Novel VEGF-independent strategies targeting tumor vasculature: clinical aspects

In the last decades, the active research in the field of tumor angiogenesis has led to the development of a class of agents providing an effective inhibition of neo-vessel formation through the blockade of VEGF related pathways. More recently, the identification of other factors involved in tumor angiogenesis, such as platelet-derived growth factor, fibroblast growth factor and Angiopoietins has emphasized the need to develop agents targeting multiple pro-angiogenic pathways. Although contrasting data are currently available regarding the clinical efficacy of multikinase inhibitors, Sunitinib, Sorafenib and Pazopanib have displayed encouraging results, and have fuelled further evaluations. Moreover, definitive data are also eagerly awaited regarding the clinical role of angiopoietins inhibitors. On the other hand, the existence of morphological, functional and architectural differences between normal and tumor vasculature has provided solid basis for the development of a novel class of compounds, known as Vascular Disrupting Agents (VDAs) able to selectively disrupt existing tumor vessels. After initial concerns related to the potential development of severe cardiovascular toxicities, further clinical investigations have shown a safe toxicity profile for these agents. Moreover, despite the discouraging data on dolostatin-10 and ASA404, several VDAs, including CAP4, Ombrabulin and Pinabulin have already shown promising activity in phase I-II clinical trials warranting more advanced evaluations. In this review we aimed at summarizing the most relevant VEGF-independent strategies targeting tumor vasculature, focusing on the clinical development of novel antiangiogenic agents including multikinase and angiopoietins inhibitors as well as VDAs.     

A novel human antitumor dimeric immunoRNase

We report on the construction, production, and characterization of a new fully human dimeric immunoRNase, obtained by fusion of Erbicin, a human anti-ErbB2 single-chain antibody fragment (scFv), with a dimeric mutant of human pancreatic RNase (HHP2-RNase). This novel immunoagent with 2 scFv moieties each fused to 1 of the 2 subunits of the dimeric RNase, called ERB-HHP2-RNase, has shown improved biologic properties with respect to the previously reported monomeric ERB-hRNase immunoRNase: it selectively binds ErbB2-positive cancer cells with an increased avidity; it is not inhibited by the cytosolic ribonuclease inhibitor; it is endowed with a more powerful cytotoxic activity.     

Mitoxantrone treatment in multiple sclerosis: a 5-year clinical and MRI follow-up

Mitoxantrone (MTX) is an antineoplastic agent approved for treatment of secondary progressive and rapidly worsening relapsing-remitting multiple sclerosis (MS). We designed a longitudinal open-label prospective study to evaluate the efficacy and toxicity of MTX over a 2-year treatment period with a further 3-year follow-up. Fifty consecutive MS patients were included and received MTX intravenously (8 mg/m² every 2 months for a total of 12 infusions). Efficacy was assessed clinically and by brain MRI performed before MTX therapy, at the end of treatment and at the end of each year of follow-up. Forty-nine patients completed the 5-year study, 44 (89.8%) completed the MTX course, five (10.2%) interrupted the treatment because of side effects. Fifteen (30.6%) patients showed Expanded Disability Status Scale (EDSS) progression on treatment and nine (18.4%) during follow-up. Seventeen (34.7%) patients had enhancing lesions at baseline, nine (18.4%) at the end of treatment, but none at the end of follow-up. In conclusion, we observed EDSS progression in about 1/3 of the patients during the treatment period and in 1/5 during the further 3-year follow-up period. This evidence suggests a delayed beneficial effect after MTX treatment is completed with only a minority of patients showing disability progression once the drug was suspended.     

Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment.

Clostridium difficile is the aetiological agent of pseudomembranous colitis, and animal studies suggest the essential role of secreted toxin A in inducing disease. This study examined the biological responses to toxin A by human intestinal epithelial cells. Confluent monolayers of Caco2, HT29, and T84 cells and primary epithelial cells in organ cultures of human colonic biopsy specimens and after detachment with EDTA were studied. Interleukin 8 was assayed using enzyme linked immunosorbent assay (ELISA). Purified C difficile toxin A induced cell rounding and detachment of monolayers of the epithelial cell lines. Cells in detached monolayers initially remained viable while adherent to each other. Subsequently, an increasing number of apoptotic cells appeared in suspension. Exposure to toxin A for 24 hours induced interleukin 8 production in T84 and HT29 cells. Toxin A also induced epithelial cell rounding, detachment, and apoptosis in organ cultures of human colonic biopsy specimens. During culture (in medium only), EDTA detached colonic epithelial cells produced interleukin 8 and cell death occurred by apoptosis. Colonic disease by C difficile may be initiated by toxin A mediated induction of epithelial cell interleukin 8 production and apoptosis after cell detachment from the basement membrane. Studies on isolated (toxin untreated) colonic epithelial cells suggest that interleukin 8 production and apoptosis occur as a consequence of cell injury and detachment.     

The N-terminal 11 amino acids of human erythrocyte band 3 are critical for aldolase binding and protein phosphorylation: implications for band 3 function

The 911 amino acid band 3 (SLC4A1) is the major intrinsic membrane protein of red cells and is the principal Cl-/HCO3- exchanger. The N-terminal cytoplasmic domain of band 3 anchors the spectrin-based membrane skeleton to the lipid bilayer through its interaction with ankyrin and also binds glycolytic enzymes and hemoglobin. We identified a son of a consanguineous marriage with severe anemia in association with marked deficiency of band 3 (12% +/- 4% of normal). Direct nucleotide sequencing of SLC4A1 gene demonstrated a single base substitution (T --> C) at position + 2 in the donor splice site of intron 2, resulting in the generation of a novel mutant protein. Biochemical characterization of the mutant protein showed that it lacked the first 11 N-terminal amino acids (band 3 Neapolis). The expression of the mutant protein resulted in the complete absence of membrane-bound aldolase, and the mutant band 3 could not be tyrosine phosphorylated. The ability of the malarial parasite P falciparum to invade these red cells was significantly decreased. The identification of a novel band 3 mutant and its structural and functional characterization enabled us to identify pivotal roles for the 11 N-terminal amino acids in several protein functions and, in turn, in red-cell physiology.     

IL‐3 synergises with basophil‐derived IL‐4 and IL‐13 to promote the alternative activation of human monocytes

Basophil‐derived IL‐4 is involved in the alternative activation of mouse monocytes, as recently shown in vivo. Whether this applies to human basophils and monocytes has not been established yet. Here, we sought to characterise the interaction between basophils and monocytes and identify the molecular determinants. A basophil‐monocyte co‐culture model revealed that IL‐3 and basophil‐derived IL‐4 and IL‐13 induced monocyte production of CCL17, a marker of alternative activation. Critically, IL‐3 and IL‐4 acted directly on monocytes to induce CCL17 production through histone H3 acetylation, but did not increase the recruitment of STAT5 or STAT6. Although freshly isolated monocytes did not express the IL‐3 receptor α chain (CD123), and did not respond to IL‐3 (as assessed by STAT5 phosphorylation), the overnight incubation with IL‐4 (especially if associated with IL‐3) upregulated CD123 expression. IL‐3‐activated JAK2‐STAT5 pathway inhibitors reduced the CCL17 production in response to IL‐3 and IL‐4, but not to IL‐4 alone. Interestingly, monocytes isolated from allergen‐sensitised asthmatic patients exhibited a higher expression of CD123. Taken together, our data show that the JAK2‐STAT5 pathway modulates both basophil and monocyte effector responses. The coordinated activation of STAT5 and STAT6 may have a major impact on monocyte alternative activation in vitro and in vivo.     

Localization of the meningococcal receptors for human transferrin.

The interaction between gold-labelled human transferrin (Au-HTF) with live meningococci after growth in vivo or in different in vitro conditions was examined by electron microscopy to localize and quantify the numbers of HTF-binding sites on the cell surface. It was clearly demonstrated that HTF binds to the surface of live meningococci (of different serogroups and serotypes) after growth in either iron-sufficient or iron-restricted cultures, although the degree of labelling was always higher (2- to 35-fold) in the latter case. The commensal Neisseria polysaccharea behaved similarly. Ultrathin sections showed that Au-HTF was localized predominantly on the outer membrane of the cells and vesicles, with hardly any internalization. Au-HTF labelling on meningococci was significantly reduced after incubation with unlabelled HTF or with rabbit antiserum containing antibodies against transferrin-binding proteins (TBPs), demonstrating the specificity of the interaction. These sera also blocked binding between HTF and outer membrane proteins on Western immunoblots. Direct evidence of the expression of the TBPs (Western blots) and localization of the HTF receptor (electron microscopy) on in vivo-grown meningococci was obtained from organisms derived without laboratory culturing from the cerebrospinal fluid of a patient. There was considerable cell-to-cell variation in the amount of labelling present on cells of the same sample (in vitro- or in vivo-grown organisms) and between different strains. The degree of binding varied with time of incubation of the cells with Au-HTF. The gold particles frequently formed discrete circles on the cell surfaces of the in vitro-grown organisms; these circles appear to be associated with outer membrane vesicle formation. The results show that the TBPs, which form part of the active components of the HTF receptor(s), are expressed in vivo and are surface exposed and immunogenic and that antibodies against them can interfere with the HTF binding of the meningococcal cells, which may affect iron utilization. This study further supports the concept of regarding the TBPs as future vaccine candidates.     

A cancer‐associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss‐of‐function

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27^Kip1, a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer‐associated germinal p27^Kip1 variant, namely glycine9‐>arginine‐p27^Kip1 (G9R‐p27^Kip1) identified in a parathyroid adenoma. We unexpectedly found that G9R‐p27^Kip1 lacks the major tumor suppressor activities of p27^Kip1 including its antiproliferative and pro‐apoptotic functions. In addition, G9R‐p27^Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild‐type p27^Kip1‐transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R‐p27^Kip1 on S12, a residue normally never found modified in p27^Kip1. The novel S12 phosphorylation appears responsible for the loss of function of G9R‐p27^Kip1 since S12AG9R‐p27^Kip1 recovers most of the p27^Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D‐p27^Kip1 recapitulates G9R‐p27^Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin‐dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27^Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post‐translational modifications due to generation of novel consensus sequences by cancer‐associated missense mutations.

Abbreviations

1D/WB

monodimensional western blotting

2D/WB

two‐dimensional western blotting

CDK

cyclin‐dependent kinase

CHX

cycloheximide

G9R‐p27

glycine9‐>arginine‐p27

IUPs

intrinsically unstructured proteins

mAbs

monoclonal antibodies

MEN

multiple endocrine neoplasia

MENX

multiple endocrine neoplasia X

PTMs

post‐translational modifications

rAbs

rabbit antibodies

TSG

tumor suppressor gene

wt‐p27

wild‐type p27