Bioinformatics analysis of miRNAs in the neuroblastoma 11q-deleted region reveals a role of miR-548l in both 11q-deleted and MYCN amplified tumour cells.

Neuroblastoma is a childhood tumour that is responsible for approximately 15% of all childhood cancer deaths. Neuroblastoma tumours with amplification of the oncogene MYCN are aggressive, however, another aggressive subgroup without MYCN amplification also exists; rather, they have a deleted region at chromosome arm 11q. Twenty-six miRNAs are located within the breakpoint region of chromosome 11q and have been checked for a possible involvement in development of neuroblastoma due to the genomic alteration. Target genes of these miRNAs are involved in pathways associated with cancer, including proliferation, apoptosis and DNA repair. We could show that miR-548l found within the 11q region is downregulated in neuroblastoma cell lines with 11q deletion or MYCN amplification. In addition, we showed that the restoration of miR-548l level in a neuroblastoma cell line led to a decreased proliferation of these cells as well as a decrease in the percentage of cells in the S phase. We also found that miR-548l overexpression suppressed cell viability and promoted apoptosis, while miR-548l knockdown promoted cell viability and inhibited apoptosis in neuroblastoma cells. Our results indicate that 11q-deleted neuroblastoma and MYCN amplified neuroblastoma coalesce by downregulating miR-548l.

Novel TPR::ROS1 Fusion Gene Activates MAPK, PI3K and JAK/STAT Signaling in an Infant-type Pediatric Glioma.

BACKGROUND/AIM: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. PATIENT AND METHODS: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. RESULTS: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. CONCLUSION: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.

Digitoxin Affects Metabolism, ROS Production and Proliferation in Pancreatic Cancer Cells Differently Depending on the Cell Phenotype.

Digitoxin has repeatedly shown to have negative effects on cancer cell viability; however, the actual mechanism is still unknown. In this study, we investigated the effects of digitoxin (1-100 nM) in four pancreatic cancer cell lines, BxPC-3, CFPAC-1, Panc-1, and AsPC-1. The cell lines differ in their KRAS/BRAF mutational status and primary tumor or metastasis origin. We could detect differences in the basal rates of cell proliferation, glycolysis, and ROS production, giving the cell lines different phenotypes. Digitoxin treatment induced apoptosis in all four cell lines, but to different degrees. Cells derived from primary tumors (Panc-1 and BxPC-3) were highly proliferating with a high proportion of cells in the S/G2 phase, and were more sensitive to digitoxin treatment than the cell lines derived from metastases (CFPAC-1 and AsPC-1), with a high proportion of cells in G0/G1. In addition, the effects of digitoxin on the rate of glycolysis, ROS production, and proliferation were dependent on the basal metabolism and origin of the cells. The KRAS downstream signaling pathways were not altered by digitoxin treatment, thus the effects exerted by digitoxin were probably disconnected from these signaling pathways. We conclude that digitoxin is a promising treatment in highly proliferating pancreatic tumors.

Inflammation suppresses DLG2 expression decreasing inflammasome formation.

PURPOSE: Loss of expression of DLG2 has been identified in a number of cancers to contribute to the disease by resulting in increased tumor cell proliferation and poor survival. In light of the previous evidence that DLG2 alters the cell cycle and affects proliferation, combined with indications that DLG2 is involved in NLRP3 inflammasome axis we speculated that DLG2 has an immune function. So far, there is no data that clearly elucidates this role, and this study was designed to investigate DLG2 in inflammatory colon disease and in colon cancer as well as its impact on inflammasome induction. METHODS: The DLG2 expression levels were established in publicly available inflammation, colon cancer and mouse model datasets. The overexpression and silencing of DLG2 in colon cancer cells were used to determine the effect of DLG2 expression on the activation of the inflammasome and subsequent cytokine release. RESULTS: The expression of DLG2 is repressed in inflammatory colon diseases IBD and Ulcerative colitis as well as colorectal cancer tissue compared to healthy individuals. We subsequently show that induction with inflammatory agents in cell and animal models results in a biphasic alteration of DLG2 with an initial increase followed by an ensuing decrease. DLG2 overexpression leads to a significant increase in expression of IL1B, IκBζ and BAX, components that result in inflammasome formation. DLG2 silencing in THP1 cells resulted in increased release of IL-6 into the microenvironment which once used to treat bystander COLO205 cells resulted in an increase in STAT3 phosphorylation and an increase proliferating cells and more cells in the G2/M phase. Restoration of DLG2 to the colon resulted in reduced AKT and S6 signaling. CONCLUSION: DLG2 expression is altered in response to inflammation in the gut as well as colon cancer, resulting in altered ability to form inflammasomes. TRIAL REGISTRATION: NCT03072641.

DLG2 impairs dsDNA break repair and maintains genome integrity in neuroblastoma.

BACKGROUND: In primary neuroblastoma, deletions on chromosome 11q are known to result in an increase in the total number of chromosomal breaks. The DNA double-strand break repair pathways mediated by NHEJ are often upregulated in cancer. DLG2, a candidate tumor suppressor gene on chromosome 11q, has previously been implicated in DNA repair. METHODS: We evaluated an association between gene expression and neuroblastoma patient outcome, risk categorization, and 11q status using publicly available microarray data from independent neuroblastoma patient datasets. Functional studies were conducted using comet assay and H2AX phosphorylation in neuroblastoma cell lines and in the fruit fly with UVC-induced DNA breaks. RESULTS: We show that the NHEJ genes PARP1 and FEN1 are over expressed in neuroblastoma and restoration of DLG2 impairs their gene and protein expression. When exposed to UVC radiation, cells with DLG2 over expression show less DNA fragmentation and induce apoptosis in a p53 S46 dependent manner. We could also confirm that DLG2 over expression results in CHK1 phosphorylation consistent with previous reports of G2/M maintenance. CONCLUSIONS: Taken together, we show that DLG2 over expression increases p53 mediated apoptosis in response to etoposide and UVC mediated genotoxicity and reduced DNA replication machinery.

Na+/K+­ATPase subunit α3 expression is associated with the efficacy of digitoxin treatment in pancreatic cancer cells.

The alpha subunits (ATP1A1-3) of Na+/K+-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin. In the present study, three pancreatic cancer cell lines, AsPC-1, Panc-1 and CFPAC-1, were used to investigate the effects of digitoxin in relation to the expression of the subunits ATP1A1 and ATP1A3. Cell viability and intracellular calcium concentrations was measured in relation to the gene and protein expression of ATP1A1 and ATP1A3. Digitoxin was used to treat the cells at concentrations of 1-100 nM, and the intracellular calcium concentrations increased in a concentration-dependent manner in the Panc-1 and in the CFPAC-1 cells with treatment at 100 nM. In the AsPC-1 cells only the supraphysiological concentration of digitoxin (100 nM) resulted in a decrease in the number of viable cells (unviable cells increased to 22%), whereas it had no effect on intracellular calcium levels. The number of viable Panc-1 and CFPAC-1 cells decreased after digitoxin treatment at 25-100 nM (unviable Panc-1 cells increased to 33-59%; unviable CFPAC-1 cells increased to 22-56%). Digitoxin treatment also affected the transcriptional expression of the ATP1A1 and ATP1A3 subunits. In Panc-1 cells, ATP1A3 gene expression was negatively associated with the digitoxin concentration (25-100 nM). In the AsPC-1 and CFPAC-1 cells, the expression of the ATP1A1 gene increased in the cells treated with the 100 nM digitoxin concentration. The protein expression of ATP1A1 and ATP1A3 was not altered with digitoxin treatment. The basal protein expression of ATP1A1 was high in the AsPC-1 and CFPAC-1 cells, compared to the Panc-1 cells, in contrast to the basal expression of ATP1A3, which was higher in the Panc-1 cells, compared to the other pancreatic cancer cells used. On the whole, the present study demonstrates that the high expression of ATP1A3 renders pancreatic cancer cells more susceptible to digitoxin-induced cell death. The findings suggest that the expression of ATP1A3 may be used as a marker for tumor sensitivity to digitoxin treatment, where a high expression of ATP1A3 is favorable for the anticancer effects of digitoxin.

Discovery of a rare GKAP1-NTRK2 fusion in a pediatric low-grade glioma, leading to targeted treatment with TRK-inhibitor larotrectinib.

Here we report a case of an 11-year-old girl with an inoperable tumor in the optic chiasm/hypothalamus, who experienced several tumor progressions despite three lines of chemotherapy treatment. Routine clinical examination classified the tumor as a BRAF-negative pilocytic astrocytoma. Copy-number variation profiling of fresh frozen tumor material identified two duplications in 9q21.32-33 leading to breakpoints within the GKAP1 and NTRK2 genes. RT-PCR Sanger sequencing revealed a GKAP1-NTRK2 exon 10-16 in-frame fusion, generating a putative fusion protein of 658 amino acids with a retained tyrosine kinase (TK) domain. Functional analysis by transient transfection of HEK293 cells showed the GKAP1-NTRK2 fusion protein to be activated through phosphorylation of the TK domain (Tyr705). Subsequently, downstream mediators of the MAPK- and PI3K-signaling pathways were upregulated in GKAP1-NTRK2 cells compared to NTRK2 wild-type; phosphorylated (p)ERK (3.6-fold), pAKT (1.8- fold), and pS6 ribosomal protein (1.4-fold). Following these findings, the patient was enrolled in a clinical trial and treated with the specific TRK-inhibitor larotrectinib, resulting in the arrest of tumor growth. The patient's condition is currently stable and the quality of life has improved significantly. Our findings highlight the value of comprehensive clinical molecular screening of BRAF-negative pediatric low-grade gliomas, to reveal rare fusions serving as targets for precision therapy.

The loss of DLG2 isoform 7/8, but not isoform 2, is critical in advanced staged neuroblastoma.

BACKGROUND: Neuroblastoma is a childhood neural crest tumor showing large clinical and genetic heterogeneity, one form displaying 11q-deletion is very aggressive. It has been shown that 11q-deletion results in decreased expression of DLG2, a gene residing in the deleted region. DLG2 has a number of different isoforms with the main difference is the presence or absence of a L27 domain. The L27 domain containing DLG proteins can form complexes with CASK/MPP and LIN7 protein family members, which will control cell polarity and signaling. METHODS: We evaluated the DLG gene family and the LIN7 gene family for their expression in differently INSS staged neuroblastoma from publically available data and primary tumors, we included two distinct DLG1 and DLG2 N-terminal transcript isoforms encoding L27 domains for their expression. Functionality of DLG2 isoforms and of LIN7A were evaluated in the 11q-deleted neuroblastoma cell line SKNAS. RESULTS: In neuroblastoma only two DLG2 isoforms were expressed: isoform 2 and isoform 7/8. Using the array data we could determine that higher expression of DLG members that contain L27 domains correlated to better survival and prognosis. Whilst DLG1 showed a decrease in both isoforms with increased INSS stage, only the full length L27 containing DLG2 transcripts DLG2-isoform 7/8 showed a decrease in expression in high stage neuroblastoma. We could show that the protein encoded by DLG2-isoform 7 could bind to LIN7A, and increased DLG2-isoform 7 gene expression increased the expression of LIN7A, this reduced neuroblastoma cell proliferation and viability, with increased BAX/BCL2 ratio indicating increased apoptosis. CONCLUSION: We have provided evidence that gene expression of the L27 domain containing DLG2-isoform 7/8 but not L27 domain lacking DLG2-isoform 2 is disrupted in neuroblastoma, in particular in the aggressive subsets of tumors. The presence of the complete L27 domain allows for the binding to LIN7A, which will control cell polarity and signaling, thus affecting cancer cell viability.

Impaired muscle morphology in a Drosophila model of myosin storage myopathy was supressed by overexpression of an E3 ubiquitin ligase.

Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/ß-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. This study shows that a higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients.

MYO5B mutations in pheochromocytoma/paraganglioma promote cancer progression.

Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important for subtyping, and may provide optimization of therapeutic regimens. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status. Immunohistochemistry and mRNA expression analysis in 30 PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a paraganglioma harboring a somatic MYO5B:p.G1611S mutation. In addition to five previously discovered MYO5B mutations, the present study of 30 PPGL (8 previous and 22 new samples) also revealed two, and hence recurrent, mutations in the gene paralog MYO5A. The three MYO5B missense mutations with the highest prediction scores (p.L587P, p.G1611S and p.R1641C) were selected and functionally validated using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293). In vitro analysis showed a significant increased proliferation rate in all three MYO5B mutated clones. The two somatically derived mutations, p.L587P and p.G1611S, were also found to increase the migration rate. Expression analysis of MYO5B mutants compared to wild type clones, demonstrated a significant enrichment of genes involved in migration, proliferation, cell adhesion, glucose metabolism, and cellular homeostasis. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved in the malignant progression in some PPGL tumors.

Low DLG2 gene expression, a link between 11q-deleted and MYCN-amplified neuroblastoma, causes forced cell cycle progression, and predicts poor patient survival.

BACKGROUND: Neuroblastoma (NB) is a childhood neural crest tumor. There are two groups of aggressive NBs, one with MYCN amplification, and another with 11q chromosomal deletion; these chromosomal aberrations are generally mutually exclusive. The DLG2 gene resides in the 11q-deleted region, thus makes it an interesting NB candidate tumor suppressor gene. METHODS: We evaluated the association of DLG2 gene expression in NB with patient outcomes, stage and MYCN status, using online microarray data combining independent NB patient data sets. Functional studies were also conducted using NB cell models and the fruit fly. RESULTS: Using the array data we concluded that higher DLG2 expression was positively correlated to patient survival. We could also see that expression of DLG2 was inversely correlated with MYCN status and tumor stage. Cell proliferation was lowered in both 11q-normal and 11q-deleted NB cells after DLG2 over expression, and increased in 11q-normal NB cells after DLG2 silencing. Higher level of DLG2 increased the percentage of cells in the G2/M phase and decreased the percentage of cells in the G1 phase. We detected increased protein levels of Cyclin A and Cyclin B in fruit fly models either over expressing dMyc or with RNAi-silenced dmDLG, indicating that both events resulted in enhanced cell cycling. Induced MYCN expression in NB cells lowered DLG2 gene expression, which was confirmed in the fly; when dMyc was over expressed, the dmDLG protein level was lowered, indicating a link between Myc over expression and low dmDLG level. CONCLUSION: We conclude that low DLG2 expression level forces cell cycle progression, and that it predicts poor NB patient survival. The low DLG2 expression level could be caused by either MYCN-amplification or 11q-deletion. Video Abstract.

Drosophila model of myosin myopathy rescued by overexpression of a TRIM-protein family member.

Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/ß-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the α-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. To investigate the mechanisms underlying the pathology of the recurrent causative MYH7 mutation (K1729del), we have developed a Drosophila melanogaster model of Laing distal myopathy by genomic engineering of the Drosophila Mhc locus. Homozygous MhcK1728del animals die during larval/pupal stages, and both homozygous and heterozygous larvae display reduced muscle function. Flies expressing only MhcK1728del in indirect flight and jump muscles, and heterozygous MhcK1728del animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of MhcK1728del mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous MhcK1728del larvae also demonstrated structural and functional impairments in heart muscle, which were not observed in heterozygous animals, indicating a dose-dependent effect of the mutated allele. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with MhcK1728del were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. Our observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy.

Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity.

BACKGROUND: Class IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is an integral mediator of insulin signaling. The p110 catalytic and p85 regulatory subunits of PI3K are the products of separate genes, and while they come together to make the active heterodimer, they have opposing roles in insulin signaling and action. Deletion of hepatic p110α results in an impaired insulin signal and severe insulin resistance, whereas deletion of hepatic p85α results in improved insulin sensitivity due to sustained levels of phosphatidylinositol (3,4,5)-trisphosphate. Here, we created mice with combined hepatic deletion of p110α and p85α (L-DKO) to study the impact on insulin signaling and whole body glucose homeostasis. METHODS: Six-week old male flox control and L-DKO mice were studied over a period of 18 weeks, during which weight and glucose levels were monitored, and glucose tolerance tests, insulin tolerance test and pyruvate tolerance test were performed. Fasting insulin, insulin signaling mediators, PI3K activity and insulin receptor substrate (IRS)1-associated phosphatidylinositol kinase activity were examined at 10 weeks. Liver, muscle and white adipose tissue weight was recorded at 10 weeks and 25 weeks. RESULTS: The L-DKO mice showed a blunted insulin signal downstream of PI3K, developed markedly impaired glucose tolerance, hyperinsulinemia and had decreased liver and adipose tissue weights. Surprisingly, however, these mice displayed normal hepatic glucose production, normal insulin tolerance, and intact IRS1-associated phosphatidylinositol kinase activity without compensatory upregulated signaling of other classes of PI3K. CONCLUSIONS: The data demonstrate an unexpectedly overall mild metabolic phenotype of the L-DKO mice, suggesting that lipid kinases other than PI3Ks might partially compensate for the loss of p110α/p85α by signaling through other nodes than Akt/Protein Kinase B.

Lowered Expression of Tumor Suppressor Candidate MYO1C Stimulates Cell Proliferation, Suppresses Cell Adhesion and Activates AKT.

Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of well-stratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition.

Verification of microRNA expression in human endometrial adenocarcinoma.

BACKGROUND: MicroRNAs are small non-coding RNAs that have been implicated in tumor initiation and progression. In a previous study we identified 138 miRNAs as differentially expressed in endometrial adenocarcinoma compared to normal tissues. One of these miRNAs was miRNA-34a, which regulates several genes involved in the Notch pathway, which is frequently altered in endometrial cancer. The aims of this study were to verify the differential expression of a subset of miRNAs and to scrutinize the regulatory role of mir-34a on the target genes NOTCH1 and DLL1. METHODS: Twenty-five miRNAs that were previously identified as differentially expressed were subjected to further analysis using qPCR. To investigate the regulation of NOTCH1 and DLL1 by mir-34a, we designed gain- and loss-of-function experiments in Ishikawa and HEK293 cell lines by transfection with a synthetic mir-34a mimic and a mir-34a inhibitor. RESULTS: Of the 25 validated miRNAs, seven were down-regulated and 18 were up-regulated compared to normal endometrium, which was fully consistent with our previous findings. In addition, the up-regulation of mir-34a led to a significant decrease in mRNA levels of NOTCH1 and DLL1, while down-regulation led to a significant increase in mRNA levels of these two genes. CONCLUSIONS: We verified both up-regulated and down-regulated miRNAs in the tumor samples, indicating various roles of microRNAs during tumor development. Mir-34a functions as a regulator by decreasing the expression of NOTCH1 and DLL1. Our study is the first to identify a correlation between mir-34a and its target genes NOTCH1 and DLL1 in endometrial adenocarcinoma.

p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity.

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.

The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo.

The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37δ, an alternatively spliced product of human PI3K p110δ, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37δ in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37δ further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37δ both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37δ prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.

High level of p37δ-mRNA relative to p110δ-mRNA in neuroblastoma tumors correlates with poor patient survival.

Alterations in the PI3K/Akt pathway, a pathway that promotes proliferation and oncogenic transformation, are common in various cancers. In neuroblastoma, activation of Akt is correlated with aggressive disease although mutations in genes of this pathway are rare. Previous findings include a few mutations in PIK3CD, the gene encoding PI3K catalytic subunit delta, p110δ. We recently reported that an alternatively spliced form of p110δ, called p37δ, had cell proliferative properties and was over-expressed in ovarian and colorectal tumors. Here, we investigated p37δ in neuroblastoma primary tumors of different stages using qPCR (TaqMan) for gene expression analysis (46 samples) and Western blot for protein analysis (22 samples). Elevated levels of both p37δ-mRNA and p110δ-mRNA were detected in metastasizing neuroblastoma tumors compared to normal adrenal gland (P < 0.05), and higher expression of p37δ-mRNA relative to p110δ-mRNA in neuroblastoma non-survivor patients compared to survivors (P < 0.01). p37δ-Protein levels but not p110δ levels correlated with increased pAKT(T308) and pERK levels. The p37δ-mRNA levels did not correlate with the protein levels, indicating major regulation at the translational/protein level. Deregulation of signaling pathways is a hallmark of cancer development. Here, we show that p37δ, a kinase-dead isoform of the PI3K catalytic subunit p110δ, is over-expressed in neuroblastoma tumors, and that it correlates with the activation of both PI3K/Akt- and RAS-signaling pathways.

Aggressive neuroblastomas have high p110alpha but low p110delta and p55alpha/p50alpha protein levels compared to low stage neuroblastomas.

BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt pathway is involved in neuroblastoma development where Akt/PKB activation is associated with poor prognosis. PI3K activity subsequently activates Akt/PKB, and as mutations of PI3K are rare in neuroblastoma and high levels of PI3K subunit p110delta is associated with favorable disease with low p-Akt/PKB, the levels of other PI3K subunits could be important for Akt activation. METHODS: Protein levels of Type IA PI3K catalytic and regulatory subunits were investigated together with levels of phosphorylated Akt/PKB and the PI3K negative regulator PTEN in primary neuroblastoma tumors. Relation between clinical markers and protein levels were evaluated through t-tests. RESULTS: We found high levels of p-Akt/PKB correlating to aggressive disease and p-Akt/PKB (T308) showed inverse correlation to PTEN levels. The regulatory isomers p55alpha/p50alpha showed higher levels in favorable neuroblastoma as compared with aggressive neuroblastoma. The PI3K-subunit p110alpha was found mainly in advanced tumors while p110delta showed higher levels in favorable neuroblastoma. CONCLUSIONS: Activation of the PI3K/Akt pathway is seen in neuroblastoma tumors, however the contribution of the different PI3K isoforms is unknown. Here we show that p110alpha is preferentially expressed in aggressive neuroblastomas, with high p-Akt/PKB and p110delta is mainly detected in favorable neuroblastomas, with low p-Akt/PKB. This is an important finding as PI3K-specific inhibitors are suggested for enrollment in treatment of neuroblastoma patients.