Abstract P2-20-06: Comparative effect of Nerlynx & HER2+ positive & negative breast cancer cell lines on the barrier function of human brain endothelial cells
Authors: Martin Tracey Amanda , Meyers Isaac , Appadurai Emily , Jiang Wen G 
Published: March 2020

Background Brain Metastasis in Breast Cancer (BMBC) is a significant contributor to mortality & presents a challenge to modern treatment of advanced breast cancer. HER2+ breast cancers have a predilection for metastasis to the brain & therefore the HER2 inhibitor Nerlynx could exhibit efficacy in treating BMBC. Previous work suggests that Nerlynx can pass through the Blood-Brain Barrier (BBB) & may alter it during treatment of BMBC. Methods The BBB was modelled using hCMEC-D3 cells. BMBC was modelled using HER2- MDA-MB-231 cells or HER2+ SK-BR-3 cells. To evaluate changes in protein expression, hCMEC-D3 cells were treated with 50 nM Nerlynx ± SK-BR-3-conditioned medium & analysed using the Kinexus microarray service. Functional changes in electrical resistance, permeability, proliferation & migration were measured using Trans-Endothelial Resistance (TEER) & Paracellular Permeability (PCP) assays. Changes in TJ protein location were assessed using immunofluorescence (IF). Results Protein microarray analysis compared to the control, co-treatment with 50 nM Nerlynx & SK-BR-3-CM was associated with increased expression of migration-associated proteins (ROCK1, +166%) & cell cycle regulatory components (Rb, +114%; CDK6, +159%; PLK1, +168%) as well as the guanine exchange factor VAV1 (+123%), intracellular kinases RSK1 (+110%) & PKCβ (+107%), & the HGF receptor MET (+74%). In contrast, decreased expression of the transcription factor STAT2 (-98%), the cell cycle regulators CDK2 (-85%) & CDK1 (-75%), focal adhesion component Paxillin 1 (-74%) & the RTK Kit (- 59%) are features of co-treatment compared to control. Compared to treatment with SK-BR-3-CM alone, co-treatment with Nerlynx was associated with an increase in the expression of Paxillin 1 (+809%), AKT1 (+458%) & MET (+449%), as well as CDK6 (+310%), PDK1 (+215%), the RTK DDR1 (+212%), lymphangiogenic receptor VEGFR3 (+203%) & Rb (+183%). Compared to treatment with SK-BR-3-CM alone, co-treatment with Nerlynx was associated with a decrease in the expression of STAT2 (-98%), IRS1 (-92%), ROCK2 (-84%), CSK (-73%), Kit (-71%), STAT3 (-68%), JAK2 (-67%) & VEGFR2 (-64%) & an increase in the expression of ILK1 (+554%), Kit (+448%), AKT1 (+337%), Rb (+261%) & VAV1 (+184%). However, compared to treatment with Nerlynx alone, co-treatment with SK-BR-3 CM was associated with a decrease in the expression of IRS1 (-99%), STAT2 (-98%), Paxillin 1 (-92%), IKKα (-78%), CDK1 (- 65%) FAK (-63%) & STAT3 (-62%). hCMEC-D3 co-treated with MDA-MB231-CM, 100 nM Nerlynx treatment exhibited increased PCP, after 60 min post-treatment & a similar effect was seen after 5 nM Nerlynx treatment at 120 min. In hCMEC-D3 monolayers co-treated with SK-BR-3- CM, 100 nM Nerlynx there was a decrease in PCP by 120 min post-treatment. Conversely, treatment with 5 nM or 50 nM Nerlynx led to an increase in PCP by 60 min or 180 min, respectively. Nerlynx treatment inhibited the proliferation of hCMEC-D3 cells in a dose-dependent manner with larger effects seen upon the addition of medium conditioned by MDA-MB-231 cells or SK-BR-3 cells. There was a concurrent change in distribution of TJ proteins as assessed using IF. Discussion Nerlynx inhibits the proliferation of brain endothelial cells but its role in altering barrier function is unclear. Nerlynx exerts a greater effect on brain endothelial cells in the presence of medium conditioned by either HER2+ or HER2- cells, suggesting it may be efficacious in the treatment of BMBC. The MET signalling pathway is implicated as a potential regulator of TJ-mediated BBB permeability in BMBC. Further work including BBB co-culture models, alternative brain endothelial lines & evaluation at the gene & protein level are warranted.

Citation Format: Tracey Amanda Martin, Isaac Meyers, Emily Appadurai, Wen G Jiang. Comparative effect of Nerlynx & HER2+ positive & negative breast cancer cell lines on the barrier function of human brain endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-06.