P85 Alpha Research Articles

PI3KR1 and AKT1 in largemouth bass (Micropterus salmoides): molecular cloning, characterization, and its involvement in the alleviation of hepatic glycogen deposition caused by insulin inclusion in vitro.

In this study, the full-length cDNA sequences of the phosphatidylinositol-3-kinase p85 alpha (PI3KR1) and serine/threonine kinase 1 (AKT1) genes in largemouth bass (Micropterus salmoides) were obtained using the rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that the cloned sequences of PI3KR1 and AKT1 are 4170 bp and 3672 bp in length, with open reading frames (ORFs) of 1389 bp and 1422 bp encoding 462 and 473 amino acids, respectively. Sequence alignment and evolutionary tree analysis indicated their close relationship to other teleosts, especially those with similar feeding habits. Tissue distribution demonstrated widespread distribution of both genes in various tissues, with the highest abundance in the liver. Further results found that the upregulation of the expression of p-PI3KR1, p-AKT1, p-FoxO1, and GLUT2 proteins by insulin, while suppressing the expression of the total FoxO1 protein, effectively triggers a significant activation of the PI3KR1-AKT1 insulin signaling pathway. Meanwhile, the mRNA levels of the key glycolytic genes, including glucokinase (gk), pyruvate kinase (pk), and phosphofructokinase liver type (pfkl), have been enhanced evidently. In contrast, the expression of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (pepck), glucose-6-phosphatase catalytic subunit (g6pc), and fructose-1,6-bisphosphatase-1 (fbp1) has been notably down-regulated. In addition, insulin treatment promoted the phosphorylation of glycogen phosphorylase (PYGL) and the dephosphorylation of glycogen synthase (GS), and the glycogen content in the insulin-treated group was remarkably reduced compared to the control group. Overall, our study indicates that the activation of PI3KR1-AKT1 insulin signaling pathway represses the hepatic glycogen deposition via the regulation of glycolysis and gluconeogenesis, which provides some new insights into nutritional strategy to effectively regulate the glucose metabolism in carnivorous fish.

The effects and mechanisms of modified Xiaoyaosan on chronic unpredictable mild stress (CUMS)-induced depressive mice based on network pharmacology

Ethnopharmacological relevanceClinical research and basic scientific experiments have shown that modified Xiaoyaosan (MXYS) has antidepressant effects, whose system mechanism however has not been thoroughly characterized. Aim of the studyThis research was aimed at evaluating the treatment effects of MXYS on chronic unpredictable mild stress (CUMS)-induced depressive mice and exploring underlying mechanisms. Materials and methodsWhether MXYS has effects on depression was investigated via the depressive behaviors of mice, electron microscopy, real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, immunofluorescence (IF) staining and the stereotaxic injection of adeno-associated viruses (AAVs). In addition, network pharmacology was applied to predict relevant molecular targets and possible mechanisms and perform further in vivo validation. ResultsMXYS is effective in ameliorating the depression-like symptoms of CUMS mice. It can stimulate autophagosome formation, activate the expression of microtubule-associated protein 1 light chain 3 (LC3B), autophagy-related gene 5 (Atg5), Atg7 and neuron-specific nuclear protein (NeuN), and decrease the protein expression sequestosome 1 (SQSTM1/p62). The autophagy-upregulating effect of MXYS was weakened by silencing. The network pharmacology analysis revealed that mitogen-activated protein kinase 1 (MAPK1), MAPK3, serine/threonine-protein kinase (AKT1), proto-oncogene tyrosine-protein kinase (SRC), PI 3 kinase p85 alpha (PIK3R1), catenin (cadherin-associated protein) beta 1 (CTNNB1) and human thrombin activator 1 (HRAS) may be of importance to treat depression by MXYS. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that metabolic and autophagy pathways, pathways in cancer and MAPK, phosphoinositide 3-kinase (PI3K)-Akt and rhoptry-associated protein 1 (Rap1) signaling pathways are involved in the antidepressant effects of MXYS. As suggested by Western blot, the anti-depression mechanism of MXYS is possibly associated with the extracellular signal-regulated protein kinase (ERK)/P38 MAPK signaling pathway. ConclusionThe findings indicate the possible antidepressant effects of MXYS on CUMS mice via triggering autophagy to alleviate neuronal apoptosis and prompting autophagy, which may involve the ERK/P38 MAPK signaling pathway.

Altered Placental Insulin Signaling in the Absence of Systemic Insulin Resistance in Hypertensive Disorders of Pregnancy among South Indian Tamil Population

A cross sectional study was conducted to explore the possible derangements in placental insulin signaling and also to investigate the presence or absence of maternal systemic insulin resistance in different types of hypertensive disorders of pregnancy (HDP). Study Design: Healthy pregnant women (n=32) and pregnant women with gestational hypertension (GH) (n=18), late onset Pre-eclampsia (LP) (n=18) and early onset Pre-eclampsia (EP) (n=19) were recruited for the study. Maternal venous blood and placental samples were collected from the study subjects at 35±7 weeks of gestation. Insulin resistance parameters such as fasting glucose, fasting insulin, homeostatic model assessment of insulin resistance, beta cell function and insulin sensitivity and glycated hemoglobin were estimated in the maternal venous blood samples. Placental protein expressions of insulin signaling molecules were analyzed by western blotting method. Results: Maternal venous blood insulin resistance parameters were not significantly different between the study groups. However, placental content of phospho insulin receptor beta was significantly decreased in GH, LP and EP groups in comparison to controls. We also found a significant decrease in placental protein expression of phosphatidyl inositol 3-kinase p85 alpha in LP group when compared to other groups.

Mutational Landscape of ER Receptor Negative Endometrial Cancer Patients Categorized as No Specific Molecular Profile (NSMP)

The endometrial cancer (EC) molecular classification introduced by The Cancer Genome Atlas (TCGA) has initiated molecular-based classification with clear prognostic value. Four major tumor subtypes exist: 1) TP53 mutation (p53abn); 2) POLE mutation (POLEmut); 3) mismatch repair protein deficient (MMRd); and 4) no specific molecular profile (NSMP). Of these subtypes, p53abn patients exhibited the worst prognosis. Recently, a prognostic refinement of NSMP EC using estrogen receptor (ER) status, from PORTEC-III trial data, showed that (ER-) NSMP patients had a strikingly poor prognosis, similar to p53abn patients. Currently, drivers of NSMP (ER-) disease are unknown and require genomic characterization. In this study, we perform an integrated molecular analysis to characterize this aggressive and poorly understood cohort. RNAseq and mutational data was downloaded from the TCGA, PanCancer Atlas, Uterine Endometrial Carcinoma dataset via the genomic data commons (GDC). All patients in this cohort demonstrated endometrioid histology. P53abn, and POLE mutated patients were filtered using mutational data. MMRd and NSMP ESR1 gene low expressors (ER Low) were filtered using Z-score cutoffs of RNAseq expression data. Survival analysis was performed using the Survival R-package. Gene expression testing was performed using the EdgeR R-package. Pathway Enrichment analysis was conducted using EnrichR software. Forty-two patients out of 529 samples met our GDC NSMP (ER low) filtering criteria (7%), similar to the 5% of PORTEC-III patients classified as NSMP (ER -). Strikingly 83% percent of GDC NSMP (ER low) patients harbored a mutation in the PI3K-AKT-mTOR signaling pathway, with 48% of GDC NSMP (ER low) patients carrying a mutation in the p110 alpha (PIK3CA) gene. Interestingly, of the patients with PIK3CA mutant tumors, 2/20 patients died in 5 years (10%), compared to 9/22 patients whose tumors were PIK3CA WT (41%) (p = 0.02). Among GDC NSMP (ER low) patients with PIK3CA WT tumors gene expression signatures were enriched for MYC target genes (p = 1.1e-51), DNA replication (p = 4.3e-30), cell cycle (p = 9.32e-20), and cell cycle checkpoints (p = 6.4e-20) pathways. Additionally, strongly in PIK3CA WT tumors are mitochondrial membrane proteins (p = 1.63e-37) and ribosomal proteins (2.9e-24). In this analysis, we show that GDC NSMP (ER low) tumors nearly all harbor a mutation in the PI3K-AKT-mTOR signaling pathway. Further we demonstrate that the most common gene mutation in the cohort, PIK3CA, is counterintuitively a marker of improved survival. Additionally, we show that within that this subpopulation, PIK3CA WT patients exhibit robustly upregulated gene expression programs dedicated to energy production, cell cycling, and division.

Hydrophobic Bile Salts Induce Pro-Fibrogenic Proliferation of Hepatic Stellate Cells through PI3K p110 Alpha Signaling.

Bile salts accumulating during cholestatic liver disease are believed to promote liver fibrosis. We have recently shown that chenodeoxycholate (CDC) induces expansion of hepatic stellate cells (HSCs) in vivo, thereby promoting liver fibrosis. Mechanisms underlying bile salt-induced fibrogenesis remain elusive. We aimed to characterize the effects of different bile salts on HSC biology and investigated underlying signaling pathways. Murine HSCs (mHSCs) were stimulated with hydrophilic and hydrophobic bile salts. Proliferation, cell mass, collagen deposition, and activation of signaling pathways were determined. Activation of the human HSC cell line LX 2 was assessed by quantification of α-smooth muscle actin (αSMA) expression. Phosphatidyl-inositol-3-kinase (PI3K)-dependent signaling was inhibited both pharmacologically and by siRNA. CDC, the most abundant bile salt accumulating in human cholestasis, but no other bile salt tested, induced Protein kinase B (PKB) phosphorylation and promoted HSC proliferation and subsequent collagen deposition. Pharmacological inhibition of the upstream target PI3K-inhibited activation of PKB and pro-fibrogenic proliferation of HSCs. The PI3K p110α-specific inhibitor Alpelisib and siRNA-mediated knockdown of p110α ameliorated pro-fibrogenic activation of mHSC and LX 2 cells, respectively. In summary, pro-fibrogenic signaling in mHSCs is selectively induced by CDC. PI3K p110α may be a potential therapeutic target for the inhibition of bile salt-induced fibrogenesis in cholestasis.

Effects of dietary berberine hydrochloride inclusion on growth, antioxidant capacity, glucose metabolism and intestinal microbiome of largemouth bass (Micropterus salmoides)

The present study was conducted to investigate the influence of berberine inclusion on growth, antioxidant capacity, glucose metabolism and intestinal microbiome of largemouth bass. Three experimental diets were formulated with 0, 100, and 400 mg/kg berberine hydrochloride, which were fed to triplicate groups of juvenile largemouth bass twice daily for 11 weeks. The results of feeding trial revealed that dietary berberine inclusion produced no significant influence on growth performance of largemouth bass. However, the inclusion of dietary berberine significantly reduced serum glucose content, and alleviated hepatic glycogen accumulation. Gene expression analysis indicated that dietary berberine inclusion significantly elevated the expression of insulin pathway related genes, such as insulin receptor a (IRa), phosphatidylinositol-3-kinase p85 alpha (PI3KR1) and serine/threonine kinase 1 (AKT1), which was partly associated with the repressed expression of glycolysis related genes, glucokinase (GK) and phosphofructokinase liver type (PFKL). Additionally, the inclusion of dietary berberine significantly decreased the content of lipid peroxidation products, MDA. Meanwhile, the antioxidant capacity was significantly elevated at both transcriptional level and enzymatic level, which was suggested to be mediated by Nrf2/Keap1. The analysis of bacterial 16S rRNA V3–4 region indicated that the intestinal microbiota profile was significantly altered at both phylum and genus level, with elevated relative abundance of Fusobacteria, and Cetobacterium, and deceased enrichment for potential pathogenic bacteria genera Plesiomonas with the inclusion of dietary berberine. In summary, the inclusion of dietary berberine improved glucose metabolism, antioxidant capacity and intestinal microbiome of largemouth bass.

PHLPP2 gene L1016S (rs61733127) and PIK3R1 gene Met326Ile (rs3730089) polymorphisms are associated with the risk of colon and breast cancers

The PIK3R1 and PHLPP2 genes encode the p85 alpha subunit of PI3K and a phosphatase for AKT, respectively, which play a direct role in regulating the PI3K-AKT pathway that promotes cell survival, growth, and differentiation. While most attention is focused on the factors that positively affect this pathway, negative regulation is equally important. The aim of this study was to investigate the association of SNPs rs61733127 (L1016S) in PHLPP2 gene and rs3730089 (Met326Ile) in PIK3R1 gene with colon and breast cancer, respectively because both SNPs have been reported to play a functional role in corresponding encoded enzymes and both genes are negatively involved in regulating the PI3K-AKT pathway. 139 colon and 149 breast cancers patients and 279 healthy controls were included in the present study. The target SNPs were genotyped using tetra- ARMS-PCR. In addition, the genotypes of 10 samples for each SNP were confirmed by sequencing. Statistical analysis was performed using SPSS 21.0 and by Fisher exact, T, χ2 and logistic regression tests. As revealed, the genotype AG (OR = 2.18, p = 0.001, CI = 1.36–3.50) and allele G (OR = 1.92, p = 0.001, CI = 1.30–2.84) of rs61733127 in the PHLPP2 gene significantly increased the risk of colon cancer. In addition, genotype AA (OR = 0.2, p = 0.001, CI = 3.00–8.00) and allele A (OR = 0.5, p = 0.001, CI = 1.00–4.00) of rs3730089 in the PIK3R1 gene significantly decreased the risk of breast cancer. The results suggest that SNPs in genes involved in regulating of PI3K-AKT pathway can be used as a marker for susceptibility to colon and breast cancers.

Abstract PS18-13: Men1611 is a pi3k α/β selective and δ sparing inhibitor with long-lasting antitumor activity in different genetic backgrounds of pik3ca mutant breast cancer models

Abstract Dysregulation of the PI3K/AKT/mTOR pathway has the potential to trigger the activation of class I phosphoinositide 3-kinases (PI3Ks) and eventually lead to cancer. Class I PI3Ks are heterodimers composed of the regulatory subunit p85 and the catalytic subunit p110. There are four isoforms of p110, alpha and beta, important in solid tumors, and delta and gamma, preferentially expressed in leukocytes, and drivers in hematological malignancies. While activation of PI3K p110alpha is the most frequent oncogenic event in solid tumors, p110beta plays an important role in PTEN deficient cancers and in mediating resistance to p110alpha selective PI3K inhibitors.MEN1611 is a PI3K inhibitor active on p110alpha (both mutants and wt), beta and gamma (8.6- and 2.2- fold less potent compared to the alpha, respectively), while sparing the delta isoform (36-fold less potent compared to the alpha). Thus, MEN1611 differs from other selective PI3K inhibitors such as alpelisib (p110alpha selective) and taselisib (p110beta sparing). MEN1611 is currently in clinical development for patients with HER2 positive advanced or metastatic breast cancer in combination with trastuzumab with/without fulvestrant (B-PRECISE-01). Patient-derived xenograft (PDX) models and breast cancer cell lines with different genetic background were used to assess the antitumor activity of MEN1611 in p110 alpha- and beta- dependent tumors. Healthy-donors derived CD69+ B-cells, which are primarily dependent on p110 delta for proliferation and survival were used to test the activity of the compound against the p110delta. According to the biochemical selectivity, MEN1611 demonstrated a lower cytotoxic potential in a p110delta-driven cellular model (CD19+ B-cells), when compared to taselisib (>150-fold) and an improved cytotoxic activity in the p110beta-driven cellular model (PTEN-null breast cancer cells) when compared to alpelisib. Moreover, MEN1611 selectively decreases the p110alpha protein levels in PIK3CA mutated breast cancer cells in a concentration-dependent manner, with a corresponding reduction in pAKT. In vivo, MEN1611 monotherapy showed significant and long-lasting anti-tumor activity, reflected in tumor-stasis or tumor regression in several trastuzumab-resistant PIK3CA-mutant HER2 positive PDXs with different concomitant genomic aberrations. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. Interestingly, MEN1611 monotherapy showed a significant tumor volume inhibition in a PTEN-null breast cancer PDX model. Overall, our data provide evidence that MEN1611 is active against HER2 positive and PTEN-null breast tumors. Moreover, its delta-sparing profile may have potential implications on MEN1611 tolerability and ability to overcome resistance to p110alpha selective inhibitors. Citation Format: Alessio Fiascarelli, Corrado Carrisi, Giuseppe Merlino, Stefania Capano, Diego Bisignano, Simone Talucci, Alessandro Bressan, Daniele Bellarosa, Mario Bigioni, Maurizio Scaltriti, Joaquin Arribas, Andrea Pellacani, Massimiliano Salerno, Monica Binaschi. Men1611 is a pi3k α/β selective and δ sparing inhibitor with long-lasting antitumor activity in different genetic backgrounds of pik3ca mutant breast cancer models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-13.

The inclusion of fenugreek seed extract aggravated hepatic glycogen accumulation through reducing the expression of genes involved in insulin pathway and glycolysis in largemouth bass, Micropterus salmoides

The poor carbohydrate utilization of carnivorous fish seriously restricts their growth and health status. The effects of fenugreek seed extract (FSE), with effectively therapeutic effect on diabetes in mammals, on growth performance, hepatic glycogen accumulation and glucose metabolism were explored in largemouth bass with initial body weight 41.62 ± 0.06 g. Triplicate groups of fish were fed diets with graded levels of FSE (0, 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30%) twice daily for 14 weeks. Results showed that the inclusion of FSE produced no statistic difference on growth performance and feed utilization of largemouth bass. However, the supplementation of FSE linearly and quadratically elevated hepatic glycogen content without statistic influence on serum glucose content. The results of gene expression analysis showed that the FSE inclusion exhibited poor regulation on the expression of gluconeogenesis related genes, glucose-6-phosphatase catalytic subunit (G6PC), fructose-1, 6-bisphosphatase-1 (FBP1) and phosphoenolpyruvate carboxykinase (PEPCK), but led to decreased expression of glycolysis related genes, ADP dependent glucokinase (ADPGK) and pyruvate kinase (PK), in both linear and quadratic manners. The reduced glycolysis at transcriptional level partly resulted from the inhibited expression of genes involved in the insulin pathway, such as insulin receptor (IR) b, insulin receptor substrate (IRS) and phosphatidylinositol-3-kinase p85 alpha (PI3KR1). In conclusion, the FSE supplementation did not improve but even reduce the carbohydrate utilization capacity of largemouth bass through the downregulated expression of genes involved in the insulin pathway and glycolysis.

Exogenous Therapeutics of Microrna-29a Attenuates Development of Hepatic Fibrosis in Cholestatic Animal Model through Regulation of Phosphoinositide 3-Kinase p85 Alpha

Recent studies have found that microRNA-29a (miR-29a) levels are significantly lower in fibrotic livers, as shown with human liver cirrhosis. Such downregulation influences the activation of hepatic stellate cells (HSC). Phosphoinositide 3-kinase p85 alpha (PI3KP85α) is implicated in the regulation of proteostasis mitochondrial integrity and unfolded protein response (UPR) and apoptosis in hepatocytes. This study aimed to investigate the potential therapeutic role of miR-29a in a murine bile duct ligation (BDL)-cholestatic injury and liver fibrosis model. Mice were assigned to four groups: sham, BDL, BDL + scramble miRs, and BDL + miR-29a-mimic. Liver fibrosis and inflammation were assessed by histological staining and mRNA/protein expression of representative markers. Exogenous therapeutics of miR-29a in BDL-stressed mice significantly attenuated glutamic oxaloacetic transaminase (GOT)/glutamic-pyruvic transaminase (GPT) and liver fibrosis, and caused a significant downregulation in markers related to inflammation (IL-1β), fibrogenesis (TGF-β1, α-SMA, and COL1α1), autophagy (p62 and LC3B II), mitochondrial unfolded protein response (UPRmt; C/EBP homologous protein (CHOP), heat shock protein 60 (HSP60), and Lon protease-1 (LONP1, a mitochondrial protease), and PI3KP85α within the liver tissue. An in vitro luciferase reporter assay further confirmed that miR-29a mimic directly targets mRNA 3′ untranslated region (UTR) of PI3KP85α to suppress its expression in HepG2 cell line. Our data provide new insights that therapeutic miR-29a improves cholestasis-induced hepatic inflammation and fibrosis and proteotstasis via blocking PI3KP85α, highlighting the potential of miR-29a targeted therapy for liver injury.

Mutation-Associated Phenotypic Heterogeneity in Novel and Canonical PIK3CA Helical and Kinase Domain Mutants.

Phosphatidylinositol 3-kinase, catalytic subunit alpha (PIK3CA) is an oncogene often mutated in colorectal cancer (CRC). The contribution of PIK3CA mutations in acquired resistance to anti-epidermal growth factor receptor (EGFR) therapy is well documented, but their prognostic and predictive value remain unclear. Domain- and exon-specific mutations are implicated in either favorable or poor prognoses, but there is paucity in the number of mutations characterized outside of the mutational hotspots. Here, two novel non-hotspot mutants—Q661K in exon 13 and C901R in exon 19—were characterized alongside the canonical exon 9 E545K and exon 20 H1047R mutants in NIH3T3 and HCT116 cells. Q661K and E545K both map to the helical domain, whereas C901R and H1047R map to the kinase domain. Results showed variable effects of Q661K and C901R on morphology, cellular proliferation, apoptosis resistance, and cytoskeletal reorganization, with both not having any effect on cellular migration. In comparison, E545K markedly promoted proliferation, survival, cytoskeletal reorganization, migration, and spheroid formation, whereas H1047R only enhanced the first three. In silico docking suggested these mutations negatively affect binding of the p85 alpha regulatory subunit to PIK3CA, thereby relieving PIK3CA inhibition. Altogether, these findings support intra-domain and mutation-specific variability in oncogenic readouts, with implications in degree of aggressiveness.

Macrophages from a type 1 diabetes mouse model present dysregulated Pl3K/AKT, ERK 1/2 and SAPK/JNK levels.

Diabetes causes dysregulation in signal transduction in immune cells leading to an impaired response to pathogens. Herein, we investigated the impact of type 1 diabetes (T1D) in bone marrow-derived macrophages (BMDM), using male non-diabetic and diabetic C57BL/6 mice (alloxan 60 mg/kg, i.v., CEUA/FCF/USP - 467). Diabetic BMDM expressed impaired phosphoinositide 3-kinase (PI3K), being lower p-PI3K p55 levels and higher levels of PI3K p110 alpha, whereas protein kinase B (PKB/Akt) (Ser-473 and Thr-308), extracellular signal-regulated kinases (ERK 1/2), and stress-activated protein kinase (SAPK/JNK) were enhanced compared to non-diabetic BMDM. Further evaluation of the responsiveness to lipopolysaccharide (LPS; 0.1 and 1 ug/mL), diabetic BMDM and peritoneal macrophage secreted dysregulated levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 levels. In 24 h, diabetic BMDM stimulated by LPS presented lower metabolic activity, with no differences in cell surveillance. Therefore, LPS re-stimulation (0.1 ug/mL) in diabetic BMDM resulted in higher secretion of TNF-α compared to non-diabetic BMDM. However, diabetic peritoneal macrophages secreted similar IL-6 levels in the first and additional 24 h of LPS stimulation. In general, our results demonstrated that diabetes exerts an impact in both BMDM and peritoneal macrophages ability to secrete cytokine under LPS stimulation.

Prostate epithelial-specific expression of activated PI3K drives stromal collagen production and accumulation.

We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3KGOF/+ ). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3KGOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF-β pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3KGOF/+ . Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF-β signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PIK3R1 gene polymorphisms are associated with type 2 diabetes and related features in the Turkish population.

The phosphatidylinositol 3-kinase p85 alpha regulatory subunit 1 gene (PIK3R1) encodes the PIK3R1 protein, which plays a direct role in insulin signaling. PIK3R1 (p85 regulatory subunit) connects firmly with the p110 catalytic subunit, and together these proteins form the phosphatidylinositol 3-kinase (PI3K) protein. PI3K is a key protein in the Akt signaling pathway, which regulates cell survival, growth, differentiation, glucose trafficking, and utilization. Defects in the insulin signaling cascade play an important role in the development of insulin resistance, which shares a common genetic basis for metabolic diseases such as type 2 diabetes (T2D), obesity and cardiovascular diseases. In our study, we investigated the effect of single nucleotide polymorphisms (SNPs) rs3756668 in 3'UTR region, rs706713 and rs3730089 in exons 1 and 6, respectively, rs7713645 and rs7709243 in intron 1, and rs1550805 in intron 6 of PIK3R1 gene on T2D. This study enrolled a total of 840 individuals, including 427 diabetic individuals (206 obese and 221 non-obese) and 413 nondiabetic individuals (138 obese and 275 non-obese). The target SNPs were analyzed using real-time polymerase chain reaction (RT-PCR). Statistical analysis was performed using SPSS18.0 (IBM Corp., Armonk, USA). The p-values ≤0.05 were considered statistically significant. The SNPs rs706713 (Tyr73Tyr) and rs3730089 (Met326Ile) located in exons, and rs7713645, rs7709243 and rs1550805 located in introns were determined to be significantly associated with T2D and phenotypic features such as obesity, insulin resistance and the lipid parameters. The association with SNP rs3756668, which is located in the 3'UTR, was not significant. Our study supports the role of PIK3R1, an important candidate gene due to its critical role in insulin signal transduction, in T2D development.

Effect of combined inhibition of p110 alpha PI3K isoform and STAT3 pathway in ovarian cancer platinum-based resistance.

BackgroundOvarian cancer is associated with poor prognostic outcome due to late diagnosis and to intrinsic and acquired resistance to platinum-based chemotherapy in a large number of patients. This chemoresistance is acquired through the peritoneal and ascites microenvironment by several released factors, such as IL-6,. Preclinical studies have implicated the activation of PI3K pathway in chemoresistance, showing it to extend tumor cell survival and modulate multidrug resistance. We aimed to evaluate the implication of the p110 alpha PI3K subunit in ovarian cancer chemoresistance acquisition, and to evaluate whether the STAT3 pathway can mediate resistance to PI3K inhibitors through secretion of IL6.ResultsHuman ovarian adenocarcinoma IGROV-1 and JHOC-5 cells cultured in ascites showed an increase in carboplatinum-based resistance. Level of chemoresistance was associated to IL6 concentration in ascites. Activation of PI3K/Akt, STAT and MAPK pathways was observed after IGROV-1 incubation with ascites and treatment with carboplatin. Neither IGROV-1 nor JHOC-5 cells exposed to ascites treated with additional IL-6 directed antibody showed any reversion of the chemoresistance.ConclusionIL6-related resistance was not abolished by the selective inhibition of PI3K alpha subunit coupled with the anti-IL6-receptor antibody tocilizumab. This dual inhibition requires further exploration in other ovarian cancer models such as clear cell carcinoma.

Abstract P2-09-18: Multiplexed (18-Plex) measurement of protein targets in trastuzumab-treated patients using imaging mass cytometry

Abstract Introduction: Recent studies have shown that the molecular heterogeneity of HER2 intracellular (ICD) and extracellular (ECD) domains, as well as overall immune infiltration, are associated with response to adjuvant trastuzumab. Traditional strategies for in situ measurement in the tumor microenvironment allow the combination of up to 6 targets, limiting our capability for in-depth interrogation of tissues. Imaging Mass Cytometry (IMC) uses metal-conjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this high-throughput multiplexing platform to perform an 18-plex assessment of HER2 ICD/ECD, cytotoxic T cell infiltration and other structural and signaling proteins in a cohort of patients treated with trastuzumab. Methods: An antibody panel for detection of 18 targets (Pancytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, beta-catenin, HER3, MET, EGFR, ERK 1-2, MEK 1-2, PTEN, PI3K p110 alpha, Akt, mTOR, Ki67 and Histone H3) was conjugated to unique metals for detection in an IMC instrument (Fluidigm). All assays were objectively standardized and validated using quantitative immunofluoresce (QIF). Finally, the IMC technique was validated against HER2 single marker assays by QIF. We used a collection of trastuzumab-treated patients from the HeCOG 10/05 trial (n=180), and identified a case:control series using 5-year recurrence events (n=19), which were matched to controls (n=41) by age and TNM stage. Formalin-fixed, paraffin embedded tissues in tissue microarray format were ablated in the IMC attachment to the CyTOF flow cytometer for simultaneous detection of markers. Image visualization was conducted using MCD Viewer (Fluidigm). Statistical analyses were performed using a range of platforms. Results: Patients that recurred after adjuvant treatment with trastuzumab showed a decreased fraction of HER2 ECD pixels over threshold in a compartment determined by CK and HER2 ICD compared to cases without recurrence (p=0.057). After exclusion of the lowest HER2 expressers (that would have fallen below the threshold for positive by current HER2 assays), 5-year recurrence events where associated with reduced total ECD/ ICD ratio intensity in tumor (p=0.044). Patients below the median for total ECD/ICD ratio showed a trend for decreased benefit from trastuzumab (p=0.066). Levels of cytotoxic T cell infiltration, depicted by total CD8 intensity, were lower in patients with recurrences (p=0.05). Conclusion: Objective measurement of highly multiplexed protein targets in routine, fixed breast cancer tissues shows that a decreased ratio of HER2 ECD/ ICD is associated with 5-year recurrence after trastuzumab treatment. This observation is consistent with our previous work using QIF but represents the first time this has been done on identical cell content (on a single tissue section). Additionally, on the same section we found that lower levels of overall cytotoxic T cell infiltration were associated with worse outcome. Further analysis of the multiplexed data, including both correlative and distance-based analyses are underway. Citation Format: Carvajal-Hausdorf DE, Stanton KP, Patsenker J, Villarroel-Espindola F, Esch A, Montgomery RR, Psyrri A, Kalogeras KT, Kotoula V, Fountzilas G, Schalper KA, Kluger Y, Rimm DL. Multiplexed (18-Plex) measurement of protein targets in trastuzumab-treated patients using imaging mass cytometry [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-18.

Inhibition of Protein Phosphatase 2A Sensitizes Mucoepidermoid Carcinoma to Chemotherapy via the PI3K-AKT Pathway in Response to Insulin Stimulus

Background/Aims: Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that mediates cell cycle regulation and metabolism. Mounting evidence has indicated that PP2A inhibition exhibits considerable anticancer potency in multiple types of human cancers. However, the efficacy of PP2A inhibition remains unexplored in mucoepidermoid carcinoma (MEC), especially in locally advanced and metastatic cases with limited systemic treatment. In this study, we demonstrated the therapeutic potency of LB100 in mucoepidermoid carcinoma. Methods: In this study, the expression of PP2A was evaluated using immunohistochemical (IHC) staining. The effects associated with LB100 alone and in combination with cisplatin for the treatment of mucoepidermoid carcinoma were investigated both in vitro, regarding metabolism, proliferation, and migration, and in vivo in a mucoepidermoid carcinoma xenograft model. In addition, with LB100 treatment and in response to an insulin stimulus, the expression levels and phosphorylation levels of targets in the PI3K-AKT pathway were determined using western blot analysis and immunoblotting. Results: The expression of protein phosphatase 2A was significantly upregulated in the clinical specimens of high-grade MECs compared with those of low-/medium-grade MECs and normal controls. In this article, we report that a small molecule PP2A inhibitor, LB100, decreased cellular viability and glycolytic activity and induced G2/M cell cycle arrest. Importantly, LB100 enhanced the efficacy of cisplatin in mucoepidermoid carcinoma cells both in vitro and in vivo. PP2A inhibition by LB100 increased the phosphorylation of insulin receptor substrate 1(IRS-1) on serine residues, downregulated the expression of phosphatidylinositol 3-kinase (PI3K) p110 alpha subunit and dephosphorylated AKT at Ser473 and Thr308 in mucoepidermoid carcinoma cells in response to insulin stimulus. Conclusion: These results highlight the translational potential of PP2A inhibition to synergize with cisplatin in mucoepidermoid carcinoma treatment.

Identification of the Binding Sites on Rab5 and p110beta Phosphatidylinositol 3-kinase

Rab5 is a small monomeric GTPase that mediates protein trafficking during endocytosis. Inactivation of Rab5 by GTP hydrolysis causes a conformational change that masks binding sites on its “switch regions” from downstream effectors. The p85 subunit of phosphatidylinositol 3-kinase (PI3K) is a GTPase activating protein (GAP) towards Rab5. Whereas p85 can bind with both Rab5-GTP and Rab5-GDP, the PI3K catalytic subunit p110β binds only Rab5-GTP, suggesting it interacts with the switch regions. Thus, the GAP functions of the catalytic arginine finger (from p85) and switch region stabilization (from p110β) may be provided by both proteins, acting together. To identify the Rab5 residues involved in binding p110β, residues in the Rab5 switch regions were mutated. A stabilized recombinant p110 protein, where the p85-iSH2 domain was fused to p110 (alpha or beta) was used in binding experiments. Eleven Rab5 mutants, including E80R and H83E, showed reduced p110β binding. The Rab5 binding site on p110β was also resolved through mutation of p110β in its Ras binding domain, and includes residues I234, E238 and Y244. This is a second region within p110β important for Rab5 binding. The Rab5-GTP:p110β interaction may be further elucidated through the characterization of these non-binding mutants in cells.

Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3-kinase signaling

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

Abstract 146: The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models

Abstract Activating mutations in PIK3CA are commonly found in a wide variety of human cancers, and the dysregulation of the phosphoinositide-3 kinase (PI3K) signaling pathway has been implicated in tumor cell growth and survival. Taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha, sparing inhibition of p110beta, is more potent in cancer cells bearing PIK3CA mutants than those with wildtype PIK3CA. Preclinical studies demonstrate that taselisib induces more apoptotic cell death in PIK3CA mutant cancer cells than other PI3K inhibitors. We have discovered that taselisib has a dual mechanism of action, both blocking PI3K signaling, and inducing a decrease in p110a protein levels. Mass spec analysis reveals that taselisib treatment leads to the specific depletion of mutant p110alpha without significant change in wildtype p110alpha protein levels. This drug-induced p110a protein depletion is rescued by E1 inhibitors and by proteasome inhibitors. Other clinical PI3K inhibitors, including p110alpha selective and pan-PI3K inhibitors, are unable to induce the depletion of mutant p110 alpha protein. Furthermore, we have discovered that taselisib more effectively maintains pathway suppression in PIK3CA mutant cells at 24 hrs in response to feedback. In comparison to other clinical-stage PI3K inhibitors administered at a maximum tolerated dose, taselisib has superior efficacy with increased tumor regressions in PIK3CA mutant xenograft models. In summary, these preclinical studies indicate that PI3K inhibitors, which have the ability to trigger degradation of mutant p110a protein, can more effectively suppress the signaling pathway, which may result in greater anti-tumor activity and improved therapeutic index in PIK3CA mutant tumors. Citation Format: Kyung W. Song, Kyle A. Edgar, Donald S. Kirkpatrick, Lilian Phu, Stephen Schmidt, Michelle Nannini, Rebecca Hong, Eric Cheng, Lisa Crocker, Amy Young, Deepak Sampath, Lori Friedman. The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 146. doi:10.1158/1538-7445.AM2017-146