BAY61-3606 Affects the Viability of Colon Cancer Cells in a Genotype-Directed Manner

Background: K-RAS mutation poses a particularly difficult problem for cancer therapy. Activating mutations in K-RAS are common in cancers of the lung, pancreas, and colon and are associated with poor response to therapy. As such, targeted therapies that abrogate K-RAS-induced oncogenicity would be of tremendous value. Methods: We searched for small molecule kinase inhibitors that preferentially affect the growth of colorectal cancer cells expressing mutant K-RAS. The mechanism of action of one inhibitor was explored using chemical and genetic approaches. Results: We identified BAY61-3606 as an inhibitor of proliferation in colorectal cancer cells expressing mutant forms of K-RAS, but not in isogenic cells expressing wild-type K-RAS. In addition to its anti-proliferative effects in mutant cells, BAY61-3606 exhibited a distinct biological property in wild-type cells in that it conferred sensitivity to inhibition of RAF. In this context, BAY61-3606 acted by inhibiting MAP4K2 (GCK), which normally activates NFκβ signaling in wild-type cells in response to inhibition of RAF. As a result of MAP4K2 inhibition, wild-type cells became sensitive to AZ-628, a RAF inhibitor, when also treated with BAY61-3606. Conclusions: These studies indicate that BAY61-3606 exerts distinct biological activities in different genetic contexts

Integrated Genomic and Gene Expression Profiling Identifies Two Major Genomic Circuits in Urothelial Carcinoma

Similar to other malignancies, urothelial carcinoma (UC) is characterized by specific recurrent chromosomal aberrations and gene mutations. However, the interconnection between specific genomic alterations, and how patterns of chromosomal alterations adhere to different molecular subgroups of UC, is less clear. We applied tiling resolution array CGH to 146 cases of UC and identified a number of regions harboring recurrent focal genomic amplifications and deletions. Several potential oncogenes were included in the amplified regions, including known oncogenes like E2F3, CCND1, and CCNE1, as well as new candidate genes, such as SETDB1 (1q21), and BCL2L1 (20q11). We next combined genome profiling with global gene expression, gene mutation, and protein expression data and identified two major genomic circuits operating in urothelial carcinoma. The first circuit was characterized by FGFR3 alterations, overexpression of CCND1, and 9q and CDKN2A deletions. The second circuit was defined by E3F3 amplifications and RB1 deletions, as well as gains of 5p, deletions at PTEN and 2q36, 16q, 20q, and elevated CDKN2A levels. TP53/MDM2 alterations were common for advanced tumors within the two circuits. Our data also suggest a possible RAS/RAF circuit. The tumors with worst prognosis showed a gene expression profile that indicated a keratinized phenotype. Taken together, our integrative approach revealed at least two separate networks of genomic alterations linked to the molecular diversity seen in UC, and that these circuits may reflect distinct pathways of tumor development

Phosphoproteomics Identifies Oncogenic Ras Signaling Targets and Their Involvement in Lung Adenocarcinomas

Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer

'Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich's ataxia'

Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (▵Ψm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure

Epithelial Tissues Have Varying Degrees of Susceptibility to KrasG12D-Initiated Tumorigenesis in a Mouse Model

Activating mutations in the Kras gene are commonly found in some but not all epithelial cancers. In order to understand the susceptibility of different epithelial tissues to Kras-induced tumorigenesis, we introduced one of the most common Kras mutations, KrasG12D, broadly in epithelial tissues. We used a mouse model in which the G12D mutation is placed in the endogenous Kras locus controlled by inducible, Cre-mediated recombination in tissues expressing cytokeratin 19 including the oral cavity, GI tract, lungs, and ducts of the liver, kidney, and the pancreas. Introduction of the KrasG12D mutation in adult mouse tissues led to neoplastic changes in some but not all of these tissues. Notably, many hyperplasias, metaplasias and adenomas were observed in the oral cavity, stomach, colon and lungs, suggesting that exposure to products of the outside environment promotes KrasG12D-initiated tumorigenesis. However, environmental exposure did not consistently correlate with tumor formation, such as in the small intestine, suggesting that there are also intrinsic differences in susceptibility to Kras activation. The pancreas developed small numbers of mucinous metaplasias with characteristics of early stage pancreatic intraepithelial neoplasms (PanINs), supporting the hypothesis that pancreatic ducts have the potential to give rise pancreatic cancer

Activating Transcription Factor 4 Confers a Multidrug Resistance Phenotype to Gastric Cancer Cells through Transactivation of SIRT1 Expression

BACKGROUND: Multidrug resistance (MDR) in gastric cancer remains a major challenge to clinical treatment. Activating transcription factor 4 (ATF4) is a stress response gene involved in homeostasis and cellular protection. However, the expression and function of ATF4 in gastric cancer MDR remains unknown. In this study, we investigate whether ATF4 play a role in gastric cancer MDR and its potential mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that ATF4 overexpression confered the MDR phenotype to gastric cancer cells, while knockdown of ATF4 in the MDR variants induced re-sensitization. In this study we also showed that the NAD(+)-dependent histone deacetylase SIRT1 was required for ATF4-induced MDR effect in gastric cancer cells. We demonstrated that ATF4 facilitated MDR in gastric cancer cells through direct binding to the SIRT1 promoter, resulting in SIRT1 up-regulation. Significantly, inhibition of SIRT1 by small interfering RNA (siRNA) or a specific inhibitor (EX-527) reintroduced therapeutic sensitivity. Also, an increased Bcl-2/Bax ratio and MDR1 expression level were found in ATF4-overexpressing cells. CONCLUSIONS/SIGNIFICANCE: We showed that ATF4 had a key role in the regulation of MDR in gastric cancer cells in response to chemotherapy and these findings suggest that targeting ATF4 could relieve therapeutic resistance in gastric cancer

A Common Role for Various Human Truncated Adenomatous Polyposis Coli Isoforms in the Control of Beta-Catenin Activity and Cell Proliferation

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of β-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR). The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of β-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of β-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5) with an up-regulation of β-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of β-catenin. This control can happen even when truncated APC lacks the β-catenin inhibiting domain (CiD) involved in targeting β-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting β-catenin signalling to the “just right” level

Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins

Sirt1 is an NAD(+)-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1. ATP suppresses Sirt1 activity by impairing the CTD's ability to bind to the deacetylase domain as well as its ability to function as the substrate recruitment site. ATP, but not NAD(+), causes a conformational shift to a less compact structure. Mutations that prevent ATP binding increase Sirt1's ability to promote stress resistance and inhibit adipogenesis under high-ATP conditions. Interestingly, the CTD can be attached to other proteins, thereby converting them into energy-regulated proteins. These discoveries provide insight into how extreme energy deprivation can impact Sirt1 activity and underscore the complex nature of Sirt1 structure and regulation

Transcriptional recapitulation and subversion of embryonic colon development by mouse colon tumor models and human colon cancer

Colon tumors from four independent mouse models and 100 human colorectal cancers all exhibited striking recapitulation of embryonic colon gene expression from embryonic days 13.5-18.5