Effect Of Phosphoinositide 3-kinase Inhibition Research Articles

Stage-Dependent Activity and Pro-Chondrogenic Function of PI3K/AKT during Cartilage Neogenesis from Mesenchymal Stromal Cells

Differentiating mesenchymal stromal cells (MSCs) into articular chondrocytes (ACs) for application in clinical cartilage regeneration requires a profound understanding of signaling pathways regulating stem cell chondrogenesis and hypertrophic degeneration. Classifying endochondral signals into drivers of chondrogenic speed versus hypertrophy, we here focused on insulin/insulin-like growth factor 1 (IGF1)-induced phosphoinositide 3-kinase (PI3K)/AKT signaling. Aware of its proliferative function during early but not late MSC chondrogenesis, we aimed to unravel the late pro-chondrogenic versus pro-hypertrophic PI3K/AKT role. PI3K/AKT activity in human MSC and AC chondrogenic 3D cultures was assessed via Western blot detection of phosphorylated AKT. The effects of PI3K inhibition with LY294002 on chondrogenesis and hypertrophy were assessed via histology, qPCR, the quantification of proteoglycans, and alkaline phosphatase activity. Being repressed by ACs, PI3K/AKT activity transiently rose in differentiating MSCs independent of TGFβ or endogenous BMP/WNT activity and climaxed around day 21. PI3K/AKT inhibition from day 21 on equally reduced chondrocyte and hypertrophy markers. Proving important for TGFβ-induced SMAD2 phosphorylation and SOX9 accumulation, PI3K/AKT activity was here identified as a required stage-dependent driver of chondrogenic speed but not of hypertrophy. Thus, future attempts to improve MSC chondrogenesis will depend on the adequate stimulation and upregulation of PI3K/AKT activity to generate high-quality cartilage from human MSCs.

For Better or Worse: The Potential for Dose Limiting the On-Target Toxicity of PI 3-Kinase Inhibitors.

The hyper-activation of the phosphoinositide (PI) 3-kinase signaling pathway is a hallmark of many cancers and overgrowth syndromes, and as a result, there has been intense interest in the development of drugs that target the various isoforms of PI 3-kinase. Given the key role PI 3-kinases play in many normal cell functions, there is significant potential for the disruption of essential cellular functions by PI 3-kinase inhibitors in normal tissues; so-called on-target drug toxicity. It is, therefore, no surprise that progress within the clinical development of PI 3-kinase inhibitors as single-agent anti-cancer therapies has been slowed by the difficulty of identifying a therapeutic window. The aim of this review is to place the cellular, tissue and whole-body effects of PI 3-kinase inhibition in the context of understanding the potential for dose limiting on-target toxicities and to introduce possible strategies to overcome these.

Abstract 2979: Dual targeting of HER3 and PIK3CA has potent anti-tumor effects in pre-clinical models of HNSCC

Abstract Background: The phosphoinositide 3-kinase (PI3K) pathway is a frequently mutated oncogenic pathway in head and neck squamous cell carcinoma (HNSCC). This pathway is often further activated by dysregulation of receptor tyrosine kinases, such as the epidermal growth factor receptor family. HER3 has a direct phosphorylation site with the regulatory subunit of PIK3CA, and is being investigated as a potential co-target to increase anti-tumor effects of PI3K inhibition. In this study, we use the human monoclonal antibody KTN3379, which blocks HER3 in the autoinhibited configuration and thus inhibits ligand-independent and ligand, neuregulin β-1,-dependent HER3 activation, and an inhibitor of PIK3CA (BYL719) to determine if HER3 and PIK3CA can be inhibited in a synergistic fashion. KTN3379 and BYL719 are clinical stage compounds and are being evaluated in HNSCC clinical trials. Methods and Results: Treatment with neuregulin β-1 significantly increased phosphorylation of HER3, AKT, and S6 across HNSCC cell lines, regardless of baseline level of HER3 expression. HER3 inhibition (KTN3379) consistently abrogated these effects and decreased baseline phosphorylation of HER3. Combined blockade of HER3 (KTN3379) and PI3K (BYL719) inhibited the growth of HNSCC cell lines in a synergistic fashion (CI range 0.1-0.5), in both cell proliferation and colony formation assays. This synergy appeared to be less pronounced in HPV-associated cancer cell lines. Combined treatment decreased tumor growth in xenograft models of HNSCC. Furthermore, treatment of some HNSCC cell lines with BYL719 led to upregulation of the HER3 protein in HNSCC cell lines by Western blotting analysis, suggesting a possible role of HER3 in PI3K pathway inhibitor resistance. Conclusion: HER3 interacts with PI3KCA, and the PI3K pathway in multiple HNSCC cell lines, and combined blockade has synergistic effects in pre-clinical HNSCC models. These findings suggest that HER3 and PI3K dual inhibition may be more effective in tumors that are resistant to mono-therapy with either HER3 or PI3K inhibition alone. Citation Format: Nayel Khan, Kara S. Davis, Neal Godse, Carolyn Kemp, Sucheta Kulkarni, Diego Alvarado, Theresa LaVallee, Jennifer R. Grandis, Umamaheswar Duvvuri. Dual targeting of HER3 and PIK3CA has potent anti-tumor effects in pre-clinical models of HNSCC. [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 2979.

Abstract 1674: HER3 inhibition potentiates anti-tumor effects of PI3K inhibitors in pre-clinical models of HNSCC

Abstract Background: The phosphoinositide 3-kinase (PI3K) pathway is thought to be the most frequently mutated oncogenic pathway in head and neck squamous cell carcinoma (HNSCC). This pathway is often further activated by deregulated receptors such as the epidermal growth factor receptor family. HER3 has a direct phosphorylation site with the regulatory subunit of PIK3CA, and is being investigated as a potential co-target to increase anti-tumor effects of PI3K inhibition. In this investigation, we use blocking antibodies against HER3 and its ligand, neuregulin β-1, (KTN3379) and PIK3CA (BYL719) to investigate the effect of HER3 in modulating response to PI3K inhibition. Methods and Results: Treatment with neuregulin β-1 significantly increased phosphorylation of HER3, AKT, and S6 across HNSCC, regardless of baseline level of HER3 expression. HER3 inhibition (KTN3379) consistently abrogated these effects, and decreased baseline phosphorylation of HER3. Combined blockade of HER3 (KTN3379) and PI3K (BYL719) inhibits the growth of HNSCC cell lines more effectively than inhibition of a single receptor alone, in both cell proliferation and colony formation assays. Combined treatment decreases phosphorylation of AKT and S6 more effectively than inhibition with either drug in some HNSCC cell lines. Treatment with neuregulin β-1 diminishes the inhibitory effect of BYL719 on HNSCC cell growth. Furthermore, treatment of some HNSCC cell lines with BYL719 led to upregulation of the HER3 pathway in HNSCC cell lines by Western blotting analysis, suggesting a possible role of HER3 in PI3K resistance. Conclusion: HER3 interacts with PI3KCA, and the PI3K pathway in multiple HNSCC cell lines, and combined blockade leads to pronounced effects in pre-clinical models of HNSCC. These findings suggest that HER3 blockade, may be efficacious when combined with PI3K pathway inhibition. Citation Format: Kara S. Davis, Nayel Khan, Carolyn Kemp, Sucheta Kulkarni, Diego Alvarado, Theresa LaVallee, Jennifer R. Grandis, Umamaheswar Duvvuri. HER3 inhibition potentiates anti-tumor effects of PI3K inhibitors in pre-clinical models of HNSCC. [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 1674. doi:10.1158/1538-7445.AM2015-1674

Abstract A33: Utilizing insulin the treatment of prostate cancer with BKM120 abrogates the therapeutic effect of PI3K pathway inhibition

Abstract Genetic aberrations in PTEN, the negative regulator of phosphoinositide 3-kinase (PI3K) signaling, are common in prostate cancers (PCA) with high Gleason Scores and correlate with adverse outcome. Currently, PI3K inhibitors, as single agents and in combination, have entered Phase I and II clinical trials for metastatic castrate resistant PCA. Alpha-specific and pan-PI3K inhibitors cause hyperglycemia as an on-target side effect by blocking insulin-dependent PI3K signaling in the liver and muscle and patients develop hyperinsulinemia in response to hyperglycemia, and insulin treatment is often needed to manage drug-induced hyperglycemia. Using human and murine prostate cancer cell lines, we examined the effect of adding insulin to BKM120 treatment on PI3K pathway activation. Using human prostate cancer cell lines LNCaP and PC3 which both express insulin receptor (IR) and IGF-1 receptor (IGF-1R), we found downregulation of the PI3K pathway by BKM120 treatment and upregulation of the PI3K pathway by insulin treatment, as assessed by AKT phosphorylation. In both cell lines, the PI3K pathway is reactivated when insulin is added to BKM120 treatment. Using cells derived from PTEN-/-P53-/- murine PCA which express low levels of IR and IGF-1R, we found minimal activation of the PI3K pathway with insulin, either alone or in combination with BKM120. Conversely, using cells derived from Myc-overexpressing mice which express high levels of IR and IGF-1R, we found robust activation of the PI3K pathway with insulin treatment. More importantly, while BKM120 abolished PI3K activity in the Myc-overexpressing cells, the addition of insulin to BKM120 resulted in the reactivation of the PI3K pathway to the level of PI3K pathway activity without BKM120 treatment. Together these findings suggest that the use of insulin in combination with BKM120 abrogates the therapeutic effect of PI3K inhibition. In addition, they indicate that PCAs that have high levels of IR and/or IGF-1 expression are particularly sensitive to PI3K inhibitors as well as insulin reactivation of the PI3K pathway. Citation Format: Lily C. Wang, Sunnie S. Kim, David M. Nanus, Lewis C. Cantley. Utilizing insulin the treatment of prostate cancer with BKM120 abrogates the therapeutic effect of PI3K pathway inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A33.

Phosphoinositide 3-kinase alpha-dependent regulation of branching morphogenesis in murine embryonic lung: evidence for a role in determining morphogenic properties of FGF7.

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.

Phosphoinositide 3-kinase (PI3K) as a New Therapeutic Target for Rheumatoid Arthritis

Dysregulated activation of immune and synovial cells and their reciprocal action play a key role in the pathogenesis of rheumatoid arthritis (RA). Various signal transduction molecules regulate cellular responses and small molecular inhibitors targeting the signal molecules, such as Janus kinase (JAK) and spleen tyrosine kinase (Syk) inhibitors, which have been developed for treating RA. Phosphoinositide 3-kinase (PI3K) is one of the signal molecules, which regulates innate and adaptive immune systems and is over-expressed in RA. PI3Ks phosphorylate phosphoinositide-4,5- bisphosphate (PI-4,5-P2) generates phosphoinositide-3,4,5- triphosphate (PI-3,4,5-P3) at the cell membrane. PI3Ks are divided into class I, II and III. Two catalytic subunits, p110γ and p110δ of PI3K, modulate cellular development, differentiation, proliferation, migration, cytokine synthesis and antibody production in both innate and adaptive immune systems. In RA synovium and synovial fibroblasts, the expression of p110γ and p110δ is increased, and their upregulation results in the abnormal activation of cellular immune responses. In preclinical animal models for RA, genetic deletion of p110γ and p110δ and selective inhibitors decrease the clinical arthritis score, synovial inflammation, cellular infiltration, bone and cartilage erosion and osteoclast activity. There is a synergistic effect for controlling arthritis by dual inhibition of PI3Kγ and PI3Kδ. Through reviewing the function of PI3K in the immune system and the effect of PI3K inhibition in preclinical arthritis animal models, we can expect the PI3K inhibition as a new therapeutic target for treatment of RA.

Phosphoinositide 3-kinase (PI3K)

Dysregulated activation of immune and synovial cells and their reciprocal action play a key role in the pathogenesis of rheumatoid arthritis (RA). Various signal transduction molecules regulate cellular responses and small molecular inhibitors targeting the signal molecules, such as Janus kinase (JAK) and spleen tyrosine kinase (Syk) inhibitors, which have been developed for treating RA. Phosphoinositide 3-kinase (PI3K) is one of the signal molecules, which regulates innate and adaptive immune systems and is over-expressed in RA. PI3Ks phosphorylate phosphoinositide-4,5- bisphosphate (PI-4,5-P2) generates phosphoinositide-3,4,5- triphosphate (PI-3,4,5-P3) at the cell membrane. PI3Ks are divided into class I, II and III. Two catalytic subunits, p110 γ and p110δ of PI3K, modulate cellular development, differentiation, proliferation, migration, cytokine synthesis and antibody production in both innate and adaptive immune systems. In RA synovium and synovial fibroblasts, the expression of p110γ and p110δ is increased, and their up-regulation results in the abnormal activation of cellular immune responses. In preclinical animal models for RA, genetic deletion of p110γ and p110δ and selective inhibitors decrease the clinical arthritis score, synovial inflammation, cellular infiltration, bone and cartilage erosion and osteoclast activity. There is a synergistic effect for controlling arthritis by dual inhibition of PI3Kγ and PI3Kδ. Through reviewing the function of PI3K in the immune system and the effect of PI3K inhibition in preclinical arthritis animal models, we can expect the PI3K inhibition as a new therapeutic target for treatment of RA.

Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus.

Event Abstract Back to Event Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus. Joshua G. Pemberton1, James L. Stafford1, Yi Yu1 and John P. Chang1* 1 University of Alberta, Biological Sciences, Canada Two endogenous gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) stimulates LH and GH secretion via Ca2+-dependent intracellular signalling pathways in goldfish, Carassius auratus. Whether phosphoinositide 3-kinase (PI3K) mediates GnRH-stimulated LH and GH release and associated intracellular Ca2+ increases ([Ca2+]i) were investigated using hormone release studies with primary cultures of dispersed goldfish pituitary and Fura2 fluorescence Ca2+ imaging in morphologically identified goldfish pituitary somatotropes and gonadotropes. Western blot showed the presence of immunoreactive PI3K p85α, the regulatory subunit for conventional Class IA PI3Ks, in goldfish pituitary and brain tissue extracts and both endogenous GnRH isoforms increased phosphorylation of PI3K p85α in excised pituitary fragments. Selective PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM) significantly reduced sGnRH- and cGnRH-II-elicited LH release responses from primary cultures of mixed pituitary cells and [Ca2+]i increases in identified gonadotropes. Surprisingly, both PI3K inhibitors reduced the GH release response to the two GnRHs but only attenuated the [Ca2+]i response to cGnRH-II. The effects of PI3K inhibition on hormone release responses were specific to GnRHs and not the result of general decreases in the releasable hormone pool or sensitivity to [Ca2+]i changes as both wortmannin and LY294002 had no effect on Ca2+ ionophore-evoked LH and GH secretion. These results indicate that PI3K is involved upstream of [Ca2+]i increases in mediating LH and GH responses in a cell- and GnRH isoform-specific manner and is the first to implicate PI3K in GnRH-induced LH and GH release in any primary pituitary cell system. Acknowledgements This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) individual Discovery Grants to J.P. Chang and J.L. Stafford. Financial support for J.G. Pemberton was provided by the Province of Alberta Queen Elizabeth II Graduate Scholarship, NSERC Alexander Graham Bell Canada Graduate Scholarship (CGS M), and the University of Alberta as a recipient of the Dr. Richard E. Peter Memorial Scholarship, Walter H. Johns Graduate Fellowship and Department of Biological Sciences Teaching Assistantship. Y. Yu was a recipient of a F.S. Chia PhD Recruitment Scholarship from the University of Alberta. We thank Alan Mar and Herman Cortes for their assistance with this study. Keywords: fura2 fluorescence calcium imaging, gonadotropes and somatotropes, gonodotropin-releasing hormone, luteinizing and growth hormone secretion, Phosphoinositide 3-kinase, wortmannin and LY294002 Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Intracellular signaling Citation: Pemberton JG, Stafford JL, Yu Y and Chang JP (2011). Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus.. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00079 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Prof. John P Chang, University of Alberta, Biological Sciences, Edmonton, Alberta, T6G2E9, Canada, john.chang@ualberta.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joshua G Pemberton James L Stafford Yi Yu John P Chang Google Joshua G Pemberton James L Stafford Yi Yu John P Chang Google Scholar Joshua G Pemberton James L Stafford Yi Yu John P Chang PubMed Joshua G Pemberton James L Stafford Yi Yu John P Chang Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Phosphoinositide-3-Kinase Catalytic Subunit Inhibition Does Not Produce Synthetic Lethality in Estrogen Receptor Positive Breast Cancer Cells with Acquired Resistance to Estrogen Deprivation.

Abstract Background: Several phosphoinositide-3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical development. However, targeting strategies for PI3K catalytic subunits for treatment in estrogen receptor positive (ER+) disease have not been fully developed. We have recently reported that targeted inhibition of the two major PI3K catalytic subunits, p110α and p110β, using RNA interference (RNAi) induces apoptosis in short term estrogen-deprived ER+ breast cancer cells. Similarly, treatment with BEZ235, a clinical grade p110α/mammalian target of rapamycin (mTOR)-selective inhibitor, also promoted apoptosis in estrogen-deprived ER+ breast cancer cells. Importantly, we found that estradiol significantly suppressed death in ER+ cells induced by inhibition of PI3K catalytic subunits through both RNA interference and BEZ235. In contrast estrogen deprivation or PI3K inhibition individually caused little or no increase in apoptosis, indicating that there is a synthetic lethal interaction with estrogen deprivation/PI3K inhibitor combination treatment in ER+ breast cancer. These data suggest that combining estrogen deprivation with PI3K inhibition may improve clinical outcome by eradicating ER+ cells through increased tumor cell kill. We have extended these studies in order to determine the effect of PI3K catalytic subunit inhibition in long-term estrogen deprived (LTED) cells, a model for acquired resistance to endocrine therapy.Methods: LTED sub-lines of the ER+ MCF7 and T47D breast cancer cell lines were derived by depriving cells of estrogen for 6–9 months in phenol red-free medium containing charcoal-stripped serum after which time cells grow readily without supplemental estrogen. The effects of PI3K inhibition using RNAi and treatment with BEZ235 or BGT226, a p110α-selective inhibitor, were compared in short-term estrogen deprived MCF7 and T47D cells and their LTED derivatives by measuring effects on cell signaling, growth and apoptosis. Cell signaling was evaluated through western blotting, cell growth was measured using resazurin reduction and apoptosis was quantified by flow cytometry using the TUNEL assay.Results: PI3K inhibition using p110α RNAi or treatment with BEZ235 inhibited the growth of MCF7 LTED cells in the absence of supplemental estrogen. However, p110α RNAi or treatment with BEZ235 or the clinical grade p110α-selective inhibitor BGT226 failed to significantly induce apoptosis in MCF7 LTED cells. The effects of PI3K inhibition in T47D LTED cells are under investigation and will be presented.Conclusions: These results suggest that that the efficacy of PI3K inhibitors will be blunted after the onset of acquired resistance to estrogen deprivation through treatment with aromatase inhibitors and that a combined ER/PI3K targeting strategy in ER+ breast cancer treatment should be employed in the endocrine therapy sensitive phase of the disease. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3131.

Effects of PI3K inhibition and low docosahexaenoic acid on cognition and behavior

Alterations in two components of the brain's insulin signaling pathway, docosahexaenoic acid (DHA) content and phosphoinositide 3-kinase (PI3K) activity, have been implicated in the insulin resistance that is central to type II diabetes mellitus (DM). A 2- to 3-fold increased risk of developing Alzheimer's disease (AD) in patients with type II DM suggests a potential link between cognition and insulin action. The current study was designed to examine the impact of DHA dietary content and PI3K activity on learning, memory, depression, and anxiety in rodents. Mice were divided into the following groups: (1) control diet and vehicle injection (control PI3K), (2) control diet and wortmannin injection (PI3K inhibition), (3) low DHA diet and vehicle, and (4) low DHA diet and wortmannin. Each group was assessed for effects on activity, cognition, depression, and anxiety. Concentrations of glucose and insulin in plasma were quantified to confirm insulin resistance. Results showed significant increases in depression, anxiety, plasma insulin and glucose, and significant decreases in activity in wortmannin-treated mice regardless of diet. The control diet/wortmannin-treated group showed a significant decrease in memory compared to all other groups. The low DHA diet/wortmannin-treated group had slightly improved memory and lower levels of depression compared to the control diet/wortmannin-treated group. Results of the present study suggest that inhibition of PI3K decreases activity and memory while increasing insulin resistance, depression, and anxiety. In addition, these results suggest a possible compensatory role of low DHA in decreasing the effects of dysfunctional PI3K in AD associated cognitive decline and depression.