Efrat Dvash, Michal Har-Tal, Sara Barak, Ofir Meir, Menachem Rubinstein
Nature Communications 6, 10112, 2015.
Endoplasmic reticulum (ER) stress and major chemotherapeutic agents damage DNA by generating reactive oxygen species (ROS). We found that ER stress and major chemotherapeutic agents induce leukotriene C4 (LTC4) biosynthesis in cells of non-hematopoietic lineage. Following ER stress or chemotherapy, an LTC4 biosynthetic machinery, consisting of microsomal glutathione-S-transferase 2 (MGST2), 5-lipoxygenase (5-LO), 5-lipoxygenase-activating protein (FLAP) and cytoplasmic phospholipase A2 (cPLA2), was activated by assembly at the nuclear envelope. ER stress and chemotherapy also triggered nuclear translocation of the two LTC4 receptors, CysLTR1 & CysLTR2. Acting in an intracrine manner, LTC4 then elicits nuclear translocation of NADPH oxidase 4 (NOX4), resulting in nuclear ROS accumulation, oxidative DNA damage and dsDNA breaks. Mgst2 deficiency, RNAi and LTC4 receptor antagonists abolished ER stress- and chemotherapy-induced ROS accumulation and DNA damage in vitro and in mouse kidneys. Cell death and mouse morbidity were also significantly attenuated. Hence, MGST2-generated LTC4 is the major mediator of ER stress- and chemotherapy-triggered oxidative stress and oxidative DNA damage, thereby augmenting cell death. Tumor cells of hematopoietic origin do not express MGST2. Indeed, they remained refractive to chemotherapy in the presence of LTC4 inhibitors. We therefore propose that LTC4 inhibitors, commonly used for the treatment of asthma, may alleviate chemotherapy-associated morbidities when used in hematologic malignancies. Our findings provide the mechanism by which these asthma drugs attenuated the neurotoxicity of amyloid β peptide, damage to heart myocytes by hypoxia-reperfusion and gentamycin-triggered kidney damage, as previously reported by several other research teams. Furthermore, NOX4 has been implicated in other major human pathologies, including metabolic diseases, neurodegeneration and osteoporosis. Therefore, inhibition of its activation by LTC4 receptor antagonists, already serving as approved asthma drugs, may be of even a broader clinical significance.
The following scheme summarises the MGST2-LTC4 mode of action:
Scheme: The stress-triggered signalling pathway leading to DNA damage and cell death.
Glossary: LTC4, leukotriene C4; CysLTR1 & CysLTR2, the two LTC4 receptors; MGST2, microsomal glutathione S transferase 2; FLAP, 5-LO activating protein; cPLA2, cytoplasmic phospholipase A2; 5-LO, 5-lipoxygenase; ER, endoplasmic reticulum; NOX4, NADPH oxidase 4; ROS, reactive oxygen species.
Menachem Rubinstein and Chiara Riganti
Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, 7610001, Israel. Department of Oncology, University of Torino, Italy.
J. Nat’l. Cancer Inst. 2015, 107(5), DJV046.
More than four decades have passed since ABC transporters were identified as the culprit of multidrug resistance (MDR). Extensive studies led to development of highly effective inhibitors of the ABC transporters, but so far these inhibitors failed in clinical trials. We explored the possible existence of MDR mechanisms other than drug transport. Major chemotherapeutic agents, including doxorubicin, 5-FU, vincristine and bortezomib trigger extensive endoplasmic reticulum (ER) stress. We observed that MDR tumor cells are also resistant to ER stress-triggered cell death, suggesting that ER stress is an important mechanism by which chemotherapy kills tumor cells. The transcription factor C/EBP beta is over-expressed in many tumor types, naturally appearing in two isoforms: a full size active form termed LAP, and a dominant negative truncated form termed LIP. Previously, we demonstrated that LAP augments tumor progression by attenuating ER stress-triggered cell death, whereas LIP attenuates tumor progression by enhancing ER stress-triggered cell death. We now find that a broad range of MDR cell lines, as well as primary MDR tumor cells, lack LIP (Fig. 1). Importantly, restoring LIP abolished the chemoresistance in all of these MDR cell types (Fig. 2). On further analysis, we discovered that LIP mRNA expression was identical in chemosensitive cells and in their drug-selected MDR subclones. However, LIP was rapidly degraded by proteasomal and lysosomal proteases in the MDR cells and not in the chemosensitive cells. We then demonstrated that proteasome and lysosome inhibitors attenuated LIP degradation in MDR cells, rendering them more sensitive to chemotherapy. A combination of such inhibitors, currently used in the treatment of myeloma (bortezomib, carfilozumib), and malaria (chloroquine), should be explored as potential means for reversing MDR.
Fig. 1. Multidrug resistant subline (HT29/MDR) of human colon carcinoma cell line (HT29) does not express C/EBP-β LIP or other ER stress markers under ER stress or chemotherapy.
Glossary: eIF2α, eukaryotic initiation factor 2α; peIF2α, phosphorylated eIF2α; C/EBPβ LAP, CCAAT/enhancer binding protein β liver-activating protein; C/EBPβ LIP, C/EBPβ liver inhibitory protein; CHOP, C/EBPβ homologous protein; Pgp, P-glycoprotein (a multidrug transporter); MRP1, MRP2, multidrug resistance proteins 1 & 2.
Fig. 2. Vector-mediated (A) or inducible (B) C/EBPβ LIP expression restores the response of HT29/MDR cells to ER stress (brefeldin A) and to chemotherapy (irinotecan, 5-fluorouracil and oxaliplatin). (live cells are stained purple with crystal violet).
Ofir Meir, Ariel Werman and Menachem Rubinstein, Dept. of Molecular genetics, The Weizmann Institute of Science, Rehovot 76100, Israel. firstname.lastname@example.org
Our group is currently studying the adaptation of tumor cells to endoplasmic reticulum (ER) stress. Nutrient shortage, hypoxia and accumulation of toxic metabolites due to limited vasculature trigger continuous ER stress in solid tumors. Radiation, chemotherapy and specifically proteasome inhibitors exert their anti-tumor activity at least in part by further increasing the tumor ER stress. In order to survive, tumor cells adapt to continuous ER stress but the mechanisms that mediate this adaptation are poorly characterized.
We found that tumor cells adapt to continuous ER stress by regulating the expression of the transcription factor C/EBP-ß. Over-expression of C/EBP-ß was reported in many tumor types, including breast cancer, colon cancer, melanoma and more but its exact mode of action as tumor promoters was not known. Our in vivo and in vitro studies show that C/EBP-ß promotes tumor progression by attenuating ER stress-triggered cell death (see Figs. 1 &2).
Currently, we are characterizing C/EBP-ß-induced genes, asking how do they contribute to tumor cell survival and to drug resistance. We hope that such studies will eventually lead to development of improved therapeutic approaches to cancer.
Figure 1. Attenuation of ER stress in a melanoma tumor. Over-expression of C/EBP-ß reduced tumor ER stress, as determined by immunostaining of the two of ER stress markers – TRIB3 and Herpud1 (red). cell nucleai were counter-stained with DAPI.
Figure 2. Attenuation of ER stress led to a fourfold increase of the average tumor mass. Mice were inoculated with melanoma tumor cells. C/EBP-ß was induced and tumor mass was determined after 8 and 13 days. Upon attenuation of the tumor ER stress a statistically significant increase in the average tumor mass (P<0.0001; N=9 per group) was observed.
Danit Finkelshtein, Ariel Werman, Daniela Novick, Sara Barak and Menachem Rubinstein
Over the years, our group has identified many previously uncharacterized receptors and binding proteins, notably TNFR1 & 2 (with Prof. D. Wallach), IFNAR2, and IL-18BP (with Prof. C. Dinarello). In 1993, we found that a naturally occurring soluble form of the LDL receptor (LDLR) inhibits infection of cells by vesicular stomatitis virus (VSV). Because LDLR-deficient cells were still infected by VSV we could not determine if LDLR is a VSV receptor (Fischer, D.G. et al, Science, 262:250-3). We now report that LDLR is the major VSV receptor, but VSV can infect cells also through other LDLR family members (PNAS online Apr. 15th, 2013).
Why is VSV an important virus?
VSV exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Utilizing this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination and viral oncolysis, and are extensively employed for laboratory gene transduction in vivo and in vitro.
What did we find?
The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Using a combination of monoclonal antibody against LDLR and recombinant Receptor Associated Protein (RAP), we completely blocked VSV entry and infectivity (Fig. 1). Addition of RAP to the culture medium completely blocked transduction of LDLR-deficient cells by VSV-G-pseudotyped lentiviral vector (Fig. 2). These results indicate that LDLR serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors.
Fig. 1. LDLR and its family members serve as VSV receptors.
Crystal violet stained WISH cells grown to confluence in 96 well plates, incubated (30 min., 37°C) with the indicated combinations of RAP (200 nM), neutralizing anti LDLR mAb 29.8, and non-neutralizing anti LDLR mAb 28.28 (50 µg/ml each); cells were then challenged with VSV at the indicated MOI. Cell viability (bar plot) was determined by reading the OD540 of cultures treated with VSV at MOI=0.06. ***P<0.002, n=4.
Fig. 2. LDLR and its other family members serve as major and minor VSV entry ports in human and mouse cells.
(A) EGFP expression in WT human FS-11 fibroblasts and LDLR-deficient GM701 fibroblasts, transduced with EGFP-encoding VSV-G-pseudotyped lentiviral vector (VSV-G-LV) in the absence (Control) or presence of RAP (100 nM). Insets: higher magnifications. Bars=500 µm. (B) Average±SD of EGFP expression shown in panel A. ***P<0.0002, n=3. *P<0.03, n=3. (C) EGFP expression in WT murine embryonic fibroblasts (WT MEFs) and LDLR-deficient MEFs, transduced with EGFP-encoding VSV-G-LV as in A. Insets: higher magnifications. Bars=500 µm. (D) Average±SD of EGFP expression shown in panel C. All fluorescence intensity values were normalized to the nuclei counts. *P<0.05, **P<0.007, ***P<0.002, n=3.
Why are these findings remarkable?
The widespread expression of LDLR family members accounts for the pantropism of VSV, and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction. Our study closes a quest for the VSV receptor that started in 1983, when phosphatidylserine was proposed as a possible VSV receptor and later shown not to be the VSV receptor.
The identification of the VSV receptor is of significant clinical importance since recombinant VSV and VSV-G-pseudotyped viral vectors are being developed for viral oncolysis, for vaccination and for gene therapy. Upregulation of LDLR in vivo, e.g., by pre-treatment with statins might increase the efficacy of such vectors. Furthermore, liver cells and certain tumor cells, which express high levels of LDLR, might be the preferred targets of VSV-G based gene therapy as well as VSV-G-based viral oncolysis.
Updated: 19/01/2016 16:45:40 Contact E-mail: Menachem.Rubinstein@weizmann.ac.il