Sandra Healy

Targeting the endoplasmic reticulum-stress response as an anticancer strategy

The endoplasmic reticulum (ER) is the site of synthesis and folding of secretory and membrane bound proteins. The capacity of the ER to process proteins is limited and the accumulation of unfolded and misfolded proteins can lead to ER stress which has been associated with a wide range of diseases including cancer. In this review we initially provide an overview of our current understanding of how cells respond to ER stress at the molecular level and the key players involved in mediating the unfolded protein response (UPR). We review the evidence suggesting that the ER stress response could be important for the growth and development of tumors under stressful growth conditions such as hypoxia or glucose deprivation, which are commonly encountered by most solid tumors, and we analyse how it may be possible to exploit the unfolded protein response as an anticancer strategy. Two approaches to target the unfolded protein response are proposed-the first involves inhibiting components of the unfolded protein response so cells cannot adapt to stressful conditions and the second involves overloading the unfolded protein response so the cell is unable to cope, leading to cell death. We focused on proteins with an enzymatic activity that can be targeted by small molecule inhibitors as this is one of the most common approaches utilized by drug discovery companies. Finally, we review drugs currently in clinical development that affect the ER stress response and that may have potential as anti-tumor agents alone or in combination with other chemotherapeutics.

Stress management at the ER: regulators of ER stress-induced apoptosis.

The endoplasmic reticulum (ER) is an elaborate cellular organelle essential for cell function and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins which are detected by ER transmembrane receptors that initiate the unfolded protein response (UPR) to restore normal ER function. If the ER stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, particularly because ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review we aim to shed light on the proteins that are not core components of the UPR signaling pathway but which can influence the course of the ER stress response by regulating the switch from the adaptive phase to apoptosis.

Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders

•  Introduction •  ER stress and the UPR ‐  The IRE1 axis: non‐conventional splicing of XBP1 mRNA ‐  The PERK axis: attenuation of translation ‐  The ATF6 axis: regulated proteolytic activation •  ER stress–induced apoptosis •  ER stress and autophagy •  The UPR and neurodegenerative disease ‐  Alzheimers disease ‐  Parkinsons disease ‐  Amyotrophic lateral sclerosis ‐  Prion diseases •  Future perspectives

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity.

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.

Mechanisms of ER Stress-Mediated Mitochondrial Membrane Permeabilization

During apoptosis, the process of mitochondrial outer membrane permeabilization (MOMP) represents a point-of-no-return as it commits the cell to death. Here we have assessed the role of caspases, Bcl-2 family members and the mitochondrial permeability transition pore on ER stress-induced MOMP and subsequent cell death. Induction of ER stress leads to upregulation of several genes such as Grp78, Edem1, Erp72, Atf4, Wars, Herp, p58ipk, and ERdj4 and leads to caspase activation, release of mitochondrial intermembrane proteins and dissipation of mitochondrial transmembrane potential (ΔΨm). Mouse embryonic fibroblasts (MEFs) from caspase-9, -2 and, -3 knock-out mice were resistant to ER stress-induced apoptosis which correlated with decreased processing of pro-caspase-3 and -9. Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of ΔΨm. However, only deficiency of caspase-9 and -2 could prevent ER stress-mediated loss of ΔΨm. Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of ΔΨm. These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

Cross platform microarray analysis for robust identification of differentially expressed genes

BackgroundMicroarrays have been widely used for the analysis of gene expression and several commercial platforms are available. The combined use of multiple platforms can overcome the inherent biases of each approach, and may represent an alternative that is complementary to RT-PCR for identification of the more robust changes in gene expression profiles.In this paper, we combined statistical and functional analysis for the cross platform validation of two oligonucleotide-based technologies, Affymetrix (AFFX) and Applied Biosystems (ABI), and for the identification of differentially expressed genes.ResultsIn this study, we analysed differentially expressed genes after treatment of an ovarian carcinoma cell line with a cell cycle inhibitor. Treated versus control RNA was analysed for expression of 16425 genes represented on both platforms.We assessed reproducibility between replicates for each platform using CAT plots, and we found it high for both, with better scores for AFFX. We then applied integrative correlation analysis to assess reproducibility of gene expression patterns across studies, bypassing the need for normalizing expression measurements across platforms. We identified 930 genes as differentially expressed on AFFX and 908 on ABI, with ~80% common to both platforms. Despite the different absolute values, the range of intensities of the differentially expressed genes detected by each platform was similar. ABI showed a slightly higher dynamic range in FC values, which might be associated with its detection system. 62/66 genes identified as differentially expressed by Microarray were confirmed by RT-PCR.ConclusionIn this study we present a cross-platform validation of two oligonucleotide-based technologies, AFFX and ABI. We found good reproducibility between replicates, and showed that both platforms can be used to select differentially expressed genes with substantial agreement. Pathway analysis of the affected functions identified themes well in agreement with those expected for a cell cycle inhibitor, suggesting that this procedure is appropriate to facilitate the identification of biologically relevant signatures associated with compound treatment. The high rate of confirmation found for both common and platform-specific genes suggests that the combination of platforms may overcome biases related to probe design and technical features, thereby accelerating the identification of trustworthy differentially expressed genes.

Structural determinants of DISC function: new insights into death receptor-mediated apoptosis signalling.

Death receptors are members of the tumour necrosis factor (TNF) receptor superfamily characterised by an ~80 amino acid long alpha-helical fold, termed the death domain (DD). Death receptors diversified during early vertebrate evolution indicating that the DD fold has plasticity and specificity that can be easily adjusted to attain additional functions. Eight members of the death receptor family have been identified in humans, which can be divided into four structurally homologous groups or clades, namely: the p75(NTR) clade (consisting of ectodysplasin A receptor, death receptor 6 (DR6) and p75 neurotrophin (NTR) receptor); the tumour necrosis factor receptor 1 clade (TNFR1 and DR3), the CD95 clade (CD95/FAS) and the TNF-related apoptosis-inducing ligand receptor (TRAILR) clade (TRAILR1 and TRAILR2). Receptors in the same clade participate in similar processes indicating that structural diversification enabled functional specialisation. On the surface of nearly all human cells multiple death receptors are expressed, enabling the cell to respond to a plethora of external signals. Activation of different death receptors converges on the activation of three main signal transduction pathways: nuclear factor-κB-mediated differentiation or inflammation, mitogen-associated protein kinase-mediated stress response and caspase-mediated apoptosis. While the ability to induce cell death is true for nearly all DRs, the FAS and TRAILR clades have specialised in inducing cell death. Here we summarise recent discoveries about the molecular regulation and structural requirements of apoptosis induction by death receptors and discuss how this information can be used to better explain the biological functions, similarities and distinguishing features of death receptors.

Controlling the unfolded protein response-mediated life and death decisions in cancer.

Cancer cells are exposed to intrinsic (oncogene) or extrinsic (microenvironmental) challenges, leading to activation of stress response pathways. The unfolded protein response (UPR) is the cellular response to endoplasmic reticulum (ER) stress and plays a pivotal role in tumor development. Depending on ER stress intensity and duration, the UPR is either pro-survival to preserve ER homeostasis or pro-death if the stress cannot be resolved. On one hand, the adaptive arm of the UPR is essential for cancer cells to survive the harsh conditions they are facing, and on the other hand, cancer cells have evolved mechanisms to bypass ER stress-induced cell death, thereby conferring them with a selective advantage for malignant transformation. Therefore, the mechanisms involved in the balance between survival and death outcomes of the UPR may be exploited as therapeutic tools to treat cancer.

Biology of the Endoplasmic Reticulum

Since its discovery in 1945, our knowledge of the structure and many functions of the endoplasmic reticulum (ER) has advanced at a phenomenal rate. Early studies focused on the structure, which was then followed by biochemical and functional studies associated with calcium storage and release from the ER, protein folding and secretion, ER associated degradation (ERAD) and ER stress responses. Currently there is a significant interest in the role of ER in such cellular processes as cell death, autophagy and cross-talk with other organelles. In this chapter we give an overview of the structural characteristics and biochemical functioning of the ER and describe its manifold roles in cellular physiology. Finally, we explain how the sensitive nature of the protein folding process in the ER enables this organelle to act as a sensor of a broad range of cellular stresses. Signals emanating from the stressed ER play central roles in differentiation processes, cellular homeostasis and cell death.

Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models

Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.