c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype Yunqing Lia,b,1, Angela Lia, Martin Glasc,d, Bachchu Lala,b, Mingyao Yinga,b, Yingying Sanga, Shuli Xiaa,b, Daniel Trageserc, Hugo Guerrero-Cázarese, Charles G. Eberhartf, Alfredo Quiñones-Hinojosae,g, Bjorn Schefflerc, and John Laterraa,b,g,h,1 aHugo W. Moser Research Institute at Kennedy Krieger, Baltimore, MD 21205; Departments of bNeurology, eNeurosurgery, fPathology, gOncology, and hNeuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21287; and cInstitute of Reconstructive Neurobiology and dDivision of Clinical Neurooncology, Department of Neurology, University of Bonn Medical Center, D-53105 Bonn, Germany Edited by George F. Vande Woude, Van Andel Research Institute, Grand Rapids, MI, and approved May 12, 2011 (received for review November 10, 2010) The tyrosine kinase c-Met promotes the formation and malignant the formation and/or maintenance of neoplastic stem-like cells. progression of multiple cancers. It is well known that c-Met hyper- However, the dynamic regulation of RFs and their influence on the activation increases tumorigenicity and tumor cell resistance to DNA neoplastic stem cell phenotype remain relatively unknown. damaging agents, properties associated with tumor-initiating stem Signaling initiated by the receptor tyrosine kinase c-Met pro- cells. However, a link between c-Met signaling and the formation motes the formation and malignant progression of multiple can- and/or maintenance of neoplastic stem cells has not been previously cers including gliomas through autocrine/paracrine mechanisms identified. Here, we show that c-Met is activated and functional in activated by c-Met overexpression and/or expression of the c-Met glioblastoma (GBM) neurospheres enriched for glioblastoma tumor- ligand hepatocyte growth factor (HGF) (8). We and others have initiating stem cells and that c-Met expression/function correlates shown that c-Met activation enhances tumor cell resistance to with stem cell marker expression and the neoplastic stem cell DNA damage and enhances the tumor-initiating capacity of phenotype in glioblastoma neurospheres and clinical glioblastoma transformed cell lines, properties that have been attributed to the specimens. c-Met activation was found to induce the expression of neoplastic stem cell phenotype (9–11). In this study, we specifi- reprogramming transcription factors (RFs) known to support em- cally examine the influence of c-Met signaling on GBM-derived bryonic stem cells and induce differentiated cells to form pluripo- neurospheres that are enriched for GBM SCs. We show that MEDICAL SCIENCES tent stem (iPS) cells, and c-Met activation counteracted the effects of c-Met is expressed and activated in GBM neurospheres and es- forced differentiation in glioblastoma neurospheres. Expression of tablish a unique functional relationship between c-Met signaling, the reprogramming transcription factor Nanog by glioblastoma cells RF expression, and the neoplastic SC phenotype. Our results is shown to mediate the ability of c-Met to induce the stem cell suggest that the capacity for c-Met to support the GBM SC phe- characteristics of neurosphere formation and neurosphere cell notype involves an endogenous dynamic mechanism analogous self-renewal. These findings show that c-Met enhances the popula- to cellular reprogramming. tion of glioblastoma stem cells (GBM SCs) via a mechanism requiring Nanog and potentially other c-Met–responsive reprogramming Results transcription factors. c-Met Signaling Is Activated in GBM-Derived Neurospheres. As a first step to determine whether c-Met regulates GBM SCs, we ex- cancer stem cell | hepatocyte growth factor | Sox2 | Oct4 | Klf4 amined c-Met receptor expression, activation, and downstream signaling in human GBM-derived neurosphere lines shown pre- lioblastomas (GBMs) are heterogeneous aggressive neo- viously by ourselves and others to be enriched in tumor-initiating Gplasms containing neoplastic stem-like cells (1). These cells neoplastic stem cells (12, 13), and in low-passage (<5) primary commonly referred to as glioblastoma stem cells (GBM SCs), neurospheres derived directly from human GBM xenograft lines exhibit the capacity for unlimited growth as multicellular spheres (developed and kindly provided by C. David James, University of in defined medium, multilineage differentiation, and efficient tu- California, San Francisco, CA) (14). As shown previously for mor initiation in immune-deficient animals. GBM SCs are cur- established neurosphere lines, the primary neurospheres used in rently believed to play a leading role in therapeutic resistance this study express the stem/progenitor cell markers Sox2, Nestin, and tumor recurrence (2). Defining the origin(s) of GBM SCs and and CD133 when maintained in serum-free neurosphere me- the biochemical/molecular pathways that support the stem-like dium containing epidermal growth factor/fibroblast growth factor tumor-initiating phenotype is of major importance. (EGF/FGF) and express the lineage-specific markers GFAP, Transcription factors such as Sox2, c-Myc, Klf4, Oct4, and Tuj1, and O4 when transferred to serum-containing medium after Nanog have an essential role in sustaining the growth and self- growth factor withdrawal (Fig. S1), consistent with their stem- renewal of embryonic stem (ES) cells. Introducing these tran- like phenotype (1, 15). All of the GBM-derived neurospheres ex- scription factors into mouse and human differentiated somatic cells amined expressed various levels of activated (phospho-Tyr1234/35) results in their reprogramming into pluripotent ES-like cells called c-Met (Fig. 1A). Stimulating neurospheres with the c-Met ligand induced pluripotent stem (iPS) cells (3). Remarkable similarities HGF increased c-Met phosphorylation and activated known exist between stem cell reprogramming and oncogenesis. Both processes are supported by alterations in the expression/function of similar collaborating genes perpetuating subpopulations of cells Author contributions: Y.L., M.G., C.G.E., B.S., and J.L. designed research; Y.L., A.L., B.L., fi M.Y., Y.S., S.X., D.T., and H.G.-C. performed research; M.G., C.G.E., A.Q.-H., and B.S. con- capable of inde nite self-renewal (4). Reprogramming transcrip- tributed new reagents/analytic tools; Y.L., M.G., B.L., B.S., and J.L. analyzed data; and Y.L. tion factors (RFs) display varying degrees of oncogenic potential, and J.L. wrote the paper. are overexpressed in human cancers, and their expression levels The authors declare no conflict of interest. have been correlated with malignant progression and poor prog- This article is a PNAS Direct Submission. nosis (5, 6). Loss of tumor suppressors such as p53 enhances the 1To whom correspondence may be addressed. E-mail: [email protected] or efficiency of iPS cell generation by RFs (7). These similarities [email protected]. implicate mechanisms by which the expression/function of en- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dogenous RFs influences the malignant phenotype by supporting 1073/pnas.1016912108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1016912108 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 c-Met Signaling Supports the GBM SC Phenotype. The capacity to form neurospheres is a biomarker of GBM cell stemness and correlates with tumor-initiating capacity (20). We evaluated the capacity of c-Met to regulate neurosphere formation, neuro- sphere cell proliferation and differentiation, and the formation of neurosphere-derived tumor xenografts. Neurospheres were dissociated to single cells and cultured ± (with or without) HGF or ± SU11274 in medium lacking EGF/FGF. HGF significantly enhanced the neurosphere forming capacity of GBM1A-derived cells by 31 ± 6%. There was a trend toward increased sphere formation in primary Mayo39-derived cells, which was not sig- nificant (Fig. 3A). Conversely, SU11274 significantly diminished the formation of neurospheres by both GBM1A and Mayo39- derived cells by 37% and 35%, respectively (Fig. 3A). Neuro- sphere formation was also inhibited by the chemically distinct c-Met inhibitor PF2341066 (Fig. S5A). Growth factor withdrawal in the presence of serum is a widely used method to force GBM SC differentiation (1, 15). To evaluate Fig. 1. c-Met is expressed and functional in GBM-derived neurospheres. (A) the capacity of c-Met activation to regulate the neurosphere- Immunoblots showing activated (phosphorylated) c-Met in multiple human forming stem cell phenotype under more stringent conditions, GBM-derived neurospheres. (B) Neurosphere cells were cultured overnight neurosphere cells were first subjected to conditions of transient without EGF/FGF and then ± (with or without) HGF for 15 min. HGF induces forced differentiation in serum-containing medium as shown in c-Met, AKT, MAPK, and Stat3 activation (phosphorylation). (C) Neurospheres Fig. S1A. HGF induced these transiently predifferentiated cells to ± fl were treated HGF for 3 h. Immuno uorescent micrographs of neurosphere form neurospheres as determined by limited dilution assay (Fig. 3B cytospins show HGF-induced translocation of Stat3 (green) to DAPI-stained and Fig. S5B). Consistent with its effect on neurosphere forming nuclei (blue). (D) Mayo39 neurosphere cells grown in neurosphere medium fi containing EGF/FGF were treated ± the c-Met inhibitor SU11274 for 1 h. capacity, HGF signi cantly induced neurosphere cell proliferation Immunoblots show