Conceptos

During the inflammatory response, the expression of many adhesion molecules is increased, including intercellular adhesion molecule (ICAM)-1. Before leukocytes can reach sites of injury or infection they bind and crawl along the endothelium, and then cross via junctions or through the body of the endothelial cells (ECs). This process is called transendothelial migration (TEM). Leukocytes crawl along endothelial cells by applying tractional forces to ICAM-1 receptors (Oh et al., 2007). Although RhoA signaling is known to be activated downstream of force on ICAM-1, little is known about its regulation and the effect of force on ECs.

To examine the effect of mechanical stimulation on neonatal human dermal blood microvascular ECs (HMVECs), we used magnetic tweezers to apply 160 pN pulses of force to anti-ICAM-1 coated beads (Fig. 4.7 A). Analysis of the average bead dis- placement over time revealed that HMVECs increased their stiffness within 10 sec of pulsatile force application on ICAM-1 (indicated by decreased displacement), and that this stiffness reponse was diminished by disrupting the actin cytoskeleton or the RhoA pathway with several pharmaceutical interventions (Lessey-Morillon et al., 2014).

Additional experiments revealed that RhoA is activated (within 1 min) in response to force on ICAM-1 (Lessey-Morillon et al., 2014), we sought to determine the GEF mechanisms by which RhoA is activated. Several candidate GEFs were tested but only LARG was observed to mediate RhoA activation downstream of force on ICAM- 1 (Lessey-Morillon et al., 2014). To investigate the effect of LARG on stiffness and

Figure 4.7: The RhoA GEF LARG regulates the stiffness response to force on ICAM-1. (A) Magnetic tweezers were used to apply 160 pN pulses of force in a 3 sec on, 5 sec off regimen to 4.5 µm anti-ICAM-1 coated beads attached to HMVECs. Typical displacements exhibited a viscoelastic response. (B) Average stiffness of HMVECs treated with a control or LARG shRNA lentivirus, and then probed with anti-ICAM-1 coated beads. (C) Relative displacement of anti-ICAM-1 coated beads on control or LARG shRNA treated HMVECs (n ≥15). (Error is SEM,p≤0.01)Data: Elizabeth Lessey

stiffness response, we used a shRNA to decrease LARG expression. We observed that loss of LARG expression reduced HMVEC stiffness (p = 0.08; Fig. 4.7 B), and that the significant stiffness response to force was lost after LARG depletion (Fig. 4.7 C). These results suggest that LARG activates RhoA in response to force on ICAM-1 to regulate a stiffening response.

Further experiments revealed that depletion of LARG reduces both neutrophil (a type of leukocyte) migration velocity across a monolayer of HMVECs and passage through the monolayer (TEM decreased ∼ 35%) (Lessey-Morillon et al., 2014). Al- though RhoA activity is known to contribute to TEM by weakening EC junctions (Aghajanian et al., 2008), this work provides the first evidence that LARG activation of RhoA may promote neutrophil TEM by increasing the stiffness of the EC surface.

4.5 HIF1α and HIF2α are sufficient to promote cancer phenotypes

During hypoxia (low oxygen conditions), transcription factors called hypoxia-inducible factors (HIFs) upregulate genes to promote cell survival. For example, cells within a growing cancerous tumor often experience hypoxic conditions, and accordingly, HIF ac- tivation has been shown in numerous cancers (Semenza, 2003). HIFs promote metastatic progression by transcriptional regulation of genes that mediate ECM degradation, the epithelial to mesenchymal transition (EMT), and cell adhesion and motility (Semenza, 2003). Here, our goal was to study HIFs in melanoma.

While initial results showed that HIF1α and HIF2α are necessary for hypoxia- dependent invadopodia formation and cell invasion (Hanna et al., 2013), we sought to determine whether these HIFs were sufficient to drive cell invasion and wanted to examine their effect on cell stiffness. Thus, we tested the invasion of human melanoma A-375 cells that were transfected with control EGFP, HIF1α (HIF1dPA), or HIF2α

(HIF2dPA). The HIF cells remain stable under normoxia (normal oxygen conditions; typically, HIFs are degraded under these conditions). Assessing invasion revealed sig- nificant increases for HIF1dPA and HIF2dPA compared to the EGFP control (Fig. 4.8

A,B). Next, we tested cell stiffness by magnetic tweezers (Fig. 4.8 C) and PBR (Fig.

4.8 D,E) and found that HIF1dPA and HIF2dPA significantly decrease stiffness com- pared to the EGFP control. Together, these results indicate that HIF1α and HIF2α

Figure 4.8: HIF1α and HIF2α are sufficient to increase cell invasion and de- crease cell stiffness. (A) Representative images of cells transfected with control EGFP, HIF1dPA, or HIF2dPA after invasion through Matrigel chambers. (B) Quan- tification of invasion assay. (C) Magnetic tweezers were used to apply a 50-100 pN pulse of force for 5 sec to 4.5 µm FN-coated beads. (D) Average MSD vs τ for cell populations. (E) Average MSD (inset: RMS displacement, stiffness) at the τ = 1sec timescale. (Error is SEM, ∗ ∗ ∗p <0.0005, ∗ ∗p <0.005, ∗p < 0.05) Data in (A,B): Sara Hanna; Data in (C): Tim O’Brien

Chapter 5: Cell Mechanics and the Epithelial to Mesenchymal Transition

5.1 Overview

Recent work has shown that invasive cancer cells have a reduced stiffness and ex- ert larger forces on their environment. Although these studies suggest a role for cell mechanics in cancer progression, little is known about the molecular mechanisms that regulate mechanics during cancer. To address this need, we turned our attention to the epithelial to mesenchymal transition (EMT). EMT is a well characterized process in embryogenesis and wound healing, and recently has been implicated as a model for the physical detachment of cancer cells before they begin to metastasize. While classic EMT hallmarks include loss of cell-cell adhesions, morphology changes, and increased invasion capacity, little is known about the associated mechanical changes. Therefore, in this chapter, we ask the following questions:

1. Is there a mechanical phenotype adopted during EMT?

2. If so, what are the biochemical mechanisms responsible for the alternations in cell mechanics?

To answer these questions, we apply a multi-assay approach to investigate, for the first time, the mechanical phenotype associated with growth factor induced EMT. We employ active and passive microrheology assays to characterize cell stiffness and stiffness

response to externally applied force before and after this cancer tranistion. Using the novel rotating magnet device discussed in Ch. 3, we are able to study the molecular mechanisms behind the mechanical characteristics that cells adopt during EMT. We carefully execute loss and gain of function experiments to reveal a novel, functional connection between cell stiffness and the increased invasion capactiy acquired after growth factor induced EMT.

EMT collaboration. In this project, I designed and performed the magnetic tweezer and PBR experiments, and analyzed the data. I developed the rotating magnet de- vice to enable biochemical analysis of force-dependent signaling pathways. George Li managed cell culture, prepared specimens for mechanical experiments, executed bio- chemical and invasion assays. The work in this chapter has been published: L.D. Osborne, G.Z. Li, T. How, E.T. O’Brien III, G. Blobe, R. Superfine, K. Mythreye. “Altered stiffness and mechanical response to force by the Rho GEFs LARG and GEF- H1 regulate cell invasion during TGF-β induced EMT”. Molecular Biology of the Cell. doi: 10.1091/mbc.E14-05-1015. (2014). Referenced in text as (Osborne et al., 2014).