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Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6 ) is frequently amplified in breast cancer, with an increased incidence in the triple‐negative breast cancer (TNBC ) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three‐dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6 ‐depleted, triple‐negative MDA‐MB ‐231 cells rearranged the actin cytoskeleton and restored epidermal growth factor‐mediated invasion. In patients with localized, lymph node‐negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse‐free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6 –fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC .

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Although sublingual allergen-specific immunotherapy has been proved to be effective in the treatment of allergic diseases, controversy surrounds the means by which such a local therapy can induce systemic immunological changes. Adhesion molecules are critical in the regulation of leukocyte traffic. It has been hypothesized that allergenic extract, administered locally, may induce an up-regulation of the mucosal vessel vascular adhesion molecules (CAMs) resulting in local recruitment of circulating inflammatory cells. In the present study we investigated whether the mite antigens, Der p1 and Der p2, can modulate CAM expression of human endothelial cells (HEC). To do this, slices of whole human umbilical cord vein underwent short-term (8 hours) cultures in the presence or absence of mite antigen (baseline, unstimulated controls). Cryostatic sections of the specimens were then evaluated immunohistochemically for expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) molecules. The results revealed that while Der p1 is capable of significantly up-regulating ICAM-1 and VCAM-1 on HEC, Der p2 antigen moderately up-regulates ICAM-1 expression but is ineffective in modulating VCAM-1. Although preliminary, these results clearly support the hypothesis that at least some of the effects of sublingual immunotherapy may derive from inflammatory cell recruitment at the site of allergen release.

The 45th Annual Meeting of the Italian Cancer Society (SIC), held at the Centro Congressi Giovanni XXIII in Bergamo, Italy on 9-12 November, 2003, attracted almost 400 participants. The Scientific Committee chaired by R Giavazzi (Mario Negri Institute, Bergamo) and the board of the Italian Cancer Society produced a packed and varied program. Plenary sessions with keynote speakers (24 lectures) were combined with oral proffered papers (20 selected presentations), providing a platform from laboratory science to clinical interventions. Two hundred and sixty-one abstracts were submitted to the conference and presented in poster and poster discussion sessions, providing a stimulating ground for discussion and a network of interactive collaboration among young investigators. The venue of Bergamo offered exceptional hospitality with social events in the beautiful old town and at the Modern Art Museum. We hope that the following report gives an idea of the event and will encourage the reader to attend the 46th meeting of the SIC, 24-26 October, 2004 in Pisa.

In neuroblastoma, high levels of mRNA for p14h trkA and p75 LNGFR neurotrophin receptors are predictive of favorable outcome. Their evaluation by Northern blot, however, requires substantial amounts of tissue and this prevents their routine evaluation as well as the possibility for multicenter studies to be easily carried out. In an attempt to overcome these limitations, the feasibility and reliability of determining both neurotrophin receptors on cryostat sections by immunohistochemistry were assessed, and these findings were compared to those obtained from Northern blot analysis. Primary tumor samples from 28 untreated patients at all stages were evaluated by using H10 anti-p140 trkA and ME20.4 anti-p75 LNGFR mAbs. Although weak, positiveimmunostaining was found in 9 of 28 tumors for p140 trkA and in 5 of 28 tumors for p75 LNGFR . As compared to Northern blot, the concordance rate was 79% (22 of 28 cases) for p140 trkA (p < 0.05) and 71% (20 of 28 cases) for p75 LNGFR (p < 0.05). No case negative for Northern blot was found to be positive with immunohistochemistry. Since only high mRNA levels for both receptors have been shown to be clinically relevant, their immunohistochemical detection, although less sensitive than Northern blot, can be just as sufficient and reliable as a prognostic tool, and possibly with a better cost-benefit ratio.

In order to identify genes relevant for melanoma development, we carried out cDNA array experiments employing an in vitro model of human melanoma progression, consisting of two cell lines: one, LP, derived from a primary melanoma and the other, LM, from its metastatic supraclavicular lymph node. Basic cDNA array data identified 26 genes as down-regulated in the LM cell line. Northern blot analysis confirmed an effective transcriptional down-regulation for five out of 13 genes analyzed. The products of these five genes belong to different functional protein types, such as transcription and translation regulators (Edg-2, eIF-3 p110, and RNPL/RBM3), extracellular communicators (PRSS11) and members of the major histocompatibility complex (β2-microglobulin). Some previously described differences in expression patterns, such as loss of HLA I, were confirmed by our array data. In addition, we identified and validated for the first time the reduced expression level of several genes during melanoma progression. In particular, reduced Edg-2 gene product expression was also confirmed in a group of 50 primary melanomas and unrelated metastases. In conclusion, comparative hybridization by means of cDNA arrays assisted in identifying a series of novel progression-associated changes in gene expression, confirming, at the same time, a number of previously described results. © Blackwell Munksgaard, 2003.

The molecular mechanisms involved in lymphangiogenesis were unknown until recently. We previously demonstrated that the endothelin-1 (ET-1) axis stimulates lymphatic endothelial cells (LEC) and lymphatic vessels to grow and invade. Here we further investigated the effect of ET-1 on lymphatic vessels and evaluated whether ET-1 actions result in the functional activation of lymphangiogenesis. Using highly purified human LEC, characterized for the expression of ET-1 axis members by quantitative real-time PCR, we found that the endothelin B receptor (ETB), upon activation by ET-1, induced matrix-metalloproteinase activation, demonstrating that ET-1 influenced the activity of the proteolytic enzymes required for LEC invasion. Functional assays performed by using intradermal lymphangiography demonstrated that ET-1 promoted the formation of lymphatic vessels and that these vessels were capable of lymphatic flow. ETB blockade with the specific antagonist BQ788 inhibited matrix-metalloproteinase activation and dye transport within the lymphatic vessels, demonstrating that ETB is involved in the regulation of the growth of and in the formation of functional vessels upon activation by ET-1. Our results suggest that ET-1 is a lymphangiogenic mediator and that targeting pharmacologically ETB may be therapeutically exploited in a variety of diseases, including cancer.

Screening of a cDNA expression library from a primary breast tumor with the autologous patient serum led to the isolation of 6 cDNA clones corresponding to 3 different genes, including a novel gene that maps to chromosome 1 and encodes the human homologue of mouse Mena (hMena, cDNA clone RMNY-BR-55), a protein of the Ena/VASP family involved in the regulation of cell motility and adhesion. A cancer-restricted antibody response against hMena was demonstrated, since 18/93 cancer patient sera, the majority (10/52) from breast cancer, showed anti-hMena-specific IgG, while no antibodies were present in healthy donors. When hMena protein expression was analyzed by Western blot and immunohistochemistry, the antigen was overexpressed in the majority of breast cancer cell lines and in 75% of primary breast tumor lesions evaluated. Furthermore, when HLA-A2-restricted peptides from the hMena sequence were used to stimulate CD8+ T cells, an hMena-specific response was found in 9 out of 12 HLA-A2+ breast cancer patients. In 4 patients, this cell-mediated immune response was concomitant with antibody response to hMena. Furthermore, an hMena-specific T-cell line was established from an HLA-A2+ breast cancer patient whose primary tumor lesion overexpressed the hMena protein. The present findings highlight the emerging role that overexpression of cytoskeleton regulatory components may have in the induction of a specific antitumor immune response.

Coexpression for c-Kit receptor and its ligand stem cell factor (SCF) has been described in neuroblastoma (NB) cell lines and tumors, suggesting the existence of an autocrine loop modulating tumor growth. We evaluated c-Kit and SCF expression by immunohistochemistry in a series of 75 primary newly diagnosed neuroblastic tumors. Immunostaining for c-Kit was found in 10/75 and for SCF in 17/75, with 5/10 c-Kit-positive tumors also expressing SCF. For both, c-Kit and SCF staining were predominantly found in the most aggressive subset of tumors, i.e., those amplified for MYCN: c-Kit was detected in 8/14 amplified vs. 2/61 single copy (p

Endothelin-1 (ET-1) and its receptors are overexpressed in human Kaposi's sarcoma lesions. Here we show that in human KS IMM cell line ET-1 increased secretion and activation of matrix-metalloproteinase-2 (MMP-2), -3, -7, -9 and -13, as well as of membrane-type 1-MMP (MT1-MMP). ET-1 and ET-3 also enhanced the expression of tissue inhibitor of MMP-2, essential for MT1-MMP-mediated MMP-2 activation. Combined addition of both ET(B) receptor (ET(B)R) and ET(A)R antagonists completely blocked the ET-1-induced MMP activity. By immunohistochemistry, we observed that ET-1 increased MMP-2 and MT1-MMP expression and their localization at the cell surface. Treatment with both antagonists resulted also in the suppression of ET-1-induced phosphorylation of focal adhesion proteins, FAK and paxillin, which are essentials for cell motility. ET-1 induced a dose-dependent enhancement in KS IMM cell migration and MMP-dependent molecule, A-182086, an orally bioavailable ET(A/B)R antagonist, completely inhibited cell proliferation and tumor growth in KS IMM xenografts. These findings demonstrate that ET-1-driven autocrine loop is crucial for enhanced invasiveness of KS IMM cells and promote tumor growth in vivo. Such activities can be blocked by the ET(A/B)R antagonists, which may be effective anti-angiogenic and anti-tumor molecules for the treatment of Kaposi's sarcoma.

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

In neuroblastoma, high levels of mRNA for p140trkA and p75LNGFR neurotrophin receptors are predictive of favorable outcome. Their evaluation by Northern blot, however, requires substantial amounts of tissue and this prevents their routine evaluation as well as the possibility for multicenter studies to be easily carried out. In an attempt to overcome these limitations, the feasibility and reliability of determining both neurotrophin receptors on cryostat sections by immunohistochemistry were assessed, and these findings were compared to those obtained from Northern blot analysis. Primary tumor samples from 28 untreated patients at all stages were evaluated by using H10 anti-p140trkA and ME20.4 anti-p75LNGFR mAbs. Although weak, positive immunostaining was found in 9 of 28 tumors for p140trkA and in 5 of 28 tumors for p75LNGFR. As compared to Northern blot, the concordance rate was 79% (22 of 28 cases) for p140trkA (p0.05) and 71% (20 of 28 cases) for p75LNGFR (p0.05). No case negative for Northern blot was found to be positive with immunohistochemistry. Since only high mRNA levels for both receptors have been shown to be clinically relevant, their immunohistochemical detection, although less sensitive than Northern blot, can be just as sufficient and reliable as a prognostic tool, and possibly with a better cost-benefit ratio.

Class II MHC antigen expression in the intestinal epithelium of 28 small bowel biopsies from 23 celiac patients were studied by means of indirect immunofluorescence and immunoperoxidase using monoclonal antibodies. Patients were divided on the basis of diet into two subgroups: 15 subjects on a gluten-containing diet (GCD) and 13 on a gluten-free diet (GFD). The control group included 10 pediatric subjects with normal intestinal mucosa who underwent intestinal biopsy for chronic diarrhea or short stature. DR antigens and invariant chain were expressed in all patients, regardless of the diet, as well as in the control subjects. DQ was found in one patient only on GCD. DP antigens were present in 12/15 patients on GCD, and in 2/13 on GFD (Fisher's exact test, p = 8.8 x 10(-4), as well as in 3/10 control subjects. In 4/5 celiac patients, DP antigens, which were undetectable on GFD, could be demonstrated after gluten challenge. The results of the study show that DR antigens are expressed by intestinal mucosa of celiac patients independently of their gluten exposure and that DQ antigens are consistently undetectable. Statistically significant differences in expression of DP antigens on enterocytes of celiac patients on GCD and their neoexpression after gluten challenge may represent a basis for further investigation.

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-14C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can be easily quantitated by an enzymatic assay.