Abstract P4-08-05: Basement membrane localized tumor cells are protected from HER2-targeted therapy in vivo.
Authors: Zoeller JJ , Bronson RT , Gilmer TM , Selfors LM , Lu Y , Apple SK , Press MF , Hurvitz SA , Slamon DJ , Mills GB , Brugge JS 
Published: August 2014

Abstract
Drug resistance compromises the efficacy of HER2-targeted therapy. Results from our laboratory, and previous reports from others, indicate that tumor cell attachment to basement membrane (BM) and other extracellular matrix (ECM) proteins may confer drug resistance. We have discovered a differential drug response between the outer, matrix-attached cells and inner matrix-deprived cells comprising 3D tumor spheroids grown in reconstituted BM (Muranen 2012). The outer matrix-attached cells are resistant to multiple drug therapies due to upregulation of a multi-factor survival program including anti-apoptotic proteins and growth factor receptors. To address whether these observations are relevant in vivo, we utilized a previously described model of human-in-mouse HER2+ ductal carcinoma in situ, which involves the intraductal transplantation of human HER2+ SUM225 tumor cells directly into the ductal network of the mouse mammary gland. The tumors are characterized by organized nests of noninvasive cells confined within a BM surrounded by ECM. We focused on the tumor cell response to short-term lapatinib monotherapy in vivo. A close examination of the tumor architecture revealed that cells closest to the BM, and nearest to the vasculature, display a striking insensitivity to lapatinib whereas the remainder of the tumor undergoes extensive cell death in response to treatment. Further characterization also revealed that cells closest to the BM largely maintain proliferative capacity despite an overall reduction in the total Ki67+ cell population. To further explore the response to treatment, we performed reverse phase protein array (RPPA) analysis on protein lysates prepared from tumor fragments following lapatinib monotherapy. RPPA profile analysis revealed a lapatinib-induced adaptive response program characterized by upregulation of multiple receptor tyrosine kinases, reactivation of AKT and ERK pathway components, and the upregulation of prosurvival BCL2 family proteins. Evaluation of BCL2 in matched SUM225 tumor sections revealed selective upregulation within the BM-localized tumor cells. We predict BCL2 upregulation in the BM-localized tumor cells may represent the critical adaptive response mechanism by which these cell populations would escape lapatinib treatment. BCL2 therefore represents a target for designing combined therapeutic approaches capable of abrogating the protected BM-localized tumor cells. Under 3D culture, lapatinib combined with the BCL2 antagonist ABT737 resulted in greater SUM225 tumor cell synthetic lethality compared to single agent treatment. The combination treatment is currently being explored in vivo. We evaluated the translational relevance of our preclinical observations within the context of HER2+ clinical disease. Patient biopsy samples collected as part of an ongoing clinical trial were assayed for BCL2 before and after short-term HER2-targeted therapy. BCL2 was clearly upregulated post-treatment in a subset of patient samples and largely correlated with a poor response to treatment. Our results suggest that resistant populations may be a source of residual disease post-therapy, therefore identifying and characterizing these cells will be crucial to the prevention of disease recurrence in the clinic.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-05.