The main function of the mammalian immune system is to monitor tissue homeostasis, to protect against invading or infectious pathogens and to eliminate damaged cells. Therefore, it is surprising that cancer occurs with such a high frequency in humans. Recent insights that have been gained from clinical studies and experimental mouse models of carcinogenesis expand our understanding of the complex relationship between immune cells and developing tumours. Here, we examine the paradoxical role of adaptive and innate leukocytes as crucial regulators of cancer development and highlight recent insights that have been gained by manipulating immune responses in mouse models of de novo and spontaneous tumorigenesis.

Nuclear factor kappa B (NF-κB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-κB activity and cell cycle regulation. NF-κB was found to stimulate transcription of cyclin D1, a key regulator of G1checkpoint control. Two NF-κB binding sites in the human cyclin D1 promoter conferred activation by NF-κB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1phase were controlled by NF-κB. Moreover, inhibition of NF-κB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-κB promotes G1-to-S-phase transition in mouse embryonal fibroblasts and in T47D mammary carcinoma cells. Impaired cell cycle progression of T47D cells expressing an NF-κB superrepressor (IκBαΔN) could be rescued by ectopic expression of cyclin D1. Thus, NF-κB contributes to cell cycle progression, and one of its targets might be cyclin D1.

The human papillomavirus (HPV) E7 protein is one of only two viral proteins that remain expressed in HPVassociated human cancers. HPV E7 proteins share structural and functional similarities with oncoproteins encoded by other small DNA tumor viruses such as adenovirus E1A and SV40 large tumor antigen. The HPV E7 protein plays an important role in the viral life cycle by subverting the tight link between cellular dierentiation and proliferation in normal epithelium, thus allowing the virus to replicate in dierentiating epithelial cells that would have normally withdrawn from the cell division cycle. The transforming activities of E7 largely re¯ect this important function. Oncogene (2001) 20, 7888 ± 7898.

Angiogenesis involves dynamic interactions between specialized endothelial tip and stalk cells that are believed to be regulated in part by VEGF and Dll4-Notch signaling. However, our understanding of this process is hampered by limited knowledge of the heterogeneity of endothelial cells and the role of different signaling pathways in specifying endothelial phenotypes. Here, we characterized by single-cell transcriptomics the heterogeneity of mouse endothelial cells and other stromal cells during active angiogenesis in xenograft tumors as well as from adult normal heart, following pharmacologic inhibition of VEGF and Dll4-Notch signaling. We classified tumor endothelial cells into three subpopulations that appeared to correspond with tip-like, transition, and stalk-like cells. Previously identified markers for tip and stalk cells were confirmed and several novel ones discovered. Blockade of VEGF rapidly inhibited cell-cycle genes and strongly reduced the proportion of endothelial tip cells in tumors. In contrast, blockade of Dll4 promoted endothelial proliferation as well as tip cell markers; blockade of both pathways inhibited endothelial proliferation but preserved some tip cells. We also phenotypically classified other tumor stromal cells and found that tumor-associated fibroblasts responded to antiangiogenic drug treatments by upregulating hypoxia-associated genes and producing secreted factors involved in angiogenesis. Overall, our findings better define the heterogeneity of tumor endothelial and other stromal cells and reveal the roles of VEGF and Dll4-Notch in specifying tumor endothelial phenotype, highlighting the response of stromal cells to antiangiogenic therapies. These findings provide a framework for defining subpopulations of endothelial cells and tumor-associated fibroblasts and their rapid changes in gene expression following antiangiogenic treatment. .

The angiopoietins Ang1 (ANGPT1) and Ang2 (ANGPT2) are secreted factors that bind to the endothelial cellspecific receptor tyrosine kinase Tie2 (TEK) and regulate angiogenesis. Ang1 activates Tie2 to promote blood vessel maturation and stabilization. In contrast, Ang2, which is highly expressed by tumor endothelial cells, is thought to inhibit Tie2 activity and destabilize blood vessels, thereby facilitating VEGF-dependent vessel growth. Here, we show that the inhibition of tumor xenograft growth caused by an Ang2-specific antibody (REGN910) is reversed by systemic administration of the Tie2 agonist Ang1. These results indicate that Ang2 blockade inhibits tumor growth by decreasing Tie2 activity, showing that Ang2 is a Tie2 activator. REGN910 treatment of tumors resulted in increased expression of genes that are repressed by Tie2 activation, providing further evidence that REGN910 inhibits Tie2 signaling. Combination treatment with REGN910 plus the VEGF blocker aflibercept reduced tumor vascularity and tumor perfusion more dramatically than either single agent, resulting in more extensive tumor cell death and more potent inhibition of tumor growth. Challenging the prevailing model of Ang2 as a destabilizing factor, our findings indicate that Ang2 plays a protective role in tumor endothelial cells by activating Tie2, thereby limiting the antivascular effects of VEGF inhibition. Thus, blockade of Ang2 might enhance the clinical benefits currently provided by anti-VEGF agents. Cancer Res; 73(1); 108-18. Ó2012 AACR.

The p53 tumor suppressor is stabilized in cells expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein. In contrast, expression of the HPV-16 E6 protein inactivates p53 by targeting it for proteasomal degradation. Since p53 activation is associated with protein accumulation we investigated the biochemical mechanisms and biological consequences of p53 stabilization in HPV-16 E7-expressing cells. Transcriptional reporter assays, expression profiling studies using cDNA arrays, and immunoblot analyses of known p53 target genes suggest that p53 remains transcriptionally inert in E7-expressing cells. The stabilized p53 in E7-expressing cells is in a wild-type conformation and the same number of phospho-forms is present. Furthermore, E7 expression does not alter p53 localization or generally block nuclear export or proteasomal degradation of p53. Moreover, the stabilized p53 remains susceptible to mdm2-induced proteasome-mediated degradation, and exogenous transfected p53 is transcriptionally active in E7-expressing cells. Taken together, these results suggest that E7 can interfere with the normal turnover of p53 but that the resulting increase of p53 has no detectable transcriptional consequences on the p53 targets that we investigated.

Anti-VEGF therapies benefit several cancer types, but drug resistance that limits therapeutic response can emerge. We generated cell lines from anti-VEGF-resistant tumor xenografts to investigate the mechanisms by which resistance develops. Of all tumor cells tested, only A431 (A431-V) epidermoid carcinoma cells developed partial resistance to the VEGF inhibitor aflibercept. Compared with the parental tumors, A431-V tumors secreted greater amounts of IL6 and exhibited higher levels of phospho-STAT3. Notably, combined blockade of IL6 receptor (IL6R) and VEGF resulted in enhanced activity against A431-V tumors. Similarly, inhibition of IL6R enhanced the antitumor effects of aflibercept in DU145 prostate tumor cells that displays high endogenous IL6R activity. In addition, post hoc stratification of data obtained from a clinical trial investigating aflibercept efficacy in ovarian cancer showed poorer survival in patients with high levels of circulating IL6. These results suggest that the activation of the IL6/STAT3 pathway in tumor cells may provide a survival advantage during anti-VEGF treatment, suggesting its utility as a source of response biomarkers and as a therapeutic target to heighten efficacious results.

In response to oncogenic insults, normal human cells execute a defense response that culminates in cellular suicide, apoptosis. Normal human diploid fibroblasts expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein are predisposed to apoptosis when they are deprived of growth factors. Even though a dominant negative p53 mutant abrogates the cell death response, it is not accompanied by p53 phosphorylation, the DNA binding capacity of p53 remains unaltered, and no activation of common p53-dependent transcriptional targets is observed. Expression of two insulin-like growth factor-1 binding proteins, IGFBP-2 and -5, is increased presumably in response to enhanced NF-kappaB activity in HPV-16 E7-expressing serum-starved cells. Phosphorylation of AKT, an important modulator of IGF-1 survival signaling, is lower in serum-starved E7-expressing cells, and exogenously added IGF-1 can partially inhibit the cell death response. This suggests that IGFBP-2 and -5 may limit IGF-1 availability thus decreasing survival signaling. Caspase 3 but not caspase 8 is activated in serum-starved HPV-16 E7-expressing cells. Caspase inhibition affects nuclear DNA fragmentation, but cell death is not inhibited. Although mitochondria play important roles in caspase-dependent as well as -independent forms of cell death, there is no evidence for cytochrome c release and thus for mitochondrial permeabilization in growth factor deprived HPV-16 E7-expressing cells.