P85 Regulatory Subunit Research Articles

Abstract 18534: TAT-Protein Blockade of PTEN After Cardiac Ischemia is Highly Protective

Introduction: Cooling protects against cardiac ischemia/reperfusion (I/R) injury by enhancing PI3K/Akt/NOS signaling. PI3K/Akt signaling depends on binding by its regulatory subunit p85 to the catalytic subunit p110 or the inhibitory phosphatase PTEN. We studied novel TAT-p85 fusion proteins that selectively inhibit these competing p85 interactions. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection in a heart cell model of I/R. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would abrogate protection. To further test TAT fusion proteins for the I/R injury of mouse cardiac arrest (CA), we studied whether a TAT-fused PTEN C-terminal 9 amino acid (TAT-PTEN9c) PTEN inhibitor would activate Akt in critical organs and improve survival. Methods: A TAT-Δp110 p85 plasmid was constructed with nucleotide deletions at amino acids 478-513. The TAT-ΔPTEN p85 plasmid deleted amino acids 1-313. TAT-PTEN9c was synthesized by Anaspec. Cardiomyocytes were isolated from 1-2-day old C57BL6/J mice and exposed to up to 90 min ischemic arrest with 3 h reperfusion (I/R). Cell viability was evaluated by propidium iodide uptake and p-Akt was measured by Western blot. C57BL6 mice were subjected to an established potassium-induced CA protocol. TAT-PTEN9c (7.8 mg/kg) was given IV after return of spontaneous circulation (ROSC) (n=10) and the 2nd dose for another 5 mice was given at 60 min after ROSC. Results: TAT-Δp110 p85 increased cell death from 44.6 +/- 2.7% to 92.5 +/- 3.4%, while TAT-ΔPTEN p85 decreased cell death caused by I/R from 47.8 +/- 1.4% to 11.9 +/- 5.3%. 30 mice were randomized to either saline or TAT-PTEN9c post-ROSC. TAT fusion proteins could be seen within heart and brain tissues within 2 minutes of IV infusion. The survival rate was significantly increased in TAT-PTEN9c treated mice compared with that of saline treated mice at 2 hrs (14/15, 93% vs. 9/15, 60%, P < 0.05) and at 4 hrs (10/15, 67% vs. 6/15, 40%, P < 0.05) after ROSC. Conclusions: TAT fusion protein inhibition of PTEN during cardiac I/R and after cardiac arrest appear to be promising strategies for enhancing Akt activation and survival.

Direct Association of Heat Shock Protein 20 (HSPB6) with Phosphoinositide 3-kinase (PI3K) in Human Hepatocellular Carcinoma: Regulation of the PI3K Activity

HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-α stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC.

Combination therapies in Myeloproliferative Neoplasms: why do we need them and how to identify potential winners?

The myeloproliferative neoplasms (MPN) are clonal myeloid disorders characterized by proliferation of mature myeloid cells, such that in polycythaemia vera (PV), the red cell proliferation dominates, platelets in essential thrombocythaemia (ET) and in myelofibrosis (MF), there may be cytopenia or proliferation, but the characteristic feature is the strikingly abnormal bone marrow stroma. These entities have a tendency to show phenotypic mimicry and may transform from one to another, for example, 20–30% of patients with PV are likely to develop MF. The significant event in this field was the recognition that Janus Kinase-2 (JAK2) activation was highly prevalent, followed by the description of the JAK2V617F mutation in 2005 (vide infra), which stimulated renewed interest in disease biology. Janus Kinase-2-targeted therapies have led to marked improvements for patients with this condition. However, it is obvious that the pathogenesis of these complex disorders reaches beyond this mutation; only 50–60% of patients with ET, for example, have the JAK2 mutation and several additional mutations have been described, which are of relevance in both the pathogenesis and clinical phenotype of these conditions.

The inhibition of p85αPI3KSer83 phosphorylation prevents cell proliferation and invasion in prostate cancer cells

Phosphoinositide 3-kinase proteins are composed by a catalytic p110 subunit and a regulatory p85 subunit. There are three classes of PI3K, named class I-III, on the bases of the protein domain constituting and determining their specificity. The first one is the best characterized and includes a number of key elements for the integration of different cellular signals. Regulatory p85 subunit shares with the catalytic p110 subunit, a N-terminal SH3 domain showing homology with the protein domain Rho-GTP-ase. After cell stimulation, all class I PI3Ks are recruited to the inner face of the plasma membrane, where they generate phosphatidylinositol-3,4,5-trisphosphate by direct phosphorylation of phosphatidylinositol-4,5-bisphosphate. All pathways trigger the control of different phenomena such as cell growth, proliferation, apoptosis, adhesion and migration through various downstream effectors. We have previously provided direct evidences that a Serine in position 83, adjacent to the N-terminal SH3 domain of regulatory subunit of PI3K, is a substrate of PKA. The aim of this work is to confirm the role of p85αPI3KSer83 in regulating cell proliferation, migration and invasion in prostate cancer cells LNCaP. To this purpose cells were transfected with mutant forms of p85, where Serine was replaced by Alanine, where phosphorylation is prevented, or Aspartic Acid, to mimic the phosphorylated residue. The findings of this study suggest that identifying a peptide mimicking the sequence adjacent to Ser 83 may be used to produce antibodies against this residue that can be proposed as usefool tool for prognosis by correlating phosphorylation at Ser83 with tumor stage.

Signal Regulatory Protein α Is Associated With Tumor-Polarized Macrophages Phenotype Switch and Plays a Pivotal Role in Tumor Progression

Macrophages (Mψ) are the major component of infiltrating leukocytes in tumors and exhibit distinct phenotypes according to the microenvironment. We have recently found that signal regulatory protein α (SIRPα), the inhibitory molecule expressed on myeloid cells, plays a critical role in controlling innate immune activation. Here, we identify that SIRPα is down-regulated on monocytes/Mψ isolated from peritumoral areas of hepatocellular carcinoma (HCC) samples, while its level is moderately recovered in intratumor Mψ. In vitro assays demonstrate that SIRPα expression is significantly reduced on Mψ when cocultured with hepatoma cells. This reduction is partly due to the soluble factors in the tumor microenvironment. Knockdown (KD) of SIRPα prolongs activation of nuclear factor kappa B (NF-κB) and PI3K-Akt pathways as Mψ encounter tumor cells, leading to an increased capacity of Mψ for migration, survival, and proinflammatory cytokine production. Enhanced Stat3 and impaired Stat1 phosphorylation are also observed in tumor-exposed SIRPα-KD Mψ. Adoptive transfer with SIRPα-KD Mψ accelerates mouse hepatoma cells growth in vivo by remolding the inflammatory microenvironment and promoting angiogenesis. SIRPα accomplishes this partly through its sequestration of the signal transducer Src homology 2-containing phosphotyrosine phosphatase (SHP2) from IκB kinase β (IKKβ) and PI3K regulatory subunit p85 (PI3Kp85). These findings suggest that SIRPα functions as an important modulator of tumor-polarized Mψ in hepatoma, and the reduction of SIRPα is a novel strategy used by tumor cells to benefit their behavior. Therefore, SIRPα could be utilized as a potential target for HCC therapy.

Specific inhibition of PI3K p110δ inhibits CSF‐1‐induced macrophage spreading and invasive capacity

Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3'-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P production. While primary bone marrow-derived macrophages and the immortalized bone marrow-derived macrophage cell line M-/-.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ-specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1-regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis.

Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling

Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection.

Phosphatase and Tensin Homolog Deficiency and Resistance to Trastuzumab and Chemotherapy

The phosphatase and tensin homolog (PTEN) gene is a tumor suppressor that is mutated in a large number of cancers at high frequency. Oncogenic receptor tyrosine kinases such as human epidermal growth factor receptor 2 (HER2 [ERBB2]) potently activate phosphatidylinositol-3 kinase (PI3K) by autophosphorylating their C-termini and/or phosphorylating associated adaptor proteins such as GAB1/2, insulin receptor substrates 1/2, and HER3. In turn, these phosphorylated tyrosines bind the p85 regulatory subunit of PI3K, thus relieving the p110 catalytic subunit of PI3K from p85-mediated inhibition. Active p110 phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), which then recruits pleckstrin homology domain– containing proteins like AKT, PDK1, steroid glucocorticoid kinase, and so on to the plasma membrane. These kinases become activated at the plasma membrane and mediate enhanced cell proliferation and survival. The protein encoded by the PTEN gene is the 3 lipid phosphatase of PIP3. Therefore, PTEN-deficient tumors lack negative regulation of PI3K signaling and, as a result, exhibit aberrant activation of this transforming pathway. This regulation is particularly important in breast cancers with HER2 gene amplification, which rely on HER2-activated PI3K for their progression and survival. Indeed, sustained inhibition of PI3K is required for the antitumor action of the HER2 inhibitors trastuzumab and lapatinib. In HER2-positive/PTEN-deficient tumors, the hyperactivity of PI3K, as a result of both HER2 overexpression and loss of PTEN function, is predicted to attenuate the action of HER2 inhibitors and, therefore, potentially lead to drug resistance, as shown in several mechanistic and clinical correlative studies. The report by Perez et al that accompanies this editorial is the largest study to date that evaluates the correlation between PTEN status and response to trastuzumab in patients with HER2overexpressing breast cancer. In this study, intensity of PTEN expression was scored by immunohistochemistry (IHC) in 1,802 HER2-positive tumors from patients who were enrolled onto the North Central Cancer Treatment Group N9831 adjuvant trial of trastuzumab. N9831 compared adjuvant chemotherapy (arm A) versus adjuvant chemotherapy followed by trastuzumab (arm B) versus adjuvant chemotherapy with concomitant trastuzumab (arm C) in patients with resected HER2-positive breast cancer. PTEN-positive tumors were those with any staining of more than 0%. Appropriate for the scoring of a tumor suppressor, tumors with 0% staining were compared with tumors that exhibited any detectable PTEN protein. A total of 1,286 tissue microarray sections with three cores per block and 516 whole-tumor sections were analyzed. The rate of PTEN loss was 26%. In comparing disease-free survival (DFS) within each treatment arm, there was no difference between patients with PTEN-positive and PTEN-negative tumors. The investigators also compared DFS between the treatment arms for both the PTEN-positive and PTENnegative groups. Hazard ratios (HRs) for concurrent trastuzumab (arm C) versus no trastuzumab (arm A) were 0.65 (P .003) for PTEN-positive and 0.47 (P .005) for PTEN-negative tumors. Comparing sequential trastuzumab (arm B) with no trastuzumab (arm A), HRs were 0.7 (P .009) for PTEN-positive cancers and 0.85 (P .44) for PTEN-negative tumors. From these data, the authors concluded that patients with HER2-positive early breast cancer with or without PTEN benefit from adjuvant trastuzumab. Inconsistent with this report, several smaller retrospective clinical studies have shown that loss or low levels of PTEN are associated with a reduced clinical benefit from trastuzumab. Furthermore, well-conducted mechanistic studies have shown that genetic inactivation of PTEN in HER2-overexpressing cells counteracts the effect of HER2 inhibitors like trastuzumab. How might the difference between this large, carefully conducted study and other clinical correlative and preclinical studies be reconciled? Several possibilities to explain these differences should be considered. First, for practical reasons, most studies in HER2-overexpressing breast cancer have assessed PTEN status (which we interpret as PTEN function) by IHC. However, we should note that not all molecular alterations that result in a loss in PTEN function are detected by IHC. Loss of PTEN function can result from mutation or deletion of the PTEN gene, gene inactivation by loss of heterozygosity at 10q23, reduced RNA/protein levels via transcriptional dysregulation, microRNA expression, and methylation, or reduced phosphatase activity by post-translational alterations in phosphorylation, acetylation, or ubiquitination of the PTEN protein. This suggests that a fraction of HER2 gene–amplified breast cancers that are PTEN positive by IHC may express otherwise nonfunctional PTEN. In the Catalogue of Somatic Mutations in Cancer database, PTEN mutations were detected in approximately 6% of all subtypes of breast cancer, but in the recent analysis of 453 breast tumors by The Cancer Genome Atlas, PTEN mutations were observed in 16 of 453 tumors (3.5%) and did not JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 17 JUNE 1

Gain of Interaction with IRS1 by p110α-Helical Domain Mutants Is Crucial for Their Oncogenic Functions

PIK3CA, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase α, is frequently mutated in human cancers. Most of these mutations occur at two hot-spots: E545K and H1047R located in the helical domain and the kinase domain, respectively. Here, we report that p110α E545K, but not p110α H1047R, gains the ability to associate with IRS1 independent of the p85 regulatory subunit, thereby rewiring this oncogenic signaling pathway. Disruption of the IRS1-p110α E545K interaction destabilizes the p110α protein, reduces AKT phosphorylation, and slows xenograft tumor growth of a cancer cell line expressing p110α E545K. Moreover, a hydrocarbon-stapled peptide that disrupts this interaction inhibits the growth of tumors expressing p110α E545K.

Genetic deregulation of the PIK3CA oncogene in oral cancer

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is one of the most commonly deregulated pathways in human cancers. PI3K comprises a catalytic (p110α) and regulatory subunit (p85), and p110α is encoded by the PIK3CA gene. Here, we summarize the known genetic alterations, including amplifications and mutations, of the PIK3CA oncogene in oral cancer. We discuss in detail PIK3CA mutations and their mutual exclusivity with pathway genes in addition to the incidence of PIK3CA mutations in relation to ethnicity. We describe the constitutive activation of PI3K signaling, oncogenicity, and the genetic deregulation of the PIK3CA gene and its association with oral cancer disease stage. We emphasize the importance of therapeutically targeting the genetically deregulated PIK3CA oncogene and its signaling. We also discuss the implications of targeting Akt and/or mTOR, which are the downstream effectors of PI3K that may possibly pave the way for molecular therapeutic targets for PIK3CA-driven oral carcinogenesis. Furthermore, this critical review provides a complete picture of the PIK3CA oncogene and its deregulation in oral cancer, which may facilitate early diagnosis and improve prognosis through personalized molecular targeted therapy in oral cancer.

Abstract 1109: c-Cbl ubiquitination of p85 is essential for EpoR downregulation.

Abstract Impaired deactivation of growth factor receptors is strongly associated with carcinogenesis. Among these receptors erythropoietin receptor (EpoR) is the main regulator for red blood cell production. EpoR−/− mice die of anemia, whereas constitutive EpoR signaling is oncogenic. Binding of Epo to the EpoR activates cytosolic JAK2 kinase, which in turn phosphorylates tyrosine residues in the EpoR cytoplasmic domain, to recruit and activate signaling proteins for erythropoiesis. Following this activation, EpoR is internalized and degraded to terminate the signaling. We have shown that Epo induces clathrin-dependent EpoR internalization and p85 regulatory subunit of PI3 Kinase (PI3K) plays a key role in this process. Interestingly, this mechanism does not require PI3K activity. One disease associated with this down-regulation mechanism is Primary familial and congenital polycythemia (PFCP). PFCP patients harbor truncated EpoRs, have increased red blood cell mass and their erythroid progenitors are hypersensitive to Epo. These truncated EpoR lack the binding site for p85 and restoring p85 binding site rescues EpoR internalization and corrects Epo hypersensitivity. However, the exact mechanism of how p85 aids in EpoR internalization is unclear. We observed that Epo induces p85 mono-ubiquitination. To test if ubiquitination of p85 regulates EpoR internalization, we generated p85 truncation mutants to identify the domains required for binding to EpoR. We found that the C-terminal SH2 domain and p110 binding domain of p85 interact with the EpoR. c-Cbl-/- mice were reported to have erythroid hyperplasia and c-Cbl was shown to bind p85. We hypothesized that upon stimulation c-Cbl ubiquitinates p85, which recruits endocytic proteins such as Epsin for EpoR down-regulation. In an in vitro kinase assay, c-Cbl can be directly phosphorylated by wild-type but not kinase-deficient JAK2. In addition, Y731 of c-Cbl became phosphorylated upon Epo stimulation which is in the pYXXM p85 binding motif. Importantly, Epo induced EpoR internalization was abolished in cells knock down of c-Cbl by siRNA or in mouse embryonic fibroblasts (MEFs) isolated from c-Cbl knockout animals. Two ligase-deficient c-Cbl mutants,ΔY368 and ΔY371 both had a dominant negative effect on the internalization of EpoR. Together, our results show that c-Cbl, and its ligase activity, play a crucial role in Epo-induced EpoR internalization. Secondly, we observed that upon Epo stimulation EpoR co-localizes with Epsin1 on the plasma membrane. We examined if PFCP EpoR recruits Epsin1 upon Epo stimulation. Consistent with our hypothesis that Epsin1 is recruited to the EpoR by ubiquitinated p85, PFCP EpoR does not co-localize with Epsin1 on the plasma membrane upon Epo stimulation. Studying molecular players and mechanisms in EpoR down-regulation will shed light onto our understanding of hematological malignancies. Citation Format: Gamze B. Bulut, Lily Huang. c-Cbl ubiquitination of p85 is essential for EpoR downregulation. [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 1109. doi:10.1158/1538-7445.AM2013-1109

Abstract 5155: Gain of direct interaction with IRS1 by the helical domain mutations of p110 alpha is crucial for their oncogenic functions.

Abstract PIK3CA, which encodes the catalytic subunit of phosphoinositide 3-kinase PI3Kα (p110α), is frequently mutated in human cancers. PI3Kα consists of a catalytic subunit p110α and one of several regulatory subunits (a major one being p85α). The p110α subunit contains an N-terminal adaptor-binding domain (ABD), a Ras-binding domain (RBD), a C2 domain, a helical domain and a catalytic domain. In the basal state, the regulatory p85 subunit stabilizes the catalytic p110α subunit and inhibits its enzymatic activity. Upon growth factor stimulation, the SH2 domains of p85 bind to the phospho-tyrosine residues on the receptor protein kinases or adaptor proteins such as insulin receptor substrate 1 (IRS1), thereby activating the lipid kinase activity of PI3Kα. Activated PI3Kα converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3). The second messenger PIP3 then activates downstream AKT signaling. The majority of these mutations occur at two hot-spots located in the helical (E545K) and the kinase (H1047R) domains. Here, we report that p110α E545K mutant proteins, but not the wild-type and H1047R mutant proteins, directly associate with IRS1 and that this interaction does not require the p85 regulatory subunit of PI3Kα. Disruption of IRS1-p110α E545K specific interaction results in reduction of AKT phosphorylation and slower xenograft tumor growth of a colon cancer cell line harboring this mutation. Moreover, IRS1- p110α E545K interaction stabilizes p110α proteins and brings it cytoplasmic membrane. Our study establishes a new paradigm whereby a mutant protein exerts its oncogenic functions through rewiring an oncogenic signaling pathway. Moreover, we designed an 18 amino acid hydrocarbon-stapled peptide encompassing the p110α E545K mutation that disrupts IRS1-p110α E545K interaction. The stapled mutant peptide reduces AKT phosphorylation in cancer cell lines with a p110α E545K mutation, whereas it has no effect on those cancer cell lines harboring a p110α H1047R mutation. When injected into xenograft tumors, the stapled peptide specifically inhibits growth of tumors with a p110α E545K mutation. Therefore, our data suggest that interference of IRS1-p110α E545K interaction may be exploited for targeted therapy of cancer patients harboring this mutation. The discovery of frequent mutations of PIK3CA in human cancer provides a strong rationale for inhibition of mutated p110α activities for targeted cancer therapy. However, it has remained a challenge to develop p110α isoform-specific inhibitors. Our data suggest that disruption of the interactions between helical domain mutations of p110α and IRS1 may be exploited as a more accessible targeted therapy approach. Citation Format: Yujun Hao, Chao Wang, Weiping Zheng, David Sedwick, Sanford Markowitz, Rob Ewing, Lili Liu, Zhenghe Wang. Gain of direct interaction with IRS1 by the helical domain mutations of p110 alpha is crucial for their oncogenic functions. [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 5155. doi:10.1158/1538-7445.AM2013-5155

Activation of PI3K/Akt pathway by CD133-p85 interaction promotes tumorigenic capacity of glioma stem cells

The biological significance of a known normal and cancer stem cell marker CD133 remains elusive. We now demonstrate that the phosphorylation of tyrosine-828 residue in CD133 C-terminal cytoplasmic domain mediates direct interaction between CD133 and phosphoinositide 3-kinase (PI3K) 85 kDa regulatory subunit (p85), resulting in preferential activation of PI3K/protein kinase B (Akt) pathway in glioma stem cell (GSC) relative to matched nonstem cell. CD133 knockdown potently inhibits the activity of PI3K/Akt pathway with an accompanying reduction in the self-renewal and tumorigenicity of GSC. The inhibitory effects of CD133 knockdown could be completely rescued by expression of WT CD133, but not its p85-binding deficient Y828F mutant. Analysis of glioma samples reveals that CD133 Y828 phosphorylation level is correlated with histopathological grade and overlaps with Akt activation. Our results identify the CD133/PI3K/Akt signaling axis, exploring the fundamental role of CD133 in glioma stem cell behavior.

Keratin 8 and 18 Loss in Epithelial Cancer Cells Increases Collective Cell Migration and Cisplatin Sensitivity through Claudin1 Up-regulation

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

Allosteric activation of PI3Kα by oncogenic mutations

The gene encoding the p110α catalytic subunit of class IA phosphoinositide 3-kinase (PIK3CA) is one of the most frequently mutated genes in human cancer [1]. Somatic mutations are widely dispersed throughout every domain of the catalytic subunit (Fig ​(Fig1A).1A). Two “hot spot” mutants have been shown biochemically and structurally to activate lipid kinase activity by distinct mechanisms [2]. Rare mutations, which have been shown to play a role in endometrial cancers [3], also increase lipid kinase activity. The mechanism of activation of rare mutations that are distant from both the active site and membrane interface have remained ambiguous. We have recently determined a set of allosteric conformational events that occur upon activation of wild type PI3Kα using hydrogen/deuterium exchange mass spectrometry (HDX-MS), and we find that all oncogenic mutants studied activated lipid kinase activity by mimicking or enhancing these specific events [4]. Figure 1 Oncogenic mutants in PI3K The lipid kinase activity of the class IA PI3Ks is tightly regulated by the p85 regulatory subunit which stabilizes, inhibits, and allows activation downstream of receptor tyrosine kinases (RTKs) (Fig. 1A, 1B). The interaction between the catalytic and regulatory subunit is mediated by the interaction of the adaptor-binding domain (ABD) of p110 and the coiled coil iSH2 domain of p85 [5]. The enzyme is inhibited by contacts between the N-terminal SH2 domain (nSH2) of p85 and the C2, helical and lipid kinase domains of the p110 subunit [5, 6]. The binding of phosphorylated tyrosine residues in RTKs and their associated proteins by the nSH2 disrupts this inhibitory interface and activates the enzyme. The enzyme is also inhibited by contacts between the C2 of p110 and the iSH2 of p85 [7]. The C-terminal SH2 (cSH2) domain of p85 which inhibits the p110δ and p110β isoforms of PI3K has no inhibitory influence on the p110α isoform [8]. To determine the mechanism of activation by oncogenic mutants we first investigated conformational changes that occur in the wild type upon binding to tyrosine-phosphorylated peptides derived from RTKs (pY), and upon binding to phospholipid membranes [4]. We identified four distinct conformational events that occur in the wild type PI3Kα upon activation (Fig. ​(Fig.1C):1C): (i) Disruption of the nSH2/helical interface, (ii) Disruption of the C2/iSH2 interface, (iii) Movement of the ABD relative to the kinase domain, and (iv) Interaction of the lipid kinase domain with membranes. We propose that these events are involved in the transition of a cytosolic, inhibited form of p110α to an open, activated form on the membrane surface. Oncogenic mutants in the ABD (G106V), ABD-RBD (Ras-binding domain) linker (G118D), C2 (N345K, C420R), and the hot spot mutants in the helical (E545K) and kinase domains (H1047R) upregulated lipid kinase activity by mimicking or enhancing one or more of the conformational events identified in the wild type. Mutations in the ABD and ABD-RBD linker caused both enhanced movement of the ABD domain relative to the kinase domain, and disruption of the C2/iSH2 interface compared to the WT enzyme. This same relationship was seen in mutations in the C2 domain, which also caused disruption of the C2/iSH2 interface along with movement of the ABD relative to the kinase domain. This shows that these two regions are allosterically linked to each other. These mutations can be activated by RTK derived pY, and are hyperactivated compared to the WT enzyme. The presence of the helical domain hotspot mutation located at the nSH2/helical interface fully disrupted this contact and made the mutant insensitive to pY activation, as predicted from previous biochemical and structural data [5, 6]. The kinase domain hotspot mutation caused conformational changes in the C-lobe of the kinase domain located at the membrane interface. Interestingly, the regions of the kinase domain that were exposed by this mutation are the exact same regions that were protected by the cSH2 domain in the p110β and p110δ isoforms [8]. This raises an interesting possibility that p110α has acquired inhibitory mutations in the kinase domain that enable it to bypass cSH2 mediated inhibition that occurs in the p110β and p110δ isoforms. The important role of oncogenic mutants of PI3Kα in the development of a variety of human cancers makes the development of novel inhibitory strategies an important goal. The discovery of these unexpected allosteric conformational changes that are enhanced in oncogenic mutants opens up a novel strategy towards designing small molecule inhibitors that function outside of the conserved ATP binding pocket.

Insulin Resistance and Muscle Metabolism in Chronic Kidney Disease

Insulin resistance is a common finding in chronic kidney disease (CKD) and is manifested by mild fasting hyperglycemia and abnormal glucose tolerance testing. Circulating levels of glucocorticoids are high. In muscle, changes in the insulin signaling pathway occur. An increase in the regulatory p85 subunit of Class I phosphatidylinositol 3-Kinase enzyme leads to decreased activation of the downstream effector protein kinase B (Akt). Mechanisms promoting muscle proteolysis and atrophy are unleashed. The link of Akt to the ubiquitin proteasome pathway, a major degradation pathway in muscle, is discussed. Another factor associated with insulin resistance in CKD is angiotensin II (Ang II) which appears to induce its intracellular effects through inflammatory cytokines or reactive oxygen species. Skeletal muscle ATP is depleted and the ability of AMP-activated protein kinase (AMPK) to replenish energy stores is blocked. How this can be reversed is discussed. Interleukin-6 (IL-6) levels are elevated in CKD and impair insulin signaling at the level of IRS-1. With exercise, IL-6 levels are reduced; glucose uptake and utilization are increased. For patients with CKD, exercise may improve insulin signaling and build up muscle. Treatment strategies for preventing muscle atrophy are discussed.

The proline‐rich tyrosine kinase Pyk2 regulates platelet integrin αIIbβ3 outside‐in signaling

The proline-rich tyrosine kinase Pyk2 is a focal adhesion kinase expressed in blood platelets, and is activated downstream of G-protein coupled receptors as well as integrin α2β1. In this study we have investigated the involvement of Pyk2 in integrin αIIbβ3 outside-in signaling in human and murine platelets. We analyzed the stimulation of intracellular signaling pathways in platelets from Pyk2 knockout mice adherent to immobilized fibrinogen. Pyk2 was rapidly phosphorylated and activated in human and murine platelets adherent to fibrinogen through integrin αIIbβ3. Activation of Pyk2 was Src-dependent, but did not require phospholipase Cγ2 activity. Platelets from Pyk2 knockout mice showed a defective ability to adhere and spread on fibrinogen, in association with a dramatic reduction of phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation. Pharmacological and genetic analysis demonstrated that integrin αIIbβ3 engagement selectively stimulated the β-isoform of PI3K (PI3Kβ), and that, as for Pyk2, PI3Kβ activation required Src family kinases activity, but not phospholipase Cγ2. In fibrinogen-adherent platelets, both Pyk2 and PI3Kβ were necessary for stimulation of the small GTPase Rap1b, a regulator of cell adhesion and spreading. Integrin αIIbβ3 engagement triggered the association of the PI3Kβ regulatory subunit p85 with the adaptor protein c-Cbl, which was mediated by the p85 SH3 domain, and was independent of c-Cbl tyrosine phosphorylation. However, p85-associated c-Cbl was tyrosine phosphorylated by activated Pyk2 in fibrinogen adherent platelets. These results identify a novel pathway of integrin αIIbβ3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen.

Targeted Genome-Wide Methylation and Gene Expression Analyses Reveal Signaling Pathways Involved in Ovarian Dysfunction after Developmental EDC Exposure in Rats1

Transient exposure to methoxychlor (MXC), an environmental endocrine-disrupting chemical, during fetal and neonatal stages causes ovarian dysfunction in pubertal, adult, and aging animals. Adult animals have reduced number of ovulations and abnormal follicular composition associated with altered gene expression and DNA methylation patterns. To test the hypothesis that the ovarian epigenomic changes induced by MXC are detectable following the exposure period, leading to altered gene expression by adulthood, we conducted a targeted genome-wide methylation study using Nimblegen 3x720K CpG Island Plus RefSeq Promoter Arrays. Control (vehicle), low-dose MXC (20 μg/kg/day), or high-dose MXC (100 mg/kg/day) treatments were administered between Embryonic Day 19 and Postnatal Day (PND) 7. Ovaries were collected at PND 7 immediately after exposure or at adulthood, PND 60. Array hybridizations were conducted with genomic DNA after methylated DNA immunoprecipitation and the array data were analyzed. DNA methylation events were functionally annotated, and candidate loci common to all the treatments or unique to some treatments were identified. Specific loci encoding signaling molecules such as the regulatory subunit p85 of phosphoinositide-3-kinase, insulin-like growth factor-1 receptor, Harvey rat sarcoma viral oncogene, insulin receptor, and forkhead box protein O3 were identified to be hypermethylated in MXC-treated ovaries at PND 7 and/or PND 60. Examination of gene expression changes with TaqMan low-density arrays revealed that nearly 25% of the genes that were assayed were downregulated. These data demonstrate that key molecules in specific signaling pathways such as PTEN signaling, IGF-1 signaling, or rapid estrogen signaling are epigenetically altered in MXC-exposed ovaries, which is associated with ovarian dysfunction and female infertility.

Gab Docking Proteins in Cardiovascular Disease, Cancer, and Inflammation

The docking proteins of the Grb2-associated binder (Gab) family have emerged as crucial signaling compartments in metazoans. In mammals, the Gab proteins, consisting of Gab1, Gab2, and Gab3, are involved in the amplification and integration of signal transduction evoked by a variety of extracellular stimuli, including growth factors, cytokines, antigens, and other molecules. Gab proteins lack the enzymatic activity themselves; however, when phosphorylated on tyrosine residues, they provide binding sites for multiple Src homology-2 (SH2) domain-containing proteins, such as SH2-containing protein tyrosine phosphatase 2 (SHP2), phosphatidylinositol 3-kinase regulatory subunit p85, phospholipase Cγ, Crk, and GC-GAP. Through these interactions, the Gab proteins transduce signals from activated receptors into pathways with distinct biological functions, thereby contributing to signal diversification. They are known to play crucial roles in numerous physiological processes through their associations with SHP2 and p85. In addition, abnormal Gab protein signaling has been linked to human diseases including cancer, cardiovascular disease, and inflammatory disorders. In this paper, we provide an overview of the structure, effector functions, and regulation of the Gab docking proteins, with a special focus on their associations with cardiovascular disease, cancer, and inflammation.

Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway

Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.