Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas.

A metastatic cancer develops by accumulation of mutations in genes that control growth, survival and spreading. The latter genes have not yet been identified. In lymph node metastases of head and neck squamous cell carcinomas (HNSCC), we found mutations in the MET oncogene, which encodes the tyrosine kinase receptor for Scatter Factor, a cytokine that stimulates epithelial cell motility and invasiveness during embryogenesis and tissue remodeling. We identified two somatic mutations: the Y1230C, known as a MET germline mutation which predisposes to hereditary renal cell carcinoma, and the Y1235D that is novel and changes a critical tyrosine, known to regulate MET kinase activity. The mutated MET receptors are constitutively active and confer an invasive phenotype to transfected cells. Interestingly, cells carrying the MET mutations are selected during metastatic spread: transcripts of the mutant alleles are highly represented in metastases, but barely detectable in primary tumors. These data indicate that cells expressing mutant MET undergo clonal expansion during HNSCC progression and suggest that MET might be one of the long sought oncogenes controlling progression of primary cancers to metastasis.

Overexpression of the RON gene in human breast carcinoma.

Constitutive activation of the RON gene, known to code for the tyrosine-kinase receptor for Macrophage Stimulating Protein (also known as Scatter Factor 2), has been shown to induce invasive-metastatic phenotype in vitro. As yet, nothing is known about the expression of this novel member of the MET-oncogene family in spontaneously occurring human cancers. Here we report that Ron is expressed at abnormally high levels in about 50% primary breast carcinomas (35/74 patients). Among these, the expression is increased more than 20-fold in 12 cases and the overexpressed protein is constitutively phosphorylated on tyrosine residues. Notably, Ron is only barely detectable in epithelial cells of the mammary gland, and its expression remains unchanged in benign breast lesions (including adenomas and papillomas). Overexpression was observed in different histotypic variants of carcinomas; it is associated with the disease at any stage and correlates with the post-menopausal status. In breast carcinoma cells grown in vitro, activation of the Ron receptor resulted in proliferation, migration and invasion through reconstituted basement membranes. Altogether, these data suggest a role for the RON gene in progression of human breast carcinomas to the invasive-metastatic phenotype.

Tamoxifen up-regulates c-erbB-2 expression in oestrogen-responsive breast cancer cells in vitro.

Expression of the c-erbB-2 proto-oncogene is inhibited by oestrogens in oestrogen-responsive human breast cancer cells, at both mRNA and protein level. Here we report that, where the regulation of c-erbB-2 is concerned, tamoxifen displays a full anti-oestrogenic activity, enhancing the expression of c-erbB-2 in oestrogen receptor-positive cells cultured with untreated fetal calf serum or reversing the inhibitory effect of added oestrogens. Meanwhile, tamoxifen strongly inhibited cell growth. Tamoxifen was inactive on both c-erbB-2 expression and growth of oestrogen receptor-negative cells. These results may have important implications to explain occasional failure of tamoxifen therapy in oestrogen receptor-positive breast cancers.

Hormonal regulation of c-erbB-2 oncogene expression in breast cancer cells.

Expression of the c-erbB-2 (neu, HER-2) oncogene is found to be subjected to hormonal and developmental regulation in normal as well as neoplastic mammary cells. We have previously reported that estrogens inhibit c-erbB-2 expression at both the mRNA and protein level in estrogen receptor (ER)-positive, but not in ER-negative, breast cancer cell lines. Reversion of c-erbB-2 inhibition is seen with tamoxifen. The effect on c-erbB-2 expression of several other hormones and factors, which influence mammary cell growth and differentiation, has been studied. Our observations indicate that, in normal and neoplastic mammary cells, c-erbB-2 expression is inversely related to cell proliferation. While estrogens, anti-estrogens and cAMP clearly regulate c-erbB-2 mRNA levels, epidermal growth factor dramatically decreases the c-erbB-2 protein without affecting the level of c-erbB-2 mRNA. Therefore, different signals converging in terms of cell proliferation regulate c-erbB-2 expression by different molecular mechanisms.

Hormonal control of growth factor receptor expression.

In this report, we have discussed a series of results obtained in our laboratory that, together with data by other authors, demonstrate that the expression of the erbB-2 tyrosine kinase receptor oncogene in breast cancer cells is regulated by multiple factors and hormones, which modulate their growth and differentiation. In particular, we have shown that estrogens specifically inhibit erbB-2 expression by transcriptional repression, which is exerted through a sequence within the erbB-2 gene promoter. Estrogens control mammary cell growth directly, by inducing early gene expression, and indirectly, by increasing autocrine growth factor production or decreasing growth inhibitors. The data presented here suggest that mammary cells respond to estrogen also by modifying the receptor array on their surface, thus setting their own sensitivity to the different autocrine and paracrine factors. As a first consequence, the modulation of erbB-2 expression level by antiestrogen may represent a point to consider when selecting breast cancer patients for hormonal therapy, in those (few) cases where estrogen receptor positivity accompanies erbB-2 amplification. On the other hand, antiestrogen-induced upregulation of erbB-2 may improve tumor targeting of drugs designed to interact or interfere with erbB-2, such as humanized antibodies, immunotoxins, or engineered ligands. These possibilities should be tested in appropriate model systems in the future.

[Evaluation of Ron and Met proto-oncogene expression in epithelial ovarian tumors]. / Valutazione dell'espressione dei proto-oncogeni Ron e Met nei tumori epiteliali ovarici.

BACKGROUND: Epithelial ovarian cancer is the most lethal gynecologic neoplasia. Up to date, little is known about its biology, and this makes even more difficult the definition of new therapies and the finding of early diagnostic methods. In this study, the expression of two oncogenes, Ron and Met, whose role in cancer progression has already been shown, and the possible clinical implication of their presence in the neoplastic tissue have been evaluated. METHODS: Forty-eight ovarian cancer specimens, 5 borderline lesions, 4 benign ovarian tumors and 2 normal ovaries were analyzed; from frozen tissue, Rna was extracted and cDna obtained by a RT-PCR (Retrotranscriptase-Polymerase Chain Reaction). Finally, the cDna was assayed for the presence of the Ron and the Met gene by another PCR. The results were correlated with clinicopathological parameters, and patient survival. RESULTS: Ron expression was shown in 56% of malignant lesions, and in 60% of borderline ones, while Met expression was detected in 54 and 60%, respectively. No statistically significant correlation was found between Ron and Met expression and clinicopathological features, such as histotype, grading, staging, residual tumor after debulking surgery, and response to chemotherapy, while a strong correlation (p = 0.001) was observed between overexpression of one of the oncogenes and the concomitant expression of the other. CONCLUSIONS: Even if residual tumor after debulking surgery was the most relevant prognostic factor, this study showed new data about the concomitant expression of Ron and Met oncogenes, which may suggest their cooperative role in ovarian cancer progression.

erbB-2 expression in estrogen-receptor-positive breast-tumor cells is regulated by growth-modulatory reagents.

It has previously been shown that, in the estrogen-receptor-positive breast-tumor cell lines T47D and ZR75.1, the erbB-2 protein and mRNA content are controlled negatively and positively by, respectively, estrogens and anti-estrogens. Since estrogens have a positive effect on cell proliferation, while anti-estrogens inhibit cell growth, the results suggested that there may be an inverse correlation between growth and erbB-2 expression. We have now examined this matter further. The effect of various growth-modulatory agents including estrogen (E2), progesterone (Pg), retinoic acid (RA), epidermal growth factor (EGF), insulin (Ins), prolactin (Prl), 12-O-tetradecanolyl-phorbol-13-acetate (TPA) and dibutyryl-3':5'-cyclic-AMP (cAMP) on c-erbB-2 promoter activity, RNA and protein expression have been examined. The growth stimulators E2 and EGF both reduced the level of erbB-2 protein. However, while E2 clearly repressed erbB-2 transcription, in the case of EGF, neither mRNA nor transcription were decreased. Of the agents which inhibit the growth of T47D and ZR75.1 cells--Pg, Prl, cAMP, RA and TPA--only Pg and cAMP caused an increase in the erbB-2 protein level. Pg and cAMP positively influenced c-erbB-2 promoter activity and RNA amount. TPA and RA also increased promoter activity but neither erbB-2 mRNA nor protein level was enhanced. The erbB-2 protein expression in cultures of T47D and ZR75.1 cells at different densities was also analyzed. Both the level of erbB-2 protein and c-erbB-2 promoter activity rose markedly in confluent cultures, suggesting a transcriptional mechanism of control. In conclusion, the data suggest that the effects of various agents on erbB-2 expression are complex and cannot be explained simply as reflecting the growth state of the cells.

Expression of the erbB-2 proto-oncogene during differentiation of the mammary gland in the rat.

The erbB-2 proto-oncogene encodes a transmembrane protein (p185) that is a tyrosine kinase sharing high homology with the epidermal growth factor receptor. Its expression in mammary cell lines is modulated by estrogens, epidermal growth factor, and several other factors at the RNA and/or protein levels. We have used in situ hybridization, immunoblot, and immunohistochemistry to study the expression of erbB-2 in the rat mammary gland at various stages of differentiation. erbB-2 RNA is present at low levels in mammary glands from virgin, mid-pregnant, and lactating female rats. Increased RNA levels can be detected in early and late pregnancy. In all samples, erbB-2 RNA has been found in all epithelial cells. Immunohistochemistry with antisera directed against the intracellular domain of p185 has shown that only a minority of cells are stained in virgin and early pregnant samples, whereas no staining is seen in late pregnant and lactating mammary glands. In contrast, immunoblot analysis has detected the highest levels of p185 in late pregnancy and during lactation. This may reflect either that the cellular content of p185 is too low to be detected by immunohistochemistry, or that the epitopes are not accessible to the antisera in situ. Taken together, our data indicate that erbB-2 is expressed by mammary epithelial cells at all physiological stages and suggest that erbB-2 expression is modulated at both the RNA and protein level in vivo.

Oestrogen and epidermal growth factor down-regulate erbB-2 oncogene protein expression in breast cancer cells by different mechanisms.

Mitogen-induced mammary cell growth is often accompanied by decreased levels of expression of the p185erbB-2 protein. We have previously reported that oestrogen inhibits erbB-2 mRNA and protein expression in breast cancer cells, while epidermal growth factor (EGF) treatment has been shown to decrease p185erbB-2 levels in normal mouse mammary epithelial cells. In the present work, we studied the effect of oestrogen and EGF on erbB-2 expression in oestrogen-responsive breast cancer cells. We observed that both oestrogen and EGF comparably down-regulated p185erbB-2 levels, while stimulating growth of T47D and ZR75.1 cells. Oestrogens, but not EGF, concomitantly down-regulated erbB-2 mRNA. Run-on analysis showed a reduced erbB-2 transcription rate in the presence of oestrogens. Furthermore, the transcriptional activity of a 219 bp proximal fragment of the human erbB-2 promoter was repressed by oestrogens, whereas it was enhanced by EGF. EGF stimulated both tyrosine phosphorylation and autokinase activity of p185erbB-2 down-regulates p185erbB-2 at a post-translational level. Thus, two factors converging in terms of effects on cell growth, display divergent mechanisms of regulation of erbB-2 expression.