RAS oncogenic activity predicts response to chemotherapy and outcome in lung adenocarcinoma.

Activating mutations in KRAS occur in 32% of lung adenocarcinomas (LUAD). Despite leading to aggressive disease and resistance to therapy in preclinical studies, the KRAS mutation does not predict patient outcome or response to treatment, presumably due to additional events modulating RAS pathways. To obtain a broader measure of RAS pathway activation, we developed RAS84, a transcriptional signature optimised to capture RAS oncogenic activity in LUAD. We report evidence of RAS pathway oncogenic activation in 84% of LUAD, including 65% KRAS wild-type tumours, falling into four groups characterised by coincident alteration of STK11/LKB1, TP53 or CDKN2A, suggesting that the classifications developed when considering only KRAS mutant tumours have significance in a broader cohort of patients. Critically, high RAS activity patient groups show adverse clinical outcome and reduced response to chemotherapy. Patient stratification using oncogenic RAS transcriptional activity instead of genetic alterations could ultimately assist in clinical decision-making.

A Kinetic Map of the Homomeric Voltage-Gated Potassium Channel (Kv) Family.

The voltage-gated potassium (Kv) channels, encoded by 40 genes, repolarize all electrically excitable cells, including plant, cardiac, and neuronal cells. Although these genes were fully sequenced decades ago, a comprehensive kinetic characterization of all Kv channels is still missing, especially near physiological temperature. Here, we present a standardized kinetic map of the 40 homomeric Kv channels systematically characterized at 15, 25, and 35°C. Importantly, the Kv kinetics at 35°C differ significantly from commonly reported kinetics, usually performed at room temperature. We observed voltage-dependent Q10 for all active Kv channels and inherent heterogeneity in kinetics for some of them. Kinetic properties are consistent across different host cell lines and conserved across mouse, rat, and human. All electrophysiology data from all Kv channels are made available through a public website (Channelpedia). This dataset provides a solid foundation for exploring kinetics of heteromeric channels, roles of auxiliary subunits, kinetic modulation, and for building accurate Kv models.

Initiation of high frequency multi-drug resistance following kinase targeting by siRNAs.

The sequential use of non cross-resistant cytotoxic agents is the standard of care for advanced solid tumors in order to enhance survival and optimise quality of life. Nevertheless, drug resistance to non cross-resistant agents is commonly witnessed, with clinical response rates to non cross-resistant regimens declining as the disease advances. Expression of ABC transporters is unlikely to fully explain this phenomenon, and a clear molecular explanation for this process remains uncertain. A statistical analysis of a recently published RNA interference screen targeting 779 kinases in three cell lines deriving from different tumor types reveals a significant correlation between resistance to paclitaxel and a non cross-resistant cytotoxic agent. Furthermore, 20% of kinases that promote resistance to paclitaxel when targeted by RNAi also promote resistance to a non cross-resistant agent within that same cell line, consistent with a tissue-type dependence of multi-drug resistance. Conversely, paclitaxel-specific resistance and sensitising kinases occur less frequently than expected. This indicates that several cell line specific kinases may regulate multi-drug resistance and provide a potential explanation for the phenomenon of drug resistance to non cross-resistant agents witnessed in oncology practice. Furthermore, this work supports efforts to identify common pathways of drug response for future drug discovery programmes.

Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs.

Cytotoxic drug resistance is a major cause of cancer treatment failure. We report an RNA interference screen to identify genes influencing sensitivity of different cancer cell types to chemotherapeutic agents. A set of genes whose targeting leads to resistance to paclitaxel is identified, many of which are involved in the spindle assembly checkpoint. Silencing these genes attenuates paclitaxel-induced mitotic arrest and induces polyploidy in the absence of drug. We also identify a ceramide transport protein, COL4A3BP or CERT, whose downregulation sensitizes cancer cells to multiple cytotoxic agents, potentiating endoplasmic reticulum stress. COL4A3BP expression is increased in drug-resistant cell lines and in residual tumor following paclitaxel treatment of ovarian cancer, suggesting that it could be a target for chemotherapy-resistant cancers.

Role of Bim in the survival pathway induced by Raf in epithelial cells.

Selective and sustained activation of the Raf/MAP kinase pathway in MCF-10A Delta Raf-ER cells, a spontaneously immortalized human mammary epithelial cell line, was previously shown to protect these cells from suspension-induced cell death, a critical feature of the Ras-transformed phenotype. Although autocrine signalling through the EGF receptor is crucial for the protection induced by Raf in these cells, we report here the existence of an additional, more direct survival mechanism, linking Raf activation to the inhibition of a proapoptotic member of the Bcl-2 family, Bim. While detachment from the matrix results in transcriptional induction of two variants of this BH3-only protein, BimEL and BimL, activation of the Raf/ERK signalling both prevents Bim upregulation specifically and leads to phosphorylation and degradation of the BimEL isoform. This represents an important route to protect epithelial cells from the proapoptotic effect of Bim.

Identification of novel isoforms of the BH3 domain protein Bim which directly activate Bax to trigger apoptosis.

Bim (Bcl-2-interacting mediator of cell death) is a member of the BH3 domain-only subgroup of Bcl-2 family members, for which three splice variants have been described. Bim is expressed in many healthy cell types, where it is maintained in an inactive conformation through binding to the microtubule-associated dynein motor complex. Upon certain apoptotic stimuli, Bim is released from microtubules and mediates caspase-dependent apoptosis through a mechanism that is still unclear. Here, we have identified and characterized novel splice variants of human Bim mRNA. In particular, we show that a newly discovered, small protein isoform, BimAD, is also able to induce apoptosis strongly in several human cell lines. BimAD and the previously characterized isoform BimS are shown to be capable of heterodimerizing in vivo with both death antagonists (Bcl-2 and Bcl-X(L)) and death agonists (Bax). Mutants of BimAD that bind to Bax but not to Bcl-2 still promote apoptosis, indicating that Bim can regulate apoptosis through direct activation of the Bax-mediated cell death pathway without interaction with antiapoptotic Bcl-2 family members. Furthermore, we have shown that the interaction of the BimS and BimAD isoforms with Bax leads to a conformational change in this protein analogous to that triggered by the BH3-only protein Bid.