mediated EGFR inhibition through compensatory activation of EGFR-related family members (ERBB2, ERBB3). Importantly, only GBM CSCs proliferate, whereas normal NSCs did not survive in the absence of EGFR signaling, implicating ERBB2 and ERBB3 compensation in GBM CSCs as a tumorigenic mechanism. The presence of this resistance mechanism in GBM CSCs, which is able to recapitulate GBM, likely contributes to the clinically observed rapid tumor recurrence and suggests that it is worthwhile to explore multi-EGFR receptor family inhibition as a component of more effective GBM treatment strategies.