PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways perturbations in non-functioning pituitary adenomas

Non-functioning pituitary adenomas (NFPAs) comprise a heterogeneous group, which are considered the most common pituitary tumor. As no clinically hormone hypersecretion is apparent, non-functioning pituitary adenomas are often diagnosed only when they are large enough to cause tumor mass effects, such as hypopituitarism, visual field defects or headaches. Efficient medical therapy for NFPAs is currently unavailable and surgical treatment of these tumors is not always satisfactory. Characterization of signaling regulatory events in the context of NFPAs may enable the development of new attractive novel strategies. Although data regarding gene expression profiling of signaling pathways in NFPAs have accumulated, studies aimed at fine-classification of NFPAs-specific signaling regulatory mechanisms and feedback loops are scarce.     

Involvement of ERK1/2, p38 and PI3K in megakaryocytic differentiation of K562 cells

Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen‐activated protein kinases (MAPKs) [extracellular signal‐regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3‐kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12‐myristate 13‐acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA‐treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbα expression, DNA endoreduplication, and formation of platelet‐like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down‐regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.     

Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways

Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways.     

Leishmania mexicana amastigotes inhibit p38 and JNK and activate PI3K/AKT: role in the inhibition of apoptosis of dendritic cells

Leishmania mexicana is the causal agent of cutaneous leishmaniasis in Mexico. Dendritic cells (DC) are one of the host cells of Leishmania parasites. Intracellular microorganisms inhibit host cell apoptosis as a strategy to ensure their survival in infected cells. We have previously shown that Leishmania mexicana promastigotes and amastigotes inhibit camptothecin‐induced apoptosis of monocyte‐derived dendritic cells (moDC), but the mechanisms underlying the inhibition of apoptosis of DC by Leishmania have not been established. MAP kinases and PI3K participate in the process of apoptosis and are modulated by different species of Leishmania. As shown in this study, the infection of moDC with L. mexicana amastigotes diminished significantly the phosphorylation of the MAP kinases p38 and JNK. The inhibition of both kinases diminished significantly DNA fragmentation in moDC stimulated with camptothecin. On the other hand, L. mexicana amastigotes were able to activate the anti‐apoptotic pathways PI3K and AKT. Our results indicate that L. mexicana amastigotes have the capacity to diminish MAP kinases activation and activate PI3K and AKT, which is probably one of the strategies employed by L. mexicana amastigotes to inhibit apoptosis in the infected moDC.     

PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF,P-Rex1

The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers.The phosphoinositide 3-kinase (PI3K) family of lipid kinases plays a prominent role in the growth and survival of several types of cancer (1). The PI3K pathway is aberrantly activated by a number of different mechanisms in cancers. These include genetic mutation and/or amplification of key pathway components, such as amplification or mutation of the PI3K catalytic subunit p110α (encoded by PIK3CA gene), mutation or deletion of the phosphatase PTEN, amplification or mutation of the gene encoding for the PI3K effector protein kinase AKT, as well as constitutive activation of receptor tyrosine kinases (RTKs) (e.g., HER2 amplification in breast cancer) or other less frequent events (2). PI3K phosphorylates the phosphoinositide PI(4,5)P2 in the 3′OH group of the inositol ring to produce PI(3,4,5)P3. PI(3,4,5)P3 directly binds to the pleckstrin homology (PH) domains of certain proteins, such as AKT, leading to their activation, which in turn transmit growth and survival signals.These findings have encouraged the development of several different PI3K inhibitors, many of which are either in or approaching clinical trial testing. Genotype-driven patient selection has been investigated to uncover patient populations that will be particularly susceptible to PI3K inhibitors. Cancers harboring mutations in the PIK3CA gene have emerged as among the most sensitive to single-agent PI3K inhibitors in several preclinical studies, although clinical activity to date has been mixed (3–6). These gain-of-function mutations in the PI3KCA gene are found in a broad range of cancers, and they are highly enriched in breast cancer, where they are observed in 20–25% of cases (7). In addition, breast cancers with amplified HER2, which comprise ∼20% of all breast cancers, (8) are also particularly sensitive to PI3K inhibition (9–11). However, even among patients whose cancers harbor PIK3CA mutations, a significant heterogeneity of responses has been observed to PI3K inhibitors currently being tested in clinical studies (3–5). There have been some patients with bona fide response evaluation criteria in solid tumors (RECIST) criteria responses, but the majority has not had similarly impressive outcomes. These early clinical results highlight the potential utility of a biomarker of sensitivity to single-agent PI3K inhibitors.Interestingly, early clinical trial reports have found that inhibition of PI3K signaling may sometimes lead to suppression of protein kinase MEK (MEK)/ERK signaling (6). Although a previous laboratory study had shown that the PI3K/mammalian target of rapamycin (mTOR) inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin can inhibit protein kinase RAF (RAF)/MEK/ERK-signaling (12), this clinical observation was initially surprising because several studies have shown that inhibitors of components of the PI3K signaling pathway (such as AKT and mTOR inhibitors) actually lead to activation of the MEK/ERK signaling in many cancer types (11, 13), and such feedback activation may impair sensitivity to PI3K pathway inhibitors (9, 11, 14). Because PIK3CA and HER2 amplified breast cancers are particularly sensitive to single-agent PI3K pathway inhibitors, we investigated how PI3K inhibitors impact MEK/ERK signaling in these genetically defined subsets of breast cancers. In our study, we found that several cell lines harboring PIK3CA mutation and/or HER2 amplification suppress MEK/ERK pathway signaling as well as the AKT pathway after treatment with PI3K inhibitors, and importantly, inhibition of both pathways is necessary for maximal antitumoral activity. Moreover we identify that the mechanistic link between PI3K and MEK/ERK is via a PI(3,4,5)P3-dependent regulation of the phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1)/ small GTPase Rac1 (Rac1)/protein kinase c-RAF (c-RAF) pathway in these cancers. Importantly, the expression levels of the Rac guanine exchange factor (Rac-GEF), P-Rex1, correlate with sensitivity to PI3K inhibitors in these breast cancer cell lines.     

Optimizing immunotherapy in multiple myeloma: Restoring the function of patients' monocyte-derived dendritic cells by inhibiting p38 or activating MEK/ERK MAPK and neutralizing interleukin-6 in progenitor cells

Previous studies demonstrated that circulating dendritic cells (DCs) in myeloma patients were functionally abnormal. However, the phenotype and function of patients' monocyte-derived DCs (MoDCs), which are commonly used for immunotherapy, were poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterparts, patient-derived, mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR and were poor at activating alloreactive T cells, presenting recall antigen, and activating autologous antigen- and myeloma-specific T cells. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6-specific antibody and, more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield but also produced MoDCs that were as functional as their normal counterparts. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy in this disease.     

PI3K/AKT/mTOR信号通路在糖皮质激素性股骨头坏死中的表达与作用

糖皮质激素性股骨头坏死(steroid-induced avascularnecrosis of femoral head,SANFH)是一类致残率较高的疾病,长期损害人类与社会健康,已成为亟待解决的社会问题。PI3K/AKT/mTOR信号通路是一条与细胞分化凋亡自噬等密切相关的转导通路。近年随着分子生物学和细胞生物学的进步发展,表明以此信号通路为切入点能够对SANFH进行有效的靶向调控,并通过促进成骨分化,抑制凋亡,修复血管内皮细胞以及调控自噬等多种途径,对骨细胞产生显著调控和影响。本文就PI3K/AKT/mTOR通路在SANFH中所发挥的调控机制和作用,以及各部分在激素性股骨头坏死中的表达作简要综述,为以后的研究治疗提供思路与依据。     

ERK and p38 MAP kinase activation are components of opioid-induced delayed cardioprotection

Opioids have been previously shown to confer acute and delayed cardioprotection against a prolonged ischemic insult. We have extensively characterized the signal transduction pathway mediating acute cardioprotection and have suggested a role for extracellular signal regulated kinase (ERK) in this cardioprotection. Therefore, we attempted to determine a role for ERK and the stress activated MAP kinase, p38, in opioid-induced delayed cardioprotection by using selective inhibitors of these pathways. All rats were subjected to 30 min of ischemia and 2 h of reperfusion (I/R). Control animals, injected with saline 48 h prior to I/R, had an infarct size/area at risk (IS/AAR) of 61.6 ± 1.6. 48-h pretreatment with TAN-67 (30 mg/kg), a δ₁-opioid receptor agonist, maximally reduced IS/AAR (31.2 ± 6.5). The involvement of ERK was examined with PD 098059, a selective pharmacological antagonist which inhibits the upstream kinase, MEK-1, that phosphorylates and activates ERK. PD 098059 (0.3 mg/kg) did not alter IS/AAR when administered alone (60.7 ± 4.9). However, PD 098059 (0.3 mg/kg) administration 30 min prior to TAN-67 (30 mg/kg) completely abolished cardioprotection (61.0 ± 7.6). The selective p38 inhibitor, SB 203580 (1.0 mg/kg), had no effect on IS/AAR in the absence of TAN-67 (53.1 ± 2.3). Additionally, SB 203580 (1.0 mg/kg) when administered prior to TAN-67 (30 mg/kg) partially abolished cardioprotection (51.3 ± 6.4). These results suggest that both ERK and p38 are integral components of opioid-induced delayed cardioprotection and may act via parallel pathways. Received: 21 August 2000, Returned for revision: 13 September 2000, Revision received: 18 September 2000, Accepted: 20 September 2000     

PI3K/AKT信号通路与心肌缺血再灌注损伤

PI3K/AKT通路是重要的抗细胞凋亡/促增殖信号通路,在细胞的生长、存活、增殖、迁移及代谢等方面有重要作用,并且越来越多的研究表明其在心肌缺血再灌注损伤的病理生理过程中也发挥重要作用,本文就此作一综述。     

Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging

Dysregulated signaling through the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways is often the result of genetic alterations in critical components in these pathways or upstream activators. Unrestricted cellular proliferation and decreased sensitivity to apoptotic-inducing agents are typically associated with activation of these pro-survival pathways. This review discusses the functions these pathways have in normal and neoplastic tissue growth and how they contribute to resistance to apoptotic stimuli. Crosstalk and commonly identified mutations that occur within these pathways that contribute to abnormal activation and cancer growth will also be addressed. Finally the recently described roles of these pathways in cancer stem cells, cellular senescence and aging will be evaluated. Controlling the expression of these pathways could ameliorate human health.     

Mechanisms of disease: PI3K/AKT signaling in gastrointestinal cancers

The lipid kinase phosphoinositide 3-OH kinase (PI3K) and its downstream target Akt, also known as protein kinase B (PKB), are crucial effectors in oncogenic signaling induced by various receptor-tyrosine kinases. In recent years, data are accumulating that PI3K/Akt signaling components are frequently altered in a variety of human malignancies. This review summarizes the major effects of PI3K/Akt signaling on proliferation, survival and resistance to apoptosis, angiogenesis and cell motility in gastrointestinal cancers. In addition, activation of PI3K/Akt by various growth factors, the modulation of downstream targets by Akt-induced phosphorylation as well as novel treatment strategies targeting this pathway in gastrointestinal tumors are discussed.     

Deguelin, A PI3K/AKT inhibitor, enhances chemosensitivity of leukaemia cells with an active PI3K/AKT pathway

Activation of the phosphoinositide 3 kinase (PI3K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its downregulation, by means of PI3K inhibitors, lowers resistance to various types of therapy in tumour cell lines. Recently, it has been reported that deguelin, a naturally occurring rotenoid, is a powerful inhibitor of PI3K. We investigated whether or not deguelin could enhance the sensitivity to chemotherapeutic drugs of human U937 leukaemia cells and acute myeloid leukaemia (AML) blasts with an activated PI3K/Akt network. Deguelin (10 nmol/l) induced S phase arrest with interference of progression to G2/M, and at 100 nmol/l significantly increased apoptotic cell death of U937. At 10-100 nmol/l concentrations, deguelin downregulated Akt phosphorylation of leukaemia cells and markedly increased sensitivity of U937 cells to etoposide or cytarabine. A 10 nmol/l concentration of deguelin did not negatively affect the survival rate of human cord blood CD34+ cells, whereas it increased sensitivity of AML blasts to cytarabine. Deguelin was less toxic than wortmannin on erythropoietin- and stem cell factor-induced erythropoiesis from CD34+ progenitor cells. Overall, our results indicate that deguelin might be used in the future for increasing sensitivity to therapeutic treatments of leukaemia cells with an active PI3K/Akt signalling network.     

Differential effects of STAT5 and PI3K/AKT signaling on effector and memory CD8 T-cell survival

During viral infection, effector CD8 T cells contract to form a population of protective memory cells that is maintained by IL-7 and IL-15. The mechanisms that control effector cell death during infection are poorly understood. We investigated how short- and long-lived antiviral CD8 T cells differentially used the survival and cell growth pathways PI3K/AKT and JAK/STAT5. In response to IL-15, long-lived memory precursor cells activated AKT significantly better than short-lived effector cells. However, constitutive AKT activation did not enhance memory CD8 T-cell survival but rather repressed IL-7 and IL-15 receptor expression, STAT5 phosphorylation, and BCL2 expression. Conversely, constitutive STAT5 activation profoundly enhanced effector and memory CD8 T-cell survival and augmented homeostatic proliferation, AKT activation, and BCL2 expression. Taken together, these data illustrate that effector and memory cell viability depends on properly balanced PI3K/AKT signaling and the maintenance of STAT5 signaling.     

Icaritin induces AML cell apoptosis via the MAPK/ERK and PI3K/AKT signal pathways

Icaritin, a hydrolytic product of icaritin, is isolated from the traditional Chinese medicinal herb epimedium. Icaritin inhibits the proliferation of several tumor cell lines, but its effect on acute myeloid leukemia (AML) and underlying mechanisms remain to be identified. In the present study, we demonstrated that icaritin inhibits the proliferation of human AML cell lines NB4, HL60, and U937, in a dose- and time-dependent manner. Importantly, icaritin showed anti-leukemia activity on bone marrow mononuclear cells from 15 newly diagnosed AML patients. Flow cytometry analyses indicated that icaritin induces AML cells apoptosis. Icaritin induced activation of caspase-9, -3, -7 and the cleavage of PARP as measured by Western blotting. Icaritin downregulates p-ERK and p-AKT and inhibits the expression of c-myc. These results suggest that icaritin is a promising candidate drug for the treatment of AML. The underlying mechanisms of icaritin anti-AML activity are associated with inhibition of the MAPK/ERK and PI3K/AKT signals and downregulation of c-myc.     

Distinct roles of JNKs/p38 MAP kinase and ERKs in apoptosis and survival of HCD-57 cells induced by withdrawal or addition of erythropoietin

Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.     

Cyclosporin A induces erythroid differentiation of K562 cells through p38 MAPK and ERK pathways

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells.