Analysis of Rca1 function at the G1-S transition in Drosophila melanogaster

Tight control of APC/C-Cdh1Fzr activity is essential for progression through mitosis and establishment of the G1 phase. Rca1 is a nuclear protein that inhibits the APC/C-Cdh1Fzr complex during G2 to allow cyclin accumulation and subsequent entry into mitosis. In this thesis, a localisation study of Rca1 was performed revealing that a nuclear localisation sequence (NLS) and other domains in the protein mediate efficient nuclear accumulation. Besides its function in G2, Rca1 expression can promote S phase entry. Rca1 belongs to the family of F-box proteins that mediate substrate specificity in SCF (Skp-Cul1-F-box) ubiquitin ligases. Functional studies demonstrated that the F-box is crucial for S phase induction by Rca1, suggesting that Rca1 has a secondary function in an SCF complex. A major part of this thesis was devoted to characterise the putative SCF/Rca1 complex and its target proteins. In a yeast two-hybrid screen, the SCF subunit SkpA was identified as a binding partner of Rca1. Coimmunoprecipitation studies confirmed this interaction and indicated moreover that SkpA binding relies on the F-box in Rca1. Furthermore, endogenous Cul1 coprecipitated with Rca1 and this also in an F-box dependent manner. Altogether, these experiments demonstrated that Rca1 forms a complex with the SCF core subunits SkpA and Cul1. The SCF/Rca1 complex could promote S phase entry by degrading a negative regulator of the G1-S transition. Cyclin A/Cdk1 is a potent S phase inducer, but its activity is normally dampened by the CKI Rux, inhibitory phosphorylation by Wee and APC/C-Cdh1Fzr dependent degradation of Cyclin A. Protein levels of these S phase inhibitors were not altered by coexpression of Rca1 suggesting that these proteins are not targets of the SCF/Rca1 complex. Overexpression of Rca1 in larval salivary glands results in impaired endoreplication and accumulation of Cyclin A, Cyclin E and Cdk1. Expression profiling revealed that mitotic genes (e.g. Cyclin A/B, Cdk1) are normally downregulated in salivary glands, but ectopic Rca1 expression promotes transcription of these genes. In addition, qRT-PCR analysis showed elevated transcript levels of the E2F1 targets Rnr2 and PCNA, suggesting that Rca1 overexpression leads to increased E2F1 activity. Cyclin E is also a known E2F1 target and hence, this might explain the marked increase in Cyclin E transcript levels. Finally, the gene expression analysis indicated that components of the APC/C and its target Geminin were present in larval salivary glands, thus supporting the idea that rereplication in endoreplicating cells is controlled by Geminin and the APC/C-Cdh1Fzr complex

Osteoblastic Response in Patients with Non-small Cell Lung Cancer with Activating EGFR Mutations and Bone Metastases during Treatment with EGFR Kinase Inhibitors

© 2010International Association for the Study of Lung Cance

Benchmarking of Mutation Diagnostics in Clinical Lung Cancer Specimens

Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy ‘Sanger’ sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r2 = 0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation

The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles

Endoreplicating cells undergo multiple rounds of DNA replication leading to polyploidy or polyteny. Oscillation of Cyclin E (CycE)-dependent kinase activity is the main driving force in Drosophila endocycles. High levels of CycE–Cdk2 activity trigger S phase, while down-regulation of CycE-Cdk2 activity is crucial to allow licensing of replication origins. In mitotic cells relicensing in S phase is prevented by Geminin. Here we show that Geminin protein oscillates in endoreplicating salivary glands of Drosophila. Geminin levels are high in S phase, but drop once DNA replication has been completed. DNA licensing is coupled to mitosis through the action of the anaphase-promoting complex/cyclosome (APC/C). We demonstrate that, even though endoreplicating cells never enter mitosis, APC/C activity is required in endoreplicating cells to mediate Geminin oscillation. Down-regulation of APC/C activity results in stabilization of Geminin protein and blocks endocycle progression. Geminin is only abundant in cells with high CycE–Cdk2 activity, suggesting that APC/C–Fzr activity is periodically inhibited by CycE–Cdk2, to prevent relicensing in S-phase cells

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the Skp–Cullin–F-box (SCF) complex. So far, no function has been ascribed to this domain. We find that the F-box of Rca1 is dispensable for APC/C–Fzr inhibition during G2. Nevertheless, we show that Rca1 has an additional function at the G1–S transition, which requires the F-box. Overexpression of Rca1 accelerates the G1–S transition in an F-box-dependent manner. Conversely, S-phase entry is delayed in cells in which endogenous Rca1 is replaced by a transgene lacking the F-box. We propose that Rca1 acts as an F-box protein in an as yet uncharacterized SCF complex, which promotes S-phase entry

Prognostic Impact of [18F]Fluorothymidine and [18F]Fluoro-D-Glucose Baseline Uptakes in Patients with Lung Cancer Treated First-Line with Erlotinib

<div><p>3′-deoxy-3′-[¹⁸F]fluoro-L-thymidine (FLT) and 2′-deoxy-2′-[¹⁸F]fluoro-D-glucose (FDG) are used to visualize proliferative and metabolic activity of tumors. In this study we aimed at evaluating the prognostic value of FLT and FDG uptake measured by positron emission tomography (PET) in patients with metastatic non-small cell lung cancer (NSCLC) prior to systemic therapy with erlotinib. FLT and FDG maximum standardized uptake (SUVmax) values per patient were analyzed in 40 chemotherapy naive patients with advanced NSCLC (stage IV) before treatment with erlotinib. Prior therapy median SUVmax was 6.6 for FDG and 3.0 for FLT, respectively. In univariate analysis, patients with an FDG SUVmax <6.6 had a significantly better overall survival (16.3 months [95% confidence interval [CI] 7.1–25.4 months]) compared to patients with an FDG SUVmax ≥6.6 (3.1 months [95% CI 0.6–5.5 months]) (p<0.001, log rank). Similarly, low FLT uptake (SUVmax <3.0) was associated with significantly longer survival (10.3 months (0–23.3 months, 95% CI) compared to high FLT uptake (3.4 months (0–8.1 months, 95% CI) (p = 0.027). The independent prognostic value of baseline FDG uptake was demonstrated in multivariate analysis (p = 0.05, Cox regression). These data suggest that baseline SUVmax values for both FDG and FLT PET might be further developed as markers for prognostic stratification of patients in advanced NSCLC treated with tyrosine kinase inhibitors (TKI) directed against the epidermal growth factor receptor (EGFR).</p> <h3>Trial Registration</h3><p>Clinicaltrials.gov, Identifier: <a href="http://clinicaltrials.gov/ct2/show/NCT00568841">NCT00568841</a></p> </div

Kaplan Meier curves showing overall survival depending on SUVmax values.

<p>A) Overall survival of patients with high (>6.7; grey) or low (<6.7; red) baseline SUVmax in FDG-PET. (B) Overall survival of patients with high (>3; grey) or low (<3; red) baseline SUVmax in FLT-PET.</p

Individual patient characteristics and PET results.

<p>Individual patient characteristics and PET results.</p

Example of two patients with low and high baseline uptake of FDG and FLT.

<p>The patient shown in figure A with low uptake is a 66-year old female patient who had an overall survival of 21.3 months, whereas the patient in B with a high uptake is a 56-year old female patient with an overall survival of only 1.5 months. In both cases, the respective most active lesion was chosen for assessment.</p