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Introduction: Naturally occurring B–cell responses in the form of serum auto-antibodies to tumor-associated antigens are frequently observed in sera of patients with malignancies. Autoimmune responses in cancer patients may reflect molecular events that lead to tumorigenesis or disease progression. The purpose of this research was to study the role of auto-antibodies to promote human breast cancer cell growth and its potential mechanism by excessive activation of signal transduction pathways. Methods: Human antibody clones were screened from a human IgG Fab cDNA library that was constructed for a patient with breast cancer. The antibody binding to tumor antigen was tested by Western blot. The human breast cancer cell lines were treated with some of the Fab clones and the cell growth was measured by 3H-Thymidine uptake and MTT assay. Signal transduction pathways was evaluated by cell proliferation assay, in which three commonly used inhibitors including PD98059, Wortmannin, and PP1 were used. The potential signal pathway was further confirmed by Western blot. Results: Western blot performed on two selected antibodies showed that both clones’ product have stronger binding ability to the breast cancer antigen than the one from the normal breast tissues, indicating that the immune recognition of tested Fabs were breast cancer specific. The antigens recognized by the two tested clone products were of the similar size, a protein of molecular about 30-35 kDa, although the amino acid sequence of two clones varied by three residues in the CDR2 domain. The functional study showed that one IgG Fab (referred as Fab1) could stimulate both MCF-7 and MDA-MB-231 breast cancer cell lines growth in vitro. Pretreatment with either PD98059, a specific inhibitor of MEK1, or PP1, a specific inhibitor of Src, remarkably inhibited Fab1-induced breast cancer cell growth. In contrast, pretreatment with of wortmannin, a specific inhibitor of PI-3 kinase, did not show any inhibition effect to the Fab1-induced tumor growth. Western blot showed that Fab1 rapidly and transiently increased the phosphorylation of ERKs in both MCF-7 and MDA-MB-231 cells, with a maximal effect seen at 15 min and a return to baseline by 30 min. In agreement with the data from the cell proliferation assay, the induction of ERK phosphorylation by Fab1 was blocked by PD98059 and PP1, indicating that ERK phosphorylation and Src kinase activity are required for the clone 1 Fab-induced tumor growth. Conclusion: Our studies indicated the possible role of human antibody to promote tumor cell growth by directly binding of IgG Fab to breast tumor antigen and the involvement of ERK signal transduction in this Fab-induced tumor cell growth. These findings provide for further speculation as to the role of auto-antibodies in activating signaling pathways that regulate tumor growth as a basis for disease progression.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]