Abstract 5149: Subclonal sociability: Interactions of HER2 and PIK3CAmutant cells in breast cancer
Authors: Waters Ian , Karthikeyan Swathi , Dennison Lauren , Chu David , Park Ben Ho 
Published: August 2019

Background: Tumor heterogeneity is a common characteristic of many cancers and plays an important role in their development and eventual metastasis. The traditional clonal evolution model describes cancer cells as descendants of a single progenitor that has accumulated a stepwise series of mutations. However, recent sequencing studies have revealed the presence of genetically distinct subclones in numerous tumors. PIK3CA is the most commonly mutated gene in breast cancer while HER2 is overexpressed in ~20% of breast cancers. Activating missense mutations in the gene that encodes HER2 have also been discovered, and our lab has previously shown the effects of these mutations and their synergy with PIK3CA mutations in a double-mutant cell. This project explores the interactions between cells with either a HER2 or a PIK3CA mutation when they are present as genetically distinct subclones in co-cultures.

Methods: Isogenic MCF-10A cells containing mutations in PIK3CA, HER2, or no mutation were co-cultured in growth factor-deprived conditions. In these conditions, PIK3CA mutant cells are known to be able to grow, while HER2 mutant and wild-type (WT) cells arrest. The cellular makeup of the co-cultures was determined using droplet digital PCR (ddPCR) with probes against the respective mutations. Cells were tagged with stable expression of either zsGreen (PIK3CA mutants) or tdTomato (HER2 and WT) and observed under fluorescent microscopy. Further imaging was performed using the IncuCyte live cell imaging system (Essen Bioscience), which allowed for periodic imaging of cells in culture. Co-cultures were treated with Neratinib or Alpelisib (BYL719), which are HER1/2/4 and PIK3CA inhibitors, respectively. Cells in each condition were counted and ddPCR was performed to determine allelic fractions of the co-cultures.

Results: When cultured together, PIK3CA mutant cells can induce proliferation in HER2 mutant cells, but not WT cells. Over time, HER2 mutant cells grow to make up &gt;50% of the co-cultures. Imaging and transwell assay data (not included on poster) implicate cell-cell contact rather than paracrine factors as the primary driving factor of this phenotype. HER2 and PIK3CA inhibitors can effectively target the co-cultures and inhibiting the growth of the PIK3CA cells appears to halt the growth of the HER2 mutants as well.

Conclusions: PIK3CA mutant cells can induce proliferation of quiescent HER2 mutant cells in growth-factor deprived conditions. This effect appears to the cell-cell contact mediated rather than driven by secreted factors. Co-cultures that contain PIK3CA and HER2 mutant cells have therapeutic vulnerabilities that can be exploited using drugs that are currently approved or in clinical trials. These results have important implications in understanding early tumor development and suggest that therapeutic approaches that target subclonal populations may be effective in certain tumors.

Citation Format: Ian Waters, Swathi Karthikeyan, Lauren Dennison, David Chu, Ben Ho Park. Subclonal sociability: Interactions of HER2 and PIK3CAmutant cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5149.