Tumor cell stemness is not only essential for tumor initiation, growth, and malignant progression, but also promotes resistance to conventional therapy [5
]. The important concept of the CSC/TIC paradigm is that tumor cells can lose their clonogenic and tumorigenic potentials via differentiation. Therefore, identifying genes and molecular pathways that regulate cancer cell stemness will facilitate the development of effective therapy, as well as further our understanding of mechanisms of malignant tumor progression.
is a member of the epidermal growth factor (EGF
)-like homeotic supergene family with homologies to members of the notch/delta/serrate
]. Also known as pref-1
, fetal antigen (FA1
and ZOG, DLK1
is a developmentally regulated gene with strong expression in immature embryonic cells [10
], suggesting an important role of DLK1
in stem cells and progenitors. Elevated expression of DLK1
is also found in a variety of tumor cells, including neuroblastoma, gliomas, breast cancer, colon cancer, pancreatic cancer, small-cell lung carcinoma, and leukemia [31
]. Studies have shown that DLK1
is capable of inhibiting in vitro
cell differentiation, including mesenchymal progenitor cells [29
] and hematopoietic stem cells [34
]. The role of DLK1 in regulating tumor cell differentiation in vitro
has also been reported in glioma cells [33
] and hematopoietic tumors [34
]. Nonetheless, the role of DLK1
in the regulation of tumor cell differentiation in vivo
had not previously been investigated.
In this study, we have demonstrated that DLK1 plays a critical role in regulating tumor cell differentiation in vivo
using xenografts derived from NB cell lines stably expressing either the wild-type full-length DLK1 or one of the following dominant-negative DLK1 mutants: DLK1-ΔCyto without the cytoplasmic domain and DLK1-DM with Y339F/S355A mutations in the cytoplasmic domain. Y339 and S355 are two putative phosphorylation sites highly conserved among mammals [9
]. Here we found that xenograft tumors derived from NB cells with stable expression of DLK1-ΔCyto or DLK1-DM showed significantly higher expression of the neuronal differentiation marker neurofilament and the glial differentiation marker GFAP. Interestingly, NB cells express DLK1 and neurofilament in a mutually exclusive manner. These observations suggest that loss of DLK1 function sensitizes NB cells to undergo differentiation in vivo
. However, it should be noted that the increased expression of neurofilament and/or GFAP in NB cells simply suggests that these tumor cells have engaged in the processes of differentiation. It does not mean that NF+
NB cells have undergone terminal differentiation like normal neuronal stem/progenitor cells because tumor cells do not follow the exact pathways of normal cell differentiation. Nonetheless, increased expression of differentiation markers strongly correlate with decreased numbers of proliferating (Ki67+
) cells and vWF+
blood vessel density, demonstrating that increased differentiation likely accounts for the reduced tumor growth in vivo
. Interestingly, overexpression of DLK1 does not significantly alter cell fate decision in vivo
, suggesting that DLK1 alone is not sufficient to overcome the pro-differentiation stresses in the tumor microenvironment.
Consistent with our findings, a recent mouse genetic study has shown that DLK1 is required for self-renewal of neuronal stem cells [40
]. Together, these findings strongly suggest that DLK1 plays a significant role in the maintenance of stemness, perhaps by functioning as a gatekeeper at “differentiation checkpoints.” Loss or inhibition of wild-type DLK1 would sensitize stem cells to engage in differentiation induced by environmental stresses. The terminal lineage-specific differentiation is likely determined by additional differentiation signals. In this study, the increased expression of both neuronal and glial differentiation markers may simply reflect an altered differentiation program in NB cells. It is also possible that the DLK1 mutants sensitize NB cells to acquire a more differentiated progenitor phenotype before terminal differentiation.
Decreased oxygenation, or hypoxia, is a common feature of the tumor microenvironment in solid tumors and is an independent prognostic factor for advanced disease progression and poor clinical outcome [41
]. Our studies and those of others have shown that hypoxia inhibits cell differentiation [20
] and is able to arrest progenitor cells in an undifferentiated state [18
]. We found that hypoxia strongly increased DLK1
expression in neuronal tumor cells in vitro
]. Here, we found that robust DLK1 expression correlated with tumor hypoxia. These observations suggest that DLK1 may synergize with hypoxia to repress tumor cell differentiation in vivo
The requirement for the DLK1 cytoplasmic domain, especially the conserved Y339 and S355 residues, suggests that DLK1 is an important signal transducer or mediator for stem cell maintenance. As shown in this study, DLK1ΔCyto and DLK1-DM can increase the basal phosphorylation of ERKs, but not that of AKT. This result is consistent with the previous reports that increased ERK phosphorylation is associated with differentiation of neuronal progenitor cells [21
] and that activation of the MEK/ERK pathway facilitates differentiation but reduces self-renewal of embryonal stem cells [12
]. Since the MEK/ERK pathway can promote growth as well as differentiation, these findings strongly suggest that DLK1 can potentially play a critical role in regulating the MEK/ERK pathway in cell fate decision-making.
Together, our current work and our previous study [9
] have provided strong evidence demonstrating a critical role of DLK1 in the maintenance of NB cell stemness. Since DLK1 is overexpressed in many types of cancers, it will be interesting to investigate whether CSCs/TICs from spontaneous tumors express higher levels of DLK1 than non-stem-like tumor cells do. In addition, our findings suggest that DLK1 may be further explored as a potential therapeutic target to induce differentiation of stem cell-like tumorigenic cells.