In biopsies from patients with GBM, Hes3 co-localizes with the putative cancer stem cell marker prominin14,17,18
(). These results agree with the observations that magnetic immunoprecipitation of cells from the rodent brain using an antibody against prominin generate a cell fraction which is enriched in Hes3+/Sox2+ cells14
. Taken together, these data suggest that Hes3 may be expressed in putative cancer stem cells in GBM and that it may mediate cell number expansion.
Hes3 is expressed in putative cancer stem cells in glioblastoma.
To address the potential value of Hes3 as a target in cancer medicine, we used primary cultured cells that were isolated from GBM biopsies. These cells were maintained in vitro using standard protocols for the establishment of non-cancerous neural stem cells cultures19,20,21
. These cells can be expanded with the support of the mitogen Epidermal Growth Factor (EGF) or basic Fibroblast Growth Factor (bFGF)21
. Our previous work has established that the same cells used in this study can be propagated in culture over long periods of time and many passages, express several markers of NSCs, including, Sox2 and nestin, as well as prominin, can be induced to differentiate into neurons, astrocytes, and oligodendrocytes, they phenocopy the tumor of origin in xenograph experiments, and they respond to many growth factors that NSCs also respond to21,22
We recently showed that cultured NSCs express Angiopoietin 2 (Ang2) as well as its receptor, Tie2, and that treatment with Ang2 increases cell number both in vitro and in vivo14,15
. Here we show that GBM cells also express Ang2 together with the more established marker Sox2 (). NSCs express a small number of GM1+ gangliosides on their cell surface which can be identified by binding to a fluorophor-conjugated B subunit of cholera toxin16
; in contrast, their differentiated progeny expresses many binding sites. In our cultured GBM cells (in the presence EGF or bFGF), a very small percentage of cells labeled with conjugated cholera toxin (). In contrast, when cells where treated with serum (which induces the differentiation of NSCs), the number of cholera toxin binding sites greatly increased. These results further support that the cultured GBM cells used express markers commonly associated with neural stem cells and that they possess the potential to differentiate into cells representing the main cell types of the nervous system.
Our previous work with NSCs showed that Hes3 expression is opposed by the actions of JAK. Its activity can be assessed by measuring STAT3 phosphorylation on tyrosine residue 705 (STAT3-Tyr), which is downstream of JAK23
. In contrast, Hes3 expression is supported in conditions where STAT3 is phosphorylated on serine residue 727 (STAT3-Ser)12
. NSCs cultured in bFGF exhibit low Hes3 mRNA levels, but these can be increased by concomitant exposure to Ciliary Neurotrophic Factor (CNTF) and JAK inhibitor (JAK I)12
, a treatment which elevates STAT3-Ser and inhibits STAT3-Tyr (Suppl. Fig. 1a,b
). Similar results can be obtained by low concentrations of CNTF24
that also elevate STAT3-Ser but not STAT3-Tyr phosphorylation (Suppl. Fig. 1c
). The potential of JAK I to promote the undifferentiated state in NSC cultures is also demonstrated by the fact that it inhibits the expression of the glial marker GFAP in the presence of bFGF (Suppl. Fig. 1d
). Low CNTF concentrations or concomitant high concentrations of CNTF and JAK I increase the number of cultured NSCs in the presence of bFGF (Suppl. Fig. 1e
We hypothesized that GBM cells cultured in the absence of serum and the presence of mitogen would induce the expression of Hes3 and, therefore, they would resemble, in that respect, the cells of their origin in the patient. We tested the following four conditions: (a) EGF, (b) EGF + JAK I, (c) bFGF, and (d) bFGF + JAK I. We found that the highest STAT3-Ser and lowest STAT3-Tyr phosphorylation state was in the FGF + JAK I condition (). In these conditions, cells were able to proliferate; in fact, their number was greater than in the other conditions, after one week of treatment (). We note that inclusion of the JAK inhibitor allowed cell cultures to reach a higher terminal cell density, as assessed by cell number counting following a 2 week treatment (). Hes3 expression varied among the four conditions; the highest incidence of Hes3+ cells was in the bFGF + JAK I condition (). Since the culture system presented here lacks serum to support cell growth, the presence of a mitogen (bFGF or EGF) is required. As a consequence, we studied the effects of treatments (e.g. JAK I) in the presence of these defined factors.
Hes3 regulates the number of GBM cells in culture.
To address whether Hes3 is a mediator of the expansion of GBM cells and, therefore, a putative anti-cancer target, we performed transfections with Hes3 siRNA. We tested two different control (“scrambled”) siRNA products and three different Hes3 siRNA products for corroboration. We performed the experiments in both EGF and bFGF + JAK I conditions to show the sturdiness of the result and to demonstrate that the effect of Hes3 interference is not an artifact of a particular cell culture system. In all cases, Hes3 interference caused the reduction of Hes3 expression, as expected, and a reduction in cell number (). We confirmed the results using two additional GBM cell cultures established from different patients (). A cell survival assay showed a reduction in viability in the Hes3 siRNA – transfected cells, suggesting that, partly, the reduction in cell number is due to a reduction in viability ().