Hypoxia-regulated delta-like 1 homologue enhances cancer cell stemness and tumorigenicity

Abstract

Reduced oxygenation, or hypoxia, inhibits differentiation and facilitates stem cell maintenance. Hypoxia commonly occurs in solid tumors and promotes malignant progression. Hypoxic tumors are aggressive and exhibit stem cell-like characteristics. It remains unclear, however, whether and how hypoxia regulates cancer cell differentiation and maintains cancer cell stemness. Here, we show that hypoxia increases the expression of the stem cell gene DLK1, or delta-like 1 homologue (Drosophila), in neuronal tumor cells. Inhibition of DLK1 enhances spontaneous differentiation, decreases clonogenicity, and reduces in vivo tumor growth. Overexpression of DLK1 inhibits differentiation and enhances tumorigenic potentials. We further show that the DLK1 cytoplasmic domain, especially Tyrosine339 and Serine355, is required for maintaining both clonogenicity and tumorigenicity. Because elevated DLK1 expression is found in many tumor types, our observations suggest that hypoxia and DLK1 may constitute an important stem cell pathway for the regulation of cancer stem cell-like functionality and tumorigenicity.

Figure 1

Hypoxia induces DLK1 expression. A, quantitative reverse transcription-PCR (left) for DLK1 mRNA. Total RNA was prepared from NB cells after 16 h of incubation at 1%, 21% O₂, or in the presence of 100 μmol/L deferoxamine. Data were normalized to HPRT mRNA that did not change under hypoxia (columns, mean from ≥3 experiments; bars, SEM; *, P < 0.05). Western blot for DLK1 (right) with HIF-1α as a control for hypoxia and β-actin or PCNA as a loading control. N.D., not determined. B, Western blot for DLK1. BE(2)C cells were transduced with retrovirus containing either the constitutively active HIF-1α, constitutively active HIF-2α, or empty vector (ctrl; left) or with lentivirus expressing shHIF1α, shHIF2α, or simultaneously infected with both shHIF1α and shHIF2α virus (right). Cells were cultured for 16 h at 1% (hypoxia, H) or 21% O₂ (N). C, ChIP using @HIF-1α or @HIF-2α antibodies. BE(2)C cells were incubated at 1% O₂ for 16 h to induce the HIF-α subunits. Two different sets of primers (DLK1-3D and DLK1-4P) were used to detect the DLK1 promoter gene with RAD51 primers as negative control and VEGFA primers as positive control. Amplicons with the predicted sizes were validated using the end-point PCR for 28 cycles (left). Relative levels of promoter binding were analyzed by qRT-PCR (right). D, Northern (left) and Western blot (right) for DLK1 in BE(2)C cells cultured at 1% (+) or 21% (−) O₂ with or without 1 μmol/L RA for 24 h.

Figure 2

DLK1 maintains an undifferentiated NB phenotype. A, BE(2)C cells were differentiated with 1 μmol/L RA or 10 μmol/L BrdUrd for 5 d. DLK1, Sox2, c-kit, and CD-133 were detected by Western blot. B, BE(2)C cells were infected with lentivirus expressing shDLK-2H, shDLK-4H, or empty lentiviral vector (ctrl). Left: Infected cells were stained using antibodies against DLK1 (red) or neuron-specific β-tubulin III (green) with Hoechst 33342 as nuclear stain (blue). Magnification, ×200. Right, expression of DLK1 was analyzed by Western blot, and cells with β-tubulin III–positive neurites were enumerated from five to six random fields (columns, mean; bars, SEM; *, P < 0.0001 versus control). C, left, fluorescence-activated cell sorting (FACS)–selected BE(2)C cells expressing the full-length DLK1 (DLK-FL) or empty retroviral vector (ctrl) were cultured for 3 d with or without 1 nmol/L RA and then stained as in B. Magnification, ×200. Right, neurite-positive cells were counted from five to six random fields (columns, mean; bars, SEM; *, P = 0.0001 versus RA-treated control). D, Western blot for CD133 and Notch1 in ER cells expressing the full-length DLK1 with the empty retroviral vector as control (ctrl).

Figure 3

DLK1 regulates tumor sphere formation. A, left, DLK1 expression in NB cell lines cultured in sphere medium (sphere) or in standard growth media (ctrl). Middle, kinetics of DLK1 expression during sphere formation in BE(2) cells cultured in sphere media (SphM) for 1 to 4 d. Right, DLK1 expression in BE(2) cells cultured for 2 d in tissue culture dishes with growth medium (GM/TC), tissue culture dishes with sphere media (SphM/TC), or polyHEMA-coated dishes with sphere media (SphM/pHEMA). B, BE(2)C cells were infected with lentivirus [shDLK-2H, shDLK-4H, or vector (ctrl); left] or transfected with siRNA oligos [siDLK-05, siDLK-07, or a nontargeting siRNA (ctrl); right]. Cells were plated at 48 h into polyHEMA-coated six-well plates and cultured for 4 d in sphere medium. Spheres were collected and then trypsinized for cell counting (columns, mean; bars, SEM; n = 6; *, P < 0.0003 versus control). DLK1 knockdown was confirmed by Western blot. C, BE(2)C cells were infected with lentivirus expressing shDLK-2H, shDLK-4H, or vector (ctrl). Cell growth was measured daily using the MTS assay (points, mean; bars, SEM; n = 4; *, P < 0.05 versus control, one-way ANOVA). D, FACS-sorted SY5Y cells [DLK-FL or empty vector (ctrl)] and uninfected cells (none) were cultured in the sphere medium for 5 d. Cells in tumor spheres were counted (columns, mean; bars, SEM; n = 6; *, P < 0.04 versus control). DLK1 expression was confirmed by Western blot.

Figure 4

DLK1 regulates clonogenic growth in vitro. A, ER cells were FACS-sorted for stable expression of DLK-FL or empty vector (ctrl). B, BE(2)C cells were infected with lentivirus expression shDLK-2H, shDLK-4H, or empty vector (ctrl). Left, representative images of colonies. Middle, plating efficiency = % colonies per input ± SEM (n = 6; *, P < 0.0001 versus respective control). Right, Western blot.

Figure 5

DLK1 cytoplasmic domain is required for clonogenic growth and tumor sphere formation. A, comparison of the DLK1 cytoplasmic domains among different mammalian species. Arrows, the putative phosphorylation sites [tyrosine (Y)-339 and serine (S)-355]. B, BE(2)C cells were FACS-sorted for DLK1 lacking cytoplasmic domain (DLK-ΔCyto; left), DLK1 with point mutation [Y339F (phenylalanine), S355A (alanine), or Y339F/S355A; right] or empty vector (ctrl). Clonogenic assays were performed as described in Fig. 4. *, P < 0.0002 versus control. C, FACS-sorted BE(2)C cells were cultured in the sphere medium for 4 d (Fig. 3B). *, P < 0.002 versus control.

Figure 6

DLK1 regulates tumorigenicity in vivo. A, BE(2)C cells were infected with lentivirus expressing shDLK-2H, shDLK-4H, or empty vector (ctrl). B and C, BE(2)C and ER cells were FACS-sorted for expression of each construct. Cells were injected s.c. (n = 10) in the bilateral anterior backs of nude mice: 2.5 × 10⁵ for ER cells (control or DLK-FL) and 4 × 10⁵ for BE(2)C cells (control, shDLK-2H, shDLK-4H, DLK-ΔCyto, Y339/S355A). Tumor take (%) = number of tumors formed/number of injection sites × 100 (left). Tumor volume (mm³) = (length × width²) × 1/2 (A, middle or B–C, right). Tumor weight = mean ± SEM; *, P < 0.05 versus control (A, right). See text for discussion of statistics.