Purpose: Oncogenic mutations in the KRAS/PI3K/AKT pathway are one of the most frequent alterations in cancer. Although PI3K or AKT inhibitors show promising results in clinical trials, drug resistance frequently emerges. We previously revealed Wnt/b-catenin signaling hyperactivation as responsible for such resistance in colorectal cancer. Here we investigate Wnt-mediated resistance in patients treated with PI3K or AKT inhibitors in clinical trials and evaluate the efficacy of a new Wnt/tankyrase inhibitor, NVP-TNKS656, to overcome such resistance.Experimental Design: Colorectal cancer patient-derived sphere cultures and mouse tumor xenografts were treated with NVP-TNKS656, in combination with PI3K or AKT inhibitors.We analyzed progression-free survival of patients treated with different PI3K/AKT/mTOR inhibitors in correlation with Wnt/b-catenin pathway activation, oncogenic mutations, clinicopathological traits, and gene expression patterns in 40 colorectal cancer baseline tumors.Results: Combination with NVP-TNKS656 promoted apoptosis in PI3K or AKT inhibitor-resistant cells with high nuclear b-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear b-catenin content and progressed earlier upon PI3K/AKT/ mTOR inhibition. Nuclear b-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not.Conclusions: High nuclear b-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear b-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/b-catenin inhibition and together with b-catenin may be predictive biomarkers of drug response providing a rationale to stratify colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/b-catenin inhibitors.
Thyroid transcription factor-1 (TTF-1) is a 43-kDa, phosphorylated member of the Nkx2 family of homeodomain-containing proteins expressed selectively in lung, thyroid, and the central nervous system. To assess the role of TTF-1 and its phosphorylation during lung morphogenesis, mice bearing a mutant allele, in which seven serine phosphorylation sites were mutated, Titf1 PM/PM , were generated by homologous recombination. Although heterozygous Titf1 PM/؉ mice were unaffected, homozygous Titf1 PM/PM mice died immediately following birth. In contrast to Titf1 null mutant mice, which lack peripheral lung tissues, bronchiolar and peripheral acinar components of the lung were present in the Titf1 PM/PM mice. Although lobulation and early branching morphogenesis were maintained in the mutant mice, abnormalities in acinar tubules and pulmonary hypoplasia indicated defects in lung morphogenesis later in development. Although TTF-1 PM protein was readily detected within the nuclei of pulmonary epithelial cells at sites and abundance consistent with that of endogenous TTF-1, expression of a number of known TTF-1 target genes, including surfactant proteins and secretoglobulin 1A, was variably decreased in the mutant mice. Vascular endothelial growth factor mRNA was decreased in association with decreased formation of peripheral pulmonary blood vessels. Genes mediating surfactant homeostasis, vasculogenesis, host defense, fluid homeostasis, and inflammation were highly represented among those regulated by TTF-1. Thus, in contrast to the null Titf1 mutation, the Titf1 PM/PM mutant substantially restored lung morphogenesis. Direct and indirect transcriptional targets of TTF-1 were identified that are likely to play important roles in lung formation and function.Lung formation begins with the outpouching of endodermal tissues from the laryngeal-tracheal-esophageal groove at embryonic day (E) 1 9 -9.5 in the mouse embryo. Epithelial lined tubules invade the splanchnic mesenchyme and undergo branching morphogenesis to form bronchi, bronchioles, and alveolar regions of the adult lung. Thyroid transcription factor-1 (genomic designation Titf1; also termed TEBP, or Nkx2
Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for -ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.Polyhydroxyalkanoates (PHAs) are a group of polyesters produced by a large number of bacteria, which accumulate them in intracellular granules as a response to environmental stress and nutrient imbalance (2, 7). These thermoplastic polymers have drawn great interest since their discovery due to their degradability and the potential to produce them from renewable carbon sources. Azotobacter sp. FA8 is an aerobic, nitrogen-fixing bacterium that accumulates poly(3-hydroxybutyrate) (PHB) when cultivated on several carbon sources, including sucrose (13). Although the capacity of Azotobacter strains to accumulate PHAs is well known (2, 9), except for a recently described -ketothiolase from Azotobacter vinelandii (18), the genes responsible for their synthesis have not yet been identified. In this paper we report the identification, cloning, and molecular analysis of the phb gene cluster of Azotobacter sp. FA8.Cloning and molecular analysis of the PHB synthase gene from Azotobacter sp. FA8. Genomic DNA of Azotobacter sp. FA8 was partially digested with XhoI and ligated to the mobilizable cosmid pVK102 (5) digested with the same enzyme and dephosphorylated. The resulting ligation mixture was packaged using an in vitro packaging system (Stratagene, La Jolla, Calif.) and used to transfect Escherichia coli S17-1, a strain that contains the tra genes of plasmid RP4 integrated into the chromosome (19). Transductants were selected on Luria-Bertani plates containing 10 g of tetracycline/ml. The library was screened for the presence of the PHB synthase (phbC) gene by complementation analysis. Ralstonia eutropha PHB-4 (17), a phbC mutant, was used as recipient. The complementation was carried out on a mineral salts medium (16) containing 1.5% (wt/vol) agar, 0.05% (wt/vol) NH 4 Cl, 0.5% (wt/vol) fructose, and 5 g of tetracycline/ml. One of the E. coli clones, C1, gave rise to opaque, PHB-producing transconjugants. The polymer produced by the recombinant was extracted from lyophilized cells with hot chloroform and ethanol precipitated, and the methyl ester derivatives were analyzed by gas chromatography (1) using a Gow Mac Series 580 gas chromatograph (Bridgewater, N.J.) equipped with a flame ionization detector and a packing column of Carbowax 20 M-TPA-Chromosorb W-AW (SUPELCO, Bellefonte, Pa.). The polymer obtained was found to be a homopolymer of 3-hydroxybutyrate.The recombinant cosmid from clo...
Genomic characterization of recurrent breast and lung tumors developed over the course of 10 years in a 29-year-old patient with a germline TP53 mutation (LiFraumeni Syndrome) identifi ed oncogenic alterations in the HER2 and EGFR genes across all tumors, including HER2 amplifi cations, an EGFR -exon 20 insertion, and the fi rst-in-humans HER2 V659E mutation showing a phenotypic convergent evolution toward HER2 and EGFR alterations. Following the identifi cation of HER2-activating events in the most recent lung carcinoma and in circulating tumor cells, we treated the reminiscent metastatic lesions with a lapatinib-based therapy. A symptomatic and radiologic clinical response was achieved. HER2 V659E sensitivity to lapatinib was confi rmed in the laboratory. SIGNIFICANCE:The precise knowledge of the genomic alterations present in tumors is critical to selecting the optimal treatment for each patient. Here, we report the molecular characterization and clinical response to a lapatinib-based therapy for the tumors of a Li-Fraumeni patient showing prevalence of HER2 and EGFR genomic alterations. Cancer Discov; 3(11); 1238-44.
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