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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.

Krüppel-like factors (KLFs) are transcription factors containing three conserved zinc finger motifs in the carboxy-terminus and have shown associations with many diseases, including cancer, diabetes, and vascular disorders. Krüppel-like factor 11 (KLF11), originally identified as the causative gene for maturity-onset diabetes of the young type 7 (MODY7), has been reported to facilitate insulin transcription in pancreatic β cells. Previous studies have found that endothelial KLF11 has a protective effect in sepsis, ischemic stroke, and abdominal aortic aneurysm. Considering the common vascular complications in diabetes patients, in this project, we aim to study further the role of KLF11 in vascular homeostasis, especially thrombosis. We applied a ferric chloride-induced thrombosis model to investigate the role of KLF11 in arterial thrombosis. We found that the occlusion time was significantly reduced in conventional Klf11 knockout (Klf11 KO) mice, while bone marrow transplantation could not rescue this phenotype. Given the increased expression of tissue factor (TF; encoded by the F3 gene) in the tunica media of aorta in Klf11 KO mice, we further applied the arterial thrombosis model in vascular smooth muscle cell (VSMC) specific Klf11 knockout mice and found a significant reduction of occlusion time. In human aortic smooth muscle cells, siRNA-mediated knockdown of KLF11 increased TF expression. Consistent results were observed upon adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates TF gene expression at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. In sum, our data demonstrate that KLF11 is a novel transcriptional suppressor of the TF gene in VSMCs, constituting a potential molecular target for inhibition of arterial thrombosis. To study the role of KLF11 in venous thrombosis, we utilized a stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs). We identified an increase in KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-alpha (TNF-α) induced TF expression. Using reporter gene assay, chromatin immunoprecipitation assay, and co-immunoprecipitation, we revealed that KLF11 could reduce TNF-α-induced binding of early growth response 1 (EGR1) to the TF gene promoter in ECs. In addition, we demonstrated that conventional Klf11 KO mice were more susceptible to developing stasis-induced DVT. These results suggest that under prothrombotic conditions, KLF11 downregulates TF transcription via inhibition of EGR1 in ECs. In conclusion, KLF11 protects against DVT, constituting a potential molecular target for treating thrombosis. In summary, KLF11 inhibits the basal TF expression in VSMCs and interrupts the induced EGR1 binding to the TF gene promoter under prothrombotic conditions. Thus KLF11 is important in maintaining vascular homeostasis and can be a promising therapeutic target for preventing or treating arterial thrombosis and DVT.

Kruppel-like factors (KLFs) are zinc finger proteins which bind to promoter or enhancer regions of genes in order to regulate their transcription. They are involved in many functions including the regeneration of retinal axons following optic nerve damage. However, studies involving optic nerve regeneration in zebrafish have only surveyed Klfs expression and function in retinal ganglion cells. In this study I used in situ hybridization to determine if expression of Klf6 and Klf7 were upregulated in the optic tract and/or optic tectum (the major target for the retinal axons) following damage to the optic nerve in adult zebrafish. Klf6 and Klf7 were both upregulated 1-day post-injury (dpi) in the optic tract, while increased Klf6 expression was also detected in the retinorecipient regions of the optic tectum. Expression levels of these two Klfs (based on staining intensity) became further increased in both the optic tract and the optic tectum of fish 2-7 dpi. In the optic tract and optic tectum of fish 14 dpi, expression of Klf6 and Klf7 continued to be elevated compared to their respective control levels, but reduced compared to that of 2-7 dpi. By 21 dpi, expression of both Klfs in the optic tectum became similar to their respective controls, but remained increased in the optic tract.