Melanoma cell morphogenesis in 3D environments

Abstract

During metastasis cancer cells invade tissues with diverse rigidities through dynamic changes in their cell shape. Most of our understanding of cancer cells shape determination comes from 2D studies on extremely stiff plastic substrates. As such, there is little insight into cancer cell morphogenesis on physiologically relevant matrices, and in 3D space. Rho GTP Exchange Factors (RhoGEFs) and Rho GTPase Activating Proteins (RhoGAPs) are large and diverse families of molecules that play a role in eukaryotic shape determination. Though most remain uncharacterized, RhoGEFs and RhoGAPs are ideal candidates for proteins capable of coupling melanoma morphogenesis to variations in matrix rigidity and geometry. To identify RhoGEFs and RhoGAPs that play a role in cell shape determination on soft matrices I performed genetic screens of cells cultured on soft collagen hydrogels in tandem with quantitative morphological analysis. From the screens I found that ARHGEF9/collybistin is essential for cell shape determination on both soft and stiff matrices, and in cells embedded in 3D. I show that ARHGEF9 promotes the formation of actin rich filopodia, which serve to establish and stabilize focal adhesions. Depletion of ARHGEF9 results in loss of tension at adhesions, decreased cell-wide contractility, and the inability to stabilize protrusions. Moreover, ARHGEF9 is required for melanoma cells to invade 3D matrices. The role of ARHGEF9 in shape determination and focal adhesion assembly is dependent on CDC42. I propose that ARHGEF9 may confer melanoma cells with the ability to explore and adhere to extracellular matrices. In addition, melanoma metastasis in 3D matrices is like how endothelial or neuronal cells migrate through the body during development.Abnormal cell cycle progression is one of the hallmarks of cancer cells (Hanahan and Weinberg, 2011). In this study, I show that ARHGEF9 influences the cell cycle progression. Melanoma cells depleted of ARHGEF9 have a slower growth rate and significantly lower levels of pRB compared to control cells. Thus, ARHGEF9 depleted cells enter the cell cycle with defects at the initial stage which influences their proliferation rate. My data demonstrates that ARHGEF9 can potentially be a target for slowing the uncontrolled growth rate of melanoma cells which results in tumorigenesis.