Abstract 1498: Fibronectin mediates mutation specific interactions between subclonal populations of mutant PIK3CA and HER2 cells in breast cancer
Authors: Karthikeyan Swathi , Waters Ian , Dennison Lauren , Chu David , Donaldson Joshua , Shinn Dong Ho , Croessmann Sarah , Park Ben Ho 
Published: September 2020

Breast cancer is the second most common cancer in the world and the most frequently occurring cancer among women. It is a heterogeneous disease and intratumor heterogeneity is one of the main drivers of disease progression, recurrence, therapeutic resistance and metastasis. Herein we have demonstrated that sub clonal populations containing PIK3CA and HER2 mutants exhibit distinct transformative properties accurately modeling a heterogenous tumor in patients.

AAV-mediated gene targeting to was used to heterozygously incorporate a PIK3CA (E545K) or a HER2 (L755S or V777L) mutation, separately, into both the MCF-10A and MCF-7 cell lines. Cocultures were carried out by mixing an equal number of cells containing the PIK3CA mutation and cells containing a HER2 mutation into a single culture. Lentiviral transduction was used to tag the cells with either green or red fluorescent proteins to visualize growth patterns in coculture. Cellular fractions in coculture were quantified using droplet digital PCR. Microarray analysis of cocultures identified fibronectin as a significant mediator of growth and fibronectin knockouts were created using the CRISPR Cas9 gene editing system.

MCF-10As carrying the PIK3CA mutation are epidermal growth factor (EGF) independent and those carrying the HER2 mutation are highly EGF dependent. We have identified that interactions between the PIK3CA mutants and the HER2 mutants imparts EGF independence to the HER2 mutants. The effect rendered is so potent that the HER2 mutants dominate the coculture kinetics despite the absence of EGF. This distinct transformation was observed both in vitro and in vivo in the tumorigenic MCF-7 cell line. Interestingly, this phenomenon is mutation specific and was not observed in either the MCF-10A and MCF-7 cells containing wildtype HER2 when co-cultured with the PIK3CA mutants. Microarray analysis demonstrated elevated fibronectin, suggesting it may be a mediator of this observation. Competitive inhibition of fibronectin-integrin interaction using synthetic peptides that mimic the RGD binding domain of fibronectin, reduced the growth of HER2 mutants in coculture. Notably, knocking out fibronectin in the PIK3CA mutants reversed the EGF independence observed in the HER2 mutants in both the MCF-10A and MCF -7 cell lines.

Our data suggests that mutation specific interactions between mutant PIK3CA and mutant HER2 cells in a genetically heterozygous environment imparts a selective growth advantage to the HER2 mutants. This effect is mediated by an extracellular matrix protein, fibronectin. These interactions could be used to therapeutically target the complications that arise due to tumor heterogeneity.

Citation Format: Swathi Karthikeyan, Ian Waters, Lauren Dennison, David Chu, Joshua Donaldson, Dong Ho Shinn, Sarah Croessmann, Ben Ho Park. Fibronectin mediates mutation specific interactions between subclonal populations of mutant PIK3CA and HER2 cells in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1498.