1 We have examined the effects of the immunosuppressive drugs cyclosporin A (CsA) and FK 506 on exocytosis in two in vitro preparations of the exocrine pancreas-lobules and dispersed acini. 2 In lobules taken from starved rats and stimulated with the secretagogue caerulein, both CsA and FK 506, given shortly before stimulation, caused a dose-dependent inhibition of amylase secretion. In lobules from rats that had been pretreated in vivo with the protease inhibitor FOY-305 to stimulate secretion maximally, both CsA and FK 506 inhibited secretion of newly synthesized proteins, whereas only FK 506 inhibited caerulein-stimulated amylase release. 3 These different effects of the immunosuppressants on amylase release were reflected in their effects on degranulation, as revealed by electron microscopy. Control acinar cells in lobules from FOY-305-treated rats were almost completely degranulated, whereas treatment with FK 506, but not CsA, caused the accumulation of zymogen granules close to the apical plasma membrane. 4 In dispersed acini, stimulated with the cholinomimetic secretagogue bethanechol, both CsA and FK 506 reduced the secretory response, to about 45% of control; IC50 values were 50 nM and 3 nM, respectively. A similar partial inhibition of exocytosis was seen in acini permeabilized with the bacterial toxin streptolysin 0 and stimulated with 1O gM Ca2".5 These results demonstrate that the immunosuppressants cause an inhibition of exocytosis in the exocrine pancreas that is both rapid in onset and potent. The loss of the inhibitory effect of CsA on amylase release in lobules taken from FOY-305-treated rats may reveal a change in the characteristics of exocytosis as a consequence of the high level of stimulation, and also indicates that CsA and FK 506 have subtly different effects on secretion. We suggest that these drugs might be useful tools in the dissection of the molecular mechanisms of exocytosis.
Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a PIK3R1W624R variant in PDXs from a high grade serous EOC. Allele frequencies of PIK3R1W624R in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of PIK3R1W624R and addiction of PIK3R1W624R carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that PIK3R1 encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The PIK3R1W624R mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer.
The pancreatic acinar cell is one of a number of cell types in which phosphoproteins are believed to be involved in the control of regulated exocytosis. We have examined the effects of three agents that affect secretion in the acinar cell on the phosphorylation states of proteins on the zymogen granule membrane. We show that Ca2+ and GTP gamma S, which stimulate secretion, also stimulate the phosphorylation of a protein of M(r) 45,000 (p45) on isolated zymogen granules. On the other hand, the protein kinase inhibitor genistein inhibits both secretion and phosphorylation of p45. For all three agents, p45 phosphorylation is affected over concentration ranges identical to those that affect secretion. The stimulatory effect of GTP gamma S and the inhibitory effect of genistein are also seen when the phosphorylation state of p45 on granules within permeabilized cells is examined. Ca2+, however, over the same concentration range, now causes dephosphorylation of p45. Furthermore, the time-course of this effect is similar to that of Ca(2+)-triggered secretion. Phosphorylation of p45 is exclusively on serine, with no detectable phosphorylation on either threonine or tyrosine. We propose that exocytosis in pancreatic acini is controlled at least in part through the phosphorylation/dephosphorylation of p45, with dephosphorylation acting as a trigger for exocytosis.
Patients with advanced ovarian cancers have experienced little improvement in overall survival with standard treatments. We used patient derived models to accelerate the discovery of treatment options. We developed a platform of Patient Derived Xenografts (PDX), by implanting and propagating patient's tumor samples collected at surgery in severely immunocompromised mice. From each PDX line we derived short term cultures of PDX Derived Tumor Cells (PDTCs). We envisioned that the weakness of PDXs and PDTCs, i.e. lack of human stromal and immune cells, might be instrumental to link tumor biomarkers to treatments. We have successfully propagated 49 PDX lines from metastatic EOC, which were fully characterized as far as histology, immunohistochemistry of epithelial and tissue specific markers and presence of TP53 and BRCA1/2 mutations. On PDTCs cultures we first assessed sensitivity to Carboplatin, currently used as first-line drug in ovarian cancer treatment. Of PDX lines derived from naïve metastatic HGS-EOC copy number variations and whole exome sequencing analyses were carried out, in order to identify putative and actionable cancer genes. Thus, on PDTCs we assayed also approved or experimental targeted drugs as monotherapy or in combinations. In one PDX line we identified a possibly loss-of-function mutation (W624R) of the PIK3R1 gene (encoding the p85alpha regulatory subunit of PI3K) with an allele frequency of 0.9, which could result in activation of the PI3K pathway. Several PI3K inhibitors were assayed on PDTCs of this PDX line harboring the PIK3R1W624R. Buparlisib (a Pan Class I PI3Ki) showed the ability to block proliferation of the PDTCs and the growth of the relevant PDXs in vivo. Altogether these data show that Patient Derived models are invaluable tools to unveil actionable pathways for the treatment of advanced/metastatic HGS-EOC. Citation Format: Concetta D'Ambrosio, Jessica Erriquez, Maddalena Arigoni, Sonia Capellero, Gloria Mittica, Eleonora Ghisoni, Fulvio Borella, Dionyssios Katsaros, Silvana Privitera, Marisa Ribotta, Elena Maldi, Giovanna Di Nardo, Enrico Berrino, Tiziana Venesio, Riccardo Ponzone, Marco Vaira, Douglas Hall, Mercedes Jimenez-Linan, Anna Paterson, Giorgio Valabrega, Raffaele Calogero, James Brenton, Mariaflavia Di Renzo, Martina Olivero. Assays of conventional chemotherapeutics and targeted drugs for ovarian cancer using patient derived models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1677.
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