Abstract A6: Sustained MAPK signaling contributes to reduced sensitivity to lapatinib

Abstract Background: Approximately 20% to 30% of metastatic breast cancers overexpress the HER2 receptor tyrosine kinase. The standard front-line therapy for HER2-overexpressing breast cancer is the HER2 monoclonal antibody trastuzumab. However, for patients whose disease progresses on trastuzumab-based therapy, the dual EGFR/HER2 small-molecule tyrosine kinase inhibitor lapatinib is available. Unfortunately, the majority of metastatic breast cancer patients who do not respond to trastuzumab also show poor response to lapatinib. The mechanisms underlying lapatinib resistance are poorly understood. Elucidating these mechanisms may ultimately allow new therapeutic strategies to be developed for patients with HER2-overexpressing breast cancer. We have found that breast cancer cells with reduced sensitivity to lapatinib exhibit sustained mTORc1 and MAPK signaling. We previously showed that genetic knockdown or pharmacologic inhibition of PI3K/mTORc1 signaling improves response to lapatinib. In our current work, we examined the role of MAPK signaling in lapatinib sensitivity. Materials and Methods: Lapatinib-sensitive BT474 and HCC1419 cells, and lapatinib-resistant MDA361 cells were purchased from American Type Culture Collection, Manassas, VA. Lapatinib-resistant JIMT1 cells were purchased from DSMZ, Germany. MAPK signaling was blocked pharmacologically and genetically, and lapatinib response was then evaluated using trypan blue exclusion and anchorage-independent growth assays. Combined inhibition of MEK and lapatinib was analyzed statistically by CalcuSyn software to generate drug combination indices. Western blotting was performed to assess the effects of combination lapatinib and MEK inhibitor on HER2 signaling. Results and Discussion: MDA361 and JIMT1 cells, which have reduced sensitivity to lapatinib, exhibited sustained MAPK signaling after lapatinib treatment relative to lapatinib-sensitive BT474 and HCC1419 cells. Pharmacologic MEK inhibition increased sensitivity to lapatinib in MDA361 and JIMT1 cells, as measured by trypan blue cell viability and anchorage-independent three-dimensional assays. The combination of MEK inhibitor plus lapatinib showed pharmacologic synergy in JIMT1 and MDA361 cells, as indicated by combination indices below 1.0 using CalcuSyn software. At the molecular level, combined inhibition of MEK plus lapatinib resulted in greater reduction in phosphorylated MAPK versus either agent alone. In addition, our initial experiments suggested that combination MEK inhibitor plus lapatinib results in induction of Foxo1a and Foxo3a and reduced expression of FoxM1. Ongoing work will confirm the regulation of these forkhead transcription factors in lapatinib-resistant cells treated with MEK inhibitor plus lapatinib using Western blots, PCR, and immunofluorescence to examine nuclear translocation. Genetic knockdown of MEK will also be tested for the ability to increase lapatinib-mediated cell cycle arrest or apoptosis in JIMT1 and MDA361 cells. Finally, future xenograft studies will determine if pharmacologic inhibition of MEK increases lapatinib sensitivity in HER2-overexpressing breast cancers that are resistant to trastuzumab and lapatinib. Conclusions: In addition to our previous data showing that sustained mTORc1 signaling contributes to lapatinib resistance, we now show that resistant cells also exhibit sustained MAPK signaling. Our data supports further study of pharmacologic inhibition of mTORc1 and MAPK to improve response to lapatinib in HER2-overexpressing breast cancer.