The role of Krüppel-like factors in breast cancer cell mechanobiology

Abstract

Mechanobiology explores the relationship between mechanical forces and biological systems, and is increasingly being realized to play fundamental roles in human health and disease. There is constant cross-talk between cells and their surrounding stroma, in which cells can perceive physical cues and modify their behavior - as well as their environment - in response. Mechanosensitive transcription factors (MSTFs) function as important effectors in this process, transforming physical stimuli into changes in gene expression. In cancer, physical changes in the tumor microenvironment can influence the activity of genes involved in proliferation, migration, and invasion, and thereby impact disease progression. This research focuses on Krüppel-like factors (KLFs), a group of transcription factors initially recognized for their importance in embryo patterning and stem cell plasticity. More recently, however, it has been shown that KLFs can also function as MSTFs in a number of diseases, including cancer. The aim of this project was to investigate whether a select group of KLFs (KLF2, KLF4, KLF5, and KLF6) function as MSTFs in breast cancer cells. To do this, we looked at patterns of KLF expression in response to both long-term adaption to different physical environments, as well as to rapid physical changes, using two-dimensional (2D) and three-dimensional (3D) cell culture model systems and the 4T1 series of heterogeneous breast cancer subclones. Signaling pathways potentially involved in transducing physical events into KLF expression changes were examined, and siRNA KLF knockdown experiments were conducted to assess the possible impacts of altered KLF expression on tumor cell morphology, adhesion, proliferation, invasion, and the ability to exert tension on the extracellular matrix. RT-qPCR results indicate that the expression of the KLFs examined exhibit both short-term (rapid), as well as longer-term (adaptive) responses to changes in the physical microenvironment. Some of the expression patterns were common among the different KLFs and tumor cell subclones, whereas certain others diverged. Pharmacological inhibition studies implicated mechanosensitive Piezo1 calcium channels and focal adhesion kinase activity in coupling physical changes and KLF expression. Cell-cell and cell-substrate adhesion, and the ability of cells to invade a collagen gel were identified as behaviors markedly affected by KLF5 and KLF6 knockdown, respectively. Overall, these results support the hypothesis that KLFs function as mechanosensitive transcription factors in breast cancer cells.