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PDF file - 589K, Figure S1. Antiproliferative effects of Compound C in glioma cells. Figure S2. Compound C induces AMPK-independent autophagy and the antiproliferative effects of Compound C are not blocked by autophagy and apoptosis inhibitors. Figure S3. Constitutively active Akt fails to block Compound C?s antiproliferative action. Figure S4. Effects of Compound C on glioma cell cycle.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor important for cell growth, proliferation, survival, and metabolic regulation. Active AMPK inhibits biosynthetic enzymes like mTOR and acetyl CoA carboxylase (required for protein and lipid synthesis, respectively) to ensure that cells maintain essential nutrients and energy during metabolic crisis. Despite our knowledge about this incredibly important kinase, no specific chemical inhibitors are available to examine its function. However, one small molecule known as compound C (also called dorsomorphin) has been widely used in cell-based, biochemical, and in vivo assays as a selective AMPK inhibitor. In nearly all these reports including a recent study in glioma, the biochemical and cellular effects of compound C have been attributed to its inhibitory action toward AMPK. While examining the status of AMPK activation in human gliomas, we observed that glioblastomas express copious amount of active AMPK. Compound C effectively reduced glioma viability in vitro both by inhibiting proliferation and inducing cell death. As expected, compound C inhibited AMPK; however, all the antiproliferative effects of this compound were AMPK independent. Instead, compound C killed glioma cells by multiple mechanisms, including activation of the calpain/cathepsin pathway, inhibition of AKT, mTORC1/C2, cell-cycle block at G2–M, and induction of necroptosis and autophagy. Importantly, normal astrocytes were significantly less susceptible to compound C. In summary, compound C is an extremely potent antiglioma agent but we suggest that caution should be taken in interpreting results when this compound is used as an AMPK inhibitor. Mol Cancer Ther; 13(3); 596–605. ©2014 AACR.

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Purpose: Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica, controls glioblastoma growth.Experimental Design: Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma.Results: We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against glioblastoma multiforme in vitro and in vivo. Azt caused cell-cycle arrest, most prominently at the G1–S stage in glioblastoma multiforme cells expressing EGFRvIII, an oncogene present in about 20% to 25% of glioblastoma multiformes. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma protein, cell-cycle arrest at G1—S, and cell death. Independent of retinoblastoma hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of glioblastoma multiforme cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor-induced phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and STAT3. These effects were specific because Azt did not affect mTOR or other cell-cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of glioblastoma multiforme growth.Conclusions: Our preclinical findings demonstrate nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo. Clin Cancer Res; 20(1); 199–212. ©2013 AACR.

PDF file - 312K, Nimbolide is more cytotoxic than many other chemotherapy agents. (A) Cell proliferation assay showing viability of GBM cells in the presence of Temozolomide (TMZ) for 72 hours. (B) Cell proliferation assay showing the cytotoxic effect of Nimbolide in comparison with other chemotherapeutic agents. Data is representative of two independent experiments. +p ≤ 0.05; *p ≤ 0.001.

Glioma stem–like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1–ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo. Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1–ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219–30. ©2016 AACR.

Supplementary Table S1. Cohort demographics of 83, 84, 157, 1016, 28, 711 and 267 glioma spheres. Supplementary Table S2. STR analysis result of 83, 84, 157, 1016, 28, 711 and 267 glioma spheres. Supplementary Table S3. Physical chemical properties of GA11 and GA23, determined by BIOVIA Discovery Studio 4.5.

Supplement Fig. S1. Original film scan of western blots in indicated figures; Supplementary Fig. S2. FOXD1 is identified as a MES GSC-representative transcription factor, related to Figure 1.; Supplementary Fig. S3. Glycolysis assay by measuring relative lactic acid levels of MES83 glioma spheres transduced with shNT control or shFOXD1#1; Supplementary Fig. S4. FOXD1 transcriptionally regulates the expression of ALDH1A3 in MES83 GSCs, related to Figure 4.; Supplementary Fig. S5. RNA-interference mediated silencing of ALDH inhibits tumorigenicity of Drosophila eye neoplasia, related to Figure 5.; Supplementary Fig. S6. Analysis of Rembrandt database showing a higher expression of ALDH1A3 associated with poor prognosis in glioma patients, related to Figure 6.; Supplementary Fig. S7. ALDH inhibitor GA11 inhibits the tumorigenicity of MES83 glioma spheres, related to Figure 6.

Supplementary methods and materials

Alzheimer's disease (AD) is the most common cause of dementia, characterized by the deposition of Amyloid-beta (Aβ) plaques in the brain. We have previously developed Amytrap peptide (the active pharmacological ingredient, API) and linked it to a sepharose bead matrix by click chemistry to form Amytrapper matrix, which was able to bind and remove Aβ from human sera and plasma spiked with biotinylated Aβ42 (bio-Aβ42) in vitro. To extend the logic of the previous studies, the current study investigates whether the Amytrap peptide coated inside a medically viable polycarbonate catheter (Amytrapper catheter) could bind and retain Aβ from the human sera. The Amytrapper matrix and the novel Amytrapper catheter were able to bind and retain spiked bio-Aβ42 from human sera or native Aβ from plasma of AD patients. Additional characteristics of the Amytrapper catheter are evaluated and presented in this study. The results presented here provide a proof-of-principle for the first time that extracorporeal Amytrapper device aids clearance of native Aβ (from plasma of AD patients). Thus, our device Amytrapper, either in the form of Sepharose matrix or catheter, could become a novel therapeutic strategy to remove Aβ from circulation in AD patients.

Stress is integral to tumor evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activate the bioenergetic sensor AMP kinase (AMPK), and tumor cells hijack an AMPK-regulated stress response pathway conserved in normal cells, to survive. Analysis of The Cancer Genome Atlas (TCGA) data revealed that AMPK isoforms are highly expressed in the lethal human cancer Glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumors. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with the cAMP response element binding protein-1 (CREB1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumor bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK supporting the utility of AMPK pharmacological inhibitors in the treatment of GBM.

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

Activation of nuclear factor κB (NF-κB) induces mesenchymal (MES) transdifferentiation and radioresistance in glioma stem cells (GSCs), but molecular mechanisms for NF-κB activation in GSCs are currently unknown. Here, we report that mixed lineage kinase 4 (MLK4) is overexpressed in MES but not proneural (PN) GSCs. Silencing MLK4 suppresses self-renewal, motility, tumorigenesis, and radioresistance of MES GSCs via a loss of the MES signature. MLK4 binds and phosphorylates the NF-κB regulator IKKα, leading to activation of NF-κB signaling in GSCs. MLK4 expression is inversely correlated with patient prognosis in MES, but not PN high-grade gliomas. Collectively, our results uncover MLK4 as an upstream regulator of NF-κB signaling and a potential molecular target for the MES subtype of glioblastomas.

Glioblastoma (GBM) remains the deadliest of all primary brain tumors with very few effective treatment options. Recently, we reported that high AXL expression is correlated with poor prognosis in GBM patients and demonstrated the therapeutic benefits of targeting AXL, a member of TAM receptor tyrosine kinase family using a novel small molecule inhibitor, BGB324 in immunocompetent mouse GBM models and xenografts of patient-derived glioma stem cells(GSCs). The promise of BGB324 in tumor burden management prompted us to develop a clinical trial with BGB324 as a single agent therapeutic with the goal to extend it as a combinatorial therapy in the future. Our surgical PK/PD clinical trial with BGB324 in recurrent GBM has been approved by the Brain Malignancy Steering Committee at the National Cancer Institute. Study treatment will consist of 2 cohorts of adult GBM patients, one (Group A) receiving the treatment pre-operatively and the other (Group B) receiving no treatment at all prior to surgery. First 5 patients recruited to Group A will be checked for the desired intra-tumoral drug concentration achieved to continue the trial. Group A will be supplemented by an additional 5 patients bringing the number to n=10 in each arm of the trial. Following surgical resection, patients in both cohorts will receive BGB324 daily in 21-day cycles. Treatment will be continued unless patients exhibit significant toxicity or substantial tumor progression. Our preclinical findings show the upregulation of AXL and its role in apoptosis induction in mesenchymal GBM as well as its association with MLK4, a serine threonine kinase we previously characterized as a mesenchymal GSC molecular target. Inhibition of phosphorylation of AXL and concomitant NF-kB activation in mesenchymal GSCs was found to be the nodal target of the drug action. An up-to-date information of the trial will be presented in detail.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1–AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002–13. ©2018 AACR.

The tumour suppressor gene p53 is mutated in approximately 50% of the human cancers. p53 is involved in genotoxic stress-induced cellular responses. The role of EGFR and ERK in DNA-damage-induced apoptosis is well known. We investigated the involvement of activation of ERK signalling as a consequence of non-functional p53, in sensitivity of cells to doxorubicin. We performed cell survival assays in cancer cell lines with varying p53 status: MCF-7 (wild-type p53, WTp53), MDA MB-468 (mutant p53, MUTp53), H1299 (absence of p53, NULLp53) and an isogenic cell line MCF-7As (WTp53 abrogated). Our results indicate that enhanced chemosensitivity of cells lacking wild-type p53 function is because of elevated levels of EGFR which activates ERK. Additionally, we noted that independent of p53 status, pERK contributes to doxorubicin-induced cell death.

BACKGROUND Finasteride and dutasteride were developed originally as 5α-reductase inhibitors to block the conversion of testosterone to dihydrotestosterone (DHT). These drugs may possess off-target effects on the androgen receptor (AR) due to their structural similarity to DHT. METHODS A total of four human prostate cancer cell models were examined: LNCaP (T877A mutant AR), 22Rv1 (H874Y mutant AR), LAPC4 (wild-type AR), and VCaP (wild-type AR). Cells were cultured in 10% charcoal-stripped fetal bovine serum, either with or without DHT added to the medium. AR activity was evaluated using the ARE-luciferase assay or the expression of AR regulated genes. RESULTS Dutasteride was more potent than finasteride in interfering with DHT-stimulated AR signaling. Disruption of AR function was accompanied by decreased cell growth. Cells that rely on DHT for protection against death were particularly vulnerable to dutasteride. Different prostate cancer cell models exhibited different sensitivities to dutasteride and finasteride. LNCaP was most sensitive, LAPC4 and VCaP were intermediate, while 22Rv1 was least sensitive. Regardless of the AR genotype, if AR was transfected into drug-sensitive cells, AR was inhibited by drug treatment; and if AR was transfected into drug-resistant cells, AR was not inhibited. CONCLUSIONS The direct inhibitory effect of dutasteride or finasteride on AR signaling is cell line specific. Mutations in the ligand binding domain of AR do not appear to play a significant role in influencing the AR antagonistic effect of these drugs. Subcellular constituent is an important factor in determining the drug effect on AR function. Prostate 73: 1483–1494, 2013. © 2013 Wiley Periodicals, Inc.

Glioma stem–like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1–ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo. Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1–ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219–30. ©2016 AACR.

The present study was designed to investigate (i) the role of AMPK activation in inducing autophagy in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia, and (ii) whether autophagy offers a survival advantage under these harsh conditions. Low androgen and low oxygen are two co-existing conditions frequently found in prostate cancer tissue following surgical or medical castration. In LNCaP cells, androgen deprivation and hypoxia together boosted AMPK activation to a higher level than that seen with either condition alone. The augmented AMPK response was associated with improved viability and the induction of autophagy. These observations suggest that a threshold of AMPK activity has to be attained in order to trigger autophagy, since neither androgen deprivation nor hypoxia by itself was capable of pushing AMPK activity past that threshold. Beclin-1 was identified as a potential downstream target of AMPK in turning on the autophagic cascade. If autophagy was blocked by chemical inhibition or RNA interference of key regulators, e.g., AMPK or beclin-1, more cells would die by apoptosis. The occurrence of autophagy is thus a survival mechanism for androgen-dependent prostate cancer cells to escape from an androgen-deprived and hypoxic subsistence.

Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild type AR. When PC-3 cells, which are AR-null, were transfected with either the wild type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy.

In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.

AMP kinase (AMPK) is an evolutionarily conserved enzyme required for adaptive responses to various physiological and pathological conditions. AMPK executes numerous cellular functions, some of which are often perceived at odds with each other. While AMPK is essential for embryonic growth and development, its full impact in adult tissues is revealed under stressful situations that organisms face in the real world. Conflicting reports about its cellular functions, particularly in cancer, are intriguing and a growing number of AMPK activators are being developed to treat human diseases such as cancer and diabetes. Whether these drugs will have only context-specific benefits or detrimental effects in the treatment of human cancer will be a subject of intense research. Here we review the current state of AMPK research with an emphasis on cancer and discuss the yet unresolved context-dependent functions of AMPK in human cancer.

The in vivo hypoglycaemic activity of a dialysed fenugreek seed extract (FSE) was studied in alloxan (AXN)-induced diabetic mice and found to be comparable to that of insulin (1.5 U kg(-1)). FSE also improved intraperitoneal glucose tolerance in normal mice. The mechanism by which FSE attenuated hyperglycaemia was investigated in vitro. FSE stimulated glucose uptake in CHO-HIRc-mycGLUT4eGFP cells in a dose-dependent manner. This effect was shown to be mediated by the translocation of glucose transporter 4 (GLUT4) from the intracellular space to the plasma membrane. These effects of FSE on GLUT4 translocation and glucose uptake were inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and bisindolylmaleimide 1, a protein kinase C (PKC)-specific inhibitor. In vitro phosphorylation analysis revealed that, like insulin, FSE also induces tyrosine phosphorylation of a number of proteins including the insulin receptor, insulin receptor substrate 1 and p85 subunit of PI3-K, in both 3T3-L1 adipocytes and human hepatoma cells, HepG2. However, unlike insulin, FSE had no effect on protein kinase B (Akt) activation. These results suggest that in vivo the hypoglycaemic effect of FSE is mediated, at least in part, by the activation of an insulin signalling pathway in adipocytes and liver cells.

Cyclophosphamide (CPA) is a widely used chemotherapeutic drug in neoplasias. It is a DNA and protein alkylating agent that has a broad spectrum of activity against variety of neoplasms including breast cancer. The therapeutic effectiveness of CPA is limited by the high-dose hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. The present study examines the potential of combining well-tolerated antibiotic doxycycline (DOX) with CPA and understanding the mechanism of cell killing. Interestingly, we found that DOX significantly enhances the tumor regression activity of CPA on xenograft mice model bearing MCF-7 cells. DOX also potentiates MCF-7 cell killing by CPA in vitro. In presence of DOX (3 microg/ml), the IC50 value of CPA decreased significantly from 10 to 2.5 mM. Additional analyses indicate that the tumor suppressor p53 and p53-regulated proapoptotic Bax were upregulated in vivo and in vitro following CPA treatment in combination with DOX, suggesting that upregulation of p53 may contribute to the enhancement of antitumor effect of CPA by DOX. Furthermore, downregulation of antiapoptotic Bcl-2 was observed in animals treated with CPA and CPA plus DOX when compared to untreated or DOX-treated groups. Our results raise the possibility that this combination chemotherapeutic regimen may lead to additional improvements in treatment of breast cancer.

As tumors evolve, they undergo metabolic state transitions in response to flux of nutrients, O2, and therapy. The evolving cancer-genome undergoes several intended and unintended mutations, amplifications, insertions and deletions. These dynamic changes create metabolic vulnerability and impose great burden on cancer cells. To thrive, evolving cancer cells acquire a remarkably altered metabolic and signaling flexibility. Identifying genes that protect tumor cells against cell-intrinsic vulnerabilities and the hostile tumor environment may provide novel therapy in human cancer. The multifunctional kinase AMPK is an evolutionarily conserved energy/stress sensor that plays important yet undefined role during tumor evolution. AMPK activated by the tumor suppressor LKB1 or the calcium-regulated kinase CAMKK2. AMPK inhibits mTOR and lipogenesis and therefore not surprisingly, many laboratories found that agents (e.g., metformin, now in several clinical trials) that activate AMPK suppress tumor growth. However, a few recent studies questioned this over-simplistic model. Using glioblastoma multiforme (GBM) as a model, we found that the AMPK pathway is constitutively active in human GBM and in a clinically relevant genetically engineered mouse model of high grade glioma. The AMPK-HIF1 alpha axis was earlier shown to play a tumor suppressive role in a mouse model of lymphoma. Unexpectedly, we uncovered that the AMPK-HIF1 alpha axis plays a tumor promoting role in GBM. Biochemical, genetic and metabolic experiments showed that AMPK positively regulates a HIF1alpha and GABPA-dependent transcriptional program of GBM bioenergetics that allows optimal energy metabolism and survival. Unfortunately, there are no specific AMPK inhibitors. Therefore, using multiple genetic tools to reduce, or express (rescue) targets of the AMPK- HIF1alpha-GABPA pathway in vivo and in vitro, we observed that AMPK is essential for GBM growth and survival.

AMP kinase (AMPK) is an evolutionarily conserved enzyme required for adaptive responses to various physiological and pathological conditions. The multisubunit structure and sensitivity to various stresses confer complexity to AMPK function which scores beyond energy sensing. Here we show that inhibition of AMPK reduces viability of patient-derived primary Glioblastoma Multiforme (GBM) lines and tumors. We demonstrate that primary GBM cells coopt the AMPK-CREB1 pathway to coordinate global bioenergetics program through control of the transcription factors HIF1alpha and GABPA. But a growing number of AMPK activators are being developed to treat human diseases such as cancer and diabetes. This may be because AMPK activity can inhibit biosynthetic kinases like mammalian target of rapamycin (mTOR) and acetyl Co-A carboxylase (ACC). We found that AMPK silencing in glioma cells did not affect mTOR or downstream targets. AMPK is not absolutely necessary for mTOR inhibition, suggesting that in certain contexts, activated AMPK and mTOR may coexist. Indeed, this is what we observed in glioblastoma suggesting that basally active AMPK may not be sufficient to put a brake on mTOR. Further analysis of our preliminary data from primary tumor cells on Cap-dependent and well reported procancer IRES dependent translation under tumor microenvironment context will demonstrate significance of AMPK in translational machinery in GBM and will benefit in finding new therapeutic targets. Citation Format: Rishi Raj Chhipa, Biplab Dasgupta. AMPK's role in glioblastoma survival: An insight into its regulation on transcription factors and mTOR. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A23.

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis-two crucial arms of cancer growth-AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor important for cell growth, proliferation, survival, and metabolic regulation. Active AMPK inhibits biosynthetic enzymes like mTOR and acetyl CoA carboxylase (required for protein and lipid synthesis, respectively) to ensure that cells maintain essential nutrients and energy during metabolic crisis. Despite our knowledge about this incredibly important kinase, no specific chemical inhibitors are available to examine its function. However, one small molecule known as compound C (also called dorsomorphin) has been widely used in cell-based, biochemical, and in vivo assays as a selective AMPK inhibitor. In nearly all these reports including a recent study in glioma, the biochemical and cellular effects of compound C have been attributed to its inhibitory action toward AMPK. While examining the status of AMPK activation in human gliomas, we observed that glioblastomas express copious amount of active AMPK. Compound C effectively reduced glioma viability in vitro both by inhibiting proliferation and inducing cell death. As expected, compound C inhibited AMPK; however, all the antiproliferative effects of this compound were AMPK independent. Instead, compound C killed glioma cells by multiple mechanisms, including activation of the calpain/cathepsin pathway, inhibition of AKT, mTORC1/C2, cell-cycle block at G2–M, and induction of necroptosis and autophagy. Importantly, normal astrocytes were significantly less susceptible to compound C. In summary, compound C is an extremely potent antiglioma agent but we suggest that caution should be taken in interpreting results when this compound is used as an AMPK inhibitor. Mol Cancer Ther; 13(3); 596–605. ©2014 AACR.

Purpose: Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica, controls glioblastoma growth. Experimental Design: Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma. Results: We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against glioblastoma multiforme in vitro and in vivo. Azt caused cell-cycle arrest, most prominently at the G1–S stage in glioblastoma multiforme cells expressing EGFRvIII, an oncogene present in about 20% to 25% of glioblastoma multiformes. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma protein, cell-cycle arrest at G1—S, and cell death. Independent of retinoblastoma hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of glioblastoma multiforme cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor-induced phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and STAT3. These effects were specific because Azt did not affect mTOR or other cell-cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of glioblastoma multiforme growth. Conclusions: Our preclinical findings demonstrate nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo. Clin Cancer Res; 20(1); 199–212. ©2013 AACR.

Finasteride and dutasteride were developed originally as 5α-reductase inhibitors to block the conversion of testosterone to dihydrotestosterone (DHT). These drugs may possess off-target effects on the androgen receptor (AR) due to their structural similarity to DHT.A total of four human prostate cancer cell models were examined: LNCaP (T877A mutant AR), 22Rv1 (H874Y mutant AR), LAPC4 (wild-type AR), and VCaP (wild-type AR). Cells were cultured in 10% charcoal-stripped fetal bovine serum, either with or without DHT added to the medium. AR activity was evaluated using the ARE-luciferase assay or the expression of AR regulated genes.Dutasteride was more potent than finasteride in interfering with DHT-stimulated AR signaling. Disruption of AR function was accompanied by decreased cell growth. Cells that rely on DHT for protection against death were particularly vulnerable to dutasteride. Different prostate cancer cell models exhibited different sensitivities to dutasteride and finasteride. LNCaP was most sensitive, LAPC4 and VCaP were intermediate, while 22Rv1 was least sensitive. Regardless of the AR genotype, if AR was transfected into drug-sensitive cells, AR was inhibited by drug treatment; and if AR was transfected into drug-resistant cells, AR was not inhibited.The direct inhibitory effect of dutasteride or finasteride on AR signaling is cell line specific. Mutations in the ligand binding domain of AR do not appear to play a significant role in influencing the AR antagonistic effect of these drugs. Subcellular constituent is an important factor in determining the drug effect on AR function.

Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells.

Androgen receptor (AR) signaling plays a critical role in the development and progression of prostate cancer. It has been reported previously that peroxiredoxin-1 (Prx1), a member of a novel family of peroxidases, interacts physically with AR to enhance AR transactivation of target genes. In the present study, we evaluated the biological significance of Prx1 in modulating dihydrotestosterone (DHT)-stimulated growth and AR target gene expression of prostate cancer cells. We also investigated the mechanism by which Prx1 might potentiate AR signaling. The contribution of Prx1 was assessed mainly by using the approach of stable Prx1 knockdown. The major observations are as follows: (a) A low level of Prx1 desensitizes cells to growth stimulation and AR target gene induction by DHT, such that exposure to a higher level of DHT is required to reach the same magnitude of response when Prx1 is depressed; (b) Prx1 increases the affinity of AR to DHT and decreases the rate of DHT dissociation from the occupied receptor; (c) Prx1 enhances the NH2 terminus and COOH terminus interaction of AR; a stronger N-C interaction is consistent with a more robust AR activation signal by keeping DHT tight in the ligand-binding pocket; (d) the stimulatory effects of Prx1 on AR ligand binding affinity and AR N-C interaction are manifested regardless of a wild-type or mutant AR. The above findings led us to believe that Prx1 may be a therapeutic target in blocking the transition of prostate cancer from an androgen-dependent to an androgen-refractory phenotype. (Mol Cancer Res 2009;7(9):1543–52)

The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury ("bystander effect" (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF-7 breast cancer cells which were coplated with 5-fluorouracil (5-FU)-treated MDA-MB-231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA-MB-231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non-metastatic MCF-7 breast cancer cells (MCF-EGFP), was used. A bystander killing effect was observed in MCF-EGFP cells cocultured with MDA-MB-231 cells pretreated with 5-FU. The striking decrease in MCF-EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF-EGFP cell death was confirmed by using antagonistic anti-FasL antibody that reverses bystander cell death by blocking FasL on MDA-MB-231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF-EGFP cells by treatment with Pifithrin-alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF-EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy-induced bystander cell death in breast cancers.

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.

The in vivo hypoglycaemic activity of a dialysed fenugreek seed extract (FSE) was studied in alloxan (AXN)-induced diabetic mice and found to be comparable to that of insulin (1.5 U kg(-1)). FSE also improved intraperitoneal glucose tolerance in normal mice. The mechanism by which FSE attenuated hyperglycaemia was investigated in vitro. FSE stimulated glucose uptake in CHO-HIRc-mycGLUT4eGFP cells in a dose-dependent manner. This effect was shown to be mediated by the translocation of glucose transporter 4 (GLUT4) from the intracellular space to the plasma membrane. These effects of FSE on GLUT4 translocation and glucose uptake were inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and bisindolylmaleimide 1, a protein kinase C (PKC)-specific inhibitor. In vitro phosphorylation analysis revealed that, like insulin, FSE also induces tyrosine phosphorylation of a number of proteins including the insulin receptor, insulin receptor substrate 1 and p85 subunit of PI3-K, in both 3T3-L1 adipocytes and human hepatoma cells, HepG2. However, unlike insulin, FSE had no effect on protein kinase B (Akt) activation. These results suggest that in vivo the hypoglycaemic effect of FSE is mediated, at least in part, by the activation of an insulin signalling pathway in adipocytes and liver cells.

DNA damaging chemotherapeutic agents like carboplatin (Carb) and 5-fluorouracil (5-FU), whose effects are mediated through diverse intracellular targets, induce apoptosis in various cancer cells including human papillomavirus (HPV) positive HEp-2 and KB cells. The present work reports the involvement of Bcl-2 in response to the exposure of HEp-2 and KB cells to Carb or 5-FU. We demonstrate that both these drugs are potent inducers of apoptosis. Apoptosis was preceded by decrease in Bcl-2 protein level accompanied by caspase-9 activation and poly(ADP-ribose) polymerase (PARP) cleavage without altering Bax expression. Further analysis revealed down-regulation of Bcl-2 mRNA as well as protein in drugs treated cells. Ectopic expression of Bcl-2 protected cells against drugs mediated DNA damage-induced apoptosis. Overall, data indicates that genotoxic stress leads to down-regulation of Bcl-2 in HEp-2 and KB cells, which plays a decisive role in the outcome of stress in these cells.

Background Cancer cells reprogram their metabolism for optimal growth and survival. Identifying the genes and their functions crucial for cancer metabolic reprograming might have therapeutic implications. The multifunctional kinase AMPK is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because while on one hand active AMPK inhibits mTOR and lipogenesis - two crucial arms of cancer growth, AMPK also ensures viability during metabolic stress. Many studies have shown that AMPK activation by two indirect AMPK agonists AICAR and metformin (now in many cancer clinical trials) reduces cancer cell proliferation - an effect that is rescued by an AMPK inhibitor Compound C in some studies. We used genetic models to scrutinize the specificity of these reagents and examine whether AMPK is a growth suppressor in glioblastoma. Materials and methods We used silencing RNA against AMPK in established GBM cell lines and examined viability in the presence or absence of the reagents to define their mechanisms of action. Results Surprisingly, we found that compared to normal tissue, AMPK is constitutively active in both human and mouse gliomas in vivo. We found that both AMPK agonists inhibited proliferation, but through discrete AMPK-independent mechanisms and both reagents reduced GBM growth in vivo independent of AMPK. Importantly, A769662, a new and direct AMPK activator had no effect on GBM cell proliferation. Metformin directly inhibited mTOR by enhancing PRAS40 association with RAPTOR, while AICAR blocked cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. In another surprise, Compound C itself proved to be a potent anti-GBM agent; however, its cell killing effects were pleiotropic and also independent of AMPK. We next examined if AMPK is required for viability of freshly established patient-derived GBM cells and found that many of these cells lose viability in vitro when AMPK was silenced. Conclusions Our results suggest that AICAR, metformin and Compound C are all AMPK-independent anti-proliferative agents. Importantly, physiologically active AMPK in GBM could be a growth promoter rather than a growth suppressor in vivo. We are taking multiple genetic approaches to determine this in mouse models of human GBM.