Catalytic Subunit Of Phosphoinositide 3-kinase Research Articles

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12wk of age. Both sexes had smaller marrow areas from 6wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Establishment of a human ovarian clear cell carcinoma cell line mutant in PIK3CB but not PIK3CA

A human ovarian clear cell carcinoma cell line was established from a 46-year-old Japanese woman. That line, designated MTC-22, has proliferated continuously for over 6 months in conventional RPMI 1640 medium supplemented with 10% foetal bovine serum and has been passaged over 50 times. MTC-22 doubling-time is ~ 18 h, which is much shorter than most ovarian clear cell carcinoma lines reported to date. Morphologically, MTC-22 cells exhibit polygonal shapes and proliferate to form a monolayer in a jigsaw puzzle-like arrangement without contact inhibition. Ultrastructurally, cells exhibit numerous intracytoplasmic glycogen granules and well-developed mitochondria. G-band karyotype analysis indicated that cells have a complex karyotype close to tetraploid. We observed that the expression pattern of a series of ovarian carcinoma-related molecules in MTC-22 cells was identical to that seen in the patient’s tumour tissue. Notably, MTC-22 cells, and the patient’s carcinoma tissue, expressed low-sulphated keratan sulphate recognised by R-10G and 294-1B1 monoclonal antibodies, a hallmark of non-mucinous ovarian carcinoma, and particularly of clear cell ovarian carcinoma. Moreover, characteristic point mutations—one in ARID1A, which encodes the AT-rich interaction domain containing protein 1A, and the other in PIK3CB, which encodes the catalytic subunit of phosphoinositide 3-kinase—were seen in the patient’s tumour tissue and retained in MTC-22 cells. Collectively, these findings indicate that MTC-22 cells could serve as a valuable tool for investigating the pathophysiology of ovarian clear cell carcinoma, particularly that harbouring PIK3CB mutations, and for developing and validating new diagnostic and therapeutic approaches to this life-threatening malignancy.

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

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

Modulation of miR-146b by N6-methyladenosine modification remodels tumor-associated macrophages and enhances anti-PD-1 therapy in colorectal cancer

PurposeMicroRNA-146b (miR-146b) alleviates experimental colitis in mice by mediating macrophage polarization and the release of inflammatory factors. Our goals were to evaluate the antitumor efficacy of miR-146b in colorectal cancer (CRC) and to investigate the underlying mechanisms.MethodsWe used murine models of CRC to evaluate whether miR-146b influenced the progression of tumors independent of tumor-associated macrophages (TAMs). RNA immunoprecipitation, N6-methyladenosine (m6A) RNA immunoprecipitation and in vitro pri-miRNA processing assays were conducted to examine whether m6A mediates the maturation of pri-miR-146b/miR-146b. In a series of in vitro and in vivo experiments, we further defined the molecular mechanisms of methyltransferase-like 3 (METTL3)/miR-146b-mediated antitumor immunity and its efficacy in combination with anti-PD-1 immunotherapy.ResultsWe found that miR-146b deletion supported tumor progression by increasing the number of alternatively activated (M2) TAMs. Mechanistically, the m6A-related “writer” protein METTL3 and “reader” protein HNRNPA2B1 controlled miR-146b maturation by regulating the m6A modification region of pri-miR-146b. Furthermore, miR-146b deletion promoted the polarization of M2-TAMs by enhancing phosphoinositide 3-kinase (PI3K)/AKT signaling, and this effect was mediated by the class IA PI3K catalytic subunit p110β, which reduced T cell infiltration, aggravated immunosuppression and ultimately promoted tumor progression. METTL3 knockdown or miR-146b deletion induced programmed death ligand 1 (PD-L1) production via the p110β/PI3K/AKT pathway in TAMs and consequently augmented the antitumor activity of anti-PD-1 immunotherapy.ConclusionsThe maturation of pri-miR-146b is m6A-dependent, and miR-146b deletion-mediated TAM differentiation promotes the development of CRC by activating the PI3K/AKT pathway, which induces upregulation of PD-L1 expression, inhibits T cell infiltration into the TME and enhances the antitumor activity of anti-PD-1 immunotherapy. The findings reveal that targeting miR-146b can serve as an adjuvant to anti-PD-1 immunotherapy.Graphical

PIK3CB as a potential target to regulate chemosensitivity in glioblastoma.

e14051 Background: Glioblastoma (GBM) is the most common primary central nervous system malignancy but exhibits universally poor outcomes in part due to chemoresistance. Temozolomide (TMZ) can significantly improve overall survival, but resistance mechanisms limit its consistent efficacy. The phosphoinositide 3 kinase (PI3K) pathway is involved in multiple TMZ resistance mechanisms and is highly druggable; however, pan-inhibition of its four grossly homologous but functionally distinct catalytic subunits (PIK3CA/B/D/G encode p110α/β/δ/γ, respectively) was unsuccessful clinically. Therefore, by understanding how the individual PI3K subunits regulate TMZ sensitivity, a more selective treatment strategy can be developed to combat chemoresistance. Methods: Differential expression of PI3K subunit genes was assessed using RNA sequencing (RNAseq) results obtained from public databases (e.g., DepMap, The Cancer Tissue Genome Atlas) for GBM cell lines and patient tumors expressing high or low O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair enzyme highly implicated in TMZ resistance. PI3K protein levels were assessed using immunohistochemistry of gliomas available at The Human Protein Atlas (THPA). PI3K gene expression was correlated with PI3K pathway activation, quantified as protein kinase B phosphorylation (pAKT). PI3K mRNAs and pAKT were then correlated with the half maximal inhibitory concentrations (IC50s) of TMZ in GBM cell lines to evaluate for a relationship between PI3K gene expression, PI3K pathway activity, and TMZ sensitivity. Finally, hazard ratios were used to compare PI3K gene expression in patient tumors with survival and response to chemotherapy to determine if expression was predictive of prognosis and/or chemosensitivity. Results: PIK3CB was the only PI3K gene enriched in both GBM cell lines and tumors, demonstrating significantly higher expression than all other PI3K genes in GBM (P < 0.0001), independent of MGMT levels. PIK3CB expression remained the highest even after controlling for GBM risk factors such as tumor recurrence, isocitrate dehydrogenase (IDH) mutations, molecular subtypes, or patient gender/age differences. Levels of the p110β protein were also significantly higher than those of other PI3K catalytic subunits (P < 0.001). PIK3CB expression was positively correlated with pAKT levels (P < 0.05), but only in cell lines and tumors with low MGMT activity. Both PIK3CB expression and pAKT levels correlated positively with TMZ IC50s (P < 0.05) in MGMT-deficient GBMs, suggesting an inverse relationship between PIK3CB and TMZ sensitivity. Finally, better prognosis and significant survival benefit with chemotherapy were both associated with lower levels of PIK3CB in MGMT-deficient patients. Conclusions: PIK3CB may selectively regulate chemoresistance in GBM, suggesting a role for p110β selective inhibition to improve chemosensitivity.

Overcoming temozolomide resistance in glioblastoma through selective inhibition of phosphoinositide 3-kinase catalytic subunit.

e14045 Background: Glioblastoma is one of medicine's most difficult-to-treat cancers due to its inevitable resistance to chemotherapies, such as temozolomide (TMZ). Tumor resistance to TMZ often comes from overactivation of phosphoinositide 3-kinase (PI3K), making this enzyme an ideal anti-cancer drug target; however, in practice, non-selective targeting of PI3K has resulted in undesirable clinical outcomes. Therefore, it is necessary to explore each subunit of PI3K to determine their effect on chemoresistance in order to develop therapeutics to overcome said resistance. PI3K has four homologous yet functionally distinct catalytic subunits: p110α, p110β, p110δ, and p110γ. Preliminary in silico analysis of each subunit has demonstrated that p110β is more highly expressed in glioblastoma than the other subunits. Here we report that TMZ resistance in glioblastoma is driven primarily by activation of p110β. Methods: Glioblastoma cell lines with high p110β levels (SF295 and U87MG) and low p110β levels (A172 and LN229) were cultured. CRISPR-Cas9 was used to knockout p110α, p110β, or p110δ in cultured cell lines. The viability of cultured cells was measured using an MTS viability assay. Newly cultured wild-type cells were treated with IC50 doses of selective p110β inhibitor TGX-221 and TMZ. Mice bearing SF295 xenograft tumors were treated with DMSO, TGX-221, and/or TMZ. A Bliss model of independence was used to assess the association between TGX-221 and TMZ with synergistic and antagonistic patterns measured using excess over Bliss (EOB) scores. Results: There was a significant reduction in viability among SF295 p110β-knockout cells (p < 0.001) and an additive cytotoxic effect following treatment with TMZ. The LN229 p110β-knockout cells treated with TMZ did not reduce cell viability. Similarly, no substantial declines in viability were observed in the p110α- or p110δ-knockout cells before or after TMZ treatment in either cell line. In newly cultured wild-type p110β-high and p110β-low glioblastoma cell lines, TGX-221 or TMZ alone were insufficient to inhibit cell viability; however, when combined, TGX-221 and TMZ synergistically decreased cell survival with an EOB of 45.5% in U87MG cells and 26.3% in SF295 cells. No additive effects were detected in the p110β-low cell lines following TGX-221 and TMZ treatment. Additionally, TGX-221 combined with TMZ resulted in a drastic reduction in xenograft tumor growth, which was significantly greater than TGX-221 (p < 0.02) or TMZ (p < 0.003) treatment alone. Conclusions: Inhibition of p110β via CRISPR-Cas9 knockout or pharmaceutical blockade restores TMZ sensitivity in p110β-high glioblastoma cells and xenograft glioblastoma tumors. These results demonstrate the varying importance of PI3K catalytic subunits and necessitate further exploration into p110β as a potential drug target to overcome chemoresistance in glioblastoma.

The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.

IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitrobinding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.

Clinical and functional characterization of germline PIK3CA variants in patients with PIK3CA-related overgrowth spectrum disorders.

Mosaic variants in the PIK3CA gene, encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), produce constitutive PI3K activation, which causes PIK3CA-related overgrowth spectrum disorders. To date, fewer than 20 patients have been described with germline alterations in PIK3CA. In this study, we describe three unrelated individuals with overgrowth and germline PIK3CA variants. These variants were discovered through whole-exome sequencing and confirmed as germline by testing multiple tissue types, when available. Functional analysis using Patient 1's fibroblast cell line and two previously reported patients' cell lines showed increased phosphorylation of AKT during cellular starvation revealing constitutive activation of the phosphoinositide-3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway. Alternatively, stimulation of the cells by fetal bovine serum produced a reduced response, indicating an activated status of the PI3K complex reducing the pathway response to further external stimulation. Additional studies utilizing Biolog Phenotype Microarray technology indicated reduced energy production when cells were exposed to growth factors stimulating the PI3K/AKT/mTOR pathway, confirming the trend observed in the AKT phosphorylation test after stimulation. Furthermore, treatment with inhibitors of the PI3K/AKT/mTOR pathway rescued the normal energy response in the patients' cells. Collectively, these data demonstrate that disease-causing germline PIK3CA variants have a functional consequence, similar to mosaic variants in the PI3K/AKT/mTOR pathway.

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

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

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression.

Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.

Abstract 3243: Synergistic effects of connexin 43 inhibitor and PI3K isoform-selective inhibitors in temozolomide-resistant glioblastoma

Abstract Glioblastoma is the most common central nervous system cancer and is responsible for approximately one-half of all malignant brain tumors. With a 5-year survival rate of 6.8% and a median observed survival time of only 8 months, recurrent glioblastoma is among the world’s most lethal malignancies. Temozolomide (TMZ) has become a standard chemotherapeutic for glioblastoma due to its alkylating effects and ability to cross the blood-brain barrier. Unfortunately, many patients with glioblastoma develop resistance to TMZ, severely limiting therapeutic options. Recent studies have found a link between ectopic connexin 43 (Cx43) and TMZ-resistant glioblastoma. Further studies have shown success in overcoming this resistance by combining TMZ with alpha carboxyl terminus 1 (αCT1) peptide, a clinically tested Cx43 carboxyl terminus mimetic peptide inhibitor; however, because of the short half-life of αCT1, therapeutic potential is limited, and new approaches are needed. In this study, we hypothesized that simultaneously targeting Cx43 with αCT1 and phosphoinositide 3-kinase (PI3K) using PI3K isoform-selective inhibitors would synergistically sensitize glioblastoma to TMZ treatment because p110β, one of the four PI3K catalytic subunits, is a prime survival factor for TMZ-resistant glioblastoma. We cultured newly dissected primary glioblastoma cells, glioblastoma cell lines, and glioblastoma stem cells and dosed them with various combinations of αCT1, TMZ, and one of two clinically tested p110β inhibitors (TGX-221 or GSK2636771). Cell viability was measured using an MTS viability assay. Glioblastoma cells that expressed high levels of Cx43 and p110β experienced a significant decrease in viability when dosed with a triple combination. The additive properties of these combinations were measured using an excess over Bliss (EOB) score derived from Bliss independence modeling, which showed synergistic effects as high as 81% for triple therapy combinations in TMZ-resistant cell lines. The EOB was significantly higher in triple therapies than the double or single therapies. To support our findings, we used the same combinations in primary glioblastoma cells and measured the activity of cleaved caspase 3/7 using a luminescence assay. There was a synergistic increase in caspase 3/7 activity in the triple therapy that was not seen in any of the other combinations. Identical treatment in non-pathological human astrocytes suggests that our drug combinations do not exacerbate non-selective TMZ toxicity in the brain. Collectively, our data demonstrate that simultaneous inhibition of Cx43 and PI3K is an effective approach for overcoming TMZ resistance in glioblastoma. Citation Format: Kasen R. Hutchings, Kevin J. Pridham, Min Liu, Joseph Owens, Zhi Sheng. Synergistic effects of connexin 43 inhibitor and PI3K isoform-selective inhibitors in temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3243.

A 5’-tiRNA fragment that inhibits proliferation and migration of laryngeal squamous cell carcinoma by targeting PIK3CD

tRNA-derived small RNAs (tsRNAs) participate in several biological processes, including carcinogenesis. The correlations between tsRNAs and human cancers are attracting substantial attention. Nevertheless, the involvement of tsRNAs in laryngeal squamous cell carcinoma (LSCC) progression remains unclear. We constructed tsRNAs expression profiles in LSCC and adjacent normal tissues by next-generation sequencing. Interestingly, we identified a specific 5′-tiRNA fragment (tRF-33-Q1Q89P9L842205) that was significantly downregulated and was closely associated with lymph node metastasis and advanced stages of LSCC. Importantly, we found that tRF-33-Q1Q89P9L842205 suppressed cell growth, proliferation, migration, invasion and induced apoptosis in LSCC by directly silencing phosphoinositide 3-kinase catalytic subunit (PIK3CD). We speculated that tRF-33-Q1Q89P9L842205 is a potential diagnostic biomarker for LSCC and acts as a tumor suppressor by directly targeting PIK3CD.

Physiological expression of PI3K H1047R mutation reveals its anti-metastatic potential in ErbB2-driven breast cancer.

p110α is a catalytic subunit of phosphoinositide 3-kinase (PI3K), a major downstream effector of receptor tyrosine kinase ErbB2, that is amplified and overexpressed in 20-30% of breast cancers, 40% of which have an activating mutation in p110α. Despite the high frequency of PIK3CA gain-of-function mutations, their prognostic value is controversial. Here, we employ a knock-in transgenic strategy to restrict the expression of an activated form of ErbB2 and p110α kinase domain mutation (p110αHR) in the mammary epithelium. Physiological levels of transgene expression under the control of their endogenous promoters did not result in a major synergistic effect. However, tumors arising in ErbB2/p110αHR bi-genic strain metastasized to the lung with significantly reduced capacity compared to tumors expressing ErbB2 alone. The reduced metastasis was further associated with retention of the myoepithelial layer reminiscent of ductal carcinoma in situ (DCIS), a non-invasive stage of human breast cancer. Molecular and biochemical analyses revealed that these poorly metastatic tumors exhibited a significant decrease in phospho-myosin light chain 2 (MLC2) associated with cellular contractility and migration. Examination of human samples for MLC2 activity revealed a progressive increase in cellular contractility between non-invasive DCIS and invasive ductal carcinoma. Collectively, these data argue that p110αHR mutation attenuates metastatic behavior in the context of ErbB2-driven breast cancer.

Analysing the cellular mechanisms underlying activated PI3K δ Syndrome (APDS) reveals differences between APDS1 and APDS2

Abstract APDS is a primary immunodeficiency condition caused by heterozygous gain-of-function mutations in PIK3CD (APDS1), which encodes the leukocyte-restricted p110-δ catalytic subunit of phosphoinositide 3-kinase (PI3K) or heterozygous loss-of-function mutations in PIK3R1 (APDS2), which encodes the regulatory subunit of PI3K. PI3K is activated downstream of many receptors expressed by lymphocytes and plays important roles in their activation and differentiation. The increased PI3K activity in cells from APDS patients leads to multiple immune manifestations including lymphoproliferation, respiratory tract infections, Th2-related pathologies, impaired Ab responses and autoimmunity. We previously dissected the cellular defects that lead to disease in APDS1 by studying patient cells and our novel mouse model. This revealed changes in lymphocytes including increased memory T cells, defective Tfh function, decreased isotype switching and a break in B cell tolerance and production of autoantibodies. We have now generated a mouse model of APDS2 (Pik3r1E11SpD) and use this together with studies of patients’ cells to determine whether increased PI3K signalling due to loss of regulatory function causes the same cellular dysregulation. While we observed many of the same changes previously observed in APDS1, including altered cytokine production and decreased isotype switching, we also identified multiple differences in the cellular phenotype in APDS2 compared to APDS1. Together, these studies reveal that although APDS1 and 2 both cause increased PI3K signalling and result in similar clinical phenotypes, there are distinctions between how these two types of mutations affect cellular function. Supported by grants from the NHMRC and an AAI Careers in Immunology Fellowship

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

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

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

Nuclear upregulation of class I phosphoinositide 3-kinase p110β correlates with high 47S rRNA levels in cancer cells

The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared with PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are, however, unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA level in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear:cytoplasmic staining ratios were detected in high-grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3] were measured in the nucleus of EC cells, and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5)P 3 were localised in the nucleolus, which correlated withhigh levels of 47S pre-rRNA. p110β inhibition led to a decrease in both 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC.This article has an associated First Person interview with Fatemeh Mazloumi Gavgani, joint first author of the paper.

The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease

Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.

Deletion of P110δ promotes the development of myocarditis in ApoE‑deficient mice by increasing mononuclear cell peritoneal infiltration.

Phosphoinositide 3-kinase catalytic subunit δ isoform (P110δ) is mainly expressed in white blood cells. It is involved in T and B lymphocyte differentiation, maturation and the neutrophil chemotaxis process. Apolipoprotein E (ApoE) is an arginine-rich alkaline protein, which is present in plasma chylomicron, low-density lipoprotein and very low-density lipoprotein. The present study aimed to determine the effects of P110δ deletion on myocarditis in ApoE−/− mice. A mouse model of ApoE and P110δ double deletion was initially constructed; hematoxylin and eosin (H&E) staining was performed to detect the histological alterations in the mouse myocardium. Systolic and diastolic alterations, and alterations in the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were examined by electrocardiogram. Blood cell of ApoE and P110δ double mice was used to detect changes in white blood cells and monocytes. Western blotting was used to detect the expression levels of apoptosis-associated proteins, whereas flow cytometry was used to detect the percentage of apoptosis. Morphological alterations in myocardial cells were observed under a microscope. The results of polymerase chain reaction demonstrated that double deletion mice were successfully constructed. H&E staining revealed that cells in the ApoE−/− mice were spindle-shaped; however, the nuclei were smaller in the double deletion mice. There was no change in cardiac contraction in normal mice; however, in double deletion mice, the systolic and diastolic contractions were markedly reduced. LVFS and LVEF were decreased compared with in the control group. Blood cell analysis indicated that the content of white blood cells and monocytes in the experimental group was significantly higher than that in the control group. Western blotting demonstrated that the expression levels of apoptotic proteins in double deletion mice were significantly higher compared with in the control group. Flow cytometry revealed that the apoptotic ratio was increased in double deletion mice compared with in the control group (42 vs. 21%). These findings suggested that deletion of P110δ may induce monocyte peritoneal infiltration and increase apoptosis, thus promoting the development of myocarditis.

PI 3‐kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3‐kinase (PI3K) and its product phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.