Woo- Jeong

Axin Inhibits Extracellular Signal-regulated Kinase Pathway by Ras Degradation via β-Catenin

Interactions between the Wnt/β-catenin and the extracellular signal-regulated kinase (ERK) pathways have been posited, but the molecular mechanisms and cooperative roles of such interaction in carcinogenesis are poorly understood. In the present study, the Raf-1, MEK, and ERK activities were concomitantly decreased in fibroblasts, which inhibit morphological transformation and proliferation by Axin induction. The inhibition of the components of the ERK pathway by Axin occurred in cells retaining wild-type β-catenin, including primary hepatocytes, but not in cells retaining non-degradable mutant β-catenin. Axin inhibits cellular proliferation and ERK pathway activation induced by either epidermal growth factor or Ras, indicating a role of Axin in the regulation of growth induced by ERK pathway activation. ERK pathway regulation by Axin occurs at least partly via reduction of the protein level of Ras. Both wild-type and mutant Ras proteins are subjected to regulation by Axin, which occurs in cells retaining wild-type but not mutant β-catenin gene. The role of β-catenin in the regulation of the Ras-ERK pathway was further confirmed by Ras reduction and subsequent inhibitions of the ERK pathway components by knock down of mutated form of β-catenin. The Ras regulation by Axin was blocked by treatment of leupeptin, an inhibitor of the lysosomal protein degradation machinery. Overall, Axin inhibits proliferation of cells at least partly by reduction of Ras protein level via β-catenin. This study provides evidences for the role of the Ras-ERK pathway in carcinogenesis caused by mutations of the Wnt/β-catenin pathway components.

NM23H2 inhibits EGF- and Ras-induced proliferation of NIH3T3 cells by blocking the ERK pathway

The NM23 family proteins are involved in a variety of biological processes including tumor metastasis, development, and differentiation; however, their functions in the regulation of cellular proliferation are poorly understood. We have investigated the role of one NM23 family protein, NM23H2, in the regulation of cellular proliferation directed by the extracellular signal regulated kinase (ERK) pathway. The activity of ERKs was enhanced by knockdown of endogenous NM23H2 and blocked by overexpression of NM23H2 in both NIH3T3 and HEK293 cells. Additionally, the epidermal growth factor (EGF)- and oncogenic Ras(G12R)-induced proliferation of both HEK293 and NIH3T3 cells was reduced by NM23H2 overexpression. Furthermore, activation of Raf-1, MEK and the ERKs by either EGF or Ras(G12R) was inhibited by NM23H2 overexpression. Together, our data indicate that NM23H2 is a negative regulator of cellular proliferation stimulated by EGF- and Ras-mediated activation of the ERK pathway.

Akt is involved in the inhibition of cell proliferation by EGF

Axin is a negative regulator of the Wnt/β-catenin pathway and is involved in the regulation of axis formation and proliferation. Involvement of Axin in the regulation of other signaling pathways is poorly understood. In this study, we investigated the involvement of Akt in growth regulation by Axin in L929 fibroblasts stimulated by EGF. Akt activity was increased by EGF treatment and Ras activation, respectively. Both the EGF- and Ras-induced Akt activations were abolished by Axin induction, as revealed by both Western blot and immunocytochemical analyses. The proliferation and Akt activation induced by EGF were decreased by Axin induction, and the effects of EGF were abolished by treatment of an Akt-specific inhibitor. Therefore, Axin inhibits EGF-induced proliferation of L929 fibroblasts by blocking Akt activation.

Interaction between Wnt/β-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway

Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli (APC) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

Abstract A45: A potential therapeutic strategy for treatment of colorectal cancers through regulating Ras stability via inhibition of Wnt/β-catenin pathway

Ras is a well-characterized proto-oncogene, and its various mutations contribute to transformation and tumorigenesis in a wide variety of human cancers. Although the GTP/GDP loading switch is known as a major regulatory mechanism of Ras, other mechanisms affecting Ras activity are poorly understood. Here, we report a mechanism for controlling Ras activity by regulating its degradation and describe this regulatory mechanism in the suppression of cellular transformation and tumors induced by Ras mutations. We found that negative regulators of Wnt/β-catenin signaling contributed to the polyubiquitin-dependent degradation of Ras following phosphorylation by glycogen synthase kinase 3β (GSK3β) and subsequent recruitment of β-TrCP-E3 ligase. We found a positive association between tumorigenesis and Ras stabilization due to the aberrant activation of Wnt/β-catenin signaling. Our results indicate an essential role for phosphorylation and degradation of Ras and suppression of Ras-induced transformation via the Wnt/β-catenin pathway and provide insight into the role of this novel regulatory mechanism for Ras in tumorigenesis of colorectal cancers (CRCs). Because both Ras and Wnt/β-catenin pathways are frequently activated in CRCs, these results suggest that the regulation of Ras stability through inhibition of the Wnt/β-catenin pathway could be an ideal therapeutic approach for treatment of CRC. Citation Format: Pu-Hyeon Cha, Yong-Hee Cho, Woo-Jeong Jeong, Kang-Yell Choi. A potential therapeutic strategy for treatment of colorectal cancers through regulating Ras stability via inhibition of Wnt/β-catenin pathway. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A45. doi: 10.1158/1557-3125.RASONC14-A45

Abstract A50: Oncogenic K-Ras accelerates cancer stem cell activation via aberrant Wnt/β-catenin signaling

Oncogenic K-Ras is known to play an important role in tumor growth, but their involvement in the activation of cancer stem cells (CSCs) is not clear. Here, we show the relationship between K-Ras mutation and CSC activation in the tumorigenesis of colorectal cancer (CRC) by systematic in vitro and in vivo studies as well as by clinical evaluations of human specimens. K-Ras mutation is revealed as an important factor for CSC activation through initial APC loss. The critical role of APC mutation in tumorigenesis involving the activation of CSCs by K-Ras mutation was confirmed by genetic studies using APCMin/+ and K-RasG12D(LA2) mice. We therefore propose that CSC activation by both APC and K-Ras mutations was induced via the Wnt/β-catenin signaling pathway, which was initially activated by APC loss and further enhanced through the Ras-ERK pathway. Citation Format: Byoung-San Moon, Woo-Jeong Jeong, Kang-Yell Choi. Oncogenic K-Ras accelerates cancer stem cell activation via aberrant Wnt/β-catenin signaling. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A50. doi: 10.1158/1557-3125.RASONC14-A50

Abstract 2685: Regulation of Ras stability via inhibition of Wnt/β-catenin pathway as a potential therapeutic strategy to treat colorectal cancers

Ras is a well-characterized proto-oncogene, and its various mutations contribute to transformation and tumorigenesis in a wide variety of human cancers. Although the GTP/GDP loading switch is known as a major regulatory mechanism of Ras, other mechanisms affecting Ras activity are poorly understood. Here, we report a mechanism for controlling Ras activity by regulating its degradation and describe this regulatory mechanism in the suppression of cellular transformation and tumors induced by Ras mutations. We found that negative regulators of Wnt/β-catenin signaling contributed to the polyubiquitin-dependent degradation of Ras following phosphorylation by glycogen synthase kinase 3β (GSK3β) and subsequent recruitment of β-TrCP-E3 ligase. We found a positive association between tumorigenesis and Ras stabilization due to the aberrant activation of Wnt/β-catenin signaling. Our results indicate an essential role for phosphorylation and degradation of Ras and suppression of Ras-induced transformation via the Wnt/β-catenin pathway and provide insight into the role of this novel regulatory mechanism for Ras in tumorigenesis of colorectal cancers (CRCs). Because both Ras and Wnt/β-catenin pathways are frequently activated in CRCs, these results suggest that the regulation of Ras stability through inhibition of the Wnt/β-catenin pathway could be an ideal therapeutic approach for treatment of CRC. Citation Format: Pu-Hyeon Cha, Yong-Hee Cho, Woo-Jeong Jeong, Hyun-Yi Kim, Kang-Yell Choi. Regulation of Ras stability via inhibition of Wnt/β-catenin pathway as a potential therapeutic strategy to treat colorectal cancers. [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 2685. doi:10.1158/1538-7445.AM2014-2685

Abstract 4164: Regulatory mechanism for Ras degradation and its role in the suppression of cellular transformation and tumorigenesis

Ras is a well-characterized proto-oncogene, and its various mutations contribute to transformation and tumorigenesis in a wide variety of human cancers. Although the GTP/GDP loading switch is known as a major regulatory mechanism of Ras, other mechanisms affecting Ras activity, especially those related to cancer, are poorly understood. Here, we report a novel regulatory mechanism for Ras degradation and its role in suppression of cellular transformation and tumors induced by Ras mutations. We show that the negative regulators of Wnt/β-catenin signaling are associated with polyubiquitin-dependent degradation of Ras following phosphorylation and subsequent recruitment of α-TrCP-E3 ligase. The interaction of Ras with the WD40 domain of α-TrCP targeted Ras for polyubiquitination and degradation. This process stimulated by Axin or adenomatous polyposis coli (Apc) and was inhibited by Wnt3a via modulating the phosphorylation status of Ras. Ras-mediated cellular transformation was also inhibited by the expression of α-TrCP and/or Axin. In vivo regulation of Ras stability by Wnt/β-catenin signaling was indicated via measurements of the status of Ras in the intestines of mice stimulated with recombinant Wnt3a by intravenous tail vein injection. The Ras deregulation by negative Wnt/β-catenin signaling was also revealed in human cancers such as colorectal cancer and HCCs etc. Together, our results indicate an essential role for phosphorylation and degradation of Ras and suppression of Ras-induced transformation via the Wnt/β-catenin pathway and provide insight into the role of this novel regulatory mechanism for Ras in colorectal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4164. doi:1538-7445.AM2012-4164