Building: Arthur and Rochelle Belfer Building for Biomedical Research

Tel: 972-8-934-2313

Fax: 972-8-934-4108

e-mail: menachem.rubinstein@weizmann.ac.il

ER stress and tumor progression

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 several genes. Over-expression of these genes was reported in many tumor types, including breast cancer, colon cancer, melanoma and more but their exact mode of action as tumor promoters was not known. Our in vivo and in vitro studies show that these genes promote tumor progression by attenuating ER stress-triggered cell death (see Figs. 1 &2). Currently, we are deciphering the mode of action of these 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 a gene 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. The ER stress-attenuating gene 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.

Comparison of closely related genomes reveals that speciation has largely resulted from evolution of gene regulatory regions, whereas the exons have been more conserved. Indeed, regulation of gene expression is a complex field, whose study is crucial in every area of biological research.
In view of our long-term interest in cytokines and their associated proteins, we recently focused on studying the regulation of their gene expression. In the past, we have identified various components of the cytokine network, including the interferon alpha/beta receptor and more recently, an IL-18 binding protein (IL-18BP), which regulates pro-inflammatory Th1 responses. IL-18BP is specifically induced by IFNg. We found that the IL-18BP promoter includes within its proximal region (bases -1 to -122) a gamma-activated sequence (GAS) followed by an IRF-1 response element (IRF-E) that are essential for basal and IFNg -induced promoter activity. Our studies further revealed that IFNg induces the association of IRF-1 and C/EBPb, and the resulting heterodimer translocated from the cytoplasm to the nucleus. Electrophoretic mobility shift assays (EMSA) revealed that this heterodimer binds to the proximal GAS-IRF-E pair. Further characterization of the promoter revealed a clockwork of silencers and enhancers that mediated responses to various cytokines (Hurgin, V. et al, 2002).

In addition to the GAS-IRF-E pair, the proximal promoter contained two AP-1 sites, acting as silencers, two additional C/EBP-Es and two NF-kB-Es, all acting as enhancers. Mutation of any one of these elements affected both basal and IFNg-induced transcription of the IL-18BP gene by orders of magnitude. The NF-k B-E probably mediated responses to IL-1b and TNF a, as these cytokines synergized with IFNg in inducing IL-18BP, but were unable to induce the gene in the absence of IFNg. Indeed, SN50, a specific NF- kB inhibitor, significantly reduced IL-18BP mRNA induction. Furthermore, we found that IFNg induced a complex between NF- kB and the proximal AP-1-E, suggesting a possible mechanism by which IFNg may overcome the AP-1-mediated repression of IL-18BP gene expression. Further analysis has identified a distal silencer/enhancer pair within bases -1081 to -1272, that was physically associated with the proximal IRF-1. Chromatin immunoprecipitation confirmed the presence of these separate proximal and distal promoter regions, which merged into a single transcription-activation complex following induction by IFNg (See scheme).

Expression profiling revealed several late IFNg-induced genes whose proximal promoter regions contain adjacent IRF-E and C/EBP-E (Hurgin V., et al in preparation). Indeed, EMSA demonstrated binding of the IRF-1-C/EBPb heterodimer to these elements, suggesting that this heterodimer is a general transcription- activator of many IRF-1-dependent IFNg-induced genes (Hernandez A. et al ., in preparation).
These studies reveal the complexity of transcription-regulation, even in the case of a gene product such as IL-18BP, which has a rather narrow spectrum of biological activities.

Selected Publications

Novick, D. Kim, S-H., Fantuzzi, G., Reznikov, L.L., Dinarello, C.A. and Rubinstein, M. Interleukin-18 Bind ing Protein: A Novel modulator of the Th1 cytokine Response. Immunity, 10, 127-136, 1999 . PDF Version

Liu, B., Novick, D., Kim, S-H. and Rubinstein, M. Production of a biologically active human interleukin 18 requires its prior synthesis as PRO-IL-18. Cytokine, 12,1519-1525, 2000. PDF Version

Novick, D., Nabioullin, R.R., Ragsdale, W., McKenna, S., Weiser, W., Garone, L., Burkins, C., Kim, S-H., Rubinstein, M., Tepper, M.A. and Arulanandam, A.R. The neutrzlization of type I IFN biologic actions by anti-IFNAR-2 monoclonal antibodies is not entirely due to inhibition of Jak-Stat tyrosine phosphorylation. J. Interferon Cytokine Res., 20, 971-982, 2000. PDF Version

Cohen, B., Barkan, D., Levy, Y., Goldberg, I, Fridman E., Kopolovic, J. and Rubinstein, M. Leptin induces angiopoietin-2 expression in adipose tissues. J. Biol. Chem., 276, 7697-7700, 2001. PDF Version

Kim, S-H., Azam, T., Yoon, D.Y., Reznikov, L.L., Novick, D., Rubinstein, M. and Dinarello, C.A. Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein. Proc. Natl. Acad. Sci. USA., 98, 3304-3309, 2001. PDF Version

Stuyt, R.J., Netea, M.G., Kim, S-H., Novick, D., Rubinstein, M., Kullberg, B.J., Van der Meer, J.W. and Dinarello, C.A. Differential roles of interleukin-18 (IL-18) and IL 12 for induction of gamma interferon by staphylococcal cell wall components and superantigens. Infect. Immun., 69, 5025-5030, 2001. PDF Version

Novick, D., Schwartsburd, B., Pinkus, R., Suissa, D., Belzer, I., Sthoeger, Z., Keane, W.F., Chvatchko, Y., Kim, S-H., Fantuzzi, G., Dinarello, C.A. and Rubinstein, M. A novel IL-18BP ELISA shows IL-18BP in sepsis and extensive decrease of free IL-18. Cytokine, 14, 334-342, 2001. PDF Version

Zecchina, G., Novick, D., Rubinstein, M., Barak, V., Dinarello, C.A. and Nagler, A. Interleukin-18 binding protein in acute graft versus host disease and engraftment following allogeneic peripheral blood stem cell transplants. J. Hematother. Stem. Cell. Res. 10: 769-776, 2001. PDF Version

Kim, S-H., Azam, T., Novick, D., Yoon, D.Y., Reznikov, L.L., Bufler, P., Rubinstein, M. and Dinarello, C.A. Identification of amino acid residues critical for biological activity in human Interleukin-18. J. Biol. Chem. 277: 10998-11003, 2002. PDF Version

Hurgin, V., Novick, D. and Rubinstein, M. The promoter of IL-18 binding protein: activation by an IFN-g induced complex of IFN regulatory factor 1 and CCAAT/ enhancer binding protein b. Proc. Natl. Acad. Sci. USA., 99, 16957- 16962, 2002. PDF Version

Kaser, A., Novick, D., Rubinstein, M., Siegmund, B., Enrich, B., Koch, R.O., Vogel, W., Kim, S-H., Dinarello, C.A. and Tilg, H. Interferon-a induces interleukin-18 binding protein in chronic hepatitis C patients. Clin. Exp. Immunol. 129, 332-338, 2002. PDF Version

Ariel, A., Novick, D., Rubinstein, M., Dinarello, C.A., Lider, O. and Hershkoviz, R. IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components. J. Leukoc. Biol. 72, 192-198, 2002. PDF Version

Ludwiczek, O., Kaser, A., Novick, D., Dinarello, C.A., Rubinstein, M., Vogel, W. and Tilg, H. Plasma levels of Interleukin-18 and interleukin-18 binding protein are elevated in patiesnts with chronic liver disease. J. Clin. Immunol. 22, 331-337, 2002. PDF Version

Stuyt, R.J., Kim, S.H., Reznikov, L.L., Fantuzzi, G., Novick, D., Rubinstein, M., Kullberg, B.J., van der Meer, J.W., Dinarello, C.A. and Netea MG. Regulation of Staphylococcus epidermidis-induced IFN-g in whole human blood: the role of endogenous IL-18, IL-12, IL-1, and TNF. Cytokine. 21, 65-73, 2003. PDF Version

Dinarello, C.A., Novick, D., Rubinstein, M. and Lonnemann, G. Interleukin 18 and interleukin 18 binding protein: possible role in immunosuppression of chronic renal failure. Blood Purif. 21, 258-270, 2003. PDF Version

Gangemi, S., Basile, G., Merendino, R.A., Minciullo, P.L., Novick, D., Rubinstein, M., Dinarello, C.A., Lo Balbo C., Franceschi, C., Basili, S., D'Urbano, E., Davi, G., Nicita-Mauro, V. and Romano, M. Increased circulating Interleukin-18 levels in centenarians with no signs of vascular disease: another paradox of longevity? Exp Gerontol. 38, 669-672, 2003. PDF Version

Shevah, O., Borrelli, P., Rubinstein, M. and Laron, Z. Identification of two novel mutations in the human growth hormone receptor gene. J Endocrinol Invest. 26, 604-608, 2003.

Feezor, R.J., Oberholzer, C., Baker, H.V., Novick, D., Rubinstein, M., Moldawer, L.L., Pribble, J., Souza, S., Dinarello, C.A., Ertel, W. and Oberholzer, A. Molecular characterization of the acute inflammatory response to infections with gram-negative versus gram-positive bacteria. Infect Immun. 71, 5803-5813, 2003. PDF Version

Lonnemann, G., Novick, D., Rubinstein, M. and Dinarello, C.A. Interleukin-18, interleukin-18 binding protein and impaired production of interferon-g in chronic renal failure. Clin Nephrol. 60, 327-334, 2003.

Azam, T., Novick, D., Bufler, P., Yoon, D.Y., Rubinstein, M., Dinarello, C.A. and Kim, S.H. Identification of a critical Ig-like domain in IL-18 receptor a and characterization of a functional IL-18 receptor complex. J Immunol. 171, 6574-6580, 2003. PDF Version

Novick, D. and Rubinstein, M. Receptor isolation and characterization: from protein to gene. Methods Mol Biol. 249, 65-80, 2004.

Last Updated: 18 October 2009