Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.
Defects in insulin-receptor function have been associated with insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). Several types of mutations in the insulin-receptor gene have been identified in patients with genetic syndromes of extreme insulin resistance. In some patients, insulin resistance results from a decrease in the number of insulin receptors on the cell surface. In one patient with leprechaunism (leprechaun/Minn-1), there is greater than 90% decrease in the levels of insulin-receptor mRNA. This patient is a compound heterozygote for two mutations in the insulin-receptor gene, both of which act in a cis-dominant fashion to decrease levels of mRNA transcribed from that allele. In one allele, there is a nonsense mutation at codon 897. All 22 exons of the other allele have a normal sequence, so that the mutation in this allele appears to map outside the coding sequence of the gene. Impaired insertion in the plasma membrane also causes insulin resistance. In two sisters (patients A-5 and A-8) with type A extreme insulin resistance, there is an 80-90% decrease in the number of insulin receptors expressed on the surface of their cells. Both sisters, whose parents are first cousins, are homozygous for a point mutation in which valine is substituted for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. This mutation retards the posttranslational processing of the receptor and impairs the transport of receptors to the cell surface. Another patient with leprechaunism (leprechaun/Ark-1) is a compound heterozygote with two different mutant alleles of the insulin-receptor gene. In the allele derived from the father, there is a nonsense mutation at codon 672 that truncates the insulin receptor by deleting the COOH-terminal of the alpha-subunit and the entire beta-subunit. This truncated receptor, lacking a transmembrane domain, appears not to be expressed at the plasma membrane. In leprechaun/Ark-1, there is a missense mutation in the allele of the insulin-receptor gene derived from the mother. This point mutation results in substitution of glutamic acid for lysine at position 460 in the COOH-terminal half of the alpha-subunit. This mutation increases receptor affinity and impairs the ability of acid pH to dissociate insulin from the receptor within the endosome. There is a defect in recycling the receptor back to the plasma membrane associated with this defect. This results in an accelerated rate of receptor degradation and a consequent decrease in the number of receptors on the cell surface in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Correlations between germline APC mutation sites and colorectal pathophenotypes, as evaluated by the direct count of adenomas at colectomy, were investigated analysing colectomy specimens from 29 FAP patients carrying one mis-sense (codon 208) and 14 frame-shift or non-sense APC mutations (codons 232, 367, 437, 623, 876, 995, 1061, 1068, 1075, 1112, 1114, 1309, 1324, 1556). The mis-sense mutation at codon 208 was associated with a relatively mild colorectal pathophenotype. The mutation at codon 367, subject to alternative splicing, was associated with attenuated FAP. The mutation at codon 1309 was associated with the profuse colorectal adenomatosis. For 13 mutations, predicted to result in null alleles or truncated APC proteins, we correlated density and distribution of colorectal adenomas with the predicted functional effects of the mutation. The most severe colorectal pathophenotype was significantly associated with the truncating mutation at codon 1309, which is located downstream to the I β-catenin binding domain but upstream II β-catenin-binding domain. Mutations between codons 867 and 1114, which affect the I β-catenin binding domain, as well as mutations occurring in exons 6 and 9, predicted to result in null alleles, were associated with a less severe colorectal pathophenotype. Overall, the highest number of adenomas was detected in the right colon, followed by the left colon, transverse colon sigma and rectum. However, the highest density of adenomas was observed in the left colon, followed by the right colon, sigma, transverse colon and rectum. Colorectal carcinomas, observed in only five patients, were all in the left colon. © 2000 Cancer Research Campaign
Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles’ heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.Electronic supplementary materialThe online version of this article (10.1007/s00401-017-1799-2) contains supplementary material, which is available to authorized users.
BackgroundPancreatic cancer (PC) is the fourth most common cause of cancer death. Combination therapies with classical chemotherapeutic agents improved treatment of advanced PC at the cost of a relevant toxicity, but the 5-year survival rate remains below 5%. Consequently, new therapeutic options for this disease are urgently needed. In this study, we explored the effect of two repurposed drug candidates nelfinavir and nitroxoline, approved for non-anticancer human use, in PC cell lines. Nelfinavir and nitroxoline were tested as single agents, or in combinations with or without erlotinib, a targeted drug approved for PC treatment.MethodsThe effects of the drugs on the viability of AsPC-1, Capan-2 and BxPC-3 PC cell lines were assessed by MTT. The impact of the treatments on cell cycle distribution and apoptosis was analyzed by flow cytometry. The effects of treatments on proteins relevant in cell cycle regulation and apoptosis were evaluated by western blot. Self-renewal capacity of PC cell lines after drug treatments was assessed using a clonogenic assay.ResultsWhen used as single agents, nelfinavir and nitroxoline decreased viability, affected cell cycle and reduced the expression of relevant cell cycle proteins. The effects on apoptosis were variable among PC cell lines. Moreover, these agents drastically impaired clonogenic activity of the three PC cell lines. Combinations of nelfinavir and nitroxoline, with or without erlotinib, resulted in dose- and cell-dependent synergistic effects on cell viability. These effects were paralleled by cell cycle alterations and more consistent apoptosis induction as compared to single agents. Treatments with drug combinations induced drastic impairment of clonogenic activity in the three cell lines.ConclusionsThis study shows that two non-antitumor drugs, nelfinavir and nitroxoline, as single agents or in combination have antitumor effects that appear comparable, or in some case more pronounced than those of erlotinib in three PC cell lines. Our results support repurposing of these approved drugs as single agents or in combination for PC treatment.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0904-2) contains supplementary material, which is available to authorized users.
Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The “NOTCH signaling pathway” was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1165-y) contains supplementary material, which is available to authorized users.
The etiology of breast cancer in Africa is scarcely investigated. Breast cancer was responsible for 456/2,233 cancer patients (20.4%) ascertained between 1999 and 2004 at Gezira University, Central Sudan. Male breast cancer accounted for 16/456 patients (3.5%), 275/440 female patients (62.5%) were premenopausal and 150/440 cases (34%) occurred in women with > or =5 childbirths. We characterized for germline BRCA1/2 mutations a one-year series of patients (34 females, 1 male) selected by diagnosis within age 40 years or male gender. Overall 33/35 patients were found to carry 60 BRCA1/2 variants, of which 17 (28%) were novel, 22 (37%) reported in populations from various geographic areas and 21 (35%) reported worldwide. Detected variants included 5 truncating mutations, one of which (in BRCA2) was in the male patient. The 55 non-truncating variants included 3 unclassified variants predicted to affect protein product and not co-occurring with a truncating mutation in the same gene. Patients were from different tribes but AMOVA showed that most BRCA1/2 variation was within individuals (86.41%) and patients clustered independently of tribe in a phylogenetic tree. Cluster analysis based on age at cancer diagnosis and reproductive variables split female patients in two clusters that, by factor analysis, were explained by low versus high scores of the total period occupied by pregnancies and lactation. The cluster with low scores comprised all 4 patients with truncating mutations and 3 of the 4 carriers of unclassified variants predicted to affect protein product. Our findings suggest that in Central Sudan BRCA1/2 represent an important etiological factor of breast cancer in males and young women less exposed to pregnancy and lactation. Factors other than BRCA1/2 may contribute to breast cancer in young highly multiparous women who breast-fed for prolonged periods.
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