EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis

Abstract

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.

Fig. 1

Characterization of invasive, non-angiogenic and angiogenic human glioblastoma xenografts. a Hematoxylin and eosin (H&E) and immunohistochemical staining of sections from invasive (P17) and angiogenic (P13) xenograft tumors with antibodies against CD31, an endothelial marker. Arrows point to an angiogenic area. Arrowheads show the sharp demarcation of angiogenic tumors. N depicts a necrotic area and the asterisk indicates microvascular proliferation. b Area fraction of vascular elements immunostained with vWF in invasive versus angiogenic tumors from two different animals in each group. Quantification was performed at ×200 magnification. P < 0.001; n = 20. c T2- and T1-weighted MRIs with and without contrast show demarcated tumors with contrast enhancement in the angiogenic group (P13), while invasive tumors (P6) have ill-defined borders and no contrast enhancement. d Western blot shows high expression of angiogenic factors in angiogenic (P6, P8, P22) compared to invasive tumors (P1, P3, P13). e Immunohistochemical staining of sections from both groups with antibodies against nestin. Arrows point at the white matter/cortex border demonstrating less invasion into the cortex by angiogenic tumors. f Quantification of invasive cells in cortical areas from two different animals in each group. HPF high microscopic view field (×400 magnification). P < 0.001; n = 10. Values represent mean ± SD. Scale bars 100 μm

Fig. 2

EGFR amplification and activation promote non-angiogenic tumor growth in glioblastoma xenografts. a aCGH of invasive (P17) and angiogenic (P13) tumors. Red circle highlights EGFR amplification. FISH with an EGFR/chromosome 7 probe in red and green, respectively and immunohistochemical staining of sections from invasive and angiogenic tumors with antibodies against wtEGFR and phosphorylated EGFR. Amplification, expression and activation of wtEGFR are present only in the invasive phenotype. b FISH with an EGFR/chromosome 7 probe in red and green, respectively (patient/xenograft labels correspond to Table 1). The majority of tumor cells in xenograft tumors show high EGFR amplification. In contrast, tumor cells from patient biopsies show variable amounts of EGFR amplification. c In vivo passaged EGFR-amplified tumor that stays stably invasive (patient 8) shows also stable wtEGFR and pEGFR expression. In contrast, an in vivo passaged EGFR-amplified tumor that switches to angiogenesis (P17) shows downregulation of wtEGFR and pEGFR in the angiogenic center. N depicts a necrotic area and the arrowheads indicate microvascular proliferation. All pictures are taken from the tumor center. Scale bars 100 μm. d wtEGFR and EGFRvIII western blot of stably invasive xenografts and xenografts that switch to angiogenesis (xenograft labels correspond to Table 1). EGFRvIII expression is lost in stably invasive xenografts, while it is upregulated upon the angiogenic switch. e Immunhistochemical staining with antibodies against wtEGFR and EGFRvIII. EGFRvIII is expressed in a xenograft (A3) that switches to angiogenesis, while wtEGFR is downregulated. Scale bars 100 μm

Fig. 3

EGFR activation promotes invasive/non-angiogenic tumor growth in GBM patient biopsies. Tissue microarray (TMA) of GBM biopsies. a EGFR-amplified GBM biopsies as verified by FISH with an EGFR/chromosome 7 probe in red and green, respectively. H&E sections and angiopoietin2 stainings indicate non-angiogenic (upper panel) versus angiogenic areas (lower panel) in EGFR-amplified tumors. High pEGFR expression is only found in non-angiogenic areas (upper panel). b Immunohistochemical staining of pEGFR positive biopsies selected from the TMA with antibodies against pEGFR and vWF. pEGFR positive tumor areas are non-/less angiogenic compared to angiogenic, pEGFR negative areas within the same biopsies. Scale bars 100 μm. c Area fraction of vascular elements immunostained with vWF from pEGFR positive versus angiogenic, pEGFR negative areas from five different patients. Quantification was performed at ×200 magnification. P < 0.001; n = 10. Values represent mean ± SD

Fig. 4

Cetuximab inhibits growth and invasion of EGFR-amplified tumors. EGFR-amplified xenograft tumors (P8) were treated with cetuximab intracerebrally for 4 weeks using osmotic mini-pumps 6 weeks after tumor implantation. a T2- and T1-weighted MRIs of control and treated tumors with and without contrast. Treated tumors are smaller compared to control tumors. b Quantification of tumor volumes from MRI pictures. P < 0.001, n = 3 (controls), n = 4 (cetuximab). Values represent mean ± SD. c Quantification of proliferating tumor cells from Ki67 immunostained sections at ×400 magnification. P < 0.001, n = 10. Values represent mean ± SD. d Immunohistochemical staining with antibodies against human-specific nestin, used as a tumor cell marker. Asterisk marks the injection site (IS). Scale bars 100 μm. e Quantification of invasive, nestin positive tumor cells. HPF high microscopic view field (×400 magnification). P < 0.001 except: cetuximab proximal to IS versus cetuximab distal to IS, P < 0.01 and control versus 2 weeks post cetuximab, P = 0.196; n = 10. Values represent mean ± SD

Fig. 5

Stable expression of EGFR-CD533 induces an angiogenic switch in EGFR-amplified tumors. Tumor spheroids from EGFR-amplified tumors (P8) were mock infected or infected with lentiviral vectors carrying EGFR-CD533. Infected spheroids were implanted into the brain of nude rats. a Western blot of a control tumor and a tumor transduced with EGFR-CD533 with antibodies against EGFR. b Kaplan–Meier survival curve of EGFR-CD533 and control tumors. The difference in survival is statistically significant (log-rank; P < 0.05). c T2- and T1-weighted MRIs with and without contrast show more demarcated tumors with contrast enhancement in the EGFR-CD533 group, while the control tumors have ill-defined borders and are devoid of contrast enhancement. d Macroscopic, coronal view of rat brains with control and EGFR-CD533 tumors. H&E sections show non-angiogenic versus angiogenic tumor growth in control versus EGFR-CD533 tumors. Immunohistochemical staining with antibodies against pEGFR, vWF and pimonidazole, a marker for hypoxia. N depicts necrotic areas and the arrowheads indicate microvascular proliferation. Scale bars 100 μm. e Quantification of pEGFR positive cells in tumors from two different animals in each group. Quantification was performed at ×400 magnification. P < 0.001; n = 10. f Western blot with antibodies against HIF1A and VEGF. g Western blot with antibodies against pStat3, pAkt and pMAPK

Fig. 6

Expression of EGFR-CD533 promotes a mesenchymal to epithelial-like transition in EGFR-amplified tumors. a Quantification of invasive cells in cortical areas from two different animals in each group. HPF high microscopic view field (×400 magnification). Asterisk P < 0.001; n = 10. Values represent mean ± SD. b Hematoxylin and eosin (H&E) and immunohistochemical staining against nestin show mesenchymal shape of cells in control versus epithelial-like shape in EGFR-CD533 tumors. Scale bars 100 μm. c Western blot with antibodies against vimentin, snail and twist