Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma

Abstract

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.

Figure 1.

Tumor growth enhancement induced by mixing of wtEGFR and ΔEGFR-expressing cells. (A, top) H&E at day 12 after intracranial injection of U87wt (wt), U87Δ-LacZ (Δ-LacZ), or U87wt mixed with U87Δ-LacZ at 90:10 or 99:1 ratios (wt + Δ-LacZ 90%–10% or wt + Δ-LacZ 99%–1%; 100% = 5 × 10⁵ cells). (Bottom) Whole-brain sections and X-Gal staining of tumor samples; Fast red counterstain. LacZ-positive percentage mean of each tumor sample is indicated below X-Gal staining pictures. (B) Tumor volume after subcutaneous injection of U87wt cells alone or mixed with U87Δ-LacZ, U87Par-LacZ, or U87DK-LacZ at ratios of 90:10 or 99:1 (100% = 1 × 10⁶ cells). Error bars represent mean ± SEM; n = 6. (*) P < 0.05. (C, top) H&E of mouse brains 22 d after intracranial injection of mAstr-Ink4/Arf^−/−-wtEGFR-GFP astrocytes (wt-GFP) alone, mAstr-Ink4/Arf^−/−-ΔEGFR (Δ) alone, or mAstr-Ink4/Arf^−/−-wtEGFR-GFP astrocytes and mAstr-Ink4/Arf^−/−-ΔEGFR mixed at a 90:10 ratio (100% = 5 × 10⁵ cells). (Bottom) mAstr-Ink4/Arf^−/−-wtEGFR-GFP cells as GFP immunofluorescence. DAPI (blue) labels nuclei.

Figure 2.

ΔEGFR cells enhance wtEGFR cell growth in vivo and in vitro. (A) Soft agar colony formation assay quantification of U87Δ cells treated with normal media (Neg), U87Par CM (ParCM), U87wt CM (wtCM), or U87Δ CM (ΔCM). (B) Soft agar colony formation assay quantification of U87wt cells treated with normal media (Neg), U87Par CM (ParCM), U87wt CM (wtCM), or U87Δ CM (ΔCM). (C) Soft agar colony formation assay quantification of mAstr-Ink4/Arf^−/−-wtEGFR treated with mAstr-Ink4/Arf^−/− CM (ParCM) or mAstr-Ink4/Arf^−/−-ΔEGFR CM (ΔCM). (D) Tumor growth kinetics after subcutaneous injection of U87wt-LacZ (wt-LacZ), U87Δ (Δ), or U87wt-LacZ mixed with U87Δ at ratios of 90:10, 50:50, or 10:90 (100% = 2 × 10⁵ cells). (E) Representative X-Gal staining images of subcutaneous tumors obtained at day 22 after subcutaneous injection of U87wt-LacZ (wt-LacZ), U87Δ (Δ), or U87wt-LacZ mixed with U87Δ at ratios of 90:10, 50:50, or 10:90. Fast red counterstain. (F) Relative tumor volume after analysis of tumor composition by X-Gal staining and Image Pro-Analyzer 6.2 software of tumors obtained at day 22 after subcutaneous injection of indicated mixtures of U87wt and U87Δ. Error bars in all experiments represent mean ± SEM. One-way ANOVA and two-tail t-test were used to compare samples. (**) P < 0.001. n = 6 for subcutaneous injection; n = 3 for soft agar assay.

Figure 3.

EGFR and STAT3 are activated in mixed tumors and after in vitro treatment of wtEGFR cell with ΔEGFR cell CM. (A) Analysis of EGFR and STAT3 phosphorylation by Western blot (left) and densitometry (right) of tumor lysates obtained after intracranial injection of indicated U87 cell line derivative mixtures. Every lane represents a different tumor sample. Arrows indicate wtEGFR or ΔEGFR bands. Error bars represent mean ± SEM (n = 3). Mann-Whitney U-test was used to compare results. (*) P < 0.05. (B) Western blot analysis of U87wt lysates untreated (−) or treated with 50 ng/mL EGF, or U87Par CM (ParCM) or U87Δ CM (ΔCM) showing activation of EGFR, ERK, Akt, and STAT3 pathways when treated with ΔCM. (C) Western blot analysis of mAstr-Ink4/Arf^−/−-wtEGFR lysates untreated (−) or treated with mAstr-Ink4/Arf^−/−-Par CM (ParCM), mAstr-Ink4/Arf^−/−-ΔEGFR CM (ΔCM), or 2.5 ng/mL EGF. (D) Western blot analysis of lysates of U87wt treated with U87Δ CM (ΔCM), with or without pretreatment with 2 μM EGFR inhibitors gefitinib (G) or erlotinib (E). (E) Western blot analysis of lysates of U87wt untreated (−) or treated with serum-free medium (SFM), EGF, or U87Δ CM (ΔCM), with or without preincubation with EGFR ligand trap (501-Fc). Actin and/or total protein were used as loading controls.

Figure 4.

IL-6 and LIF are up-regulated in ΔEGFR-expressing cells, and IL-6 overexpression enhances wtEGFR tumor growth and soft agar colony formation. Real-time PCR for IL-6 (A) and LIF (B) expression in U87MG and mAstr-Ink4/Arf^−/− (Par) cells, or cells engineered to overexpress wtEGFR (wt), ΔEGFR (Δ), or ΔEGFR with a dead kinase domain (DK). (C) ELISA quantification of IL-6 (top) and multiplex quantification of LIF (bottom) in supernatants of U87Par (Par), U87wt (wt), U87Δ (Δ), and U87DK (DK) cultures after 48 h of starvation. (D) Soft agar colony formation assay quantification of U87wt cells treated with normal media (Neg) U87Δ CM (ΔCM), U87Par CM (ParCM), or U87Par-IL6 CM (Par-IL-6 CM). (E) Tumor growth kinetics after subcutaneous injection of U87wt 100%, or U87wt mixed with U87Par or U87Par-IL6 at a ratio of 90%:10% (100% = 1 × 10⁶ cells). Inserted in the graph is the tumor growth kinetics for U87Par 10% and U87Par-IL6 10%. Error bars in all experiments represent mean ± SEM. One-way ANOVA and two-tail t-test were used to compare samples. (*) P < 0.05; (**) P < 0.001. n = 3 for soft agar assay; n = 6 for subcutaneous injections.

Figure 5.

IL-6 and LIF are responsible for wtEGFR stimulation and growth enhancement via gp130–EGFR interaction. (A) Western blot analysis of EGFR and STAT3 phosphorylation in U87wt lysates untreated (−) or treated with recombinant IL-6, LIF, or both, or with U87Δ CM (ΔCM) pretreated or not with anti-IL-6-neutralizing and anti-LIF-neutralizing antibodies (IL-6n + LIFn). (B) Western blot analysis of EGFR and STAT3 phosphorylation in mAstr-Ink4/Arf^−/−-wtEGFR lysates untreated (−) or treated with recombinant LIF or with mAstr-Ink4/Arf^−/−-ΔEGFR CM (ΔCM) pretreated or not with anti-LIF-neutralizing (LIFn) or anti-gp130-neutralizing (gp130n) antibodies. (C) Western blot analysis of U87wt lysates after stimulation with U87Δ CM (ΔCM) pretreated or not with anti-IL-6-neutralizing (IL-6n) or anti-gp130-neutralizing (gp130n) antibodies. (D) Coimmunoprecipitation of EGFR and gp130 in U87wt lysates untreated (−) or treated with U87Δ CM (ΔCM). Total lysates were loaded as controls. IgG immunoprecipitation was used as negative control. (E) Tumor volumes 17 d after subcutaneous injection of U87wt (wt) nontransfected or transfected with 25 nM luciferase siRNA (wt-Luc siRNA) or gp130 siRNA (wt-gp130 siRNA) alone or mixed with U87Δ at a ratio 90:10, respectively. (F,G) Tumor volumes 24 d (F) or 21 d (G) after subcutaneous injection of U87wt alone or mixed with U87Δ transfected with 25 nM luciferase siRNA (Δ-Luc siRNA) and IL-6 siRNA (Δ-IL6 siRNA) (F), or LIF siRNA (Δ-LIF siRNA) (G) at a ratio of 90:10, respectively (100% = 1 × 10⁶ cells). Error bars represent mean ± SEM. One-way ANOVA and two-tail t-test were used for statistical analysis; n = 6. (*) P < 0.05; (**) P < 0.001.

Figure 6.

IL-6 and/or LIF expression correlates with ΔEGFR-positive human GBM clinical samples and glioma stem cells. (A) Analysis of ΔEGFR and IL-6 (left) or LIF (right) expression by real-time PCR in 19 human GBM samples. (B) Analysis of ΔEGFR and IL-6 (left) or LIF (right) expression by real-time PCR in nine human glioma stem cell lines. Error bars in all experiments represent mean ± SEM. Expression levels three times or more the expression in normal brain were considered as high expression. (*) P < 0.05., Fisher's exact test.

Figure 7.

Model of paracrine-mediated tumor enhancement and maintenance of glioma heterogeneity. (Top panel) Cells expressing ΔEGFR (stippled) secrete elevated levels of IL-6 family cytokines IL-6 and/or LIF, which promote the in vivo growth of cells with high levels of wtEGFR (dark grey), thus maintaining the heterogenic composition of the tumor. At the molecular level, IL-6 and LIF bind to their respective receptors, IL-6R and LIFR, which form oligomeric complexes with two or one subunits, respectively, of the common signal transducer gp130. Upon ligand binding, gp130 complexes recruit and activate JAK kinases (not shown), which phosphorylate gp130, LIFR, and STAT3 at Tyr705, inducing its transcriptional activation function. Activated gp130 interacts with and transactivates EGFR, leading to receptor-mediated signaling pathway activation (Akt and MAPK activation). (?) The participation of IL-6R and LIFR, as well as their ligands, in gp130:EGFR complexes has yet to be determined. Coordinate activation of STAT3, Akt, and MAPK enhance the survival and proliferation of cells expressing amplified wtEGFR.