Seung Bae Rho

Dkk3, downregulated in cervical cancer, functions as a negative regulator of β‐catenin

The Wnt/β‐catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt‐induced stabilization of the β‐catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real‐time PCR revealed that the Dkk3 gene is frequently downregulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. This promoter methylation was associated with reduced expression of Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction of Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated β‐catenin‐responsive luciferase activity in a dose‐dependent manner and decreased the β‐catenin levels. By utilizing a yeast two‐hybrid screen, βTrCP, a negative regulator of β‐catenin was identified as a novel Dkk3‐interacting partner. Coexpression with βTrCP synergistically enhanced the inhibitory function of Dkk3 on β‐catenin. The stable expression of Dkk3 blocks the nuclear translocation of β‐catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator of β‐catenin and its downregulation contribute to an activation of the β‐catenin signaling pathway.

Anti-apoptotic protein TCTP controls the stability of the tumor suppressor p53

MINT‐8057126: p53 (uniprotkb:P04637) physically interacts (MI:0915) with TCTP (uniprotkb:P13693) by anti tag coimmunoprecipitation (MI:0007) MINT‐8057160: TCTP (uniprotkb:P13693) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by two hybrid (MI:0018)

Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

Expression of carbonic anhydrase IX (CA9) was shown to be strongly involved in high incidences of metastasis and poor prognosis in various human tumors. In this study, we investigated the possible role for CA9 in tumor metastases in vitro, using a gene transfection tool in the human cervical carcinoma cell line C33A. Gene expression profiling of CA9-transfected cells (C33A/CA9) and vector-transfected cells (C33A/Mock) was investigated by DNA microarray. The biological functions of differentially expressed genes between the C33A/CA9 and C33A/Mock cells included cell growth, regulation of cell–cell and cell–extracellular matrix adhesion and cytoskeletal organization. Immunofluorescent stain and Matrigel culture showed cytoskeletal remodeling, disassembled focal adhesion, weakened cell-cell adhesion and increased motility in C33A/CA9 cells. These invasive and metastatic phenotypes were associated with Rho-GTPase-related epithelial-mesenchymal transition. Inhibition of the Rho/Rho kinase pathway by a ROCK inhibitor (Y27632) and si-Rho (short interference RNA against RhoA) showed that Rho-GTPase signaling was involved in cellular morphologic and migratory changes. The effect of CA9 on Rho-GTPase signaling was also confirmed by silencing CA9 expression. Our results suggest that CA9 overexpression induces weakening of cell adhesions and augmented cell motility by aberrant Rho-GTPase signal transduction. Our study shows an underlying mechanism of CA9-related enhanced metastatic potential of tumor cells.

Thioridazine induces apoptosis by targeting the PI3K/Akt/mTOR pathway in cervical and endometrial cancer cells

Recently, thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-methylthiophenothiazine), a well-known anti-psychotic agent was found to have anti-cancer activity in cancer cells. However, the molecular mechanism of the agent in cellular signal pathways has not been well defined. Thioridazine significantly increased early- and late-stage apoptotic fraction in cervical and endometrial cancer cells, suggesting that suppression of cell growth by thioridazine was due to the induction of apoptosis. Cell cycle analysis indicated thioridazine induced the down-regulation of cyclin D1, cyclin A and CDK4, and the induction of p21 and p27, a cyclin-dependent kinase inhibitor. Additionally, we compared the influence of thioridazine with cisplatin used as a control, and similar patterns between the two drugs were observed in cervical and endometrial cancer cell lines. Furthermore, as expected, thioridazine successfully inhibited phosphorylation of Akt, phosphorylation of 4E-BP1 and phosphorylation of p70S6K, which is one of the best characterized targets of the mTOR complex cascade. These results suggest that thioridazine effectively suppresses tumor growth activity by targeting the PI3K/Akt/mTOR/p70S6K signaling pathway.

Insulin-like growth factor binding protein-5 (IGFBP-5) acts as a tumor suppressor by inhibiting angiogenesis

Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth control, induction of apoptosis and other IGF-stimulated signaling pathways. In this study, we focused on characterizing the specific function of IGFBP-5 as novel antiangiostatic factor. Overexpression of IGFBP-5 suppressed the tube formation as well as the biological functions of angiostatic activity in vivo. This result is due to the reduced expressions of phosphorylated protein kinase B and phosphorylated endothelial NO synthase, which plays important roles in the regulation of angiogenesis when stimulated by vascular endothelial growth factor. Further, IGFBP-5 expression prevented tumor growth and inhibited tumor vascularity in a xenograft model of human ovarian cancer. These results are the first evidence showing that IGFBP-5 plays a role as tumor suppressor by inhibiting angiogenesis.

Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.

IFN-γ/IRF-1-induced p27kip1 down-regulates telomerase activity and human telomerase reverse transcriptase expression in human cervical cancer

Telomerase activation is regulated by the expression of human telomerase reverse transcriptase (hTERT) and is a key step in the development of human cancers. Interferon‐γ (IFN‐γ) signaling induces growth arrest in many tumors through multiple regulatory mechanisms. The p27 tumor suppressor protein inhibits the formation of tumors through the induction of cell cycle arrest and/or apoptosis. We demonstrate here that p27 Kip1 inhibits hTERT mRNA expression and telomerase activity through post‐transcriptional up‐regulation by IFN‐γ/IRF‐1 signaling. The ectopic expression of p27 suppressed hTERT expression and telomerase activity in human cervical cancer cell lines, HeLa and HT3. Furthermore, hTERT promoter activity of mouse embryonic fibroblasts (MEFs) deficient in p27 (p27−/− MEFs) was significantly higher than that of wild‐type MEFs. Overexpression of p27 suppressed hTERT promoter activity and telomerase activity of p27−/− MEFs. In addition p27 down‐regulated E7 protein expression and in transiently transfected HeLa cells, E7 increased hTERT promoter activity. In conclusion, we propose that inhibition of the hTERT expression and telomerase activity may be a novel tumor suppressor function of p27.

Programmed cell death 6 (PDCD6) inhibits angiogenesis through PI3K/mTOR/p70S6K pathway by interacting of VEGFR-2.

Programmed cell death 6 (PDCD6) was originally found as a pro-apoptotic protein, but its molecular mechanism is not well understood. In this study, we have attempted to investigate the effects of PDCD6 on the inhibition of angiogenesis-mediated cell growth as a novel anti-angiogenic protein. Purified recombinant human PDCD6 inhibited cell migration in a concentration-time-dependent manner. We also found that overexpressed PDCD6 suppressed vascular endothelial growth factor (VEGF)-induced proliferation, invasion, and capillary-like structure tube formation in vitro. PDCD6 suppressed phosphorylation of signaling regulators downstream from PI3K, including Akt, mammalian target of rapamycin (mTOR), glycogen synthase kinase-3β(GSK-3β), ribosomal protein S6 kinase (p70S6K), and also decreased cyclin D1 expression. We found binding PDCD6 to VEGFR-2, a key player in the PI3K/mTOR/P70S6K signaling pathway. Taken together, these data suggest that PDCD6 plays a significant role in modulating cellular angiogenesis.

Elevated level of SUMOylated IRF-1 in tumor cells interferes with IRF-1-mediated apoptosis

SUMOylation of transcription factors often attenuates transcription activity. This regulation of protein activity allows more diversity in the control of gene expression. Interferon regulatory factor-1 (IRF-1) was originally identified as a regulator of IFN-α/β, and its expression is induced by viral infection or IFN stimulation. Accumulating evidence supports the theory that IRF-1 functions as a tumor suppressor and represses the transformed phenotype. Here we report that the level of SUMOylated IRF-1 is elevated in tumors. Site-directed mutagenesis experiments disclose that the SUMOylation sites of IRF-1 are identical to the major ubiquitination sites. Consequently, SUMOylated IRF-1 displays enhanced resistance to degradation. SUMOylation of IRF-1 attenuates its transcription activity, and SUMOylated IRF-1 inhibits apoptosis by repression of its transcriptional activity. These data support a mechanism whereby SUMOylation of IRF-1 inactivates its tumor suppressor function, which facilitates resistance to the immune response.

The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity

The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.