Hypoinsulinaemic, hypoketotic hypoglycaemia due to mosaic genetic activation of PI3-kinase.
ABSTRACT: OBJECTIVE:Genetic activation of the insulin signal-transducing kinase AKT2 causes syndromic hypoketotic hypoglycaemia without elevated insulin. Mosaic activating mutations in class 1A phospatidylinositol-3-kinase (PI3K), upstream from AKT2 in insulin signalling, are known to cause segmental overgrowth, but the metabolic consequences have not been systematically reported. We assess the metabolic phenotype of 22 patients with mosaic activating mutations affecting PI3K, thereby providing new insight into the metabolic function of this complex node in insulin signal transduction. METHODS:Three patients with megalencephaly, diffuse asymmetric overgrowth, hypoketotic, hypoinsulinaemic hypoglycaemia and no AKT2 mutation underwent further genetic, clinical and metabolic investigation. Signalling in dermal fibroblasts from one patient and efficacy of the mTOR inhibitor Sirolimus on pathway activation were examined. Finally, the metabolic profile of a cohort of 19 further patients with mosaic activating mutations in PI3K was assessed. RESULTS:In the first three patients, mosaic mutations in PIK3CA (p.Gly118Asp or p.Glu726Lys) or PIK3R2 (p.Gly373Arg) were found. In different tissue samples available from one patient, the PIK3CA p.Glu726Lys mutation was present at burdens from 24% to 42%, with the highest level in the liver. Dermal fibroblasts showed increased basal AKT phosphorylation which was potently suppressed by Sirolimus. Nineteen further patients with mosaic mutations in PIK3CA had neither clinical nor biochemical evidence of hypoglycaemia. CONCLUSIONS:Mosaic mutations activating class 1A PI3K cause severe non-ketotic hypoglycaemia in a subset of patients, with the metabolic phenotype presumably related to the extent of mosaicism within the liver. mTOR or PI3K inhibitors offer the prospect for future therapy.
Project description:Information on the genetic events leading to thyroid cancer in dogs is lacking.Upregulation of the PI3K/Akt pathway has an important role in the tumorigenesis of thyroid carcinoma in dogs.Fifty-nine dogs with thyroid carcinoma and 10 healthy controls.Quantitative RT-PCR was performed for VEGFR-1, VEGFR-2, EGFR, PIK3CA, PIK3CB, PDPK1, PTEN, AKT1, AKT2, COX-2, and CALCA. Mutation analysis was performed for known hotspots of RAS (N, K, H), PIK3CA, BRAF, RET, and for the entire coding region of PTEN.Forty-three dogs (73%) had follicular cell thyroid carcinoma (FTC) and 16 dogs (27%) had medullary thyroid carcinoma (MTC). The relative mRNA expressions of VEGFR-1 (P < .001), VEGFR-2 (P = .002), PDPK1 (P < .001), AKT1 (P = .009), and AKT2 (P < .001) were increased in FTC, and those of EGFR (P < .001), VEGFR-1 (P = .036), and PIK3CA (P = .019) were increased in MTC when compared to normal thyroid glands. Mutation analysis of K-RAS identified 2 activating missense mutations, which also have been described in thyroid cancer of humans. A G12R substitution was present in 1 FTC and an E63K substitution was present in 1 MTC. No functional mutations were found in the sequenced regions of H-RAS, N-RAS, PIK3CA, BRAF, RET, and PTEN.The increased expression of several genes associated with PI3K/Akt signaling suggests the involvement of this pathway in the pathogenesis of thyroid carcinoma in dogs, warranting further research on pathway activation and gene amplification. The mutations most frequently associated with thyroid cancer in humans are rare in dogs.
Project description:Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3? by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3?. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN.
Project description:Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110? isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.
Project description:The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110? catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.
Project description:The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway is critical for cellular growth and metabolism. Recently, mosaic or segmental overgrowth, a clinical condition caused by heterozygous somatic activating mutations in PIK3CA, was established as PIK3CA-related overgrowth spectrum (PROS). In this study, we report a Japanese female diagnosed with PROS, who presented with hyperplasia of the lower extremities, macrodactyly, multiple lipomatosis, and sparse hair. Sequencing and mutant allele frequency analysis of PIK3CA from affected tissues revealed that the patient had a heterozygous mosaic mutation (c.3140A>G [p.H1047R]) in PIK3CA and that there were higher mutant allele frequencies from samples with a larger amount of subcutaneous adipose tissue. We established two fibroblast cell lines from the patient, harboring high and low frequencies of the mosaic mutation, in which AKT and S6 showed higher level of phosphorylation compared with three control fibroblasts, indicating that PI3K/AKT/mTOR signaling is activated. We assessed the therapeutic effects of four compounds (rapamycin, NVP-BEZ235, aspirin, and metformin) on PI3K/AKT/mTOR signaling pathway and cell growth. All four compounds suppressed S6 phosphorylation and inhibited cell growth of the patient-derived fibroblast cell lines. However, only metformin mildly inhibited the growth of the control fibroblast cell lines. Since PROS is a congenital disorder, drugs for therapy should take into consideration the natural growth of children. Thus, metformin is a candidate drug for treating PROS in growing children.
Project description:The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is activated in multiple cancers including ovarian carcinoma (OC). However, the relative contribution of the single components within the PI3K pathway to AKT activation in OC is still unclear. We examined 98 tumor samples from Italian OC patients for alterations in the members of the PI3K pathway. We report that AKT is significantly hyperactive in OC compared to normal tissue (n?=?93; p<0.0001) and that AKT activation is preferentially observed in the elderly (>58 years old; n?=?93; p<0.05). The most frequent alteration is the overexpression of the p110? catalytic subunit of PI3K (63/93, ?68%); less frequent alterations comprise the loss of PTEN (24/89, 27%) and the overexpression of AKT1 (18/96, 19%) or AKT2 (11/88,12.5%). Mutations in the PIK3CA or KRAS genes were detected at lower frequency (12% and 10%, respectively) whereas mutations in AKT1 or AKT2 genes were absent. Although many tumors presented a single lesion (28/93, of which 23 overexpressed PIK3CA, 1 overexpressed AKT and 4 had lost PTEN), many OC (35/93) presented multiple alterations within the PI3K pathway. Apparently, aberrant PI3K signalling was mediated by activation of the canonical downstream AKT-dependent mTOR/S6K1/4EBP1 pathway and by regulation of expression of oncogenic transcription factors that include HMGA1, JUN-B, FOS and MYC but not by AKT-independent activation of SGK3. FISH analysis indicated that gene amplification of PIK3CA, AKT1 and AKT2 (but not of PI3KR1) and the loss of PTEN are common and may account for changes in the expression of the corresponding proteins. In conclusion, our results indicate that p110? overexpression represents the most frequent alteration within the PI3K/AKT pathway in OC. However, p110? overexpression may not be sufficient to activate AKT signalling and drive ovarian tumorigenesis since many tumors overexpressing PI3K presented at least one additional alteration.
Project description:In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform ? (PI3K-C2?) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2? produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2? does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2?-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2? supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis.
Project description:Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85?/p55?/p50? subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85? with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85? overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome.
Project description:BACKGROUND: The present study focused on the prognostic roles of PIK3CA and PIK3R1 genes and additional PI3K pathway-associated genes in breast cancer. METHODS: The mutational and mRNA expression status of PIK3CA, PIK3R1 and AKT1, and expression status of other genes involved in the PI3K pathway (EGFR, PDK1, PTEN, AKT2, AKT3, GOLPH3, WEE1, P70S6K) were assessed in a series of 458 breast cancer samples. RESULTS: PIK3CA mutations were identified in 151 samples (33.0%) in exons 1, 2, 9 and 20. PIK3R1 mutations were found in 10 samples (2.2%) and underexpression in 283 samples (61.8%). AKT1 mutations were found in 15 samples (3.3%) and overexpression in 116 samples (25.3%). PIK3R1 underexpression tended to mutual exclusivity with PIK3CA mutations (p = 0.00097). PIK3CA mutations were associated with better metastasis-free survival and PIK3R1 underexpression was associated with poorer metastasis-free survival (p = 0.014 and p = 0.00028, respectively). By combining PIK3CA mutation and PIK3R1 expression status, four prognostic groups were identified with significantly different metastasis-free survival (p = 0.00046). On Cox multivariate regression analysis, the prognostic significance of PIK3R1 underexpression was confirmed in the total population (p = 0.0013) and in breast cancer subgroups. CONCLUSIONS: PIK3CA mutations and PIK3R1 underexpression show opposite effects on patient outcome and could become useful prognostic and predictive factors in breast cancer.
Project description:Lymphatic malformations (LMs) are congenital, nonneoplastic vascular malformations associated with postzygotic activating PIK3CA mutations. The mutation spectrum within LMs is narrow, with the majority having 1 of 3 hotspot mutations. Despite this relative genetic homogeneity, clinical presentations differ dramatically. We used molecular inversion probes and droplet digital polymerase chain reaction to perform deep, targeted sequencing of PIK3CA in 271 affected and unaffected tissue samples from 81 individuals with isolated LMs and retrospectively collected clinical data. Pathogenic PIK3CA mutations were identified in affected LM tissue in 64 individuals (79%) with isolated LMs, with variant allele fractions (VAFs) ranging from 0.1% to 13%. Initial analyses revealed no correlation between VAF and phenotype variables. Recognizing that different mutations activate PI3K to varying degrees, we developed a metric, the genotype-adjusted VAF (GVAF), to account for differences in mutation strength, and found significantly higher GVAFs in LMs with more severe clinical characteristics including orofacial location or microcystic structure. In addition to providing insight into LM pathogenesis, we believe GVAF may have broad applicability for genotype-phenotype analyses in mosaic disorders.