Expression of epidermal growth-factor receptor correlates with metastatic potential of 13762nf rat mammary adenocarcinoma cells.

Increased expression of EGFR in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive correlation, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of receptor. This was demonstrated in Northern blot analysis, in immunoprecipitation studies using metabolically labeled whole cell lysates and in Western blot analysis of membrane fractions. Cross-linking of radiolabeled ligand to intact cells identified on both cell types specific binding to a 170 kd protein, however, at much lower levels on low-metastatic MTC cells and not in sufficient amounts to estimate receptor numbers by Scatchard analysis. In contrast, Scatchard plot analysis of I-125-EGF binding to MTLn3 cells revealed the expression of about 10,000 high and 46,000 low affinity sites. Both cell lines expressed the ligand in comparable amounts as was demonstrated by using a specific rat TGFalpha cDNA probe in Northern blot and an antibody recognising membrane bound TGF in FACS analysis. Adhesion of MTC cells to immobilized collagen or fibronectin was rapid reaching 50% after 30 min while control MTLn3 cells demonstrated lower adhesion to collagen. Addition of 10 ng/ml EGF increased the rate and the maximal adhesion of MTLn3 cells to collagen G, while the adhesion kinetics of MTC cells to collagen G or fibronectin were unaffected.

Rapid effects of EGF on cytoskeletal structures and adhesive properties of highly metastatic rat mammary adenocarcinoma cells.

In the highly metastatic rat mammary adenocarcinoma cell clone MTLn3, EGF induced increased adhesion to fibronectin while in the human epidermoid carcinoma cell line A431 EGF induced diminished adhesive properties. Flattening of cells with extensive formation of filopodia was observed in MTLn3 cells within 5 min of EGF addition, while in A431 cells EGF induced rounding up and only occasional formation of filopodia. Immunofluorescent analysis revealed extension of microtubules (MT) into the filopodia and Western blot analysis demonstrated an EGF-induced 2- to 3-fold increase in the amount of assembled tubulin in MTLn3 but not in A431 cells. In MTLn3, but only marginally in A431 cells, EGF treatment resulted in phosphorylation of a 280 kD cytoskeleton-associated protein, which was rapid and dose-dependent. These results suggest differential signal transduction pathway of cytoskeleton-associated EGFRs in highly metastatic MTLn3 as compared with A431 cells.

Ligand-induced activation of epidermal growth factor receptor in intact rat mammary adenocarcinoma cells without detectable receptor phosphorylation.

Expression and function of epidermal growth factor receptor (EGFR) was investigated in a metastatic cell clone (MTLn3) derived from the 13762NF rat mammary adenocarcinoma. No receptor phosphorylation could be identified in intact cells or in membrane preparations, while EGF-dependent phosphorylation of substrates occurred in intact cells. Indications for active suppression of receptor phosphorylation came from the fact that EGFRs bound in immunocomplexes or associated with the cytoskeleton of detergent treated cells were able to undergo basal and EGF-induced phosphorylation in vitro. Cross-linking experiments with 125I-EGF, as well as [35S]methionine labeling followed by immunoprecipitation with receptor specific antibodies readily detected in MTLn3 cells the expected 170-kDa EGFR protein. In addition, two proteins with molecular masses of 420-480 and 95 kDa specifically bound 125I-EGF on intact MTLn3 and sparse cultures of A431 cells. Phosphorylation of the 420-480 kDa molecule could be identified in immunocomplexes of EGFRs isolated from MTLn3 and sparse A431 cells, but the 95-kDa receptor molecule was never phosphorylated. While the presence of alternative forms of EGFR in the highly metastatic cell clone MTLn3 was unexpected, our observations of inefficient receptor autophosphorylation are in agreement with other recent reports and suggest that in MTLn3 cells EGFR-mediated signal transduction can be an event independent from receptor autophosphorylation.