Mingyao Ying

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.

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 48 h 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.

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

Kruppel-like Factor-9 (KLF9) Inhibits Glioblastoma Stemness through Global Transcription Repression and Integrin α6 Inhibition

Background: Cancer cell stemness determines tumor propagation and malignancy by poorly defined mechanisms. Results: The KLF9 transcription factor regulates cell fate and oncogenic pathways by repressing gene transcription in glioblastoma stem cells. Conclusion: KLF9 inhibits glioblastoma cell stemness and tumor growth by directly repressing genes, including ITGA6. Significance: KLF9 and its transcriptional network are potential targets for regulating cancer stem cells and glial malignancies. It is increasingly important to understand the molecular basis for the plasticity of neoplastic cells and their capacity to transition between differentiated and stemlike phenotypes. Kruppel-like factor-9 (KLF9), a member of the large KLF transcription factor family, has emerged as a regulator of oncogenesis, cell differentiation, and neural development; however, the molecular basis for the diverse contextual functions of KLF9 remains unclear. This study focused on the functions of KLF9 in human glioblastoma stemlike cells. We established for the first time a genome-wide map of KLF9-regulated targets in human glioblastoma stemlike cells and show that KLF9 functions as a transcriptional repressor and thereby regulates multiple signaling pathways involved in oncogenesis and stem cell regulation. A detailed analysis of one such pathway, integrin signaling, showed that the capacity of KLF9 to inhibit glioblastoma cell stemness and tumorigenicity requires ITGA6 repression. These findings enhance our understanding of the transcriptional networks underlying cancer cell stemness and differentiation and identify KLF9-regulated molecular targets applicable to cancer therapeutics.

Regulation of Glioblastoma Tumor-Propagating Cells by the Integrin Partner Tetraspanin CD151

Glioblastoma (GBM) stem cells (GSCs) represent tumor-propagating cells with stem-like characteristics (stemness) that contribute disproportionately to GBM drug resistance and tumor recurrence. Understanding the mechanisms supporting GSC stemness is important for developing therapeutic strategies for targeting GSC-dependent oncogenic mechanisms. Using GBM-derived neurospheres, we identified the cell surface tetraspanin family member CD151 as a novel regulator of glioma cell stemness, GSC self-renewal capacity, migration, and tumor growth. CD151 was found to be overexpressed in GBM tumors and GBM neurospheres enriched in GSCs. Silencing CD151 inhibited neurosphere forming capacity, neurosphere cell proliferation, and migration and attenuated the expression of markers and transcriptional drivers of the GSC phenotype. Conversely, forced CD151 expression promoted neurosphere self-renewal, cell migration, and expression of stemness-associated transcription factors. CD151 was found to complex with integrins α3, α6, and β1 in neurosphere cells, and blocking CD151 interactions with integrins α3 and α6 inhibited AKT phosphorylation, a downstream effector of integrin signaling, and impaired sphere formation and neurosphere cell migration. Additionally, targeting CD151 in vivo inhibited the growth of GBM neurosphere-derived xenografts. These findings identify CD151 and its interactions with integrins α3 and α6 as potential therapeutic targets for inhibiting stemness-driving mechanisms and stem cell populations in GBM.

Abstract 3476: Hyaluronan-mediated motility receptor as a novel target for inhibiting glioblastoma stem cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Glioblastoma stem cells (GSCs) are a subpopulation of tumor cells that promote tumor invasion and angiogenesis, therapeutic resistance and tumor recurrence. Here, we study the function of hyaluronan-mediated motility receptor (HMMR) in human GSCs. HMMR is hyper-expressed in human glioblastoma samples relative to lower-grade glioma and non-neoplastic specimens. Hyper-expression of HMMR also correlates with poor survival in glioma patients. HMMR is a multifunctional oncogenic protein that plays essential roles in tumorgenesis. Extracellular HMMR forms a complex with CD44 that upon binding to hyaluronan activates intracellular signaling pathways that regulate tumor cell survival, proliferation and invasion. Intracellular HMMR associates with microtubules, interacts with the mitotic spindle and contributes to tumor progression by promoting genomic instability. HMMR and CD44 are two ubiquitous receptors for hyaluronan, which is a prominent component of the microenvironment in most malignant tumors. CD44 has been identified as a cancer stem cell marker and CD44 directly regulates cancer stem cells in a variety of cancers including glioblastoma. Critical evidence regarding the function of HMMR in actual glioblastoma tumors and in the context of tumor-initiating GSCs is still lacking. Results: We found that HMMR is ubiquitously expressed in a panel of human glioblastoma-derived neurosphere cultures that are enriched for GSCs. Targeting HMMR by lentivirus-mediated shRNA delivery potently disrupted in vitro self-renewal of GSCs and inhibited the expression of key GSC markers, such as CD133, SOX2 and Olig2. HMMR knockdown also blocked the in vivo tumorigenic potential of GSCs in mouse orthotopic xenograft model. Notably, disruption of HMMR in GSCs attenuated the activity of Notch signaling, which supports the cancer stem cell hierarchy in glioblastoma and various other cancer types. Significance: This research identifies HMMR as a novel therapeutic target in the treatment of glioblastoma by depleting the tumor-initiating GSCs. It also introduces a novel hypothesis regarding the mechanistic crosstalk between HMMR-mediated signaling and Notch signaling pathway. Understanding this crosstalk between HMMR and Notch signaling will have a significant impact on current efforts to target Notch driven mechanisms for GBM therapy. Overall, these studies will lay an essential foundation for the development of HMMR-targeting strategies, such as HMMR inhibitors or monoclonal antibodies, as new therapeutic approaches targeting glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3476. doi:1538-7445.AM2012-3476

Krüppel-like factor 9 and histone deacetylase inhibitors synergistically induce cell death in glioblastoma stem-like cells

BackgroundThe dismal prognosis of patients with glioblastoma (GBM) is attributed to a rare subset of cancer stem cells that display characteristics of tumor initiation, growth, and resistance to aggressive treatment involving chemotherapy and concomitant radiation. Recent research on the substantial role of epigenetic mechanisms in the pathogenesis of cancers has prompted the investigation of the enzymatic modifications of histone proteins for therapeutic drug targeting. In this work, we have examined the function of Krüppel-like factor 9 (KLF9), a transcription factor, in chemotherapy sensitization to histone deacetylase inhibitors (HDAC inhibitors).MethodsSince GBM neurosphere cultures from patient-derived gliomas are enriched for GBM stem-like cells (GSCs) and form highly invasive and proliferative xenografts that recapitulate the features demonstrated in human patients diagnosed with GBM, we established inducible KLF9 expression systems in these GBM neurosphere cells and investigated cell death in the presence of epigenetic modulators such as histone deacetylase (HDAC) inhibitors.ResultsWe demonstrated that KLF9 expression combined with HDAC inhibitor panobinostat (LBH589) dramatically induced glioma stem cell death via both apoptosis and necroptosis in a synergistic manner. The combination of KLF9 expression and LBH589 treatment affected cell cycle by substantially decreasing the percentage of cells at S-phase. This phenomenon is further corroborated by the upregulation of cell cycle inhibitors p21 and p27. Further, we determined that KLF9 and LBH589 regulated the expression of pro- and anti- apoptotic proteins, suggesting a mechanism that involves the caspase-dependent apoptotic pathway. In addition, we demonstrated that apoptosis and necrosis inhibitors conferred minimal protective effects against cell death, while inhibitors of the necroptosis pathway significantly blocked cell death.ConclusionsOur findings suggest a detailed understanding of how KLF9 expression in cancer cells with epigenetic modulators like HDAC inhibitors may promote synergistic cell death through a mechanism involving both apoptosis and necroptosis that will benefit novel combinatory antitumor strategies to treat malignant brain tumors.

Abstract 2892: A MYC-driven medulloblastoma model derived from human induced pluripotent stem cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Brain tumors are the most common cause of childhood oncological death, and medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current MB treatments yield five-year survival rates of 60-70%, but usually result in significant neurological, intellectual and physical disabilities. Recent gene expression studies have identified four MB subgroups, many of which have unique clinical and histopathological features. Patients with Group 3 MB are more likely to have aggressive and invasive tumors with large cell/anaplastic histology, and have the worst prognosis. Group 3 MB is characterized by amplification and overexpression oncogenic transcription factor MYC, herein referred to as MYC-driven MB. Modeling MYC-driven MB is critical for developing and testing potential therapies for this highly aggressive MB. Recently, murine MYC-driven MB models have been developed using mouse neural stem cells (NSCs) or neuronal precursor cells (NPCs). But human MB models derived from individual-specific cells are still lacking. Human induced pluripotent stem cells (iPSCs) can be differentiated into various types of cells and hold great promise for developing individual-specific disease models. It is valuable to develop MB models using human iPSCs from both MB patients and unaffected persons. In comparison with mouse-cell-derived MB models, human-iPSC-derived MB models will provide a unique and high-impact platform not only for personalized drug discovery but also for studying the role of individuals distinctive genetic background in carcinogen sensitivity and MB susceptibility. Transcription factor Atoh1 governs the development of cerebellar granule neurons and is essential for MB formation. Here, we induced Atoh1 in human iPSCs to differentiate these cells into NPCs. We further infected these Atoh1-induced NPCs with lentiviruses encoding a stabilized form of MYC (MYCT58A) and dominant-negative p53 (DNp53). These NPCs generated aggressive tumors after being transplanted into mouse cerebellum. NPCs infected by DNp53 alone did not form tumors after 90 days. These MYC-driven tumors were comprised of poorly differentiated, medium to large size cells which showed nuclear molding, prominent nucleoli and numerous mitotic (Ki67+) and apoptotic (Cleaved-Caspase-3+) cells. These tumors also expressed early neuronal lineage marker (β-tubulin III). All these features closely mimic human Group 3 MB. Moreover, we also established neurosphere cultures from these MYC-driven tumors to enrich cancer stem-like cells that have the capability for long-term self-renewal and tumor initiation upon serial transplantations. In summary, we established a novel human-iPSC-drived cancer model for modeling MYC-driven MB. Our results support the feasibility to recapitulate human cancers using progenies derived from human iPSCs. The iPSC-derived MB model we established will facilitate mechanistic studies and drug testing for human aggressive MB. Citation Format: Jonathan Sagal, Charles G. Eberhart, Mingyao Ying. A MYC-driven medulloblastoma model derived from human induced pluripotent stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2892. doi:10.1158/1538-7445.AM2015-2892

Abstract 3014: Hyaluronan-mediated motility receptor maintains stemness and tumorigenic potential of glioblastoma stem cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Glioblastoma (GBM) stem cells (GSCs) are a subpopulation of tumor cells that display stem-like characteristics (stemness) and play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is becoming increasingly important to identify novel therapeutic targets in GSC for anti-GBM therapies. Here, we demonstrate that hyaluronan-mediated motility receptor (HMMR) is hyper-expressed in GBM tumors and supports the self-renewal and tumorigenic potential of GSCs. HMMR silencing impairs GSC self-renewal and inhibits the expression of GSC markers and regulators. HMMR silencing suppresses GSC-derived tumor growth and extends the survival of mice bearing GSC xenografts. HMMR overexpression promotes GSC self-renewal and intracranial tumor propagation. In human GBM tumor specimens, HMMR expression is positively correlated with the expression of cell-stemness-associated markers and regulators. Our findings identify HMMR as a potential therapeutic target for inhibiting GSCs, suggesting therapeutic applications against GBM. Citation Format: Jessica Tilghman, John Laterra, Mingyao Ying. Hyaluronan-mediated motility receptor maintains stemness and tumorigenic potential of glioblastoma stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3014. doi:10.1158/1538-7445.AM2014-3014

Abstract 3310: Regulation of reprogramming factors by c-Met signaling in glioblastoma stem cells

Reprogramming transcription factors such as Sox2, c-Myc, Klf4, Oct4 and Nanog have an essential role in sustaining the growth and self-renewal of embryonic stem cells. Forced expression of these reprogramming factors can reprogram differentiated somatic cells into pluripotent stem cells. Reprogramming factor overexpression has also been found in human cancer stem-like cells including glioblastoma stem cells (GBM SCs). This suggests that reprogramming factors support the self-renewing and tumor-initiating phenotype of cancer stem cells. Hence, the molecular signaling pathways that regulate reprogramming factor and their support of the cancer stem-like phenotype in GBM and other solid tumors are of considerable interest. We recently found that the tyrosine kinase c-Met oncogene up-regulates a network of reprogramming factors consisting of Oct4. Sox2, Klf4, c-Myc and Nanog in GBM-derived neurospheres that are enriched in GBM SCs. Reprogramming factor induction by c-Met was observed under serum-free culture conditions that support neurosphere-formation and in cells that were pre-differentiated in serum-containing medium. Using flow cytometry and quantitative reverse transcription-PCR, we also found that GBM neurosphere cell subpopulations expressing c-Met at high levels also express high levels of Nanog and Sox2 in comparison to low c-Met expressing cells. Using specific pharmacological inhibitors of PI(3)kinase (LY294002, wortmannin), MAPkinase (PD98059, U0126), and Stat3 (Stattic, Janus Kinase JSI-124), we found that c-Met signaling regulates reprogramming factors mainly via two parallel pathways: the Jak-Stat3 and PI(3)K-Akt pathways. Inhibiting Oct4 induction abrogated the capacity of c-Met to induce Sox2, c-Myc, and Nanog but not KLF4 expression. Inhibiting Nanog induction significantly inhibited (84% inhibition) the capacity of c-Met signaling to stimulate GBM SC self renewal as measured by neurosphere formation and Edu incorporation, 84% and 61% inhibition, respectively. Together, these findings indicate that c-Met signaling upregulates an Oct4- and Nanog-dependent reprogramming factor network that supports GBM SCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3310. doi:10.1158/1538-7445.AM2011-3310