A R T I C L E Inhibition of NF- B in cancer cells converts inflammation- induced tumor growth mediated by TNF to TRAIL-mediated tumor regression Jun-Li Luo,1 Shin Maeda,1 Li-Chung Hsu,1 Hideo Yagita,2 and Michael Karin1,* 1Laboratory of Gene Regulation and Signal Transduction, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093 2 Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan *Correspondence: karinoffice@ucsd.edu Summary We used an experimental murine cancer metastasis model in which a colon adenocarcinoma cell line generates lung metastases, whose growth is stimulated in response to injection of bacterial lipopolysaccharide (LPS), to investigate the role of NF- B in inflammation-induced tumor growth. We found that LPS-induced metastatic growth response in this model depends on both TNF production by host hematopoietic cells and NF- B activation in tumor cells. Inhibition of NF- B in both colon and mammary carcinoma cells converts the LPS-induced growth response to LPS-induced tumor regression. The latter response is TNF -independent, but depends on another member of the TNF superfamily, TRAIL, whose receptor is induced in NF- B-deficient cancer cells. Introduction and Kohn, 2001). Whereas certain cytokines and chemokines promote cancer cell proliferation and survival, others can inhibit tumor growth and kill cancer cells (Coussens and Werb, 2002 Invasion and metastasis are the main clinical phenomena that distinguish malignant from benign tumors, and are the leading Dunn et al., 2002). In addition to deregulated cell growth and survival, many tumors have acquired the ability to subvert im- causes of death in cancer patients. Accumulating evidence sug- gests that tumor progression is governed not only by genetic mune and inflammatory responses to their benefit (Pollard, 2004). It is therefore of great importance to identify the molecular changes intrinsic to the cancer cell (Moody et al., 2002 Steeg, 2003), but also by epigenetic and environmental factors (Cous- mechanisms through which the immune/inflammatory system promotes tumor cell proliferation, survival, and invasiveness as sens and Werb, 2002 Pollard, 2004). Chronic infection and the ensuing inflammation are considered to be some of the most well as those responsible for tumor cell killing. A better under- standing of these mechanisms may eventually lead to develop- important epigenetic and environmental factors contributing to tumorigenesis and tumor progression (Coussens and Werb, ment of new therapeutic strategies that will allow conversion of the tumor-promoting effect of the immune/inflammatory system 2002 Pollard, 2004). Although the adaptive immune system reduces tumor incidence through immune-surveillance mecha- to a strong tumoricidal effect. Using a mouse model of inflam- mation-induced tumor growth, we explored this possibility nisms (Dunn et al., 2002), the innate immune system can pro- mote tumor development and progression through inflamma- through the manipulation of NF- B activity within the cancer cell. tion-dependent mechanisms (Coussens and Werb, 2002). It was also observed that bacterial infection following surgery can pro- The NF- B family of transcription factors plays a central role in regulation of immune and inflammatory responses, apoptosis, mote growth of metastases in experimental animals and human patients (Harmey et al., 2002 Pidgeon et al., 1999 Taketomi and oncogenesis (Baldwin, 2001 Karin et al., 2002). A wide range of stimuli, including cytokines and viral and bacterial prod- et al., 1997). Immune and inflammatory cells and their secreted chemokines and cytokines have dramatic effects both on the ucts, activate NF- B, mostly through I B kinase (IKK)-depen- dent phosphorylation and subsequent degradation of specific host���s physiology and on cancer cell behavior, modulating the growth, migration, and differentiation of many cell types within inhibitors, the I Bs, that retain NF- B in the cytoplasm (Ghosh and Karin, 2002). Upon activation, NF- B dimers enter the nu- the tumor microenvironment (Coussens and Werb, 2002 Liotta S I G N I F I C A N C E NF- B transcription factors play a central role in immune and inflammatory responses. Some NF- B-regulated genes are associated with tumor progression and metastasis, processes known to be induced by inflammation. Our work explains how NF- B converts inflammatory stimuli into tumor growth signals and consequently speeds up metastatic tumor growth. Inhibition of NF- B in cancer cells converts inflammation-induced tumor growth to inflammation-induced tumor regression. While the first response is mediated by TNF , the second response is mediated by TRAIL. Our results suggest that inhibition of NF- B in cancer cells can strongly potentiate the efficacy of TRAIL or TRAIL inducers in cancer therapy. CANCER CELL : SEPTEMBER 2004 �� VOL. 6 �� COPYRIGHT ��� 2004 CELL PRESS 297

A R T I C L E Figure 1. NF- B inhibition converts LPS-induced tumor growth to tumor regression A: Establishment of an NF- B-deficient cell line. Mouse colon adenocarcinoma CT26 cells were stably transfected with either an empty vector or a vector encoding the I B (AA) super-repressor. Cells were stimulated with TNF (10 ng/ml), and expression of endogenous I B and I B (AA) was analyzed by immunoblotting. NF- B DNA binding activity was examined by EMSA. B: NF- B inhibition results in LPS-induced tumor regression. 1 105 cells (CT, CT/vector, or CT/ I B [AA]) were injected into mice that after 9 days were administered PBS (vehicle) or LPS. Seven days later, mice were sacrificed and lungs were removed, weighed, and photographed, and the numbers of tumor nodules detectable on the lung surface were determined and statisti- cally analyzed (*, p 0.05 **, p 0.01). C: Histology of lung metastatic tumors. Tumors generated by injection of the indicated cell lines were exposed to PBS (vehicle) or LPS in vivo, and 7 days later the lungs were removed, formalin fixed, sectioned, and H&E stained. Magnifica- tion: 10 . cleus, where they modulate transcription of many genes encod- al., 2002 Pidgeon et al., 1999), and demonstrated that NF- B activation in cancer cells is responsible for inflammation-induced ing cytokines, growth factors, cell adhesion molecules, and anti- apoptotic proteins. Some NF- B-regulated gene products, tumor growth. Importantly, inhibition of NF- B converts LPS- induced tumor growth to LPS-induced tumor regression. We including those encoding intercellular adhesion molecule 1 (ICAM-1), the extracellular matrix protein tenascin C, vascular found that the mediators through which LPS induces these opposite outcomes are TNF and TRAIL, respectively. Our re- endothelial growth factor (VEGF), the chemokine IL-8 and its mouse homologs, the proinflammatory enzyme cyclooxygenase sults suggest a novel strategy for cancer therapy, based on combining the inhibition of NF- B in cancer cells with induction 2 (COX2), and matrix metalloprotease 9 (MMP9), are associated with tumor progression and metastasis (Coussens and Werb, of TRAIL, by the host immune system. 2002 Karin et al., 2002). Thus, it seems that NF- B can convert inflammatory stimuli into tumor growth signals. However, NF- B Results may also control the expression of apoptosis-promoting cyto- kines (death cytokines) such as TNF (Zhu et al., 2000) and NF- B activation is required for LPS-induced FAS ligand (FASL) (Kasibhatla et al., 1998). Nonetheless, the tumor growth ability of NF- B to inhibit apoptosis appears to be stronger We modified a murine metastasis model in which a mouse colon than its ability to promote apoptosis (Karin and Lin, 2002), and cancer cell line, CT26, originally induced in BALB/c mice by exposure to 1,2-dimethylhydrazine, was used to generate lung therefore, inhibition of NF- B was suggested to be a useful strategy for cancer therapy (Baldwin, 2001 Karin et al., 2002). metastases (Harmey et al., 2002 Pidgeon et al., 1999) whose growth is stimulated in response to LPS administration. To ex- Here we modified an experimental murine metastasis model in which bacterial lipopolysaccharide (LPS), a byproduct of gram- amine the role of NF- B in this process, we transfected CT26 cells with a vector encoding an I B ���super-repressor��� mutant negative bacteria, induces metastatic tumor growth (Harmey et 298 CANCER CELL : SEPTEMBER 2004