Protein kinase C iota in mammalian cell polarity and cancer

Protein Kinase C iota (PKCɩ) is a serine/threonine kinase that is involved in epithelial polarity and malignancy where polarity is typically abrogated. The aim of this thesis was to characterise the role of PKCɩ in oncogenic signaling in the context of polarity loss. This work set out to identify activators, binding partners and downstream effectors of PKCɩ with a view to informing rational therapeutic design. Madin Darby Canine Kidney (MDCK) cells, grown as polarised cysts containing a central lumen in Matrigel, developed a multiple-lumen phenotype on perturbation of PKCɩ, suggesting a threshold requirement for normal lumenogenesis. When transfected with oncogenic Ras, MDCK cells formed large spheres lacking a lumen and polarity. Upon PKCɩ inhibition with siRNA or small molecules, polarity was partially rescued indicating that PKCɩ lies downstream of Ras in this polarity loss pathway. Structural studies have enabled two distinct approaches to identify effectors of PKCɩ. A novel protein-protein interaction motif (RIPR) within PKCɩ was identified and shown here to be responsible for interaction with a subset of target proteins, including LLGL2. MDCK cysts expressing a PKCɩ mutated in this motif developed a multiple-lumen phenotype. In a second approach, use was made of an inhibitor bound complex of PKCɩ to design a drug insensitive mutant. Expression of the resistant mutant in HEK293 cells led to resistance to inhibitor induced dephosphorylation of LLGL2 and protected MDCK cells from developing inhibitor induced multiple lumens and loss of polarity. This tool has been used to screen for PKCɩ substrates. In summary, PKCɩ lies downstream of H-Ras in MDCK polarity signalling. The site at which PKCɩ binds LLGL2 is defined and shown to contribute to normal lumenogenesis. A drug insensitive PKCɩ mutant is described that can be utilised for substrate and biomarker identification

Massive Supergravity and Deconstruction

We present a simple superfield Lagrangian for massive supergravity. It comprises the minimal supergravity Lagrangian with interactions as well as mass terms for the metric superfield and the chiral compensator. This is the natural generalization of the Fierz-Pauli Lagrangian for massive gravity which comprises mass terms for the metric and its trace. We show that the on-shell bosonic and fermionic fields are degenerate and have the appropriate spins: 2, 3/2, 3/2 and 1. We then study this interacting Lagrangian using goldstone superfields. We find that a chiral multiplet of goldstones gets a kinetic term through mixing, just as the scalar goldstone does in the non-supersymmetric case. This produces Planck scale (Mpl) interactions with matter and all the discontinuities and unitarity bounds associated with massive gravity. In particular, the scale of strong coupling is (Mpl m^4)^1/5, where m is the multiplet's mass. Next, we consider applications of massive supergravity to deconstruction. We estimate various quantum effects which generate non-local operators in theory space. As an example, we show that the single massive supergravity multiplet in a 2-site model can serve the function of an extra dimension in anomaly mediation.Comment: 24 pages, 2 figures, some color. Typos fixed and refs added in v

Addition of nintedanib or placebo to neoadjuvant gemcitabine and cisplatin in locally advanced muscle-invasive bladder cancer (NEOBLADE) : a double-blind, randomised, phase 2 trial

Background Recurrence is common after neoadjuvant chemotherapy and radical treatment for muscle-invasive bladder cancer. We investigated the effect of adding nintedanib to neoadjuvant chemotherapy on response and survival in muscle-invasive bladder cancer. Methods NEOBLADE was a parallel-arm, double-blind, randomised, placebo-controlled, phase 2 trial of neoadjuvant gemcitabine and cisplatin chemotherapy with nintedanib or placebo in locally advanced muscle-invasive bladder cancer. Patients aged 18 years or older, with an Eastern Cooperative Oncology Group performance status of 0–1, were recruited from 15 hospitals in the UK. Patients were randomly assigned (1:1) to nintedanib or placebo using permuted blocks with random block sizes of two or four, stratified by centre and glomerular filtration rate. Treatments were allocated using an interactive web-based system, and patients and investigators were masked to treatment allocation throughout the study. Patients received oral nintedanib (150 mg or 200 mg twice daily for 12 weeks) or placebo, in addition to usual neoadjuvant chemotherapy with intravenous gemcitabine 1000 mg/m2 on days 1 and 8 and intravenous cisplatin 70 mg/m2 on day 1 of a 3-weekly cycle. The primary endpoint was pathological complete response rate, assessed at cystectomy or at day 8 of cycle 3 (plus or minus 7 days) if cystectomy did not occur. Primary analyses were done in the intention-to-treat population. The trial is registered with EudraCT, 2012-004895-01, and ISRCTN, 56349930, and has completed planned recruitment. Findings Between Dec 4, 2014, and Sept 3, 2018, 120 patients were recruited and were randomly allocated to receive nintedanib (n=57) or placebo (n=63). The median follow-up for the study was 33·5 months (IQR 14·0–44·0). Pathological complete response in the intention-to-treat population was reached in 21 (37%) of 57 patients in the nintedanib group and 20 (32%) of 63 in the placebo group (odds ratio [OR] 1·25, 70% CI 0·84–1·87; p=0·28). Grade 3 or worse toxicities were observed in 53 (93%) of 57 participants who received nintedanib and 50 (79%) of 63 patients in the placebo group (OR 1·65, 95% CI 0·74–3·65; p=0·24). The most common grade 3 or worse adverse events were thromboembolic events (17 [30%] of 57 patients in the nintedanib group vs 13 [21%] of 63 patients in the placebo group [OR 1·63, 95% CI 0·71–3·76; p=0·29]) and decreased neutrophil count (22 [39%] in the nintedanib group vs seven [11%] in the placebo group [5·03, 1·95–13·00; p=0·0006]). 45 treatment-related serious adverse events occurred in the nintedanib group and 43 occurred in the placebo group. One treatment-related death occurred in the placebo group, which was due to myocardial infarction. Interpretation The addition of nintedanib to chemotherapy was safe but did not improve the rate of pathological complete response in muscle-invasive bladder cancer