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e13021Background: We have previously shown that protein phosphatase 2A (PP2A) may mediate acquired lapatinib resistance and inhibition of PP2A with okadaic acid enhances lapatinib response in vitro...

e12092Background: We have previously reported that lapatinib (L) induces reversible senescence in HCC1419 HER2 positive breast cancer cells (McDermott et al, ASCO 2011). In this study we examined i...

632 Background: HER-2, a member of the transmembrane receptor tyrosine kinase ErbB family, is over-expressed in approximately 25% of BC. HER-2 targeted therapies, in particular, T, a monoclonal antibody targeting HER-2, and lapatinib (L), a reversible HER-2 tyrosine kinase inhibitor, have been shown to significantly improve the prognosis for HER-2 positive BC patients. However, resistance to T and/or L is a significant clinical problem. The aim of this study is to assess the activity of N (HKI-272), an irreversible HER-2 tyrosine kinase inhibitor, in HER-2 overexpressing BC cell lines, including T and/or L resistant cells. Methods: Using proliferation assays, the effect of N was assessed alone and in combination with T in HER-2 positive BC cell lines, including T and/or L resistant cell lines. The effect of N on HER-2 and downstream signalling molecules, Erk and Akt, was determined by immunoblotting. Results: HER-2 positive BC cell lines, including T and/or L resistant cells, are sensitive to N alone with IC50 values (concentration which inhibits 50% of growth) ranging from 1 to 280 nM. The combination of N and T has additive effects in SkBR3 and BT474 which are sensitive to T and also in SKBR3-Lwhich are resistant to L. In the cell lines HCC1954, HCC1954-L, MDA-MB-453, JIMT1 and SKBR3-HL which are resistant to T, combined treatment with T and N showed no enhancement compared to N alone. Finally, N decreased phosphorylation of HER-2, Erk and Akt in all cell lines tested. Conclusions: Our results suggest that N should be studied in patients with HER-2 positive BC, including patients with T and/or L resistant BC. We also demonstrate that N in combination with T may be more effective than either agent alone in T sensitive cells.

617 Background: Not all HER2-positive breast cancer patients respond to HER2-targeted drugs and some, who initially respond, subsequently relapse. There is a need to identify biomarkers for improved patient selection. Here we aimed to identify gene expression changes associated with response to HER2-targeted drugs. Methods: To identify mRNA changes associated with resistance, whole genome microarrays were used to profile conditioned medium (CM) from HER2-positive cell lines (SKBR3, HCC1954) and their lapatinib (L)-resistant variants (SKBR3-LR, HCC1954-LR). Neuromedin U (NmU) over-expression, identified in this way, was confirmed in the L-resistant cells and corresponding CM by qPCR and ELISA. Pooled data from 21 published expression datasets (n=3489 patients) was mined to relate NmU mRNA to tumour subtype and patients' outcome. NmU cDNA and siRNAs were used to over-express and knock-down expression of NmU, respectively, prior to assessing it’s association with response to L, Trastuzumab (T) and Neratinib (N). Results: Analysis of the tumour data showed NmU expression to be particularly associated with poor outcome for patients with HER2-positive tumours (p=0.000005). In cell lines, we identified NmU mRNA and protein to be at significantly higher levels in L-resistant cells and corresponding CM, compared to that of L-sensitive SKBR3 and HCC1954. This trend was observed for T-resistant and N-resistant SKBR3 cells (SKBR3T, SKBR3N) and their CM, compared to sensitive parent SKBR3 cells and CM. NmU cDNA over-expression in SKBR3 and HCC1954 increased resistance to L, T and N. Knock-down of NmU endogenous levels in SKBR3-LR and innately L-resistant MDA-MB-361 and T47D sensitised these cells to L, T and N. Further analysis of our NmU over-expressing and knock-down cells indicated NmU to also be associated with increased motility, invasion and anoikis resistance. Conclusions: NmU expression is prognostic of poor outcome in HER2-positive breast tumours. In cell line models, NmU over-expression is associated with resistance to L, T and N. Taken together, we propose NmU as a possible prognostic and predictive biomarker for HER2-positive cancers, with potential also as a co-target to help circumvent resistance to these drugs. [SFI: 08/SRC/B1410]

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

583 Background: Resistance to HER2 targeted therapies is a significant clinical problem. To study acquired resistance to the dual EGFR/HER2 tyrosine kinase inhibitor lapatinib (L) in breast cancer,...