Jae-Sik Shin

Foxp3 Expression in p53-dependent DNA Damage Responses

The forkhead transcription factor, Foxp3, is thought to act as a master regulator that controls (suppresses) expression of the breast cancer oncogenes, SKP2 and HER-2/ErbB2. However, the mechanisms that regulate Foxp3 expression and thereby modulate tumor development remain largely unexplored. Here, we demonstrate that Foxp3 up-regulation requires p53 function, showing that Foxp3 expression is directly regulated by p53 upon DNA damage responses in human breast and colon carcinoma cells. Treatment with the genotoxic agents, doxorubicin or etoposide, induced Foxp3 expression in p53-positive carcinoma cells, but not in cells lacking p53 function. Furthermore, knock down of endogenous wild-type p53 using RNA interference abrogated Foxp3 induction by genotoxic agents, and exogenous expression of p53 in cells lacking p53 restored the responsiveness of Foxp3 to DNA-damaging stresses. In addition, Foxp3 knock down blunted the p53-mediated growth inhibitory response to DNA-damaging agents. These results suggest that induction of Foxp3 in the context of tumor suppression is regulated in a p53-dependent manner and implicate Foxp3 as a key determinant of cell fate in p53-dependent DNA damage responses.

p34SEI-1 Inhibits Apoptosis through the Stabilization of the X-Linked Inhibitor of Apoptosis Protein: p34SEI-1 as a Novel Target for Anti–Breast Cancer Strategies

The p34(SEI-1) protein exerts oncogenic effects via regulation of the cell cycle, which occurs through a direct interaction with cyclin-dependent kinase 4. Such regulation can increase the survival of various types of tumor cells. Here, we show that the antiapoptotic function of p34(SEI-1) increases tumor cell survival by protecting the X-linked inhibitor of apoptosis protein (XIAP) from degradation. Our findings show that p34(SEI-1) inhibits apoptosis. This antiapoptotic effect was eliminated by the suppression of p34(SEI-1) expression. We also determined that direct binding of p34(SEI-1) to the BIR2 domain prevents ubiquitination of XIAP. Interestingly, p34(SEI-1) expression is absent or weak in normal tissues but is strongly expressed in tissues obtained from patients with breast cancer. Furthermore, the expression levels of p34(SEI-1) and XIAP seem to be coordinated in human breast cancer cell lines and tumor tissues. Thus, our findings reveal that p34(SEI-1) uses a novel apoptosis-inhibiting mechanism to stabilize XIAP.

Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF)

Background: BRAF is a downstream effector kinase of Ras. Results: RNF149, a RING finger domain-containing E3 ubiquitin ligase, is one of the several proteins shown to interact with wild-type BRAF by tandem affinity purification. Conclusion: RNF149 induces ubiquitination and subsequent proteasomal degradation of wild-type but not mutant BRAF. Significance: This is the first ubiquitin ligase shown to degrade wild-type BRAF in a proteasome-dependent manner. Members of the RAF family (ARAF, BRAF, and CRAF/RAF-1) are involved in a variety of cellular activities, including growth, survival, differentiation, and transformation. An oncogene encodes BRAF, the function of which is linked to MEK activation. BRAF is the most effective RAF kinase in terms of induction of MEK/ERK activity. However, the mechanisms involved in BRAF regulation remain unclear. In the present work, we used a tandem affinity purification approach to show that RNF149 (RING finger protein 149) interacts with wild-type BRAF. The latter protein is a RING domain-containing E3 ubiquitin ligase involved in control of gene transcription, translation, cytoskeletal organization, cell adhesion, and epithelial development. We showed that RNF149 bound directly to the C-terminal kinase-containing domain of wild-type BRAF and induced ubiquitination, followed by proteasome-dependent degradation, of the latter protein. Functionally, RNF149 attenuated the increase in cell growth induced by wild-type BRAF. However, RNF149 did not bind to mutant BRAF or induce ubiquitination thereof. Thus, we show that RNF149 is an E3 ubiquitin ligase active on wild-type BRAF.

p34SEI-1 Inhibits Doxorubicin-Induced Senescence through a Pathway Mediated by Protein Kinase C-δ and c-Jun-NH2-Kinase 1 Activation in Human Breast Cancer MCF7 Cells

In this study, we describe a novel function of the p34SEI-1 protein, which is both an oncogenic protein and a positive regulator of the cell cycle. The p34SEI-1 protein was found to inhibit doxorubicin-induced senescence. We investigated the molecular mechanisms of the inhibitory effect of p34SEI-1 on senescence. First, we found that the activation of protein kinase C-δ (PKC-δ), which is cleaved into a 38 kDa active form from a 78 kDa pro-form, induced after doxorubicin treatment, was inhibited by p34SEI-1. Furthermore, p34SEI-1 induced the ubiquitination of PKC-δ. Yet, there is no interaction between p34SEI-1 and PKC-δ. We also found that the phosphorylation of c-Jun-NH2-kinase 1 (JNK1) induced after doxorubicin treatment was suppressed by p34SEI-1, but not in JNK2. Consistently, pharmacologic or genetic inactivation of either PKC-δ or JNK1 was found to inhibit doxorubicin-induced senescence. In addition, the genetic inactivation of PKC-δ by PKC-δ small interfering RNA resulted in an inhibition of JNK1 activation, but PKC-δ expression was not inactivated by JNK1 small interfering RNA, implying that the activation of JNK1 could be dependently induced by PKC-δ. Therefore, p34SEI-1 inhibits senescence by inducing PKC-δ ubiquitination and preventing PKC-δ–dependent phosphorylation of JNK1. [Mol Cancer Res 2009;7(11):1845–53]

p34SEI-1 inhibits ROS-induced cell death through suppression of ASK1

Apoptosis signal-regulating kinase 1 (ASK1) is a key factor for controlling several cellular events including the cell cycle, senescence and apoptosis, in response to reactive oxygen species (ROS). However, the mechanisms that regulate ASK1 protein levels remain largely unexplored. In this study, we demonstrate that p34SEI-1, a positive cell cycle regulator with an oncogenic potential, inhibits ROS-induced cell death by suppressing ASK1. We first found that p34SEI-1-expressing cells have enhanced resistance to hydrogen peroxide (H2O2). Moreover, ectopic expression of p34SEI-1 clearly inhibited H2O2-induced phosphorylation of ASK1 in the colon cancer cell lines- HCT116 and SW620-in association with a decrease in ASK1 protein levels. Interestingly, p34SEI-1 induced ubiquitination of ASK1, however, no direct interaction was found between p34SEI-1 and ASK1. These results suggest that p34SEI-1 inhibits ROS-induced cell death through by indirectly inducing ubiquitination of ASK1.

A novel small-molecule IAP antagonist, AZD5582, draws Mcl-1 down-regulation for induction of apoptosis through targeting of cIAP1 and XIAP in human pancreatic cancer

Inhibitor of apoptosis proteins (IAPs) plays an important role in controlling cancer cell survival. IAPs have therefore attracted considerable attention as potential targets in anticancer therapy. In this study, we investigated the anti-tumor effect of AZD5582, a novel small-molecule IAP inhibitor, in human pancreatic cancer cells. Treating human pancreatic cancer cells with AZD5582 differentially induced apoptosis, dependent on the expression of p-Akt and p-XIAP. Moreover, the knockdown of endogenous Akt or XIAP via RNA interference in pancreatic cancer cells, which are resistant to AZD5582, resulted in increased sensitivity to AZD5582, whereas ectopically expressing Akt or XIAP led to resistance to AZD5582. Additionally, AZD5582 targeted cIAP1 to induce TNF-α-induced apoptosis. More importantly, AZD5582 induced a decrease of Mcl-1 protein, a member of the Bcl-2 family, but not that of Bcl-2 and Bcl-xL. Interestingly, ectopically expressing XIAP and cIAP1 inhibited the AZD5582-induced decrease of Mcl-1 protein, which suggests that AZD5582 elicits Mcl-1 decrease for apoptosis induction by targeting of XIAP and cIAP1. Taken together, these results indicate that sensitivity to AZD5582 is determined by p-Akt-inducible XIAP phosphorylation and by targeting cIAP1. Furthermore, Mcl-1 in pancreatic cancer may act as a potent marker to analyze the therapeutic effects of AZD5582.

Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) Stability Contributes to YM155 Resistance in Human Gastric Cancer Cells

Background: YM155, a survivin suppressant, has been shown anticancer efficacy in various cancer cells. Results: Human gastric cancer cells differentially displayed the sensitivity to YM155 dependent to degradation of cIAP1. Conclusion: Cellular inhibitor of apoptosis protein 1 (cIAP1) interacts with survivin to control their mutual stability and determine sensitivity to YM155. Significance: The regulation of cIAP1 enhances the efficacy for YM155 treatment in gastric cancer. YM155, which blocks the expression of survivin, a member of the inhibitor of apoptosis (IAP) family, induces cell death in a variety of cancer types, including prostate, bladder, breast, leukemia, and non-small lung cancer. However, the mechanism underlying gastric cancer susceptibility and resistance to YM155 is yet to be specified. Here, we demonstrate that cIAP1 stability dictates resistance to YM155 in human gastric cancer cells. Treatment of human gastric cancer cells with YM155 differentially induced cell death dependent on the stability of cIAP1 as well as survivin. Transfection with cIAP1 expression plasmids decreased cell sensitivity to YM155, whereas knockdown of endogenous cIAP1 using RNA interference enhanced sensitivity to YM155. In addition, double knockdown of survivin and cIAP1 significantly induced cell death in the YM155-resistant cell line, MKN45. We also showed that YM155 induced autoubiquitination and proteasome-dependent degradation of cIAP1. Surprisingly, survivin affected the stability of cIAP1 through binding, contributing to cell sensitivity to YM155. Thus, our findings reveal that YM155 sensitizes human gastric cancer cells to apoptotic cell death by degrading cIAP1, and furthermore, cIAP1 in gastric cancer cells may act as a PD marker for YM155 treatment.

Iris Nertschinskia ethanol extract differentially induces cytotoxicity in human breast cancer cells depending on AKT1/2 activity.

Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.

Abstract A65: A novel small-molecule IAP antagonist, AZD5582, draws Mcl-1 down-regulation for induction of apoptosis through targeting of cIAP1 and XIAP in human pancreatic cancer

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Inhibitor of apoptosis proteins (IAPs) plays an important role in controlling cancer cell survival. IAPs have therefore attracted considerable attention as potential targets in anticancer therapy. In this study, we investigated the anti-tumor effect of AZD5582, a novel small-molecule IAP inhibitor, in human pancreatic cancer cells. Treating human pancreatic cancer cells with AZD5582 differentially induced apoptosis, dependent on the expression of p-Akt and p-XIAP. Moreover, the knockdown of endogenous Akt or XIAP via RNA interference in pancreatic cancer cells, which are resistant to AZD5582, resulted in increased sensitivity to AZD5582, whereas ectopically expressing Akt or XIAP led to resistance to AZD5582. Additionally, AZD5582 targeted cIAP1 to induce TNF-α-induced apoptosis. More importantly, AZD5582 induced a decrease of Mcl-1 protein, a member of the Bcl-2 family, but not that of Bcl-2 and Bcl-xL. Interestingly, ectopically expressing XIAP and cIAP1 inhibited the AZD5582-induced decrease of Mcl-1 protein, which suggests that AZD5582 elicits Mcl-1 decrease for apoptosis induction by targeting of XIAP and cIAP1. Taken together, these results indicate that sensitivity to AZD5582 is determined by p-Akt-inducible XIAP phosphorylation and by targeting cIAP1. Furthermore, Mcl-1 in pancreatic cancer may act as a potent marker to analyze the therapeutic effects of AZD5582. Citation Format: Eun-Yeung Gong, Eun Young Lee, Jai-Hee Moon, Jae-Sik Shin, Seung-Woo Hong, Soo-A Jung, Ih-Yeon Hwang, Jeong Hee Kim, Dong-Hoon Jin, Tae Won Kim. A novel small-molecule IAP antagonist, AZD5582, draws Mcl-1 down-regulation for induction of apoptosis through targeting of cIAP1 and XIAP in human pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A65.

Abstract 5486: Combined targeting of the PI3K and MAPK pathways is required for efficacy in treating colorectal cancer of the wild and mutant types

The KRAS oncogene is commonly mutated in human colon cancer. The downstream pathways of mutant KRAS, including the RAF/MEK/ERK, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)-AKT signaling are indispensible in order to understand how to pharmacologically target them when treating colon cancer with the KRAS mutant. However, the heterogeneous responses of these effector pathways remain unclear. Recent studies have indicated that mutational activation of PI3K (PIK3CA) in KRAS mutant tumor cells induces resistance to the inhibition of MEK, one of the MAPK signal cascades. Wild-type p53 also suppresses the oncogenic potential of the MAPK pathway. Our study shows that bortezomib inhibits the PI3K-AKT pathway and that nutlin-3A used as a p53-inducing agent blocks the MAPK pathway. The high doses of bortezomib induced apoptosis through complete suppression of AKT phosphorylation in KRAS and PI3K mutant colon cancer cells. Treating patients using high doses of nutlin-3A resulted in apoptosis by inducing p53 phosphorylation in colon cancer cells with wild-type p53. We also showed that the combination of bortezomib and nutlin-3A exhibited a synergistic effect on KRAS mutant colon cancer cells with PI3K mutant and wild-type p53, whereas there are no combined effect in KRAS mutant colon cancer cells with the p53 mutant. In addition, bortezomib and nutlin-3A exhibited combined effect in KRAS wild-type colon cancer cells with PI3K mutant and wild-type p53, although not in p53 mutant cells. We consistently noted that treatment with bortezomib and nutlin-3A after p53 silencing using small, interfering RNA (siRNA) in p53 wild-type colon cancer cells, resulted in a decrease of the populations of dead cells. Therefore, these results suggest that a combination of bortezomib and nutlin-3A can overcome the heterogeneous responses of the effector pathway in colon cancer with KRAS mutant; they also provide a new basis for the clinical testing of PI3K- and MAPK-targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2011-5486