The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non–Small Cell Lung Cancer (NSCLC) address all aspects of management for NSCLC. These NCCN Guidelines Insights focus on recent updates to the NCCN Guidelines regarding targeted therapies, immunotherapies, and their respective biomarkers.

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Lung RF ablation appears to be safe and linked with promising long-term survival and local tumor progression outcomes, especially given the patient population treated.

Protein kinase C (PKC) has been implicated in integrinmediated spreading and migration. In mammary epithelial cells there is a partial co-localization between β1 integrin and PKCα. This reflects complexes between these proteins as demonstrated by fluorescense resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy and also by coprecipitation. Constitutive complexes are observed for the intact PKCα and also form with the regulatory domain in an activation-dependent manner. Expression of PKCα causes upregulation of β1 integrin on the cell surface, whereas stimulation of PKC induces internalization of β1 integrin. The integrin initially traffics to an endosomal compartment in a Ca 2ϩ /PI 3-kinase/ dynamin I-dependent manner and subsequently enters an endocytic recycling pathway. This induction of endocytosis by PKCα is a function of activity and is not observed for the regulatory domain. PKCα, but not PKCα regulatory domain expression stimulates migration on β1 integrin substrates. This PKCα-enhanced migratory response is inhibited by blockade of endocytosis.

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non–Small Cell Lung Cancer (NSCLC) provide recommended management for patients with NSCLC, including diagnosis, primary treatment, surveillance for relapse, and subsequent treatment. Patients with metastatic lung cancer who are eligible for targeted therapies or immunotherapies are now surviving longer. This selection from the NCCN Guidelines for NSCLC focuses on targeted therapies for patients with metastatic NSCLC and actionable mutations.

Although closely related, RhoA and RhoC have distinct molecular targets and functional roles in cell migration and invasion.

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.

Fö rster resonance energy transfer (FRET) microscopy is a powerful technique that enables the visualization of signaling intermediates, protein interactions, and protein conformational and biochemical status. With the availability of an ever-increasing collection of fluorescent proteins, pairs of spectrally different variants have been used for the study of FRET in living cells. However, suitable spectral overlap, necessary for efficient FRET, is limited by the requirement for proper emission separation. Currently used FRET pairs represent compromises between these opposing spectral demands that reduce the maximally attainable FRET sensitivity. We present a previously undescribed FRET acceptor, a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). REACh allows the use of the photophysically superior FRET donor EGFP, with which it exhibits optimal spectral overlap, which obviates the need for narrow spectral filtering and allows additional fluorescent labels to be used within the same cell. The latter allows the generation of sophisticated bioassays for complex biological questions. We show that this dark acceptor is ideally suited for donor fluorescence lifetime imaging microscopy (FLIM) and confirm these measurements with an independent intensity-based donor fluorescence quenching resonance energy transfer (FqRET) assay. REACh also can be used in donor photobleaching kinetics-based FRET studies. By detecting FRET between a GFP-tagged ubiquitination substrate and REACh-labeled ubiquitin, we imaged the active ubiquitination machinery inside cells. This assay therefore can be used to study proteins whose function is regulated by ubiquitination.biosensor ͉ fluorescence lifetime imaging microscopy ͉ ubiquitin ͉ proteasome F luorescent protein-based Förster resonance energy transfer (FRET) (1) assays allow the detection and quantification of a variety of cellular biochemical events, e.g., GTPase activity status, protein phosphorylation, degradation, conformational changes, and interactions (2, 3). Spectral contamination, i.e., donor emission bleed-through and direct acceptor excitation, complicates the measurement of FRET between fluorescent protein conjugates and reduces the dynamic range and sensitivity even when both fluorophores are included in the same reporter construct. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption but separated emission spectra to allow their selective imaging. Because of the relatively broad emission spectra and small Stokes shift, fluorescent proteins generally fail to fulfill these criteria.The most used FRET pair is a cyan fluorescent protein (CFP) donor and a yellow fluorescent protein (YFP) acceptor (3, 4). CFP furthermore suffers from a reduced fluorescence yield when compared with most members of the fluorescent protein family (5). Moreover, its excitation at low wavelengths causes substantial autofluorescence in cells and is not compatible with commonly used ...