NOTCH1 and PIK3CA mutation are related to HPV-associated vulvar squamous cell carcinoma.

NOTCH1 and PIK3CA are members of important cell signalling pathways that are deregulated in squamous cell carcinomas of various organs. Vulvar squamous cell carcinomas (vulvSCC) are classically divided into two pathways, HPV-associated or HPV-independent, but the effect of NOTCH1 and PIK3CA mutations in both groups is unclear. We analysed two different cohorts of vulvSCC using Hybrid Capture-based Comprehensive Genomic Profiling and identified NOTCH1 and PIK3CA mutations in 35% and 31% of 48 primary vulvSCC. In this first cohort, PIK3CA and NOTCH1 mutations were significantly correlated with HPV infection (p < 0.01). Furthermore, mutations in both genes were associated with an advanced tumor stage and poorly differentiated status (p < 0.05). PIK3CA and NOTCH1 mutations were also associated with shorter patient survival which did not reach significance. In the second cohort of 735 advanced vulvSCC from metastatic site biopsies or from sites of unresectable loco-regional disease, NOTCH1 and PIK3CA mutations were reported in 14% and 20.3%, respectively. 4 of 48 (8%) and 22 of 735 vulvSCC (3.0%) featured genomic alterations (short variants and/or copy number changes and/or rearrangements) in both NOTCH1 and PIK3CA. NOTCH1 mutations were mostly located in the extracellular EGF-like domains, were inactivating and indicated that NOTCH1 functions predominantly as a tumor suppressor gene in vulvSCC. In contrast, PIK3CA mutations favored hotspot codons 1624 and 1633 of the gene, indicating that PIK3CA acts as an oncogene in vulvar carcinogenesis. In conclusion, NOTCH1 and PIK3CA mutations are detectable in a substantial proportion of vulvSCC and are related to HPV infection and more aggressive tumor behaviour.

Improving the turnaround time of molecular profiling for advanced non-small cell lung cancer: Outcome of a new algorithm integrating multiple approaches.

BACKGROUND: Molecular tumor profiling to identify oncogenic drivers and actionable mutations has a profound impact on how lung cancer is treated. Especially in the subgroup of non-small cell lung cancer (NSCLC), molecular testing for certain mutations is crucial in daily clinical practice and is recommended by international guidelines. To date, a standardized approach to identify druggable genetic alterations are lacking. We have developed and implemented a new diagnostic algorithm to harmonize the molecular testing of NSCLC. PATIENTS AND METHODS: In this retrospective analysis, we reviewed 119 patients diagnosed with NSCLC at the University Hospital Zurich. Tumor samples were analyzed using our standardized diagnostic algorithm: After the histological diagnosis was made, tissue samples were further analyzed by immunohistochemical stainings as well as the real-time PCR test Idylla™. Extracted DNA was further utilized for comprehensive genomic profiling (FoundationOne®CDx, F1CDx). RESULTS: Out of the 119 patients were included in this study, 100 patients were diagnosed with non-squamous NSCLC (nsqNSCLC) and 19 with squamous NSCLC (sqNSCLC). The samples from the nsqNSCLC patients underwent testing by Idylla™ and were evaluated by immunohistochemistry (IHC). F1CDx analysis was run on 67 samples and 46 potentially actionable genomic alterations were detected. Ten patients received the indicated targeted treatment. The median time to test results was 4 days for the Idylla test, 5 days for IHC and 13 days for the F1CDx. CONCLUSION: In patients with NSCLC, the implementation of a standardized molecular testing algorithm provided information on predictive markers for NSCLC within a few working days. The implementation of broader genomic profiling led to the identification of actionable targets, which would otherwise not have been discovered.

Purified retinal nitric oxide synthase enhances ADP-ribosylation of rod outer segment proteins.

Nitric oxide synthase is present in different cell layers of vertebrate retina and seems to have neuromodulatory functions in the outer retina. The enzyme, when purified from a bovine retina extract, has an apparent molecular mass of 160 kDa and resembles the neuronal constitutive NOS type I with respect to Ca(2+)-calmodulin sensitivity, Km value and inhibition by analogues of L-arginine. Retinal NOS is present in a preparation of rod outer segments attached to parts of the inner segments, but not in pure outer segments. We describe the enhancement of specific ADP-ribosylation of outer segment proteins by purified retinal NOS.

Distinct molecular recognition of calmodulin-binding sites in the neuronal and macrophage nitric oxide synthases: a surface plasmon resonance study.

The neuronal nitric oxide synthase and the macrophage nitric oxide synthase are differently regulated by Ca2+/calmodulin. We investigated the dynamics of calmodulin binding to the putative calmodulin-binding sites in both nitric oxide synthases. Peptides derived from the putative calmodulin-binding sites were synthesized and immobilized to a dextran layer of a biosensor chip. Complex formation of calmodulin and the peptides was monitored by surface plasmon resonance spectroscopy and recorded as sensorgrams. We determined a dissociation constant KD of 5.0 x 10(-9) M for the neuronal nitric oxide synthase and calmodulin. The association rate constant and the dissociation rate constant were ka = 1.58 x 10(5) M-1 s-1 and kd = 7.87 x 10(-4) s-1, respectively. Sensorgrams obtained with the macrophage nitric oxide synthase peptide were remarkably different. Calmodulin, once bound to the peptide, did not dissociate. Association of calmodulin to the peptide occurred with the same rate constants (ka = 3 x 10(4) M-1 s-1) regardless of the presence or absence of Ca2+. The affinity was in the subnanomolar range (KD) < 0.1 x 10(-9) M). We conclude that the extremely tight binding of calmodulin to the NOS-II is solely controlled by the calmodulin-binding segment and not by other parts of the protein.

Introduction of a phosphate at serine741 of the calmodulin-binding domain of the neuronal nitric oxide synthase (NOS-I) prevents binding of calmodulin.

The calmodulin-binding domain of neuronal nitric oxide synthase (NOS-I) is represented by a segment of 26 amino acids. We tested whether the phosphorylation of a serine in the calmodulin-binding domain changes the affinity of calmodulin for this binding site. We monitored the binding of calmodulin to synthetic peptides by surface plasmon resonance spectroscopy, an electrophoretic mobility assay, circular dichroism spectroscopy and competitive inhibitory studies. All four experimental approaches showed that binding of calmodulin to the calmodulin-binding site is blocked by introduction of a phosphate. Phosphorylation of the calmodulin-binding domain of NOS-I could be a negative feedback loop to turn off NOS-I activity.

Calmodulin controls the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the beta-subunit.

Calmodulin (CaM) controls the activity of the rod cGMP-gated ion channel by decreasing the apparent cGMP affinity. We have examined the mechanism of this modulation using electrophysiological and biochemical techniques. Heteromeric channels, consisting of alpha- and beta-subunits, display a high CaM sensitivity (EC50

Ligand sensitivity of the 2 subunit from the bovine cone cGMP-gated channel is modulated by protein kinase C but not by calmodulin.

1. Homomeric cyclic nucleotide-gated (CNG) channels composed of alpha2 subunits from bovine cone photoreceptors were heterologously expressed in the human embryonic kidney (HEK) 293 cell line. Modulation of cGMP sensitivity by protein kinase C (PKC)-mediated phosphorylation and by binding of calmodulin (CaM) was investigated in inside-out patches. 2. A peptide encompassing the putative CaM-binding site within the N-terminus of the channel protein binds Ca(2+)-CaM with high affinity, yet the ligand sensitivity of alpha2 channels is not modulated by CaM. 3. PKC-mediated phosphorylation increased the activation constant (K(1/2)) for cGMP from 19 to 56 microM and decreased the Hill coefficient (from 2.5 to 1.5). The change in ligand sensitivity involves phosphorylation of the serine residues S577 and S579 in the cGMP-binding domain. The increase in K(1/2) was completely abolished in mutant channels in which the two serine residues were replaced by alanine. 4. An antibody specific for the delta isoform of PKC strongly labels the cone outer segments. 5. Modulation of cGMP affinity of bovine alpha2 CNG channels by phosphorylation could play a role in the regulation of photoreceptor sensitivity.