Cho-Rok Jung

E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis

The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1α (HIF-1α), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1α. UCP is detected coincidently with HIF-1α in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1α and may be a new molecular target for therapeutic intervention in human cancers.

Adenovirus‐mediated transfer of siRNA against PTTG1 inhibits liver cancer cell growth in vitro and in vivo

The pituitary tumor transforming (PTTG) gene family comprises PTTG1, 2, and 3. Forced expression of PTTG1 (securin) induces cellular transformation and promotes tumor development in animal models. PTTG1 is overexpressed in various human cancers. However, the expression and pathogenic implications of the PTTG gene family in hepatocellular carcinoma are largely unknown. Gene silencing using short interfering RNA (siRNA) has become an efficient means to study the functions of genes and has been increasingly used for cancer gene therapy approaches. We report that PTTG1, but not PTTG2 and 3, was highly and frequently expressed in liver cancer tissues from patients and highly in SH‐J1, SK‐Hep1, and Huh‐7 hepatoma cell lines. Adenoviral vector encoding siRNA against PTTG1 (Ad.PTTG1‐siRNA) depleted PTTG1 specifically and efficiently in SH‐J1 hepatoma cells, which resulted in activation of p53 that led to increased p21 expression and induction of apoptosis. The depletion of PTTG1 in HCT116 colorectal cancer cells exhibited a cytotoxic effect in a p53‐dependent manner. Ad.PTTG1‐siRNA‐mediated cytotoxic effect was dependent on expression levels of PTTG1 and p53 in hepatoma cell lines. Huh‐7 hepatoma cells, once transduced with Ad.PTTG1‐siRNA, displayed markedly attenuated growth potential in nude mice. Intra‐tumor delivery of Ad.PTTG1‐siRNA led to significant inhibition of tumor growth in SH‐J1 tumor xenograft established in nude mice. In conclusion, PTTG1 overexpressed in hepatoma cell lines negatively regulates the ability of p53 to induce apoptosis. PTTG1 gene silencing using siRNA may be an effective modality to treat liver cancer, in which PTTG1 is abundantly expressed. (HEPATOLOGY 2006;43:1042–1052.)

Enigma negatively regulates p53 through MDM2 and promotes tumor cell survival in mice

The human E3 ubiquitin ligase murine double minute 2 (MDM2) targets the tumor suppressor p53 for ubiquitination and degradation but also promotes its own ubiquitination and subsequent degradation. As the balance between MDM2 and p53 levels plays a crucial role in regulating cell proliferation and apoptosis, we sought to identify factors selectively inhibiting MDM2 self-ubiquitination. Here we have shown that the LIM domain protein Enigma directly interacts with MDM2 to form a ternary complex with p53 in vitro and in human hepatoma and colon carcinoma cell lines and mouse embryonic fibroblasts. We found that Enigma elicited p53 degradation by inhibiting MDM2 self-ubiquitination and increasing its ubiquitin ligase activity toward p53 in cells. Moreover, mitogenic stimuli such as serum, FGF, and HGF increased Enigma transcription via induction of serum response factor (SRF), leading to MDM2 stabilization and subsequent p53 degradation. We observed similar results in the livers of mice treated with HGF. In humans, we found SRF and Enigma coexpressed with MDM2 but not p53 in several liver and stomach tumors. Finally, we showed that Enigma promoted cell survival and chemoresistance by suppressing p53-mediated apoptosis in both cell lines and a mouse xenograft model. Our findings suggest a role for Enigma in tumorigenesis and uncover a mechanism whereby mitogens attenuate p53 antiproliferative activity through an SRF/Enigma/MDM2 pathway.

PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex

Protein arginine methyltransferase 3 (PRMT3) comprises a region not required for catalytic activity in its amino-terminus and the core domain catalyzing protein arginine methylation. PRMT3 has been shown to interact with the 40S ribosomal protein S2 (rpS2) and methylate arginine residues in the arginine-glycine (RG) repeat region in the amino-terminus of rpS2. We investigated the biological implications of this interaction by delineating the domains that mediate binding between PRMT3 and rpS2. The rpS2 (100-293 amino acids) domain, but not the amino-terminus of rpS2 that includes the RG repeat region was essential for binding to PRMT3 and was susceptible to degradation. The amino-terminus of PRMT3, but not its catalytic core was required for binding to and the stability of rpS2. Overexpressed rpS2 was ubiquitinated in cells, but expression of PRMT3 reduced this ubiquitination and stabilized the rpS2 protein. Recombinant PRMT3 formed an active enzyme complex with endogenous rpS2 in vitro. Recombinant rpS2 in molar excess modestly increased the enzymatic activity of PRMT3 in vitro. Our results suggest that in addition to its catalytic function, PRMT3 may control the level of rpS2 protein in cells by inhibiting ubiquitin-mediated proteolysis of rpS2, while rpS2 may regulate the enzymatic activity of PRMT3 as a likely non-catalytic subunit.

Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno‐associated virus (rAAV) vectors have a number of advantages including in vivo long‐term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector.

Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells

The leucine‐rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinsons disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature.

Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway.

E2‐EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel‐Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia‐inducible factor (HIF)‐1α. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response‐1 (Egr‐1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12‐myristate 13‐acetate induced UCP expression following early induction of Egr‐1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence‐specific DNA‐binding of Egr‐1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr‐1 or SRF increased UCP expression. RNA interference‐mediated depletion of endogenous Egr‐1 or SRF impaired EGF‐ or serum‐mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr‐1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF‐1α protein level under non‐hypoxic conditions, suggesting that the Egr‐1/SRF‐UCP‐VHL pathway is in part responsible for the increased HIF‐1α protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr‐1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth. J. Cell. Biochem. 105: 1117–1127, 2008.

A Liver-specific Gene Expression Panel Predicts the Differentiation Status of in vitro Hepatocyte Models

Alternative cell sources, such as three‐dimensional organoids and induced pluripotent stem cell–derived cells, might provide a potentially effective approach for both drug development applications and clinical transplantation. For example, the development of cell sources for liver cell–based therapy has been increasingly needed, and liver transplantation is performed for the treatment for patients with severe end‐stage liver disease. Differentiated liver cells and three‐dimensional organoids are expected to provide new cell sources for tissue models and revolutionary clinical therapies. However, conventional experimental methods confirming the expression levels of liver‐specific lineage markers cannot provide complete information regarding the differentiation status or degree of similarity between liver and differentiated cell sources. Therefore, in this study, to overcome several issues associated with the assessment of differentiated liver cells and organoids, we developed a liver‐specific gene expression panel (LiGEP) algorithm that presents the degree of liver similarity as a “percentage.” We demonstrated that the percentage calculated using the LiGEP algorithm was correlated with the developmental stages of in vivo liver tissues in mice, suggesting that LiGEP can correctly predict developmental stages. Moreover, three‐dimensional cultured HepaRG cells and human pluripotent stem cell–derived hepatocyte‐like cells showed liver similarity scores of 59.14% and 32%, respectively, although general liver‐specific markers were detected. Conclusion: Our study describes a quantitative and predictive model for differentiated samples, particularly liver‐specific cells or organoids; and this model can be further expanded to various tissue‐specific organoids; our LiGEP can provide useful information and insights regarding the differentiation status of in vitro liver models. (Hepatology 2017;66:1662–1674).

In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells

Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine‐specific gene promoters Kriippel‐like factor 5 monomeric Cherry (KLF5mCherry) and intestine‐specific homeobox enhanced green fluorescence protein (ISXeGFP). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry‐ or eGFP‐expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell‐based therapies and preclinical screenings in the intestinal field.—Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.‐S., Lee, M.‐O., Oh, J.‐H., Lee, J., Kim, S., Jung, C.‐R., Kim, J., Son, M.‐Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. FASEB J. 32,111‐122 (2018). www.fasebj.org

Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial–mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.