Mutations In P110α Research Articles

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.

Molecular insights into regulation of PI3Kα

PIK3CA, the gene for the lipid kinase p110α is one of the most frequently mutated oncogenes across all types of cancer. p110α is an enzyme that catalyzes the formation of phosphatidylinositol 3, 4, 5 Triphosphate (PIP3). PIP3 recruits effector proteins which regulate growth, proliferation and motility. Due to this role as a master cell regulator, the activity of p110α is maintained in an inactive confirmation and is only activated downstream of Receptor Tyrosine Kinases (RTKs) and RAS family of GTPases. p110α is maintained in an inactive confirmation through interactions with its regulatory subunit as well as inhibitory contacts with the C‐terminus. However, oncogenic mutations in p110α breaks these inhibitory interactions and drives hyperactivity without the need for activation signals leading to uncontrolled cell growth. We provide molecular insights into the regulation of oncogenic mutants of p110α using Hydrogen‐Deuterium Exchange Mass Spectrometry (HDX‐MS). HDX‐MS reveals the dynamic changes between the natural cytosolic state and fully‐active membrane bound states of the enzyme. We find unique molecular mechanisms regulating how mutants at the c‐terminus activate lipid kinase activity (H1047R, M1043L, G1049R and N1068KLKR). Using extensive biophysical tools, biochemical assays, and MD simulations, we have tested the oncogenic potential of these mutations. Our results elucidates a unifying theory towards the regulation of PI3Kα and how oncogenic mutations drive hyperactivity. This will aid in understanding the regulation of PI3Kα and in developing isoform specific inhibitors.

Cancer-associated mutations in the p85α N-terminal SH2 domain activate a spectrum of receptor tyrosine kinases

The phosphoinositide 3-kinase regulatory subunit p85α is a key regulator of kinase signaling and is frequently mutated in cancers. In the present study, we showed that in addition to weakening the inhibitory interaction between p85α and p110α, a group of driver mutations in the p85α N-terminal SH2 domain activated EGFR, HER2, HER3, c-Met, and IGF-1R in a p110α-independent manner. Cancer cells expressing these mutations exhibited the activation of p110α and the AKT pathway. Interestingly, the activation of EGFR, HER2, and c-Met was attributed to the ability of driver mutations to inhibit HER3 ubiquitination and degradation. The resulting increase in HER3 protein levels promoted its heterodimerization with EGFR, HER2, and c-Met, as well as the allosteric activation of these dimerized partners; however, HER3 silencing abolished this transactivation. Accordingly, inhibitors of either AKT or the HER family reduced the oncogenicity of driver mutations. The combination of these inhibitors resulted in marked synergy. Taken together, our findings provide mechanistic insights and suggest therapeutic strategies targeting a class of recurrent p85α mutations.

336P PI3K pathway biomarkers and clinical response in a phase I/Ib study of GDC-0077 in hormone receptor-positive/HER2-negative breast cancer (HR+/HER2– BC)

Mutations in p110α (gene, PIK3CA) occur in ∼40% of HR+/HER2– BC. GDC-0077 is a potent p110α-isoform selective inhibitor that degrades mutant (mut) p110α and elicits anti-tumour activity in PIK3CAmut preclinical models alone and with endocrine therapy (ET). We present a preliminary co-analysis of PI3K pathway biomarkers and clinical outcomes from patients (pts) with PIK3CAmut HR+/HER2– BC in an ongoing open-label, phase I/Ib dose-escalation study of GDC-0077, with ET (letrozole/fulvestrant) +/– palbociclib (NCT03006172; data cut 1 Jan 2020).

Abstract NG16: Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors

Abstract NG16: Double <i>PIK3CA</i> mutations in <i>cis</i> increase oncogenicity and sensitivity to PI3Kα inhibitors

Impact of p85α Alterations in Cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in the regulation of cell signaling, proliferation, survival, migration and vesicle trafficking in normal cells and is frequently deregulated in many cancers. The p85α protein is the most characterized regulatory subunit of the class IA PI3Ks, best known for its regulation of the p110-PI3K catalytic subunit. In this review, we will discuss the impact of p85α mutations or alterations in expression levels on the proteins p85α is known to bind and regulate. We will focus on alterations within the N-terminal half of p85α that primarily regulate Rab5 and some members of the Rho-family of GTPases, as well as those that regulate PTEN (phosphatase and tensin homologue deleted on chromosome 10), the enzyme that directly counteracts PI3K signaling. We highlight recent data, mapping the interaction surfaces of the PTEN–p85α breakpoint cluster region homology (BH) domain, which sheds new light on key residues in both proteins. As a multifunctional protein that binds and regulates many different proteins, p85α mutations at different sites have different impacts in cancer and would necessarily require distinct treatment strategies to be effective.

Abstract IA26: Clinical development of PI3K/AKT pathway inhibitors in breast cancer

Abstract Targeting the PI3K/AKT/mTOR pathway has been a focus for drug development since the discovery of PI3K-activating mutations in p110α over 10 years ago. Breast cancer has been a primary indication for development of this class of drugs because of the high prevalence of these mutations, up to 45% in hormone receptor-positive breast cancer, and the synergy observed in combinations with endocrine therapies. An mTOR inhibitor, everolimus, was approved in breast cancer in combination with exemestane in 2012, indicating the synergy between ER and PI3K pathway translates to the clinic. Apitolisib, a dual PI3K pan-isoform and mTOR inhibitor, has discontinued development due to toxicities, despite compelling preclinical activity across multiple tumor types regardless of PI3K and PTEN status. Pictilisib, a pan-isoform PI3K inhibitor, has shown some evidence of activity, but in phase II studies has demonstrated that the activity is limited by dose intensity. Buparlisib, a pan-isoform PI3K inhibitor, has demonstrated efficacy in a large phase III study, but was not deemed sufficiently active relative to the toxicity in HR+ breast cancer to be submitted for regulatory approval. Second-generation PI3K and AKT inhibitors with higher selectivity and mutant specificity are in clinical development and hope to thread a better therapeutic index by dosing higher for more profound inhibition, and/or selectively inhibiting mutated forms of PI3K. Taselisib (GDC-0032) is a beta-sparing and PI3K-mutant selective inhibitor that leads to degradation of only the mutant form of PI3K and is currently in phase III. More recently, taselisib met the primary endpoint of improved response rates in a randomized, neoadjuvant phase II trial, LORELEI. Alpelisib is an alpha-selective PI3K inhibitor that is also in phase III. Recent data with ipatasertib, an AKT inhibitor, has demonstrated better tolerability and efficacy in randomized phase II trials of both prostate cancer and triple-negative breast cancer, where the PI3K/AKT signaling pathway is activated by either loss of PTEN or PI3K mutations, and may offer a better therapeutic index given its unique mechanism of action and place in the PI3K signaling pathway. Overall, the PI3K/AKT pathway is still considered an important pathway to target for certain cancers; however, target selectivity, dosing, and the right combination are critical to find a therapeutic index to provide a good option for patients. Citation Format: Mika Derynck. Clinical development of PI3K/AKT pathway inhibitors in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA26.

Patient-derived mutations within the N-terminal domains of p85\u03b1 impact PTEN or Rab5 binding and regulation

The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.

ErbB2-positive mammary tumors can escape PI3K-p110α loss through downregulation of the Pten tumor suppressor.

Breast cancer is the most common cancer among women and 30% will be diagnosed with an ErbB2-positive cancer. Forty percent of ErbB2-positive breast tumors have an activating mutation in p110α, a catalytic subunit of phosphoinositide 3-kinase (PI3K). Clinical and experimental data show that breast tumors treated with a p110α-specific inhibitor often circumvent inhibition and resume growth. To understand this mechanism of resistance, we crossed a p110α conditional (p110αflx/flx) mouse model with mice that overexpresses the ErbB2/Neu-IRES-Cre transgene (NIC) specifically in the mammary epithelium. Although mammary-specific deletion of p110α dramatically delays tumor onset, tumors eventually arise and are dependent on p110β. Through biochemical analyses we find that a proportion of p110α-deficient tumors (23%) display downregulation of the Pten tumor suppressor. We further demonstrate that loss of one allele of PTEN is sufficient to shift isoform dependency from p110α to p110β in vivo. These results provide insight into the molecular mechanism by which ErbB2-positive breast cancer escapes p110α inhibition.

Insights into the pathological mechanisms of p85α mutations using a yeast-based phosphatidylinositol 3-kinase model.

In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, β, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85–p110 interactions typical of cancer.

Haploinsufficiency of Klippel-Trenaunay syndrome geneAggf1inhibits developmental and pathological angiogenesis by inactivating PI3K and AKT and disrupts vascular integrity by activating VE-cadherin

Aggf1 is the first gene identified for Klippel-Trenaunay syndrome (KTS), and encodes an angiogenic factor. However, the in vivo roles of Aggf1 are incompletely defined. Here we demonstrate that Aggf1 is essential for both physiological angiogenesis and pathological tumour angiogenesis in vivo. Two lines of Aggf1 knockout (KO) mice showed a particularly severe phenotype as no homozygous embryos were observed and heterozygous mice also showed embryonic lethality (haploinsufficient lethality) observed only for Vegfa and Dll4. Aggf1+/- KO caused defective angiogenesis in yolk sacs and embryos. Survived adult heterozygous mice exhibit frequent haemorrhages and increased vascular permeability due to increased phosphorylation and reduced membrane localization of VE-cadherin. AGGF1 inhibits VE-cadherin phosphorylation, increases plasma membrane VE-cadherin in ECs and in mice, blocks vascular permeability induced by ischaemia-reperfusion (IR), restores depressed cardiac function and contraction, reduces infarct sizes, cardiac fibrosis and necrosis, haemorrhages, edema, and macrophage density associated with IR. Mechanistically, AGGF1 promotes angiogenesis by activating catalytic p110α subunit and p85α regulatory subunit of PI3K, leading to activation of AKT, GSK3β and p70S6K. AKT activation is significantly reduced in heterozygous KO mice and isolated KO ECs, which can be rescued by exogenous AGGF1. ECs from KO mice show reduced capillary angiogenesis, which is rescued by AGGF1 and AKT. Tumour growth/angiogenesis is reduced in heterozygous mice, which was associated with reduced activation of p110α, p85α and AKT. Together with recent identification of somatic mutations in p110α (encoded by PIK3CA), our data establish a potential mechanistic link between AGGF1 and PIK3CA, the two genes identified for KTS.

Abstract 1141: The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells

Abstract INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and Inositol polyphosphate-4-phosphatase, type II (INPP4B) are two important enzymes maintaining homeostasis of phosphoinositides in cells. Class IA PI3K produces Phosphatidylinositol-3,4,5-bisphosphate (PIP3) by phosphorylation of Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at D3 position, while INPP4B dephosphorylate Phosphatidylinositol-4,5-bisphosphate (PI(3,4)P2) at the D4 position to generate Phosphatidylinositol 3-phosphate (PI(3)P). All of these phospholipids are important second messengers. PI3K activates its downstream substrate Protein Kinase B (Akt) by PIP3. INPP4B, however, blocks Akt activities in epithelial cells through the turnover of PI(3,4)P2. PI3K/Akt pathway is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. Gain of function mutations in the catalytic subunit p110α of PI3K occur in up to 1/3 of human colorectal cancers (CRC). The kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α. Our previous report showed that this mutation in p110α results in an increase in cellular levels of PIP3 and in turn an enhancement of Akt activity. Since the H1047R mutation and INPP4B have an opposite effect on Akt activities, the interesting question is if there is crosstalk between the two or if H1047R mutation can affect cellular INPP4B expression. We hypothesize that H1047R mutated p110α down-regulates INPP4B and therefore results in increased Akt activity. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous protein expression level of INPP4B was detected by immune-blotting analysis in parental, WT and MUT HCT116 cells. To further investigate the effect on INPP4B of the H1047R mutation, we conducted RNA-sequencing for profiling, quantifying RNA transcripts in WT and MUT HCT116 cells, and compared RNA transcript level of INPP4B between the two isogenic cell lines of HCT116 cells. RESULTS: 1) Endogenous expression level of INPP4B protein in HCT116WT cells was over 10-times higher than in HCT116MUT cells. 2) INPP4B RNA was down regulated by the H1047R mutation in p110α (PIK3CA) of Class 1A PI3K. CONCLUSION: Our results suggested that hyperactivity of Akt in H1047R mutation bearing cells may be also related to low level of cellular INPP4B, and that the H1047R mutated p110α may increase cell migration and cancer metastasis through down regulation of tumor suppressor gene INPP4B. Although further studies are needed to reveal the mechanism of H1047R mutated PI3K mediated INPP4B-regulation, INPP4B expression level may be a potential maker to predict PI3K mutation induced CRC metastasis. Citation Format: Guanghua Wan, Scott A. Ness, Ashwani Rajput. The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1141.

The H1047R point mutation in p110 alpha changes the morphology of human colon HCT116 cancer cells.

The class IA phosphatidylinositol 3-kinases (PI3K) is involved in controlling changes in cell morphology, which is a highly coordinated cellular event. This event is powered by actin filament polymerization and remodeling. The gain-of-function mutations in the catalytic subunit of p110α of class IA PI3K, which occur in up to one-third of human colorectal cancers (CRCs), are capable of causing dysregulation of cell signaling and thus may result in the alteration in cell morphology and motility and in turn cause cancer metastasis. In vivo studies have demonstrated that cell lines bearing the H1047R point mutation, the most frequent cancer-specific mutation in the kinase domain of p110α, are more metastatic than cells carrying wild-type p110α. In the current study, we show that the H1047R in p110α of PI3K decreases F-actin polymerization, increases the formation of filopodia and significantly changes the cell morphology in HCT116 cancer cells. The anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), which is also involved in actin polymerization and cell migration, is downregulated by the H1047R mutation in p110α. Our data suggest that the H1047R mutation in PI3K is responsible for the rearrangement of the cytoskeleton, alteration in cell morphology and enhancing cell motility, and that Bcl-2 may be involved in the H1047R mutation-mediated morphological changes and increased migratory capability.

P110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity.

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.

Abstract 5008: The H1047R point mutation in p110 alpha of PI3K decrease Bcl2 expression in human colon cancer HCT116 cells

Abstract INTRODUCTION: PI3K and anti-apoptotic protein Bcl2 are two important players in regulating cell survival through two distinct cell signaling pathways. Beside the survival function, PI3K is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. At the same time, Bcl2 has also been reported to be involved in actin polymerization and cell motility dissociate from its anti-apoptotic function. Inhibition of PI3K has been shown to result in a 45% decreases in Bcl2 promoter activity. Several reports have shown that loss of Bcl2 expression correlates with tumor recurrence in CRC and high levels of Bcl2 are predictive of relapse-free survival in stage II CRC. Mutations in the catalytic subunit p110α (PIK3CA) of Class 1A PI3K occur in up to 1/3 of human colorectal cancers (CRC) and the kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α and results in a gain of function. In vivo studies have demonstrated that cell lines bearing the p110α mutation are more metastatic than the cells with wild type p110α. The aim of current study was to investigate the effect of H1047R mutation in PI3K on Bcl2 regulation. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous expression level of Bcl2 was detected by immune-blotting analysis in WT and MUT HCT116 cells. To further investigate the effect of WT and MUT p110α on Bcl2 regulation, Bcl2 expression was tested under the condition of ether overexpression or inhibition of p110α. For inhibition experiment, HCT116 WT and MUT cells were treated by the specific inhibitor of p110α A66. While, overexpression of p110α was conducted by stable transfection into HCT116 WT cells with Tet-On inducible plasmids, which contains the whole sequences of coding region of either wild type (PIK3CAWT) or H1047R-p110α (PIK3CAA3140G). RESULTS: 1) Endogenous expression level of Bcl2 in HCT116WT cells was at least 3-times higher than it in HCT116MUT cells. 2) The increase of Bcl2 expression is doxycline dose-dependent in the HCTWT cells which were transfected with pTRE3G-PIK3CAWT. However, overexpression of H1047R-p110α did not affect Bcl2 levesl in HCT116WT cells with pTRE3G-PIK3CAA3166C transfection. 3) Expression level of Bcl2 in HCT116 MUT cells was increased by A66 dose-dependent inhibition of p110α. The down-regulation of Bcl2, however, was observed in HCT WT cells. CONCLUSION: Our results suggested that the wild type and the H1047R p110α have the opposite effect in the regulation of Bcl2 expression: up-regulation by WT- p110α and down- regulation by H1047R p110α. Both PI3K and Bcl2 cannot be ignored in cancer prognosis and therapy. Further studies are needed to reveal the mechanism of PI3K mediated Bcl2-regulation, and the effect of the interaction of Bcl2 and PI3K on cell migration and CRC metastasis. Citation Format: Guanghua Wan, Ashwani Rajput. The H1047R point mutation in p110 alpha of PI3K decrease Bcl2 expression in human colon cancer HCT116 cells. [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 5008. doi:10.1158/1538-7445.AM2015-5008

Regulation of the PI3K pathway through a p85α monomer-homodimer equilibrium.

The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer-dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development.

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects.

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP2) to phosphoinositide-3,4,5-trisphosphate (PIP3), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP2, a membrane lipid.

Targeting the Protein–Protein Interaction between IRS1 and Mutant p110α for Cancer Therapy

Phosphoinositide-3-kinase, catalytic, alpha polypeptide, which encodes the catalytic p110α subunit of phosphatidylinositol 3-kinase α, is the most frequently mutated oncogene in human cancers. Targeting mutant p110α holds great promise for cancer therapy. However, it is challenging to develop p110α isoform-specific inhibitors. Most p110α mutations occur at two hot spot regions: an acidic cluster (E542, E545, and Q546) in the helical domain and a histidine residue (H1047) in the kinase domain. We recently discovered that p110α helical domain mutant proteins, but not the kinase domain mutant proteins, directly associate with insulin receptor substrate 1 (IRS1). Moreover, we demonstrated that disruption of protein-protein interaction between p110α helical domain mutant and IRS1 inhibits the growth of tumors with such mutations. The direct protein interaction between IRS1 and p110α helical domain mutants may provide a more accessible target for developing novel precision cancer therapy.

Oncogenic Mutations of p110α Isoform of PI 3-Kinase Upregulate Its Protein Kinase Activity

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 βc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 βc receptor (βic). We find p110α, p110β and p110γ all phosphorylate βic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards βic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn2+ dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110β and p110γ all display significant activity in the presence of Mg2+. Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.

Abstract 2636: The H1047R point mutation in PI3KCA changes morphology in human colon cancer cells.

Abstract INTRODUCTION: The PI3K signaling pathway involves a wide range of cell signaling phenomena, including cell proliferation, differentiation, angiogenesis, survival, adhesion, cytoskeletal rearrangement, invadopodia formation and motility. Mutations in the catalytic subunit p110α (PIK3CA) of Class 1A phosphatidyinositol 3-kinase (PI3K) occur in up to 1/3 of human colorectal cancers (CRC). The kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α and results in a gain of function. In vivo studies have demonstrated that cell lines bearing the p110α mutation are more metastatic than the cells with wild type p110α. We have previously shown that inhibition of PI3K-p110α significantly delayed or abrogated the migration of HCT116 parental cell line, which is a CRC cell line that is heterozygous for the PIK3CA mutation and thus contains both the H1047R mutation in p110α and a wild type allele of PIK3CA. The aim of our study was to investigate the mechanism of how the H1047R mutation of PI3K results in the increased cell motility in HCT116 cell line. We hypothesize that the rearrangement of the cytoskeleton may be responsible for the changes in cell morphology and cell motility in the presence of the H1047R mutation of PI3K. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Cell morphology was observed by microscopic approaches and F-actin, the major component of cytoskeleton, was stained with Alexa Fluor® 488 Phalloidin. Effect of PI3K-p110α inhibition on cell morphology was tested at a series of time points with 1.0 μM of A66 and PIK75, which are specific inhibitors of p110α. RESULTS: The morphological appearance of MUT cells was considerably different as compared to WT cells. The WT HCT116 cells normally show a rounded and more clumped morphology. However, the MUT HCT116 cells become elongated and actin filaments appear to align along the length of the cell. The cells also adopt less clumped and more fibroblastic morphology. Furthermore, when the MUT cells were treated with the p110α specific inhibitors, the cell morphology then again resembled the WT cells. The morphological changes in MUT HCT116 cells possibly contribute to enhanced migratory capacity. CONCLUSION: The morphologic appearance of MUT PIK3CA bearing colon cancer cells is markedly different from WT cells. The inhibition of mutant PIK3CA function results in morphologic changes that resemble the WT allele bearing cells. Thus, the interaction of PI3K and the cell cytoskeleton appears to be an important aspect in regulating cell motility and subsequently, metastases. The PI3K pathway remains a desirable target in CRC therapeutics. Citation Format: Guanghua Wan, Essence Hand, Judy Lin Cannon, Ashwani Rajput. The H1047R point mutation in PI3KCA changes morphology in human colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2636. doi:10.1158/1538-7445.AM2013-2636