Targeting autophagy: a novel anticancer strategy with therapeutic implications for imatinib resistance

Autophagy is an ancient, intracellular degradative system which plays important roles in regulating protein homeostasis and which is essential for survival when cells are faced with metabolic stress. Increasing evidence suggests that autophagy also functions as a tumor suppressor mechanism that harnesses the growth and/or survival of cells as they transition towards a rapidly dividing malignant state. However, the impact of autophagy on cancer progression and on the efficacy of cancer therapeutics is controversial. In particular, although the induction of autophagy has been reported after treatment with a number of therapeutic agents, including imatinib, this response has variously been suggested to either impair or contribute to the effects of anticancer agents. More recent studies support the notion that autophagy compromises the efficacy of anticancer agents, where agents such as chloroquine (CQ) that impair autophagy augment the anticancer activity of histone deacetylase (HDAC) inhibitors and alkylating agents. Inhibition of autophagy is a particularly attractive strategy for the treatment of imatinib-refractory chronic myelogenous leukemia (CML) since a combination of CQ with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) compromises the survival of even BCR-ABL-T315I+ imatinib-resistant CML. Additional studies are clearly needed to establish the clinical utility of autophagy inhibitors and to identify patients most likely to benefit from this novel therapeutic approach

Prostaglandin E2 drives cyclooxygenase-2 expression via cyclic AMP response element activation in human pancreatic cancer cells.

Cyclooxygenase-2 (COX-2) is constitutively expressed in most human primary carcinomas and with its synthesized product, prostaglandin E2 (PGE2), appears to play important roles in tumor invasion, angiogenesis, resistance to apoptosis and suppression of host immunity. However, the molecular mechanisms that control COX-2 expression are unclear. The purpose of this study was to clarify the mechanism of basal and PGE2-mediated COX-2 expression in the highly metastatic L3.6pl human pancreatic cancer cell line. Using RNA interference to disrupt the expression of CREB and the NF-kappaB p65 subunit, we found that both are involved in maintaining basal COX-2 expression in L3.6pl cells. We also demonstrated that PGE2 increased the cyclic AMP concentration, thereby activating protein kinase A (PKA), which in turn phosphorylated the cyclic AMP response element binding protein (CREB), leading to interaction with the cyclic AMP response element in the promoter region of the COX-2 gene. Immunocytochemical analysis confirmed that PGE2 stimulated the translocation of PKA to the nucleus and increased the immuno-reactivity of phosphorylated CREB. Pretreatment with the PKA selective inhibitor H 89 and the E-prostanoid receptor 2 inhibitor AH 6809 reduced COX-2 upregulation by PGE2. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay results further suggested a role for CREB in COX-2 transcriptional control. Understanding the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate the development of novel therapeutic approaches

Rational cotargeting of HDAC6 and BET proteins yields synergistic antimyeloma activity

Inhibition of bromodomain and extra terminal (BET) protein family members, including BRD4, decreases the expression of c-MYC and other key oncogenic factors and also significantly induces histone deacetylase 6 (HDAC6) expression. On the basis of the role of HDAC6 in malignant pathogenesis, we hypothesized that rational cotargeting of HDAC6 and BET family proteins may represent a novel approach that yields synergistic antimyeloma activity. We used genetic and pharmacologic approaches to selectively impair HDAC6 and BET function and evaluated the consequential impact on myeloma pathogenesis. These studies identified HDAC6 upregulation as an efficacy reducing mechanism for BET inhibitors because antagonizing HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of multiple myeloma (MM) cell lines and primary CD138+ cells obtained from patients with MM. The synergy of this therapeutic combination was linked to significant reductions in c-MYC expression and increases in apoptosis induction. Administration of the clinical HDAC6 inhibitor ricolinostat was very well tolerated and significantly augmented the in vivo antimyeloma activity of JQ1. Ex vivo pharmacodynamic analyses demonstrated that the combination of JQ1 and ricolinostat led to significantly lower MM cell proliferation and increased apoptosis and diminished expression of c-MYC and BCL-2. These data demonstrate that cotargeting of HDAC6 and BET family members is a novel and clinically actionable approach to augment the efficacy of both classes of agents that warrants further investigation.National Institutes of Health, National Cancer Institute [R01CA190789, R01CA172443]; University of Arizona Cancer Center Support Grant [P30CA023074]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

ELR510444 Inhibits Tumor Growth and Angiogenesis by Abrogating HIF Activity and Disrupting Microtubules in Renal Cell Carcinoma

Background: Hypoxia-inducible factor (HIF) is an attractive therapeutic target for renal cell carcinoma (RCC) as its high expression due to the loss of von Hippel-Lindau (VHL) promotes RCC progression. Considering this, we hypothesized that ELR510444, a novel orally available small molecule inhibitor of HIF activity, would reduce angiogenesis and possess significant activity in RCC. The mechanism of action and therapeutic efficacy of ELR510444 were investigated in in vitro and in vivo models of RCC. Principal Findings: ELR510444 decreased HIF-1a and HIF-2a levels, reduced RCC cell viability and clonogenic survival, and induced apoptosis. VHL-deficient RCC cells were more sensitive to ELR510444-mediated apoptosis and restoration of VHL promoted drug resistance. Higher concentrations of ELR51044 promoted apoptosis independently of VHL status, possibly due to the microtubule destabilizing properties of this agent. ELR510444 significantly reduced tumor burden in the 786-O and A498 RCC xenograft models. These effects were associated with increased necrosis and apoptosis and inhibition of angiogenesis. Conclusions: ELR510444 is a promising new HIF inhibitor that reduced RCC cell viability, induced apoptosis, and diminished tumor burden in RCC xenograft models. ELR510444 also destabilized microtubules suggesting that it possesses vascula

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Autophagy: New Insights into Its Roles in Cancer Progression and Drug Resistance

Autophagy is a mechanism of lysosomal proteolysis that is utilized to degrade damaged organelles, proteins, and other cellular components. Although key studies demonstrate that autophagy functions as a mechanism of tumor suppression via the degradation of defective pre-malignant cells, autophagy can also be used as a mechanism to break down cellular components under stress conditions to generate the required metabolic materials for cell survival. Autophagy has emerged as an important mediator of resistance to radiation, chemotherapy, and targeted agents. This series of articles highlight the role of autophagy in cancer progression and drug resistance and underscores the need for new and more effective agents that target this process