Ilaria Rossi

Immunoprevention of Mammary Carcinoma in HER-2/neu Transgenic Mice Is IFN-γ and B Cell Dependent

A vaccine combining IL-12 and allogeneic mammary carcinoma cells expressing p185neu completely prevents tumor onset in HER-2/neu transgenic BALB/c mice (NeuT mice). The immune protection elicited was independent from CTL activity. We now formally prove that tumor prevention is mainly based on the production of anti-p185neu Abs. In the present studies, NeuT mice were crossed with knockout mice lacking IFN-γ production (IFN-γ−/−) or with B cell-deficient mice (μMT). Vaccination did not protect NeuT-IFN-γ−/− mice, thus confirming a central role of IFN-γ. The block of Ab production in NeuT-μMT mice was incomplete. About one third of NeuT-μMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-μMT mice that responded to the vaccine with a robust production of anti-p185neu Ab displayed a markedly delayed tumor onset. In these NeuT-μMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-μMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2q molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice.

p185neu protein is required for tumor and anchorage-independent growth, not for cell proliferation of transgenic mammary carcinoma

Transgenic FVB‐NeuN mice (N202) bearing the rat neu protooncogene driven by the mouse mammary tumor virus promoter/enhancer develop focal mammary carcinomas overexpressing the neu‐encoded p185neu protein. In vitro expression of p185neu among mammary carcinoma cultures was heterogeneous, and we could establish some cell lines and clones displaying a complete loss of p185neu expression, along with others with very high p185neu protein level. Upon in vivo injection, p185neu‐positive cells gave rise to fast‐growing tumors with a short latency, while p185neu‐negative cells required a very long latency time, and the resulting tumors were invariably p185neu‐positive. The lower growth ability of p185neu‐negative cells in vivo was also confirmed in athymic nude mice. In vitro, analysis of anchorage‐independent growth in soft agar revealed colony formation from p185neu‐positive but not p185neu‐negative cells. The direct involvement of p185neu in clonogenicity was demonstrated by the inhibition of p185neu‐positive colony growth in soft agar in the presence of an anti‐p185neu monoclonal antibody. By contrast, a higher level of anchorage‐dependent clonogenic growth and proliferation was observed in p185neu‐negative cells as compared to p185neu‐positive cells, thus explaining the relative ease with which p185neu‐negative cell lines and clones were established in vitro. Together, the results indicate that p185neu expression can lead to tumor formation and metastasis through the modification of intrinsic properties of cells related to anchorage‐independent growth ability rather than to proliferation or host‐dependent mechanisms. Int. J. Cancer 87:186–194, 2000.

The Metastatic Ability of Ewing’s Sarcoma Cells Is Modulated by Stem Cell Factor and by Its Receptor c-kit

Ewings sarcoma is a primitive highly malignant tumor of bone and soft tissues usually metastasizing to bone, bone marrow, and lung. Growth factor receptors and their ligands may be involved in its growth and dissemination. We analyzed the expression of c-kit and its ligand stem cell factor (SCF) in a panel of six Ewings sarcoma cell lines. All cell lines exhibited substantial levels of surface c-kit expression, and five of six displayed transmembrane SCF on the cell surface. Expression of c-kit was down-modulated in all lines by exposure to exogenous SCF. The SCF treatment was able to confer to cells a growth advantage in vitro, due both to an increase in cell proliferation and to a reduction in the apoptotic rate. When used in the lower compartment of a migration chamber, SCF acted as a strong chemoattractant for Ewings sarcoma cells. The pretreatment of cells with SCF reduced their chemotactic response to SCF. In athymic nude mice, Ewings sarcoma cells injected intravenously metastasized to the lung and to a variety of extrapulmonary sites, including bone and bone marrow. Metastatic sites resembled those observed in Ewings sarcoma patients and corresponded to SCF-rich microenvironments. The in vitro pretreatment of cells with SCF strongly reduced the metastatic ability of Ewings sarcoma cells, both to the lung and to extrapulmonary sites. This could be dependent on the down-modulation of c-kit expression observed in SCF-pretreated cells, leading to a reduced sensitivity to the chemotactic and proliferative actions of SCF. Our results indicate that the response to SCF mediated by c-kit may be involved in growth, migration, and metastatic ability of Ewings sarcoma cells.

Down regulation of major histocompatibility complex class I expression in mammary carcinoma of HER-2/neu transgenic mice

Transgenic mice carrying the HER‐2/neu proto‐oncogene under tissue‐specific transcriptional control of a mammary tumor virus long terminal repeat (Tg‐MMTVneu mice) spontaneously develop mammary carcinomas. HER‐2/neu is a tumor antigen that can be recognized by cytotoxic T lymphocytes if tumor cells present the appropriate major histocompatibility complex (MHC) class I glycoproteins. The purpose of this work was to assess whether mammary carcinomas arising in Tg‐MMTVneu mice correctly expressed MHC (H‐2q) class I gene products. We analyzed by flow cytometry 51 primary tumors from 19 transgenic mice. About one‐half of the tumors showed a reduced expression of class I antigens. All tumors were highly positive for membrane neu. Some mice had multiple mammary carcinomas with widely different MHC expression levels, and most mice had at least one tumor with a low expression. Treatment with γ‐interferon of carcinoma cells cultured in vitro induced a strong re‐expression of H‐2q antigens. Our results suggest that the immune response activated in vivo by HER‐2/neu‐positive tumors can lead to the emergence of escape variants characterized by a down‐regulation of MHC class I products. Int. J. Cancer 77:937–941, 1998.© 1998 Wiley‐Liss, Inc.

Expression of HER/erbb family of receptor tyrosine kinases and induction of differentiation by glial growth factor 2 in human rhabdomyosarcoma cells

Five human rhabdomyosarcoma cell lines were used to investigate the surface expression of HER/erbB receptors, particularly of HER‐2, HER‐3 and HER‐4, by flow cytometry. HER‐2 was expressed in 3/5 rhabdomyosarcoma cell lines. HER‐3 was expressed in all rhabdomyosarcoma cell lines. None of the rhabdomyosarcoma cell lines investigated showed HER‐4 surface expression. To study the biological activity of HER/erbB receptors in rhabdomyosarcomas, cells were cultured in the presence of glial growth factor 2 (GGF‐2), a specific ligand of HER‐3 that is able to stimulate myogenesis in normal myoblasts. In 3 of 3 human rhabdomyosarcoma cell lines positive for both HER‐2 and HER‐3 receptors, the treatment with GGF‐2 induced a significant increase in myogenic differentiation. Int. J. Cancer 87:29–36, 2000.

HER/erbB receptors as therapeutic targets of immunotoxins in human rhabdomyosarcoma cells.

Human rhabdomyosarcoma cells express HER/erbB growth factors receptors. Receptors belonging to this family are overexpressed and play a role in many types of epithelial and neural cancer and have been selected as targets for cancer therapy. In this paper EGF-R, HER-2 and HER-3 receptors were tested as therapeutic targets of immunotoxins in human rhabdomyosarcoma. Rhabdomyosarcoma cells were treated with indirect immunotoxins consisting in primary specific murine monoclonal antibodies recognizing EGF-R, HER-2 and HER-3 followed by secondary F(ab´)2 antimouse immunoglobulin linked to saporin-S6, a type 1 ribosome-inactivating protein (RIP) from the seeds of Saponaria officinalis. The indirect immunotoxin targeting EGF-R caused a significant inhibition in cell growth and protein synthesis and a strong increase in apoptosis in rhabdomyosarcoma cells, whereas indirect immunotoxins against HER-2 and HER-3 were ineffective. The toxic activity of anti-EGF-R immunotoxin was also observed on rhabdomyosarcoma cells expressing low level of EGF-R. EGF-R could be a novel therapeutic target of immunotoxins in human rhabdomyosarcoma.

Expression of interleukin 15 (IL-15) in human rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma.

Interleukin 15 (IL‐15) is a recently discovered cytokine that stimulates lymphocyte proliferation and migration via a trimeric receptor sharing the β and γ signal transducing chains with the IL‐2 receptor. IL‐15 is typically produced by normal cells that do not release IL‐2, but little information is currently available on human tumors. To assess whether human musculo‐skeletal sarcomas produce IL‐15, we analyzed surgical specimens and cell lines obtained from rhabdomyosarcoma, osteosarcoma and Ewings sarcoma. IL‐15 mRNA was present in 9/9 surgical specimens (3 Ewings sarcomas, 5 osteosarcomas and 1 rhabdomyosarcoma). The analysis of a panel of cell lines (7 derived from Ewings sarcoma, 12 from osteosarcoma and 5 from rhabdomyosarcoma) showed that all rhabdomyosarcoma and osteosarcoma cell lines expressed IL‐15 mRNA at levels ranging from low to high, while Ewings sarcoma cells contained little or no IL‐15 message. ELISA assays showed IL‐15 release in a subset of rhabdomyosarcomas and osteosarcomas, but not in Ewings sarcoma. The highest production of IL‐15, in the picogram/ml range, was found in rhabdomyosarcoma cell lines RH30 and RD. Int. J.Cancer 71:732‐736, 1997.

An anti-apoptotic role for NGF receptors in human rhabdomyosarcoma

The expression and biological function of Nerve Growth Factor (NGF) receptors was studied in a panel of rhabdomyosarcoma cell lines derived from embryonal and alveolar histotype. All the cell lines expressed both the high affinity receptor TrkA and the low affinity receptor p75(NTR). Treatment with exogenous NGF did not considerably alter rhabdomyosarcoma cell growth or differentiation, but significantly inhibited spontaneous apoptosis as well as apoptosis, and induced by serum starvation or apoptosis induced by treatment with cycloheximide (CHX). Rhabdomyosarcoma cell lines expressed NGF and other neurotrophins and trace amounts of NGF protein were found in the supernatants of rhabdomyosarcoma cell cultures. Blocking the putative autocrine loop with an anti-NGF antibody resulted in an increase in apoptosis compared with control cultures. These data suggest that the simultaneous presence of both high and low affinity NGF receptors engaged by endogenous or exogenous NGF might contribute to the escape from apoptosis exhibited by the rhabdomyosarcoma cells.

Production of stem cell factor and expression of c‐kit in human rhabdomyosarcoma cells: Lack of autocrine growth modulation

Human rhabdomyosarcoma cells produce autocrine and paracrine growth factors that can sustain their growth and malignancy. Here we report constitutive production of stem cell factor (SCF) by 5 of 5 human rhabdomyosarcoma cell lines both of alveolar and embryonal histotype. SCF production, ranging from 30 to 162 pg/ml, was independent from the degree of myogenic differentiation and was not modulated by exogenous addition of retinoic acid (RA) or tumor necrosis factor‐α (TNF‐α). Four of 5 rhabdomyosarcoma cell lines expressed the mRNA for SCF receptor c‐kit, while the 5th cell line became weakly positive for c‐kit mRNA only after stimulation with retinoic acid. On the cell surface, c‐kit protein was detectable at very low levels in only 1 of 5 rhabdomyosarcoma cell lines and was not up‐regulated by RA or TNF‐α. Addition of anti‐c‐kit and anti‐SCF blocking antibodies, or of exogenous SCF did not alter the in vitro growth ability of rhabdomyosarcoma cells. In conclusion, our data show that rhabdomyosarcoma cells produce consistent amounts of SCF but did not demonstrate autocrine growth modulation. SCF secretion may thus have a paracrine, rather than an autocrine activity in this tumor. Int. J. Cancer 78:441–445, 1998.

Wild-type p53-mediated down-modulation of interleukin 15 and interleukin 15 receptors in human rhabdomyosarcoma cells

We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression.