Eva M. Valverius

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-rasH, v-rasKi, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-rasH + SV40T and v -rasH + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.

Modulation by Estrogen and Growth Factors of Transforming Growth Factor-Alpha and Epidermal Growth Factor Receptor Expression in Normal and Malignant Human Mammary Epithelial Cells

A number of well-characterized growth factors, such as epidermal growth factor (EGF) and the insulin-like growth factors (IGFs), can stimulate the proliferation and modify the differentiation of normal and malignant rodent and human mammary epithelial cells (Salomon et al. 1986 a, c). For example, EGF or transforming growth factor-alpha (TGFa) can stimulate the lobulo-alveolar development of mouse mammary glands in organ culture or in vivo (Tonelli and Soroff 1980; Okamoto and Oka 1984; Vonderhaar 1987). In addition, EGF may perform a physiological role in the development of the mouse mammary gland during pregnancy and lactation and in the spontaneous formation of mammary tumors in mice (Okamoto and Oka 1984; Oka et al. 1987; Tsutsumi et al. 1987). Several of these growth factors, such as IGF-I, platelet-derived growth factor (PDGF), TGFa, and TGFs, have been found in the conditioned medium (CM) from several human breast cancer cell lines (Salomon et al. 1984; Dickson et al. 1986; Huff et al. 1986; Bronzert et al. 1987; Knabbe et al. 1987; Peres et al. 1987). The production of TGFa, IGF-I, and TGF/3 can be modulated by estrogens or anti-estrogens, suggesting that the growth-promoting effects of estrogen may be mediated in part by TGFa and/or IGF-I and that the growth-inhibitory effects of anti-estrogen may be mediated by TGF/3 (Dickson et al. 1985; Perroteau et al. 1986; Knabbe et al. 1987; Liu et al. 1987; Peres et al. 1987; Huff et al. 1988). However, it is still unclear whether these or other growth factors are present in mammary tumor cells in vivo and, if so, whether they are involved in the etiology of tumor development and progression. Likewise, there is little information on the distribution and level of expression of similar growth factors in normal mammary epithelial cells at specific times during the development of the mammary gland.

Modulation of EGF receptor expression by differentiating agents in human colon carcinoma cell lines.

The existence of an autocrine loop for self-stimulation of growth in malignant cells has been proposed for transforming growth factor-alpha (TGF alpha) and its receptor, the epidermal growth factor (EGF) receptor, in a variety of malignant cell types. Expression of both has been described in colon carcinoma. In order to investigate whether there is a correlation between TGF alpha and EGF receptor mRNA expression and differentiation, we studied the effects of differentiating agents on seven human colon carcinoma cell lines. All of the lines responded to the differentiating agents. In four of the seven lines there was increased EGF receptor mRNA two to five days after treatment with 2 mM sodium butyrate. In three of these lines TGF alpha mRNA and protein were also increased. In the one cell line treated with the differentiating agents DMF and DMSO, EGF receptor mRNA was also increased. [125I]-EGF binding to the cells was measured before and after treatment with butyrate. In two of three cell lines, increased EGF receptor mRNA was accompanied by a 2.4-fold increase in the number of binding sites per cell. In SW620 cells, no EGF receptor binding was detected before or after butyrate treatment. In the two cell lines in which butyrate increased EGF receptor binding, simultaneous treatment with EGF did not enhance growth. These data demonstrate increased expression of the TGF alpha/EGF receptor system after differentiation of colon carcinoma cell lines and suggest that their expression may be characteristic of a differentiated phenotype.

Transforming growth factors in human breast cancer.

Transforming growth factors alpha and beta (TGF alpha and TGF beta) are two growth factors which are frequently associated with a number of human breast cancer cell lines and with primary human breast carcinomas. Expression of TGF alpha protein and specific TGF alpha mRNA transcripts (4.8 and 1.6 kb) can be induced by estrogens in estrogen-responsive breast cancer cells, suggesting that the mitogenic effects of estrogen may in part be mediated through this potential autocrine growth factor. In contrast, anti-estrogens such as tamoxifen can increase the secreted levels of TGF beta, which is a potent growth-inhibitor for some human breast cancer cell lines. Anti-estrogens generally decrease TGF alpha production. TGF alpha mRNA expression has been detected in approximately 40-70% of primary human breast tumors, while expression of a 2.6 kb TGF beta mRNA transcript can be detected in 70-80% of breast tumors. Interference with (e.g. TGF alpha) or augmentation of (e.g. TGF beta) the effects of these two growth factors may have some potential clinical applications in the treatment of breast cancer.

The role of ras gene expression and transforming growth factor α production in the etiology and progression of rodent and human breast cancer

Cancer represents a spectrum of different diseases that generally arise through a series of multiple yet discrete steps that include initiation, promotion, transformation, progression, and metastasis [1]. Genetic alterations to specific host cellular genes or sets of genes are associated with some or all of these stages [1–3]. Damage to DNA can result in somatic changes due to the action of chemical carcinogens, mutagens, viruses, or radiation. A large body of evidence suggests that certain endogenous regulatory genes are likely targets for insult from exogenous environmental agents. These cellular regulatory genes or proto-oncogenes have been implicated in the control of cellular proliferation and/or differentation [3–7]. Activation of these genes through processes such as point mutation, amplification, rearrangement, insertional mutagenesis, chromosomal translocation, or overexpression can lead to neoplastic transformation in vitro and tumorigenicity in vivo [3,5,7]. Approximately 35–40 different proto-oncogenes have been identified to date and can be stratified in some cases into families based on their structure and function [3, 7]. These gene families were originally detected as the dominantly transforming genes that were associated with the genome of different acutely transforming retroviruses and that were transduced or captured as cellular proto-oncogenes [3,4,7]. DNA transfection assays have also demonstrated the presence of other additional groups of activated cellular proto-oncogenes that are associated with the DNA obtained from a number of rodent tumors and from a small fraction of primary human tumors and that are capable of transforming immortalized rodent fibroblasts, such as NIH-3T3 cells, in vitro [8–15]. In addition to this group of genes, there exists a second major group of genes, the tumor suppressor or antioncogenes, which are genes that give rise to tumors after allelic deletion or inactivation [16–18]. These recessive genes, such as the Rb—1 (retinoblastoma) gene, can code for nuclear phosphoproteins that can negatively regulate cell growth and that have the potential to interact with and to be subsequently inactivated by other specific nuclear oncogene proteins, such as the SV40T antigen protein and the Adenovirus E1A protein [19–22].