Induces Cell Survival Research Articles

Differential activation of MAPKs by individual and combined ochratoxin A and citrinin treatments in porcine kidney PK15 cells

The aim of this study was to investigate the underlying mechanisms of OTA and CTN individual and combined toxicity in porcine kidney PK15 cells of proximal tubule origin. Activation and expression of mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 were determined by Western blot analysis. MAPKs were differentially activated by single or dual OTA and CTN treatments. Single OTA and CTN stimulated transient ERK and prolonged JNK activation, while phospho-p38 signal was more persistent after OTA treatment. Mycotoxin mixture provoked significant down-regulation of ERK activation, more prolonged phospho-p38 signal, and two-stage JNK phosphorylation pattern. In order to define the role of particular MAPKs in mycotoxin(s) cytotoxicity, we performed MTT assay with specific MAPKs inhibitors. In both individual and combined treatments JNK and p38 inhibition significantly induced cell survival. When cells were exposed to toxin mixture, inhibition of ERK also promoted cell survival, although to a lesser extent that JNK and p38 inhibition. Next we investigated the association between calcium (Ca2+) and MAPKs after OTA and/or CTN treatments, and we employed Ca2+ chelator BAPTA-AM. We demonstrated that p38 activation was significantly down-regulated in cells treated with CTN alone or OTA + CTN suggesting the role of Ca2+ in mycotoxin-induced cell death.

Targeting survival and cell trafficking in multiple myeloma and Waldenstrom macroglobulinemia using pan-class I PI3K inhibitor, buparlisib.

The phosphatidylinositol-3 kinase (PI3K) pathway is activated in multiple myeloma (MM) and Waldenstrom Macroglobulenima (WM), and plays a crucial role in tumor progression and drug resistance. In this study, we characterized the role of pan-class I PI3K inhibition on cell trafficking and survival of MM and WM cells. We tested the effect of pan-class I PI3K inhibition by siRNA silencing or pharmacologic inhibition with buparlisib on MM cell survival, apoptosis and cell cycle in vitro and tumor growth and mobilization of MM cells in vivo. We then evaluated buparlisib-dependent mechanisms of induced MM cell mobilization. Moreover, the effect of buparlisib on cell survival, apoptosis, and adhesion of WM cells to bone marrow stromal cells (BMSCs) has been evaluated. We showed that buparlisib induced toxicity in MM cells, supported by induction of apoptosis and cell cycle arrest. Buparlisib was also found to reduce tumor progression in vivo. Importantly, buparlisib enhanced MM cell mobilization in vivo which was driven by decreased adhesion of MM cells to BMSCs and increased chemotaxis via up-regulation of CXCR4 expression. Similar to its effects on MM cells, buparlisib also induced cell survival and apoptosis, and decreased adhesion in WM cells. These data highlight the critical contribution of class I PI3K signaling to the regulation of survival and cell dissemination in B-cell malignancies.

Enforced expression of miR-101 enhances cisplatin sensitivity in human bladder cancer cells by modulating the cyclooxygenase-2 pathway.

Alterations in microRNA (miRNA) expression have been shown to be involved in the tumor response to chemotherapy. However, the possible role of miR‑101 in cisplatin sensitivity in human bladder cancer cells remains unclear. In this study, quantitative polymerase chain reaction and western blotting were utilized to determine the expression profiles of miR‑101 and cyclooxygenase‑2 (COX‑2) in human bladder cancer cells. The effect of miR‑101 and small interfering RNA (siRNA) against COX‑2 on cell viability was evaluated using MTT assays, and apoptosis levels were determined using fluorescence‑activated cell sorting analysis of Annexin V/propidium iodide‑stained cells. Luciferase reporter plasmids were constructed to confirm direct targeting. This study found that the expression of miR‑101 was downregulated in the cisplatin‑resistant cell line T24/CDDP as compared with that in the parental line, T24. Furthermore, overexpression of miR‑101 significantly increased the anti‑proliferative effects and apoptosis induced by cisplatin, whereas knockdown of miR‑101 significantly decreased the anti‑proliferative effects and apoptosis induced by cisplatin. In addition, downregulation of miR‑101 induced cell survival and cisplatin resistance through the upregulation of COX‑2 expression. Luciferase gene reporter assays confirmed that COX‑2 was a direct target gene of miR‑101. Inhibition of COX‑2 using COX‑2 siRNA abrogated the cisplatin resistance induced by miR‑101 downregulation. These results suggest that miR‑101 may provide a novel mechanism for understanding cisplatin resistance in bladder cancer by modulating the COX‑2 pathway.

Establishment condition and characterization of heart-derived cell culture in Siberian sturgeon (Acipenser baerii).

This study was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture. Total 48 trials of culture employing low and middle cell densities of less than 5.5 × 10(4) cells/cm(2) in initial seeding did not induce cell survivals (0%, 0/48), but the trials in high cell density of more than 5.5 × 10(5) cells/cm(2) could induce cell survival and primary cell attachment on the plate (88.9%, 24 in 27 trials). When all initially attached cell populations were continuously cultured in two different media, only five cell populations that were enzymatically isolated and cultured under Leibovitz's L-15 medium could grow up to more than 40th subculture. Each cell population was stably cultured according to its own growth rate and all showed normal diploid DNA contents. Two morphologically different cell types that has an elongated shape or a round shape were identified in culture, which was subsequently identified that two cell types are considered as a fibroblast (an elongated shape) and a vascular endothelial cell (a round shape) on the basis of the results of gene and protein expression analysis. Additionally, the sufficient number of viable cells could be successfully retrieved after freezing and thawing from all five cell populations suggesting the feasibility of long-term cryopreservation of the cells. The data and cells obtained from this study will contribute to development of in vitro model for basic biological studies using sturgeon species.

Abstract 264: TNFR2-Bmx Signaling in Cardiac Stem Cells and Cardiac Repair

While cytokine TNF via TNFR1 induces inflammation and apoptosis, it through its second receptor TNFR2 induces cell survival and migration by activating bone marrow non-receptor tyrosine kinase Bmx. Since Bmx has been implicated in self-renewal of stem cells, we hypothesize that TNF via TNFR2 activates Bmx in cardiac stem cells (CSCs) to mediate cardiac repair. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+) /CD45(-) /VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA (references: Stem Cells 2013;31:1881-1892). In the present study, we detect the c-kit(+)Lin28(+) CSCs populations in a mouse coronary artery ligation ischemic model. Furthermore, the c-kit(+) CSCs are reduced in TNFR2-KO and Bmx-KO mice. Mechanistically, we show a crosstalk between the TNFR2-Bmx and the c-Kit signaling pathways to mediate CSC proliferation, survival and migration. These observations suggest that TNFR2-Bmx signaling in c-kit(+) CSCs induces cardiac repair, providing a potential strategy to stimulate cardiac regeneration by TNFR2-specific agonists.

The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity

The transcriptional network regulating antibody-secreting cell (ASC) differentiation has been extensively studied, but our current understanding is limited. The mechanisms of action of known "master" regulators are still unclear, while the participation of new factors is being revealed. Here, we identify Zbtb20, a Bcl6 homologue, as a novel regulator of late B cell development. Within the B cell lineage, Zbtb20 is specifically expressed in B1 and germinal center B cells and peaks in long-lived bone marrow (BM) ASCs. Unlike Bcl6, an inhibitor of ASC differentiation, ectopic Zbtb20 expression in primary B cells facilitates terminal B cell differentiation to ASCs. In plasma cell lines, Zbtb20 induces cell survival and blocks cell cycle progression. Immunized Zbtb20-deficient mice exhibit curtailed humoral responses and accelerated loss of antigen-specific plasma cells, specifically from the BM pool. Strikingly, Zbtb20 induction does not require Blimp1 but depends directly on Irf4, acting at a newly identified Zbtb20 promoter in ASCs. These results identify Zbtb20 as an important player in late B cell differentiation and provide new insights into this complex process.

Ab initio calculations on the (1)O2 quenching mechanism by trans-resveratrol.

trans-Resveratrol has shown to play an important role in a variety of biological and medical processes such as reactive oxygen species (ROS) scavenging, inhibition of apoptosis and induction of cell survival. In light of the fact that resveratrol and its oligomers were found to be selective singlet oxygen (1)O2 quenchers, we report here a systematic study on the reactivity of trans-resveratrol toward molecular oxygen in acetone simulated media. On the basis of the controversial hypotheses reported in the literature we explored, at density functional levels of theory, two different mechanisms. The first one leads to a resveratrol quinone product via an endoperoxide intermediate by attack of (1)O2 on the resorcinol ring, assisted (pathway (b)) or not (pathway (a)) by a water molecule. The second mechanism, in which the singlet oxygen reacts with the double bond connecting the two resveratrol rings leading to benzaldehyde products, involves the formation of a dioxetane intermediate. As the outcomes of our computational analysis show that the latter mechanism is kinetically more favorable than the former one, it is likely that when trans-resveratrol reacts with singlet oxygen a dioxetane intermediate is formed.

Tissue factor/factor VIIa induces cell survival and gene transcription by transactivation of the insulin-like growth factor 1 receptor

The insulin-like growth factor 1 receptor (IGF-1R) is known to promote survival and has also been implicated in the pathogenesis of several disease states, including cardiovascular disorders and cancer. Recently, we showed that binding of coagulation factor VIIa (FVIIa) to its receptor tissue factor (TF) protects cancer cells from TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis. Here we present evidence that this biological function of TF/FVIIa is dependent on the IGF-1R. IGF-1R inhibitors AG1024 and PPP as well as siRNA-mediated downregulation of IGF-1R, abolished the TF/FVIIa-mediated protection against TRAIL-induced apoptosis. Moreover, FVIIa rapidly induced a time- and concentration-dependent tyrosine phosphorylation of the IGF-1R in MDA-MB-231 breast cancer cells and in primary human monocytes, an event that was accompanied by IGF-1R chromatin binding and gene transcription. We hereby present novel evidence of a cross-talk between the coagulation and IGF-1R signalling systems, and propose that the IGF-1R is a key player in mediating TF/FVIIa-induced cell survival.

Emerging Targets in Pituitary Adenomas: Role of the CXCL12/CXCR4-R7 System.

Chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation, infection, and cancer. Several chemokines and cognate receptors are constitutively expressed in the central nervous system, not only in glial and endothelial cells but also in neurons, controlling neurogenesis, neurite outgrowth, and axonal guidance during development. In particular, the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, form a functional network that controls plasticity in different brain areas, influencing neurotransmission, neuromodulation, and cell migration, and the dysregulation of this chemokinergic axis is involved in several neurodegenerative, neuroinflammatory, and malignant diseases. CXCR4 primarily mediates the transduction of proliferative signals, while CXCR7 seems to be mainly responsible for scavenging CXCL12. Importantly, the multiple intracellular signalling generated by CXCL12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions, although a direct modulation of pituitary functioning via autocrine/paracrine mechanisms was also reported. Both CXCL12 and CXCR4 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation, as well as hormonal hypersecretion. In this review we focus on the physiological and pathological functions of immune-related cyto- and chemokines, mainly focusing on the CXCL12/CXCR4-7 axis, and their role in pituitary tumorigenesis. Accordingly, we discuss the potential targeting of CXCR4 as novel pharmacological approach for pituitary adenomas.

Aggressive Mature Natural Killer Cell Neoplasms: From EBV-Infection to Disease Etiopathogeny

Mature Natural Killer (NK) cell neoplasms are rare tumors with higher prevalence in Asia, Central and South America, which are related to Epstein Barr Virus (EBV) infection. Nature killer /T cell lymphoma, nasal type, presents as a localized or generalized destructive tumor, affecting the nose, the upper aero digestive tract or any organ or tissue, whereas aggressive NK-cell leukemia manifests as a systemic disease that preferentially affects the bone marrow, the spleen and the liver, and rapidly evolve to multiorgan failure resulting in death. Both NK-cell neoplasms arise as a consequence of the inability of the immune system to control EBV infection and of the transforming potential of multiple EBV gene products. Chronic active EBV infection and EBV-related lymphoproliferative disorders of NK-cells are predisposing conditions. The tumor NK-cells do express an EBV infection type II latency pattern, specific EBV-encoded latent membrane proteins and early region EBV RNAs being detected on lymphoma cells. The EBV encoded proteins and non-coding EBV RNAs and micro-RNAs expressed on the infected cells are involved in immune deregulation and play a crucial role in cell transformation and oncogenesis. This review addresses the mechanisms used by EBV to infect the cells and to evade the immune-surveillance as well as to induce cell survival and transformation, and characterizes the spectrum of the clinical manifestations associated with chronic EBV-infection and related T-and NK-lymphoproliferative disorders. Improving the knowledge in this subject will help to develop new therapeutic approaches for chronic EBV-infection and even prevention strategies for the aggressive NK-cell malignancies.

Heat Shock Protein 90 Inhibitor NVP-AUY922 Has Potent Anti-Tumor Activity With Adult T-Cell Leukemia-Lymphoma Cells

BackgroundAdult T-cell leukemia-lymphoma (ATL) is a chemo-resistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. The HSP90 inhibitor 17-AAG, derived from geldanamycin, has potent antitumor activity against ATL. However, geldanamycin derivatives have several limitations, including poor solubility, formulation difficulties, and severe hepatotoxicity in clinical settings, which have prompted development of second generation synthetic HSP90 inhibitors including NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor that inhibits the ATPase activity of HSP90. AUY922 has shown nanomolar efficacy against a wide range of human cancer cells in vitro and also inhibits progression of a variety of tumors in vivo. Phase I/II studies of AUY922 with advanced solid tumors and hematological malignancies are presently underway. Here, we studied the effects of AUY922 on ATL in vitro and in vivo. ResultsWe initially analyzed the effects of AUY922 (Novartis Pharmaceuticals) on survival of ATL-derived cell lines (KK1, SO4, LM-Y1, KOB, ST1) and HTLV-I-infected T-cell lines (MT2, HuT102). Cells cultured with various concentrations of AUY922 for 72 hours showed survival suppression in a dose-dependent manner in MTS assay findings. The concentrations of AUY922 required to inhibit cell survival by 50% (IC50) varied from 12.5 to 25.0 nM. We also found that the inhibitory effect of AUY was superior to that of 17-AAG. We further assessed AUY922-induced cell survival inhibition with peripheral blood mononuclear cells (PBMCs) obtained from patients with ATL and healthy donors. AUY922 induced apparent cell survival suppression in primary ATL cells, but not in normal PBMCs, while FACS analysis revealed that AUY922 induced cell-cycle arrest and apoptosis in these cell lines. Interestingly, AUY922 induced down-regulation of PIM kinases, which was confirmed by DNA microarray, qRT-PCR, and WB analysis results. Furthermore, SGI-1776, a PIM kinase inhibitor, successfully induced cell survival suppression in ATL and HTLV-1 infected cell lines in both dose- and cell-dependent manners. To elucidate the molecular mechanisms of cytotoxicity, we also examined the expressions of several client proteins using WB analysis. AUY922 treatment led to strong up-regulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease of HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including p-Akt, Akt, IκBα, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. In a xenograft model created with C.B-17/Icr-SCID mice, intraperitoneal administration of the vehicle or AUY922 was given after injection of HuT102 cells. In the control mice, bulky tumors grew within 4 weeks, whereas daily administrations of AUY922 significantly impaired tumor growth. ConclusionTogether, our findings suggest that AUY922 may be an effective therapeutic agent for ATL and PIM kinases are a novel therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Pellino3 targets RIP1 and regulates the pro-apoptotic effects of TNF-α

Tumour necrosis factor-α (TNF) can activate NF-κB to induce pro-inflammatory genes but can also stimulate the caspase cascade to promote apoptosis. Here we show that deficiency of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis without inhibiting the NF-κB pathway. Suppressed expression of Pellino3 leads to enhanced formation of the death-induced signalling complex, complex II, in response to TNF. We show that Pellino3 targets RIP1, in a TNF-dependent manner, to inhibit TNF-induced complex II formation and caspase 8-mediated cleavage of RIP1 in response to TNF/cycloheximide co-stimulation. Pellino3-deficient mice also show increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and an important determining factor in dictating whether TNF induces cell survival or death.

The roles of cyclic AMP–ERK–Bad signaling pathways on 6-hydroxydopamine-induced cell survival and death in PC12 cells

The roles of cyclic AMP (cAMP)–ERK1/2–Bad signaling pathways in 6-hydroxydopamine (6-OHDA)-induced cell survival and death were investigated. In PC12 cells, 6-OHDA (10–100μM) concentration-dependently increased the intracellular levels of cAMP mediated by the Ca2+-CaMKII-adenylyl cyclase system. 6-OHDA at the non-toxic level (10μM) induced transient ERK1/2 phosphorylation and BadSer112 phosphorylation, which maintained cell survival. In contrast, the high levels of cAMP induced by toxic levels (50 and 100μM) of 6-OHDA induced sustained ERK1/2 phosphorylaton and BadSer155 phosphorylation. The cells then moved to cell death process through Bcl2 phosphorylation and caspase-3 activation. BadSer155 phosphorylation by 6-OHDA was inhibited by PKA (H89) and MEK (U0126) inhibitors, indicating that it was mediated via the cAMP–PKA-sustained ERK1/2 system. In SK-N-BE(2)C cells, the non-toxic level of 6-OHDA also showed transient ERK1/2 phosphorylation and BadSer112 phosphorylation, and toxic levels of 6-OHDA exhibited sustained ERK1/2 phosphorylation and BadSer155 phosphorylation. These results suggest that ERK1/2 phosphorylation by 6-OHDA shows biphasic functions on cell survival and death in PC12 cells. It is, therefore, proposed that the cAMP–ERK1/2–Bad signaling pathways incurred by toxic levels of 6-OHDA play a role in dopamine neuron death of animal models of Parkinson’s disease.

Xenon Treatment Protects Against Cold Ischemia Associated Delayed Graft Function and Prolongs Graft Survival in Rats

Prolonged hypothermic storage causes ischemia-reperfusion injury (IRI) in the renal graft, which is considered to contribute to the occurrence of the delayed graft function (DGF) and chronic graft failure. Strategies are required to protect the graft and to prolong renal graft survival. We demonstrated that xenon exposure to human proximal tubular cells (HK-2) led to activation of range of protective proteins. Xenon treatment prior to or after hypothermia–hypoxia challenge stabilized the HK-2 cellular structure, diminished cytoplasmic translocation of high-mobility group box (HMGB) 1 and suppressed NF-κB activation. In the syngeneic Lewis-to-Lewis rat model of kidney transplantation, xenon exposure to donors before graft retrieval or to recipients after engraftment decreased caspase-3 expression, localized HMGB-1 within nuclei and prevented TLR-4/NF-κB activation in tubular cells; serum pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were reduced and renal function was preserved. Xenon treatment of graft donors or of recipients prolonged renal graft survival following IRI in both Lewis-to-Lewis isografts and Fischer-to-Lewis allografts. Xenon induced cell survival or graft functional recovery was abolished by HIF-1α siRNA. Our data suggest that xenon treatment attenuates DGF and enhances graft survival. This approach could be translated into clinical practice leading to a considerable improvement in long-term graft survival.

NTRK3 Is a Potential Tumor Suppressor Gene Commonly Inactivated by Epigenetic Mechanisms in Colorectal Cancer

NTRK3 is a member of the neurotrophin receptor family and regulates cell survival. It appears to be a dependence receptor, and thus has the potential to act as an oncogene or as a tumor suppressor gene. NTRK3 is a receptor for NT-3 and when bound to NT-3 it induces cell survival, but when NT-3 free, it induces apoptosis. We identified aberrantly methylated NTRK3 in colorectal cancers through a genome-wide screen for hypermethylated genes. This discovery led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. NTRK3 is methylated in 60% of colon adenomas and 67% of colon adenocarcinomas. NTRK3 methylation suppresses NTRK3 expression. Reconstitution of NTRK3 induces apoptosis in colorectal cancers, if NT-3 is absent. Furthermore, the loss of NTRK3 expression associates with neoplastic transformation in vitro and in vivo. We also found that a naturally occurring mutant NTRK3 found in human colorectal cancer inhibits the tumor suppressor activity of NTRK3. In summary, our findings suggest NTRK3 is a conditional tumor suppressor gene that is commonly inactivated in colorectal cancer by both epigenetic and genetic mechanisms whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.

Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells

BackgroundAll-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance.ResultsWe performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RARα to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RARα-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RARβ2 and p53. In contrast, over-expression of the inactive form of Akt restores RARβ2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes.ConclusionOur results demonstrate that rapid activation of Akt blocks transcription-dependent mechanism of ATRA, promotes invasion and cell survival and confers resistance to retinoic acid treatment in lung cancer cells. These findings provide an incentive for the design and clinical testing of treatment regimens that combine ATRA and PI3k inhibitors for lung cancer treatment.

LCK Is an Important Mediator of B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia Cells

B-cell receptor (BCR) signals promote survival of chronic lymphocytic leukemia (CLL) cells, and it is believed that overexpressed and constitutively active Lyn mediates this signaling. Here, we show that CLL cells express lymphocyte-specific protein tyrosine kinase (LCK) and that inhibition of this Src family tyrosine kinase with the specific inhibitor [4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2-d]pyrimidin-7-yl-cyclopentane (Lck-i)], or reduction of its expression with siRNA, blocks the induction of CD79a, Syk, inhibitor of IκB kinase (IKK), Akt, and extracellular signal-regulated kinase (ERK) phosphorylation by BCR cross-linking in these cells. Furthermore, we show that CLL cells with high levels of LCK expression have higher levels of BCR-mediated IKK, Akt, and ERK phosphorylation as well as cell survival than CLL cells with low levels of LCK expression. We also show that treatment of CLL cells with Lck-i inhibits BCR cross-linking-induced cell survival. Taken together, these data show a major role for LCK in proximal and distal BCR-mediated signaling in CLL cells and suggest that LCK expression is important in the pathogenesis of this disease. On a clinical level, these studies advocate the use of specific LCK inhibitors in the treatment of progressive CLL.

β2 integrin–derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis

Spleen tyrosine kinase (Syk) induces cell survival and proliferation in a high proportion of acute myeloid leukemia (AML) blasts, but the underlying molecular events of Syk signaling have not been investigated. Proteomic techniques have allowed us to identify the multiprotein complex that is nucleated by constitutively active Syk in AML cells. This complex differs from the B-lymphoid Syk interactome with respect to several proteins, especially the integrin receptor Mac-1, the Fc-γ receptor I (FcγRI), and the transcription factors STAT3 and STAT5. We show in several AML cell line models that tonic signals derived from the Fc-γ chain lead to Syk-dependent activation of STAT3 and STAT5, which in turn induces cell survival and proliferation. Moreover, stimulation of Mac-1 or FcγRI intensifies the constitutive Syk-mediated STAT3/5 activation in AML cells, a scenario likely to take place in the bone marrow niche. In accordance with these findings, we observed that β2 integrins, including Mac-1, trigger proliferation of AML cells in an AML cell/stroma coculture model. Taken together, we identified an oncogenic integrin/Syk/STAT3/5 signaling axis that might serve as a therapeutic target of AML in the future.

Abstract 1107: β2-integrin-derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis.

Abstract Constitutive activation of certain kinase-dependent signal transduction pathways is critical for the pathogenesis of acute myeloid leukemia (AML). Apart from FLT3-ITD the spleen tyrosine kinase (Syk) induces cell survival and proliferation in a high proportion of AML blasts, but the underlying molecular events of Syk signaling have not been investigated so far. Using a phosphoproteomic approach we elucidated the AML-specific phosphorylation state of Syk and found that the kinase is constitutively phosphorylated at all known activation inducing tyrosine residues. Moreover, we have identified the multi-protein complex that is nucleated by constitutively active Syk in AML cells by quantitative proteomic techniques. This complex differs from the B lymphoid Syk interactome regarding several proteins, in particular the β2-integrin receptor Mac-1 and also the oncogenic transcription factors STAT3 and STAT5. Our functional studies in primary AML cell cultures revealed that tonic signals derived from the integrin-associated Fc-γ chain lead to Syk-dependent activation of STAT3 and STAT5 that in turn induces AML cell proliferation. Moreover, stimulation of Mac-1 intensifies the constitutive Syk-mediated STAT3/5 activation in AML cells, a scenario likely to take place in the bone marrow niche. In accordance with these findings, we observed that β2-integrins including Mac-1 induce proliferation of AML cells in an AML cell/stroma co-culture model. Taken together, we identified an oncogenic integrin/Syk/STAT signaling axis that might serve as a therapeutic target of AML in the future. Citation Format: Thomas Oellerich, Mark Oellerich, Michael Engelke, Silvia Muench, Sebastian Mohr, He-Hsuan Hsiao, Jing Zhang, Hanibal Bohnenberger, Tobias Berg, Michael Rieger, Juergen Wienands, Gesine Bug, Christian Brandts, Henning Urlaub, Hubert Serve. β2-integrin-derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1107. doi:10.1158/1538-7445.AM2013-1107

Abstract 3191: The Akt-mTOR axis determines cell fate through the regulation of eIF2α phosphorylation pathway.

Abstract Metazoans respond to environmental stress by inducing the phosphorylation of the α subunit of translation initiation factor eIF2 at S51 (eIF2αP), a modification that leads to protein synthesis inhibition. We demonstrate that eIF2αP is induced by pharmacological inhibition or genetic ablation of the PI3K-Akt-mTOR pathway (1). Increased eIF2αP is an evolutionary conserved process that involves the endoplasmic reticulum (ER)-resident protein kinase PERK, which is negatively regulated by Akt dependent phosphorylation at T799. The PERK-eIF2αP arm is downregulated by Akt in cells exposed to ER stress or oxidative stress leading to the induction of cell survival or death respectively. In unstressed cells, the PERK-eIF2αP pathway guards survival and facilitates adaptation to the deleterious effects of PI3K, Akt or mTOR inactivation. Inactivation of the PERK-eIF2αP arm sensitizes tumor death from pharmacological inhibition of the PI3K-Akt-mTOR pathway. The PERK-eIF2αP pathway links Akt and mTOR signaling to translational control with implications in tumor treatment with pharmacological inhibitors of PI3K-Akt-mTOR pathway. Citation Format: Clara Tenkerian, Zineb Mounir, Jothi Krishnamoorthy, Antonis E. Koromilas. The Akt-mTOR axis determines cell fate through the regulation of eIF2α phosphorylation pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3191. doi:10.1158/1538-7445.AM2013-3191 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.