PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

Abstract

Poor prognosis of glioblastoma (GBM) is attributable to the propensity of tumor cells to infiltrate into the brain parenchyma. Protein kinase C (PKC) isozymes are highly expressed or aberrantly activated in GBM. However, how this signaling node translates to GBM cell invasiveness remains unknown. Here, we report that among PKC isoforms, PKCδ is strongly associated with infiltration of GBM cells. Notably, PKCδ enhanced Tyr418 phosphorylation of the non-receptor tyrosine kinase SRC, which in turn activated STAT3 and subsequent NOTCH2 signaling, ultimately leading to GBM cell invasiveness. Furthermore, we showed that PKCδ was aberrantly activated in GBM cells by c-MET, a receptor tyrosine kinase hyperactivated in GBM. In agreement, inhibition either component in the c-MET/PKCδ/SRC/STAT3 signaling axis effectively blocked the NOTCH2 signaling and invasiveness of GBM cells. Taken together, our findings shed a light on the signaling mechanisms behind the constitutive activation of PKCδ signaling in GBM.

Keywords: NOTCH2; PKCδ; c-MET; glioblastoma; infiltration.

Figure 1. Effect of PKCδ on infiltration of GBM cells through mesenchymal transformation

A. Migration and invasion assay in GBM cells transfected with control or PKC isoform siRNAs as indicated. B. Migration and invasion assay in GBM cells transfected with control or PKCδ siRNAs. C. Effect of PKCδ depletion on infiltration of U87 GBM cells in collagen-based matrix three-dimensional (3D) culture system. Scale bar, 100 μm. D. Western blot analysis for mesenchymal markers and regulators in U87 GBM cells transfected with control or PKCδ siRNAs. E. Immunocytochemistry for CDH2 and VIM in U87 GBM cells transfected with control or PKCδ siRNAs. F. Migration and invasion assay in GBM cells transduced with MFG or HA-PKCδ. G. Effect of PKCδ on infiltration of U87 GBM cells in collagen-based matrix 3D culture system. Scale bar, 100 μm. H. Western blot analysis for mesenchymal markers and regulators in U87 GBM cells transduced with MFG or HA-PKCδ. I. Immunocytochemistry for CDH2 and VIM in U87 GBM cells transduced with MFG or HA-PKCδ. J, K. Immunohistochemistry for p-PKCδ (J), and CDH2, VIM (K) in orthotopic U87 cell-xenograft tumors. U87 GBM cells were transduced with pSuper or PKCδ shRNA prior to injection to mice. Scale bar, 200 μm. L, M. q-RT PCR (L) and Western blot analysis (M) for mesenchymal markers and regulators in the orthotopic xenograft tumors. β-actin was used for a loading control. *, P < 0.05 versus control; **, p<0.01 versus control.

Figure 2. PKCδ promotes mesenchymal transformation through activation of SRC and STAT3

A, B. Western blot analysis for phosphorylation status of SRC and STAT3 in GBM cells transfected with control or PKCδ siRNAs (A), or transduced with MFG or HA-tagged PKCδ (B). C, D. Migration and invasion assay in GBM cells transfected with control or SRC (C), or STAT3 siRNAs (D). E. Infiltration of GBM cells transfected with control siRNAs or siRNAs against SRC or STAT3 in collagen-based matrix 3D culture system. Scale bar, 100 μm. F, G. Immunohistochemistry (F) and western blot analysis (G) for p-SRC and p-STAT3 in orthotopic U87 GBM cell-xenograft tumors. U87 GBM cells were transduced with control or PKCδ shRNA prior to orthotopic injection to mice. Scale bar, 200 μm. (H, I) Western blot analysis for CDH2, SNAI2 and ZEB1 H., and immunocytochemistry for CDH2 I. in U87 GBM transfected with control or SRC siRNAs. (J, K) Western blot analysis for CDH2, SNAI2 and ZEB1 J., and immunocytochemistry for CDH2 K. in U87 GBM cells transfected with control or STAT3 siRNAs. L. Western blot analysis for p-STAT3 in U87 GBM cells transfected with control or SRC siRNAs. M. Western blot analysis for p-SRC in U87 GBM cells transfected with control or STAT3 siRNAs. β-actin was used for a loading control. *, P < 0.05 versus control; **, p<0.01 versus control.

Figure 3. NOTCH2 is required for PKCδ-associated mesenchymal transformation

A, B. qRT-PCR for NOTCH isoforms (A) and the ligands (B) in U87 GBM cells transfected with control or PKCδ siRNAs. C, D. Immunocytochemistry for NOTCH2 (C) and ligands JAG1 and -2 (D) in U87 GBM cells transfected with control or PKCδ siRNAs. E. Western blot analysis for NOTCH2 and its ligands JAG1 and -2 in U87 GBM cells transduced with MFG or HA-PKCδ. F, G. Migration and invasion assay in U87 GBM cells transfected with NOTCH2 siRNAs (F) or treated with γ-secretase inhibitor (GSI) (G), as compared to control. H. Infiltration of U87 GBM cells transfected with control or NOTCH2 siRNAs in collagen-based matrix 3D culture system. Scale bar, 100 μm. I. Migration and invasion assay in U87 GBM cells transfected with control siRNAs or siRNAs against JAG1 or -2. J. Western blot analysis for NICD2 in U87 GBM cells transfected with control siRNAs or siRNAs against JAG1 or -2. K, L. Western blot analysis for CDH2, SNAI2 and ZEB1 (K), and immunocytochemistry for CDH2 (L) in U87 GBM cells transfected with NOTCH2 siRNAs or treated with GSI. M, N. Immunohistochemical staining (M) and qRT-PCR (N) for NOTCH-2, JAG1 and -2 in orthotopic xenograft tumors formed by U87 GBM cells transduced with control (pSuper) or PKCδ shRNAs. Scale bar, 200 μm. O, P. qRT-PCR for NOTCH-2, JAG1 and -2 in U87 GBM cells transfected by control siRNAs or siRNAs against SRC (O) or STAT3 (P). Q. Western blot analysis for NICD2 in U87 GBM cells transfected by control siRNAs or siRNAs against SRC or STAT3. β-actin was used for a loading control. *, P < 0.05 versus control; **, p<0.01 versus control.

Figure 4. PKCδ is activated by c-MET

A. Western blot analysis for activation status of PKCδ in U87 GBM cells transfected with control, c-MET, EGFR or EGFRvIII siRNAs. B. Migration and invasion assay in U87 GBM cells transfected with control or c-MET siRNAs. C. Infiltration of X01 GBM cells transfected with control or c-MET siRNAs in collagen-based matrix 3D culture system. Scale bar, 100 μm. D, E. Western blot analysis for activation status of PKCδ, SRC, STAT3 and NOTCH2 (D) or for CDH2, SNAI2 and ZEB1 (E) in U87 GBM cells transfected with control or c-MET siRNAs. F. qRT-PCR for NOTCH-2, JAG1 and -2 in U87 GBM cells transfected by control or c-MET siRNAs. G. Kinase assay of immunoprecipitated c-MET using GSC-PKCδ as a substrate and western blot analysis for p-PKCδ in U87 GBM cells transfected with control or c-MET siRNAs. β-actin was used for a loading control. *, P < 0.05 versus control; **, p<0.01 versus control.

Figure 5. Clinical relevance of PKCδ in GBM patients

A. Immunohistochemistry for PKCδ and p-PKCδ in human normal brain tissues and GBM patients (n = 30). Scale bar, 200 μm. B. PKCδ kinase activity in normal brain tissue and 20 cases of human GBM patients. C. Immunohistochemistry for co-staining of PKCδ and NICD2 in human GBM. Scale bar, 100 μm. D. Kaplan-Meier survival curves of high and low levels of PKCδ in human brain tumor patients with REMBRANDT database. E. Schematic model illustrating the PKCδ-associated signaling pathways leading to infiltration of GBM.