Amphiregulin as an autocrine growth factor for c-Ha-ras- and c-erbB-2-transformed human mammary epithelial cells.

Abstract

Amphiregulin (AR), a member of the epidermal growth factor (EGF) family, was found to be as potent as EGF in stimulating the anchorage-dependent growth (ADG) of immortalized, nontransformed human mammary epithelial MCF-10A cells. MCF-10A cells transformed by either an activated human c-Ha-ras protooncogene (MCF-10A ras) or by overexpression of a nonactivated rat c-neu gene (MCF-10A neu) exhibited a 35% reduction in the response to AR in ADG when compared to MCF-10A cells, but AR was still as potent as EGF in these transformants. Exogenous AR exhibited only 15-20% of the activity of EGF in stimulating the anchorage-independent growth, a response that is normally dependent upon exogenous EGF, of the oncogene-transformed MCF-10A cells. MCF-10A cells express low levels of a 1.4-kb AR mRNA transcript, while MCF-10A ras and MCF-10A neu cells display a 15- to 30-fold increase in the levels of AR mRNA and endogenous AR protein as determined by Western blot analysis. Exogenous EGF was found to induced both the AR mRNA and protein in the MCF-10A parental and transformed cells. A 20-mer phosphorothioate antisense deoxyoligonucleotide complementary to the 5' sequence of AR mRNA was able to significantly reduce the levels of endogenous AR protein and to inhibit the EGF-stimulated ADG and anchorage-independent growth of MCF-10A ras and MCF-10A neu cells. These data suggest that AR may function as an EGF-dependent autocrine growth factor in mammary epithelial cells that have been transformed by either a point-mutated c-Ha-ras or c-neu.

The induction of apoptosis in human mammary luminal epithelial cells by expression of activated c-neu and its abrogation by glucocorticoids

“…However, neither the N4.1 cell line nor a variant of the HB4a cell line transformed with point mutated c-Ha-ras (R4.2) (data not shown) produced tumours in nude mice. This failure to acquire a fully malignant phenotype in vivo has also been observed in studies with these oncogenes using the spontaneously immortalised, non-transformed human mammary epithelial cell line, MCF-1OA (Ciardellio et al, 1992;Normanno et al, 1994). This is in contrast to with the findings of D'souza et al (1993), and shows that the role of c-erbB-2 overexpression (or mutationally activated neu-T) in the acquisition of tumorigenicity in human mammary epithelial cells is as yet unresolved.…”

Evidence for the transforming activity of a truncated Int6 gene, in vitro

“…Colony numbers (at least ®ve separate samples), 50.1 mm, are plotted versus cell type. Doubly-transformed DBL/NIH3T3 cells and Harvey Ras-transformed MCF10A cells (MCF10AneoT) were provided by Dr David Salomon, LTIB, NCI as a positive control for anchorage independent growth in soft agar (Ciardiello et al, 1990a(Ciardiello et al, ,b 1992Normanno et al, 1994) Figure 6 PCR Analysis of in vivo growths. Genomic DNA was extracted from para n embedded tissue and analysed for human Alu sequences.…”

“…Afterwards, these cell lines were maintained in 350 mg/ml Neomycin sulphate. Doubly-transformed DBL/NIH3T3 cells and Harvey Ras-transformed MCF10A cells (MCF10AneoT) were provided by Dr David Salomon, LTIB, NCI as a positive control for anchorage independent growth in soft agar (Ciardiello et al, 1990a(Ciardiello et al, ,b, 1992Normanno et al, 1994) These transformed cell lines were cultured in conditions identical to that of their originating cell lines, NIH3T3 and MCF10A respectively.…”

“…Cells were seeded into soft agar and allowed to grow for 6 weeks. Harvey Ras-transformed MCF10A cells (MCF10AneoT), which have been shown to form colonies in soft agar (a kind gift from Dr David Salomon, LTIB, NCI, USA), were used as a positive control for growth in soft agar (Ciardiello et al, 1990a(Ciardiello et al, 1992Normanno et al, 1994). Int6sh transfected MCF10A cells produced a larger number of colonies compared with MCF10A cells and were found to be equivalent in colony-forming ability with the Harvey Ras-transformed MCF10A cells (MCF10AneoT) (see Figure 5).…”

Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells

“…These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system. Whereas the expression of AR in a variety of both nontransformed and tumoral cells appears to be stimulated in the presence of EGF (Normanno et al, 1994b;Sehgal et al, 1994), the potential regulation of AR by growth factors in normal breast epithelial cells has never been examined. Because previous studies suggest that dysregulated expression of AR may be a component of mammary tumorigenesis we proposed to analyse and to compare the regulation of amphiregulin gene expression and protein secretion by EGF in normal and tumoral breast epithelial cells.…”