Androgen antagonists or androgen deprivation is a primary therapeutic modality for the treatment of prostate cancer. Invariably, however, the disease becomes progressive and unresponsive to androgen ablation therapy (hormone refractory). The molecular mechanisms by which the androgen antagonists inhibit prostate cancer proliferation are not fully defined. In this report, we demonstrate that SIRT1, a nicotinamide adenosine dinucleotide-dependent histone deacetylase linked to the regulation of longevity, is required for androgen antagonist-mediated transcriptional repression and growth suppression. Androgen antagonist-bound androgen receptor (AR) recruits SIRT1 and NCoR to AR-responsive promoters and deacetylates histone H3 locally at the PSA promoter. Furthermore, SIRT1 down-regulation by siRNA or by pharmacological means increased the sensitivity of androgen-responsive genes to androgen stimulation, enhanced the sensitivity of prostate cancer cell proliferative responses to androgens, and decreased the sensitivity of prostate cancer cells to androgen antagonists. In this study, we demonstrate the ligand-dependent recruitment of a class III HDAC into a co-repressor transcriptional complex, and a necessary functional role for a class III HDAC as a transcriptional co-repressor in AR antagonist-induced transcriptional repression. Collectively, these findings identify SIRT1 as a co-repressor of AR and elucidate a new molecular pathway relevant to prostate cancer growth and approaches to therapy.
Androgen Antagonists; Androgen Receptor; SIRT1; Histone Deacetylase; Prostate cancer
RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D1, cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.
DNA oligonucleotides with sequence homology to human telomeric DNA (T-oligo) induce cell cycle arrest, followed by apoptosis, senescence, or autophagy in a human cancer cell type-specific manner. T-oligo has potential as a new therapeutic strategy in oncology because of its ability to target certain types of tumor cells while sparing normal ones. In the present study, we demonstrate the T-oligo-induced S-phase cell cycle arrest in four pancreatic cancer cell lines. To further contribute to the mechanistic understanding of T-oligo, we also identify cyclin dependent kinase 2 (cdk2) as a functional mediator in the T-oligo-induced cell cycle arrest of pancreatic cancer cells. Ectopic expression of a constitutively-active cdk2 mutant abrogates T-oligo-induced cell cycle arrest in these tumor cells while knockdown of cdk2 expression alone recapitulates the T-oligo effect. Finally, we demonstrate the dispensability of T-oligo-induced ATM/ATR-mediated DNA damage response-signaling pathways, which have long been considered functional in the T-oligo signaling mechanism.
T-oligo; Pancreatic Cancer; Cell Cycle Arrest; Pancreatic Cancer Stem Cells
RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serumstarved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2Fregulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.
RING3; Nuclear translocation; Immunolocalisation; Confocal microscopy; Leukaemia
A subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.
shRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student’s t tests or ANOVA.
We demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.
These findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.
Protein Kinase C isozymes; Synthetic lethal interaction; Cancer-initiating cell; Xenograft tumor model
Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide.
thalassemia; short chain fatty acids; butyrates; erythropoiesis; fetal globin; quantitative trait loci
Inhibition of PKC activity in transformed cells and tumor cells containing activated p21Ras results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21Ras oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21Ras. Dominant-negative mutants of PKC, siRNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKCδ isozyme demonstrated that PKCδ plays a critical role in p21Ras-mediated apoptosis. An activating p21Ras mutation, or activation of the PI3K Ras effector pathway, increased the levels of PKCδ protein and activity in cells, whereas inhibition of p21Ras activity decreased the expression of PKCδ protein. Activation of the Akt survival pathway by oncogenic Ras required PKCδ activity. Akt activity was dramatically decreased after PKCδ suppression in cells containing activated p21Ras. Conversely, constitutively-activated Akt rescued cells from apoptosis induced by PKCδ inhibition. Collectively, these findings demonstrate that p21Rasthrough its downstream effector PI3K, induces PKCδ expression and that this increase in PKCδ activity, acting through Akt, is required for cell survival. The p21Ras effector molecule responsible for the initiation of the apoptotic signal after suppression of PKCδ activity was also determined to be PI3K. PI3K (p110-CAAX), was sufficient for induction of apoptosis after PKCδ inhibition. Thus, the same p21Ras effector, PI3K, is responsible for delivering both a pro-apoptotic signal, and a survival signal, the latter being mediated by PKCδ and Akt. Selective suppression of PKCδ activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21Ras.
Pharmacologic induction of fetal globin synthesis is an accepted therapeutic strategy for treatment of the beta hemoglobinopathies and thalassemias, as even small increases in hemoglobin F (HbF) levels reduce clinical severity in sickle cell disease and reduce anemia in beta thalassemia. Prior generation short chain fatty acid therapeutics, arginine butyrate and phenylbutyrate, increased fetal and total hemoglobin levels in patients, but were limited by high doses or intravenous infusion. A fetal globin-inducing therapeutic with convenient oral dosing would be an advance for these classic molecular diseases.
Healthy adult human subjects were treated with a novel SCFA derivative, sodium 2,2 dimethylbutyrate (SDMB), or placebo, with one of four single dose levels (2, 5, 10 and 20 mg/kg) or daily doses (5, 10, or 15 mg/kg) over 14 days, and monitored for adverse clinical and laboratory events, drug levels, reticulocytes, and HbF assays. SDMB was well-tolerated with no clinically significant adverse events related to study medication. The terminal half-life ranged from 9–15 hours. Increases in mean absolute reticulocytes were observed at all dose levels in the 14-day study. The favorable PK profiles and safety findings indicate that SDMB warrants further investigation for treatment of anemic subjects with beta hemoglobinopathies.
short chain fatty acids; anemias; pharmacokinetic profiles; erythropoiesis; fetal hemoglobin
In prostate and breast cancer, the androgen and estrogen receptors mediate induction of androgen- and estrogen-responsive genes respectively, and stimulate cell proliferation in response to the binding of their cognate steroid hormones. Sirtuin 1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD+)-dependent class III histone deacetylase (HDAC) that has been linked to gene silencing, control of the cell cycle, apoptosis and energy homeostasis. In prostate cancer, SIRT1 is required for androgen-antagonist-mediated transcriptional repression and growth suppression of prostate cancer cells. Whether SIRT1 plays a similar role in the actions of estrogen or antagonists had not been determined. We report here that SIRT1 represses the transcriptional and proliferative response of breast cancer cells to estrogens, and this repression is estrogen receptor-alpha (ERα)-dependent. Inhibition of SIRT1 activity results in the phosphorylation of ERα in an AKT-dependent manner, and this activation requires phosphoinositide 3-kinase (PI3K) activity. Phosphorylated ERα subsequently accumulates in the nucleus, where ERα binds DNA ER-response elements and activates transcription of estrogen-responsive genes. This ER-dependent transcriptional activation augments estrogen-induced signaling, but also activates ER-signaling in the absence of estrogen, thus defining a novel and unexpected mechanism of ligand-independent ERα-mediated activation and target gene transcription. Like ligand-dependent activation of ERα, SIRT1 inhibition-mediated ERα activation in the absence of estrogen also results in breast cancer cell proliferation. Together, these data demonstrate that SIRT1 regulates the most important cell signaling pathway for the growth of breast cancer cells, both in the presence and the absence of estrogen.
Sirtuin; estrogen receptor; SIRT1; ligand-independent; breast cancer
Epstein-Barr virus (EBV) is the causal agent in the etiology of Burkitt's lymphoma and nasopharyngeal carcinoma and is also associated with multiple human malignancies, including Hodgkin's and non-Hodgkin's lymphoma, and posttransplantation lymphoproliferative disease, as well as sporadic cancers of other tissues. A causal relationship of EBV to these latter malignancies remains controversial, although the episomic EBV genome in most of these cancers is clonal, suggesting infection very early in the development of the tumor and a possible role for EBV in the genesis of these diseases. Furthermore, the prognosis of these tumors is invariably poor when EBV is present, compared to their EBV-negative counterparts. The physical presence of EBV in these tumors represents a potential “tumor-specific”
target for therapeutic approaches. While treatment options for other types of herpesvirus infections have evolved and improved over the last two decades, however, therapies directed at EBV have lagged. A major constraint to pharmacological intervention is the shift from lytic infection to a latent pattern of gene expression, which persists in those tumors associated with the virus. In this paper we provide a brief account of new virus-targeted therapeutic approaches against EBV-associated malignancies.
MMP2 has been shown to play an important role in cancer cell invasion and the expression of MMP2 is associated with the poor prognosis of prostate cancer; however, the mechanism of MMP2 expression is largely unknown.
SIRT1 is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase (class III HDAC) that has recently been shown to have implications in regulating cancer cell growth and apoptosis. The purpose of this study is to determine the role of SIRT1 in regulating MMP2 expression and tumor invasion in prostate cancer cells.
The interfering RNAi was used to knockdown SIRT1 from prostate cancer cells. Immunoblots, RT-PCR, zymographic assays, co-immunoprecipitation analysis and transwell assays were used to examine the effects of SIRT1 silencing on MMP2 expression and activity, on SIRT1 and MMP2 interaction, and on prostate cancer cell invasion. The immuno-histochemical assay was performed to study SIRT1 expression in prostate cancer tissues.
We show that SIRT1 associates and deacetylates MMP2 and SIRT1 regulates MMP2 expression by controlling MMP2 protein stability through the proteosomal pathway. Thus, we demonstrated a novel mechanism in that MMP2 expression can be regulated at the posttranslational level by SIRT1. Furthermore, we determined that SIRT1 inhibition reduced prostate cancer cell invasion and SIRT1 is highly expressed in advanced prostate cancer tissues.
SIRT1 is an important regulator of MMP2 expression, activity, and prostate cancer cell invasion. Overexpressed SIRT1 in advanced prostate cancer may play an important role in prostate cancer progression.
SIRT1; MMP2; protein stability; prostate cancer; invasion
Ovarian cancer remains a leading cause of death among women worldwide, and current treatment regimens for advanced disease are inadequate. Oligonucleotides with sequence homology to telomeres (called T-oligos) have been shown to mimic DNA damage responses in cells and induce cytotoxic effects in certain tumor cell lines. We studied the effects of 2 distinct 16 mer T-oligos in 4 human ovarian epithelial carcinoma cell lines. A T-oligo with perfect homology to the telomere overhang region demonstrated some cytotoxic activity in half of the cell lines. A G-rich T-oligo derivative showed more potency and broader cytotoxic activity in these lines than the parental T-oligo. Activation of apoptotic pathways in ovarian cancer cells by exposure to the T-oligo was demonstrated by multiple independent assays. T-oligo was shown to have additive, or more than additive, activity in combination with 2 different histone deacetylase drugs currently in clinical testing. T-oligos may therefore provide a new and tumor-targeted approach to ovarian cancers.
The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulate specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFκB, and because the antiapoptotic and growth promoting activities of NFκB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFκB signaling. Here we demonstrate that FeLV U3-LTR indeed upregulates NFκB signaling pathway via activation of Ras-Raf-IκB kinase (IKK) and degradation of IκB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR−/− mouse embryo fibroblasts we further demonstrate that although dsRNA activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFκB by the LTR. Our study thus demonstrates involvement of a TLR3 dependent but PKR independent dsRNA mediated signaling pathway for NFκB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.
Leukemia virus; LTR; transactivation; NFκB; TLR3
The Ras proteins (K-Ras, N-Ras, H-Ras) are GTPases that function as molecular switches for a variety of critical cellular activities and their function is tightly and temporally regulated in normal cells. Oncogenic mutations in the RAS genes, which create constitutively-active Ras proteins, can result in uncontrolled proliferation or survival in tumor cells.
The paper discusses three therapeutic approaches targeting the Ras pathway in cancer: 1) Ras itself, 2) Ras downstream pathways, and 3) synthetic lethality. The most adopted approach is targeting Ras downstream signaling, and specifically the PI3K-AKT-mTOR and Raf-MEK pathways, as they are frequently major oncogenic drivers in cancers with high Ras signaling. Although direct targeting of Ras has not been successful clinically, newer approaches being investigated in preclinical studies, such as RNA interference-based and synthetic lethal approaches, promise great potential for clinical application.
The challenges of current and emerging therapeutics include the lack of “tumor specificity” and their limitation to those cancers which are “dependent” upon aberrant Ras signaling for survival. While the newer approaches have the potential to overcome these limitations, they also highlight the importance of robust preclinical studies and bidirectional translational research for successful clinical development of Ras-related targeted therapies.
The U3-LTR region of leukemia viruses transactivates cancer-related signaling pathways through the production of a non-coding RNA transcript although the role of this transcript in virus infection remains unknown. In this study we demonstrate for the first time that an LTR-specific small non-coding RNA is produced from a FeLV-infected feline cell line. RNA cloning identified this as a 104 base transcript that originates from the U3-LTR region. We also demonstrate that in in vitro assays this LTR RNA transcript activates NFκB signaling. Taken together, our findings suggest a possible role for this LTR transcript in FeLV pathogenesis.
FeLV; LTR; non-coding RNA
Protein kinase C δ (PKC δ) modulates cell survival and apoptosis in diverse cellular systems. We recently reported that PKCδ functions as a critical anti-apoptotic signal transducer in cells containing activated p21Ras and results in the activation of AKT, thereby promoting cell survival. How PKCδ is regulated by p21Ras, however, remains incompletely understood. In this study, we show that PKCδ, as a transducer of anti-apoptotic signals, is activated by phosphotidylinositol 3′ kinase/Phosphoinositide-dependent kinase 1 (PI3K-PDK1) to deliver the survival signal to Akt in the environment of activated p21Ras. PDK1 is upregulated in cells containing an activated p21Ras. Knockdown of PDK1, PKCδ, or AKT forces cells containing activated p21Ras to undergo apoptosis. PDK1 regulates PKCδ activity, and constitutive expression of PDK1 increases PKCδ activity in different cell types. Conversely, expression of a kinase-dead (dominant-negative) PDK1 significantly suppresses PKCδ activity. p21Ras-mediated survival signaling is therefore regulated by via a PI3K-AKT pathway, which is dependent upon both PDK1 and PKCδ, and PDK1 activates and regulates PKCδ to determine the fate of cells containing a mutated, activated p21Ras.
PKCδ; PI3K; Akt; Protein kinase C; Apoptosis; Proliferation
Androgen antagonists or androgen deprivation are the primary therapeutic modalities for the treatment of prostate cancer. Invariably, however, the disease becomes progressive and unresponsive to androgen ablation therapy (hormone refractory). The molecular mechanisms by which androgen antagonists inhibit prostate cancer proliferation are not fully defined. In this study, we identify two molecules which are required for effective prostate cancer cell responsiveness to androgen antagonists. We establish that androgen receptor (AR)-dependent transcriptional suppression by androgen antagonists requires the tumor suppressor prohibitin. This requirement for prohibitin was demonstrated using structurally-distinct androgen antagonists, stable and transient knockdown of prohibitin and transfected and endogenous AR-responsive genes. The SWI–SNF complex core ATPase BRG1, but not its closely-related counterpart ATPase BRM, is required for this repressive action of prohibitin on AR-responsive promoters. Androgen antagonists induce recruitment of prohibitin and BRG1 to endogenous AR-responsive promoters and induce a physical association between AR and prohibitin and BRG1. The recruitment of prohibitin to endogenous AR-responsive promoters is dependent upon antagonist-bound AR. Prohibitin binding in the prostate-specific antigen (PSA) promoter results in the recruitment of BRG1 and the dissociation of p300 from the PSA promoter. These findings suggest that prohibitin may function through BRG1-mediated local chromatin remodeling activity and the removal of p300-mediated acetylation to produce androgen antagonist-mediated transcriptional repression. Furthermore, in addition to its necessary role in AR-mediated transcriptional repression, we demonstrate that prohibitin is required for full and efficient androgen antagonist-mediated growth suppression of prostate cancer cells.
Antiviral drugs alone have been unsuccessful in the treatment of Epstein Barr virus (EBV)-associated malignancies because the virus maintains a latent state of replication in these tumors. In recent years, novel therapeutic approaches are being investigated wherein lytic replication of the virus is induced prior to the use of cytotoxic antiviral drugs. The choice of suitable agents to induce lytic replication has been a critical step in this novel approach. We have previously demonstrated that butyrate derivatives induce a lytic pattern of EBV gene expression in patient-derived EBV-positive lymphoblastoid cell lines and, together with nucleoside analog ganciclovir, effectively reduce or eliminate tumor growth in humans. Butyrate has drawbacks as a therapeutic agent, however, as constant intravenous infusion is required to achieve detectable plasma levels of this drug. In this study, we investigated whether discontinuous exposure to butyrate is capable of initiating lytic-phase gene expression and thymidine kinase induction, and sensitizing EBV-positive lymphoma cells to ganciclovir-mediated cell growth arrest and apoptosis. We demonstrate that multiple daily 6 hr exposures of the EBV-positive Burkitt’s lymphoma cell line P3HR1 to butyrate induced sustained expression of the EBV lytic-phase protein BMRF. Viral thymidine kinase was also induced by intermittent exposure, although to a lower level than with continuous exposure treatment. However, discontinuous exposure to butyrate in combination with ganciclovir induced a similar level of tumor cell growth inhibition as did continuous treatment, as measured by serial enumeration of viable cells, MTT cell proliferation assays, and measurement of cellular DNA content. We further demonstrated that those cells which survived initial exposure to butyrate plus ganciclovir remained susceptible to further cycles of combination treatment. These findings suggests that continuous infusion of butyrate may not be necessary for maintaining viral thymidine kinase gene expression and sensitization to anti-viral agents in EBV-associated tumors, and that therapeutic regimens which employ more convenient, discontinuous exposure to butyrate may also be effective clinically.
Epstein-Barr Virus; Butyrate; Thymidine Kinase; Ganciclovir; Growth inhibition; Apoptosis
The concept of targeting cancer therapeutics towards specific mutations or abnormalities in tumor cells which are not found in normal tissues has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21Ras-signaling pathways. Carcinoid and other neuroendocrine tumors similarly have been demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI3-Kinase and Raf/MAP kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed, and that PKCδ suppression is not toxic to cells with normal levels of p21Ras signaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24 hr is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines.
Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations which contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.
carcinoid; Ras; apoptosis; cancer
The β-hemoglobinopathies and thalassemias are serious genetic blood disorders affecting the β-globin chain of hemoglobin A (α2βA2). Their clinical severity can be reduced by enhancing expression of fetal hemoglobin (γ-globin), producing HbF (α2γ2,). In studies reported here, γ-globin induction by 23 novel, structurally-unrelated compounds, which had been predicted through molecular modeling and in silico screening of a 13,000 chemical library, was evaluated in vitro in erythroid progenitors cultured from normal subjects and β-thalassemia patients, and in vivo in transgenic mice or anemic baboons. Four predicted candidates were found to have high potency, with 4- to 8-fold induction of HbF. Two of these compounds have pharmacokinetic profiles favorable for clinical application. These studies thus effectively identified high potency γ-globin inducing candidate therapeutics and validated the utility of in silico molecular modeling.
Butyrate; fetal hemoglobin; hemoglobinopathy; small molecules
In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.
acylcarnitine; butyrate; butyrylcarnitine; carnitine; histone deacetylase
The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for β-type globin gene switching, and specifically activates transcription of the adult β-globin gene promoter. We sought to determine if EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials.
The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators.
Results and conclusions
Knockdown of EKLF prevents SCFA-induced expression of the γ-globin promoter in a stably expressed μLCRβprRlucAγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous γ-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for γ-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous γ-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes.
erythropoiesis; fetal hemoglobin; gamma globin; globin gene switching; hemoglobinopathy; sickle cell disease; thalassemia
1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has shown strong promise as an anti-proliferative agent in several malignancies, yet its therapeutic use has been limited by its toxicity leading to search for analogs with anti-tumor property and low toxicity. In this study we evaluated the in vitro and in vivo properties of 1,25-dihydroxyvitamin D3-3-bromoacetate (1,25(OH)2D3-3-BE), an alkylating derivative of 1,25(OH)2D3 as a potential therapeutic agent for renal cancer. Dose-response of 1,25(OH)2D3-3-BE in two kidney cancer cell-lines was evaluated for its antiproliferative and apoptotic properties, and mechanisms were evaluated by Western Blot and FACS analyses. Therapeutic potential of 1,25(OH)2D3-3-BE was assessed by determining its stability in human serum, and evaluating its efficacy in a mouse xenograft model of human renal tumor. We observed that 1,25(OH)2D3-3-BE is significantly more potent than an equivalent concentration of 1,25(OH)2D3 in inhibiting growth of A498 and Caki 1 human kidney cancer cells. 1,25(OH)2D3-3-BE-mediated growth inhibition was promoted through inhibition of cell cycle progression by down-regulating cyclin A and induction of apoptosis by stimulating caspase activity. Moreover, 1,25(OH)2D3-3-BE strongly inhibited Akt phosphorylation and phosphorylation of its downstream target, caspase 9. 1,25(OH)2D3-3-BE appeared to be stable in human serum. In xenograft mouse model of human renal tumor, 1,25(OH)2D3-3-BE was more potent at reducing tumor size compared to 1,25(OH)2D3 which was accompanied by an increase in apopotosis and reduction of cyclin A staining in the tumors. These results suggest a translational potential of this compound as a therapeutic agent in renal cell carcinoma. Data from this study and extensive studies of vitamin D for the prevention of many malignancies support the potential of 1,25(OH)2D3-3-BE for preventing renal cancer and the development of relevant in-vivo prevention models for assessing this potential, which do not exist at present.
vitamin D; renal cancer; anti-proliferative effect; apoptosis; anti-tumor effect
Human B- or T-cell lymphoma lines and primary murine lymphomas were treated with DNA oligonucleotides homologous to the telomere (TTAGGG repeat; “T-oligo”), either alone or in combination with standard, widely-used anticancer chemotherapeutic agents. T-oligo induces cell cycle arrest and apoptosis in cultured human or murine B or T-lymphoma cell lines and primary tumor cells, but exerts no detectable toxicity on normal human or murine primary lymphocytes. Exposure to T-oligo is hypothesized to mimic exposure of the 3′ telomere repeat sequence, activating the ataxia telangiectasia mutated kinase, which phosphorylates downstream effectors such as p53, but effects are not dependent solely on functional p53. T-oligo causes early S-phase arrest and cooperates well with G2- or M-phase-specific anticancer agents; when combined at 1/10th of the conventional dose, vincristine and T-oligo produce greater-than-additive killing of human or murine lymphoma cells (78% of cells undergoing apoptosis after 6 hr vs. 5% of control cells). In mice, 1/10th of the conventional dose of a standard combination of cyclophosphamide, adriamycin, vincristine and prednisone is twice as effective when used in combination with low dose T-oligo. Thus, T-oligo sensitizes tumors to traditional anticancer agents and represents a potentially important new addition to the therapeutic arsenal for aggressive lymphomas.
lymphoma; cancer therapy; apoptosis; oligonucleotide; telomere
Transgenic mice with lymphoid-restricted overexpression of the double bromodomain protein bromodomain-containing 2 (Brd2) develop splenic B-cell lymphoma and, upon transplantation, B-cell leukemia with leukemic infiltrates in liver and lung. Brd2 is a nuclear-localized transcription factor kinase that is most closely related to TATA box binding protein–associated factor, 250 kDa (TAFII250) and the Drosophila developmental protein female sterile homeotic. Constitutive expression of BRD2 in the lymphoid compartment increases cyclin A transcription, “priming” transgenic B cells for proliferation. Mice stochastically develop an aggressive B-cell lymphoma with the features of B-1 cells, including CD5 and surface IgM expression. The B-cell lymphoma is monoclonal for immunoglobulin gene rearrangement and is phenotypically stable. The lymphoblasts are very large and express a transcriptome that is similar to human non-Hodgkin lymphomas. Both a wild-type BRD2 transgene and a kinase-null point mutant drive lymphomagenesis; therefore we propose that, rather than kinase activity, Brd2-mediated recruitment of E2 promoter binding factors (E2Fs) and a specific histone acetyltransferase to the cyclin A promoter by both types of transgene is a mechanistic basis for neoplasia. This report is the first to describe a transgenic mouse model for constitutive expression of a protein with more than one bromodomain.