Lynn Ueno

Akt-mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3‐kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor‐κB (NFκB) by inducing phosphorylation and subsequent degradation of inhibitor of κB (IκB). We show here that NFκB‐driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFκB‐dependent transcription. The degradation of the IκB protein is strongly enhanced in Akt‐transformed cells, and the loss of NFκB activity by introduction of a super‐repressor of NFκB, IκBSR, interferes with PI3K‐ and Akt‐induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFκB at serine 534 is also upregulated in Akt‐transformed cells. Our data suggest that the stimulation of NFκB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IκB kinase) α and β. Akt phosphorylates IKKα on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKα and β. Our results demonstrate two separate functions of the IKK complex in NFκB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IκB and the phosphorylation of p65. The data further support the conclusion that NFκB activity is essential for PI3K‐ and Akt‐induced oncogenic transformation.

MicroRNA-135b Promotes Cancer Progression by Acting as a Downstream Effector of Oncogenic Pathways in Colon Cancer

Summary MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment.

Inhibitor of MYC identified in a Krohnke pyridine library.

Significance MYC is an essential transcriptional regulator that controls cell proliferation. Elevated MYC is a driving force in most human cancers, yet MYC has been an exceedingly challenging target for small-molecule inhibitors. Here we describe a novel MYC inhibitor that interacts directly with MYC and interferes with its transcriptional and oncogenic activities. In a fluorescence polarization screen for the MYC–MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC–MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.

The butterfly effect in cancer: A single base mutation can remodel the cell

Significance A single base substitution in one allele of the PIK3CA gene (encoding the catalytic subunit p110α of PI3K) in a human breast epithelial cell induces a gene expression profile that closely resembles the gene expression profile of basal breast cancer. The mutation also causes extensive remodeling of gene signatures that are not known to be connected to the activity of PI3K. The data show that a cancer-specific mutation that induces a gain of function in PI3K has an unexpectedly deep and broad impact on the phenotypic properties of the cell. We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110α of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.

Synthesis and evaluation of a cyclic imine derivative conjugated to a fluorescent molecule for labeling of proteins

A cyclic imine conjugated to a fluorescent dansyl group was synthesized and used for covalent labeling of proteins. The covalent attachment to proteins was confirmed by gel electrophoresis and mass analysis.

Phosphatidylinositol 4,5-bisphosphate-specific AKT1 is oncogenic.

The protein kinase AKT1 (v‐akt murine thymoma viral oncogene homolog 1), also referred to as protein kinase B (PKB), is an essential mediator of the phosphatidylinositol 3‐kinase signaling pathway. Elevated activity of AKT1 is common in human cancer. Localization at the plasma membrane, leading to enhanced phosphorylation and activation of AKT1, is an important factor determining the oncogenicity of this kinase. Although the phosphatidylinositol 3‐kinase signaling pathway is frequently upregulated in cancer, cancer‐specific mutations in AKT1 are not common. Recently, such a mutation has been identified in breast, colon and ovarian cancers. The mutation is located in the pleckstrin homology (PH) domain of AKT1 and results in a glutamic acid to lysine substitution at residue 17. The resultant change in the conformation of the PH domain facilitates membrane binding of the mutant protein. Here we show that exchange of the PH domain leading to preferential binding of phosphatidylinositol 4,5‐bisphosphate (PIP2) over phosphatidylinositol 3,4,5‐trisphosphate (PIP3) constitutively activates AKT1. AKT1 with this altered PIP affinity induces oncogenic transformation in cultures of chicken embryo fibroblasts and causes neoplastic growth and angiogenesis in the chorioallantoic membrane of the chicken embryo. Gain‐of‐function mutants of AKT1 may not be affected by PI3K inhibitors that are currently in development. Therefore, AKT1 remains a distinct and important cancer target.

Oncogenic activity of the regulatory subunit p85β of phosphatidylinositol 3-kinase (PI3K)

Significance PI3K is a lipid kinase that regulates a broad spectrum of cellular activities. It is a dimeric enzyme consisting of a catalytic and a regulatory subunit. The regulatory subunit stabilizes and inhibits the catalytic subunit. The two major regulatory subunits p85α and p85β have overlapping and unique functions. The data presented here show that in contrast to p85α, the native, wild-type p85β has oncogenic potential and suggest that p85β is a less effective inhibitor of p110 than p85α. These findings are in accord with emerging information that assigns p85β a specific role in human cancer. Expression of the regulatory subunit p85β of PI3K induces oncogenic transformation of primary avian fibroblasts. The transformed cells proliferate at an increased rate compared with nontransformed controls and show elevated levels of PI3K signaling. The oncogenic activity of p85β requires an active PI3K-TOR signaling cascade and is mediated by the p110α and p110β isoforms of the PI3K catalytic subunit. The data suggest that p85β is a less effective inhibitor of the PI3K catalytic subunit than p85α and that this reduced level of p110 inhibition accounts for the oncogenic activity of p85β.

Synthetic molecules for disruption of the MYC protein-protein interface

MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity, downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new compounds aimed at improving these properties. This blind SAR campaign has produced a new lead compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure. This probe represents a significant advancement that will not only enable biophysical characterization of this interaction and further SAR, but also contribute to advances in understanding of MYC biology.

Abstract 3339: The regulatory subunit of PI3K, p85β, induces cellular transformation, enhanced cell proliferation and increased PI3K signaling

We have expressed the human p85β in chicken embryo fibroblasts (CEF) using the RCAS retroviral vector. At low concentrations, RCAS-p85β induces the formation of distinct foci of transformed cells. CEF transfected with high concentrations of RCAS-p85β show enhanced cell proliferation and PI3K signaling. RCAS-p85β shares this growth promoting potential with the cancer-derived mutants of p85α, but the wild type p85α protein expressed by the RCAS vector lacks this potential. The transforming and signaling activities of p85β depend on the PI3K catalytic subunit p110. We have used p110 isoform-specific inhibitors to show that the transforming and signaling activities of p85β can be mediated by p110α or p110β or both. This is in contrast to the transforming mutants of p85α which depend almost exclusively on p110α for their growth-promoting effect. p85β can be phosphorylated at Y655. Mutations of the phosphorylation site Y655A or Y655E lose almost all of their transforming and enhanced signaling activity. This result indicates that p85β could be subject to control by tyrosine kinases. Our data document important differences between p85α and p85β and suggest that p85β could carry out special functions in signaling and in cancer. This work was supported by NIH Grant R01 CA078230. This is abstract 26049 of The Scripps Research Institute. Citation Format: Yoshihiro Ito, Jonathan R. Hart, Lynn Ueno, Peter K. Vogt. The regulatory subunit of PI3K, p85β, induces cellular transformation, enhanced cell proliferation and increased PI3K signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3339. doi:10.1158/1538-7445.AM2014-3339

Abstract 4479: PF-04691502, a potent and selective PI3K/mTOR dual inhibitor with antitumor activity

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The PI3K pathway, which regulates cell growth, proliferation and survival, is activated in many types of human tumors by mutational activation of PI3Kα, loss of function of PTEN or activation of receptor tyrosine kinases. Inhibition of key signaling proteins in the pathway, such as PI3K, AKT and mTOR, therefore represents a high value targeting strategy for diverse cancers. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in in vitro biochemical, cell and xenograft models. In in vitro biochemical assays PF-04691502 inhibited recombinant PI3Kα, β, γ and δ isoforms with Kis of 1.2-2.2 nM and recombinant mTOR with a Ki of 9.1 nM. PF-04691502 demonstrated a high degree of selectivity for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the Class III PI3K hVps34. PF-04691502 also inhibited transformation of avian fibroblasts mediated by PI3K γ, δ, mutant PI3Kα E545K or membrane-localized AKT with IC50s of ∼100nM. In cell assays PF-04691502 inhibited PI3K/mTOR signaling in SKOV3 ovarian cancer cells with PI3Kα mutations and in U87MG glioblastoma cells with PTEN alteration, as indicated by reduced levels of phosphorylation of AKT(T308), AKT(S473) and S6 ribosomal proteins. Functional studies for anti-proliferative effects suggest PF-04691502 has broad efficacy across tumor types. In SKOV3 and U87MG xenograft models PF-04691502 treatment resulted in dose-dependent tumor growth inhibition (TGI) with maximum TGI of ∼70% at the maximum tolerated dose of 10 mg/kg, by once daily oral dosing. Inhibition of AKT(S473) phosphorylation and S6RP(S235/236)/PRAS40(T246)/4EBP1(T37/46) phosphorylation were used as quantitative and qualitative pharmacodynamic (PD) endpoints, respectively; a clear pharmacokinetic (PK)/PD relationship was established in both models after multiple dose oral administration. In the U87MG xenograft model AKT(S473) phosphorylation was inhibited with an estimated EC50 of 5.7 nM (free plasma concentration) based on PK/PD modeling. The free plasma Area Under Curve was estimated to be 850 nM*hr for 70% TGI at 10mg/kg and was found to be similar in the SKOV3 model. The projected human efficacious dose of 10 mg once daily oral dosing provides Caverage steady state exposure of 22.4 nM (free plasma concentration) which is sufficient for 50-80% inhibition of pAKT S473, and corresponds to 74% TGI. Phase 1 clinical trials of PF-04691502 as a single agent are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4479.