Ilan Stein

NF-κB functions as a tumour promoter in inflammation-associated cancer

The causes of sporadic human cancer are seldom recognized, but it is estimated that carcinogen exposure and chronic inflammation are two important underlying conditions for tumour development, the latter accounting for approximately 20% of human cancer. Whereas the causal relationship between carcinogen exposure and cancer has been intensely investigated, the molecular and cellular mechanisms linking chronic inflammation to tumorigenesis remain largely unresolved. We proposed that activation of the nuclear factor κB (NF-κB), a hallmark of inflammatory responses that is frequently detected in tumours, may constitute a missing link between inflammation and cancer. To test this hypothesis, we studied the Mdr2-knockout mouse strain, which spontaneously develops cholestatic hepatitis followed by hepatocellular carcinoma, a prototype of inflammation-associated cancer. We monitored hepatitis and cancer progression in Mdr2-knockout mice, and here we show that the inflammatory process triggers hepatocyte NF-κB through upregulation of tumour-necrosis factor-α (TNFα) in adjacent endothelial and inflammatory cells. Switching off NF-κB in mice from birth to seven months of age, using a hepatocyte-specific inducible IκB-super-repressor transgene, had no effect on the course of hepatitis, nor did it affect early phases of hepatocyte transformation. By contrast, suppressing NF-κB inhibition through anti-TNFα treatment or induction of IκB-super-repressor in later stages of tumour development resulted in apoptosis of transformed hepatocytes and failure to progress to hepatocellular carcinoma. Our studies thus indicate that NF-κB is essential for promoting inflammation-associated cancer, and is therefore a potential target for cancer prevention in chronic inflammatory diseases.

Translation of Vascular Endothelial Growth Factor mRNA by Internal Ribosome Entry: Implications for Translation under Hypoxia

ABSTRACT Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5′ untranslated region (5′UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5′UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5′UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5′UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long “improved” IRES element was abrogated, however, following substitution of a few bases near the 5′ terminus as well as substitutions close to the translation start codon. Both the full-length 5′UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.

Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes.

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is upregulated in response to a hypoxic or hypoglycemic stress. Here we show that the increase in steady-state levels of VEGF mRNA is partly due to transcriptional activation but mostly due to increase in mRNA stability. Both oxygen and glucose deficiencies result in extension of the VEGF mRNA half-life in a protein synthesis-dependent manner. Viewing VEGF as a stress-induced gene, we compared its mode of regulation with that of other stress-induced genes. Results showed that under nonstressed conditions, VEGF shares with the glucose transporter GLUT-1 a relatively short half-life (0.64 and 0.52 h, respectively), which is extended fourfold and more than eightfold, respectively, when cells are deprived of either oxygen or glucose. In contrast, the mRNAs of another hypoxia-inducible and hypoglycemia-inducible gene, grp78, as well as that of HSP70, were not stabilized by these metabolic insults. To show that VEGF and GLUT-1 are coinduced in differentially stressed microenvironments, multicell spheroids representing a clonal population of glioma cells in which each cell layer is differentially stressed were analyzed by in situ hybridization. Cellular microenvironments conducive to induction of VEGF and GLUT-1 were completely coincidental. These findings show that two different consequences of tissue ischemia, namely, hypoxia and glucose deprivation, induce VEGF and GLUT-1 expression by similar mechanisms. These proteins function, in turn, to satisfy the tissue needs through expanding its vasculature and improving its glucose utilization, respectively.

Lyn Is a Target Gene for Prostate Cancer Sequence-Based Inhibition Induces Regression of Human Tumor Xenografts

The Src-related protein kinase Lyn plays an important role in B-cell activation. However, several lines of evidence suggest that it is also involved in the control of cellular proliferation and the inhibition of apoptosis. We have discovered that Lyn is expressed in normal prostate epithelia, in 95% of primary human prostate cancer (PC) specimens examined, and in all of the PC cell lines that we assayed. Moreover, Lyn knockout mice display abnormal prostate gland morphogenesis, which suggests that Lyn plays an important role in prostate epithelium development and implies that Lyn is a candidate target for specific therapy for PC. Using a drug-design strategy to construct sequence-based peptide inhibitors, a Lyn-specific inhibitor, KRX-123, targeting a unique interaction site within Lyn, was synthesized. KRX-123 was found to inhibit cellular proliferation in three hormone-refractory PC cell lines, DU145, PC3, and TSU-Pr1 with IC50 values of 2–4 μm. In vivo, tumor volume of DU145 explants in nude mice was significantly reduced after once-a-week injections of KRX-123, at a dose of 10 mg/kg, for a period of 5 weeks. Histological analyses of the treated tumors indicated extensive apoptosis. Thus, we suggest that Lyn inhibition may serve as a prime target for the treatment of hormone-refractory PC.

S100A8 and S100A9 are novel nuclear factor kappa B target genes during malignant progression of murine and human liver carcinogenesis

The nuclear factor‐kappaB (NF‐κB) signaling pathway has been recently shown to participate in inflammation‐induced cancer progression. Here, we describe a detailed analysis of the NF‐κB–dependent gene regulatory network in the well‐established Mdr2 knockout mouse model of inflammation‐associated liver carcinogenesis. Expression profiling of NF‐κB–deficient and NF‐κB–proficient hepatocellular carcinoma (HCC) revealed a comprehensive list of known and novel putative NF‐κB target genes, including S100a8 and S100a9. We detected increased co‐expression of S100A8 and S100A9 proteins in mouse HCC cells, in human HCC tissue, and in the HCC cell line Hep3B on ectopic RelA expression. Finally, we found a synergistic function for S100A8 and S100A9 in Hep3B cells resulting in a significant induction of reactive oxygen species (ROS), accompanied by enhanced cell survival. Conclusion: We identified S100A8 and S100A9 as novel NF‐κB target genes in HCC cells during inflammation‐associated liver carcinogenesis and provide experimental evidence that increased co‐expression of both proteins supports malignant progression by activation of ROS‐dependent signaling pathways and protection from cell death. (HEPATOLOGY 2009.)

Intercellular communication between vascular smooth muscle and endothelial cells mediated by heparin-binding epidermal growth factor-like growth factor and vascular endothelial growth factor

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB‐EGF when introduced subcutaneously into nude mice. HB‐EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti‐vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor‐α (TGF‐α) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT‐PCR analysis demonstrated that VEGF165 mRNA levels were increased in vascular SMC 4–10‐fold by 0.35–2 nM of HB‐EGF, respectively. Our data suggest that HB‐EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.

The Chemokine CXCL16 and Its Receptor, CXCR6, as Markers and Promoters of Inflammation-Associated Cancers

Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes.

Human and Mouse VEGFA-Amplified Hepatocellular Carcinomas Are Highly Sensitive to Sorafenib Treatment

UNLABELLED Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib-the first-line drug in advanced HCC-targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs. SIGNIFICANCE Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib-the first-line treatment of advanced HCC-which has an overall moderate therapeutic efficacy.

Regulation of the polycomb protein Ring1B by self-ubiquitination or by E6-AP may have implications to the pathogenesis of Angelman syndrome

The polycomb repressive complex (PRC) 1 protein Ring1B is an ubiquitin ligase that modifies nucleosomal histone H2A, a modification which plays a critical role in regulation of gene expression. We have shown that self-ubiquitination of Ring1B generates multiply branched, “noncanonical” polyubiquitin chains that do not target the ligase for degradation, but rather stimulate its activity toward histone H2A. This finding implies that Ring1B is targeted by a heterologous E3. In this study, we identified E6-AP (E6-associated protein) as a ligase that targets Ring1B for “canonical” ubiquitination and subsequent degradation. We further demonstrated that both the self-ubiquitination of Ring1B and its modification by E6-AP target the same lysines, suggesting that the fate of Ring1B is tightly regulated (e.g., activation vs. degradation) by the type of chains and the ligase that catalyzes their formation. As expected, inactivation of E6-AP affects downstream effectors: Ring1B and ubiquitinated H2A levels are increased accompanied by repressed expression of HoxB9, a PRC1 target gene. Consistent with these findings, E6-AP knockout mice display an elevated level of Ring1B and ubiquitinated histone H2A in various tissues, including cerebellar Purkinje neurons, which may have implications to the pathogenesis of Angelman syndrome, a neurodevelopmental disorder caused by deficiency of E6-AP in the brain.

Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma

Ectopic lymphoid-like structures (ELSs) are often observed in cancer, yet their function is obscure. Although ELSs signify good prognosis in certain malignancies, we found that hepatic ELSs indicated poor prognosis for hepatocellular carcinoma (HCC). We studied an HCC mouse model that displayed abundant ELSs and found that they constituted immunopathological microniches wherein malignant hepatocyte progenitor cells appeared and thrived in a complex cellular and cytokine milieu until gaining self-sufficiency. The egress of progenitor cells and tumor formation were associated with the autocrine production of cytokines previously provided by the niche. ELSs developed via cooperation between the innate immune system and adaptive immune system, an event facilitated by activation of the transcription factor NF-κB and abolished by depletion of T cells. Such aberrant immunological foci might represent new targets for cancer therapy.