Abstract P6-14-01: Invasively distinct subpopulations cooperate via a laminin-332/Rac1 axis in triple-negative breast cancer

Abstract

Abstract Intratumoral heterogeneity poses a significant hurdle for cancer treatment, yet is under-characterized in the context of tumor invasion. Cancer cells from solid tumors can invade through two predominant modes: collective invasion, whereby cancer cells invade in multi-cellular packs or streams marked by intact cell-cell junctions; and single-cell invasion, whereby cells invade independently without intercellular adhesion. We have observed that collective and single-cell invasion co-occur within the same tumor microenvironment in triple-negative breast cancer, suggesting that invasive heterogeneity supports cooperative behavior between tumor subpopulations. To test this, we used a novel, published technique developed by the lab (SaGA) to isolate pure subpopulations of 4T1 cells that collectively invade (collectives) or single cells that invade alone (singles). 3-D spheroids of SaGA-purified collectives and singles exhibited almost exclusively collective and single-cell invasion, respectively, and these invasive phenotypes were retained over multiple passages. Integration of RNA sequencing and methylation array data obtained from RNA and DNA isolates, respectively, of collectives and singles revealed that collectives exhibit drastic overexpression and promoter hypomethylation of two laminin genes that form the laminin-332 complex, Lama3 and Lamc2. Additionally, an unbiased proteomic analysis of secreted proteins also revealed an overabundance of these laminins in collectives media. We found that singles have increased expression of integrin α6 and β4, which together have been found to specifically bind to laminin-332 to activate the Rac1 GTPase. Interestingly, our RNA sequencing data revealed a binary overexpression of a Rac1 GTPase, Prex1 in singles, suggesting that singles have enhanced Rac1 activation when compared to collectives with the potential for hyperactivation upon laminin-332 binding. Indeed, laminin-332 resulted in higher GTP-bound Rac1 in singles and subsequently increased invasion of singles in 3-D models. Additionally, laminin-332 induced cell elongation at the leading edge of singles spheroids, which was reversable by treatment with a Rac1 inhibitor. Together, our data suggests that distinct subpopulations amidst a heterogeneous tumor cooperate via laminin-332 and Rac1 to facilitate tumor invasion in metastatic triple-negative breast cancer. Citation Format: Sung Bo (Joseph) Yoon, Janna Mouw, Luxiao Chen, Hao Wu, Adam Marcus. Invasively distinct subpopulations cooperate via a laminin-332/Rac1 axis in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-01.