Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: Local stimulation using magnetic microspheres as assessed by quantitative digital microscopy

Abstract

Background: ErbB2 (HER-2), a member of the epidermal growth factor (EGF) receptor family, is a class I transmembrane receptor tyrosine kinase. Although erbB2 has no known physiologic ligand, it can form complexes with other members of the family and undergo transactivation of its very potent kinase activity, thereby initiating downstream signaling and cell proliferation. ErbB2 is a frequent pathologic marker in ductal invasive breast carcinomas and is targeted by using a specific humanized monoclonal antibody, trastuzumab (Herceptin). The antibody is effective in only 20% to 50% of erbB2-positive tumors, and this resistance, as yet poorly understood, constitutes a major therapeutic challenge. Methods: Magnetic microspheres coated with ligands or antibodies are widely used for separation of proteins and cells and allow localized, high intensity, and precisely timed stimulation of cells. We used EGF-and trastuzumabcovered paramagnetic microspheres, quantitative confocal laser scanning microscopy, and digital image processing to investigate the (trans)activation of and local signal propagation from erbB1 and erbB2 on trastuzumab sensitive and resistant carcinoma cell lines expressing these receptors at high levels. Results: On A431 cells expressing high levels of endogenous erbB1 and transfected erbB2-mYFP (A4-erbB2-mYFP F4 cell line), EGF-coupled-microspheres activated

Trastuzumab causes antibody-dependent cellular cytotoxicity–mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance

“…Many groups, including ours, have characterized breast cancer cell lines and clinical tumor specimens for intrinsic cellular features such as ErbB2 protein turnover, autocrine production of EGF-related ligands (19), activation of the IGF-I receptor pathway (20), loss of PTEN function (23), steric hindrance of ErbB2 on the cell surface (22,41). Although these intrinsic mechanisms may partly underlie nonresponsiveness to trastuzumab, our results clearly stress the importance of immune-mediated mechanisms, the capacity of which may vary depending on tumor volume, either as a function of gross cell numbers, or of accessibility of tumor cells by effector cells.…”

Hsp90 inhibitor 17-AAG reduces ErbB2 levels and inhibits proliferation of the trastuzumab resistant breast tumor cell line JIMT-1

“…The molecular mechanisms of resistance to trastuzumab, similarly to its mode of action, are largely unknown and possibly include various factors. Autocrine production of EGF-like ligands [22] or overexpression of insulin-like growth factor 1 receptor (IGF1R) [23], leading to an ErbB2-independent means for the constitutive activation of the PI3K pathway, as well as blocking of trastuzumab binding by MUC4, a cell surface mucin [24,25], have been implicated in trastuzumab resistance.…”

ErbB-directed immunotherapy: Antibodies in current practice and promising new agents

“…MUC4 expression pattern in JIMT-1 cells was shown to inversely correlate with the trastuzumab binding pattern, and down-regulation of MUC4 by RNA interference resulted in increased sensitivity to trastuzumab [141]. Also, trastuzumab at high local densities bound to paramagnetic microbeads proved to be efficient in activating ErbB2 in JIMT-1 cells [23]. These data suggest that MUC4 masks the trastuzumab binding site on ErbB2 resulting in steric hindrance and trastuzumab resistance.…”

“…This property suggests that ErbB2 alone can cause transformation if overexpressed; however, biophysical studies failed to detect significant ErbB2 homodimerization in solution [20]. On the other hand, in the cellular environment where ErbB2 may be regulated in manifold intricate ways, ErbB2 homodimerization [21,22] and autophosphorylation [23] is often detected in tumor cells overexpressing this protein.…”

“…It was previously suggested that trastuzumab resistance is caused by masking of ErbB2 by MUC4 [23,140,141] or hyaluronan [145]. These molecules may not only block the binding of trastuzumab, but could also prevent the recruitment of Fc receptor bearing immune effector cells.…”