The interference of Notch1 target Hes1 affects cell growth, differentiation and invasiveness of glioblastoma stem cells through modulation of multiple oncogenic targets

Abstract

The invasive and lethal nature of Glioblastoma multiforme (GBM) necessitates the continuous identification of molecular targets and search of efficacious therapies to inhibit GBM growth. The GBM resistance to chemotherapy and radiation it is attributed to the existence of a rare fraction of cancer stem cells (CSC) that we have identified within the tumor core and in peritumor tissue of GBM. Since Notch1 pathway is a potential therapeutic target in brain cancer, earlier we highlighted that pharmacological inhibition of Notch1 signalling by γ-secretase inhibitor-X (GSI-X), reduced cell growth of some c-CSC than to their respective p-CSC, but produced negligible effects on cell cycle distribution, apoptosis and cell invasion. In the current study, we assessed the effects of Hes1-targeted shRNA, a Notch1 gene target, specifically on GBM CSC refractory to GSI-X. Depletion of Hes1 protein induces major changes in cell morphology, cell growth rate and in the invasive ability of shHes1-CSC in response to growth factor EGF. shHes1-CSC show a decrease of the stemness marker Nestin concurrently to a marked increase of neuronal marker MAP2 compared to pLKO.1-CSC. Those effects correlated with repression of EGFR protein and modulation of Stat3 phosphorylation at Y705 and S727 residues. In the last decade Stat3 has gained attention as therapeutic target in cancer but there is not yet any approved Stat3-based glioma therapy. Herein, we report that exposure to a Stat3/5 inhibitor, induced apoptosis either in shHes1-CSC or control cells. Taken together, Hes1 seems to be a favorable target but not sufficient itself to target GBM efficaciously, therefore a possible pharmacological intervention should provide for the use of anti-Stat3/5 drugs either alone or in combination regimen.

Keywords: Hes1; Notch1; differentiation; glioblastoma stem cells; self-renewal.

Figure 1. Downmodulation of Hes1 expression affects Notch1 signaling, self-renewal, oncogenic signaling pathways and cell growth rate in shHes1-CSC

(A) RT-qPCR analyses reveal a significant decrease of Notch1 signaling components including conventional Hes1 targets. (B) Western blot analyses confirm the downmodulation of Notch1 signaling gene profile and highlight the neural differentiation of CSC via upregulation of MAP2 and GFAP and loss of Nestin. (C) Depletion of Hes1 diminishes the phosphorylation levels of Stat3 at Y705 but induces those at S727 residue. Furthermore, noteworthy are a remarkable reduction of EGFR protein the upregulation of PDGFRβ and the downmodulation of expression of angiogenic markers (CD31 and VE-cadherin). (D) Knockdown of Hes1 expression was associated with a highly significant inhibition of the proliferation rate of shHes1-CSC clone 7152 and 7153 vs pLKO.1 cells. Data are expressed as mean ± SD (n = 3), and are representative of three independent experiments. We denote the significant difference between cell clones and control cells (***P ≤ 0.001).

Figure 2. Targeting Hes1 expression induces morphological changes and negatively affects the cell cycle profile in shHes1-CSC

(A–C) Phase-contrast images captured at 200× magnification after 6hs and 48hs in growth conditions, reveal substantial cell changes with attachment of shHes1-CSC on plastic dishes and formation of neuron-like cells (arrows in B,C), contrary to pLKO.1 cells which formed classical not-adherent neurospheres. (D–F) FACS analyses of cell cycle profiles reveal a substantial shift from S phase to G1 fraction of shHes1-CSC clones compared to pLKO.1 cells. Data of flow cytometry are representative of two independent experiments. The difference of percentages in cell cycle distribution between cell clones and p-LKO.1-CSC is consistent and can be considered biologically significant.

Figure 3. Modulation of neural differentiation in shHes1-CSC by immunofluorescence assay

(A–C) Immunostaining for Nestin reveals its upregulation in control cells vs Hes1-depleted cells (arrows). (D–F) Immunostaining for MAP2 reveals its upregulation in Hes1-depleted cells vs control cells (arrows). Images were captured at 400X magnification.

Figure 4. Targeting Hes1 impairs cell invasive abilities, modulates epithelial mesenchymal transition and inflammatory cytokines gene expression

(A) Addition of EGF promoted a significant migration of pLKO.1 cells in cell invasion assay compared to stem medium, contrary to that occurs in shHes1-CSC. PDGFAA and PDGFBB promoted a significant migration of shHes1-CSC clones in comparison to stem medium, instead pLKO.1-CSC are not significantly influenced by PDGFAA and PDGFBB. Error bars represent the mean ± SD of two independent experiments performed in triplicate. *P values < 0.05, **P < 0.01**, ***P < 0.001 vs. control stem medium were considered statistically significant. (B) RT-qPCR analyses of key genes for angiogenesis, EMT, immunomodulation and cell invasion. mRNA expression for cell-substrate molecules interaction (CDH1, CDH5) were drastically downmodulated in shHes1-CSC vs pLKO.1 cells. Few EMT markers (TWIST1, FN1, VIM), together with immunomodulators (NOS2, IL6, IL1B, IL23, NF-κB2) and matrix metalloproteinases (MMP7, MMP9) were similarly downmodulated in shHes1-CSC in comparison to pLKO.1 cells.

Figure 5. Stat3/5 are critical factors for survival of CSC

(A) MTS assay was assessed to evaluate cells sensitivity to SH-4-54, a Stat3/5 inhibitor. CSC undergone three doses of SH-4-54 (0.5–2–5 μM) and tested for cell vitality after 24 h of treatment. Data are represented as mean ± SD (n = 3) and are representative of three independent experiments. ***P < 0.001 vs control was considered highly significant. (B) Western blot analyses reported the effects of SH-4-54 on Caspase-3 activation and PARP1 cleavage (arrowheads). Deleterious effects were observed for Akt, Stat3 and Stat5 and Erk1/2 signaling pathways, Bcl2 and CyclinD1.