Juyong Yoon

MEK1/2 inhibitors AS703026 and AZD6244 may be potential therapies for KRAS mutated colorectal cancer that is resistant to EGFR monoclonal antibody therapy.

Epidermal growth factor receptor (EGFR) monoclonal antibodies (mAb) are used widely to treat metastatic colorectal cancer (mCRC) patients, but it is now clear that patients harboring K-ras mutation are resistant to EGFR mAbs such as cetuximab (Erbitux) and panitumumab (Vectibix). For this reason, current recommendations for patient care involve diagnosing the K-ras mutational status of patients prior to EGFR mAb therapy. In this study, we investigated the ability of two MEK inhibitors currently in clinical trials, AS703026 and AZD6244, to address the challenge posed by the resistance of K-ras mutated colorectal cancers to EGFR mAb. AS703026 and AZD6244 were tested in various cell-based assays and tumor xenograft studies, focusing on isogenic human colorectal tumor cell lines that expressed only WT or mutant K-Ras (D-WT or D-MUT). The EGFR mAb cetuximab inhibited the Ras-ERK pathway and proliferation of D-WT cells in vitro and in vivo, but it did not inhibit proliferation of D-MUT cells in either setting. In contrast, AS703026 and AZD6244 effectively inhibited the growth of D-MUT cells in vitro and in vivo by specific inhibition of the key MEK downstream target kinase ERK. Inhibition of MEK by AS703026 or AZD6244 also suppressed cetuximab-resistant colorectal cancer cells attributed to K-ras mutation both in vitro and in vivo. Our findings offer proof-of-concept for the use of MEK inhibitors as an effective therapy in K-ras mutated CRC.

Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway.

BackgroundValproic acid (VPA), a commonly used mood stabilizer that promotes neuronal differentiation, regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3β (GSK3β). However, the mechanism by which VPA promotes differentiation is not understood.ResultsWe report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects, however, are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through β-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the β-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos.ConclusionWe propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos.

Ras Stabilization Through Aberrant Activation of Wnt/β-Catenin Signaling Promotes Intestinal Tumorigenesis

Crosstalk between the Wnt/β-catenin and the MAPK pathways contributes to colon cancer. Blocking Ras Degradation in Colon Cancer Both the Wnt/β-catenin pathway and the Ras-activated mitogen-activated protein kinase (MAPK) pathway can contribute to cancer. Jeong et al. report an interaction between these two pathways through the β-catenin destruction complex and the E3 ubiquitin ligase adaptor β-TrCP. Ras was phosphorylated by glycogen synthase kinase 3β, a component of the β-catenin destruction complex, and this enabled the phosphorylation-dependent recruitment of β-TrCP, which increased ubiquitin- and proteasome-mediated degradation of Ras, thereby providing a brake on Ras-mediated activation of the MAPK pathway. Conditions that compromised the function of the destruction complex, such as the presence of Wnt or genetic mutations or deficiency in components of the destruction complex, stabilized Ras and increased MAPK pathway activity. The importance of this regulatory crosstalk was verified in colon cancer samples from patients and mouse models of colon cancer, suggesting that targeting both the hyperactive Wnt pathway and the Ras pathway may be an effective combination therapy. Although the guanosine triphosphate/guanosine diphosphate loading switch is a major regulatory mechanism that controls the activity of the guanosine triphosphatase Ras, we report a distinct mechanism for regulating Ras activity through phosphorylation-mediated degradation and describe the role of this second 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 after its phosphorylation by glycogen synthase kinase 3β (GSK3β) and the subsequent recruitment of β-TrCP–E3 ligase. We found a positive association between tumorigenesis and Ras stabilization resulting from the aberrant activation of Wnt/β-catenin signaling in adenomas from two mouse models of colon cancer, human colonic tumors from various stages, and colon polyps of patients with familial adenomatous polyposis. Our results indicated that GSK3β plays an essential role in Ras degradation and that inhibition of this degradation pathway by aberrant Wnt/β-catenin signaling may contribute to Ras-induced transformation in colorectal tumorigenesis.

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.

Valproic Acid Induces Hair Regeneration in Murine Model and Activates Alkaline Phosphatase Activity in Human Dermal Papilla Cells

Background Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo. Methodology/ Principal Findings Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl2 also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. Conclusions/ Significance Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.

Bone morphogenetic protein 4 stimulates neuronal differentiation of neuronal stem cells through the ERK pathway

Bone morphogenic protein 4 (BMP4), a member of the TGF-β superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker β-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/β-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3β (GSK3β) activating the Wnt/β-catenin pathway. The BMP4-mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.

Persicaria hydropiper (L.) spach and its flavonoid components, isoquercitrin and isorhamnetin, activate the Wnt/β-catenin pathway and inhibit adipocyte differentiation of 3T3-L1 cells.

Obesity, which is related to metabolic syndrome and is associated with liver disease, represents an epidemic problem demanding effective therapeutic strategies. Evidence shows that the Wnt/β‐catenin pathway is closely associated with obesity and that small molecules regulating the Wnt/β‐catenin pathway can potentially control adipogenesis related to obesity. Eleven plant extracts activating the Wnt/β‐catenin pathway were screened by using HEK 293‐TOP cells retaining the Wnt/β‐catenin signaling reporter gene. An extract of Persicaria hydropiper (L.) Spach was found to activate Wnt/β‐catenin signaling. P. hydropiper is grown worldwide in temperate climates and is found widely in Southeast Asia. The P. hydropiper extract inhibited the differentiation of adipocyte 3T3‐L1 cells. Isoquercitrin and isorhamnetin, constituents of P. hydropiper, also activated Wnt/β‐catenin signaling and suppressed the differentiation of 3T3‐L1 cells. These results indicate that isoquercitrin in P. hydropiper suppresses the adipogenesis of 3T3‐L1 cells via the inhibition of Wnt/β‐catenin signaling. P. hydropiper and isoquercitrin may therefore be potential therapeutic agents for obesity and its associated disorders. Copyright

KR-POK Interacts with p53 and Represses Its Ability to Activate Transcription of p21WAF1/CDKN1A

Transcriptional regulation by p53 is thought to play a role in its ability to suppress tumorigenesis. However, there remain gaps in understanding about how p53 regulates transcription and how disrupting this function may promote cancer. Here we report a role in these processes for the kidney cancer-related gene KR-POK (ZBTB7C), a POZ domain and Krüppel-like zinc finger transcription factor that we found to physically interact with p53. Murine embryonic fibroblasts isolated from genetically deficient mice (Kr-pok(-/-) MEFs) exhibited a proliferative defect relative to wild-type mouse embryonic fibroblasts (MEF). The zinc finger domain of Kr-pok interacted directly with the DNA binding and oligomerization domains of p53. This interaction was essential for Kr-pok to bind the distal promoter region of the CDKN1A gene, an important p53 target gene encoding the cell-cycle regulator p21WAF1, and to inhibit p53-mediated transcriptional activation of CDKN1A. Kr-pok also interacted with the transcriptional corepressors NCoR and BCoR, acting to repress histone H3 and H4 deacetylation at the proximal promoter region of the CDKN1A gene. Importantly, Kr-pok(-/-) MEFs displayed an enhancement in CDKN1A transactivation by p53 during the DNA damage response, without any parallel changes in transcription of either the p53 or Kr-pok genes themselves. Furthermore, Kr-pok promoted cell proliferation in vitro and in vivo, and its expression was increased in more than 50% of the malignant human kidney cancer cases analyzed. Together, our findings define KR-POK as a transcriptional repressor with a pro-oncogenic role that relies upon binding to p53 and inhibition of its transactivation function.

Small-molecule binding of the axin RGS domain promotes β-catenin and Ras degradation.

Both the Wnt/β-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both β-catenin and Ras, via targeting the Wnt/β-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating β-catenin and Ras degradation through enhancement of the β-catenin destruction complex activating GSK3β. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both β-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/β-catenin and Ras pathways.