Tae-Aug Kim

Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells

Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are “cured” of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications.

Definition of Smad3 Phosphorylation Events That Affect Malignant and Metastatic Behaviors in Breast Cancer Cells

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer.

The Smad7–Skp2 complex orchestrates Myc stability, impacting on the cytostatic effect of TGF-β

ABSTRACT In most human cancers the Myc proto-oncogene is highly activated. Dysregulation of Myc oncoprotein contributes to tumorigenesis in numerous tissues and organs. Thus, targeting Myc stability could be a crucial step for cancer therapy. Here we report Smad7 as a key molecule regulating Myc stability and activity by recruiting the F-box protein, Skp2. Ectopic expression of Smad7 downregulated the protein level of Myc without affecting the transcription level, and significantly repressed its transcriptional activity, leading to inhibition of cell proliferation and tumorigenic activity. Furthermore, Smad7 enhanced ubiquitylation of Myc through direct interaction with Myc and recruitment of Skp2. Ablation of Smad7 resulted in less sensitivity to the growth inhibitory effect of TGF-&bgr; by inducing stable Myc expression. In conclusion, these findings that Smad7 functions in Myc oncoprotein degradation and enhances the cytostatic effect of TGF-&bgr; signaling provide a possible new therapeutic approach for cancer treatment.

Abstract LB-304: Smad7-Skp2 complex orchestrates c-Myc stability

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In most of human cancer, the c-Myc proto-oncogene is highly activated. Dysregulation of c-Myc oncoprotein contributes to drive tumorigenesis in numerous tissues and organs. Thus, targeting c-Myc stability can be a critical step for cancer therapy. Here we report Smad7 as a key molecule to regulate c-Myc stability and activity by recruiting F-box protein, Skp2. Ectopic expression of Smad7 down-regulated the protein level of c-Myc without affecting transcription level and significantly repressed its transcriptional activity, leading to inhibition of cell proliferation and tumorigenic activity. Furthermore, Smad7 enhanced ubiquitination of c-Myc through direct interaction with c-Myc and recruitment of Skp2. Ablation of Smad7 resulted in less sensitivity to the growth inhibitory effect of TGF-β by inducing stable c-Myc expression. In conclusion, these findings that Smad7 functions as a transductory role in c-Myc oncoprotein degradation and enhances the cytostatic effect of TGF-β signaling provide new insightful therapeutic approach for cancer treatment. Citation Format: Tae-Aug Kim, Jin-Muk Kang, John Niderhuber, Seong-Jin Kim. Smad7-Skp2 complex orchestrates c-Myc stability. [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 LB-304. doi:10.1158/1538-7445.AM2014-LB-304

Abstract 5178: Smad7 is integrated mediator for the proteolysis of Myc.

Myc, a pleiotropic transcription factor, plays a prominent role in human cancer progression. The regulation of Myc stability, therefore, is critical to understand the molecular mechanism of tumorigenesis in various cancers. Myc stability is tightly regulated by the activity of ubiqutin ligases, Skp2 and Fbw7. However, a key mediator of this process has not been addressed yet. Here, we have identified Smad7 as an intermediary regulating Myc stability by recruiting F-box protein, Skp2. Microarray analysis using the cell lines stably expressed Smad7, indicates that Smad7 markedly inhibits transcription of, c-myc-target genes including Id1, Id2, and Id3 without affecting c-myc gene expression. The expression of Myc protein, however, is down-regulated by induction of Smad7 in vitro, and in vivo, Smad7 transgene inducible system in primary epidermal keratinocyte. Conversely, the loss of Smad7 is implicated in the increase of endogenous Myc and Id2 expression, indicating that Smad7 is involved in post-translational regulation of Myc. Indeed, Smad7 ubiquitylates Myc through Smad7 PY motif, showing that mutation or deletion on PY motif diminishes the ubiquitylation of Myc. Treatment of proteasome inhibitor abates Smad7-mediated Myc degradation in HaCaT cell line that conditionally express level of human c-myc. Mapping of Smad7 interactions with Myc and Skp2 indicates that c-terminus, MH2 domain, of Smad7 binds to 3LRR/7LRR domain (aa142-390) of Skp2, and also interacts with Fbw7. Furthermore, intermediate domain (aa 162-236) of Myc has been identified to interact with c-terminus of Smad7. In the presence of Skp2 and Fbw7, Smad7 facilitated the degradation of Myc, thus impairing Myc stability. Smad7 also markedly represses Myc-mediated transcriptional activity and its target gene expression. These findings provide a new insight of Smad7 functions as a key mediator for the proteolysis of Myc proto-oncoprotein. Citation Format: Jin-Muk Kang, Ja-Shil Hyun, Bona Lee, Ja-Kyung Kim, Ji-Hee Lee, Seong-Jin Kim, Tae-Aug Kim. Smad7 is integrated mediator for the proteolysis of Myc. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5178. doi:10.1158/1538-7445.AM2013-5178

Abstract 3310: Human artificial chromosome (HAC) vector with a conditional centromere for correction of human genetic deficiencies.

Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. Due to their unique maintenance features and unlimited cloning capacity, HACs avoid the lack of copy number control and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are “cured” of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications. Citation Format: Jung-Hyun Kim, Artem Kononenko, Indri Erliandri, Tae-Aug Kim, Megumi Nakano, Yuichi Iida, J. Carl Barrett, Mitsuo Oshimura, Hiroshi Masumoto, William C. Earnshaw, Vladimir Larionov, Natalay Kouprina. Human artificial chromosome (HAC) vector with a conditional centromere for correction of human genetic deficiencies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3310. doi:10.1158/1538-7445.AM2013-3310

Abstract 4866: The dynamic interaction of nuclear Smads with peri- and centromeric heterochromatin is involved in the modulation of histone modification and chromatin structure

Smads are intracellular signaling transducers of TGF-β family ligands. Through TGF-β activation, phosphorylated R-Smads (Smad2/3) form active heteromeric complexes with co-Smad (Smad4) and translocate to the nucleus where they interact with chromatin to assemble the transcriptional machinery of various target genes. Evidence also indicates that Smads are involved in chromatin modulation via interactions with histone modifiers. The role of pre-existing nuclear Smads in chromatin modulation, however, has not been described, nor have the molecular mechanisms of their dynamic interaction with heterochromatin been elucidated. Here we report that nuclear Smads directly bind to specific sites on heterochromatin, altering chromatin architecture upon TGF-β activation. Subcellular fractionation indicates that both active and inactive nuclear Smad 2/3 remains in the biochemically insoluble fraction. We found that only non-phosphorylated Smad3 interacted with HP1α, a key component of heterochromatin under transcriptionally inactive conditions. The level of HP1α, but not HP1β and γ, was regulated in a TGF-β dependent manner in both Smad 2/3 KO MEFs and mouse mammary epithelial cells. Localization of the Smad3 in the pericentromeric heterochromatin and centromere regions of the human chromosomes suggests Smad3 may function as an organizer of chromatin architecture and chromosome assembly during mitosis. ChIP assay indicated that pericentromeric and centromeric heterochromatin bound Smads were dissociated by TGF-β activation and translocated to target gene promoter regions to activate gene transcription. Interestingly, TGF-β enhanced pericentromeric heterochromatin binding of Smad3 in a Smad3 overexpressed human breast cancer cell line, MCF10Ca1, but dramatically decreased in a cell line with deletion of MH2 domain in Smad3. Furthermore, deletion of MH2 domain markedly increased by more than 100 folds both H3 lys4 dimethylation and lys9 dimethylation in centromeric heterochromatin but diminished them in pericentromeric heterochromatin. This suggests that the MH2 domain of the Smad3 may play a critical role on modulation of heterochromatin structure in a manner that activates or represses genome wide gene transcription activity. In conclusion, these results indicate that nuclear Smads are involved in the modulation of chromatin structure through the regulation of HP1α and their direct interaction with peri- and centromeric heterochromatin. Therefore dysregulation of nuclear Smad expression may increase genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4866.

Mechanisms of TGF-β-Induced Apoptosis in Cancer Cells

Apoptosis is a common regulatory process of multicellular organisms. Transforming growth factor beta (TGF-β) has essential roles in a variety of apoptotic pathways including the mitochondrial apoptotic, death receptor, and other intracellular signaling pathways. The TGF-β-mediated apoptotic process involves not only intracellular proapoptotic responses but also anti-apoptotic signals. Resistance to TGF-β regulatory signals is the most indicative characteristic of many cancer cells during tumorigenesis. Therefore, controlling the homeostatic balance of these regulatory signals is critical for the prevention of tumorigenesis. Understanding the mechanisms of TGF-β-induced apoptosis in cancer cells will provide new insight of anticancer therapy.