Hyuk-Jin Cha

Inhibition of pluripotent stem cell-derived teratoma formation by small molecules

Significance We found that quercetin/YM155-induced selective cell death is sufficient to completely inhibit teratoma formation after transplantation of human pluripotent stem cell (hPSC)-derived cells. These data provide the first “proof of concept” that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy. The future of safe cell-based therapy rests on overcoming teratoma/tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together, these results provide the “proof of concept” that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy.

Genetic modification of human adipose-derived stem cells for promoting wound healing

BACKGROUNDnDiverse growth factors secreted from human adipocyte-derived stem cells (hASCs) that support or manage adjacent cells have been studied for therapeutic potentials to a variety of pathological models. However, senescent growth arrest in hASCs during in vitro culture and subsequent defective differentiation potential, have been technical barriers to further genetic modification of hASCs for functional improvement.nnnOBJECTIVEnWe investigated the feasibility of long-term hASC culture to enhance their therapeutic use.nnnMETHODSnWe used a MYC variant to generate hASCs expressing v-myc and determined their growth potential and growth factor secretion profile. We further introduced an AKT variant to generate constitutively active (CA)-Akt/v-myc hASCs. Finally, we tested the ability of promoting wound healing of medium conditioned with CA-Akt/v-myc hASCs.nnnRESULTSnThe v-myc hASCs actively proliferated longer than control hASCs. Increased secretion of vascular endothelial growth factor (VEGF) by v-myc hASCs promoted the migration potential of hASCs and vasculogenesis in co-cultured endothelial cells. Additional genetic modification of v-myc hASCs using CA-Akt further increased VEGF secretion. In addition, injection of CA-Akt/v-myc hASCs-CM into wound-mice model promoted wound healing compared to normal hASCs-CM.nnnCONCLUSIONnGenetic modification of hASCs to stimulate secretion of growth factors is a novel strategy to maximize their paracrine effect and improve their therapeutic potential.

Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity.

Summary Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs) to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR) as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs) were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies.

Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression

Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up‐regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress‐mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell‐cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle‐dependent manner, which is directly governed by APC/CCdh1 at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline‐inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/CCdh1 is important for normal mitotic progression. J. Cell. Biochem. 116: 1602–1612, 2015.

Technical approaches to induce selective cell death of pluripotent stem cells

Despite the recent promising results of clinical trials using human pluripotent stem cell (hPSC)-based cell therapies for age-related macular degeneration (AMD), the risk of teratoma formation resulting from residual undifferentiated hPSCs remains a serious and critical hurdle for broader clinical implementation. To mitigate the tumorigenic risk of hPSC-based cell therapy, a variety of approaches have been examined to ablate the undifferentiated hPSCs based on the unique molecular properties of hPSCs. In the present review, we offer a brief overview of recent attempts at selective elimination of undifferentiated hPSCs to decrease the risk of teratoma formation in hPSC-based cell therapy.

Induction of integrin β3 by sustained ERK activity promotes the invasiveness of TGFβ-induced mesenchymal tumor cells

The emerging roles of integrin β3 in the epithelial-mesenchymal transition (EMT) and drug resistance underline its significance in cancer metastasis and recurrence. However, the molecular mechanism underlying the distinctive expression of integrin β3 is less understood. In the present report, we demonstrated that repetitive exposure to transforming growth factor β (TGFβ), a potent inducer of the EMT, significantly increased the expression of integrin β3 in A549 lung cancer cells with distinct mesenchymal properties, such as actin filament reorganization and invasiveness. Notably, integrin β3 expression was associated to cancer cell invasion and migration, and was determined not by Smad4-dependent pathways but by sustained ERK1/2 activity in the mesenchymal cancer cells. These data suggest that ERK1/2 plays an important role in mediating non-canonical TGFβ signal pathways for integrin β3 expression. Therefore, the targeting of the MEK/ERK activity seems to be a promising therapeutic approach to suppressing EMT-associated cancer progression that potentially occurs in TGFβ-enriched microenvironments, which would lead to the suppression of the metastatic potential of integrin β3-positive cancer cells.

Intact wound repair activity of human mesenchymal stem cells after YM155 mediated selective ablation of undifferentiated human embryonic stem cells

BACKGROUNDnRisk of teratoma formation during human pluripotent stem cell (hPSC)-based cell therapy is one of the technical hurdles that must be resolved before their wider clinical application. To this end, selective ablation of undifferentiated hPSCs has been achieved using small molecules whose application should be safe for differentiated cells derived from the hPSCs.nnnOBJECTIVEnHowever, the functional safety of such small molecules in the cells differentiated from hPSCs has not yet been extensively validated.nnnMETHODnWe used the survivin inhibitor YM155, which induced highly selective cell death of hPSCs for ablating undifferentiated hESCs after differentiation to human mesenchymal stem cells (hMSCs) and examined whether hMSCs remained fully functional after being exposed by YM155.nnnRESULTSnWe demonstrated that human mesenchymal stem cells (hMSCs) derived from human embryonic stem cells (hESCs) remained fully functional in vitro and in vivo, while hESCs were selectively ablated.nnnCONCLUSIONnThese results suggest that a single treatment with YM155 after differentiation of hMSCs would be a valid approach for teratoma-free cell therapy.

Suppression of SIRT2 and altered acetylation status of human pluripotent stem cells: possible link to metabolic switch during reprogramming

Primed human pluripotent stem cells (hPSCs) are highly dependent on glycolysis rather than oxidative phosphorylation, which is similar to the metabolic switch that occurs in cancer cells. However, the molecular mechanisms that underlie this metabolic reprogramming in hPSCs and its relevance to pluripotency remain unclear. Cha et al. (2017) recently revealed that downregulation of SIRT2 by miR-200c enhances acetylation of glycolytic enzymes and glycolysis, which in turn facilitates cellular reprogramming, suggesting that SIRT2 is a key enzyme linking the metabolic switch and pluripotency in hPSCs.

Role of MEK partner-1 in cancer stemness through MEK/ERK pathway in cancerous neural stem cells, expressing EGFRviii

BackgroundGlioma stem cells (GSCs) are a major cause of the frequent relapse observed in glioma, due to their high drug resistance and their differentiation potential. Therefore, understanding the molecular mechanisms governing the ‘cancer stemness’ of GSCs will be particularly important for improving the prognosis of glioma patients.MethodsWe previously established cancerous neural stem cells (CNSCs) from immortalized human neural stem cells (F3 cells), using the H-Ras oncogene. In this study, we utilized the EGFRviii mutation, which frequently occurs in brain cancers, to establish another CNSC line (F3.EGFRviii), and characterized its stemness under spheroid culture.ResultsThe F3.EGFRviii cell line was highly tumorigenic in vitro and showed high ERK1/2 activity as well as expression of a variety of genes associated with cancer stemness, such as SOX2 and NANOG, under spheroid culture conditions. Through meta-analysis, PCR super-array, and subsequent biochemical assays, the induction of MEK partner-1 (MP1, encoded by the LAMTOR3 gene) was shown to play an important role in maintaining ERK1/2 activity during the acquisition of cancer stemness under spheroid culture conditions. High expression of this gene was also closely associated with poor prognosis in brain cancer.ConclusionThese data suggest that MP1 contributes to cancer stemness in EGFRviii-expressing glioma cells by driving ERK activity.

BCL2 induced by LAMTOR3/MAPK is a druggable target of chemoradioresistance in mesenchymal lung cancer

Mesenchymal-type cancers after epithelial mesenchymal transition (EMT) were recently shown to acquire chemoresistance through expressing EMT specific transcription factors. However, druggable (or actionable) target(s) for chemoresistance in mesenchymal-type lung cancers remain unidentified. Here, we used a public clinical genomic database and mesenchymal lung cancer cells (MLCC) model derived from the A549 lung adenocarcinoma cell line to demonstrate that BCL2 expression, which is highly induced in mesenchymal-type lung cancers, as a predictor of poor prognosis in mesenchymal lung cancer patients and association with acquired chemoradioresistance. Thereby, combination treatment with BH3 mimetics, such as ABT-263 and ABT-737, clearly attenuated chemoresistance in MLCCs. BCL2 expression in MLCCs was induced by ERK1 activity through the upregulation of the MEK1/ERK1 scaffold protein MEK partner-1 (MP1). Interfering with the MEK1/MP1/ERK1 axis using a MEK1 inhibitor or MP1 depletion repressed BCL2 expression and sensitized MLCCs to chemoradiotherapy. Taken together, our results suggest that targeting druggable proteins in the MEK1/MP1/ERK1/BCL2 axis, such as MEK1 or BCL2, with currently available FDA approved drugs is a currently feasible approach to improve clinical outcomes of mesenchymal lung cancer patients.