3845

KRAS

Plexiform and/or dermal neurofibromas are nerve sheath tumors of the peripheral nervous system that are usually present in individuals with neurofibromatosis type 1 (NF1). Neurofibromas arise from Schwann cells with biallelic inactivation of NF1, the gene that encodes neurofibromin. This protein is responsible for regulation of the Ras-mediated pathway, which has been shown to play a crucial role in epithelial-to-mesenchymal transition (EMT). EMT is a biological process that occurs during embryogenesis and wound healing and is involved in pathological processes such as organ fibrosis and cancer metastasis. However, the relationship between neurofibromin and EMT has not been elucidated. We investigated whether the EMT-related signaling pathway was upregulated in NF1-associated neurofibromas and Schwann cells by assessing the expression levels of the EMT-related transcription factors Snail, Slug, Twist, ZEB1 and ZEB2. Immunohistochemical studies and quantitative reverse transcription polymerase chain reaction revealed an increase in the expression levels of EMT-related transcription factors in neurofibroma specimens and NF1-derived Schwann cells (sNF96.2). In addition, the silencing of NF1 by siRNA induced the expression of EMT-related transcription factors in normal human Schwann cells and in epithelial-like breast cancer cells. Our findings suggest that the loss of neurofibromin activated the EMT-related signaling pathway and that the excessive mesenchymal reaction may play a key role in the development of NF1-associated neurofibromas.

Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC-->AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal.

Cyclin-dependent kinase 8 (CDK8), a gene encoding the cyclin-dependent kinase (CDK) component of the Mediator complex, is known as a colon cancer oncogene. Our recent study showed that CDK8 plays an important role in the formation of pancreatic cancer, but the CDK8 expression levels were not completely identical in different pancreatic cancer samples. The level of CDK8 expression depended on whether the K-ras gene was mutated; its expression was much higher in samples carrying a K-ras mutation than in wild-type K-ras samples. Moreover, CDK8 expression was reduced following mutated K-ras knockdown in K-ras-mutated pancreatic cancer cells, whereas CDK8 expression was increased following expression of mutated K-ras in wild-type K-ras cells. Our study demonstrates that mutated K-ras stimulates CDK8 expression, possibly by regulating HIF-1alpha, and both CDK8 and mutated K-ras were confirmed to promote cell proliferation and prevent apoptosis in vitro. Additionally, we found that both CDK8 and mutated K-ras promote the invasion and migration of pancreatic cancer cells via the positive regulation of the Wnt/beta-catenin signaling pathway, thereby increasing the expression of Snail1 and ZEB1, which act as important stimulating factors of the epithelial-to-mesenchymal transition (EMT). Finally, knockdown of either CDK8 or mutated K-ras contributed to attenuated pancreatic cancer growth in BALB/c nude mice. In conclusion, these findings demonstrate that mutated K-ras promotes CDK8 expression and that the regulatory effects of CDK8 on the EMT are partially attributed to the Wnt/beta-catenin signaling pathway.CI - Copyright (c) 2014 Elsevier Ireland Ltd. All rights reserved.

Despite the screening program, breast cancer is the commonest cause of cancer death in women in the industrialized world. In this study, we investigate the correlation among poorly differentiated carcinoma, epithelial to mesenchymal transition (EMT) phenomenon, and expression of NF-kB, Sonic Hedgehog (SHH), K-RAS, and PTX3 in breast cancer in 100 breast biopsies. Samples were classified as follows: 30 benign lesions (BL), 30 ductal infiltrating carcinomas low grade (MLG1), and 40 ductal infiltrating carcinomas high grade (MLG3). expression of vimentin, CD44, beta-catenin, NF-kB, SHH, K-RAS, CD44, and PTX3 was studied by immunohistochemistry. The different rate of cells with vimentin, nuclear beta-catenin, and CD44 expression in MLG3 as compared with MLG1 and BL suggested that the process of de-differentiation of breast cancer cells could be related to the EMT. Our results showed a significant increase in NF-kB signal in MLG3 (2.33 +/- 0.77) with respect to MLG1 (1.26 +/- 0.55) and BL (0.86 +/- 0.52). SHH expression appeared low in BL (1.00 +/- 0.41) and homogenously widespread in MLG1 (1.23 +/- 0.63) and MLG3 (1.56 +/- 0.54). An important increase in K-RAS signal was observed in MLG3 compared to that in BL (2.20 +/- 0.69 vs 0.82 +/- 0.59). As regards PTX3, we observed a strong expression in MLG3 (2.00 +/- 0.78) with respect to BL (0.58 +/- 0.55) and MLG1 (1.53 +/- 0.76). The recurring expression of NF-kB, SHH, K-RAS, and PTX3 in vimentin- and CD44-positive breast cancer cells allows to speculate that breast cells acquire the ability to express these molecules in concomitance to EMT phenomenon.

APC (80-90%) and K-Ras (40-50%) mutations frequently occur in human colorectal cancer (CRC) and these mutations cooperatively accelerate tumorigenesis including metastasis. In addition, both beta-catenin and Ras levels are highly increased in CRC, especially in metastatic CRC (mCRC). Therefore, targeting both the Wnt/beta-catenin and Ras pathways could be an ideal therapeutic approach for treating mCRC patients. In this study, we characterized the roles of KY1022, a small molecule that destabilizes both beta-catenin and Ras via targeting the Wnt/beta-catenin pathway, in inhibiting the cellular events, including EMT, an initial process of metastasis, and apoptosis. As shown by in vitro and in vivo studies using APCMin/+/K-RasG12DLA2 mice, KY1022 effectively suppressed the development of mCRC at an early stage of tumorigenesis. A small molecular approach degrading both beta-catenin and Ras via inhibition of the Wnt/beta-catenin signaling would be an ideal strategy for treatment of mCRC.

K-ras mutation is involved in cancer progression including invasion and migration, but the underlying mechanism is not yet clear. Cathepsin L is a lysosomal cysteine protease and has recently been associated with invasion and migration in human cancers when it is overexpressed. Our recent studies have shown that ionizing radiation (IR) enhanced expression of cathepsin L and increased invasion and migration of tumor cells, but the molecular mechanism is still unclear. In the present study, the effects of K-ras mutation and IR induced invasion and migration of lung cancer as well as the underlying mechanisms were investigated both in vitro and in vivo. Firstly, the levels of cathepsin L and epithelial mesenchymal transition (EMT) marker proteins remarkably changed in A549 (K-ras mutant) after irradiation compared with H1299 (K-ras wild), thereby promoting invasion and migration. Additionally, cathepsin L and its downstream transcription factor CUX1/p110 were increased after irradiation in A549 transfected with CUX1/p200, and the proteolytic processing of CUX1 by cathepsin L was remarkably increased after co-transfection of CUX1/p200 and cathepsin L-lentivirus in H1299. In addition, delivery of a mutant K-ras (V12) into HEK 293 cells stimulated EMT after irradiation due to the accumulation of cathepsin L. Moreover, mutated K-ras was associated with IR-induced cathepsin L and EMT in BALB/c nude mice. Finally, the level of cathepsin L expression was higher in samples carrying a K-ras mutation than in wild-type K-ras samples and the mesenchymal markers were upregulated in the samples of mutant K-ras, whereas the epithelial marker E-cadherin was downregulated in non-small cell lung cancers tissues. In conclusion, the findings demonstrated that mutated K-ras promotes cathepsin L expression and plays a pivotal role in EMT of human lung cancer. The regulatory effect of IR-induced cathepsin L on lung cancer invasion and migration was partially attributed to the Cathepsin L /CUX1-mediated EMT signaling pathway. This study will provide cathepsin L as a potential target for tumor therapy.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Mucin1 (MUC1) is an epithelial glycoprotein overexpressed in ovarian cancer and actively involved in tumor cell migration and metastasis. Using novel in vitro and in vivo MUC1-expressing conditional (Cre-loxP) ovarian tumor models, we focus here on MUC1 biology and the roles of Kras activation and Pten deletion during cell transformation and epithelial-to-mesenchymal transition (EMT). We generated several novel murine ovarian cancer cell lines derived from the ovarian surface epithelia (OSE) of mice with conditional mutations in Kras, Pten or both. In addition, we also generated several tumor-derived new cell lines that reproduce the original tumor phenotype in vivo and mirror late stage metastatic disease. Our results demonstrate that de novo activation of oncogenic Kras does not trigger increased proliferation, cellular transformation or EMT, and prevents MUC1 upregulation. In contrast, Pten deletion accelerates cell proliferation, triggers cellular transformation in vitro and in vivo, and stimulates MUC1 expression. Ovarian tumor-derived cell lines MKP-Liver and MKP-Lung cells reproduce in vivo EMT and represent the first immune competent mouse model for distant hematogenous spread. Whole genome microarray expression analysis using tumor and OSE-derived cell lines reveal a 121 gene signature associated with EMT and metastasis. When applied to n=542 cases from The cancer Genome Atlas (TCGA) ovarian cancer dataset, the gene signature identifies a patient subset with decreased survival (P=0.04). Using an extensive collection of novel murine cell lines we have identified distinct roles for Kras and Pten on MUC1 and EMT in vivo and in vitro. The data has implications for future design of combination therapies targeting Kras mutations, Pten deletions and MUC1 vaccines.