MEK Inhibitor PD98059 Research Articles

Abstract 5453: Dual mTOR/PI3K inhibitors induce ERK pathway overactivation in pancreatic cell lines: Rationale for combinatory therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC), which comprises 90% of all human pancreatic cancers, is a devastating disease, with overall 5-year survival rate of only 3-5%. As the current therapies offer very limited survival benefits, novel molecular therapeutic targets and strategies are urgently needed to treat this aggressive disease. The complementary activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1 and MEK/ERK signaling pathways in many cancer cells, including PDAC, potently stimulates their proliferation and survival. However, mTORC1 also mediates negative feedback loops that restrain upstream signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feedback loops by selective mTORC1 inhibitors, e.g. by rapamycin and its analogs, unleashes over-activation of the PI3K/Akt pathway that potentially opposes the anti-proliferative effects of mTOR inhibitors. This prompted the development of active-site mTORC1/2 kinase inhibitors (TOR-KIs) and dual PI3K and mTOR inhibitors (PI3K/TOR-KIs). It is not known whether PI3K/TOR-KIs induce any effect on the activity of the ERK pathway in PDAC cells. Consequently, we treated serum-starved cultures of PDAC cells (MiaPaca-2 and PANC-1) with increasing concentrations of the dual PI3K/TOR-KI NPV-BEZ235 followed by stimulation with insulin and neurotensin, a potent mitogenic combination for these cells. As expected, prior exposure to NPV-BEZ235 potently inhibited mTORC1 activation (scored by S6 phosphorylation at Ser-240/244) and mTORC2-mediated Akt phosphorylation at Ser-473, in a concentration-dependent manner. Strikingly, we also demonstrate, for the first time, that exposure to NPV-BEZ235 markedly enhanced the phosphorylation of ERK at Thr-202 and Tyr-204. Maximal ERK over-activation coincided with complete inhibition of Akt phosphorylation at Ser-473 (produced at 100-500 ηM NPV-BEZ235). ERK over-activation was also seen when PDAC cells were stimulated with 2% fetal bovine serum and when a different PI3K/TOR-KI (PKI-587) was used instead of NPV-BEZ235. Treatment with the MEK inhibitors U126 or PD0325901 (1-5 μM) prevented ERK over-activation induced by PI3K/TOR-KIs. Crucially, a combination of NPV-BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor added individually. Thus, the elucidation of novel feedbacks loops and crosstalk mechanisms can assist in developing rational and effective strategies for developing combination therapies in PDAC. Citation Format: Heloisa P. Soares, Ming Ming, Michelle Mellon, James Sinnet-Smith, Enrique Rozengurt. Dual mTOR/PI3K inhibitors induce ERK pathway overactivation in pancreatic cell lines: Rationale for combinatory therapy. [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 5453. doi:10.1158/1538-7445.AM2014-5453

7,8-Dihydroxyflavone protects PC12 cells against 6-hydroxydopamine-induced cell death through modulating PI3K/Akt and JNK pathways

We have recently shown that 7,8-dihydroxyflavone (7,8-DHF) protects PC12 cells against 6-OHDA-induced cytotoxicity through its antioxidant activity. In the present study, we investigated the molecular mechanisms underlying the neuronal protective activity of 7,8-DHF. Western blot analysis showed that 6-OHDA (100μM, 24h) enhanced the phosphorylation of JNK and ERK1/2, but it markedly suppressed the expression of p-Akt, implying that 6-OHDA induces PC12 cell death through activating the pro-apoptotic MAPKs pathway but suppressing the survival PI3K/Akt pathway. More importantly, addition of 7,8-DHF fully prevented the activation of JNK and suppression of Akt induced by 6-OHDA. Interestingly, pretreatment with the PI3K-specific inhibitor LY294002 largely blocked 7,8-DHF function in protecting PC12 cells from 6-OHDA-induced cell death. In contrast, the MEK inhibitor PD98059 showed little effect on the protective activity of 7,8-DHF. These results suggest that 7,8-DHF might protect PC12 cells from 6-OHDA-induced cell death through activating PI3K/Akt pathway and inhibiting JNK pathway.

ERK activation is required for CCK-mediated pancreatic adaptive growth in mice.

High levels of cholecystokinin (CCK) can stimulate pancreatic adaptive growth in which mature acinar cells divide, leading to enhanced pancreatic mass with parallel increases in protein, DNA, RNA, and digestive enzyme content. Prolonged release of CCK can be induced by feeding trypsin inhibitor (TI) to disrupt normal feedback control. This leads to exocrine growth in a CCK-dependent manner. The extracellular signal-related kinase (ERK) pathway regulates many proliferative processes in various tissues and disease models. The aim of this study was to evaluate the role of ERK signaling in pancreatic adaptive growth using the MEK inhibitors PD-0325901 and trametinib (GSK-1120212). It was determined that PD-0325901 given two times daily by gavage or mixed into powdered chow was an effective and specific inhibitor of ERK signaling in vivo. TI-containing chow led to a robust increase in pancreatic mass, protein, DNA, and RNA content. This pancreatic adaptive growth was blocked in mice fed chow containing the MEK inhibitors. PD-0325901 blocked TI-induced ERK-regulated early response genes, cell-cycle proteins, and mitogenesis by acinar cells. It was determined that ERK signaling is necessary for the initiation of pancreatic adaptive growth but not necessary to maintain it. PD-0325901 blocked adaptive growth when given before cell-cycle initiation but not after mitogenesis had been established. Furthermore, GSK-1120212, a chemically distinct inhibitor of the ERK pathway that is now approved for clinical use, inhibited growth similar to PD-0325901. These data demonstrate that the ERK pathway is required for CCK-stimulated pancreatic adaptive growth.

Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger.

Epidermal growth factor (EGF) is linked to the pathogenesis of polycystic kidney disease (PKD). We explored signaling pathways activated by EGF in orpk cilia (-) collecting duct cell line derived from a mouse model of PKD (hypomorph of the Tg737/Ift88 gene) with severely stunted cilia, and in a control orpk cilia (+) cell line with normal cilia. RT-PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na(+)/H(+) exchangers (NHE), NHE-1, NHE-2, NHE-3, NHE-4, and NHE-5 in both cell lines. EGF stimulated proton efflux in both cell lines. This effect was significantly attenuated by MIA, 5-(n-methyl-N-isobutyl) amiloride, a selective inhibitor of NHE-1 and NHE-2, and orpk cilia (-) cells were more sensitive to MIA than control cells (P < 0.01). EGF significantly induced extracellular signal-regulated kinase (ERK) phosphorylation in both cilia (+) and cilia (-) cells (63.3 and 123.6%, respectively), but the effect was more pronounced in orpk cilia (-) cells (P < 0.01). MIA significantly attenuated EGF-induced ERK phosphorylation only in orpk cilia (-) cells (P < 0.01). EGF increased proliferation of orpk cilia (+) cells and orpk cilia (-) cells, respectively, and MIA at 1-5 μM attenuated EGF-induced proliferation in orpk cilia (-) cells without affecting proliferation of orpk cilia (+) cells. EGF-induced proliferation of both cell lines was significantly decreased by the EGFR tyrosine kinase inhibitor AG1478 and MEK inhibitor PD98059. These results suggest that EGF exerts mitogenic effects in the orpk cilia (-) cells via activation of growth-associated amiloride-sensitive NHEs and ERK.

Loss of oncogenic Notch1 with resistance to a PI3K inhibitor in T-cell leukaemia.

Mutations that deregulate Notch1 and Ras/phosphoinositide 3 kinase (PI3K)/Akt signalling are prevalent in T-cell acute lymphoblastic leukaemia (T-ALL), and often coexist. Here we show that the PI3K inhibitor GDC-0941 is active against primary T-ALLs from wild-type and Kras(G12D) mice, and addition of the MEK inhibitor PD0325901 increases its efficacy. Mice invariably relapsed after treatment with drug-resistant clones, most of which unexpectedly had reduced levels of activated Notch1 protein, downregulated many Notch1 target genes, and exhibited cross-resistance to γ-secretase inhibitors. Multiple resistant primary T-ALLs that emerged in vivo did not contain somatic Notch1 mutations present in the parental leukaemia. Importantly, resistant clones upregulated PI3K signalling. Consistent with these data, inhibiting Notch1 activated the PI3K pathway, providing a likely mechanism for selection against oncogenic Notch1 signalling. These studies validate PI3K as a therapeutic target in T-ALL and raise the unexpected possibility that dual inhibition of PI3K and Notch1 signalling could promote drug resistance in T-ALL.

A Combination of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2) and MEK (MAP Kinase/ERK Kinase) Inhibitor PD0325901 Increases Bone Formation in a Murine Model of Neurofibromatosis Type I Pseudarthrosis.

Congenital tibial dysplasia is a severe pediatric condition that classically results in a persistent pseudarthrosis. A majority of these cases are associated with neurofibromatosis type I (NF1), a genetic disorder in which inactivation of the NF1 gene leads to overactivity of the Ras-MEK-MAPK (mitogen-activated protein kinase) signaling pathway. We therefore hypothesized that pharmaceutical inhibition of MEK-MAPK may be a beneficial therapeutic strategy. In vitro methods were used to demonstrate a role for the MEK inhibitor PD0325901 in promoting osteogenic differentiation in Nf1-/- calvarial osteoblasts. Local applications of rhBMP-2 and/or PD0325901 were then tested in a mouse model of NF1 tibial pseudarthrosis featuring localized double inactivation of the Nf1 gene in a fracture. Mice received no treatment, PD0325901 (10 mg/kg/day from two days before fracture to ten days after fracture), rhBMP-2 (10 μg), or a combination of rhBMP-2 and PD0325901. Animals treated with the delivery vehicle alone, PD0325901, rhBMP-2, or the PD0325901 + rhBMP-2 combination showed union rates of 0%, 8%, 69% (p < 0.01), or 80% (p < 0.01), respectively, at twenty-one days after fracture. Mice treated with the rhBMP-2 + PD0325901 combination displayed a callus volume sixfold greater than the vehicle controls and twofold greater than the group receiving rhBMP-2 alone. Although MEK inhibition combined with rhBMP-2 led to increases in bone formation and union, the proportion of fibrous tissue in the callus was not significantly reduced. The data suggest that MEK inhibition can promote bone formation in combination with rhBMP-2 in the context of an NF1 pseudarthrosis. However, PD0325901 did not promote substantive bone anabolism in the absence of an exogenous anabolic stimulus and did not suppress fibrosis. This study examines a signaling pathway-based approach to treating poor bone healing in a model of NF1 pseudarthrosis.

Generation of tetraploid complementation mice from embryonic stem cells cultured with chemical defined medium

Mouse embryonic stem cells (mESCs) derived from inner cell mass (ICM) of pre-implantation embryos, can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3 inhibitor CHIR99021 and MEK inhibitor PD0325901 ("2i") and leukemia inhibitor factor (LIF). Compare to conventional culture medium, all components of this medium are defined. With the N2B27 medium, “2i” and LIF, mESCs can contribute to the germline of the chimeric embryos, however, whether the “all-ES cells” mice can been generated by tetraploid complementation is unclear yet, while the tetraploid complementation serve as a golden standard to assess the pluripotency of ES cells. Here, our study showed that mESCs derived and cultured with the N2B27 complete medium could generate fertile mice by tetraploid complementation. In addition, the survival rate of tetraploid complementation mice produced by inbred mES cell lines is higher than the conventional culture condition, and increased the percentage of Oct4 positive cells contrast to conventional medium either. Therefore, the N2B27 medium supplemented with “2i” and LIF is an alternative choice for the derivation and long-term culture of mouse embryonic stem cells.

Chemokine CXCL1 enhances inflammatory pain and increases NMDA receptor activity and COX-2 expression in spinal cord neurons via activation of CXCR2

Recent studies have shown that CXCL1 upregulation in spinal astrocytes is involved in the maintenance of neuropathic pain. However, whether and how CXCL1 regulates inflammatory pain remains unknown. Here we show that intraplantar injection of CFA increased mRNA and protein expressions of CXCL1 and its major receptor CXCR2 in the spinal cord at 6h and 3days after the injection. Immunofluorescence double staining showed that CXCL1 and CXCR2 were expressed in spinal astrocytes and neurons, respectively. Intrathecal injection of CXCL1 neutralizing antibody or CXCR2 antagonist SB225002 attenuated CFA-induced mechanical and heat hypersensitivity on post-CFA day 3. Patch-clamp recordings showed that CXCL1 potentiated NMDA-induced currents in lamina II neurons via CXCR2, and this potentiation was further increased in CFA-treated mice. Furthermore, intrathecal injection of CXCL1 increased COX-2 expression in dorsal horn neurons, which was blocked by pretreatment with SB225002 or MEK (ERK kinase) inhibitor PD98059. Finally, pretreatment with SB225002 or PD98059 decreased CFA-induced heat hyperalgesia and COX-2 mRNA/protein expression and ERK activation in the spinal cord. Taken together, our data suggest that CXCL1, upregulated and released by spinal astrocytes after inflammation, acts on CXCR2-expressing spinal neurons to increase ERK activation, synaptic transmission and COX-2 expression in dorsal horn neurons and contributes to the pathogenesis of inflammatory pain.

Tu1919 Diagnostic Accuracy of Carcinoembryonic Antigen (CEA) in Cyst Fluid Analysis in Histologically Confirmed Pancreatic Cysts: Results From a Large, Multicenter Cohort Study

Background: The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway plays a pivotal role in the proliferation and survival of pancreatic ductal adenocarcinoma (PDAC) cells and is aberrantly activated in pancreatic cancer tissues. In addition to growth-promoting signaling, mTORC1/S6K also mediates negative feedback loops that restrain upstream signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feedback loops by selective mTORC1 inhibitors, e.g. by rapamycin and its analogs, unleashes overactivation of the PI3K/Akt pathway that potentially oppose the anti-proliferative effects of mTOR inhibitors. This prompted the development of active-site mTORC1/2 kinase inhibitors (TOR-KIs) and dual PI3K and mTOR inhibitors (PI3K/TOR-KIs). Recently, we reported that TOR-KIs induce an unexpected increase in the activity of the ERK pathway in PDAC cells through a PI3K-independent pathway. Here, we examined whether PI3K/TOR-KIs also induce ERK pathway over-activation in PDAC cells. Results: To determine the effect of dual PI3K/TOR inhibition on ERK activation, we treated serum-starved cultures of PDAC cells (MiaPaca-2 and PANC-1) with increasing concentrations of the dual PI3K/TOR-KI NPVBEZ235 for 2 h followed by stimulation with insulin and neurotensin, a potent mitogenic combination for these cells. As expected, prior exposure to NPV-BEZ235 potently blocked mTORC1 activation (scored by S6 phosphorylation at Ser-240/244) and mTORC2-mediated Akt phosphorylation at Ser-473, in a concentration-dependent manner. Strikingly, we also demonstrate, for the first time that exposure to NPV-BEZ235 markedly enhanced the increase in the phosphorylation of ERK at Thr-202 and Tyr-204. Maximal ERK over-activation coincided with complete inhibition of Akt phosphorylation at Ser-473 (produced at 100500 ηM NPV-BEZ235). ERK over-activation was also seen when PDAC cells were stimulated with 2% fetal bovine serum instead of insulin and neurotensin and when a different PI3K/ TOR-KI (PKI-587) was used instead of NPV-BEZ235. Treatment with the MEK inhibitors U126 or PD0325901 (1-5 μM) prevented ERK over-activation induced by PI3K/TOR-KIs. In order to examine whether the over-activation of the ERK pathway counterbalances the growth-suppressive effect of PI3K/TOR-KIs, PDAC cells were treated with NPV-BEZ235, PD0325901 or a combination of NPV-BEZ235 and PD0325901. The combination of these drugs caused a more pronounced inhibition of cell growth than that produced by each inhibitor added individually. Conclusion: Collectively, our results highlight the importance of discovering novel signaling crosstalk to anticipate mechanisms of tumor resistance to new drugs. The capability of predicting drug resistance can assist in developing rational and effective strategies for developing combination therapies in PDAC.

Tu1915 Suppression of the mTOR/PI3K Pathway Promotes ERK Pathway Activation in Human Pancreatic Cancer Cells

Background: The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway plays a pivotal role in the proliferation and survival of pancreatic ductal adenocarcinoma (PDAC) cells and is aberrantly activated in pancreatic cancer tissues. In addition to growth-promoting signaling, mTORC1/S6K also mediates negative feedback loops that restrain upstream signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feedback loops by selective mTORC1 inhibitors, e.g. by rapamycin and its analogs, unleashes overactivation of the PI3K/Akt pathway that potentially oppose the anti-proliferative effects of mTOR inhibitors. This prompted the development of active-site mTORC1/2 kinase inhibitors (TOR-KIs) and dual PI3K and mTOR inhibitors (PI3K/TOR-KIs). Recently, we reported that TOR-KIs induce an unexpected increase in the activity of the ERK pathway in PDAC cells through a PI3K-independent pathway. Here, we examined whether PI3K/TOR-KIs also induce ERK pathway over-activation in PDAC cells. Results: To determine the effect of dual PI3K/TOR inhibition on ERK activation, we treated serum-starved cultures of PDAC cells (MiaPaca-2 and PANC-1) with increasing concentrations of the dual PI3K/TOR-KI NPVBEZ235 for 2 h followed by stimulation with insulin and neurotensin, a potent mitogenic combination for these cells. As expected, prior exposure to NPV-BEZ235 potently blocked mTORC1 activation (scored by S6 phosphorylation at Ser-240/244) and mTORC2-mediated Akt phosphorylation at Ser-473, in a concentration-dependent manner. Strikingly, we also demonstrate, for the first time that exposure to NPV-BEZ235 markedly enhanced the increase in the phosphorylation of ERK at Thr-202 and Tyr-204. Maximal ERK over-activation coincided with complete inhibition of Akt phosphorylation at Ser-473 (produced at 100500 ηM NPV-BEZ235). ERK over-activation was also seen when PDAC cells were stimulated with 2% fetal bovine serum instead of insulin and neurotensin and when a different PI3K/ TOR-KI (PKI-587) was used instead of NPV-BEZ235. Treatment with the MEK inhibitors U126 or PD0325901 (1-5 μM) prevented ERK over-activation induced by PI3K/TOR-KIs. In order to examine whether the over-activation of the ERK pathway counterbalances the growth-suppressive effect of PI3K/TOR-KIs, PDAC cells were treated with NPV-BEZ235, PD0325901 or a combination of NPV-BEZ235 and PD0325901. The combination of these drugs caused a more pronounced inhibition of cell growth than that produced by each inhibitor added individually. Conclusion: Collectively, our results highlight the importance of discovering novel signaling crosstalk to anticipate mechanisms of tumor resistance to new drugs. The capability of predicting drug resistance can assist in developing rational and effective strategies for developing combination therapies in PDAC.

Loss of NF1 in Cutaneous Melanoma Is Associated with RAS Activation and MEK Dependence

Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition.

Bisphenol AF-induced endogenous transcription is mediated by ERα and ERK1/2 activation in human breast cancer cells.

Bisphenol AF (BPAF)-induced transcriptional activity has been evaluated by luciferase reporter assay. However, the molecular mechanism of BPAF-induced endogenous transcription in human breast cancer cells has not been fully elucidated. In the present study, we investigated the effect and mechanism of BPAF-induced endogenous transcription detected by real-time PCR in human breast cancer cells. We found that BPAF stimulated transcription of estrogen responsive genes, such as trefoil factor 1 (TFF1), growth regulation by estrogen in breast cancer 1 (GREB1) and cathepsin D (CTSD), through dose-dependent and time-dependent manners in T47D and MCF7 cells. Gene-silencing of ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER) by small interfering RNA revealed that BPAF-induced endogenous transcription was dependent on ERα and GPER, implying both genomic and nongenomic pathways might be involved in the endogenous transcription induced by BPAF. ERα-mediated gene transcription was further confirmed by inhibition of ER activity using ICI 182780 in ERα-positive T47D and MCF7 cells as well as overexpression of ERα in ERα-negative MDA-MB-231 breast cancer cells. Moreover, we utilized Src tyrosine kinase inhibitor PP2 and two MEK inhibitors PD98059 and U0126 to elucidate the rapid nongenomic activation of Src/MEK/ERK1/2 cascade on endogenous transcription. Our data showed that BPAF-induced transcription could be significantly blocked by PP2, PD98059 and U0126, suggesting activation of ERK1/2 was also required to regulate endogenous transcription. Taken together, these results indicate that BPAF-induced endogenous transcription of estrogen responsive genes is mediated through both genomic and nongenomic pathways involving the ERα and ERK1/2 activation in human breast cancer cells.

EGFR Signaling Promotes β-Cell Proliferation and Survivin Expression during Pregnancy

Placental lactogen (PL) induced serotonergic signaling is essential for gestational β-cell mass expansion. We have previously shown that intact Epidermal growth factor –receptor (EGFR) function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced β-cell mass compensation. Islets were isolated from wild-type and β-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for β-cell proliferation and expression of genes involved in gestational β-cell growth. β-cell mass dynamics were analyzed both with traditional morphometrical methods and three-dimensional optical projection tomography (OPT) of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5) when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5). PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin (Birc5) mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of β-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.

Reversal of Bortezomib Resistance in Myelodysplastic Syndrome Cells by MAPK Inhibitors

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of malignant neoplasms with distinctive clinicopathological features. Currently, there is no specific approach for the treatment of MDS. Here, we report that bortezomib (BTZ), a proteasome inhibitor that has been used to treat plasma cell myeloma, induced G2/M phase cycle arrest in the MDS cell line SKM-1 through upregulation of Wee1, a negative regulator of G2/M phase transition. Treatment by BTZ led to reduced SKM-1 cell viability as well as increased apoptosis and autophagy. The BTZ-induced cell death was associated with reduced expression of p-ERK. To elucidate the implications of downregulation of p-ERK, we established the BTZ resistant cell line SKM-1R. Our data show that resistance to BTZ-induced apoptosis could be reversed by the MEK inhibitors U0126 or PD98059. Our results suggest that MAPK pathway may play an important role in mediating BTZ resistance.

Cotargeting the PI3K and RAS Pathways for the Treatment of Neuroendocrine Tumors

The precise involvement of the PI3K/mTOR and RAS/MEK pathways in carcinoid tumors is not well defined. Therefore, the purpose of our study was to evaluate the role these pathways play in carcinoid cell proliferation, apoptosis, and secretion and to determine the effects of combined treatment on carcinoid tumor inhibition. The human neuroendocrine cell lines BON (pancreatic carcinoid), NCI-H727 (lung carcinoid), and QGP-1 (somatostatinoma) were treated with either the pan-PI3K inhibitor, BKM120, or the dual PI3K-mTOR inhibitor, BEZ235, alone or in combination with the MEK inhibitor, PD0325901; proliferation, apoptosis, and protein expression were assessed. Peptide secretion was evaluated in BON and QGP-1 cells. The antiproliferative effect of BEZ235, alone or combined with PD0325901, was then tested in vivo. Both BKM120 and BEZ235 decreased proliferation and increased apoptosis; combination with PD0325901 significantly enhanced the antineoplastic effects of either treatment alone. In contrast, neurotensin peptide secretion was markedly stimulated with BKM120 treatment, but not BEZ235. The combination of BEZ235 + PD0325901 significantly inhibited the growth of BON xenografts without systemic toxicity. Both BKM120 and BEZ235 effectively inhibited neuroendocrine tumor (NET) cell proliferation and stimulated apoptosis. However, inhibition of the PI3K pathway alone with BKM120 significantly stimulated neurotensin peptide secretion; this did not occur with the dual inhibition of both PI3K and mTOR using BEZ235 suggesting that this would be a more effective treatment regimen for NETs. Moreover, the combination of BEZ235 and the MEK inhibitor PD0325901 was a safe and more effective therapy in vivo compared with single agents alone.

HBx Protein Promotes Oval Cell Proliferation by Up-Regulation of Cyclin D1 via Activation of the MEK/ERK and PI3K/Akt Pathways

Growing evidence has shown that hepatic oval cells, also named liver progenitor cells, play an important role in the process of liver regeneration in various liver diseases. Oval cell proliferation has been reported in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and chronic liver disease. Studies have found expression of HBV surface and core antigens in oval cells in the livers of patients with HCC, suggesting that HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. In addition, there is evidence of multiplication of HBV in oval cell culture. However, little research has been performed to explore the role of HBV-encoded proteins in the proliferation of hepatic oval cells. Previously, we successfully transfected the HBV x (HBx) gene, one of the four genes in the HBV genome, into a rat LE/6 oval cell line. In this study, we tested whether or not the transfected HBx gene could affect oval cell proliferation in vitro. Our results show that overexpression of HBx promotes the proliferation of oval cells and increases cyclin D1 expression, assessed at both the mRNA and protein levels. We also found that HBx activated the PI-3K/Akt and MEK/ERK1/2 pathways in HBx-transfected oval cells. Furthermore, the HBx-induced increases in cyclin D1 expression and oval cell proliferation were completely abolished by treatment with either MEK inhibitor PD184352 or PI-3K inhibitor LY294002. These results demonstrated that HBx has the ability to promote oval cell proliferation in vitro, and its stimulatory effects on cell proliferation and expression of cyclin D1 depend on the activation of the MEK/ERK and PI3K/Akt signaling pathways in cultured oval cells.

CD44 targets Na+/H+ exchanger 1 to mediate MDA-MB-231 cells’ metastasis via the regulation of ERK1/2

Background:CD44, a transmembrane glycoprotein expressed in a variety of cells and tissues, has been implicated in tumour metastasis. But the molecular mechanisms of CD44-mediated tumour cell metastasis remain to be elucidated.Methods:The downregulation of CD44 was determined by immunofluorescence. Moreover, the motility of breast cancer cells was detected by wound-healing and transwell experiments. Then the spontaneous metastasis of CD44-silenced MDA-MB-231 cells was tested by histology with BALB/c nude mice.Results:A positive correlation between CD44 and Na+/H+ exchanger isoform 1 (NHE1) was found in two breast cancer cells. CD44 downregulation could inhibit the metastasis of MDA-MB-231 cells and the expressions of Na+/H+ exchanger 1. Moreover, CD44 overexpression upregulated the metastasis of MCF-7 cells, but the elevated metastatic ability was then inhibited by Cariporide. Interestingly, during these processes only the p-ERK1/2 was suppressed by CD44 downregulation and the expression of matrix metalloproteinases and metastatic capacity of MDA-MB-231 cells were greatly inhibited by the MEK1 inhibitor PD98059, which even had a synergistic effect with Cariporide. Furthermore, CD44 downregulation inhibits breast tumour outgrowth and spontaneous lung metastasis.Conclusions:Taken together, this work indicates that CD44 regulates the metastasis of breast cancer cells through regulating NHE1 expression, which could be used as a novel strategy for breast cancer therapy.

Stress-induced stimulation of choline transport in cultured choroid plexus epithelium exposed to low concentrations of cadmium

The choroid plexus epithelium forms the blood-cerebrospinal fluid barrier and accumulates essential minerals and heavy metals. Choroid plexus is cited as being a "sink" for heavy metals and excess minerals, serving to minimize accumulation of these potentially toxic agents in the brain. An understanding of how low doses of contaminant metals might alter transport of other solutes in the choroid plexus is limited. Using primary cultures of epithelial cells isolated from neonatal rat choroid plexus, our objective was to characterize modulation of apical uptake of the model organic cation choline elicited by low concentrations of the contaminant metal cadmium (CdCl₂). At 50-1,000 nM, cadmium did not directly decrease or increase 30-min apical uptake of 10 μM [(3)H]choline. However, extended exposure to 250-500 nM cadmium increased [(3)H]choline uptake by as much as 75% without marked cytotoxicity. In addition, cadmium induced heat shock protein 70 and heme oxygenase-1 protein expression and markedly induced metallothionein gene expression. The antioxidant N-acetylcysteine attenuated stimulation of choline uptake and induction of stress proteins. Conversely, an inhibitor of glutathione synthesis l-buthionine-sulfoximine (BSO) enhanced stimulation of choline uptake and induction of stress proteins. Cadmium also activated ERK1/2 MAP kinase. The MEK1 inhibitor PD98059 diminished ERK1/2 activation and attenuated stimulation of choline uptake. Furthermore, inhibition of ERK1/2 activation abated stimulation of choline uptake in cells exposed to cadmium with BSO. These data indicate that in the choroid plexus, exposure to low concentrations of cadmium may induce oxidative stress and consequently stimulate apical choline transport through activation of ERK1/2 MAP kinase.

The activation of P2Y2 receptors increases MCF-7 breast cancer cells migration through the MEK-ERK1/2 signalling pathway

Adenosine 5'-triphosphate (ATP) is found in high concentrations in the extracellular microenvironment of tumours and is postulated to play critical roles in cancer progression. In the present study, we found that stimulation of human MCF-7 breast cancer cells with 30 µM ATP increased their migration by 140 ± 31%, whereas it had minor or no effect on their proliferation. This effect was prevented by the ectonucleotidase apyrase and was antagonized by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, consistently with the participation of P2 receptors. MCF-7 cells expressed messenger RNA for all known P2Y receptors and for P2X2, P2X4, P2X5, P2X6 and P2X7 receptors. Brief applications (20 s) of external ATP resulted in a 50 pA P2X-like inward current. ATP, but not adenosine diphosphate or uridine diphosphate, increased the intracellular calcium concentration in absence of extracellular calcium, and this effect was prevented by the inhibition of phospholipase C. Uridine triphosphate (UTP) (10 µM) and 2-thio-UTP (10 µM) increased intracellular calcium concentration and cell migration to the same extent as ATP. The UTP-dependent increase in cell migration was absent in cells knocked-down for P2Y2. It was inhibited by MEK inhibitor PD98059. UTP induced a time-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which was prevented by the incubation with PD98059. Taken together, these results highlight the importance of the purinergic signalling in cancer cells and indicate that the activation of P2Y2 receptors enhances breast cancer cells migration through the activation of a MEK-ERK1/2-dependent signalling pathway.

Neuroprotective properties of ciliary neurotrophic factor on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells.

Ciliary neurotrophic factor (CNTF) is a neurocytokine, which could promote survival and/or differentiation in many cell types. In this study, the biological effects of CNTF on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells and the underlying molecular mechanism of this effect were investigated for the first time. The results showed that RA was able to increase cells susceptibility to CNTF via regulating the expression levels of CNTF receptors. A further study revealed that CNTF could induce phosphorylation of STAT3, Akt and ERK1/2 in RA-predifferentiated SH-SY5Y neuroblastoma cells, while the promoting activity of CNTF on survival and neurite growth of cells was attenuated by co-treatment with JAK2 inhibitor AG490 (25 μM), STAT3 inhibitor Curcumin (50 μM), PI3K inhibitor LY-294002 (50 µM), but not by co-treatment with MEK inhibitor PD98059 (50 μM). These findings suggested that JAK2/STAT3, as well as PI3K/Akt, play important roles in mediating the survival and neurite growth response of RA-predifferentiated cells to CNTF. Our study may be useful to further understand the functional role of CNTF and offer a convenient model to explore the therapeutic potential of CNTF in neurodegenerative diseases.