Abstract Glioblastomas (GBMs) maintain their cellular heterogeneity with glioma stem cells (GSCs) producing a variety of tumor cell types. Here, we interrogated the oncogenic roles of Lim domain only 2 (LMO2) in GBM and GSCs in mice and human. High expression of LMO2 was found in human patient-derived GSCs compared to the differentiated progeny cells. LMO2 is required for GSC growth both in vitro and in vivo, as shRNA-mediated LMO2 silencing attenuated tumor growth derived from human GSCs. Further, LMO2 is sufficient to induce stem cell characteristics (stemness) in mouse pre-malignant astrocytes, as forced LMO2 expression facilitated in vitro and in vivo growth of astrocytes derived from Ink4a/Arf null mice and acquisition of GSC phenotypes. A subset of mouse and human GSCs converted into vascular endothelial-like tumor cells both in vitro and in vivo, which phenotype was attenuated by LMO2 silencing and promoted by LMO2 overexpression. Mechanistically, the action of LMO2 for induction of glioma stemness is mediated by transcriptional regulation of Jagged1 resulting in activation of the Notch pathway, whereas LMO2 directly occupies the promoter regions of the VE-cadherin gene for a gain of endothelial cellular phenotype. Subsequently, selective ablation of human GSC-derived VE-cadherin-expressing cells attenuated vascular formation in mouse intracranial tumors, thereby significantly prolonging mouse survival. Clinically, LMO2 expression was elevated in GBM tissues and inversely correlated with prognosis of GBM patients. Taken together, our findings describe novel dual roles of LMO2 to induce tumorigenesis and angiogenesis, and provide potential therapeutic targets in MGs. Citation Format: Sunghak Kim, Eun-Jung Kim, Park Cheol Gyu, Se-Yeong Oh, Minata Mutsuko, Hyunggee Kim, Ichiro Nakano. A hematopoietic stem cell factor drives brain tumor initiating cell genesis through Notch signaling. [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 2321. doi:10.1158/1538-7445.AM2015-2321

Abstract Glioma stem cells (GSCs), which reside within the perivascular niche (PVN) to maintain self-renewal capacity, are responsible for glioblastoma initiation, progression, and recurrence. However, the molecular mechanisms controlling GSCs and endothelial cells in the PVN are poorly understood. Here, we report that PDGF-driven activation of nitric oxide (NO) synthase increases NO-dependent ID4 expression, which promotes Jagged-Notch activity through miR-129 suppression in GSCs and endothelial cells. ID4 also increases PDGF-PDGFR signaling in a direct or miR-129-dependent manner, which drives the PDGFR-NO-ID4 signaling in GSCs and endothelial cells. This signaling circuit promotes tumor progression along with increased GSC self-renewal and growth of tumor vasculature in the PVN, which is dramatically suppressed by Notch inhibitor. ID4 levels correlate positively with NOS2, Hes1, and Hey1 and negatively with miR-129 in primary GSCs. Thus, targeting the PDGFR-NO-ID4 signaling circuit and Notch activity in the PVN might serve as an efficacious therapeutic modality for glioblastoma. Citation Format: Hye-Min Jeon, Hyunggee Kim. PDGFR-ID4-Notch axis is a common signaling pathway activated in glioma stem cells and endothelial cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B34.

Glioblastomas multiforme (GBM) contain highly tumorigenic, self-renewing populations of stem/initiating cells [glioblastoma stem cells (GSC)] that contribute to tumor propagation and treatment resistance. However, our knowledge of the specific signaling pathways that regulate GSCs is limited. The MET tyrosine kinase is known to stimulate the survival, proliferation, and invasion of various cancers including GBM. Here, we identified a distinct fraction of cells expressing a high level of MET in human primary GBM specimens that were preferentially localized in perivascular regions of human GBM biopsy tissues and were found to be highly clonogenic, tumorigenic, and resistant to radiation. Inhibition of MET signaling in GSCs disrupted tumor growth and invasiveness both in vitro and in vivo, suggesting that MET activation is required for GSCs. Together, our findings indicate that MET activation in GBM is a functional requisite for the cancer stem cell phenotype and a promising therapeutic target.

Aberrant activation of receptor tyrosine kinases (RTK) is causally linked to the pathobiological traits of glioblastoma and genesis of glioma stem-like cells (GSC), but the underlying mechanism is still unknown. Here, we show that epidermal growth factor receptor (EGFR) signaling regulates the proliferation, angiogenesis, and acquisition of GSC characteristics by inducing inhibitor of differentiation 3 (ID3) and ID3-regulated cytokines [GRO1 and interleukins (IL)-6 and 8] induction. We found that EGFR-mediated ID3 expression was regulated by Smad5, which was directly phosphorylated by AKT. Furthermore, ID3 alone imparted GSC features to primary astrocytes derived from Ink4a/Arf-deficient mouse, and EGFR-ID3-IL-6 signaling axis gave rise to tumor cell heterogeneity. Conversely, EGFR inhibitors suppressed EGFR-AKT-Smad5-driven induction of ID3, which led to a decrease in the tumorsphere forming ability of GSCs and U87MG cells that possess an active mutant EGFR, EGFRvIII, without obvious cytotoxic effects. However, these cells seemed to regain colonogenic ability after removal of the EGFR inhibitors. Together, the results delineate a novel integrative molecular mechanism in which the RTK-ID signaling pathway governs genesis and maintenance of GBM histopathologic features, such as GSCs-based tumor initiation, progression, and angiogenesis.

Abstract Glioblastomas (GBMs) maintain their cellular heterogeneity with glioma stem cells (GSCs) producing a variety of tumor cell types. Here, we interrogated the oncogenic roles of Lim domain only 2 (LMO2) in GBM and GSCs in mice and human. High expression of LMO2 was found in human patient-derived GSCs compared to the differentiated progeny cells. LMO2 is required for GSC growth both in vitro and in vivo, as shRNA-mediated LMO2 silencing attenuated tumor growth derived from human GSCs. Further, LMO2 is sufficient to induce stem cell characteristics (stemness) in mouse pre-malignant astrocytes, as forced LMO2 expression facilitated in vitro and in vivo growth of astrocytes derived from Ink4a/Arf null mice and acquisition of GSC phenotypes. A subset of mouse and human GSCs converted into vascular endothelial-like tumor cells both in vitro and in vivo, which phenotype was attenuated by LMO2 silencing and promoted by LMO2 overexpression. Mechanistically, the action of LMO2 for induction of glioma stemness is mediated by transcriptional regulation of Jagged1 resulting in activation of the Notch pathway, whereas LMO2 directly occupies the promoter regions of the VE-cadherin gene for a gain of endothelial cellular phenotype. Subsequently, selective ablation of human GSC-derived VE-cadherin-expressing cells attenuated vascular formation in mouse intracranial tumors, thereby significantly prolonging mouse survival. Clinically, LMO2 expression was elevated in GBM tissues and inversely correlated with prognosis of GBM patients. Taken together, our findings describe novel dual roles of LMO2 to induce tumorigenesis and angiogenesis, and provide potential therapeutic targets in MGs. Citation Format: Sunghak Kim, Eun-Jung Kim, Park Cheol Gyu, Se-Yeong Oh, Minata Mutsuko, Hyunggee Kim, Ichiro Nakano. A hematopoietic stem cell factor drives brain tumor initiating cell genesis through Notch signaling. [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 2321. doi:10.1158/1538-7445.AM2015-2321

Abstract Glioma stem cells (GSCs), which reside within the perivascular niche (PVN) to maintain self-renewal capacity, are responsible for glioblastoma initiation, progression, and recurrence. However, the molecular mechanisms controlling GSCs and endothelial cells in the PVN are poorly understood. Here, we report that PDGF-driven activation of nitric oxide (NO) synthase increases NO-dependent ID4 expression, which promotes Jagged-Notch activity through miR-129 suppression in GSCs and endothelial cells. ID4 also increases PDGF-PDGFR signaling in a direct or miR-129-dependent manner, which drives the PDGFR-NO-ID4 signaling in GSCs and endothelial cells. This signaling circuit promotes tumor progression along with increased GSC self-renewal and growth of tumor vasculature in the PVN, which is dramatically suppressed by Notch inhibitor. ID4 levels correlate positively with NOS2, Hes1, and Hey1 and negatively with miR-129 in primary GSCs. Thus, targeting the PDGFR-NO-ID4 signaling circuit and Notch activity in the PVN might serve as an efficacious therapeutic modality for glioblastoma. Citation Format: Hye-Min Jeon, Hyunggee Kim. PDGFR-ID4-Notch axis is a common signaling pathway activated in glioma stem cells and endothelial cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B34.

Glioblastomas multiforme (GBM) contain highly tumorigenic, self-renewing populations of stem/initiating cells [glioblastoma stem cells (GSC)] that contribute to tumor propagation and treatment resistance. However, our knowledge of the specific signaling pathways that regulate GSCs is limited. The MET tyrosine kinase is known to stimulate the survival, proliferation, and invasion of various cancers including GBM. Here, we identified a distinct fraction of cells expressing a high level of MET in human primary GBM specimens that were preferentially localized in perivascular regions of human GBM biopsy tissues and were found to be highly clonogenic, tumorigenic, and resistant to radiation. Inhibition of MET signaling in GSCs disrupted tumor growth and invasiveness both in vitro and in vivo, suggesting that MET activation is required for GSCs. Together, our findings indicate that MET activation in GBM is a functional requisite for the cancer stem cell phenotype and a promising therapeutic target.

Aberrant activation of receptor tyrosine kinases (RTK) is causally linked to the pathobiological traits of glioblastoma and genesis of glioma stem-like cells (GSC), but the underlying mechanism is still unknown. Here, we show that epidermal growth factor receptor (EGFR) signaling regulates the proliferation, angiogenesis, and acquisition of GSC characteristics by inducing inhibitor of differentiation 3 (ID3) and ID3-regulated cytokines [GRO1 and interleukins (IL)-6 and 8] induction. We found that EGFR-mediated ID3 expression was regulated by Smad5, which was directly phosphorylated by AKT. Furthermore, ID3 alone imparted GSC features to primary astrocytes derived from Ink4a/Arf-deficient mouse, and EGFR-ID3-IL-6 signaling axis gave rise to tumor cell heterogeneity. Conversely, EGFR inhibitors suppressed EGFR-AKT-Smad5-driven induction of ID3, which led to a decrease in the tumorsphere forming ability of GSCs and U87MG cells that possess an active mutant EGFR, EGFRvIII, without obvious cytotoxic effects. However, these cells seemed to regain colonogenic ability after removal of the EGFR inhibitors. Together, the results delineate a novel integrative molecular mechanism in which the RTK-ID signaling pathway governs genesis and maintenance of GBM histopathologic features, such as GSCs-based tumor initiation, progression, and angiogenesis.

Glioma stem cells (GSC) possess tumor-initiating potential and are relatively resistant to conventional chemotherapy and irradiation. Thus, they are considered to be major drivers for glioma initiation, progression, and recurrence. However, the precise mechanism governing acquisition of their drug resistance remains to be elucidated. Our previous study has shown that inhibitor of differentiation 4 (ID4) dedifferentiates Ink4a/Arf(-/-) mouse astrocytes and human glioma cells to glioma stem-like cells (induced GSCs or iGSCs). In this article, we report that ID4-driven iGSCs exhibit chemoresistant behavior to anticancer drugs through activation of ATP-binding cassette (ABC) transporters. We found that ID4 enhanced SOX2 protein expression by suppressing microRNA-9* (miR-9*), which can repress SOX2 by targeting its 3'-untranslated region. Consequently, ID4-mediated SOX2 induction enhanced ABCC3 and ABCC6 expression through direct transcriptional regulation, indicating that ID4 regulates the chemoresistance of iGSCs by promoting SOX2-mediated induction of ABC transporters. Furthermore, we found that short hairpin RNA-mediated knockdown of SOX2 in ID4-driven iGSCs resulted in loss of cancer stemness. Moreover, ectopic expression of SOX2 could dedifferentiate Ink4a/Arf(-/-) astrocytes and glioma cells to iGSCs, indicating a crucial role of SOX2 in genesis and maintenance of GSCs. Finally, we found that the significance of the ID4-miR-9*-SOX2-ABCC3/ABCC6 regulatory pathway is recapitulated in GSCs derived from patients with glioma. Together, our results reveal a novel regulatory mechanism by which ID4-driven suppression of miR-9* induces SOX2, which imparts stemness potential and chemoresistance to glioma cells and GSCs.

Melanoma is a serious type of skin cancer that originates from melanocytes. Rodent melanoma models have provided valuable insights into melanoma pathology; however, they often lack applicability to humans owing to genetic, anatomical, physiological, and metabolic differences. Herein, we developed a transgenic porcine melanoma model that closely resembles humans via somatic cell nuclear transfer (SCNT). Our model features the conditional oncogenes cassettes, TP53^R167H and human BRAF^V600E, controlled by melanocyte-specific CreER recombinase. After SCNT, transgenic embryos developed normally, with the capacity to develop porcine embryonic stem cells. Seven transgenic piglets with oncogene cassettes were born through embryo transfer. We demonstrated that Cre recombination-mediated oncogene activation remarkably triggered the mitogen-activated protein kinase pathway in vitro. Notably, intradermal injection of 4-hydroxytamoxifen activated oncogene cassettes in vivo, resulting in melanocytic lesions resembling hyperpigmented nevi with increased proliferative properties similar to early human melanomas. This melanoma-inducing system, heritably transmitted to offspring, supports large-scale studies. The novel porcine model provides a valuable tool for elucidating melanoma development and metastasis mechanism, advancing translational medicine, and facilitating preclinical evaluation of new anticancer drugs.

To elucidate the complex interplay of undifferentiated cancer cells in malignancy, we focus on the crucial mechanisms that maintain the undifferentiated state of cancer stem-like cells, which drive tumor growth and therapy resistance. Here, we identify a protein called dehydrogenase/reductase 13 (DHRS13) that is abundant in undifferentiated glioblastoma cells. DHRS13 is primarily located in the mitochondria and functions as a retinaldehyde reductase, converting all-trans-retinaldehyde to all-trans-retinol with high affinity for NADPH. Mechanistically, DHRS13 prevents glioma stem-like cells from differentiating by blocking retinoic acid signaling, thereby maintaining their undifferentiated state. Remarkably, the depletion of DHRS13 results in mitochondrial reactive oxygen species-driven mitophagy and cell death. Consequently, loss of DHRS13 leads to a significant decrease in tumor initiation and progression. These findings hold promise for the development of strategies that target undifferentiated cancer cells, potentially leading to improved treatment outcomes.