PI3K Regulatory Subunit P85 Research Articles

GRK2-mediated AKT activation controls cell cycle progression and G2 checkpoint in a p53-dependent manner

Cell cycle checkpoints, activated by stressful events, halt the cell cycle progression, and prevent the transmission of damaged DNA. These checkpoints prompt cell repair but also trigger cell death if damage persists. Decision-making between these responses is multifactorial and context-dependent, with the tumor suppressor p53 playing a central role. In many tumor cells, p53 alterations lead to G1/S checkpoint loss and the weakening of the G2 checkpoint, rendering cell viability dependent on the strength of the latter through mechanisms not fully characterized. Cells with a strong pro-survival drive can evade cell death despite substantial DNA lesions. Deciphering the integration of survival pathways with p53-dependent and -independent mechanisms governing the G2/M transition is crucial for understanding G2 arrest functionality and predicting tumor cell response to chemotherapy. The serine/threonine kinase GRK2 emerges as a signaling node in cell cycle modulation. In cycling cells, but not in G2 checkpoint-arrested cells, GRK2 protein levels decline during G2/M transition through a process triggered by CDK2-dependent phosphorylation of GRK2 at the S670 residue and Mdm2 ubiquitination. We report now that this downmodulation in G2 prevents the unscheduled activation of the PI3K/AKT pathway, allowing cells to progress into mitosis. Conversely, higher GRK2 levels lead to tyrosine phosphorylation by the kinase c-Abl, promoting the direct association of GRK2 with the p85 regulatory subunit of PI3K and AKT activation in a GRK2 catalytic-independent manner. Hyperactivation of AKT is conditioned by p53’s scaffolding function, triggering FOXO3a phosphorylation, impaired Cyclin B1 accumulation, and CDK1 activation, causing a G2/M transition delay. Upon G2 checkpoint activation, GRK2 potentiates early arrest independently of p53 through AKT activation. However, its ability to overcome the G2 checkpoint in viable conditions depends on p53. Our results suggest that integrating the GRK2/PI3K/AKT axis with non-canonical functions of p53 might confer a survival advantage to tumor cells.

Abstract 2030: N-cadherin-mediated activation of PI3K/Akt pathway following application of tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) therapy is FDA-approved for the treatment of glioblastoma and unresectable pleural mesothelioma, and has recently demonstrated benefit (together with an immune checkpoint inhibitor) for the treatment of metastatic non-small cell lung carcinoma (NSCLC) progressing on or after platinum-based therapy. The current study aimed to uncover cellular response mechanisms to TTFields. Methods: A2780 ovarian cancer cells and H1299 NSCLC cells were treated with TTFields for 72 h (200 and 150 kHz, respectively; 1.7 V/cm RMS). Changes in signaling pathways were analyzed using the Luminex multiplex assay and validated by protein expression. Fluorescence microscopy, calcium switch assay, antibody neutralization assay, and immunoprecipitation were employed to investigate the mechanism of action. The efficacy of concomitant treatment with TTFields and a potential inhibitor was tested in cell lines (cytotoxic and clonogenic effects) and two orthotopic mouse models: MOSE-L-FFL ovarian cancer and LL/2 lung cancer. Immunohistochemistry was performed on tumor sections from both models. Results: Increased AKT phosphorylation was observed in the cancer cells treated with TTFields. N-cadherin, known to be involved in the activation of the PI3K/AKT pathway, was elevated in the membranes of cells treated with TTFields. Elimination of calcium ions required for N-cadherin homophilic ligation abrogated TTFields-induced AKT phosphorylation, whereas calcium supplementation restored AKT phosphorylation. Inhibition of N-cadherin-mediated cell-cell contacts by neutralizing antibody resulted in a significant reduction in TTFields-induced AKT phosphorylation. Pull-down assays with an anti-N-cadherin antibody demonstrated increased recruitment of the p85 regulatory subunit of PI3K to N-cadherin complexes following TTFields application. TTFields-induced activation of AKT could be mitigated in cell cultures as well as in animal models by application of the PI3K inhibitor alpelisib. Co-treatment with alpelisib also enhanced the effectiveness of TTFields in vitro and in vivo. Conclusions: The PI3K/AKT signaling pathway, activated via cell-cell N-cadherin interactions, plays a role in the cancer cell response to TTFields treatment. Inhibiting the PI3K/AKT pathway may potentially sensitize tumors to TTFields. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Kerem Wainer-Katsir, Alexandra Volodin, Bella Koltun, Boris Brant, Yiftah Barsheshet, Tal Kan, Cfir David, Tharwat Haj Khalil, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. N-cadherin-mediated activation of PI3K/Akt pathway following application of tumor treating fields (TTFields) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2030.

Abstract B023: Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion

Abstract New therapies targeting metastatic spread are greatly needed to improve breast cancer patient survival. Recently, obscurin, an ~800kDa protein localized to the cell membrane, has risen to the spotlight as an overlooked metastasis suppressor commonly lost in breast cancer. Specifically, breast epithelial cells lacking obscurin undergo epithelial-mesenchymal transition (EMT) and demonstrate increased migration and invasion. Biochemical studies in our lab have linked breast epithelial obscurin loss to increased activation of the pro-metastatic PI3K/Akt signaling pathway. Mechanistically, the obscurin pleckstrin homology (PH)-domain directly binds the SH3-domain of the p85 regulatory subunit of PI3K. Thus far, due to obscurin’s massive size, gain of function/rescue experiments of obscurin have not been feasible. To combat this challenge, we have generated a mini-obscurin protein construct composed of an obscurin core functional unit: a myristilated obscurin PH-domain (Myr-PH). We hypothesized that expression of Myr-PH in obscurin-low breast cancer cells will reduce PI3K/Akt pathway activity and suppress metastasis. Remarkably, we report that ectopic expression of Myr-PH in the obscurin-low MDA-MB-231 (triple negative) and SKBr3 (HER2 positive) breast cancer cell lines diminishes PI3K/Akt pathway activity. Furthermore, Myr-PH sequesters p85 to the cell membrane, anchoring actin at the cell periphery. Functionally, we demonstrate that expression of Myr-PH in both cell line receptor subtypes: 1) reduces cellular migration collectively through decreased scratch assay wound closure and decreased transwell assay 8um pore migration or at the single cell level, through reduced 3um diameter microchannel migration, 2) diminishes cell seeding on multiple extracellular matrix (ECM) substrates known to populate metastatic microenvironments, and 3) decreases cell dissemination and tumorsphere invasion into a 3D collagen-1 matrix. Underlying these functional changes, we show that Myr-PH inhibits actin-dependent filopodia and invadopodia formation and decreases the expression of downstream PI3K/Akt target matrix metalloproteinases, all of which are potent drivers of metastatic dissemination. To this end, we are currently delivering the obscurin-PH domain in animals via lipid nanoparticles, aiming to examine its anti-metastatic efficacy in vivo. If successful, this work would pioneer the first gene therapy-based, targeted use of a single metastasis suppressor protein domain to block PI3K signaling and inhibit breast cancer metastasis. Citation Format: Matthew K Eason, Se-Jong Lee, Poornima Dubey, Anthony Kim, Konstantinos Konstantopoulos, Aikaterini Kontrogianni-Konstantopoulos. Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B023.

Whole-genome CRISPR screening identifies PI3K/AKT as a downstream component of the oncogenic GNAQ–focal adhesion kinase signaling circuitry

G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies.

ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryos.

ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.

Disordered Region in PI3K Regulatory Subunit p85 Drives Clathrin‐dependent Endocytosis and Regulates Cell Motility

Class 1A PI3Ks are lipid kinases composed of catalytic subunit p110 and regulatory subunit p85. Besides its catalytic activity of PI(3,4,5)P3 production, PI3K has been implicated in the process of receptor endocytosis. However, it remained unclear if and how directly class 1A PI3K engages endocytosis machinery. We found that a disordered region in the inter-SH2 (iSH2) domain of regulatory subunit p85 has binding motifs for AP-2, an adaptor protein complex for clathrin, and its plasma membrane targeting drives clathrin-mediated endocytosis. Remarkably, this endocytosis induction is independent of lipid kinase activity. Mutation of the endocytosis motifs in p85 caused its focal adhesion accumulation and increased random migration of mouse embryonic fibroblasts (MEFs). This newly discovered aspect of p85 would be a central piece to comprehensively understand the regulation of PI3K and its effect on cell dynamics.

Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling.

Chronic obstructive pulmonary disease (COPD) is a severe public health threat world-wide. Cigarette smoke (CS)-induced airway epithelial cell death is a major pathway of pathogenesis in emphysema, a subtype of COPD. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that catalyzes mono- and di-methylation on arginine residues within histone and non-histone proteins to modulate a variety of life processes, such as apoptosis. However, its role in CS-induced lung epithelial death has not been fully elucidated. Here we report that PRMT6 was decreased in mouse lung tissues from a cigarette smoke extract (CSE)-mediated experimental emphysematous model and in CSE treated or cigarette smoke exposed lung epithelial cells. Depletion of PRMT6 increased the protein levels of phosphatase PTEN and PI3K regulatory subunit p85 but decreased a downstream kinase PDK1, resulting in AKT dephosphorylation and thereafter, lung epithelial cell death. Knockout of PRMT6 inhibited epithelial survival and promoted CSE-mediated epithelial cell death, while ectopic expression of PRMT6 protein partially reversed epithelial cell death via PI3K/AKT-mediated cell survival signaling in CSE cellular models. These findings demonstrate that PRMT6 plays a crucial role in CS-induced bronchial epithelial cell death that may be a potential therapeutic target against the airway cell death in CS-induced COPD.

Abstract GS6-06: A neoadjuvant trial with letrozole identifies PRR11 in the 17q23 amplicon as a mechanism of resistance to endocrine therapy in ER-positive breast cancer

Abstract Although the 17q23 amplicon has been associated with luminal B breast cancer (BC) and high risk of recurrence, a specific gene or genes in this region that would be causal to endocrine resistance have not yet been uncovered. We performed whole transcriptome analysis on RNA extracted from 58 estrogen receptor (ER)+ BCs treated with neoadjuvant letrozole for median 7.2 months. PRR11 (Proline rich 11), located in 17q23, was upregulated in non-responding tumors as defined by relapse after a median follow up of 5 years and/or a preoperative endocrine prognostic index (PEPI) ≥4. Differential gene expression analysis between tumors expressing low vs high PRR11 mRNA showed that BC signatures associated with proliferation, IGF-1 and PI3K signaling were enriched in tumors with high PRR11 expression. Rate of PRR11 amplification is 15.2% in the Metastatic Breast Cancer project, but 9.5% and 9.4% in METABRIC and The Cancer Genome Atlas (TCGA), respectively. Gene Set Enrichment Analysis revealed an enrichment of hallmark gene sets associated with proliferation in PRR11-amplified ER+ BCs in METABRIC and TCGA. Integrated analysis of gene expression with on-treatment Ki67 levels from three independent studies with operable ER+ BCs treated with neoadjuvant aromatase inhibitor (ACOSOG-Z1031, NCT00651976, Llombart-Cussac et al.) showed that PRR11 was the only gene in 17q23 with a significant correlation with a high Ki67 levels across all studies. PRR11 knockdown inhibited E2-independent growth of HCC1428 LTED (long-term estrogen deprived) and MCF7 LTED cells in culture and MCF7 xenografts. PRR11 siRNA also inhibited growth of fulvestrant-resistant and tamoxifen-resistant MCF7 cells. Conversely, PRR11 transduction induced MDA-MB-134VI cell growth under estrogen-depleted conditions. Using a PCR array with 84-cell cycle genes, we identified SKP2, CDKN1A, CCNB2, CCNA2, CKS2 and CCNB1 as genes downregulated by PRR11 knockdown. Except for SKP2 and CDKN1A, expression of all those genes was elevated in PRR11-amplifiedER+ BCs in TCGA and METABRIC. Suggesting a link to activation of PI3K signaling, we found the proline-rich motif of PRR11 associates with the SH3 domain of the p85 regulatory subunit of PI3K. We hypothesized that this association suppresses p85 homodimer formation, thus facilitating binding of PI3Kα (p110α)-p85 dimers to IRS1, retention of p110α at the plasma membrane and, hence, activation of PI3K/AKT. To test this, we co-transfected HEK293T cells with HA-p85 and FLAG-p85. Forced expression of PRR11 reduced HA-p85 and FLAG-p85 homodimers as shown by HA and FLAG pulldowns followed by FLAG and HA immunoblots, respectively. PRR11 overexpression enhanced insulin-stimulated association of IRS1 to p110α and activation of AKT. PRR11 knockdown reduced insulin/IGF-1/2-stimulated p-AKT. In METABRIC and TCGA, PRR11 amplification and PIK3CA mutations are exclusive of each other, suggesting these alterations would be functionally linked with the same pathway. Connectivity map analysis with the list of genes significantly overexpressed in ER+/PRR11-amplified BCs predicted PI3K inhibitors as perturbations that suppress such gene list. In the MGH/Sanger dataset, PRR11-amplified BC cell lines displayed significantly higher sensitivity to the pan-PI3K inhibitor pictilisib compared to cell lines without PRR11 amplification. Finally, inhibition of PI3Kα by siRNA or alpelisib abrogated E2-independent growth and insulin-stimulated growth of PRR11-transduced MDA-MB-134VI and MCF10A cells, respectively, suggesting p110α is required for the growth promoting effects of PRR11. These data suggest that 1) PRR11 is a mediator of resistance to antiestrogens via amplification of PI3K/AKT signaling, and 2) PI3Kα is a potential therapeutic target in ER+ BCs harboring PRR11 amplification. Citation Format: Kyung-min Lee, Angel Guerrero-Zotano, Ariella Hanker, Alberto Servetto, Dhivya Sudhan, Luigi Formisano, Valerie Jansen, Paula González-Ericsson, Melinda Sanders, Thomas Stricker, Lewis Cantley, Carlos Arteaga. A neoadjuvant trial with letrozole identifies PRR11 in the 17q23 amplicon as a mechanism of resistance to endocrine therapy in ER-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS6-06.

Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway.

Nerves infiltrate the tumor microenvironment and stimulate the growth of cancer cells through the secretion of neurotransmitters. However, the contributions of nerves to the self-renewal capacity of cancer stem cells (CSCs) remain largely unknown. In this study, we found that CD133+cancer cells were responsible for the initiation of thyroid cancer. Neurons were juxtaposed with CD133+cells in thyroid cancer tissues. Acetylcholine, one of the most abundant neurotransmitters, promoted CD133 Y828 phosphorylation, and subsequently increased the interaction between CD133 and PI3K regulatory subunit p85, resulting in the activation of the PI3K-Akt pathway. Acetylcholine increased the self-renewal ability of CD133+thyroid cancer cells through activation of CD133-Akt pathway. Furthermore, acetylcholine promoted the expression of the immune regulator PD-L1 through the activation of the CD133-Akt pathway, resulting in the resistance of CD133+thyroid cancer cells to CD8+ T cells. However, acetylcholine receptor antagonist 4-DAMP blocked the positive effects of acetylcholine on the self-renewal and immune escape of CD133+thyroid cancer cells. Taken together, these data suggest that acetylcholine increases the self-renewal and immune escape abilities of CD133+thyroid cancer cells through the activation of the CD133-Akt pathway.

The Small GTPase ARF6 Activates PI3K in Melanoma to Induce a Prometastatic State.

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. SIGNIFICANCE: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2892/F1.large.jpg.

Abstract PD7-10: Neoadjuvant trial with letrozole identifies PRR11 in 17q21-23 amplicon as a resistance mechanism to endocrine therapy in ER-positive breast cancer

Abstract Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy relapse with metastatic disease. Previously, we identified 3 amplicons (11q11.3, 8p11.23, and 17q21-23) associated with endocrine-resistance (Giltnane et al. Sci Transl Med 2017). The 17q21-23 amplicon has been associated with highly proliferative luminal B tumors and cancers with high genomic instability. A causal role of this region in endocrine resistance is unclear. We performed whole transcriptome analysis on RNA extracted from 58 ER+ breast cancers of patients treated with letrozole for 5.4-9.2 months (median 7.2 months). PRR11 (Proline rich 11), located in 17q21-23, was significantly upregulated in non-responding tumors as defined by cancer relapse after a median follow up of 5 years and/or a preoperative endocrine prognostic index (PEPI) ≥4. Differential gene expression analysis between tumors expressing low vs high PRR11 mRNA showed that BC signatures associated with proliferation, cell cycle, IGF-1 and PI3K signaling were enriched in tumors with high PRR11 expression. In the Metastatic Breast Cancer Project and TCGA, PRR11 amplification was higher in metastatic vs. primary BCs (16.5% and 8.5%, respectively; Fisher's p=0.0088). Gene Set Enrichment Analysis of mRNA expression in METABRIC and TCGA revealed significant enrichment of hallmark gene sets associated with proliferation in PRR11 amplified ER+ BCs. Genome-scale RNAi screening in Project Achilles showed that among all genes in the 17q21-23 amplicon, PRR11 knockdown results in the 4th strongest anti-proliferative effect in MCF7 cells. PRR11 knockdown with siRNA inhibited proliferation, cell cycle progression, and RB phosphorylation in HCC1428 LTED (long-term estrogen deprived), MCF7 LTED, and fulvestrant-resistant MCF7 cells. Using a PCR array with 84-cell cycle genes, we identified SKP2, CDKN1A, CCNB2, CCNA2, CKS2 and CCNB1 as genes downregulated by PRR11 knockdown. Except for CDKN1A, expression of all those genes was elevated ER+ BCs with PRR11 gain or amplification in TCGA. PRR11 associates with the p85 regulatory subunit of PI3K via its SH3 domain. We speculated this association would suppress p85 homodimers, thus permitting binding of PI3Kα (p110α)-p85 dimers to IRS1 and, hence, activating PI3K/AKT. To test this, we co-transfected HEK293T cells with HA-p85 and FLAG-p85. Transfection of PRR11 into these HEK293T cells reduced HA-p85 and FLAG-p85 homodimers as shown by HA and FLAG pulldowns followed by FLAG and HA immunoblots, respectively. Finally, PRR11 knockdown resulted in a reduction of p110a and S473 P-AKT levels and inhibition of IGF-1/2 stimulated P-AKT. Not inconsistent with these data, PRR11 amplification and PIK3CA mutations in METABRIC and TCGA are exclusive of each other, suggesting these alterations are functionally linked with the same signaling pathway. These data support a role of PRR11 in PI3K/AKT activation that may be causal to resistance to estrogen deprivation. We propose PRR11, located in the 17q21-23 amplicon, is a potential mediator of resistance to antiestrogen therapy by amplifying PI3K/AKT signaling, suggesting PI3K may be a therapeutic target in ER+ BCs harboring PRR11 amplification. Citation Format: Lee K-M, Guerrero-Zotano A, Formisano L, Jansen V, Gonzalez Ericsson P, Arteaga C. Neoadjuvant trial with letrozole identifies PRR11 in 17q21-23 amplicon as a resistance mechanism to endocrine therapy in ER-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-10.

Phosphorylation of PI3K regulatory subunit p85 contributes to resistance against PI3K inhibitors in radioresistant head and neck cancer

Objectives: PI3K/Akt/mTOR pathway is commonly activated in most cancers and is correlated with resistance to anticancer therapies such as radiotherapy. Therefore, PI3K is an attractive target for treating PI3K-associated cancers. Material and Methods: We investigated the basal expression and the expression after treatment of PI3K inhibitor or Src inhibitor of PI3K/Akt pathway-related proteins in AMC-HN3, AMC-HN3R, HN30 and HN31 cells by performing immunoblotting analysis. The sensitivity to PI3K inhibitors or Src inhibitor was analyzed by MTT assay and clonogenic assay. To determine the antitumoral activity of combination treatment with PI3K inhibitor and Src inhibitor, we used using xenograft mouse model. Results: We found that PI3K regulatory subunit p85 was predominantly phosphorylated in radioresistant head and neck cancer cell line (HN31), which showed resistance to PI3K inhibitors. Next, we investigated mechanism through which PI3K p85 phosphorylation modulated response to PI3K inhibitors. Of note, constitutive activation of Src was found in HN31 cells and upon PI3K inhibitor treatment, restoration of p-Src was occurred. Src inhibitor improved the efficacy of PI3K inhibitor treatment and suppressed the reactivation of both Src and PI3K p85 in HN31 cells. Furthermore, downregulation of PI3K p85 expression by using a specific siRNA suppressed Src phosphorylation. Conclusions: Together, our results imply the novel role of the PI3K regulatory subunit p85 in the development of resistance to PI3K inhibitors and suggest the presence of a regulatory loop between PI3K p85 and Src in radioresistant head and neck cancers with constitutively active PI3K/Akt pathway.

Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes.

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease.

The quinic acid derivative KZ-41 prevents glucose-induced caspase-3 activation in retinal endothelial cells through an IGF-1 receptor dependent mechanism.

Retinal microaneurysms, an early disease manifestation of diabetic retinopathy, are associated with retinal endothelial cell (REC) death and macular edema. We previously demonstrated that a quinic acid (QA) analog, KZ-41, promoted REC survival by blunting stress-induced p38 MAPK activation. Herein, we sought to expand our understanding of the pro-survival signal transduction pathways actuated by KZ-41. Using human RECs exposed to high glucose (25 mM, 72 hours), we demonstrated that KZ-41 blocks caspase-3 activation by triggering phosphorylation of the PI3K regulatory subunit (p85; Tyr458) and its downstream target Akt (Ser473). Akt signal transduction was accompanied by autophosphorylation of the receptor tyrosine kinase, insulin growth factor-1 receptor (IGF-1R). IGF-1R knockdown using either the tyrosine kinase inhibitor AG1024 or silencing RNA abolished KZ-41’s pro-survival effect. Under high glucose stress, caspase-3 activation correlated with elevated ERK1/2 phosphorylation and decreased insulin receptor substrate-1 (IRS-1) levels. KZ-41 decreased ERK1/2 phosphorylation and reversed the glucose-dependent reduction in IRS-1. To gain insight into the mechanistic basis for IGF-1R activation by KZ-41, we used molecular modeling and docking simulations to explore a possible protein:ligand interaction between the IGF-1R kinase domain and KZ-41. Computational investigations suggest two possible KZ-41 binding sites within the kinase domain: a region with high homology to the insulin receptor contains one potential allosteric binding site, and another potential site on the other side of the kinase domain, near the hinge domain. These data, together with previous proof-of-concept efficacy studies demonstrating KZ-41 mitigates pathologic retinal neovascularization in the murine oxygen-induced retinopathy model, suggests that QA derivatives may offer therapeutic benefit in ischemic retinopathies.

Abstract 1772: PIK3CA C2 domain deletions hyperactivate PI3K, generate oncogene dependence and are exquisitely sensitive to PI3Kα inhibitors

Abstract Background: A 63 year old postmenopausal woman with advanced ER+ breast cancer resistant to endocrine therapy exhibited an exceptional response to the PI3Kα inhibitor alpelisib (BYL719) and the aromatase inhibitor letrozole (Mayer et al. Clin Cancer Res 2016). Targeted capture next generation sequence (NGS) of DNA from a liver metastasis identified a P447_L455 deletion in the C2 domain of PIK3CA. About 80% of PIK3CA activating mutations are in ‘hot spots’ within the helical and kinase domains. C2 domain mutations make up ~10% of all PIK3CA mutations in breast cancer (TCGA, Foundation Medicine) and are frequently not reported by tumor and plasma cell-free DNA NGS panels. Deletions and mutations in this domain cluster in a region encompassing amino acids 446-460 of PIK3CA. We investigated herein the functional role of PIK3CA C2 domain mutations and their response to PI3K inhibitors. Methods: V5-tagged lentiviral vectors encoding wild type, delP447-L455, and delH450-P458 were stably transduced into MCF10A non-tumorigenic human breast epithelial cells. Cell viability and acini formation in 3D Matrigel were examined in media ± EGF or insulin or alpelisib. Markers of PI3K activation were examined by immunoblot and phosphoflow analysis. The Rosetta software suite was used to construct a structural model that would predict the change in stability of the p85/p110α complex. Physical association of p85α and p110α was determined by precipitation with V5 antibodies and immunoblot analysis. Results: MCF10A cells stably expressing V5-tagged PIK3CAdelP447-L455 and PIK3CAdelH450-P458 exhibited EGF- and insulin-independent growth and higher phosphorylation of AKT, ERK and S6 when compared to parental MCF10A cells. In 3D Matrigel, MCF10A cells with PIK3CA C2 domain deletions formed invasive acini with increased protrusions, spindling, and bridging between acini. All these changes were ablated upon the addition of 1 µM of alpelisib whereas parental MCF10A cells were unaffected by alpelisib. We hypothesized that delP447-L455 would reduce the binding affinity of p110α with the p85 regulatory subunit of PI3K. A structural model of PIK3CAdelP447-L455 in the context of the regulatory complex revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Consistent with this structural analysis, coimmunoprecipitation of p85 with V5 antibodies showed reduced binding of the C2 domain deletion mutants with p85 compared to wild type p110α. Conclusions: These data suggest that C2 domain deletions in PIK3CA are activating mutations and generate oncogene dependence. As a result, tumors expressing these mutations are exquisitely sensitive to PI3Kα inhibitors. Thus, in addition to PIK3CA ‘hot spot’ mutations, C2 domain mutations should also be considered biomarkers of sensitivity to PI3K inhibitors. Citation Format: Sarah Croessmann, Jonathan Sheehan, Gregory Sliwoski, Nalin Leelatian, Jie He, Rebecca Nagy, Justin M. Balko, Ingrid A. Mayer, Richard B. Lanman, Vincent Miller, Lewis C. Cantley, Jonathan M. Irish, Jens Meiler, Carlos L. Arteaga. PIK3CA C2 domain deletions hyperactivate PI3K, generate oncogene dependence and are exquisitely sensitive to PI3Kα inhibitors [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 1772. doi:10.1158/1538-7445.AM2017-1772

Cytokine Receptor Endocytosis: New Kinase Activity-Dependent and -Independent Roles of PI3K.

Type I and II cytokine receptors are cell surface sensors that bind cytokines in the extracellular environment and initiate intracellular signaling to control processes such as hematopoiesis, immune function, and cellular growth and development. One key mechanism that regulates signaling from cytokine receptors is through receptor endocytosis. In this mini-review, we describe recent advances in endocytic regulations of cytokine receptors, focusing on new paradigms by which PI3K controls receptor endocytosis through both kinase activity-dependent and -independent mechanisms. These advances underscore the notion that the p85 regulatory subunit of PI3K has functions beyond regulating PI3K kinase activity, and that PI3K plays both positive and negative roles in receptor signaling. On the one hand, the PI3K/Akt pathway controls various aspects downstream of cytokine receptors. On the other hand, it stimulates receptor endocytosis and downregulation, thus contributing to signaling attenuation.

Abstract 989: A novel function of LncRNA AK023948—regulation of PI3K signaling

Abstract The human genome expresses a large number of long non-coding RNAs (lncRNAs) and increasing evidences suggest their physiological functions.However, the roles vast majority of lncRNAs are elusive in breast cancer. In the present study, we identified a set of lncRNAs that were differentially expressed in breast cancer tissues as compared to normal tissue. AK023948 is one of such lncRNAs which is up-regulated in breast cancer tissue and in breast cancer cell lines. Ectopic expression of AK023948 in breast cancer MCF-7 cells promotes proliferation and anchorage independent cell growth along with activation of AKT phosphorylation. On the other hand, knockdown or knockout of AK023948 in MCF-7 cells reduces the proliferation of cells and phosphorylation of AKT. Furthermore, there is a positive correlation between AK023948 and pAKT expression in breast cancer tissue microarray. To determine the underlying mechanism of AK023948-mediated activation of AKT, we performed RNA precipitation assays using biotin-labeled AK023948 RNA probe combined with mass spectrometry analysis and identified that ATP dependent RNA helicase A (RHA/DHX9) is AK023948. The interaction of AK023948 with DHX9 was confirmed by Western blot and RNA immunoprecipitation (IP), respectively. Co-IP indicated that DHX9 interacts with PI3K regulatory subunit p85. Further characterization revealed that AK023948 works as scaffold for p85-DHX9 interaction, which is essential for stability of p85 and AKT activity. Finally, we showed that AK023948 is essential to AKT activation induced by growth factors and acidosis, but it has no effect on activation of ERK phosphorylation, suggesting that AK023948 specifically regulate AKT signaling via PI3K. Together, these results suggest that AK023948 contributes to breast cancer pathogenesis by activating AKT and AK023948 may serve as a novel target for breast cancer therapy. Citation Format: Pratirodh Koirala, Yin Yuan Mo. A novel function of LncRNA AK023948—regulation of PI3K signaling. [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 989.

The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity.

Growth of noninjured axons in the adult nervous system contributes to adaptive and maladaptive plasticity, and dysfunction of this process may contribute to neurologic pathologies. Functional screening of genes regulated during growth of noninjured axons revealed CD2AP as a positive regulator of axon outgrowth. A novel association of CD2AP with TrkA and p85 suggests a distinct intracellular signaling pathway regulating growth of noninjured axons. This may also represent a novel mechanism of generating specificity in multifunctional NGF signaling. Divergent regulation of CD2AP in different axon growth conditions suggests that separate mechanisms exist for different modes of axon growth. CD2AP is the first signaling molecule associated with adult sensory axonal collateral sprouting, and this association may offer new insights for NGF/TrkA-related Alzheimer's disease mechanisms.

Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth.

The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with β-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and β-catenin and inhibited the activation of β-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies.

Dysregulated Macrophage Pro-Inflammatory Cytokine Production and Chronic Colitis in Mice Lacking Both Gab2 and Gab3

Macrophages are responsible for protecting the body against foreign invaders. We have been studying the role of Grb2-associated binding proteins (Gabs) in macrophage biology. In mice, Gabs are adaptor proteins that include three family members (Gab1, Gab2, and Gab3) that play critical regulatory roles in modulating cytokine receptor signaling. Gab2 knockout mice have no developmental defects but have impaired allergic responses, osteoclast defects, altered mast cell development, and altered hematopoiesis. Gab3 knockout mice have no defined phenotypes alone and although highly expressed in macrophages, a functional role was not found despite considerable focus on this cell type. Therefore, we set out to determine the combined role of Gab2 and Gab3 to determine whether they performed redundant functions not observable in single knockout mice. To analyze regulation of macrophage cytokine production, a Gab2/3 deficient mouse model was generated on the C57BL/6 background. Bone Marrow Derived Macrophages (BMDM) were expanded from the bone marrow (BM) of wild-type (WT), Gab2 and Gab3 single knockout and Gab2/3 knockout mice and found to similarly co-express CD11b and F4/80. However, Gab2/3 knockout BM produced only 30% of wild-type BMDM numbers. Despite reductions in BMDM absolute numbers, isolated BMDM demonstrated significant induction of pro-inflammatory cytokines TNF-α and IL-12 and anti-inflammatory cytokine IL-10 mRNA at baseline. Interestingly, after LPS stimulation (100ng/ml) we detected much greater induction of TNF-α and IL-12 mRNA and protein expression. Interestingly, despite increased IL-10 mRNA induction in Gab2/3 knockout BMDM, no IL-10 protein expression could be detected by Luminex assay. No changes were observed in production of interferon or STAT1 activation in these BMDM. Studies have shown that rapamycin treatment of macrophages suppresses mTORC1 and subsequently reduces IL-10 production and promotes pro-inflammatory cytokine production. Gab2 is known for its role in regulating the PI3K pathway through interactions with the p85 regulatory subunit of PI3K. Therefore, we also examined whether mTOR activation was effected by Gab2/3 deficiency causing altered cytokine expression. Deletion of Gab2/3 in BMDMs treated with LPS showed an inhibition of 4EBP1 phosphorylation and increased AKT phosphorylation. These results suggest that Gabs may play a critical role in modulating mTOR activation and potentially causing defects in protein translation that reflect in reduced IL-10 cytokine levels in Gab2/3 knockout cells. IL-10 has a critical immunoregulatory role that is dysregulated in patients with inflammatory bowel disease. IL-10 deficient mice develop colitis due to loss of mucosal immune tolerance. Strikingly, as early as two months of age in vivo 12/32 (37.5%) Gab2/3 knockout mice developed rectal prolapse and suffered from diarrhea within a six month period. Histological analysis of isolated colons using a scoring system confirmed spontaneous development of colitis in Gab2/3 knockout mice compared to no phenotypes observed in WT and single knockout controls. To determine whether the BM was directly involved in the disease, BM chimeras were generated using irradiated WT mice as recipients and Gab2/3 knockout mice as donors. Susceptible recipients receiving Gab2/3 knockout BM showed a more invasive colitis phenotype than the spontaneous disease and resulted in forced euthanization due to body weight decreases greater than 25%. Multiple ulcerations were present in most of the colon proximal region, with extensive epithelial damage, transmural inflammation, and in some mice adenocarcinoma. Notably, we did not observe adenocarcinoma in untransplanted Gab2/3 knockout mice, suggesting that epithelial deletion of Gab2/3 may suppress cancer whereas in the bone marrow chimera model, the epithelial cells are WT and can be transformed. Similar phenotypes were also observed in secondary transplant recipients. Lastly, treatment of Gab2/3 knockout mice with dextran-sodium-sulfate (DSS) induced rapid severe colitis that resulted in death of 80% and 40% of Gab2/3 knockout and WT mice respectively. Overall, these observations demonstrate a major redundant role for Gab2 and Gab3 in macrophage immune surveillance required for the prevention of colitis in mice. DisclosuresNo relevant conflicts of interest to declare.