Shuli Xia

c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype

The tyrosine kinase c-Met promotes the formation and malignant progression of multiple cancers. It is well known that c-Met hyperactivation increases tumorigenicity and tumor cell resistance to DNA damaging agents, properties associated with tumor-initiating stem cells. However, a link between c-Met signaling and the formation and/or maintenance of neoplastic stem cells has not been previously identified. Here, we show that c-Met is activated and functional in glioblastoma (GBM) neurospheres enriched for glioblastoma tumor-initiating stem cells and that c-Met expression/function correlates with stem cell marker expression and the neoplastic stem cell phenotype in glioblastoma neurospheres and clinical glioblastoma specimens. c-Met activation was found to induce the expression of reprogramming transcription factors (RFs) known to support embryonic stem cells and induce differentiated cells to form pluripotent stem (iPS) cells, and c-Met activation counteracted the effects of forced differentiation in glioblastoma neurospheres. Expression of the reprogramming transcription factor Nanog by glioblastoma cells is shown to mediate the ability of c-Met to induce the stem cell characteristics of neurosphere formation and neurosphere cell self-renewal. These findings show that c-Met enhances the population of glioblastoma stem cells (GBM SCs) via a mechanism requiring Nanog and potentially other c-Met–responsive reprogramming transcription factors.

DNA methylation presents distinct binding sites for human transcription factors

DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI: http://dx.doi.org/10.7554/eLife.00726.001

Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48u2009h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell–matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.

Targeting the c-Met Pathway Potentiates Glioblastoma Responses to γ-Radiation

Purpose: Resistance to current cytotoxic therapies limits the treatment of most solid malignancies. This results, in part, from the overactivation of receptor tyrosine kinases and their downstream pathways in tumor cells and their associated vasculature. In this report, we ask if targeting the multifunctional mitogenic, cytoprotective, and angiogenic scatter factor/hepatocyte growth factor (SF/HGF)/c-Met pathway potentiates antitumor responses to γ-radiation. Experimental Design: Endogenous expression of SF/HGF and c-Met was targeted in U87 MG human malignant glioma cells and xenografts using chimeric U1/ribozymes. The effects of U1/ribozymes ± γ-radiation on glioma cell proliferation, apoptosis, xenograft growth, and animal survival were examined. Results: U1/ribozymes knocked down SF/HGF and c-Met mRNA and protein levels, sensitized cells to γ-radiation (P < 0.005), and enhanced radiation-induced caspase-dependent cytotoxicity in vitro (P < 0.005). Intravenous U1/ribozyme therapy as liposome/DNA complexes or radiation alone modestly and transiently inhibited the growth of s.c. U87 xenografts. Combining the therapies caused tumor regression and a 40% tumor cure rate. In animals bearing intracranial xenografts, long-term survival was 0% in response to radiation, 20% in response to intratumoral adenoviral-based U1/ribozyme delivery, and 80% (P < 0.0005) in response to combining U1/ribozymes with radiation. This apparent synergistic antitumor response was associated with a ∼70% decrease in cell proliferation (P < 0.001) and a ∼14- to 40-fold increase in apoptosis (P < 0.0001) within xenografts. Conclusions: Targeting the SF/HGF/c-Met pathway markedly potentiates the antiglioma response to γ-radiation. Clinical trials using novel SF/HGF/c-Met pathway inhibitors in glioma and other malignancies associated with c-Met activation should ultimate include concurrent radiation and potentially other cytotoxic therapeutics.

Construction of human activity-based phosphorylation networks.

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Brutons tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

Transcription-Dependent Epidermal Growth Factor Receptor Activation by Hepatocyte Growth Factor

The mechanisms and biological implications of coordinated receptor tyrosine kinase coactivation remain poorly appreciated. Epidermal growth factor receptor (EGFR) and c-Met are frequently coexpressed in cancers, including those associated with hepatocyte growth factor (HGF) overexpression, such as malignant astrocytoma. In a previous analysis of the HGF-induced transcriptome, we found that two EGFR agonists, transforming growth factor-α and heparin-binding epidermal growth factor–like growth factor (HB-EGF), are prominently up-regulated by HGF in human glioma cells. We now report that stimulating human glioblastoma cells with recombinant HGF induces biologically relevant EGFR activation. EGFR phosphorylation at Tyr845 and Tyr1068 increased 6 to 24 h after cell stimulation with HGF and temporally coincided with the induction of transforming growth factor-α (∼5-fold) and HB-EGF (∼23-fold) expression. Tyr845 and Tyr1068 phosphorylation, in response to HGF, was inhibited by cycloheximide and actinomycin D, consistent with a requirement for DNA transcription and RNA translation. Specifically, blocking HB-EGF binding to EGFR with the antagonist CRM197 inhibited HGF-induced EGFR phosphorylation by 60% to 80% and inhibited HGF-induced S-G2-M transition. CRM197 also inhibited HGF-induced anchorage-dependent cell proliferation but had no effect on HGF-mediated cytoprotection. These findings establish that EGFR can be activated with functional consequences by HGF as a result of EGFR ligand expression. This transcription-dependent cross-talk between the HGF receptor c-Met and EGFR expands our understanding of receptor tyrosine kinase signaling networks and may have considerable consequences for oncogenic mechanisms and cancer therapeutics. (Mol Cancer Res 2008;6(1):139–50)

DNER, an epigenetically modulated gene, regulates glioblastoma-derived neurosphere cell differentiation and tumor propagation.

Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem‐like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone‐modifying agents to elucidate mechanisms by which the phenotype and tumor‐promoting capacity of GBM‐derived neoplastic stem‐like cells are regulated. Using established GBM‐derived neurosphere lines and low passage primary GBM‐derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch‐like epidermal growth factor‐related receptor (DNER), inhibited the growth of GBM‐derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor‐promoting effects of canonical Notch signaling in brain cancer stem‐like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem‐like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies. STEM CELLS 2009;27:1473–1486

Krüppel‐Like Family of Transcription Factor 9, a Differentiation‐Associated Transcription Factor, Suppresses Notch1 Signaling and Inhibits Glioblastoma‐Initiating Stem Cells

Tumor‐initiating stem cells (alternatively called cancer stem cells, CSCs) are a subpopulation of tumor cells that plays unique roles in tumor propagation, therapeutic resistance, and tumor recurrence. It is becoming increasingly important to understand the molecular signaling that regulates the self‐renewal and differentiation of CSCs. Transcription factors are critical for the regulation of normal and neopolastic stem cells. Here, we examined the expression and function of the Krüppel‐like family of transcription factors (KLFs) in human glioblastoma (GBM)‐derived neurosphere lines and low‐passage primary GBM‐derived neurospheres that are enriched for tumor‐initiating stem cells. We identify KLF9 as a relatively unique differentiation‐induced transcription factor in GBM‐derived neurospheres. KLF9 is shown to induce neurosphere cell differentiation, inhibit neurosphere formation, and inhibit neurosphere‐derived xenograft growth in vivo. We also show that KLF9 regulates GBM neurosphere cells by binding to the Notch1 promoter and suppressing Notch1 expression and downstream signaling. Our results show for the first time that KLF9 has differentiating and tumor‐suppressing functions in tumor‐initiating stem cells. STEM CELLS 2011;29:20–31

EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumors in adults. Uncontrolled proliferation and abnormal cell migration are two prominent spatially and temporally disassociated characteristics of GBMs. In this study, we investigated the role of the receptor tyrosine kinase EphB2 in controlling the proliferation/migration dichotomy of GBM. We studied EphB2 gain of function and loss of function in glioblastoma-derived stem-like neurospheres, whose in vivo growth pattern closely replicates human GBM. EphB2 expression stimulated GBM neurosphere cell migration and invasion, and inhibited neurosphere cell proliferation in vitro. In parallel, EphB2 silencing increased tumor cell proliferation and decreased tumor cell migration. EphB2 was found to increase tumor cell invasion in vivo using an internally controlled dual-fluorescent xenograft model. Xenografts derived from EphB2-overexpressing GBM neurospheres also showed decreased cellular proliferation. The non-receptor tyrosine kinase focal adhesion kinase (FAK) was found to be co-associated with and highly activated by EphB2 expression, and FAK activation facilitated focal adhesion formation, cytoskeleton structure change and cell migration in EphB2-expressing GBM neurosphere cells. Taken together, our findings indicate that EphB2 has pro-invasive and anti-proliferative actions in GBM stem-like neurospheres mediated, in part, by interactions between EphB2 receptors and FAK. These novel findings suggest that tumor cell invasion can be therapeutically targeted by inhibiting EphB2 signaling, and that optimal antitumor responses to EphB2 targeting may require concurrent use of anti-proliferative agents.

Sensitization of glioma cells to Fas-dependent apoptosis by chemotherapy-induced oxidative stress.

A prominent feature of glioblastoma is its resistance to death from Fas pathway activation. In this study, we explored the modulation of Fas-induced glioblastoma death with chemotherapeutic agents. Camptothecin significantly increased the glioblastoma cell death response to Fas receptor activation regardless of p53 status. Sublethal concentrations of camptothecin reduced the IC50 of agonistic anti-Fas antibody (CH-11) 10-fold, from 500 to 50 ng/mL, in human U87 glioblastoma cells (p53 wild-type). Cell viability in response to camptothecin, CH-11 alone, and the combination of camptothecin + CH-11 was found to be 84%, 85%, and 47% (P < 0.001), respectively. A similar pattern of relative cytotoxicity was found in U373 cells (p53 mutant). We further examined the pathways and mechanisms involved in this apparent synergistic cytotoxic response. Cell death was found to be predominantly apoptotic involving both extrinsic and intrinsic pathways as evidenced by annexin V staining, cleavage of caspases (3, 8, and 9), increased caspase activities, Smac release, and cytoprotection by caspase inhibitors. Expression of Fas-associated death domain, and not Fas, Fas ligand, or caspase proteins, increased following cell treatment with camptothecin + CH-11. Camptothecin treatment enhanced c-jun-NH2-kinase activation in response to CH-11, but inhibition of c-jun-NH2-kinase did not prevent cell death induced by the combination treatment. Reactive oxygen species, especially H2O2, were elevated following camptothecin treatment; and H2O2 enhanced cell death induced by CH-11. The antioxidants glutathione and N-acetyl-cysteine prevented cell death induced by camptothecin + CH-11. These findings show that camptothecin synergizes with Fas activation to induce glioblastoma apoptosis via a mechanism involving reactive oxygen species and oxidative stress pathways.