CD24 promotes tumor cell invasion by suppressing tissue factor pathway inhibitor-2 (TFPI-2) in a c-Src-dependent fashion.

CD24 is a glycosyl-phosphatidylinositol-anchored protein with mucin-type structure that resides exclusively in membrane microdomains. CD24 is often highly expressed in carcinomas and correlates with poor prognosis. Experimentally, the over-expression or depletion of CD24 alters cell proliferation, adhesion, and invasion in vitro and tumor growth in vivo. However, little is known about the mechanisms by which CD24 mediates these cellular effects. Here we have studied the mechanism of CD24-dependent cell invasion using transient CD24 knock-down or over-expression in human cancer cell lines. We show that CD24 depletion reduced tumor cell invasion and up-regulated expression of Tissue Factor Pathway Inhibitor 2 (TFPI-2), a potent inhibitor of extracellular matrix degradation that can block metastases formation and tumor cell invasion. Over-expression of CD24 in A125 cells resulted in reduced TFPI-2 expression and enhanced invasion. We provide evidence that the activity of c-Src is reduced upon CD24 knock-down. The silencing of c-Src, similar to CD24, was able to enhance TFPI-2 expression and reduce tumor cell invasion. An inverse expression of CD24 and TFPI-2 was observed by immunohistochemical analysis of primary breast cancers (N = 1,174). TFPI-2 expression was highest in CD24 negative samples and lowered with increasing CD24 expression. Patients with a CD24 low/TFPI-2 high phenotype showed significantly better survival compared to CD24 high/TFPI-2 low patients. Our results provide evidence that CD24 can regulate cell invasion via TFPI-2 and suggests a role of c-Src in this process.

Proteasomal and genetic inactivation of the NF1 tumor suppressor in gliomagenesis.

Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However, complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity.

Vascular targeting of anti-CD40 antibodies and IL-2 into autochthonous tumors enhances immunotherapy in mice.

Current anticancer therapy is a delicate balance between elimination of malignant cells and harmful side effects for the host. In this study, we used a tumor-homing peptide to engineer anti-CD40 agonist antibodies and recombinant IL-2 such that they were selectively delivered into spontaneously arising tumors in a transgenic mouse model of islet cell carcinogenesis. Intravenous injection of these agents, either separately or together, led to accumulation in the vicinity of tumor neovessels without toxic side effects. Although both molecules are critical for adaptive immunity, the most profound effects were seen in endothelial cells. Combined, local anti-CD40 and IL-2 therapy reduced tumor vascularity and significantly delayed tumor growth in mice. Remarkably, tumor-bearing mice remained disease-free long-term when targeted anti-CD40 and IL-2 were combined with transfers of preactivated antitumor immune cells. In this therapeutic setting, triggering of CD40 on endothelial cells induced an inflammatory response of the vessel wall and facilitated effector cell accumulation in the tumor parenchyma while IL-2 promoted antigen-specific immune cell persistence. We believe this is a novel and highly effective anticancer approach, whereby tumor stroma is "conditioned" for enhanced immune cell entry and survival, facilitating immune-mediated tumor destruction and leading to a sustained antitumor response.

Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO.

CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions.

Frequent downregulation of DMBT1 and galectin-3 in epithelial skin cancer.

DMBT1 and galectin-3 are potential interacting proteins with presumably complex roles in tumorigenesis. While at present a variety of mechanisms are discussed for DMBT1 and its participation in cancer, galectin-3 is commonly known to exert tumor-promoting effects. However, in vitro studies in a rodent system have suggested that DMBT1/galectin-3 interaction in the ECM triggers epithelial differentiation, which would point to tumor-suppressive properties. To improve the understanding of DMBT1/galectin-3 action in cancer, we carried out studies in skin cancer of different origins. Mutational analyses of DMBT1 identified a missense mutation in 1 of 13 melanoma cell lines. It led to an exchange of an evolutionary conserved proline residue for serine and located within the second CUB domain of DMBT1. Immunohistochemical analyses demonstrated absence of DMBT1/galectin-3 expression from melanocytes but induction of DMBT1 expression in 1 of 8 nevi and 1 of 11 melanomas and of galectin-3 expression in 3 of 8 nevi and 4 of 8 melanomas. These data suggest that DMBT1 and galectin-3 are unlikely to act as classical tumor suppressors in melanomas. DMBT1 and galectin-3 appear to be secreted to the ECM by epithelial cells within the epidermis and the hair follicle. Compared to the flanking normal epidermis, skin tumors of epithelial origin frequently displayed downregulation of DMBT1 (18 of 19 cases) and galectin-3 (12 of 12 cases). Thus, loss of DMBT1/galectin-3 expression may play a role in the genesis of epithelial skin cancer. This would support the view that galectin-3 can exert tumor-suppressive effects in certain scenarios, and DMBT1/galectin-3-mediated differentiation represents a candidate mechanism for this effect.