John B. Patterson

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

Vascular Targeting and Antiangiogenesis Agents Induce Drug Resistance Effector GRP78 within the Tumor Microenvironment

Therapeutic targeting of the tumor vasculature that destroys preexisting blood vessels of the tumor and antiangiogenesis therapy capitalize on the requirement of tumor cells on an intact vascular supply for oxygen and nutrients for growth, expansion and metastasis to the distal organs. Whereas these classes of agents show promise in delaying tumor progression, they also create glucose and oxygen deprivation conditions within the tumor that could trigger unintended prosurvival responses. The glucose-regulated protein GRP78, a major endoplasmic reticulum chaperone, is inducible by severe glucose depletion, anoxia, and acidosis. Here we report that in a xenograft model of human breast cancer, treatment with the vascular targeting agent, combretastatin A4P, or the antiangiogenic agent, contortrostatin, promotes transcriptional activation of the Grp78 promoter and elevation of GRP78 protein in surviving tumor cells. We further show that GRP78 is overexpressed in a panel of human breast cancer cells that has developed resistance to a variety of drug treatment regimens. Suppression of GRP78 through the use of lentiviral vector expressing small interfering RNA sensitizes human breast cancer cells to etoposide-mediated cell death. Our studies imply that antivascular and antiangiogenesis therapy that results in severe glucose and oxygen deprivation will induce GRP78 expression that could lead to drug resistance.

Stress Chaperone GRP78/BiP Confers Chemoresistance to Tumor-Associated Endothelial Cells

The tumor vasculature is essential for tumor growth and survival and is a key target for anticancer therapy. Glioblastoma multiforme, the most malignant form of brain tumor, is highly vascular and contains abnormal vessels, unlike blood vessels in normal brain. Previously, we showed that primary cultures of human brain endothelial cells, derived from blood vessels of malignant glioma tissues (TuBEC), are physiologically and functionally different from endothelial cells derived from nonmalignant brain tissues (BEC) and are substantially more resistant to apoptosis. Resistance of TuBEC to a wide range of current anticancer drugs has significant clinical consequences as it represents a major obstacle toward eradication of residual brain tumor. We report here that the endoplasmic reticulum chaperone GRP78/BiP is generally highly elevated in the vasculature derived from human glioma specimens, both in situ in tissue and in vitro in primary cell cultures, compared with minimal GRP78 expression in normal brain tissues and blood vessels. Interestingly, TuBEC constitutively overexpress GRP78 without concomitant induction of other major unfolded protein response targets. Resistance of TuBEC to chemotherapeutic agents such as CPT-11, etoposide, and temozolomide can be overcome by knockdown of GRP78 using small interfering RNA or chemical inhibition of its catalytic site. Conversely, overexpression of GRP78 in BEC rendered these cells resistant to drug treatments. Our findings provide the proof of principle that targeting GRP78 will sensitize the tumor vasculature to chemotherapeutic drugs, thus enhancing the efficacy of these drugs in combination therapy for glioma treatment. (Mol Cancer Res 2008;6(8):1268–75)

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

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.

Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

Global downregulation of microRNAs (miRNAs) is a common feature of human tumors and has been shown to enhance cancer progression. Several components of the miRNA biogenesis machinery (XPO5, DICER and TRBP) have been shown to act as haploinsufficient tumor suppressors. How the deregulation of miRNA biogenesis promotes tumor development is not clearly understood. Here we show that loss of miRNA biogenesis increased resistance to endoplasmic reticulum (ER) stress-induced cell death. We observed that HCT116 cells with a DICER hypomorphic mutation (Exn5/Exn5) or where DICER or DROSHA were knocked down were resistant to ER stress-induced cell death. Extensive analysis revealed little difference in the unfolded protein response (UPR) of WT compared to Exn5/Exn5 HCT116 cells upon ER stress treatment. However, analysis of the intrinsic apoptotic pathway showed that resistance occurred upstream of the mitochondria. In particular, BAX activation and dissipation of mitochondrial membrane potential was attenuated, and there was altered expression of BCL-2 family proteins. These observations demonstrate a key role for miRNAs as critical modulators of the ER stress response. In our model, downregulation of miRNA biogenesis delays ER stress-induced apoptosis. This suggests that disrupted miRNA biogenesis may contribute to cancer progression by inhibiting ER stress-induced cell death.

Dual IRE1 RNase functions dictate glioblastoma development

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1‐dependent decay (RIDD) of RNA. Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1‐dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.

Adaptation of the secretory pathway in cancer through IRE1 signaling.

The unfolded protein response (UPR) was originally identified as a signaling network coordinating adaptive and apoptotic responses to accumulation of unfolded proteins in the endoplasmic reticulum (ER). More recent work has shown that UPR signaling can be triggered by a multitude of cellular events and that the UPR plays a critical role in the prevention of cell transformation but also in tumor development. This has been particularly well illustrated with studies on one of the three major ER stress sensors, IRE1. This ER resident type I transmembrane protein senses luminal ER stress and transduce signals through its cytosolic RNase activity. IRE1 signaling has been shown to contribute to the progression of solid tumors through pro-angiogenic mechanisms. Herein, we expose the methodologies for investigating IRE1 signaling in tumor cells and in tumors. Moreover, we show that selective pharmacological inhibition of IRE1 RNase activity sensitizes tumor cells to ER stress.

Autophagy and the Unfolded Protein Response Promote Pro-fibrotic Effects of TGFβ1 in Human Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-β1 (TGF-β1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-β1 induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-β1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-β1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

In this Issue: Sensing Immune Danger through Unfolded Protein Response plus Pathogen Recognition Receptors; and Immune Modulation for Cancer and HIV-1 Disease

In this issue, we are hosting several reviews focusing on: (1) the interface between the immune system and endogenous or exogenous immune danger sensed through the unfolded protein response (UPR) and pattern recognition receptors (PRRs), respectively; (2) the immune suppressing role of soluble, classical, and non-classical major histocompatibility molecules (MHC I and MICs) in cancer and their consideration as new therapeutic targets; and (3) the immune protective and modulating effects of intravenous polyclonal immunoglobulin therapy, as a complementary approach within the therapeutic armamentarium of HIV disease. One of the most dynamic areas of research in the cellular biology area and beyond is focused on understanding the role of the unfolded protein response (UPR) during physiological and pathological processes. UPR is an ancient changemanagement, molecular mechanism that emerged within eukaryotic unicellular organisms to ensure the homeostasis of the protein production and folding in the secretory pathway as a response to internal and external insults (“stress”). That “stress” results from imbalances of protein accumulation, degradation, and aggregation within cellular organelles. Nevertheless, with the integration of single cells within multi-cellular organisms and evolution to increasingly complex entities that have a need for multi-faceted, highly integrated homeostatic mechanisms, the UPR also co-evolved to undertake roles beyond stress management at the cellular level. Since immunity also evolved to sense effectively, rapidly, and redundantly ‘danger’ associated with microbial infection, it has been just a question of time to elucidate how the UPR interfaces with immunity. In this issue, Dr. Masanori Kitamura outlines major evidence to date, that links the UPR to immunity through the pivotal node, NF-kB, that regulates production of a large array of proinflammatory molecules. NF-kB’s function is substantially modulated by various arms of the UPR, such as IRE1, PERK, and ATF6. Interestingly, based on the evidence discussed and independent literature, it seems that the UPR has a dual, Janus-like role vis-à-vis NF-kB directed inflammatory response (Fig. 1): (i) a rapidly activating role enhanced