Effect of acquired resistance to lapatinib in HER2-positive breast cancer cells on the Bcl-2 family members MCL-1 and BAX.

Abstract

633 Background: Lapatinib (L) is approved for the treatment of trastuzumab (T) resistant HER2 positive metastatic breast cancer. Not all HER2 positive tumors respond to L and patients who initially respond frequently relapse, due to the development of resistance to L. Understanding the molecular changes associated with L resistance may lead to the identification of targets to overcome resistance. Methods: SKBR3 cells were treated with either 250 nM L, or 125nM L and 5 µg/ml T twice weekly for 6 months to establish the resistant cell lines SKBR3-L and -TL. We measured by TUNEL assay the effect of L on apoptotic induction in SKBR3, -L and -TL cells. Gene array analysis of the SKBR3, -L and -TL cells identified differences in the expression of apoptosis-related genes, which were validated by immunoblotting. Finally using combinations of obatoclax (O) and L were tested in L resistant cells. Results: In SKBR3 cells, L (500 nM) induces apoptosis (15.8 ± 2.0%) compared to untreated controls (4.6±2.7%), whilst in SKBR3-L and -TL cells L did not induce significant apoptosis compared to controls. Gene array analysis showed that BAX mRNA is down regulated 3.2 fold in SKBR3-L cells compared to the parental cells. In SKBR3-TL cells, MCL-1 mRNA expression is increased 2.1 fold, whilst BAX mRNA is down regulated 2.1 fold compared to the parental cells. Immunoblotting confirmed that BAX protein expression was reduced in the SKBR3-L (1.5 fold, p=0.058) and significantly reduced in SKBR3-TL cells (2.0 fold, p=0.039) compared to SKBR3. MCL-1 protein expression was significantly increased in the SKBR3-L (1.6 fold, p=0.035) and -TL cells (2.3 fold, p=0.031) compared to SKBR3. Combining O (200 nM) and L (500 nM) in both SKBR3 and SKBR3-L cells produced greater growth inhibition than either drug on its own (SKBR3: 86.9±1.0% vs 73.5±3.1% for L (p<0.01) and 44.7±7.7% for O, (p<0.01); SKBR3-L: 54.2± 8.6% vs 26.9±2.4% for L (p=0.027) and 33.8±10.7% for O (p=0.04)). Conclusions: Extended exposure to L in SKBR3-L and –TL cells alters the apoptotic response to L. O alone and in combination with L results in growth inhibition in L resistant cells.