HIF-2alpha maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells

Abstract

High hypoxia-inducible factor-2alpha (HIF-2alpha) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2alpha as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2alpha reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2alpha-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2alpha maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2alpha is an attractive target for neuroblastoma therapy.

Fig. 1.

Normoxic HIF-2α protein levels are high in cultured neuroblastoma TICs. (A) Neuroblastoma specimen stained by immunohistochemistry for HIF-2α and TH. *, Magnified areas as shown in the two panels to the right. Note the virtual lack of TH signal in the HIF-2α-positive cells. (B–F) HIF-2α is expressed in neuroblastoma TICs at normoxia, as determined by qPCR (B), Western blot analysis (C), and immunohistochemistry (F). HIF-1α is expressed in neuroblastoma TICs, as determined by qPCR (D), but protein is generally very low at normoxia (E). HIF-2α but not TH protein levels are high in neuroblastoma TICs, as demonstrated by immunohistochemistry (F). (G) HIF-2α is expressed in TICs at normoxia with or without EGF and bFGF in the culture medium, as shown by immunohistochemistry. In C and E, actin serves as loading control. (Scale bars: A, 50 μm; F and G, 10 μm.) The qPCR data are mean values of three independent experiments performed in triplicate. Error bars represent SEM. Immunohistochemistry and Western blot analysis data were repeated at least three times. Dividing lines in C and E indicate that two areas of the same film or two different films have been merged.

Fig. 2.

HIF stabilization/activation in neuroblastoma TICs in response to hypoxia. (A and B) HIF protein levels as determined by Western blot analysis in TICs grown for 72 h at 1% O₂ with actin as loading control. (C and D) Expression determined by qPCR of the known hypoxia-driven genes VEGF and BNIP3 at 21% and 1% O₂. *, P < 0.05; **, P < 0.01 (Student's t test, 2-sided). (E) HIF-2α protein down-regulation upon rapamycin treatment in TICs as determined by Western blot analysis. (F–H) Expression of PHDs in TICs as determined by qPCR with SK-N-BE (2)c neuroblastoma cell expression data as comparison. The qPCR data are mean values of three independent experiments performed in triplicate. Error bars represent SEM. Western blot analysis data were repeated at least three times. Dividing lines in A and B indicate that two areas of the same film have been merged.

Fig. 3.

Human neuroblastoma TICs are immature and express neural crest/stem cell markers. (A) Neuroblastoma TICs lack or have very low expression of sympathetic neuronal differentiation markers compared with the established neuroblastoma SK-N-BE (2)c cells, as determined by qPCR. (B) Genes associated with a neural crest/stem cell phenotype are expressed in neuroblastoma TICs. (C) The activated Notch protein (icNotch) is abundant in neuroblastoma TICs but not in SK-N-BE (2)c cells, as determined by Western blotting. (D) Inhibition of γ-secretase activity by DAPT abolishes icNotch in TICs. The qPCR data are mean values of three independent experiments performed in triplicate. Error bars represent SEM. Western blotting data were repeated at least three times.

Fig. 4.

HIF-2α knockdown induces early sympathetic differentiation in TICs. (A) HIF-2α knockdown at the protein level assessed by Western blot analysis. Dividing lines indicate that two areas of the same film have been merged. (B) HIF2A knockdown at the mRNA level assessed by qPCR. (C) Down-regulation of VEGF mRNA upon HIF-2α knockdown. (D) icNotch-1 is not affected by HIF-2α knockdown, as measured by Western blot analysis of nuclear extracts from shC and shHIF2A TICs. (E–G) Down-regulation of Notch pathway components upon HIF-2α knockdown assessed by qPCR. (H–J) Up-regulation of sympathetic neuronal differentiation markers at the mRNA level upon HIF-2α knockdown assessed by qPCR. (K) Up-regulation of chromogranin A protein by HIF-2α knockdown as shown by immunohistochemistry. (L–N) Up-regulation of sympathetic neuronal differentiation markers at the mRNA level upon rapamycin treatment in TICs, as determined by qPCR. Error bars represent SD. (O–Q) Neuronal differentiation markers are not induced by rapamycin in shHIF2A cells, as determined by qPCR. The qPCR data are mean values of three independent experiments performed in triplicate or representative data of three independent experiments performed in triplicate (L–Q). Error bars represent SEM. Immunohistochemistry and Western blot analysis data were repeated at least three times.

Fig. 5.

Enhanced differentiation and reduced stromal support and vascularization in xenograft tumors derived from shHIF2A TICs. Immunohistochemistry showing HIF-2α (A), HIF-1α (B), TH (C), and CD34 (D–F) protein in shC and shHIF2A tumors. Note the high number of blood vessels in shC compared with shHIF2A tumors (D). +, Stromal regions. *, Necrotic regions. (Scale bars: A–C, E, and F, 50 μm; D, 100 μm.)

Fig. 6.

HIF-1α correlates negatively to high stage but not to patient outcome. HIF-1α protein levels were assayed by using immunohistochemistry analyses on two tissue microarrays representing a total of 93 neuroblastoma cases. A fraction of positive cells was dichotomized into four groups as described previously (6). (A) Patient stage, as determined by the International Neuroblastoma Staging System (INSS), was divided into three groups of increasing stage (INSS 1, 2, 4s; INSS 3; and INSS 4). A weak, significant correlation was found between a high number of HIF-1α-positive cells (fraction) and low patient stage (ρ = −0.27, P = 0.02). (B) For Kaplan–Meier survival analysis HIF-1α fraction scores were pooled into two groups: low fraction (0–25% positive cells) and high fraction (26–100% positive cells). There was no significant correlation between level of HIF-1α staining fraction and patient outcome (P = 0.42, log-rank test).