Katarzyna Mnich

The integrated stress response

In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro‐survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.

Loss of cannabinoid CB1 receptor expression in the 6-hydroxydopamine-induced nigrostriatal terminal lesion model of Parkinson’s disease in the rat

The endocannabinoid system is emerging as a potential alternative to the dopaminergic system for the treatment of Parkinsons disease. Like all emerging targets, validation of this systems potential for treating human Parkinsonism necessitates testing in animal models of the condition. However, if components of the endocannabinoid system are altered by the induction of a Parkinsonian state in animal models, this could have an impact on the interpretation of such preclinical experiments. This study sought to determine if expression of the CB(1) subtype of cannabinoid receptor is altered in the two most commonly used rat models of Parkinsons disease. Parkinsonian lesions were induced by stereotaxic injection of 6-hydroxydopamine into the axons (medial forebrain bundle) or terminals (striatum) of the nigrostriatal pathway. On days 1, 3, 7, 14 and 28 post-lesion, rats were sacrificed and brains were processed for tyrosine hydroxylase and CB(1) receptor immunohistochemistry. The CB(1) receptor was expressed strongly in the substantia nigra pars reticulata, minimally overlapping with tyrosine hydroxylase immunoreactivity in the pars compacta. Interestingly, while there was little change in CB(1) receptor expression following axonal lesion, expression of the receptor was significantly reduced following terminal lesion. Loss of CB(1) receptor expression in the pars reticulata correlated significantly with the loss of striatal and nigral volume after terminal lesion indicating this may have been due to 6-hydroxydopamine-induced non-specific damage of striatonigral neurons which are known to express CB(1) receptors. Thus, this result has implications for the choice of model and interpretation of studies used to investigate potential cannabinoid-based therapies for Parkinsons disease as well as striatonigral diseases such as Huntingtons disease and Multiple Systems Atrophy.

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.

Endoplasmic reticulum stress-mediated induction of SESTRIN 2 potentiates cell survival

Upregulation of SESTRIN 2 (SESN2) has been reported in response to diverse cellular stresses. In this study we demonstrate SESTRIN 2 induction following endoplasmic reticulum (ER) stress. ER stress-induced increases in SESTRIN 2 expression were dependent on both PERK and IRE1/XBP1 arms of the unfolded protein response (UPR). SESTRIN 2 induction, post ER stress, was responsible for mTORC1 inactivation and contributed to autophagy induction. Conversely, knockdown of SESTRIN 2 prolonged mTORC1 signaling, repressed autophagy and increased ER stress-induced cell death. Unexpectedly, the increase in ER stress-induced cell death was not linked to autophagy inhibition. Analysis of UPR pathways identified prolonged eIF2α, ATF4 and CHOP signaling in SESTRIN 2 knockdown cells following ER stress. SESTRIN 2 regulation enables UPR derived signals to indirectly control mTORC1 activity shutting down protein translation thus preventing further exacerbation of ER stress.

Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.

Inhibition by anandamide of 6-hydroxydopamine-induced cell death in PC12 cells.

6-hydroxydopamine (6-OHDA) is a selective neurotoxin that is widely used to investigate cell death and protective strategies in models of Parkinsons disease. Here, we investigated the effects of the endogenous cannabinoid, anandamide, on 6-OHDA-induced toxicity in rat adrenal phaeochromocytoma PC12 cells. Morphological analysis and caspase-3 activity assay revealed that anandamide inhibited 6-OHDA-induced apoptosis. The protection was not affected by antagonists of either cannabinoid receptors (CB1 or CB2) or the vanilloid receptor TRPV1. Anandamide-dependent protection was reduced by pretreatment with LY294002 (inhibitor of phosphatidylinositol 3-kinase, PI3K) and unaffected by U0126 (inhibitor of extracellularly-regulated kinase). Interestingly, phosphorylation of c-Jun-NH2-terminal kinase (JNK) in cells exposed to 6-OHDA was strongly reduced by anandamide pre-treatment. Furthermore, 6-OHDA induced c-Jun activation and increased Bim expression, both of which were inhibited by anandamide. Together, these data demonstrate antiapoptotic effects of anandamide and also suggest a role for activation of PI3K and inhibition of JNK signalling in anandamide-mediated protection against 6-OHDA.

Heat shock preconditioning protects against ER stress‐induced apoptosis through the regulation of the BH3‐only protein BIM

A mild heat shock (HS) preconditioning and acquisition of thermotolerance protects cells against a variety of cytotoxic agents that otherwise induce apoptosis. Here we tested whether there is a molecular link between HS preconditioning and endoplasmic reticulum (ER) stress‐induced apoptosis. ER stress results from a loss of ER lumen homeostasis, culminating in an accumulation of unfolded/misfolded proteins in the ER and activation of unfolded protein response (UPR). Unresolved, ER stress leads to activation of BH3‐only proteins, mitochondrial membrane permeabilization, caspase activation and apoptotic cell death. HS preconditioning (1 h at 42 °C) induced a rapid increase in HSPA1 (HSP70) levels which remained elevated for at least 48 h post‐HS. HS preconditioning significantly reduced BAX, caspase activation and apoptosis in cell cultures treated with the ER stress‐inducing agents thapsigargin (TG) and tunicamycin (TM). HS‐mediated protection was found to be due to regulation of the BH3‐only protein BIM. Further, overexpression of HSPA1 could not mimic the effect of HS on BIM expression, suggesting that other HS factors may play a role in inhibiting ER stress‐induced apoptosis by regulating BIM.

HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants.

Nerve growth factor (NGF)-mediated regulation of p75(NTR) expression contributes to chemotherapeutic resistance in triple negative breast cancer cells

Triple negative breast cancer [TNBC] cells are reported to secrete the neurotrophin nerve growth factor [NGF] and express its receptors, p75 neurotrophin receptor [p75(NTR)] and TrkA, leading to NGF-activated pro-survival autocrine signaling. This provides a rationale for NGF as a potential therapeutic target for TNBC. Here we show that exposure of TNBC cells to NGF leads to increased levels of p75(NTR), which was diminished by NGF-neutralizing antibody or NGF inhibitors [Ro 08-2750 and Y1086]. NGF-mediated increase in p75(NTR) levels were partly due to increased transcription and partly due to inhibition of proteolytic processing of p75(NTR). In contrast, proNGF caused a decrease in p75(NTR) levels. Functionally, NGF-induced increase in p75(NTR) caused a decrease in the sensitivity of TNBC cells to apoptosis induction. In contrast, knock-down of p75(NTR) using shRNA or small molecule inhibition of NGF-p75(NTR) interaction [using Ro 08-2750] sensitized TNBC cells to drug-induced apoptosis. In patient samples, the expression of NGF and NGFR [the p75(NTR) gene] mRNA are positively correlated in several subtypes of breast cancer, including basal-like breast cancer. Together these data suggest a positive feedback loop through which NGF-mediated upregulation of p75(NTR) can contribute to the chemo-resistance of TNBC cells.

Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy

Triple-negative breast cancer (TNBC) lacks targeted therapies and has a worse prognosis than other breast cancer subtypes, underscoring an urgent need for new therapeutic targets and strategies. IRE1 is an endoplasmic reticulum (ER) stress sensor, whose activation is predominantly linked to the resolution of ER stress and, in the case of severe stress, to cell death. Here we demonstrate that constitutive IRE1 RNase activity contributes to basal production of pro-tumorigenic factors IL-6, IL-8, CXCL1, GM-CSF, and TGFβ2 in TNBC cells. We further show that the chemotherapeutic drug, paclitaxel, enhances IRE1 RNase activity and this contributes to paclitaxel-mediated expansion of tumor-initiating cells. In a xenograft mouse model of TNBC, inhibition of IRE1 RNase activity increases paclitaxel-mediated tumor suppression and delays tumor relapse post therapy. We therefore conclude that inclusion of IRE1 RNase inhibition in therapeutic strategies can enhance the effectiveness of current chemotherapeutics.IRE1/XBP-1 activation has a major role in Triple negative breast cancer (TNBC). Here, the authors show that inhibition of IRE1’s RNase activity attenuates autocrine and paracrine signaling of pro-tumorigenic cytokines and synergizes with paclitaxel to confer potent anti-tumor effects in TNBC.