ABSTRACT
INTRODUCTION: Acute pancreatitis (AP) is a healthcare challenge with considerable mortality. Treatment is limited to supportive care, highlighting the need to investigate disease drivers and prognostic markers. Activin A is an established mediator of inflammatory responses, and its serum levels correlate with AP severity. We hypothesized that activin A is independent of body mass index (BMI) and is a targetable promoter of the AP inflammatory response. METHODS: We assessed whether BMI and serum activin A levels are independent markers to determine disease severity in a cohort of patients with AP. To evaluate activin A inhibition as a therapeutic, we used a cerulein-induced murine model of AP and treated mice with activin A-specific neutralizing antibody or immunoglobulin G control, both before and during the development of AP. We measured the production and release of activin A by pancreas and macrophage cell lines and observed the activation of macrophages after activin A treatment. RESULTS: BMI and activin A independently predicted severe AP in patients. Inhibiting activin A in AP mice reduced disease severity and local immune cell infiltration. Inflammatory stimulation led to activin A production and release by pancreas cells but not by macrophages. Macrophages were activated by activin A, suggesting activin A might promote inflammation in the pancreas in response to injury. DISCUSSION: Activin A provides a promising therapeutic target to interrupt the cycle of inflammation and tissue damage in AP progression. Moreover, assessing activin A and BMI in patients on hospital admission could provide important predictive measures for screening patients likely to develop severe disease.
Subject(s)
Activins/metabolism , Anti-Inflammatory Agents/pharmacology , Pancreas/pathology , Pancreatitis/diagnosis , Severity of Illness Index , Activins/antagonists & inhibitors , Activins/blood , Activins/immunology , Animals , Anti-Inflammatory Agents/therapeutic use , Biomarkers/blood , Biomarkers/metabolism , Body Mass Index , Cell Line , Ceruletide/administration & dosage , Ceruletide/toxicity , Cohort Studies , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Humans , Macrophage Activation/immunology , Macrophages , Mice , Pancreas/drug effects , Pancreas/immunology , Pancreatitis/blood , Pancreatitis/drug therapy , Pancreatitis/immunology , Patient Admission , Predictive Value of TestsABSTRACT
BACKGROUND: Colorectal cancer remains a deadly cancer due to metastatic disease. To understand the molecular mechanisms of metastasis in colon cancer, we investigated whether the copper chaperone antioxidant-1 (Atox1) protein plays a role in this process. Recent findings indicate that Atox1 protein has transcription factor activities and plays a vital role in cell proliferation in cancer cells. However, the role of Atox1 in metastasis has not been examined. METHODS: Atox1 expression was determined by immunofluorescence in a tissue microarray generated from a spectrum of CRC patients. Subcellular fractionation of colon cancer cell lines SW480 and SW620 cells was used to examine the cellular location of Atox1 in the face of activin A, a cytokine that stimulates colon cancer metastasis. Atox1 expression was genetically manipulated and cellular migration measured through trans-well assay and proliferation measured by colony formation assays. RESULTS: Here we demonstrate that in patients with metastatic colon cancer, there is a significant increase in the expression of nuclear Atox1. Interestingly, the metastatic CRC cell line SW620 has increased nuclear localization of Atox1 compared to its related non-metastatic cell line SW480. Further, inhibition of endogenous Atox1 by siRNA in SW620 decreased colony formation and reactive oxygen species generation via decreased expression of Atox1 targets cyclin D1 and NADPH oxidase subunit p47 phox, respectively. Additionally, overexpression of nuclear-targeted but not copper binding domain-mutated Atox1 in SW480 cells increased colony formation and cell migration that was further augmented by activin A stimulation, a known enhancer of colon cancer metastasis. CONCLUSIONS: Our findings suggest that nuclear Atox1 might be a new therapeutic target as well as a new biomarker for metastatic colorectal cancer.
Subject(s)
Activins/metabolism , Carcinoma , Cell Movement , Colonic Neoplasms , Copper Transport Proteins/physiology , Molecular Chaperones/physiology , Carcinoma/metabolism , Carcinoma/pathology , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , HumansABSTRACT
Although overall survival in colorectal cancer (CRC) is increasing steadily due to progress in screening, therapeutic options and precise diagnostic tools remain scarce. As the understanding of CRC as a complex and multifactorial condition moves forward, the tumor microenvironment has come into focus as a source of diagnostic markers and potential therapeutic targets. The role of TGFß in shifting the epithelial cancer compartment towards invasiveness and a pro-migratory phenotype via stromal signaling has been widely investigated. Accordingly, recent studies have proposed that CRC patients could be stratified into distinct subtypes and have identified one poor prognosis subset of CRC that is characterized by high stromal activity and elevated levels of TGFß. The TGFß superfamily member activin A is crucial for the pro-metastatic properties of the TGFß pathway, yet it has been under-researched in CRC carcinogenesis. In this review, we will elucidate the signaling network and interdependency of both ligands in the context of the tumor microenvironment in CRC.
ABSTRACT
Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB (TGFß) signaling are growth suppressive pathways that exert non-canonical pro-metastatic effects late in CRC carcinogenesis. We have recently shown that activin downregulates p21 via ubiquitination and degradation associated with enhanced cellular migration independent of SMADs. To investigate the mechanism of metastatic activin signaling, we examined activated NFkB signaling and activin ligand expression in CRC patient samples and found a strong correlation. We hypothesize that activation of the E3 ubiquitin ligase MDM2 by NFkB leads to p21 degradation in response to activin treatment. To dissect the link between activin and pro-carcinogenic NFkB signaling and downstream targets, we found that activin but not TGFB induced activation of NFkB leading to increased MDM2 ubiquitin ligase via PI3K. Further, overexpression of wild type p65 NFkB increased MDM2 expression while the NFkB inhibitors NEMO-binding domain (NBD) and Bay11-7082 blocked the activin-induced increase in MDM2. In conclusion, in colon cancer cell migration, activin utilizes NFkB to induce MDM2 activity leading to the degradation of p21 in a PI3K dependent mechanism. This provides new mechanistic knowledge linking activin and NFkB signaling in advanced colon cancer which is applicable to targeted therapeutic interventions.
Subject(s)
Activins/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Colorectal Neoplasms/metabolism , NF-kappa B/metabolism , Carcinogenesis , Cell Line, Tumor , Cell Movement , Colorectal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , NF-kappa B/genetics , Nitriles/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Signal Transduction , Sulfones/pharmacology , Ubiquitin-Protein Ligases/metabolismABSTRACT
Glucocorticoids (GC) are a cornerstone of combination therapies for multiple myeloma. However, patients ultimately develop resistance to GCs frequently based on decreased glucocorticoid receptor (GR) expression. An understanding of the direct targets of GC actions, which induce cell death, is expected to culminate in potential therapeutic strategies for inducing cell death by regulating downstream targets in the absence of a functional GR. The specific goal of our research is to identify primary GR targets that contribute to GC-induced cell death, with the ultimate goal of developing novel therapeutics around these targets that can be used to overcome resistance to GCs in the absence of GR. Using the MM.1S glucocorticoid-sensitive human myeloma cell line, we began with the broad platform of gene expression profiling to identify glucocorticoid-regulated genes further refined by combination treatment with phosphatidylinositol-3'-kinase inhibition (PI3Ki). To further refine the search to distinguish direct and indirect targets of GR that respond to the combination GC and PI3Ki treatment of MM.1S cells, we integrated 1) gene expression profiles of combination GC treatment with PI3Ki, which induces synergistic cell death; 2) negative correlation between genes inhibited by combination treatment in MM.1S cells and genes over-expressed in myeloma patients to establish clinical relevance and 3) GR chromatin immunoprecipitation with massively parallel sequencing (ChIP-Seq) in myeloma cells to identify global chromatin binding for the glucocorticoid receptor (GR). Using established bioinformatics platforms, we have integrated these data sets to identify a subset of candidate genes that may form the basis for a comprehensive picture of glucocorticoid actions in multiple myeloma. As a proof of principle, we have verified two targets, namely RRM2 and BCL2L1, as primary functional targets of GR involved in GC-induced cell death.
Subject(s)
Molecular Targeted Therapy , Multiple Myeloma/drug therapy , Receptors, Glucocorticoid/metabolism , Apoptosis/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Dexamethasone/pharmacology , Down-Regulation/drug effects , Drug Resistance, Neoplasm/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Genomics , Humans , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Nucleotides/metabolism , Phosphoinositide-3 Kinase Inhibitors , Ribonucleoside Diphosphate Reductase/metabolism , bcl-X Protein/metabolismABSTRACT
Cutaneous T-cell lymphomas (CTCLs) represent a group of hematopoietic malignancies that home to the skin and have no known molecular basis for disease pathogenesis. Sézary syndrome (SS) is the leukemic variant of CTCL. Currently, CTCL is incurable, highlighting the need for new therapeutic modalities. We have previously observed that combined small-molecule inhibition of protein kinase C-ß (PKCß) and glycogen synthase kinase 3 (GSK3) causes synergistic apoptosis in CTCL cell lines and patient cells. Through microarray analysis of a SS cell line, we surveyed global gene expression following combined PKCß-GSK3 treatment to elucidate therapeutic targets responsible for cell death. Clinically relevant targets were defined as genes differentially expressed in SS patients that were modulated by combination-drug treatment of SS cells. Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways. Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment. This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells. These data establish p38 as a SS biomarker and a potential therapeutic target for the treatment of CTCL.
Subject(s)
Mitogen-Activated Protein Kinase 11/antagonists & inhibitors , Sezary Syndrome/drug therapy , Skin Neoplasms/drug therapy , Apoptosis/drug effects , Cell Line, Tumor , Glycogen Synthase Kinase 3/antagonists & inhibitors , Humans , MAP Kinase Signaling System , Protein Kinase C beta/antagonists & inhibitors , Sezary Syndrome/pathology , Skin Neoplasms/pathologyABSTRACT
Pre-messenger RNA splicing is significantly changed in cancer cells leading to the expression of cancer-specific transcripts. These transcripts have the potential to be used as cancer biomarkers and also as targets for new therapeutic approaches. In addition, the cancer-specific transcripts have the potential to alter the drug response of the cancer cells creating a chemo-resistant state. This later property of alternative splicing presents a challenge to clinicians in the design of effective therapeutic regimens. When a patient's cancer relapses it is frequently refractory to standard chemotherapies resulting in a poor clinical outcome. Therefore, understanding the mechanisms of how alternative splicing can lead to chemo-resistance is critical to the effective delivery of treatment. Here, we will discuss the impact of alternative splicing variants on drug metabolism and activation; on drug interactions with cell signaling pathways; and on cell death pathways in cancer therapeutics. In addition to the initial characterization of splicing variants, the mechanisms leading to alterations in splicing are being studied in the setting of chemo-resistance and will be discussed here. The promise of therapeutic intervention to obviate the impact of these splicing variants will significantly enhance treatment options for cancer patients.
Subject(s)
Alternative Splicing , Neoplasms , Biomarkers, Tumor/genetics , Humans , Neoplasms/genetics , RNAABSTRACT
8-Amino-adenosine (8-NH(2)-Ado) is a ribose sugar nucleoside analogue that reduces cellular ATP levels and inhibits mRNA synthesis. Estrogen receptor-negative (ER-) metastatic breast cancers often contain mutant p53; therefore, we asked if 8-NH(2)-Ado could kill breast cancer cells without activating the p53-pathway. Regardless of the breast cancer subtype tested or the p53 status of the cells, 8-NH(2)-Ado was more cytotoxic than either gemcitabine or etoposide. 8-NH(2)-Ado treatment inhibited cell proliferation, activated cell death, and did not activate transcription of the p53 target gene p21 or increase protein levels of either p53 or p21. This occurred in the estrogen receptor-positive (ER+) MCF-7 cells that express wild-type p53, the ER+ T47-D cells that express mutant p53, and the ER- MDA-MB-468 cells or MDA-MB-231 cells that both express mutant p53. 8-NH(2)-Ado induced apoptotic death of MCF-7 cells and apoptosis was not inhibited by knockdown of functional p53. Moreover, the pan-caspase inhibitor Z-VAD blocked the 8-NH(2)-Ado-induced MCF-7 cell death. Interestingly, 8-NH(2)-Ado caused the MDA-MB-231 cells to detach from the plate with only limited evidence of apoptotic cell death markers and the cell death was not inhibited by Z-VAD. Inhibition of MDA-MB-231 cell autophagy, by reduction of ATG7 or 3-methyladenine treatment, did not block this 8-NH(2)-Ado-mediated cytotoxicity. Importantly 8-NH(2)-Ado was highly cytotoxic to triple-negative breast cancer cells and worked through a pathway that did not require wild-type p53 for cytoxicity. Therefore, 8-NH(2)-Ado should be considered for the treatment of triple-negative breast cancers that are chemotherapy resistant.
Subject(s)
Adenosine/analogs & derivatives , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Adenosine/pharmacology , Antineoplastic Agents/pharmacology , Autophagy/drug effects , Caspase Inhibitors/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , Doxycycline/chemistry , Doxycycline/pharmacology , Female , Humans , Mutant Proteins/metabolism , Neoplasm Metastasis , Oligopeptides/pharmacology , Receptors, Estrogen/metabolism , Signal Transduction/drug effects , Transcription, Genetic/drug effectsABSTRACT
The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including G6PD which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce de novo resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of G6PD expression may be particularly efficacious.
Subject(s)
2-Chloroadenosine/analogs & derivatives , Adenosine/analogs & derivatives , Drug Resistance, Neoplasm , Gene Expression Profiling , Glucosephosphate Dehydrogenase/biosynthesis , Hematologic Neoplasms , Leukemia, Lymphocytic, Chronic, B-Cell , Lymphoma, Mantle-Cell , Multiple Myeloma , Neoplasm Proteins/metabolism , 2-Chloroadenosine/pharmacology , Adenosine/pharmacology , Cell Line, Tumor , Clinical Trials, Phase I as Topic , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Screening Assays, Antitumor/methods , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Glucosephosphate Dehydrogenase/genetics , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/enzymology , Hematologic Neoplasms/genetics , Hematologic Neoplasms/pathology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/enzymology , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/pathology , Multiple Myeloma/drug therapy , Multiple Myeloma/enzymology , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Proteins/genetics , Pentose Phosphate Pathway/drug effects , Pentose Phosphate Pathway/genetics , RNA, Neoplasm/antagonists & inhibitors , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolismABSTRACT
Multiple myeloma is one of numerous malignancies characterized by increased glucose consumption, a phenomenon with significant prognostic implications in this disease. Few studies have focused on elucidating the molecular underpinnings of glucose transporter (GLUT) activation in cancer, knowledge that could facilitate identification of promising therapeutic targets. To address this issue, we performed gene expression profiling studies involving myeloma cell lines and primary cells as well as normal lymphocytes to uncover deregulated GLUT family members in myeloma. Our data demonstrate that myeloma cells exhibit reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption, maintenance of Mcl-1 expression, growth, and survival. We also establish that the activities of the enigmatic transporters GLUT8 and GLUT11 are required for proliferation and viability in myeloma, albeit because of functionalities probably distinct from whole-cell glucose supply. As proof of principle regarding the therapeutic potential of GLUT-targeted compounds, we include evidence of the antimyeloma effects elicited against both cell lines and primary cells by the FDA-approved HIV protease inhibitor ritonavir, which exerts a selective off-target inhibitory effect on GLUT4. Our work reveals critical roles for novel GLUT family members and highlights a therapeutic strategy entailing selective GLUT inhibition to specifically target aberrant glucose metabolism in cancer.
Subject(s)
Glucose Transport Proteins, Facilitative/physiology , Glucose Transporter Type 4/physiology , Molecular Targeted Therapy , Multiple Myeloma/genetics , Multiple Myeloma/therapy , Biological Availability , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , Cells, Cultured , Disease Progression , Drug Evaluation, Preclinical , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Glucose/metabolism , Glucose/pharmacokinetics , Glucose Transport Proteins, Facilitative/antagonists & inhibitors , Glucose Transport Proteins, Facilitative/genetics , Glucose Transport Proteins, Facilitative/metabolism , Glucose Transporter Type 4/antagonists & inhibitors , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , HIV Protease Inhibitors/pharmacology , Humans , Molecular Targeted Therapy/methods , Molecular Targeted Therapy/trends , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Off-Label Use , Primary Cell Culture , Ritonavir/pharmacologyABSTRACT
Glucocorticoids (GCs) are widely used in the treatment of hematological malignancies such as multiple myeloma. However, the development of resistance to GCs limits their clinical utility. Response to GCs is dependent on an active glucocorticoid receptor, GR-α, expressed at wild-type levels in the GC-sensitive cell line (MM.1S). GC-resistant derivative cell lines MM.1Re and MM.1RL display significant downregulation of GR-α transcripts. In this study, we report that a luciferase reporter containing the 3'-UTR of GR-α is significantly repressed in MM.1R cells when compared to MM.1S cells, suggesting that one or several microRNAs that are upregulated in MM.1R maybe in part responsible for the downregulation of the GR-α transcript. To examine posttranscriptional mechanisms of GR regulation, we examined miRNAs that have complimentary binding sites in the 3'-UTR of GR-α and found miR-130b, miR-181a, and miR-636 to be differentially expressed between GC-sensitive and GC-resistant MM.1 cell lines. Overexpression of miR-130b in MM.1S cells results in decreased expression of endogenous GR protein and decreased activity of the luciferase reporter. In addition, in MM.1S cells, the downstream GC response of glucocorticoid-induced leucine zipper induction is decreased by the overexpression of miR-130b, and further miR-130b inhibits GC-induced apoptosis and causes resistance to GCs.
Subject(s)
Drug Resistance, Neoplasm/genetics , Gene Expression Regulation/genetics , Glucocorticoids/therapeutic use , MicroRNAs/genetics , Multiple Myeloma/genetics , Receptors, Glucocorticoid/biosynthesis , Cell Line , Gene Expression , Humans , Immunoblotting , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Polymerase Chain Reaction , Receptors, Glucocorticoid/genetics , Transcription, GeneticABSTRACT
Cutaneous T-cell lymphomas (CTCL) represent a spectrum of several distinct non-Hodgkin's lymphomas that are characterized by an invasion of the skin by malignant, clonal lymphocytes. Our laboratory has previously demonstrated that the protein kinase C (PKC) ß inhibitor Enzastaurin increases apoptosis in malignant lymphocytes of CTCL. These results directly led to a clinical trial for Enzastaurin in CTCL in which it was well tolerated and showed modest activity. To ascertain a means of improving the efficacy of Enzastaurin, we investigated complementary signaling pathways and identified glycogen synthase kinase-3 (GSK3) as important in survival signaling in CTCL. Enzastaurin combined with GSK3 inhibitors demonstrated an enhancement of cytotoxicity. Treatment with a combination of Enzastaurin and the GSK3 inhibitor AR-A014418 resulted in upregulation of ß-catenin total protein and ß-catenin-mediated transcription. Inhibition of ß-catenin-mediated transcription or small hairpin RNA (shRNA) knockdown of ß-catenin decreased the cytotoxic effects of Enzastaurin plus AR-A014418. In addition, treatment with Enzastaurin and AR-A014418 decreased the mRNA levels and surface expression of CD44. shRNA knockdown of ß-catenin also restored CD44 surface expression. Our observations provide a rationale for the combined targeting of PKC and GSK3 signaling pathways in CTCL to enhance the therapeutic outcome.
Subject(s)
Antineoplastic Agents/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Indoles/pharmacology , Lymphoma, T-Cell, Cutaneous/drug therapy , Protein Kinase Inhibitors/pharmacology , Skin Neoplasms/drug therapy , Cells, Cultured , Glycogen Synthase Kinase 3/physiology , Humans , Hyaluronan Receptors/genetics , Lymphoma, T-Cell, Cutaneous/pathology , Protein Kinase C/physiology , Skin Neoplasms/pathology , TCF Transcription Factors/physiology , Thiazoles/pharmacology , Urea/analogs & derivatives , Urea/pharmacology , beta Catenin/physiologyABSTRACT
8-Aminoadenosine (8-NH(2)-Ado), a ribosyl nucleoside analog, in preclinical models of multiple myeloma inhibits phosphorylation of proteins in multiple growth and survival pathways, including Akt. Given that Akt controls the activity of mammalian target of rapamycin (mTOR), we hypothesized that 8-NH(2)-Ado would be active in mantle cell lymphoma (MCL), a hematological malignancy clinically responsive to mTOR inhibitors. In the current study, the preclinical efficacy of 8-NH(2)-Ado and its resulting effects on Akt/mTOR and extracellular-signal-regulated kinase signaling were evaluated using 4 MCL cell lines, primary MCL cells, and normal lymphocytes from healthy donors. For all MCL cell lines, 8-NH(2)-Ado inhibited growth and promoted cell death as shown by reduction of thymidine incorporation, loss of mitochondrial membrane potential, and poly (adenosine diphosphate-ribose) polymerase cleavage. The efficacy of 8-NH(2)-Ado was highly associated with intracellular accumulation of 8-NH(2)-adenosine triphosphate (ATP) and loss of endogenous ATP. Formation of 8-NH(2)-ATP was also associated with inhibition of transcription and translation accompanied by loss of phosphorylated (p-)Akt, p-mTOR, p-Erk1/2, p-phosphoprotein (p)38, p-S6, and p-4E-binding protein 1. While normal lymphocytes accumulated 8-NH(2)-ATP but maintained their viability with 8-NH(2)-Ado treatment, primary lymphoma cells accumulated higher concentrations of 8-NH(2)-ATP, had increased loss of ATP, and underwent apoptosis. We conclude that 8-NH(2)-Ado is efficacious in preclinical models of MCL and inhibits signaling of Akt/mTOR and Erk pathways.
Subject(s)
Adenosine/analogs & derivatives , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Lymphoma, Mantle-Cell/pathology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , Adenosine/pharmacology , Apoptosis/drug effects , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Flow Cytometry , Humans , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/metabolism , Phosphorylation/drug effects , Protein Biosynthesis/drug effects , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , TOR Serine-Threonine Kinases/metabolism , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
PURPOSE OF REVIEW: Steroid hormone receptors (SHR) are crucial regulators of disease and the basis for clinical intervention in cancers. Recent evidence confirms that microRNAs (miRNAs) impact the pathobiology of hormone-regulated malignancies. Therefore, elucidating miRNA regulation of SHR expression and modulation of miRNAs by SHRs may provide diagnostic biomarkers or therapeutic targets. RECENT FINDINGS: Estrogen receptor status has been established as a key factor in breast cancer prognosis and treatment. Recent studies detail the interactions between estrogen receptor and miRNAs in cancers. New evidence indicates involvement of miRNAs in the regulation of androgen receptor, progesterone receptor, glucocorticoid receptor in hormone responsive cancers. Several miRNAs regulate the expression of the SHRs, while other miRNAs are themselves regulated by SHR signaling in cancer. SUMMARY: Cancers have distinct miRNA expression profiles that contribute to the pathobiology of the disease. In hormone-responsive cancers, the regulatory interactions between the SHR and miRNA may contribute to disease progression. The miRNA regulation of estrogen receptor in cancer has been established in estrogen-dependent cancers. The role of miRNAs in regulating progesterone receptor, androgen receptor and glucocorticoid receptor is under investigation with new insights emerging. These interactions can provide prognostic utility as well as the potential for therapeutic intervention in the future.
Subject(s)
Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Neoplasms/genetics , Receptors, Steroid/genetics , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Hematologic Neoplasms/genetics , Hematologic Neoplasms/metabolism , Hematologic Neoplasms/pathology , Humans , Male , MicroRNAs/biosynthesis , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms, Hormone-Dependent/genetics , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Receptors, Steroid/biosynthesisABSTRACT
BACKGROUND: Gene-list annotations are critical for researchers to explore the complex relationships between genes and functionalities. Currently, the annotations of a gene list are usually summarized by a table or a barplot. As such, potentially biologically important complexities such as one gene belonging to multiple annotation categories are difficult to extract. We have devised explicit and efficient visualization methods that provide intuitive methods for interrogating the intrinsic connections between biological categories and genes. FINDINGS: We have constructed a data model and now present two novel methods in a Bioconductor package, "GeneAnswers", to simultaneously visualize genes, concepts (a.k.a. annotation categories), and concept-gene connections (a.k.a. annotations): the "Concept-and-Gene Network" and the "Concept-and-Gene Cross Tabulation". These methods have been tested and validated with microarray-derived gene lists. CONCLUSIONS: These new visualization methods can effectively present annotations using Gene Ontology, Disease Ontology, or any other user-defined gene annotations that have been pre-associated with an organism's genome by human curation, automated pipelines, or a combination of the two. The gene-annotation data model and associated methods are available in the Bioconductor package called "GeneAnswers " described in this publication.
Subject(s)
Arsenic , Drug Carriers/chemistry , Drug Compounding , Lung Neoplasms/drug therapy , Platinum , Arsenic/chemistry , Arsenic/therapeutic use , Cell Line, Tumor , Drug Delivery Systems , Drug Screening Assays, Antitumor , Humans , Liposomes/chemistry , Liposomes/therapeutic use , Molecular Structure , Platinum/chemistry , Platinum/therapeutic useABSTRACT
Multiple myeloma, an incurable plasma cell malignancy, is characterized by altered cellular metabolism and resistance to apoptosis. Recent connections between glucose metabolism and resistance to apoptosis provide a compelling rationale for targeting metabolic changes in cancer. In this study, we have examined the ability of the purine analogue 8-aminoadenosine to acutely reduce glucose consumption by regulating localization and expression of key glucose transporters. Myeloma cells counteracted the metabolic stress by activating autophagy. Co-treatment with inhibitors of autophagy results in marked enhancement of cell death. Glucose consumption by drug-resistant myeloma cells was unaffected by 8-aminoadenosine, and accordingly, no activation of autophagy was observed. However, these cells can be sensitized to 8-aminoadenosine under glucose-limiting conditions. The prosurvival autophagic response of myeloma to nutrient deprivation or to nucleoside analogue treatment has not been described previously. This study establishes the potential of metabolic targeting as a broader means to kill and sensitize myeloma and identifies a compound that can achieve this goal.
Subject(s)
Adenosine/analogs & derivatives , Autophagy/drug effects , Cell Proliferation/drug effects , Glucose/metabolism , Adenosine/pharmacology , Adenosine Triphosphate/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Chloroquine/pharmacology , Cytoplasm/metabolism , Dose-Response Relationship, Drug , Drug Synergism , Glucose Transporter Type 1 , Glucose Transporter Type 4/metabolism , Humans , Immunoblotting , Microscopy, Fluorescence , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Time Factors , Tumor Cells, CulturedABSTRACT
8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F(O) intermembrane base and F1 domain, containing alpha and beta subunits. Crystal structures of both alpha and beta subunits that bind to the substrate, ADP, are known in tight binding (alpha(dp)beta(dp)) and loose binding (alpha(tp)beta(tp)) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the alpha(tp)beta(tp) state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (alpha(dp)beta(dp)) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF(3)(-). Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.
Subject(s)
2-Chloroadenosine/analogs & derivatives , ATP Synthetase Complexes/drug effects , Adenosine/pharmacology , 2-Chloroadenosine/chemistry , 2-Chloroadenosine/pharmacology , ATP Synthetase Complexes/chemistry , Adenosine/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Glycolysis/drug effects , Glycolysis/physiology , Halogenation , Hydrolysis , Models, Molecular , Oxygen Consumption/drug effects , Protein Binding , Protein Conformation/drug effectsABSTRACT
Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.