Presence Of PI3K Inhibitors Research Articles

FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that forms the two different protein complexes, known as mTORC1 and mTORC2. mTOR signaling is activated in a variety of tumors, including glioma that is one of the malignant brain tumors. FilGAP (ARHGAP24) is a negative regulator of Rac, a member of Rho family small GTPases. In this study, we found that FilGAP interacts with mTORC1/2 and is involved in tumor formation in glioma. FilGAP interacted with mTORC1 via Raptor and with mTORC2 via Rictor and Sin1. Depletion of FilGAP in KINGS-1 glioma cells decreased phosphorylation of S6K and AKT. Furthermore, overexpression of FilGAP increased phosphorylation of S6K and AKT, suggesting that FilGAP activates mTORC1/2. U-87MG, glioblastoma cells, showed higher mTOR activity than KINGS-1, and phosphorylation of S6K and AKT was not affected by suppression of FilGAP expression. However, in the presence of PI3K inhibitors, phosphorylation of S6K and AKT was also decreased in U-87MG by depletion of FilGAP, suggesting that FilGAP may also regulate mTORC2 in U-87MG. Finally, we showed that depletion of FilGAP in KINGS-1 and U-87MG cells significantly reduced spheroid growth. These results suggest that FilGAP may contribute to tumor growth in glioma by regulating mTORC1/2 activities.

Elongation of Long-Chain Fatty Acids Is Crucial for Hematopoietic Stem Cell Engraftment and Leukemia Propagation

Lipid metabolism plays a crucial role in regulating physiological stem cells and cancer cells. Variations in length or degree of desaturation of fatty acid (FA) chains greatly contribute to lipid diversity, but whether it significantly regulates hematopoietic cell biology remains unknown. The enzyme ELOVL6 converts C16 saturated and monounsaturated FAs to C18 species. Elovl6 gene disruption (Elo6KO) in mice decreases the proportions of stearate (C18:0) and oleate (C18:1n-9) and increases those of palmitate (C16:0) and palmitoleate (C16:1n-7). Here, we report that ELOVL6 is essential for hematopoietic stem cells (HSC) engraftment after bone marrow (BM) transplantation and acute myeloid leukemia (AML) development in a mouse model. To determine whether hematopoietic cells of Elo6KO mice exhibit changes in FAs composition, we analyzed FAs in BM cell lysates. We found that the ratio of stearate to palmitate (C18:0/C16:0 ratio) was significantly decreased in Elo6KO cells compared to WT cells. To assess the effects of Elovl6 loss on the mouse HSC, we performed competitive repopulation assays. Using control or Elo6KO donor BM cells mixed in a 1:1 ratio with competitor BM cells, the chimerism of peripheral blood (PB) was about 50% in mice transplanted with control cells but remained at < 0.2% in those with Elo6KO cells at 12 weeks after transplantation. Even when transplanted Elo6KO donor cells with the competitor in a 10:1 ratio, the chimerism of PB was similarly low. Next, we investigated the roles of Elovl6 in leukemogenesis. We infected lineage-negative and c-Kit+ cells from WT or Elo6KO BM with MLL-AF9(MA9) retrovirus and transplanted lethally irradiated mice with either WT or Elo6KO MA9 cells. All mice receiving WT MA9 cells developed AML; however, AML propagation in Elo6KO MA9 cell-transplanted recipients was blocked. Elo6KO MA9 cell-transplanted mice survived until 6 months after transplantation. To assess the mechanisms underlying these phenotypes, we performed RNA-seq and gene enrichment analysis (GSEA) using CD34-, lineage-negative, Sca-1+ and c-Kit+ (CD34-LSK) cells and MA9 cells from Elo6KO or control mice. Chemotaxis, CXCR4 pathways, and their related target gene sets were enriched in Elo6KO cells, relative to control in both CD34-LSK cells and MA9 cells. We tested the chemotaxis of Elo6KO CD34-LSK and MA9 cells toward CXCL12 in a migration assay. A greater fraction of WT CD34-LSK and MA9 cells than that of these Elo6KO cells migrated to the CXCL12-containing chamber. We then examined the pharmacologic effects of inhibiting the PI3K pathway on chemotaxis. In a migration assay, the presence of PI3K inhibitor, Copanlisib, decreased the migration rate in WT MA9 cells, but not significantly in Elo6KO MA9 cells, which was already compromised. By contrast, AKT inhibitor, MK2206, did not alter WT or Elo6KO MA9 cell migration. In GSEA, Rac1-related pathways were enriched in Elo6KO cells. We thus investigated whether Rac1 activation was blocked by Elovl6 loss. The levels of the GTP-bound Rac1 were markedly increased in WT MA9 cells following CXCL12 stimulation compared to control, while those were not significantly different with/wo CXCL12 stimulation in Elo6KO MA9 cells. Given impaired chemotaxis and CXCR4-PI3K-Rac1 signaling in Elo6KO cells, we then investigated whether these cells exhibited altered actin remodeling in response to CXCL12. As expected, CXCL12 treatment induced lamellipodia formation in WT CD34-LSK cells, but not in Elo6KO CD34-LSK cells. Thus, ELOVL6 may regulate a CXCL12-CXCR4 pathway, resulting in PI3K-Rac1-mediated cytoskeletal remodeling and chemotaxis. Finally, to determine whether exogenously expressed Elovl6 could rescue Elo6KO MA9 cell phenotypes, we generated Elo6KO MA9 cells expressing Flag-tagged ELOVL6 (Elo6KO MA9Elo6) and mock controls (Elo6KO MA9mock). To evaluate the rescue of AML propagation, we transplanted lethally irradiated syngeneic mice with either Elo6KO MA9mock or Elo6KO MA9Elo6 cells. Among Elo6KO MA9mock transplanted mice, few recipients developed AML, while 10 out of 14 recipients had died due to AML after Elo6KO MA9Elo6 cell transplant. In summary, this study demonstrates novel and unexpected outcomes in normal HSCs and AML driver-transformed cells following lipid changes induced by Elovl6 deficiency. Altering lipid diversity could be a potential strategy to treat AML.

Knockdown of NOLC1 Inhibits PI3K-AKT Pathway to Improve the Poor Prognosis of Esophageal Carcinoma.

Objective Esophageal carcinoma (ESCA) is a common malignant gastrointestinal tumor. The abnormal expression of NOLC1 is involved in the tumorigenesis of various human tumors, whereas the function and mechanism of NOLC1 in ESCA remain unclear. In this study, we explored the relationship between NOLC1 and poor prognosis of ESCA, and its role and mechanism in the occurrence of ESCA. Methods The NOLC1 expression in ESCA tissues and cell lines was determined by qRT-PCR, immunohistochemistry, or western blot. The Kaplan–Meier method was conducted to estimate the overall survival. Cox regression analysis was carried out to examine the association between patient characteristics and prognosis. A recombined lentiviral vector containing NOLC1 was applied for transfecting ESCA cells (Eca109 and TE-13) and established a stable cell line with low NOLC1 expression or high NOLC1 expression, in the absence or presence of PI3K inhibitor (LY294002) treatment. Cell proliferation, apoptosis rate, invasion ability, migration ability, and PI3K/AKT pathway were detected by CCK8 assay, flow cytometry, Transwell assay, wound-healing assay, and western blot. Results NOLC1 overexpression was observed in ESCA tissues and ESCA cell lines (EC9706, Eca109, TE-13, Kyse170, T.TN) compared with adjacent normal tissues and normal esophageal cell line HEEC. NOLC1 overexpression was markedly associated with bigger tumor size, lymph node metastasis, and advanced TNM stage. Patients with NOLC1 overexpression have shorter overall survival than that of those with low NOLC1 expression. NOLC1 overexpression was considered to be an independent poor prognostic factor affecting overall survival. NOLC1 knockdown inhibited proliferation, migration, invasion, and cyclin B1 expression and promoted the apoptosis and cleaved-caspase-3 expression of Eca109 and TE-13 cells. NOLC1 overexpression accelerated proliferation, migration, invasion, and cyclin B1 expression and inhibited the apoptosis and cleaved-caspase-3 expression of ESCA cells via activating PI3K/AKT pathway. Rescue experiments showed that PI3K inhibitor (LY294002) could reverse the phenomenon caused by NOLC1 overexpression. Conclusion NOLC1 may be a marker for poor prognosis. It can participate in the occurrence and development of ESCA via the PI3K/AKT pathway.

CS nerve growth factor regulates sevoflurane anesthesiainduced nerve injury in nerve cells

Purpose: Sevoflurane is the most commonly used anesthetic agent for surgery. However, it is associated with deficiency in learning and memory abilities. The study was aimed at investigating the role of nerve growth factor (NGF) in sevoflurane anesthesia-induced nerve injury.Methods: RT-qPCR assay was applied to measure expressions of NGF, miR-98-5p and other factors related to apoptosis. CCK-8 assay was used for detecting cell viability while luciferase reporter assay was employed to measure binding condition between miR-98-5p and NGF. Expressions of proteins in PI3K/AKT/mTOR signaling pathway was measured with western blot.Results: Sevoflurane reduced cell viability of RGC-5 cells, promoted apoptosis and reduced the expression of NGF. In sevoflurane-induced RGC-5 cells, over-expression of NGF promoted cell viability with reduced apoptosis. Also, there was reduction in the protein expression of PI3K/AKT/mTOR signaling pathway by sevoflurane, while up-regulation of NGF promoted the expressions of these proteins. In the presence of PI3K inhibitor, reduction cell viability was reduced but apoptosis increased. Luciferase reporter assay detected MiR-98-5p as the target gene of NGF and its overexpression restored high cell viability in the over-expressed NGF. The rate of apoptosis and expressions of proteins was also restored with up-regulation of miR-98-5p.Conclusion: Sevoflurane caused damage to nerve cells, while over-expression of NGF reduced the injury through PI3K/AKT/mTOR signaling pathway and suppression of miR-98-5p.&#x0D; Keywords: Nerve growth factor, Sevoflurane, Nerve injury, Anesthesia, miR-98-5p

Pan-HDAC inhibitor panobinostat, as a single agent or in combination with PI3K inhibitor, induces apoptosis in APL cells: An emerging approach to overcome MSC-induced resistance.

The emergence of the drugs targeting epigenetic alterations has brought an optimistic outlook for cancer patients and probably put an end into the devastating effects of the disease. Given to the prominent involvement of histone deacetylase (HDAC) enzymes in the formation of neoplastic nature of acute promyelocytic leukemia (APL), this study was aimed to evaluate the suppressive effect of pan-HDAC inhibitor panobinostat on both NB4 and primary APL patients cells, either in the context of mono- or co-culture with mesenchymal stem cells (MSC). Panobinostat effectively reduced the survival of APL cells; however, as compared to NB4, the viability of primary cells was inhibited at higher concentrations. Our results also showed that although HDAC inhibition could merely block the survival signals transduced from MSC, the presence of PI3K inhibitor could robustly reinforce panobinostat cytotoxicity; suggesting that MSC-induced activation of PI3K may attenuate, at least partly, the efficacy of HDAC inhibition in APL cells. In addition, cellular and molecular investigations on NB4 revealed that not only panobinostat induced p21-mediated G1 arrest and ROS-mediated apoptosis, but also exerted a superior cytotoxicity when combined with c-Myc and autophagy inhibitors. Last but not least, we found that panobinostat combined with arsenic trioxide (ATO) prompted a synergistic effect and provided an improved therapeutic value in NB4; proposing that the abrogation of HDAC using panobinostat might be a befitting approach in APL, either as a single agent or in a combined-modal strategy.

Leukotriene B4 induces proliferation of rat pulmonary arterial smooth muscle cells via modulating GSK-3β/β-catenin pathway

Leukotriene B4 (LTB4) has been found to contribute to pulmonary arterial smooth muscle cells (PASMCs) proliferation and pulmonary arterial remodeling therefore the development of pulmonary arterial hypertension (PAH). Yet, the underlying molecular mechanisms remain poorly understood. The present study aims to address this issue. Our results demonstrate that LTB4 dose- and time-dependently induced proliferation of primary cultured rat PASMCs, this was accompanied with the activation of phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, and consequent inactivation of glycogen synthase kinase-3β (GSK-3β), up-regulation of β-catenin and induction of cyclin D1 expression. The presence of PI3K inhibitor (LY294002) or MEK inhibitor (U0126) or prior silencing of β-catenin with siRNA suppressed LTB4-induced cyclin D1 up-regulation and PASMCs proliferation. In addition, inactivation or lack of GSK-3β up-regulated β-catenin and cyclin D1 in PASMCs. Taken together, our study indicates that activation of PI3K/Akt and ERK1/2 pathways mediates LTB4-induced PASMCs proliferation by modulating GSK-3β/β-catenin/cyclin D1 axis and suggests that targeting this pathway might have potential value in alleviating vascular remodeling and benefit PAH.

Inhibition of PI3K pathway using BKM120 intensified the chemo-sensitivity of breast cancer cells to arsenic trioxide (ATO)

Although conventional therapeutic approaches have brought remarkable advantages for the treatment of breast cancer (BC), drug-resistance still remains as a leading cause of tumor recurrence in this malignancy. In the present study, we designed experiments to evaluate the therapeutic value of PI3K inhibition in combination with Arsenic trioxide (ATO) in MCF-7 cells. The results of our study manifested that BKM120 sensitized MCF-7 cells to the lower concentrations of ATO. The significant anti-cancer effect of PI3K inhibition became even more evident when we found that BKM120, either as a single agent or in combination with ATO, reduced clonogenic ability of anoikis-resistant stem-like MCF-7 cells. Our findings also showed that BKM120 augmented ATO-induced anti-proliferative effects through inducing G1 arrest and reducing the incorporation of BrdU into the synthesized DNA of drugs-treated cells, which was coupled with c-Myc-mediated suppression of hTERT expression. Moreover, we found that in the presence of PI3K inhibitor, ATO is able to more profoundly induce apoptosis in MCF-7 cells, as revealed by the increment in the percentage of haplodiploid sub-G1 cells and the externalization of phosphatidylserine. Real-time PCR analysis also revealed that probably down-regulation of survivin coupled with up-regulation of forkhead family transcription factors is responsible for the enhancive effect of drugs in this cell line. Conclusively, this study shed lights on the effect of PI3K inhibition in chemo-sensitivity of MCF-7 cells, disclosing that combination of BKM120 and ATO could be a promising therapeutic approach in BC.

Abstract 675: Network modeling of drug resistance mechanisms and drug combinations in breast cancer

Abstract Durable control of invasive solid tumors is thwarted by the lack of knowledge of effective drug combinations and of the acquired and intrinsic resistance mechanisms of drugs. In an effort to tackle this problem, the SU2C-NSF-TVF Drug Combination Convergence Team is using mechanistic models of cancer cell signaling based on therapeutic and cell line data in order to identify elements within cancer cells that might eventually be exploited through therapeutic combinations. Here we present a comprehensive mechanistic network model of signal transduction in ER+ PIK3CA-mutant breast cancer. Focusing on PI3K inhibitors, the model recapitulates known resistance mechanisms and predicts other possibilities for resistance: loss of RB1, FOXO3, P27, or PRAS40. To test these predictions, we analyzed genome-wide CRISPR screens of two breast cell lines in the presence of PI3K inhibitors (BYL719 and GDC0032) and found that the predicted genes (RB1, FOXO3, P27, and PRAS40) were significantly enriched in the screens. Some of these resistance genes (e.g. loss of RB1) were found to be cell-line specific and follow-up experiments in RB1-KO cells confirmed the cell-line-specific nature of PI3K-inhibitor resistance. The model also reveals known and novel combinatorial interventions that are more effective than PI3K inhibition alone. For example, the model predicts that the combination of PI3K inhibitors with inhibitors of anti-apoptotic protein MCL1 would be effective. Follow up experiments in cell lines using cell viability assays, cell death analyses, and dynamic BH3 profiling experiments to determine increases in apoptotic priming upon treatment confirmed that MCL1 inhibitors (S63845) enhance the effect of PI3K inhibitors (BYL719) and that this combinatorial effect is cell-line-specific, similarly to what was found in the resistance genes case. In conclusion, the model predicted drug resistance mechanisms and effective drug combinations, some of which were verified experimentally and found to be cell-line-specific. Next iterations of the model will incorporate the identified discrepancies and newly identified resistance mechanisms to drugs of clinical interest. Citation Format: Jorge Gómez Tejeda Zañudo, Pingping Mao, Joan Montero, Guotai Xu, Kailey J. Kowalski, Gabriela N. Johnson, José Baselga, Maurizio Scaltriti, Anthony G. Letai, Nikhil Wagle, Reka Albert. Network modeling of drug resistance mechanisms and drug combinations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 675.

Mulberry leaf extract displays antidiabetic activity in db/db mice via Akt and AMP-activated protein kinase phosphorylation.

BackgroundAugmenting glucose utilization in skeletal muscle via the phosphatidylinositol-3 kinase (PI3 kinase)/protein kinase B (Akt) pathway or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway is necessary to regulate hyperglycemia in patients with type 2 diabetes mellitus.ObjectiveWe investigated the effect of mulberry leaf extract (MLE) on glucose uptake in skeletal muscle cells and explored its in vivo antidiabetic potential.DesignMale db/db mice were treated with either MLE (50 mg/kg, 100 mg/kg, and 250 mg/kg) or metformin (100 mg/kg) for 8 weeks.ResultsMLE treatment stimulated glucose uptake, driven by enhanced translocation of glucose transporter 4 to cell membranes in L6 myotubes. These effects of MLE were synergistic with those of insulin and were abolished in the presence of PI3K inhibitor or AMPK inhibitor. In db/db mice, supplementation with MLE decreased fasting blood glucose and insulin levels and enhanced insulin sensitivity, with increases of p-Akt and p-AMPK in skeletal muscle. Moreover, MLE improved blood lipid parameters and attenuated hepatic steatosis in diabetic db/db mice.DiscussionThese findings suggest that MLE exerts antidiabetic activity through stimulating glucose disposal in skeletal muscle cells via the PI3K/Akt and AMPK pathways.ConclusionsMLE can potentially improve hyperglycemia and hepatic steatosis in patients with type 2 diabetes.

PIM kinases as therapeutic targets against advanced melanoma.

Therapeutic strategies for the treatment of metastatic melanoma show encouraging results in the clinic; however, not all patients respond equally and tumor resistance still poses a challenge. To identify novel therapeutic targets for melanoma, we screened a panel of structurally diverse organometallic inhibitors against human-derived normal and melanoma cells. We observed that a compound that targets PIM kinases (a family of Ser/Thr kinases) preferentially inhibited melanoma cell proliferation, invasion, and viability in adherent and three-dimensional (3D) melanoma models. Assessment of tumor tissue from melanoma patients showed that PIM kinases are expressed in pre- and post-treatment tumors, suggesting PIM kinases as promising targets in the clinic. Using knockdown studies, we showed that PIM1 contributes to melanoma cell proliferation and tumor growth in vivo; however, the presence of PIM2 and PIM3 could also influence the outcome. The inhibition of all PIM isoforms using SGI-1776 (a clinically-available PIM inhibitor) reduced melanoma proliferation and survival in preclinical models of melanoma. This was potentiated in the presence of the BRAF inhibitor PLX4720 and in the presence of PI3K inhibitors. Our findings suggest that PIM inhibitors provide promising additions to the targeted therapies available to melanoma patients.

Abstract 1994: Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells

Abstract Prostate Apoptosis Response-4 (Par-4) is a tumor suppressor protein whose expression level in cancer is frequently decreased; however, it is rarely mutated nor suppressed. Par-4 is not only regulated at the expression level, but also post-translationally. Most recently, we reported the cisplatin-induced caspase3-dependent cleavage of Par-4 resulting in the accumulation of a 25kd cleaved-Par-4 (cl-Par-4) fragment. In the present study, we investigated the mechanisms regulating this fragment using cl-Par-4-expressing stable clones derived from ovarian and endometrial cancer cell lines. Briefly, we used cancer cell lines in which we produced stable clones expressing the cleaved form of PAR-4 (cl.PAR-4) using lentiviral particles. Using these models, we studied the regulation of cl.PAR-4 at the protein level using Western blots. Intriguingly, cl-Par-4 protein was weakly expressed in all stable clones despite constitutive transgene expression. However, upon cisplatin treatment, cl-Par-4 levels increased up to 50-fold relative to baseline conditions. We assessed the subcellular localization of cl-Par-4 in baseline and cisplatin-treated conditions using cytoplasmic/nuclear fractionation. Results showed that cl-Par-4 is localized in both the cytoplasm and nucleus compartments, but cisplatin treatment did not alter its localization. Treatment of stable clones with proteasome and translation inhibitors revealed that cisplatin exposure may in fact protect cl-Par-4 from proteasome-dependent degradation. To confirm proteasome involvement in this process, potential ubiquitination sites were predicted using bioinformatic tools. Interestingly, we showed that PI3K and MAPK pathways are also implicated in this post-translational regulation, as evidenced by an increase of cl-Par-4 in the presence of PI3K inhibitors (Wortmannin and NVP-BEZ235) and a decrease of cl-Par-4 using MAPK inhibitors (U0126 and PD98059). Finally using bioinformatics resources, we found diverse datasets showing similar results to those we observed with the proteasome and cl-Par-4. Indeed, we have showed, using data from a published study, that the use of Bortezomib (a potent proteasome inhibitor) significantly increased Par-4 expression in MCF7 cancer cells. These new findings add to the complex mechanisms regulating Par-4 expression and activity, and justify further studies addressing the biological significance of this phenomenon in gynecological cancer cells. Citation Format: Kevin Brasseur, Pascal Adam, Laurence Tardif, François Fabi, Sophie Parent, Éric Asselin. Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1994.

Abstract 4591: Estrogen receptor-positive breast cancer: from genomic landscape to treatment approach

Abstract Background: Estrogen receptor (ER)-positive or luminal breast tumors represent around two-thirds of all breast cancers. Luminal breast cancer is a highly heterogeneous disease comprising different histologies, gene-expression profiles and mutational patterns, with very diverse clinical courses and responses to systemic treatment. Despite adjuvant endocrine therapy and chemotherapy treatment for patients at high risk of relapse, both early and late relapses still occur, a fact that highlights the unmet medical needs of these patients. A catalogue of molecular aberrations in ER+ breast cancer (BC) is critical for developing and deploying therapies that will improve patients’ lives. Methods: Comprehensive genomic profiling from 128 BC patients (February 2014 through October, 2015) were analyzed. Patients were biopsied after consultation and samples were characterized (ER, PR, and HER2 by IHC; FFPE samples for genomic [Foundation One] and proteomic analyses [Theranostics]). We also evaluated mutation distribution in cell free DNA via digital NGS using Guardant 360 panel. Results: Total 97 genes were altered in 83 ER+BC patients and most frequent genetic alterations are PIK3CA (40%), p53 (27.7%) and CCND1 (24%). The PI3K-AKT pathway specific genes (AK1, AKT2, PIK3CA, PIK3CB, PIK3R1, PTEN, MTOR, RICTOR, RAPTOR, TSC2, STK11, MDM2 and MDM4) were altered in 80% of patients. Analyzing the composite alterations in individual ER+BC patients, we observed that 20% of patients had alterations in two or more nodes of the PI3K pathway while alterations of cell cycle pathway genes (CCND1, CCNE1, CDK4, CDKN1B, CDKN2A/B and RB1) were observed in 36% of patients. Interestingly, concurrent alterations of both PI3K and cell cycle pathways were 24%. Hence we tested the anti-tumor efficacy of the combination of isoform-specific PI3K inhibitor (alpha or beta) and LEE011 using ER+ BC cell lines (PIK3CA mutated MCF7, T47D, &amp; PTEN-null MDA-MB415 cells) based model. Data show a synergistic effect of PI3K alpha or beta inhibitor and CDK4/6 inhibitor as determined from the changes in proliferative and apoptotic signals as well as cell cycle arrest. Conclusion: Since a persistent expression of the CDK4-Cyclin D1 pathway activation enables tumor cells survival even in the presence of PI3K inhibitor (Cancer Cell 2014), results of our study demonstrated that the anti-tumor effect of isoform-specific PI3Ki and CDK4/6i be beneficial in RB wt ER+BC patients with a concurrent genetic alterations of these pathways. Citation Format: Pradip De, Casey Williams, Amy Krie, Kirstin Williams, Jessica Klein, Jennifer H. Carlson, Nandini Dey, Brian Leyland-Jones. Estrogen receptor-positive breast cancer: from genomic landscape to treatment approach. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4591.

PI3K pathway is involved in ERK signaling cascade activation by histamine H2R agonist in HEK293T cells

BackgroundHistamine, through histamine H2 receptor (H2R), modulates different biological processes, involving the modulation of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Many evidences have demonstrated the existence and importance of the crossregulation between these two signaling pathways. The aim of the present work was to determine the molecular mechanisms leading to PI3K and ERK pathways modulation induced by the H2R agonist amthamine and to evaluate the possible interplay between them. MethodsPhosphorylation levels of ERK and Akt were examined by Western blot in HEK293T cells expressing the human H2R, in the presence of H2R agonist and dominant negative mutants or pharmacological inhibitors of different proteins/pathways. Transcriptional activity assays were assessed to determine SRE activity. Amthamine-mediated cellular proliferation was investigated in MA-10A cells in the presence of PI3K inhibitor. ResultsH2R agonist inhibits PI3K/Akt/mTOR and stimulates Ras/MEK/ERK pathways. Moreover, PI3K/Akt/mTOR signaling inhibition is necessary to achieve H2R mediated ERK activation. In the presence of a constitutive active mutant of Akt, amthamine is not able to mediate ERK activation. This crosstalk affects classical ERK downstream targets such as Elk1 phosphorylation and the transcriptional activity of the SRE, classically associated to proliferation. We further demonstrate that amthamine-induced proliferation in Leydig MA-10 tumor cells, is enhanced by LY294002, a PI3K inhibitor. ConclusionsThese results describe a crosstalk between PI3K/AKT/mTOR and Ras/MEK/ERK pathways induced by H2R stimulation with implications in cell proliferation. General significanceThis work indicates that the modulation of PI3K/AKT/mTOR pathway by H2R in turn regulates Ras/MEK/ERK activation conditioning the proliferative capacity of the cells.

Tropoelastin enhances nitric oxide production by endothelial cells.

This study aimed to characterize the role of tropoelastin in eliciting a nitric oxide response in endothelial cells. Nitric oxide production in cells was quantified following the addition of known nitric oxide synthase pathway inhibitors such as LNAME and 1400W. The effect of eNOS siRNA knockdowns was studied using western blotting and assessed in the presence of PI3K-inhibitor, wortmannin. Tropoelastin-induced nitric oxide production was LNAME and wortmannin sensitive, while being unaffected by treatment with 1400W. Tropoelastin acts through a PI3K-specific pathway that leads to the phosphorylation of eNOS to enhance nitric oxide production in endothelial cells. This result points to the benefit of the use of tropoelastin in vascular applications, where NO production is a characteristic marker of vascular health.

Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells.

We recently reported the caspase3-dependent cleavage of Par-4 resulting in the accumulation of a 25kDa cleaved-Par-4 (cl-Par-4) fragment and we investigated in the present study the mechanisms regulating this fragment using cl-Par-4-expressing stable clones derived from ovarian and endometrial cancer cell lines.Cl-Par-4 protein was weakly express in all stable clones despite constitutive expression. However, upon cisplatin treatment, cl-Par-4 levels increased up to 50-fold relative to baseline conditions. Treatment of stable clones with proteasome and translation inhibitors revealed that cisplatin exposure might in fact protect cl-Par-4 from proteasome-dependent degradation. PI3K and MAPK pathways were also implicated as evidenced by an increase of cl-Par-4 in the presence of PI3K inhibitors and a decrease using MAPK inhibitors. Finally using bioinformatics resources, we found diverse datasets showing similar results to those we observed with the proteasome and cl-Par-4 further supporting our data.These new findings add to the complex mechanisms regulating Par-4 expression and activity, and justify further studies addressing the biological significance of this phenomenon in gynaecological cancer cells.

Abstract 2111: Regulation and localization of the cleaved form of PAR-4 in ovarian and endometrial cancers

Abstract Prostate Apoptosis Response-4 (PAR-4) is a tumor suppressor protein whose expression level in cancer is frequently decreased; however, it is neither mutated nor suppressed. A unique feature of PAR-4 is that it can induce apoptosis selectively in cancer cells without destroying normal cells, giving it an interest in anti-cancer targeted therapy. PAR-4 is not only regulated at the expression level, but also post-translationally by phosphorylation and protein cleavage. Most recently, the caspase-3 cleavage of PAR-4 has been demonstrated by our laboratory and this cleaved fragment might be responsible for PAR–induced apoptosis. In the present study, we have investigated the mechanisms regulating PAR-4 expression/activity and its cleaved form in ovarian and endometrial cancer cell lines. Briefly, we used chemo-sensitive and resistant cancer cell lines in which we produced stable clones expressing the cleaved form of PAR-4 (cl.PAR-4) using lentiviral particles. Using these models, we studied the regulation of cl.PAR-4 at the protein and genomic level. Treated with cisplatin, sensitive cancer cells showed a decrease of PAR-4 and an important increase of endogenous cl.PAR-4. Using our stable clones, we observed that upon cisplatin treatment, the normally constitutively expressed cl.PAR-4 protein level was increased while the cDNA transgene was constitutively expressed, indicating a possible involvement of post-translational mechanisms. Our results indicate that degradation of our protein by the proteasome could be implicated because of the significant increase of cl.PAR-4 in the presence of a proteasome inhibitor (MG-132). PI3k could also be linked to this regulation because of an increase of cl.PAR-4 in the presence of PI3k inhibitors (LY294002, Wortmannin and BEZ235). Conversely, when using MAPK inhibitors (U0126 and PD98059), cl.PAR-4 was negatively regulated. We also investigated the localization of cl.PAR-4 using cytoplasmic/nuclear fractionationnation and immunofluorescence. The results indicated that cl.PAR-4 is mainly localized to the cytoplasm while being weakly present in the nucleus. Cisplatin treatment does not seem to influence its localization. This nuclear localization of cl.PAR-4 may have a role in its ability to induce apoptosis. Finally, bioinformatic analyses suggests possible ubiquitination and phosphorylation sites responsible for the regulation of cl.PAR-4. Further analyses of the post-translational mechanisms involved in cl.PAR-4 activity are still needed to better understand the regulation of this potential tumor suppressor. By better understanding the mechanisms of PAR-4 and its cleaved form, we may be able to target this protein to sensitize chemoresistant gynecological cancer cells to chemotherapy. Citation Format: Kevin Brasseur, Valérie Leblanc, Sophie Parent, Éric Asselin. Regulation and localization of the cleaved form of PAR-4 in ovarian and endometrial cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2111. doi:10.1158/1538-7445.AM2015-2111

Efficient Hematopoietic Differentiation of Common Marmoset Embryonic Stem Cells by the Inhibition of Their Self-Renewal Pathway.

Abstract 2311The human regenerative medicine by the transplantation of the functional cells differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have great potential of contributing to the treatments for various diseases, and thus have attracted huge public attention. However, the risk of unwelcome tumor formation originated from transplanted cells in recipients remains to be solved. Therefore the safety and efficacy of ESC/iPSC-based therapies should be carefully evaluated using reliable animal disease models before their clinical application. Among experimental animal models, common marmoset (CM, Callithrix jacchus), one of NEW WORLD monkeys, has recently been recognized as a useful non-human primate because they are small, easy to handle, highly reproductive and genetically very similar to humans. We have continuously investigated the characteristics of ESCs and iPSCs derived from CM.Understanding the self-renewal pathways in ESCs/iPSCs is crucial for the development of improved technology to culture and differentiate them into functional cells of potential therapeutic use. It has been reported that the maintenance of self-renewal in human or mouse ESCs/iPSCs require basic fibroblast growth factor (bFGF) or leukemia Inhibitory factor (LIF) respectively, however the growth factors required for the culture of CM ESCs/iPSCs have not been clearly determined.To clarify whether LIF or bFGF is more appropriate to maintain self-renewal of CM ESCs in culture, we examined the proliferation rate of CM40, a CM ESC line, maintained in the presence or absence of LIF or bFGF. CM ESCs were passaged at a ratio of 1:3 every 3 to 4 days. We found that the number of OCT3/4+cells was significantly increased by the addition of bFGF but not of LIF compared to control (w/o cytokines). Similar results were obtained when Cj11, another CM ESC line, was used. These results indicate that bFGF is essential for culturing CM ESCs, but LIF is dispensable.It has been reported that bFGF and its downstream PI3K-AKT and MEK-ERK pathways are important for maintenance of ESCs in human. Thus we examined whether PI3K-AKT and MEK-ERK pathway play crucial roles in the maintenance of self-renewal in CM ESCs. CM40 was cultured in the medium containing bFGF in the presence of PI3K inhibitor (LY294002) or MEK inhibitor (PD0325901). We found that the percentage and number of OCT3/4+ cells were gradually decreased in the presence of LY294002 (10 μM or 20 μM), suggesting that PI3K-AKT pathway is essential for the self-renewal of CM ESCs. Furthermore, the percentage and number of OCT3/4+cells were gradually decreased by addition of PD0325901 (1 μM or 5 μM) in the course of 4 passages, indicating that MEK-ERK pathway also plays a role in the self-renewal of CM ESCs.Next we examined if inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK promote hematopoietic differentiation in CM ESCs. One of methods for inducing hematopoietic cells from ESCs is embryoid body (EB) formation which is a conventional technique frequently used for in vitro differentiation of ESCs. Thus to induce hematopoietic differentiation, we performed EB formation assay by plating single-cell suspension of CM ESCs (3 × 105 cells) in StemLine II supplemented with 50 ng/ml BMP4 and 50 ng/ml VEGF with or without 10 μM LY294002 or 5 μM PD0325901 for 2 days. Then we removed half the medium and added fresh medium with the same final concentrations of BMP4, VEGF, LY294002 and PD0325901, plus 25 ng/ml SCF, 25 ng/ml TPO and 25 ng/ml FLT3L to expand the hematopoietic progenitors. We found that addition of LY294002 or PD0325901 increased the population of cells positive for CD34, a marker for hematopoietic stem/progenitor and endothelial cells, in day4-EBs. These CD34+cells showed hematopoietic differentiation potential proved by colony forming unit (CFU) assayTaken together, inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK in CM ESCs is thought to promote their hematopoietic differentiation by EB formation. Our findings might be useful to develop a better technology of the culture and hematopoietic differentiation of CM ESCs as well as to test efficacy and safety of ESC-derived hematopoietic cells using CM disease models for the future ESC/iPSC-based human regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

Neuroprotection by urate on 6‐OHDA‐lesioned rat model of Parkinson's disease: linking to Akt/GSK3β signaling pathway

Higher plasma urate level is reported to be associated with a reduced risk and slower progression of Parkinson's disease (PD). In this study, we explored the effects of urate on dopaminergic neurons in nigrostriatal pathway in the 6-hydroxydopamine (6-OHDA) unilaterally lesioned rats. Uric acid (UA), when given twice daily at 200 mg/kg intraperitoneally for 10 consecutive days, elevated urate (the anionic form of UA) in plasma and striatum by 55% and 36.8%, respectively, as compared with vehicle group. This regimen of UA was found to ameliorate the behavioral deficits, dopaminergic neuron loss as well as dopamine depletion in the nigrostriatal system. Moreover, UA administration was capable of increasing glutathione level and superoxide dismutase activity while decreasing malondialdehyde accumulation in striatum. In addition, the phosphorylation of both protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) in the lesioned striata of 6-OHDA-lesioned rats was dramatically reduced as compared with sham-operated rats. This reduction was attenuated in the Parkinsonian rats receiving UA treatment. Similarly, in vitro findings showed that UA alleviated the decrease in Akt activation and the increase in GSK3β activity caused by 6-OHDA. Furthermore, neuroprotection by urate and its regulation on GSK3β phosphorylation at Ser9 was found to be abolished in the presence of PI3K inhibitor. Therefore, our findings demonstrated that urate was able to protect dopaminergic neurons in rat nigrostriatal pathway against the neurotoxicity of 6-OHDA, and showed that its beneficial effects may be related to its regulation on Akt/GSK3β signaling.

Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF-TrkB-PI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF-TrkB-PI3K/Akt signaling pathway.

Combined Inhibition of PI3K and mTOR Exerts Synergistic Antiproliferative Effect, but Diminishes Differentiative Properties of Rapamycin in Acute Myeloid Leukemia Cells

A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining mammalian target of rapamycin (mTOR)-inhibitors with an inhibitor of the phosphatydilinositol 3-kinase PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. However, recent study demonstrated the potentiating effect of rapamycin on all-trans-retinoic acid (ATRA)-mediated differentiation of acute myelogenous leukemia (AML) cells, prompting us to investigate the effects of longitudinal inhibition of PI3K/Akt/mTOR signaling pathway on both proliferation and differentiative capacity of AML. In NB4, HL-60, U937 and K562 cell lines, rapamycin exerted minimal antiproliferative effects, and combining PI3K inhibitor LY 294002 and rapamycin inhibited proliferation more than LY 294002 alone. Rapamycin potentiated differentiation of ATRA-treated NB4 cells, but the combination of rapamycin and LY 294002 inhibited the expression of CD11b in both ATRA- and phorbol myristate acetate (PMA)-stimulated cells more than PI3K inhibitor alone. These results demonstrate that, although the combination of PI3K inhibitor and rapamycin is more effective in inhibiting proliferation of AML, the concomitant inhibition of PI3K and mTOR by LY 294002 and rapamycin has more inhibitory effects on ATRA-mediated differentiation than the presence of PI3K-inhibitor alone, and diminishes positive effects of rapamycin on leukemia cell differentiation.