P38 Beta Research Articles

Visible light accelerates skin wound healing and alleviates scar formation in mice by adjusting STAT3 signaling

During the wound healing process, the activation of signal transducer and activator of transcription 3 (STAT3) is considered crucial for the migration and proliferation of epithelial cells, as well as for establishing the inflammatory environment. However, an excessive STAT3 activation aggravates scar formation. Here we show that 450 nm blue light and 630 nm red light can differentially regulate the phosphorylation of STAT3 (p-STAT3) and its downstream cytokines in keratinocytes. Further mechanistic studies reveal that red light promotes wound healing by activating the PI3 kinase p110 beta (PI3Kβ)/STAT3 signaling axis, while blue light inhibits p-STAT3 at the wound site by modulating cytochrome c-P450 (CYT-P450) activity and reactive oxygen species (ROS) generation. In a mouse scar model, skin wound healing can be significantly accelerated with red light followed by blue light to reduce scar formation. In summary, our study presents a potential strategy for regulating epithelial cell p-STAT3 through visible light to address skin scarring issues and elucidates the underlying mechanisms.

Interleukin 33-mediated inhibition of A-type K+ channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice.

Background: Interleukin-33 (IL-33) has been implicated in nociceptive pain behaviors. However, the underlying molecular and cellular mechanisms remain unclear.Methods: Using electrophysiological recording, immunoblot analysis, immunofluorescence labeling, reverse transcription-PCR, siRNA-mediated knockdown approach and behavior tests, we determined the role of IL-33 in regulating sensory neuronal excitability and pain sensitivity mediated by A-type K+ channels.Results: IL-33 decreased A-type transient outward K+ currents (IA) in small-sized DRG neurons in a concentration-dependent manner, whereas the delayed rectifier currents (IDR) remained unaffected. This IL-33-induced IA decrease was dependent on suppression of the tumorigenicity 2 (ST2) receptor and was associated with a hyperpolarizing shift in the steady-state inactivation. Antagonism of Syk abrogated the IL-33-induced IA response, while inhibition of JAK2 and PKA elicited no such effect. Exposure of DRG cells to IL-33 increased the activity of Akt, but surprisingly, neither Akt nor PI3K influenced the IL-33-induced IA response. IL-33 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK). Chemical inhibition of p38 and genetic siRNA knockdown of p38 beta (p38β), but not p38α, abrogated the IA response induced by IL-33. Moreover, IL-33 increased neuronal excitability of DRG neurons and facilitated peripheral pain sensitivity in mice; both of these effects were occluded by IA blockade.Conclusions: Our present study reveals a novel mechanism by which IL-33/ST2 suppresses IA via a Syk-dependent p38β signaling pathway. This mechanism thereby increases DRG neuronal excitability and pain sensitivity in mice. Targeting IL-33/ST2-mediated p38β signaling may represent a therapeutic approach to ameliorate pain behaviors.

P38 MAPK Isoforms as Modulators of Oncogenic Phenotypes in Inflammatory Breast Cancer

Inflammatory Breast Cancer (IBC) is a rare, aggressive, and highly metastatic subtype of breast cancer; accounting for approximately 1–6% of all breast cancer diagnoses in the United States and 7–10% of breast cancer‐associated deaths in Western countries. The aggressiveness of this disease is generally attributed to the formation of diffuse tumor cell clusters, known as tumor emboli, that block the dermal lymphatic vessels near the chest wall, a key hallmark of IBC. Clinically, it has been documented that most of IBC cases are either HER2 amplified or Triple Negative (TN‐IBC), the latter being the most difficult to treat. As a result, there is a dire need to understand the molecular profile of IBC to further develop more efficient targeted therapies. It has been reported that 17β‐Estradiol (E2) exerts non‐genomic signaling in ER‐negative IBC and non‐IBC cells using estrogen receptors GPR30 and estrogen receptor alpha variant 36 (ERα‐36), which activate the PI3K/AKT/ERK pathways involved in oncogenic phenotypes. p38 isoforms alpha, beta, gamma, and delta have been demonstrated to modulate proliferation, migration, apoptosis, and cell senescence via stress‐induced pathways or mitogen‐activated pathways. Also, it has been shown that these isoforms have context‐dependent functions depending on tissue type, expression and activation level, and the pathways involved. Nevertheless, the effects of these isoforms have yet to be elucidated in IBC. Preliminary data suggest that estrogen mediates p38 activation when IBC cells are treated with E2. For this reason, we hypothesize that estrogenmediated activation is involved in p38‐induced oncogenesis via non‐genomic signaling in an isoform‐dependent manner in IBC cell lines. A comparison of p38 isoforms expression was done on both IBC and non‐IBC cell lines via Western blot and qRT‐PCR, presenting differential expression profiles. This data was then used to design a knockdown model to study the effects of the most predominant isoforms on oncogenic phenotypes. Treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines when performing a dose response curve. Ongoing studies include assessing the effects of p38 inhibition and knockdown on proliferation, invasion, migration, and tumor emboli formation under basal and endocrine conditions. To recapitulate, understanding E2 responsiveness by ER‐negative cell lines may provide great insight into novel targeted therapies using the non‐genomic signaling pathway; in particular, p38 signaling appears to be a key effector of prooncogenesis in IBC.

MAP KINASE-ACTIVATED PROTEIN KINASE-2 IS NOT ESSENTIAL DURING THE INFLAMMATORY PHASE OF WOUND REPAIR POST-MYOCARDIAL INFARCTION

Inflammation is a key element in the early phase of reparative fibrosis following myocardial infarction (MI). Altering the onset, intensity, or duration of inflammation can dramatically impact the heart's structure and function. A weak inflammatory response may lead to fatal myocardial rupture due to excessive ECM degradation or impaired scar formation. An exaggerated or prolonged inflammatory response could cause excessive scar formation, resulting in increased LV stiffness. MAP kinase-activated protein kinase-2 (MK2), a protein serine/threonine kinase, is activated by p38 alpha and beta in response to cellular stress.

Abstract P2-03-06: PTEN expression at the nexus of oncogenic signals in TNBC: Testing combination of p110beta-isoform-specific inhibitor with five PARP inhibitors

Abstract Background: TNBC is the most aggressive form of BRCA-associated BC subtype. The loss of PTEN is a common “first event” associated with basal-like subtype (Martins et al., 2012) and this mode of PI3K-pathway activation (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA (5-10%) mutations in TNBC (Ellis & Perou, 2013). Several reports suggest upregulation of PI3K/AKT mediated by PTEN loss depends on the PI3Kbeta-isoform. PARP1 is identified as a target of BRCA-defined cancers. Inhibitors of PARP1 have been recently approved by the FDA as targeted agents in BRCA-defined breast cancers. Failure to repair damaged DNA upon PARP inhibition causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Also, nuclear PTEN controls DNA repair (Bassi et al., 2013). Here we hypothesized that a combination of PARP inhibitor with p110beta-isoform-specific inhibitor would sensitize the effect of PARP inhibitor(s) in PTEN-deprived TNBC model. Methods: We tested five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) in combination with p110beta specific inhibitor, AZD6482. Four PTEN-null TNBC cell lines (BRCA WT/null), MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines were used for the study. Proliferative, apoptotic and PARylation signals following drug combinations were demonstrated by WB in a dose and time-dependent manner. Pro-apoptotic and anti-proliferative effects were verified using complementary 3D ON-TOP assay, real-time proliferation (Incucyte), AnnexinV and cl-caspase3 analyses. As an internal control, we also compared anti-proliferative signals of GDC-0032 (p110 beta sparing inhibitor) and GDC-0941 (pan PI3K inhibitor) with that of AZD6482 at 3/6 hours in MDA-MB468 and SUM149 cells. Mode of apoptosis was tested using triple fluorescence staining in live cells. Results: A dose of 500 nM BMN673 alone was effective in slowing cell proliferation and induced apoptosis. AZD6482 (5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. When compared, combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 abrogated 3D growth in BT549 and MDA-MB468 TNBC cells in a time-dependent manner. The effect of PARP inhibitor was tested by PAR signals which were abrogated either alone or in combination with carboplatin in both BRCA1/2 WT and mutated cells. The most pronounced anti-tumor effect was observed with the combination of B and AZD6482 which was mechanistically explained by the robust increase of AnnexinV positive cells following a single 500nM dose of B at both 48 and 72 hours. Pan PI3K inhibitor, GDC-0941, and AZD6482 blocked the activation of PI3K and its downstream effectors in contrast to p110 alpha-specific inhibitor, GDC-0032 in MDA-MB468 and SUM149 cells. Summary: We demonstrated a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identified PTEN-nullness as a potential predictive biomarker for a possible co-targeting of the PI3K pathway to further sensitize TNBC to PARP inhibitors. Citation Format: Dey N, Carlson JH, De P, Leyland-Jones B. PTEN expression at the nexus of oncogenic signals in TNBC: Testing combination of p110beta-isoform-specific inhibitor with five PARP inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-06.

Functions of p38 mitogen-activated protein kinase alpha and beta in human pancreatic cancer cells

Objective To elucidate possible functions of p38α and p38β in human pancreatic cancer. Methods Expression of p38α, p38β, p38γ and p38δ were determined by immunoblot in 7 cultured human pancreatic cancer cell lines. To selectively inhibit p38α or p38β, cell clones were established in a stable manner expression either p38α short hairpin RNA (shRNA) or p38β shRNA. Proliferation, single cell movement, in vitro migration, colony forming ability as well as in vivo tumorigenicity were determined by methyl thiazol tetrazolium (MTT) assay, video-time laps microscopy, boyden-chamber, soft-agar assay, tumor formation and growth in nude mice respectively. Results All pancreatic cancer cells showed p38 isoforms protein expression at various levels. Selective inhibition of p38α or p38β in Mia Paca-2 and Panc-1 cells revealed that both kinases enhance cell proliferation and anchorage-independent growth. Single cell movement and cell invasion were markedly reduced in p38α shRNA expressing clones, but not altered in p38β shRNA expressing clones. Inhibition of p38α enhanced in vivo tumorigenicity profoundly in Mia Paca-2 and Panc-1 cells. In Mia Paca-2 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 18.3% and 50.0% respectively, which decreased significantly comparing to the wild type cell and null vector transfection cell. The tumor volume decreased significantly either (P<0.05). In Panc-1 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 25.0% and 12.5% respectively. Conclusion p38 mitogen activated protein kinases (p38MAPKs) are expressed in pancreatic cancer cells and the isoforms may exert different functions. Key words: p38 mitogen-activated protein kinase; Pancreatic cancer; p38α; p38β

Low-molecular weight keratins with anti-skin aging activity produced by anaerobic digestion of poultry feathers with Fervidobacterium islandicum AW-1

Bioactive peptides contribute to various cellular processes including improved skin physiology. Hence, bioactive keratins have attracted considerable attention as active cosmetic ingredients for skin health. Here, we obtained low molecular weight (LMW) keratins from native chicken feathers by anaerobic digestion with an extremely thermophilic bacterium Fervidobacterium islandicum AW-1, followed by stepwise fractionation through ultrafiltration. To assess the effects of the feather keratins on skin health, we performed in vitro and ex vivo assays to investigate their inhibitory effects on matrix metalloproteinases (MMPs). As results, LMW feather keratins marginally inhibited collagenase, elastase, and radical scavenging activities. On the other hand, LMW feather keratins significantly suppressed the expression of ultraviolet B (UVB)-induced MMP-1 and MMP-13 in human dermal fibroblasts. Furthermore, phospho-kinase antibody array revealed that LMW feather keratins suppressed UVB-induced phosphorylation of Akts, c-Jun N-terminal kinases 1, p38 beta, and RSK2, but not ERKs in human dermal fibroblast. Overall, these results suggest that LMW feather keratins are potential candidates as cosmeceutical peptides for anti-skin aging.

Identification of the Binding Sites on Rab5 and p110beta Phosphatidylinositol 3-kinase

Rab5 is a small monomeric GTPase that mediates protein trafficking during endocytosis. Inactivation of Rab5 by GTP hydrolysis causes a conformational change that masks binding sites on its “switch regions” from downstream effectors. The p85 subunit of phosphatidylinositol 3-kinase (PI3K) is a GTPase activating protein (GAP) towards Rab5. Whereas p85 can bind with both Rab5-GTP and Rab5-GDP, the PI3K catalytic subunit p110β binds only Rab5-GTP, suggesting it interacts with the switch regions. Thus, the GAP functions of the catalytic arginine finger (from p85) and switch region stabilization (from p110β) may be provided by both proteins, acting together. To identify the Rab5 residues involved in binding p110β, residues in the Rab5 switch regions were mutated. A stabilized recombinant p110 protein, where the p85-iSH2 domain was fused to p110 (alpha or beta) was used in binding experiments. Eleven Rab5 mutants, including E80R and H83E, showed reduced p110β binding. The Rab5 binding site on p110β was also resolved through mutation of p110β in its Ras binding domain, and includes residues I234, E238 and Y244. This is a second region within p110β important for Rab5 binding. The Rab5-GTP:p110β interaction may be further elucidated through the characterization of these non-binding mutants in cells.

Abstract 1348: GDC-0032, a p110 beta sparing PI3K inhibitor is highly efficient on PIK3CA-mutated and HER2-amplified breast cancer model

Abstract Background: HER2 amplification/overexpression frequently co-occurs with PI3K pathway activation in breast tumors. Mutations in PIK3CA are known to be involved in a wide range of human cancers including HER2+ breast cancer, and mutant PIK3CA is thought to act as an oncogene. Here, we evaluate the efficacy of GDC0032, a p110 beta-sparing PI3K inhibitor against HER2+ or HER2+/PIK3CA mutated breast cancer cells. MATERIALS AND METHODS: Four HER2+ breast cancer (BC) cell lines consisting of three activating (either helical or kinase domain) PIK3CA mutation (HER2+/ER+ and HER2+/ER-) were analyzed for proliferation, apoptosis, cell cycle and signaling pathway activation assays. Preclinical efficacy of GDC-0032 was also evaluated in vivo in a mouse model. RESULTS: 1) All HER2+ and HER2+/PIK3CA mutant cell lines exhibited low IC50 values (ranging from 0.1 µM-1.5 µM) irrespective of ER-positive or not. 2) GDC-0032 caused a strong differential growth inhibition in both HER2+ and HER2+/ /PIK3CA mutated breast cancer cell lines when compared to lines that were HER2- and PIK3CA wild type by 3D-ON-TOP clonogenic assay. 3) Administration of GDC-0032 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner. Furthermore, induction of apoptosis was more with GDC-0032 when combined with RAD001. 4) GDC0032 also blocked expression of CYCLIN D1. 5) Investigation of the signal transduction revealed that the treatment of GDC-0032 reduced the level of p-AKT (Ser473 and Thr308), and p-S6 expression is indicating the down-regulation of downstream signaling of PI3K and mTOR pathway. 6) GDC-0032 was highly active at reducing established tumor growth in vivo in BC mouse xenografts harboring PIK3CA mutation and HER2 amplification. CONCLUSION: Our in vitro or in vivo data showed that GDC-0032 was highly efficient either as a single agent as well as with RAD001 in HER2+/PIK3CA mutated breast cancer cell lines. Our data suggest that GDC-0032 represents a novel therapeutic option in patients harboring PIK3CA mutations and/or HER2/neu gene amplification in breast cancer. Citation Format: Pradip De, Yuliang Sun, Jennifer Carlson, Lori Friedman, Casey Williams, Nandini Dey, Brian Leyland-Jones. GDC-0032, a p110 beta sparing PI3K inhibitor is highly efficient on PIK3CA-mutated and HER2-amplified breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1348. doi:10.1158/1538-7445.AM2017-1348

Potential Effects of javamide‐I found in coffee on inflammatory chemokine MCP‐1 in rats fed a high fat diet.

Javamide‐I is a phenolic amide found in coffee with potential health effects. Monocyte chemoattractant protein‐1 (MCP‐1) is a well‐known chemokine and its reduction is considered beneficial in suppressing the pathophysiological progression of cardiovascular, kidney and other diseases, commonly found in the obese. Therefore, in this paper, we investigated javamide‐I as a potential p38 MAP kinase inhibitor, because the kinase is critically involved in the expression of MCP‐1. At the concentration of 20 μM, javamide‐I significantly inhibited p38 MAP kinase alpha, beta, gamma and delta by 15–50% (P &lt; 0.05), particularly p38 MAP kinase alpha (IC50 = 19.2 μM) and beta (IC50 =19.5 μM). We also found that monocytic THP‐1 cells treated with javamide‐I (10, 20 and 40 μM) led to significant inhibition of MCP‐1 production by 17–49% (P &lt; 0.05) in the cells, suggesting that javamide‐I may decrease MCP‐1 expression via inhibiting p38 MAP kinase. Due to the positive in vitro data, the potential effect of javamide‐I on MCP‐1 expression was also investigated in an obesity‐induced rat model. For this study, animals were divided into three groups (n=10): a normal control diet (C), a high fat diet (HF), or a high fat diet supplemented with javamide‐I (2 mg per day) (HFS). After 16 weeks, blood samples were collected from the animals in each group and the plasma levels of MCP‐1 protein were determined. As expected, the average MCP‐1 level of the HFS group was significantly lower than that of the HF group (P &lt; 0.05), although the average MCP‐1 level of the HF group was found to be higher than that of the C group (P &lt; 0.05). In summary, the data suggest that javamide‐I found in coffee may inhibit MCP‐1 expression, possibly via inhibiting p38 MAP kinase and its supplementation could suppress plasma MCP‐1 expression critically involved in several inflammatory diseases.Support or Funding InformationUSDA (1235‐51000‐054‐00D)

Abstract B12: Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC

Abstract Background: TNBC is a highly aggressive form of BRCA-associated BC subtype for which chemotherapy still remains a major form of treatment to which patients initially respond yet a majority of them inevitably relapse. PARP1 has been identified as a target in BRCA-defined cancers and inhibitors of PARP1 has been recently approved by FDA as targeted agents in BRCA-defined cancers. The loss of PTEN is the most common “first event” associated with basal-like subtype (Martins et al., 2012) and this PI3K-pathway activating event (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA mutations in this subtype of BC (Ellis &amp; Perou, 2013). In the cytoplasm, PTEN-loss mediated upregulation of PI3K/AKT signal is known to depend on the PI3Kbeta-isoform. In contrast nuclear PTEN controls DNA repair (Bassi et al., 2013). A failure to repair the damaged DNA upon inhibition of PARP causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Thus we hypothesized that a combination of p110beta-isoform specific inhibitor and PARP inhibitor will sensitize PARP inhibitors in PTEN-null TNBC model. Methods: Five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) were tested in four PTEN-null TNBC cell lines, MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines in combination with p110beta specific inhibitor, AZD6482. Proliferative, apoptotic and PARylation signals following drug combinations were demonstrated by WB in a dose and time dependent manner. Pro-apoptotic and anti-proliferative effects were verified using complementary 3D ON-TOP assay, real time proliferation (Incucyte), AnnexinV and cl-caspase3 analyses. In order to understand the initial effects of isoform-specific inhibitors of PI3K we also compared anti-proliferative signals of GDC-0032 (p110 beta sparing inhibitor) and GDC-0941 (pan PI3K inhibitor) with that of AZD6482 at 3/6 hours in MDA-MB468 and SUM149 cells. Results: In PTEN null TNBC cells 500 nM BMN673 alone was effective in slowing cell proliferation and inducing apoptosis while AZD6482 (both 5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. Combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 blocked 3D growth of PTEN null BT549 and MDA-MB468 TNBT cells in a time dependent manner (96 hours &amp; 7 days). PARP inhibitors abrogated PAR signals either alone or in combination with carboplatin in both BRCA1/2 WT and mutated cells. The most pronounced anti-tumor effect was observed with the combination of B and AZD6482 which was mechanistically explained by the robust increase of AnnexinV positive cells following a single 500nM dose of B at both 48 and 72 hours. In both MDA-MB468 and SUM149 cells although panPI3K inhibitor, GDC-0941 downregulated pAKT (S473, T308), pP70S6K (T389), pS6RP and p4EBP1 (T37/46) levels, p110 alpha-specific inhibitor GDC-0032 failed to perturb these signals while AZD6482 blocked them all. In summary, we demonstrate a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identify that PTEN-nullness as a potential predictive context for a possible co-targeting of PI3K pathway to further sensitize TNBC to PARP inhibitors. Significance: Our findings squarely place PTEN expression at the nexus of oncogenic signals in TNBC and indicate that TNBC patients with no PTEN activity in their tumors might benefit from combined p110beta-isoform specific inhibitor and PARP inhibitor. Citation Format: Nandini Dey, Jennifer H. Carlson, Pradip De, Brian Leyland-Jones. Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B12.

Abstract B26: PI3K class I and mTOR regulate distinct steps in Met dependent tumorigenesis

Abstract The Receptor Tyrosine Kinase (RTK) Met, overexpressed or mutated in cancer, plays a major role in cancer progression and represents an attractive target for cancer therapy. Although several Met inhibitors are in clinical trials, resistance may occur as experienced in the clinic with other RTKs. Thus, a need for alternative / complementary therapy may be required to target Met driven tumors efficiently. The aim of this study was to investigate whether PI3K plays a role in Met oncogenic activity, in view of designing appropriate therapy. The study was performed on two cell models suitable to study Met driven NIH3T3 cells expressing Met Wt or the constitutively active mutant M1268T and the glioblastoma U87MG cell line, with Met constitutively activated due to an autocrine loop. Transwell cell migration and anchorage independent growth in soft agar were studied in presence of inhibitors targeting pan-PI3K (LY294002 and wortmannin), isoform-specific PI3K (GDC0941, pan-PI3K class I, A66, p110 alpha, TGX221, p110 beta) and mTOR (rapamycin), or following siRNA knock down of PI3K isoforms. Tumor growth of cells grafted to nude mice was assessed in presence or absence of rapamycin. LY294002 reduced the migration and the anchorage independent growth of Met mutant M1268T expressing cells to the same level as with Met Wt expressing cells, while no difference was detected in Met Wt expressing cells. Inhibitions / knock downs of PI3K class I reduced the migration of Met M1268T expressing cells and UMG87 cells. Interestingly, it was determined that p110 alpha and beta together are responsible for Met dependent cell migration. Moreover, the phosphorylation (Ser473) of Akt, the downstream effector of PI3K class I, was significantly increased in Met M1268T versus Wt Met expressing cells. It was inhibited by Met pharmacological inhibition, indicating that Met M1268T activates Akt in these cells. Moreover, the inhibition of Met M1268T internalization using the dynamin inhibitor dynasore led to a significant reduction of Akt phosphorylation in these cells, suggesting further that Met M1268T activates Akt on endosome. Interestingly, the endosomal activation of Akt seems to be Appl1 independent. Surprisingly, inhibitions / knock downs of PI3K class I did not decrease the anchorage independent growth of Met M1268T expressing cells and of U87MG cells. Wortmannin also had no effect. However, our results using the mTOR inhibitor indicate that in fact, mTOR is responsible for Met dependent anchorage independent growth in Met M1268T expressing cells and U87MG cells. Moreover, rapamycin significantly reduced Met dependent tumor growth in vivo. Our results suggest that while PI3K class I (p110 alpha and beta) regulate oncogenic Met dependent cell migration, the anchorage independent growth and tumor growth are regulated by mTOR. These results may have implication for therapeutical intervention. Citation Format: Alexia Hervieu, Stephanie Kermorgant. PI3K class I and mTOR regulate distinct steps in Met dependent tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B26.

P38 and interleukin-1 beta pathway via toll-like receptor 4 contributed to the skin and muscle incision and retraction-induced allodynia

BackgroundPersistent postsurgical pain, as an important clinical problem, seriously affects the quality of life in patients. However, the mechanism underlying persistent postsurgical pain remains largely unclear. The present study aims to elucidate the involvement of toll-like receptor 4 (TLR4) and its interaction with p38 and interleukin [IL]-1β signaling in dorsal root ganglion (DRG) in the persistent postsurgical pain. MethodsSkin and muscle incision and retraction (SMIR) surgery-induced paw withdrawal threshold (PWT) change was determined by applying mechanical stimuli to the plantar surface of the hind paw using von Frey hairs. The PE-10 catheter intrathecal placement was used to deliver LPS-RS, interleukin-1 receptor antagonist, or SB203580. Western blot analysis was performed to measure the expression of the TLR4, mitogen-activated protein kinases family, and IL-1β in ipsilateral L3 and L4 DRG. Immunofluorescence staining was performed to further investigate the cell type of TLR4 expression. All data were expressed as means ± standard error of the mean and analyzed using SPSS 13.0. ResultsThe results showed that the SMIR surgery, a rat model of persistent postoperative pain, decreased the ipsilateral 50% PWT, and the decrease lasted for at least 20 d. The expression of TLR4 and phosphorylation of p38 were upregulated in ipsilateral L3 and L4 DRG neurons after SMIR surgery. Pretreatment with LPS-RS, an established TLR4 antagonist, prevented p38 activation and attenuated mechanical allodynia induced by SMIR surgery. In addition, the expression of IL-1β was significantly increased after SMIR surgery. Blocking IL-1β by interleukin-1 receptor antagonist significantly improved the decreased PWT evoked by SMIR. Moreover, inhibition of TLR4 or p38 pathway prevented the IL-1β upregulation and mechanical allodynia induced by SMIR. ConclusionsThese findings suggest that the activation of p38 and IL-1β signaling pathway via TLR4 mediate mechanical allodynia after SMIR surgery.

Profiling of phosphatidylinositol 3-kinase (PI3K) proteins in insulin signaling pathway.

Phosphoinositide 3-kinase (PI3K) enzyme plays a vital role in the insulin signaling pathway as well as in other pathways that are involved in the growth, migration, and survival of cells. In the insulin signaling pathway, PI3K proteins that include p50α, p85α, p85β, p55γ, p110α, p110β, and p110γ are associated with the critical node-2. This study has used bioinformatic tools to understand phylogenetics, conservation patterns, conserved domains, orientation of residues, and interactions among PI3K proteins. The phylogenetic analysis showed p110α and p110γ with a common origin while p50α and p85α sharing an evolutionary history. The sequence alignment showed the highest score (97) between p85α and p50α. Several highly conserved amino acid residues were found high in p110 beta (n = 102). Subsequently, the number of highly conserved amino acid restudies was low in p50alpha and p55γ (n = 15). The PI3K proteins are evidentially linked to other proteins and pathways as well.

MicroRNA 126 Inhibits the Transition of Endothelial Progenitor Cells to Mesenchymal Cells via the PIK3R2-PI3K/Akt Signalling Pathway

AimsEndothelial progenitor cells (EPCs) are capable of proliferating and differentiating into mature endothelial cells, and they have been considered as potential candidates for coronary heart disease therapy. However, the transition of EPCs to mesenchymal cells is not fully understood. This study aimed to explore the role of microRNA 126 (miR-126) in the endothelial-to-mesenchymal transition (EndMT) induced by transforming growth factor beta 1 (TGFβ1). Methods and ResultsEndMT of rat bone marrow-derived EPCs was induced by TGFβ1 (5 ng/mL) for 7 days. miR-126 expression was depressed in the process of EPC EndMT. The luciferase reporter assay showed that the PI3K regulatory subunit p85 beta (PIK3R2) was a direct target of miR-126 in EPCs. Overexpression of miR-126 by a lentiviral vector (lenti-miR-126) was found to downregulate the mRNA expression of mesenchymal cell markers (α-SMA, sm22-a, and myocardin) and to maintain the mRNA expression of progenitor cell markers (CD34, CD133). In the cellular process of EndMT, there was an increase in the protein expression of PIK3R2 and the nuclear transcription factors FoxO3 and Smad4; PI3K and phosphor-Akt expression decreased, a change that was reversed markedly by overexpression of miR-126. Furthermore, knockdown of PIK3R2 gene expression level showed reversed morphological changes of the EPCs treated with TGFβ1, thereby giving the evidence that PIK3R2 is the target gene of miR-126 during EndMT process. ConclusionsThese results show that miR-126 targets PIK3R2 to inhibit EPC EndMT and that this process involves regulation of the PI3K/Akt signalling pathway. miR-126 has the potential to be used as a biomarker for the early diagnosis of intimal hyperplasia in cardiovascular disease and can even be a therapeutic tool for treating cardiovascular diseases mediated by the EndMT process.

Abstract B218: Drug-drug interaction assessment of GDC-0032, a beta-sparing inhibitor of phosphoinositide 3-kinase (PI3K), using midazolam, a sensitive CYP3A4 substrate.

Abstract Background: GDC-0032 is an orally-bioavailable, potent, and selective inhibitor of Class IA PI3K alpha, delta, gamma, and p110 beta isoforms, with 30 fold less inhibition of the p110 beta isoform. GDC-0032 has shown promising preliminary clinical activity in PIK3CA-mutant cancers and is being explored in solid tumors and in combination with endocrine therapies in breast cancer. In vitro, GDC 0032 was not a potent inhibitor of CYP isoforms 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4 (IC50 &amp;gt; 30 µM) but displayed weak time-dependent inhibition of CYP3A4, with KI and kinact values of 77 µM and 0.030 min-1, respectively. In addition, GDC-0032 induced CYP3A4, but not CYP1A2 or CYP2B6 in human hepatocytes. Based on these data, the potential for GDC-0032 to impact the pharmacokinetics (PK) of midazolam (MDZ), a sensitive in vivo marker of CYP3A4 enzymatic activity, was assessed in a Phase I trial in patients with cancer. Methods: Twelve patients with advanced or metastatic solid tumors were enrolled. All patients received 5 mg MDZ on Day 1 (D1), followed by continuous daily dosing of GDC-0032 (9 mg) starting on D2. A second MDZ dose was given on D16. Predose, 0.5, 1, 1.5, 2, 3, 4, 8, and 24 hr post-dose plasma samples were collected on D1 and D16. Maximum concentration (Cmax) and area under the concentration-time curve (AUC0-24) of MDZ were estimated by non-compartmental methods using WinNonlin (version 5.2.1). MDZ plasma Cmax and AUC0-24 without GDC-0032 (D1) and after 15 daily doses of GDC-0032 (D16) were compared. The geometric mean ratios (GMRs) of Cmax and AUC0-24 and the corresponding 90% CIs were estimated (WinNonlin version 5.2.1). Physiologically-Based Pharmacokinetic (PBPK) modeling was used to prospectively predict the DDI potential of GDC-0032 (Simcyp version 11.0 and Gastroplus version 8.0). Results: No trend in individual Cmax or AUC0-24 of MDZ was observed post-GDC-0032 treatment relative to pre-treatment. The change in MDZ exposure (Cmax and AUC0-24) before and after GDC-0032 treatment was less than 2-fold for all patients (median: 1.1 fold; range: 0.4-1.9). The GMRs (90% CIs) for MDZ+GDC-0032 relative to MDZ alone for Cmax and AUC0-24 were 0.98 (0.70-1.37) and 1.04 (0.68-1.60), respectively. Prospective PBPK modeling accurately predicted a lack of marked DDI between GDC-0032 and MDZ (1-1.4 fold change). Conclusions: Despite in vitro results suggesting that GDC-0032 may impact the PK of concomitant medications which are metabolized by CYP3A4, co-administration with MDZ did not change the PK of MDZ in patients with cancer. PBPK modeling predicted little to no impact of GDC-0032 on the PK of MDZ, consistent with the in vivo observation. These results indicate that GDC-0032 may be given concomitantly with CYP substrates with minimal risk of a PK drug-drug interaction. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B218. Citation Format: Richard A. Graham, Sravanthi Cheeti, Laurent Salphati, Tong Lu, Carla Kurkjian, Daniel Von Hoff, Jasgit Sachdev, Dejan Juric, Jose Baselga, Hema Parmer, Jerry Hsu, Jeffrey Infante. Drug-drug interaction assessment of GDC-0032, a beta-sparing inhibitor of phosphoinositide 3-kinase (PI3K), using midazolam, a sensitive CYP3A4 substrate. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B218.

Expression of Developmental-Stage-Specific Genes in the Gilthead Sea Bream Sparus aurata L.

The mechanism of early fish development as well as the control of egg quality is of great importance for the ability of the oocyte to develop after fertilization. Embryonic development is initially regulated by maternally provided mRNAs and later by the zygotic genome. Maternal mRNAs have an important role in initiating processes crucial to patterning the developing fish embryo. Furthermore, it has been shown that maternal RNA plays an important role in egg quality. The identification and characterization of candidate maternal genes in non-model fish species with important aquaculture interest like the gilthead sea bream Sparus aurata L. is of importance for future studies related to egg quality. The broodstock of the gilthead sea bream produces large quantities of eggs with a high and non-controllable quality variation. In the present study, we have studied the gene expression of 16 genes (gapdh 1 and 2, cathepsin D, L, S and Z, erk1, jnk1, p38 alpha and p38 delta, ppar alpha, beta and gamma, tubulin beta, ferritin M, cyclinA2) of different functional categories in seven developmental stages. The 16 genes were chosen based on their putative involvement in egg quality and regulation of early development. In total, 11 showed a characteristic gene expression pattern pinpointing to the possible function as maternal genes and thus may function as molecular biomarker for egg quality.

Abstract 1639: Targeting the p38 MAPK pathway inhibits irinotecan resistance in colon adenocarcinoma

Abstract Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from the colon adenocarcinoma HCT-116 and SW48 cell lines. These clones show various levels (6 to 60 fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared to the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both alpha and beta isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which specifically inhibits p38 alpha and beta, enhanced the cytotoxic activity of SN38, but not of 5-FU or oxaliplatin. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared to non-responder patients. This indicates that enhanced level of expression of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show for the first time that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2011-1639

Chapter Nine - PI 3-Kinase Regulatory Subunits as Regulators of the Unfolded Protein Response

The endoplasmic reticulum (ER) consists of an interconnected, membranous network that is the major site for the synthesis and folding of integral membrane and secretory proteins. Within the ER lumen, protein folding is facilitated by molecular chaperones and a variety of enzymes that ensure that polypeptides obtain their appropriate, tertiary conformation (Dobson, C. M. (2004). Principles of protein folding, misfolding and aggregation. Semin. Cell Dev. Biol. 15, 3-16; Ni, M., and Lee, A. S. (2007). ER chaperones in mammalian development and human diseases. FEBS Lett. 581, 3641-3651.). Physiological conditions that increase protein synthesis or stimuli that disturb the processes by which proteins obtain their native conformation, create an imbalance between the protein-folding demand and capacity of the ER. This results in the accumulation of unfolded or improperly folded proteins in the ER lumen and a state of ER stress. The cellular response, referred to as the unfolded protein response (UPR), results in activation of three linked signal transduction pathways: PKR-like kinase (PERK), inositol requiring 1 α (IRE1α), and activating transcription factor 6α (ATF6α) (Ron, D., and Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell. Biol. 8, 519-529; Schroder, M., and Kaufman, R. (2005). ER stress and the unfolded protein response. Mutat. Res./Fundam. Mol. Mech. Mutagen. 569, 29-63.). Collectively, the combined actions of these signaling cascades serve to reduce ER stress through attenuation of translation to reduce protein synthesis and through activation of transcriptional programs that ultimately serve to increase ER protein-folding capacity. Recently, we and Park et al. have characterized a novel function for the p85α and p85β subunits as modulators of the UPR by virtue of their ability to facilitate the nuclear entry of XBP-1s following induction of ER stress (Park, S. W., Zhou, Y., Lee, J., Lu, A., Sun, C., Chung, J., Ueki, K., and Ozcan, U. (2010). Regulatory subunits of PI3K, p85alpha and p85 beta, interact with XBP1 and increase its nuclear translocation. Nat. Med. 16, 429-437; Winnay, J. N., Boucher, J., Mori, M. A., Ueki, K., and Kahn, C. R. (2010). A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box-binding protein-1 to modulate the unfolded protein response. Nat. Med. 16, 438-445.). This chapter describes the recently elucidated role for the regulatory subunits of PI 3-kinase as modulators of the UPR and provides methods to measure UPR pathway activation.

Influenza A viruses induce PI3-kinase activation by two interdependent mechanisms late in the infection cycle

We have previously shown that the cellular PI3K/Akt pathway is strongly activated upon influenza A virus infection in later stages of the infection cycle and that this activation was dependent on the expression of the viral non-structural protein 1 (A/NS1) [1]. Later it was demonstrated by us and others that activation occurs upon direct interaction of the A/NS1 to the regulatory subunits of PI3K, p85 alpha and beta [2,3]. Several reports proposed that two src homology (SH)-binding motifs within A/NS1 (aa 89 [YXXXM] (SH2-binding motif) and aa 164–167 [PXXP] (SH3-binding motif 1)) may mediate binding to p85 beta. Our work confirms that tyrosine 89 within the A/NS1 is required for the interaction of A/NS1 with p85 beta, subsequent PI3K-activation. However, mutant viruses that carry a phenylalanine instead of the tyrosine at position 89 of the NS1 only showed marginal differences to wt viruses with regard to their replication fitness. More detailed analysis revealed that both, wt type and mutant viruses induced similar PI3K activation levels late in infection, suggesting that besides expression of the NS1 there are alternative virus-induced mechanisms to activate the kinase. Here we demonstrate that this additional inducer is viral 5'triphosphate RNA that accumulates late in the infection cycle. Thus, PI3K activity is regulated by a NS1 protein-dependent as well as a vRNA-dependent mechanism, presumably via the RIG-I sensory pathway. Since NS1 is also a negative regulator of RIG-I, we suggest that influenza viruses have developed multiple mechanisms to achieve a well-balanced PI3K activation at later phases of the infection cycle.