Autophagy has been emerging as a novel cytoprotective mechanism to increase 
tumor cell survival under conditions of metabolic stress and hypoxia as well as 
to escape chemotherapy-induced cell death. To elucidate whether autophagy might 
also protect cancer cells from the growth inhibitory effects of targeted 
therapies, we evaluated the autophagic status of preclinical breast cancer 
models exhibiting auto-acquired resistance to the anti-HER2 monoclonal antibody 
trastuzumab (Tzb). We first examined the basal autophagic levels in Tzb-naive 
SKBR3 cells and in two pools of Tzb-conditioned SKBR3 cells (TzbR), which 
optimally grow in the presence of Tzb doses as high as 200 microg/ml Tzb. 
Fluorescence microscopic analyses revealed that the number of punctate LC3 
structures -a hallmark of autophagy- was drastically higher in Tzb-refractory 
cells than in Tzb-sensitive SKBR3 parental cells. Immunoblotting analyses 
confirmed that the lipidation product of the autophagic conversion of LC3 was 
accumulated to high levels in TzbR cells. High levels of the LC3 lipidated form 
in Tzb-refractory cells were accompanied by decreased p62/sequestosome-1 protein 
expression, a phenomenon characterizing the occurrence of increased autophagic 
flux. Moreover, increased autophagy was actively used to survive Tzb therapy as 
TzbR pools were exquisitely sensitive to chemical inhibitors of autophagosomal 
formation/function. Knockdown of LC3 expression via siRNA similarly resulted in 
reduced TzbR cell proliferation and supra-additively interacted with Tzb to 
re-sensitize TzbR cells. Sub-groups of Tzb-naive SKBR3 parental cells 
accumulated LC3 punctate structures and decreased p62 expression after treatment 
with high-dose Tzb, likely promoting their own resistance. This is the first 
report showing that HER2-overexpressing breast cancer cells chronically exposed 
to Tzb exhibit a bona fide up-regulation of the autophagic activity that 
efficiently works to protect breast cancer cells from the growth-inhibitory 
effects of Tzb. Therapeutic targeting autophagosome formation/function might 
represent a novel molecular avenue to reduce the emergence of Tzb resistance in 
HER2-dependent breast carcinomas.