Regulatory Subunit Of Phosphoinositide 3-kinase Research Articles

Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Abstract 5841: A genome wide CRISPR-Cas9 screen identifies mediators of resistance to dual PI3K/mTOR inhibition in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor with a devastating 5-year survival rate of 6.8%. The current standard of care, consisting of surgery, radiation, and temozolomide (TMZ), has remained unchanged for over the past 15 years despite its limited efficacy and the serious therapy-related adverse events associated to this regimen. Although some frequent genetic alterations have been identified in GBM, no targeted therapies have shown efficacy in these patients to date. Among these, the phosphoinositide 3-kinase (PI3K) pathway is a signaling network that regulates cell growth, proliferation, migration, and invasion. Extensive molecular characterization of GBM has identified alterations in the components of the PI3K pathway in 80% of primary GBMs, including amplification of receptor tyrosine kinases (RTKs), activating mutations in the PI3K catalytic subunit (PIK3CA), loss-of-function mutations in the PI3K regulatory subunit (PIK3R1), and loss of the major tumor suppressor of the pathway, phosphatase and tensin homologue (PTEN). Despite the universal upregulation of PI3K pathway signaling associated with these genetic alterations, clinical trials investigating PI3K inhibitors in GBM have failed due to insufficient antitumor activity and dose-limiting toxicities. To elucidate mechanisms of resistance to PI3K inhibition in GBM, we used a genome-wide functional CRISPR-Cas9 knockout screen to identify genes that mediate resistance to the dual PI3K/mTOR inhibitor XL765 in PTEN-null SF295 GBM cells. We identified reactivation of the PI3K pathway and inhibition of autophagy as potential resistance mechanisms to dual PI3K/mTOR inhibition. Using orthogonal approaches, we will functionally validate our candidate hits through mechanistic studies, with patient-derived models, and with in vivo orthotopic xenografts. Our findings will elucidate novel mechanisms of resistance to PI3K/mTOR inhibition in GBM and will streamline rational drug combinations aimed to overcome resistance, thus eventually maximizing therapeutic responses to PI3K inhibitors in GBM. Citation Format: Annalisa V. Ferrotta, Arianna Izawa-Ishiguro, Meaghan Grogan, Ingo K. Mellinghoff. A genome wide CRISPR-Cas9 screen identifies mediators of resistance to dual PI3K/mTOR inhibition in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5841.

Immunodeficiency due to a novel variant in PIK3CD: a case report

BackgroundPrimary immunodeficiencies are immunological disorders caused by gene mutations involved in immune system development and activation. Recently, activated phosphoinositide 3-kinase delta syndrome (APDS) due to mutations in the phosphoinositide 3-kinase (PI3K), phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit delta gene (PIK3CD), and phosphoinositide 3-kinase regulatory subunit 1 (PIK3R1) genes have been reported to induce a combined immunodeficiency syndrome leading to senescent T cells, lymphadenopathy, and immunodeficiency. The exact diagnosis of these deficiencies is essential for treatment and prognosis. In recent years, targeted treatment with selective PI3Kd inhibitors has had a significant effect on controlling the symptoms of these patients.Case presentationIn this case report, we represent a 27-month-old girl with recurrent fever, an increased level of inflammatory markers, and erythema nodosum, who was referred to the rheumatology clinic. In the course of evaluations, because of the lack of clinical improvement with usual treatments, and a history of frequent respiratory infections, combined immunodeficiency was diagnosed in the immunological investigations. Moreover, whole-exome sequencing was performed for her.ConclusionThe genetic analysis found a novel variant of PIK3CD (c.1429 G > A) in the patient. Following daily antibiotic prophylaxis and monthly IV therapy, the patient’s frequent infections and fevers were controlled.

DDDR-19. NEXT-GENERATION PI3K INHIBITOR FOR GLIOBLASTOMA TREATMENT

Abstract High-grade gliomas are especially problematic because of the limited treatment options available to patients. The phosphoinositide 3-kinase (PI3K) pathway has become a significant research target within the field of cancer drug discovery due to the pathway’s promotion of cell proliferation and inhibition of apoptosis. While many new PI3K inhibitory medications show promise in preventing cancer growth, many are toxic because they inhibit multiple PI3K catalytic subunit isoforms making these drugs ineffective treatment options. Our novel peptide drug, Selectide-18, overcomes this toxicity by specifically targeting the p110β isoform of PI3K, which was shown to be the most impactful isoform in many cancer types, including several high-grade gliomas. We designed Selectide-18 after discovering an 18-residue motif unique to p110β. Selectide-18 is a mimetic peptide modeled after this motif. Our in silico analyses show that our drug works by binding to the PI3K regulatory subunit, p85α, thus preventing the binding of catalytic subunit p110β and leaving PI3K inactive. These findings were supported by coimmunoprecipitation. Using a PI3K cell-free kinase assay, we confirmed that our drug specifically targets p110β and does not inhibit p110α, p110δ, or p110γ. The IC50 of Selectide-18 was determined to be approximately 100nM using the same PI3K cell-free kinase assay. We tested several drugs in an MTS viability assay, including Selectide-18, a scrambled 18-residue negative control peptide, and PI3K inhibitor GSK2636771 to compare levels of cancer cell viability. Selectide-18 successfully killed glioblastoma cells within lines with high p110β activity. The drug had no effect in cell lines with low p110β activity. In conclusion, Selectide-18 is explicitly tailored for p110β-high gliomas and has shown promise to treat high-grade glioma effectively. We believe this model of peptide drug can usher in a new generation of p110β specific PI3K inhibitors.

Differential effects of single fatty acids and fatty acid mixtures on the phosphoinositide 3-kinase/Akt/eNOS pathway in endothelial cells

ScopeDietary fat composition is an important modulator of vascular function. Non-esterified fatty acids (NEFA) enriched in saturated fatty acids (SFA) are thought to reduce vascular reactivity by attenuating insulin signalling via vasodilator pathways (phosphoinositide 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS)) and enhancing signalling via pro-inflammatory pathways.MethodsTo examine the effects of fatty acids on these pathways, human aortic endothelial cells were incubated with single fatty acids, and mixtures of these fatty acids to mimic typical NEFA composition and concentrations achieved in our previous human study. RNA was extracted to determine gene expression using real-time RT-PCR and cell lysates prepared to assess protein phosphorylation by Western blotting.ResultsOleic acid (OA, 100 µM) was shown to down regulate expression of the insulin receptor, PTEN and a PI3K catalytic (p110β) and regulatory (p85α) subunit compared to palmitic, linoleic and stearic acids (P < 0.04), and promote greater eNOS phosphorylation at Ser1177. Both concentration and composition of the SFA and SFA plus n-3 polyunsaturated fatty acids (PUFA) mixtures had significant effects on genes involved in the PI3K/Akt pathway. Greater up-regulation was found with 800 than 400 µM concentration (respective of concentrations in insulin resistant and normal individuals), whereas greater down-regulation was evident with SFA plus n-3 PUFA than SFA mixture alone.ConclusionOur findings provide novel insights into the modulation of the PI3K/Akt/eNOS pathway by single fatty acids and fatty acid mixtures. In particular, OA appears to promote signalling via this pathway, with further work required to determine the primary molecular site(s) of action.

Cancer-associated mutations in the p85α N-terminal SH2 domain activate a spectrum of receptor tyrosine kinases

The phosphoinositide 3-kinase regulatory subunit p85α is a key regulator of kinase signaling and is frequently mutated in cancers. In the present study, we showed that in addition to weakening the inhibitory interaction between p85α and p110α, a group of driver mutations in the p85α N-terminal SH2 domain activated EGFR, HER2, HER3, c-Met, and IGF-1R in a p110α-independent manner. Cancer cells expressing these mutations exhibited the activation of p110α and the AKT pathway. Interestingly, the activation of EGFR, HER2, and c-Met was attributed to the ability of driver mutations to inhibit HER3 ubiquitination and degradation. The resulting increase in HER3 protein levels promoted its heterodimerization with EGFR, HER2, and c-Met, as well as the allosteric activation of these dimerized partners; however, HER3 silencing abolished this transactivation. Accordingly, inhibitors of either AKT or the HER family reduced the oncogenicity of driver mutations. The combination of these inhibitors resulted in marked synergy. Taken together, our findings provide mechanistic insights and suggest therapeutic strategies targeting a class of recurrent p85α mutations.

Molecular Determination of mirRNA-126 rs4636297, Phosphoinositide-3-Kinase Regulatory Subunit 1-Gene Variability rs7713645, rs706713 (Tyr73Tyr), rs3730089 (Met326Ile) and Their Association with Susceptibility to T2D.

Type 2 diabetes is a metabolic disease characterized by elevated blood sugar. It has serious complications and socioeconomic impact. The MicroRNAs are short single-stranded and non-coding RNA molecules. They regulate gene expression at the post-transcriptional levels. They are important for many physiological processes including metabolism, growth, and others. The phosphoinositide 3-kinase (PI3K) is important for insulin signaling and glucose uptake. The genome wide association studies have identified the association of certain loci with diseases including T2D. In this study we have examined the association of miR126 rs4636297 and Phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) gene Variations rs7713645, rs706713 (Tyr73Tyr), and rs3730089 (Met326Ile) with T2D using the amplification refractory mutation system PCR. Results indicated that there was a significant different (p-value < 0.05) in the Mir126 rs4636297 genotypes distribution between cases and controls, and the minor allele of the rs4636297 was also associated with T2D with OR = 0.58, p-value < 0.05. In addition results showed that there were significant differences (p-value < 0.05) of rs4636297 genotype distribution of patients with normal and patient with abnormal lipid profile. Results also showed that the PIK3R1 rs7713645 and rs3730089 genotype distribution was significantly different between cases and controls with a p-values < 0.05. In addition, the minor allele of the rs7713645 and rs3730089 were associated with T2D with OR = 0.58, p-value < 0.05. We conclude that the Mir126 rs4636297 and PIK3R1 SNPs (rs7713645 and rs3730089) were associated with T2D. These results need verification in future studies with larger sample sizes and in different populations. Protein-protein interaction and enzyme assay studies are also required to uncover the effect of the SNPs on the PI3K regulatory subunit (PI3KR1) and PI3K catalytic activity.

Epigenetic down-regulation of microRNA-126 in scleroderma endothelial cells is associated with impaired responses to VEGF and defective angiogenesis.

Impaired angiogenesis in scleroderma (SSc) is a critical component of SSc pathology. MicroRNA‐126 (miR‐126) is expressed in endothelial cells (MVECs) where it regulates VEGF responses by repressing the negative regulators of VEGF, including the sprouty‐related protein‐1 (SPRED1), and phosphoinositide‐3 kinase regulatory subunit 2 (PIK3R2). MVECs were isolated from SSc skin and matched subjects (n = 6). MiR‐126 expression was measured by qPCR and in situ hybridization. Matrigel‐based tube assembly was used to test angiogenesis. MiR‐126 expression was inhibited by hsa‐miR‐126 inhibitor and enhanced by hsa‐miR‐126 Mimic. Epigenetic regulation of miR‐126 expression was examined by the addition of epigenetic inhibitors (Aza and TSA) to MVECs and by bisulphite genomic sequencing of DNA methylation of the miR‐126 promoter region. MiR‐126 expression, as well as EGFL7 (miR‐126 host gene), in SSc‐MVECs and skin, was significantly down‐regulated in association with increased expression of SPRED1 and PIK3R2 and diminished response to VEGF. Inhibition of miR‐126 in NL‐MVECs resulted in reduced angiogenic capacity, whereas overexpression of miR‐126 in SSc‐MVECs resulted in enhanced tube assembly. Addition of Aza and TSA normalized miR‐126 and EGFL7 expression levels in SSc‐MVECs. Heavy methylation in miR‐126/EGFL7 gene was noted. In conclusion, these results demonstrate that the down‐regulation of miR‐126 results in impaired VEGF responses.

Thyroid Hormone Receptor Beta Inhibits PI3K-Akt-mTOR Signaling Axis in Anaplastic Thyroid Cancer via Genomic Mechanisms.

Thyroid cancer is the most common endocrine malignancy, and the global incidence has increased rapidly over the past few decades. Anaplastic thyroid cancer (ATC) is highly aggressive, dedifferentiated, and patients have a median survival of fewer than 6 months. Oncogenic alterations in ATC include aberrant phosphoinositide 3 kinase (PI3K) signaling through receptor tyrosine kinase (RTK) amplification, loss of phosphoinositide phosphatase expression and function, and protein kinase B (Akt) amplification. Furthermore, the loss of expression of the tumor suppressor thyroid hormone receptor beta (TRβ) is strongly associated with ATC. TRβ is known to suppress PI3K in follicular thyroid cancer and breast cancer by binding to the PI3K regulatory subunit p85α. However, the role of TRβ in suppressing PI3K signaling in ATC is not completely delineated. Here we report that TRβ indeed suppresses PI3K signaling in ATC cell lines through unreported genomic mechanisms, including a decrease in RTK expression and an increase in phosphoinositide and Akt phosphatase expression. Furthermore, the reintroduction and activation of TRβ in ATC cell lines enables an increase in the efficacy of the competitive PI3K inhibitors LY294002 and buparlisib on cell viability, migration, and suppression of PI3K signaling. These findings not only uncover additional tumor suppressor mechanisms of TRβ but shed light on the implication of TRβ status and activation on inhibitor efficacy in ATC tumors.

MiR-100-3p inhibits the adipogenic differentiation of hMSCs by targeting PIK3R1 via the PI3K/AKT signaling pathway.

MicroRNAs play an important role in the adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). How miR-100-3p influences such adipogenesis, however, remains uncertain. In this study, hMSC adipogenic differentiation was associated with miR-100-3p downregulation, and overexpressing this miRNA inhibited adipogenesis and the expression of adipogenic marker genes. Through bioinformatics approaches, miR-100-3p can bind the 3'-untranslated region (3′-UTR) of the mRNA encoding phosphoinositide 3-kinase regulatory subunit 1 (PIK3R1) such that miR-100-3p overexpression resulted in significant reductions in PIK3R1 expression. Importantly, overexpressing PIK3R1 was sufficient to reverse the anti-adipogenic effects of miR-100-3p overexpression. PIK3R1 is a critical component of the PI3K/AKT signaling pathway, and miR-100-3p overexpression resulted in reduced AKT phosphorylation in the context of adipogenesis. In addition, the adipogenic differentiation of hMSCs in which miR-100-3p was overexpressed was further enhanced upon treatment with the PI3K/AKT agonist 740Y-P relative to miR-100-3p overexpression alone. Taken together, these findings provide evidence that miR-100-3p inhibits the adipogenic differentiation of hMSCs by targeting PIK3R1 via the PI3K/AKT signaling pathway.

Integrated Analysis of Hub Genes and miRNAs in Dilated Cardiomyopathy.

Purpose The aim of this study is to identify hub genes and miRNAs by the miRNA-mRNA interaction network in dilated cardiomyopathy (DCM) disease. Methods The differentially expressed miRNAs (DEMis) and mRNAs (DEMs) were selected using data of DCM patients downloaded from the GEO database (GSE112556 and GSE3585). Gene Ontology (GO) pathway analysis and transcription factor enrichment analysis were used for selecting DEMis, and the target mRNAs of DEMis were filtered by using miRDB, miRTarBase, and TargetScan. Cytoscape software was used to visualize the network between miRNAs and mRNAs and calculate the hub genes. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to analyze the mRNAs in the regulatory network. Results A total of 9 DEMis and 281 DEMs were selected, from which we reconstructed the miRNA-mRNA network consisting of 7 miRNAs and 51 mRNAs. The top 10 nodes, miR-144-3p, miR-363-3p, miR-9-3p, miR-21-3p, miR-144-5p, miR-338-3p, ID4 (inhibitor of DNA binding/differentiation 4), miR-770-5p, PIK3R1 (p85α regulatory subunit of phosphoinositide 3-kinase (PI3K)), and FN1 (fibronectin 1), were identified as important regulators. Conclusions The study uncovered several important hub genes and miRNAs involved in the pathogenesis of DCM, among which, the miR-144-3p/FN1 and miR-9-3p/FN1 pathways may play an important role in myocardial fibrosis, which can help identify the etiology of DCM, and provide potential therapeutic targets.

Anthrax lethal factor cleaves regulatory subunits of phosphoinositide-3 kinase to contribute to toxin lethality.

Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85β (PIK3R2)3,4-are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.

MiR-155 promotes interleukin-1β-induced chondrocyte apoptosis and catabolic activity by targeting PIK3R1-mediated PI3K/Akt pathway.

Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degradation, in which elevated chondrocyte apoptosis and catabolic activity play an important role. MicroRNA‐155 (miR‐155) has recently been shown to regulate apoptosis and catabolic activity in some pathological circumstances, yet, whether and how miR‐155 is associated with OA pathology remain unexplored. We report here that miR‐155 level is significantly up‐regulated in human OA cartilage biopsies and also in primary chondrocytes stimulated by interleukin‐1β (IL‐1β), a pivotal pro‐catabolic factor promoting cartilage degradation. Moreover, miR‐155 inhibition attenuates and its overexpression promotes IL‐1β‐induced apoptosis and catabolic activity in chondrocytes in vitro. We also demonstrate that the PIK3R1 (p85α regulatory subunit of phosphoinositide 3‐kinase (PI3K)) is a target of miR‐155 in chondrocytes, and more importantly, PIK3R1 restoration abrogates miR‐155 effects on chondrocyte apoptosis and catabolic activity. Mechanistically, PIK3R1 positively regulates the transduction of PI3K/Akt pathway, and a specific Akt inhibitor reverses miR‐155 effects on promoting chondrocyte apoptosis and catabolic activity, phenocopying the results obtained via PIK3R1 knockdown, hence establishing that miR‐155 promotes chondrocyte apoptosis and catabolic activity through targeting PIK3R1‐mediated PI3K/Akt pathway activation. Altogether, our study discovers novel roles and mechanisms of miR‐155 in regulating chondrocyte apoptosis and catabolic activity, providing an implication for therapeutically intervening cartilage degradation and OA progression.

Comparative Analysis and Molecular Evolution of Class I PI3K Regulatory Subunit p85α Reveal the Structural Similarity Between nSH2 and cSH2 Domains

Phosphoinositide 3-kinase (PI3K) is an essential regulatory protein of the insulin signaling pathway and several other pathways that govern cell survival, cell proliferation, cell differentiation and oncogene regulation. The protein has main two subunits; regulatory p85 and catalytic p110. Other regulatory subunits are p50, p55. The catalytic activity of p110 is stabilized by the regulatory subunit p85α. This regulatory subunit is composed of five domains; the SH3, BCR-homology (BH), N-terminal SH2 (nSH2), C-terminal SH2 (cSH2), and inter-SH2 (iSH2). In the current study, we executed comparative analysis of the computational proteomic and molecular evolution of these five domains. Our results reveal that faster and more cost-effective methods for forming intricate relationships between the domains are worth pursuing according to vital proteomic parameters such as physico-chemical properties, evolution and post-translational modification (PTM). The results show variation instability, grand average of hydrophobicity (GRAVY), aliphatic index, globularity, PTM among the five domains, and strongly indicate a likeness between the nSH2 and cSH2 domains. The study provides vital information for the structural and functional aspects of PI3K regulatory subunit p85α. Many of these property changes might be defined as protein–protein interactions, protein folding structures and structure–function correlations in future.

C-SH2 point mutation converts p85\u03b2 regulatory subunit of phosphoinositide 3-kinase to an anti-aging gene

Insulin interacts with the insulin receptor, and the activated receptor promotes activity of the phosphoinositide-3 kinase (PI3K) enzyme. A decrease in insulin or insulin-like growth factor 1 (IGF-1) signaling increases the lifespan in mammalian species. We found that a point mutation in the C-SH2 domain of the p85β regulatory subunit of PI3K results in a prolonged lifespan. In p85β mutant cells, nerve growth factor (NGF) activates the longevity protein FOXO, and the mutant p85β gene produces strong resistance to oxidative stress, which contributes to aging. The p85β gene mutation causes increased serum insulin and low blood glucose in p85β mutant transgenic mice. Our results indicate that the p85β mutant allele alters the activity of downstream targets of PI3K by NGF and platelet-derived growth factor (PDGF) but not by insulin. We report that a point mutation in the C-SH2 domain of p85β transforms p85β into a novel anti-aging gene by abnormally regulating PI3K.

Effects of dietary carbohydrate sources on growth performance, glycogen accumulation, insulin signaling pathway and hepatic glucose metabolism in largemouth bass, Micropterus salmoides

The present study was conducted to investigate the effects of dietary carbohydrate sources, α-cassava-starch, potato starch, pea starch and dextrin, on growth performance, glycogen accumulation, insulin signaling pathway and hepatic glucose metabolism in largemouth bass (initial body weight: 36.34 ± 0.30 g) for 11 weeks. Results showed that pea starch significantly improved the specific growth rate (SGR), daily weight gain (DWG) and net fish yield (NFY) of largemouth bass compared to other starch, which maybe related to the depressed feed conversion ratio (FCR). Meanwhile, fish fed the diet with pea starch obtained the minimum hepatic glycogen content, which is significantly lower than that of α-cassava starch. The above result indicated that the fish was more tolerant to pea starch than other carbohydrate sources in accordance with the result of postprandial glucose content. The expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1) and phosphoinositide 3-kinase regulatory subunit 1 (PI3KR1) was significantly up-regulated in fish fed the diet with pea starch than others, indicating the significantly higher activity of insulin pathway in the pea starch group at least at the transcription level. Meanwhile, pea starch led to a significantly increased expression of glucokinase (GK) and liver-type 6-phosphofructokinase isozyme (PFKL), while dietary starch sources had no significant influence on the expression of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase-1 (FBP1). The improved glycolysis and poor inhibited gluconeogenesis may account for the reduced hepatic glycogen content of pea starch. Therefore, pea starch was most suitable starch among the sources used for largemouth bass.

ENKUR Is Involved in the Regulation of Cellular Biology in Colorectal Cancer Cells via PI3K/Akt Signaling Pathway.

Colorectal cancer is one of the most prevalent malignancies worldwide. ENKUR is a transient receptor potential canonical–binding protein that acts as a potential regulator or effector of transient receptor potential canonical channels. It also directly interacts with the p85 regulatory subunit of phosphoinositide 3-kinase. However, the role of ENKUR in colorectal cancer remains unclear. In the present study, the expression profiles of ENKUR in the The Cancer Genome Atlas and ONCOMINE databases were analyzed. Significant downregulation of ENKUR was observed in clinical tumor samples of various cancer types, including colorectal cancer. Decreased ENKUR messenger RNA expression and ENKUR protein level were detected in 6 human colorectal cancer cell lines. Silencing of ENKUR in colorectal cancer cells led to enhanced cell proliferation, migration, and invasion, while the opposite effects were achieved in ENKUR-overexpressing cells. Furthermore, ENKUR-underexpressing cells exhibited increased activity of phosphoinositide 3-kinase /Akt signaling pathway. Downregulation of the epithelial marker, E-cadherin, and upregulation of the mesenchymal markers, vimentin and N-cadherin, were detected in ENKUR-underexpressing cells, suggesting the induction of epithelial–mesenchymal transition. In conclusion, the present study demonstrates that ENKUR may be responsible for alterations in the proliferative, migratory, and invasive potential of colorectal cancer cells through possible involvement in the phosphoinositide 3-kinase /Akt signaling pathway.

Expression of miR‑542‑3p in osteosarcoma with miRNA microarray data, and its potential signaling pathways.

Osteosarcoma (OS) is the most common pediatric primary bone tumor, with high malignancy rates and a poor prognosis following metastasis. At present, the role of microRNA (miR)-542-3p in OS remains to be elucidated. The purpose of the present study was to investigate the expression level of miR-542-3p in OS, and its potential molecular mechanisms, via a bioinformatics analysis. First, the expression of miR-542-3p in OS based on the continuous variables of the Gene Expression Omnibus database and PubMed was studied. Subsequently, the potential target genes of miR-542-3p were predicted using gene expression profiles and bioinformatics software. On the basis of the Database for Annotation, Visualization and Integrated Discovery, version 6.8, a study of gene ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway knowledge base was conducted to explore the biological value of miR-542-3p in OS. Finally, the protein-protein interaction (PPI) network was completed using the STRING database. The expression of miR-542-3p in OS was revealed to be significantly higher compared with that in normal tissue. In total, 1,036 target genes of miR-542-3p were obtained. The results of the GO enrichment analysis revealed that the significant terms were ‘bone development’, ‘cell cycle arrest’ and ‘intracellular signal transduction’. The results of the KEGG analysis revealed the highlighted pathways that were targeted to miR-542-3p, including the sphingolipid signaling pathway (P=3.91×10−5), the phosphoinositide 3-kinase (PI3K)-AKT serine/threonine kinase (AKT) signaling pathway (P=3.17×10−5) and the insulin signaling pathway (P=1.04×10−5). The PPI network revealed eight hub genes: Ubiquitin-60S ribosomal protein L40, Ras-related C3 botulinum toxin substrate, mitogen-activated protein kinase 1, epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, PI3K regulatory subunit 1, AKT1, and actin-related protein 2/3 complex subunit 1A, which may be the key target genes of miR-542-3p in OS. Taken together, these results have demonstrated that miR-542-3p was overexpressed in OS. The potential target genes and biological functions of miR-542-3p may provide novel insights into the differentially expressed genes that are involved in OS.

Abstract B38: Synergistic effects of SHP2 and PI3K inhibitors in GAB2-overexpressing ovarian cancer

Abstract We previously found that the scaffold/adaptor growth factor receptor bound 2 (GRB2)-associated binding protein 2 (GAB2) is frequently amplified and overexpressed in primary high-grade serous ovarian cancers (HGSOCs) and cell lines. Ovarian cancer cell lines overexpressing GAB2 are dependent on GAB2 for activation of ERK1/2 and AKT signaling, consistent with its role in regulating the protein tyrosine phosphatase SHP2 (encoded by the PTPN11 gene) and the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K). In this study, we investigated whether ovarian cancer cell lines overexpressing GAB2 are sensitive to combinatorial SHP2 and PI3K inhibition. Analysis of gene expression profiles reveals that primary HGSOCs overexpressing GAB2 are associated with increased ERBB, RAS, and MAPK activity signatures. Inhibition of SHP2 by an allosteric inhibitor SHP099 selectively inhibits ERK1/2 activity, proliferation, and survival of ovarian cancer cell lines with GAB2 amplification/overexpression. Treatment with SHP099 has a synergistic effect with multiple PI3K pathway inhibitors such as BKM120, a pan-class I PI3K inhibitor, at suppressing proliferation and survival of ovarian cancer cells overexpressing GAB2 in part through induction of apoptosis. Combinatorial SHP2 and PI3K inhibition more effectively activates both pro-apoptotic proteins BIM and BAD and inhibits expression of transcription factor c-MYC, a master regulator of cancer stem cells, compared with individual inhibitor, and sensitized cancer cells to chemotherapeutic drugs paclitaxel and cisplatin. Combinatorial SHP2 and PI3K inhibition also suppresses spheroid formation of ALDH-positive ovarian cancer stem cells. Together, our findings identify a previously unrecognized role of SHP2 in promoting proliferation and survival of ovarian cancer cells in which GAB2 is amplified and/or overexpressed, and suggest that combinatorial SHP2 and PI3K inhibition may be a promising therapeutic approach for HGSOCs with GAB2 amplification/overexpression. Citation Format: Hiu Wing Cheung. Synergistic effects of SHP2 and PI3K inhibitors in GAB2-overexpressing ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B38.

PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.

BackgroundTrastuzumab is an active agent against human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC). This study aimed to characterize resistance to trastuzumab-based front-line chemotherapy in HER2+ GC patients and to establish factors predictive of this resistance.ResultsAmong 129 HER2+ GC patients, 25% displayed rapid disease progression within 4 months from initiation of therapy. These patients showed a higher rate of signet ring cell histology, bone metastasis, poor performance status, frequent loss of PTEN expression, and low HER2 amplification index compared with patients who were progression-free for at least 4 months. In contrast, there was no significant difference in the frequency of the PIK3R1 variant. Multivariate analyses confirmed two independent molecular predictors for trastuzumab resistance: loss of PTEN expression and low HER2 amplification index (<5). Patients with one or both molecular predictors at diagnosis exhibited worse progression-free and overall survival compared to those without risk factors (p < 0.001 and p = 0.001, respectively).ConclusionIn HER2+ GC patients, loss of PTEN expression and low HER2 AI correlated with resistance to trastuzumab-based therapy and dismal prognosis. Since patients harboring these molecular predictors are unlikely to respond to trastuzumab-based therapy, other novel therapeutic targets needed to be considered.MethodsHER2+ GC patients who were treated with trastuzumab in combination with either 5-fluorouracil/cisplatin or capecitabine/cisplatin were enrolled. Clinicopathologic features and molecular alterations of HER2, phosphoinositide 3-kinase regulatory subunit 1 (PIK3R1), and phosphatase and tensin homolog (PTEN) were correlated with treatment outcome. Factors predictive of resistance were also explored.