Supplementary Figure 2 from MEK1/2 Inhibitors AS703026 and AZD6244 May Be Potential Therapies for <i>KRAS</i> Mutated Colorectal Cancer That Is Resistant to EGFR Monoclonal Antibody Therapy
Supplementary Figure 3 from MEK1/2 Inhibitors AS703026 and AZD6244 May Be Potential Therapies for <i>KRAS</i> Mutated Colorectal Cancer That Is Resistant to EGFR Monoclonal Antibody Therapy
// Kug Hwa Lee 1, 2 , Woo-Jeong Jeong 1, 2 , Pu-Hyeon Cha 1, 2 , Sang-Kyu Lee 1, 2 , Do Sik Min 1, 3 and Kang-Yell Choi 1, 2 1 Translational Research Center for Protein Function Control, Yonsei University, Seoul, South Korea 2 Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea 3 Department of Molecular Biology, College of Natural Science, Pusan National University, Pusan, South Korea Correspondence to: Kang-Yell Choi, email: kychoi@yonsei.ac.kr Keywords: Sur8; fibroblast growth factor-2; protein kinase C α/δ; Ras signaling; colorectal cancer Received: July 15, 2017 Accepted: December 03, 2017 Published: December 14, 2017 ABSTRACT Scaffold proteins of the mitogen activated protein kinase (MAPK) pathway recruit protein kinase cascades to confer context-specificity to cellular signaling. Varying concentrations of scaffold proteins determine different aspects of signaling outputs. However, regulatory mechanisms of scaffold proteins are poorly understood. Sur8, a scaffold protein in the Ras-MAPK pathway, is known to be involved in cell transformation and migration, and is increased in human colorectal cancer (CRC) patient tissue. Here we determine that regulation of Sur8 stability mediates transformation and migration of CRC cells. Fibroblast growth factor 2 (FGF2) is identified as an external regulator that stabilizes Sur8. Protein kinase C-alpha and -delta (PKCα/δ) are also identified as specific mediators of FGF2 regulation of Sur8 stability. PKCα/δ phosphorylate Sur8 at Thr-71 and Ser-297, respectively. This phosphorylation is essential for polyubiquitin-dependent degradation of Sur8. Sur8 mutations, which mimic phosphorylation by PKCα/δ and destabilized Sur8, suppress the FGF2-induced transformation and migration of CRC cells. The clinical relevance of Sur8 regulation by PKCα/δ is indicated by the inverse relationship between PKCα/δ and Sur8 expression in human CRC patient tissues. Overall, our findings demonstrate for the first time a regulatory mechanism of Sur8 stability involving cellular transformation and migration in CRC.
The number of people suffering from hair loss is increasing, and hair loss occurs not only in older men but also in women and young people. Prostaglandin D2 (PGD2) is a well-known alopecia inducer. However, the mechanism by which PGD2 induces alopecia is poorly understood. In this study, we characterized CXXC5, a negative regulator of the Wnt/β-catenin pathway, as a mediator for hair loss by PGD2. The hair loss by PGD2 was restored by Cxxc5 knock-out or treatment of protein transduction domain–Dishevelled binding motif (PTD-DBM), a peptide activating the Wnt/β-catenin pathway via interference with the Dishevelled (Dvl) binding function of CXXC5. In addition, suppression of neogenic hair growth by PGD2 was also overcome by PTD-DBM treatment or Cxxc5 knock-out as shown by the wound-induced hair neogenesis (WIHN) model. Moreover, we found that CXXC5 also mediates DHT-induced hair loss via PGD2. DHT-induced hair loss was alleviated by inhibition of both GSK-3β and CXXC5 functions. Overall, CXXC5 mediates the hair loss by the DHT-PGD2 axis through suppression of Wnt/β-catenin signaling.
Abstract The hair follicle stem cells (HFSCs) utilize glycolytic metabolism during their activation and anagen induction. However, the role of pyruvate kinase M2 (PKM2), which catalyzes the final step of glycolysis, in hair regeneration has not been elucidated. In this study, we investigated the expression pattern and activity of PKM2 during the depilation-induced anagen progression in mice. We found that TEPP-46, a selective activator of PKM2, enhanced hair re-growth and proliferation of HFSCs. PKM2 expression was increased via up-regulation of the Wnt/β-catenin signaling, which is involved in hair regeneration. Moreover, a combined treatment with KY19382, a small molecule that activates the Wnt/β-catenin signaling, and TEPP-46 significantly enhanced hair re-growth and wound-induced hair follicle neogenesis (WIHN). These results indicate that simultaneous activation of the PKM2 and Wnt/β-catenin signaling could be a potential strategy for treating alopecia patients.
Abstract Background Most cancer cells employ the Warburg effect to support anabolic growth and tumorigenesis. Here, we discovered a key link between Warburg effect and aberrantly activated Wnt/β-catenin signalling, especially by pathologically significant APC loss, in CRC. Methods Proteomic analyses were performed to evaluate the global effects of KYA1797K, Wnt/β-catenin signalling inhibitor, on cellular proteins in CRC. The effects of APC -loss or Wnt ligand on the identified enzymes, PKM2 and LDHA, as well as Warburg effects were investigated. A linkage between activation of Wnt/β-catenin signalling and cancer metabolism was analysed in tumour of Apc min/+ mice and CRC patients. The roles of PKM2 in cancer metabolism, which depends on Wnt/β-catenin signalling, were assessed in xenograft-tumours. Results By proteomic analysis, PKM2 and LDHA were identified as key molecules regulated by Wnt/β-catenin signalling. APC -loss caused the increased expression of metabolic genes including PKM2 and LDHA , and increased glucose consumption and lactate secretion. Pathological significance of this linkage was indicated by increased expression of glycolytic genes with Wnt target genes in tumour of Apc min/+ mice and CRC patients. Warburg effect and growth of xenografted tumours-induced by APC- mutated-CRC cells were suppressed by PKM2-depletion. Conclusions The β-catenin-PKM2 regulatory axis induced by APC loss activates the Warburg effect in CRC.
Abstract Adenomatous polyposis coli (APC) loss-of-function mutations and K-Ras gain-of-function mutations are common abnormalities that occur during the initiation and intermediate adenoma stages of colorectal tumorigenesis, respectively. However, little is known about the role of these mutations in the regulation of cancer stem cells (CSCs) associated with colorectal cancer (CRC) tumorigenesis. We analyzed tissue from CRC patients (n = 49) and observed an association between K-Ras mutations and CSC activation during colorectal tumorigenesis. The oncogenic K-Ras mutations enhanced stemness of CRC cells carrying an APC mutation, as shown by comparisons of sphere formation, transforming potential, and differentiation in vitro and tumor-forming capacity in vivo between isogenic APC mutated CRC cells harboring either wild-type or mutant K-Ras. Moreover, the activation of CSC by K-Ras mutations in CRC required an additional APC mutation, as revealed by the induction of CD44, CD133, and CD166 in intestinal tumor tissues of APCMin/+/K-RasLA2 double-mutant mice, but not in K-RasLA2 mice. Overall, the progression and metastasis of CRC induced by K-Ras mutation occurs by initial activation of CSC by APC loss and further activation of Wnt/β-catenin signaling and subsequent activation of Ras-ERK signaling. Citation Format: Byoung-San Moon, Kug Hwa Lee, Kyounghwa Koo, Kang-Yell Choi. Activation of cancer stem cells by oncogenic K-Ras requires activated Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2015-2330
Adenomatous polyposis coli ( APC ) loss-of-function mutations and K-Ras gain-of-function mutations are common abnormalities that occur during the initiation and intermediate adenoma stages of colorectal tumorigenesis, respectively. However, little is known about the role these mutations play in cancer stem cells (CSCs) associated with colorectal cancer (CRC) tumorigenesis. We analyzed tissue from CRC patients (n = 49) to determine whether K-Ras mutations contributed to CSC activation during colorectal tumorigenesis. DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cells were cultured and evaluated for their ability to differentiate, form spheroids in vitro, and form tumors in vivo. Interaction between APC and K-Ras mutations in colorectal tumorigenesis was evaluated using APCMin/+ / K-RasLA2 mice and DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cell xenografts. (n = 4) Group differences were determined by Student t test. All statistical tests were two-sided. The sphere-forming capability of DLD-1-K-Ras-MT cells was statistically significantly higher than that of DLD-1-K-Ras-WT cells (DLD-1-K-Ras-MT mean = 86.661 pixel, 95% confidence interval [CI] = 81.701 to 91.621 pixel; DLD-1-K-Ras-WT mean = 42.367 pixel, 95% CI = 36.467 to 48.267 pixel; P = .003). Moreover, both the size and weight of tumors from DLD-1-K-Ras-MT xenografts were markedly increased compared with tumors from DLD-1-K-Ras-WT cells. Expression of the CSC markers CD44, CD133, and CD166 was induced in intestinal tumors from APCMin/+ / K-RasLA2 mice, but not K-RasLA2 mice, indicating that APC mutation is required for CSC activation by oncogenic K-Ras mutation. K-Ras mutation activates CSCs, contributing to colorectal tumorigenesis and metastasis in CRC cells harboring APC mutations. Initial activation of β-catenin by APC loss and further enhancement through K-Ras mutation induces CD44, CD133, and CD166 expression.
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