Abstract PD5-01: Microenvironment microarrays show that microenvironment mediated resistance mechanisms to lapatinib differ between basal and luminal HER2+ cells
Authors: Korkola JE , Watson S , Smith R , Thompson W , Dame M , Liby T , Bucher E , Sudar D , Nederlof M , Heiser L , Gray JW 
Published: March 2017

Abstract
Cell lines represent a valuable model system for the study of breast cancer, as they capture the cellular diversity, mutational spectrum, expression subtypes, and genomic alterations that are observed in clinical specimens. However, like any model system, cell lines are imperfect, particularly when it comes to capturing the effects of the myriad of signals and interactions they encounter in their microenvironment (ME). We are utilizing a technology known as microenvironment microarrays (MEMA) to begin to unravel the consequences of interactions of breast cancer cells with the ME. MEMA consist of thousands of unique combinations of insoluble matrix proteins that are printed to form growth pads with ligands added to the media. Cells are grown on the MEMA spots and the effects of the specific ME that they are exposed to can be read out using immunofluorescent stains of interest. When combined with automated imaging and sophisticated image processing and analysis, the MEMA platform enables the identification of specific ME conditions that alter the phenotypes of cells. We have applied MEMA to understand both baseline responses to the ME as well as how the ME might mediate response to therapeutics. We performed a pilot experiment to investigate the effects of the ME on the response to the HER2-targeted inhibitor lapatinib. We found that HCC1954 cells continued to proliferate robustly in the presence of HGF when treated with 500 nM lapatinib. In contrast, AU565 cells were proliferative in the presence of NRG1 and lapatinib, but not HGF. Focused follow up studies showed that HGF is effective in rescuing only basal HER2+ cells, while NRG1 is effective in rescuing only luminal subtype HER2+cells. Rescue with the relevant growth factor was also observed in 3-d matrigel studies, showing this was not an artifact of the 2-d culture system. We investigated the effects of drug combinations using lapatinib plus drugs that target either MET (Crizotinib) or HER3-HER2 dimers (pertuzumab). These drug combinations were able to overcome the resistance mediated by HGF and NRG1 in basal and luminal cells respectively. We found the effectiveness of pertuzumab particularly interesting, given that lapatinib should still be inhibiting HER2 kinase activity. Parallel studies found that inhibitors targeting other kinase receptors such as IGF1R partially restored sensitivity to HER2 in the presence of NRG1, suggesting a role for such receptors in the resistance. Immunoprecipitation studies showed that IGF1R co-immunoprecipitated with HER2/HER3 when pertuzumab was absent, but that additional of pertuzumab abrogated the binding of IGF1R to HER3, suggesting the formation of HER2-dependent higher order structures that can signal even when HER2 is inhibited. These studies highlight the importance of understanding the effects of the ME on cancer cells, and demonstrate the differences between ME factors that can confer resistance to HER2 targeted inhibitors in basal and luminal HER2+ cells. These findings suggest that both subtype and ME composition may be important in determining response to combinatorial treatments and may be useful to inform clinical decision making.
Citation Format: Korkola JE, Watson S, Smith R, Thompson W, Dame M, Liby T, Bucher E, Sudar D, Nederlof M, Heiser L, Gray JW. Microenvironment microarrays show that microenvironment mediated resistance mechanisms to lapatinib differ between basal and luminal HER2+ cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD5-01.