Her2 (erbB2/neu) is overexpressed in 25-30% of human breast cancers. Herceptin 
is a recombinant humanized Her2 antibody used to treat breast cancer patients 
with Her2 overexpression. Over a 5-month selection process, we isolated clones 
of BT474 (BT) human breast carcinoma cells (BT/Her(R)) that were resistant to 
Herceptin in vitro. In BT/Her(R) subclones, cell-surface, phosphorylated and 
total cellular Her2 protein remained high in the continuous presence of 
Herceptin. Likewise, the levels of cell-surface, phosphorylated, and total 
cellular Her3 and EGFR were either unchanged or only slightly elevated in 
BT/Her(R) subclones relative to BT cells. One BT/Her(R) subclone had 
substantially upregulated cell-surface EGFR, but this did not correlate with a 
higher relative resistance to Herceptin. In looking at the downstream PI-3K/Akt 
signaling pathway, phosphorylated and total Akt levels and Akt kinase activities 
were all sustained in BT/Her(R) subclones in the presence of Herceptin, but 
significantly downregulated in BT cells exposed to Herceptin. Whereas BT cells 
lost sensitivity to the PI-3K inhibitor LY294002 in the presence of Herceptin, 
BT/Her(R) subclones were equally sensitive to this agent in the presence and 
absence of Herceptin. This suggests that BT/Her(R) subclones acquired a 
Herceptin-resistant mechanism of PI-3K signaling. BT/Her(R) subclones were also 
sensitive to the EGFR kinase inhibitor AG1478 in the presence of Herceptin, to 
the same extent as BT cells. The BT/Her(R) subclones provide new insights into 
mechanisms of Herceptin resistance and suggest new treatment strategies in 
combination with other inhibitors targeted to signal transduction pathways.