Annamaria De Filippo

Unique Human Tumor Antigens: Immunobiology and Use in Clinical Trials

The individual, unique tumor Ags, which characterize each single tumor, were described 50 years ago in rodents but their molecular characterization was limited to few of them and obtained during the last 20 years. Here we summarize the evidence for the existence and the biological role of such Ags in human tumors, although such evidence was provided only during the last 10 years and by a limited number of studies, a fact leading to a misrepresentation of unique Ags in human tumor immunology. This was also due to the increasing knowledge on the shared, self-human tumor Ags, which have been extensively used as cancer vaccines. In this review, we highlight the biological and clinical importance of unique Ags and suggest how they could be used in clinical studies aimed at assessing their immunogenic and clinical potential both in active and adoptive immunotherapy of human tumors.

LAG-3 expression defines a subset of CD4(+)CD25(high)Foxp3(+) regulatory T cells that are expanded at tumor sites.

Human natural regulatory CD4+ T cells comprise 5–10% of peripheral CD4+T cells. They constitutively express the IL-2Rα−chain (CD25) and the nuclear transcription Foxp3. These cells are heterogeneous and contain discrete subsets with distinct phenotypes and functions. Studies in mice report that LAG-3 has a complex role in T cell homeostasis and is expressed in CD4+CD25+ T regulatory cells. In this study, we explored the expression of LAG-3 in human CD4+ T cells and found that LAG-3 identifies a discrete subset of CD4+CD25highFoxp3+ T cells. This CD4+CD25highFoxp3+LAG-3+ population is preferentially expanded in the PBMCs of patients with cancer, in lymphocytes of tumor-invaded lymph nodes and in lymphocytes infiltrating visceral metastasis. Ex vivo analysis showed that CD4+CD25highFoxp3+LAG-3+ T cells are functionally active cells that release the immunosuppressive cytokines IL-10 and TGF-β1, but not IL-2. An in vitro suppression assay using CD4+CD25highLAG-3+ T cells sorted from in vitro expanded CD4+CD25high regulatory T cells showed that this subset of cells is endowed with potent suppressor activity that requires cell-to-cell contact. Our data show that LAG-3 defines an active CD4+CD25highFoxp3+ regulatory T cell subset whose frequency is enhanced in the PBMCs of patients with cancer and is expanded at tumor sites.

SKI-606 Decreases Growth and Motility of Colorectal Cancer Cells by Preventing pp60(c-Src)–Dependent Tyrosine Phosphorylation of β-Catenin and Its Nuclear Signaling

Inhibition of deregulated protein tyrosine kinases represents an attractive strategy for controlling cancer growth. However, target specificity is an essential aim of this strategy. In this report, pp60(c-Src) kinase and beta-catenin were found physically associated and constitutively activated on tyrosine residues in human colorectal cancer cells. The use of specific small-interfering RNAs (siRNA) validated pp60(c-Src) as the major kinase responsible for beta-catenin tyrosine phosphorylation in colorectal cancer. Src-dependent activation of beta-catenin was prevented by SKI-606, a novel Src family kinase inhibitor, which also abrogated beta-catenin nuclear function by impairing its binding to the TCF4 transcription factor and its trans-activating ability in colorectal cancer cells. These effects were seemingly specific, as cyclin D1, a crucial beta-catenin/TCF4 target gene, was also down-regulated by SKI-606 in a dose-dependent manner accounting, at least in part, for the reduced growth (IC50, 1.5-2.4 micromol/L) and clonogenic potential of colorectal cancer cells. Protein levels of beta-catenin remained substantially unchanged by SKI-606, which promoted instead a cytosolic/membranous retention of beta-catenin as judged by immunoblotting analysis of cytosolic/nuclear extracts and cell immunofluorescence staining. The SKI-606-mediated relocalization of beta-catenin increased its binding affinity to E-cadherin and adhesion of colorectal cancer cells, with ensuing reduced motility in a wound healing assay. Interestingly, the siRNA-driven knockdown of beta-catenin removed the effect of SKI-606 on cell-to-cell adhesion, which was associated with prolonged stability of E-cadherin protein in a pulse-chase experiment. Thus, our results show that SKI-606 operates a switch between the transcriptional and adhesive function of beta-catenin by inhibiting its pp60(c-Src)-dependent tyrosine phosphorylation; this could constitute a new therapeutic target in colorectal cancer.

CCN3/nephroblastoma overexpressed matricellular protein regulates integrin expression, adhesion, and dissemination in melanoma.

CCN3/nephroblastoma overexpressed belongs to the CCN family of genes that encode secreted proteins associated with the extracellular matrix (ECM) and exert regulatory effects at the cellular level. Overexpression of CCN3 was shown in metastatic melanoma cells compared with cells of the primary tumor from the same patient. Analysis of short-term cultures from 50 primary and metastatic melanomas revealed a heterogeneous expression pattern of both the 46-kDa full-length cytoplasmic/secreted protein and the 32-kDa nuclear-truncated form. The different protein expression patterns were not associated with gene alterations or polymorphisms. Like the metastatic cells expressing high levels of the 46-kDa CCN3, cells transfected to overexpress CCN3 showed increased adhesion to ECM proteins, whereas inhibition of CCN3 expression by small interfering RNA decreased adhesion to laminin and vitronectin. CCN3 overexpression induced increased expression of laminin and vitronectin integrin receptors alpha 7 beta 1 and alpha v beta 5 by increasing their mRNA production. Moreover, CCN3 secreted by melanoma cells acted as an adhesion matrix protein for melanoma cells themselves. Analysis of CCN3 protein expression with respect to melanoma progression detected the protein in all visceral metastases tested and in most nodal metastases from relapsing patients but in only a few nodal metastases from nonrelapsing patients and cutaneous metastases. Consistently, xenotransplantation in immunodeficient mice showed a higher metastatic potential of melanoma cells overexpressing CCN3. Together, these data indicate a role for CCN3 in melanoma cell interaction with the ECM by regulating integrin expression, resulting in altered cell adhesion and leading melanoma progression to aggressive disease.

PTPRK negatively regulates transcriptional activity of wild type and mutated oncogenic β-catenin and affects membrane distribution of β-catenin/E-cadherin complexes in cancer cells

Previous reports showed that receptor-type protein-tyrosine phosphatase PTPRK co-localizes with beta-catenin at adherens junctions, and in vitro experiments suggested that beta-catenin could be substrate of PTPRK-mediated phosphatase activity. beta-catenin is a molecule endowed with a dual function being involved both in cell adhesion and in Wnt signaling pathway. Here we provide evidence for the role of PTPRK in negatively regulating the beta-catenin transcriptional activity by modulating its intracellular and membrane distribution. Expression of PTPRK protein in HEK293 cells and in PTPRK-null melanoma cell lines, one of which harbors a mutated oncogenic beta-catenin, impairs nuclear accumulation of wild type and oncogenic forms of beta-catenin, limits cytosolic levels of tyrosine-phosphorylated beta-catenin, and leads to re-localization of E-cadherin/beta-catenin complexes in ordered membrane phase along cell-cell contacts. This re-modulation of beta-catenin cellular distribution results in the inhibition of cyclin D1 and c-myc protein expression, whose genes are targets of beta-catenin. Tumor cells upon re-expression of PTPRK have a reduced proliferative and migration capacity. Moreover we show that PTPRK is also active in negatively regulating the transactivating function of beta-catenin in normal melanocytes as confirmed by experiments with silenced PTPRK by specific siRNA. Our data show that PTPRK influences transactivating activity of beta-catenin in non-tumoral and neoplastic cells by regulating the balance between signaling and adhesive beta-catenin, thus providing biochemical basis for the hypothesis of PTPRK as a tumor suppressor gene.

Human plasmacytoid dendritic cells interact with gp96 via CD91 and regulate inflammatory responses.

Glucose-regulated stress protein gp96 is known to be involved in the host response to pathogens and to cancer. Our study explored the relationships between gp96 and human blood plasmacytoid dendritic cells (pDC) and proved that gp96 directly targets pDC by a receptor-dependent interaction. Competition studies identified CD91 as a gp96 receptor on pDC, and laser confocal imaging indicated that CD91 triggering was followed by gp96 endocytosis and trafficking into early endosomes and later into the endoplasmic reticulum compartment. Using two alternative Abs, we showed that human blood pDC reproducibly expressed CD91, although different levels of expression were detectable among the analyzed donors. Moreover, CpG-matured pDC displayed CD91 receptor up-regulation that correlated with an increased gp96 binding. Functionally, gp96-pDC interaction activated the NF-κB pathway, leading to the nuclear translocation of the NF-κB complex. gp96-treated pDC maintained an immature phenotype, while they down-modulated the release of IL-8, suggesting an anti-inflammatory role of this pathway, and they strongly up-regulated the cell surface expression of the gp96 receptor CD91. CpG-matured or gp96-treated pDC, expressing high levels of the gp96 receptor CD91, antagonized the gp96-induced activation of monocyte-derived dendritic cells in terms of cell surface phenotype and cytokine production. Altogether, these results suggest that gp96-pDC interaction might represent an active mechanism controlling the strength of the immune response to free, extracellular available gp96; this mechanism could be particularly relevant in wounds and chronic inflammation.

Alternative Activation of Human Plasmacytoid DCs In Vitro And in Melanoma Lesions: Involvement of LAG-3

Plasmacytoid dendritic cells (pDCs) at tumor sites are often tolerogenic. Although pDCs initiate innate and adaptive immunity upon Toll-like receptor (TLR) triggering by pathogens, TLR-independent signals may be responsible for pDC activation and immune suppression in the tumor inflammatory environment. To identify molecules that are potentially involved in alternative pDC activation, we explored the expression and function of lymphocyte activation gene 3 (LAG-3) in human pDCs. In this report, we showed the expression of LAG-3 on the cell surface of a subset of circulating human pDCs. LAG-3+ pDCs exhibited a partially mature phenotype and were enriched at tumor sites in samples from melanoma patients. We found that LAG-3 interacted with major histocompatibility complex class II (MHC-II) to induce TLR-independent activation of pDCs with limited IFNα and enhanced IL-6 production. This in vitro cytokine profile of LAG-3-activated pDCs paralleled that of tumor-associated pDCs analyzed ex vivo. By confocal microscopy, LAG-3+ pDCs detected in melanoma-invaded lymph nodes (LNs) stained positive for IL-6 and preferentially localized near melanoma cells. These results suggest that LAG-3-mediated activation of pDCs takes place in vivo at tumor sites, and it is in part responsible for directing an immune-suppressive environment.

Heat shock proteins gp96 as immunogens in cancer patients

Heat shock proteins have been the focus of many experimental studies during the last few years in order to understand their biology and their imunologic features. We conducted pre-clinical experiments showing that gp96 purified from human melanoma lines can represent melanoma antigens and stimulate T cells known to recognize such antigens. Clinical studies of vaccination were then initiated by our group by using heat-shock protein gp96 purified from autologous tumor tissues in patients with melanoma and colorectal carcinoma. The results of these trials in metastatic melanoma patients with measurable disease showed that a melanoma-specific T cell response can be generated or increased in approximately 50% of vaccinated patients. Moreover, signs of clinical responses were obtained consisting of two complete responses and three long-lasting stabilizations. Similar results were obtained in patients with liver metastases of colorectal cancer made disease-free by surgery. In both studies a clear association was found between T cell immune response induced by the vaccine and clinical response both in the trial of melanoma (tumor response) and in that of colorectal cancer patients (disease-free and overall survival at 5 years).

CCN3 Promotes Melanoma Progression by Regulating Integrin Expression, Adhesion and Apoptosis Induced by Cytotoxic Drugs

Overexpression of CCN3 was detected in metastatic melanoma cells as compared to cells of the primary tumor from the same patient. Analysis of CCN3 in short-term cultures from 50 melanoma lesions revealed a heterogeneous expression of the 46 kDa full-length protein and the 32 kDa truncated form. In fact, some melanomas were negative for CCN3 expression, while others expressed both isoforms at the cellular level and secreted in the culture medium. Other melanomas displayed only the full-length or only the short isoform with or without protein secretion. The different protein expression patterns were not associated with alternative transcripts, gene alterations or polymorphisms. Cell fractions and immunofluorescence analysis demonstrated that the 46 kDa protein has a prevalent cytoplasmic localization, while the 32 kDa form has a nuclear localization and lacked the N-terminal domain.