B.1 Project: Intrinsic contractility of cancer cells dictates focal adhesion and stress fiber orientation to drive polarity and migration along wavy ECM fibrils in the tumor microenvironment. Personnel: Bob Fischer Collaborations: John Fourkas (University of Maryland, Chemistry) and Wolfgang Losert and (University of Maryland, Physics) Claudia Fischbach-Teschls (Cornell, Biomed Engineering) We tested the hypothesis that linear collagen fibrils such as those seen in obese mammary tissue and the tumor microenvironment promote contact-guidance-mediated cancer cell migration, while wavy ECM fibrils typical of healthy stroma inhibit migration. Our study showed that ECM wave architecture regulates contact guidance of tumor cells via actomyosin contractility, suggesting that oncogenic modulation of cell contractility may dictate the range of ECM waveforms that are conducive to metastatic cell migration. We propose that in addition to a physical barrier, wavy ECM bundles serve as a cell polarization barrier, with high-amplitude waves depolarizing tumor cells and preventing their directional migration and dissemination. Although tumor ECM architecture has been suggested to be predictive of cancer outcome, our results suggest that oncogenic upregulation of contractility could drive cell dissemination even on wavy collagen bundles typical of non-cancerous stroma. Thus, ECM architecture and myosin II activation level should be weighed in tandem in considering the predictive value of microenvironmental ECM architecture in cancer progression. B.2 Project: Mechanism of leader bleb-based cancer cell motility in non-adhesive confinement Personnel: Greg Adams Collaborations: Alex-Cartegena-Riviera We sought to understand how the unusual morphology of metastatic melanoma cells undergoing leader-bleb-based migration in non-adhesive confinement affects subcellular organization of organelles and cytoskeletal systems, and to probe the role of actin regulatory proteins in mediating this process. We found that membranous organelles, including components of the endo-lysosomal, secretory and metabolic pathways, exhibit a highly polarized distribution, predominantly concentrated in the cell body with only a minor fraction in the leader bleb. Similarly, the bulk of the vimentin155 and MT cytoskeletons concentrate in the cell body. These results suggest that the cell body compartment maintains most of the general cellular functions of metabolism and protein processing and turnover, while the leader bleb compartment may be highly specialized exclusively for motility. Our discovery here of the exceptional degree of organelle polarization during leader bleb-based motility, which occurs in the absence of gradients of extracellular cues, underscores the self-organization of this extreme cell asymmetry. Understanding the molecular mechanism of LBBM polarization will be an important focus in the future to allow development of anti-metastatic therapeutics. B.2.2. Role of plasma membrane biophysics in establishment of cancer cell polarity during leader bleb-based migration in non-adhesive confinement Personnel: Ankita Jha Collaborators: Jason Haugh, Abhishek Kumar Test the hypothesis that front-rear polarity of cancer cells undergoing leader bleb-based migration in non-adhesive confinement is mediated by the segregation of signaling components on the plasma membrane. We found that EGF signaling was required for leader bleb stability under non-adhesive confinement, suggesting the role of plasma membrane growth factor receptors in bleb-based migration. This work shows that during leader bleb-based migration of cancer cells in non-adhesive confinement, transmembrane receptors are depleted from the leading tip of the bleb by strong corralling at the back, likely to maintain persistent bleb polarization. We are currently performing siRNA of CD44 to determine if it is required for leader bled formation and the EGFR gradient, and are utilizing a new photoactivatable EGFR165 to determine if spatial organization of EGF activity is required for polarization of LBBM. Finally, our results predict that cells undergoing leader bleb-based migration may be insensitive to or repulsed by growth factor gradients, and we are gearing up to test this as well. B.4. Project: Upregulation of Lamin B Receptor in Metastatic Melanoma Promotes Enhanced Nuclear Envelope Fragility. Personnel: Michelle Baird Collaborators: Mehdi Pirzoona, Alex Cartegena-Riviera Goal: Determine the molecular pathways leading to nuclear envelope fragility in confined metastatic cancer cells. We found that cell lines derived from advanced prostate or breast cancers or metastatic melanoma all showed enhanced NE fragility compared to cell lines from normal prostate, breast and melanocyte origin. We found that high expression of lamin B receptor (LBR) in metastatic melanoma cells was required for enhanced nuclear envelope fragility in confinement and increased nuclear deformability as measured by AFM. We discovered that LBR over-expression was sufficient to increase NE fragility and deformability in normal melanocytes. Together our results indicate that upregulation of LBR during cancer progression promotes disruption of cholesterol organization in the NE to mediate increased nuclear deformability and fragility in cancer cells subject to confinement, and suggests sterol reductase activity as a possible anti-metastatic drug target. We are currently seeing if LBR expression or its sterol reductase activity are required for NE failure in metastatic melanoma cells in physiological settings including tumor models in vitro and in mice, and whether they promote genetic heterogeneity caused by repeated bouts of confined migration. B.5. Project: The LINC complex mediates Unlocking of Neurectoderm Lineage Gene (Sox1) During Early Differentiation in Mouse Embryonic Stem Cells Personnel: Mehdi Hamouda Collaborators: Kevin Chalut, Kate Miroshnikova Goal: Test the hypothesis that cytoskeletal forces generated in nave pluripotent stem cells during morphogenesis of the peri-implantation blastocyst are transmitted through the LINC complex to epigenetically effect lineage commitment during exit from nave pluripotency. We studied the role of the LINC complex and actomyosin activity in mouse embryonic stem cells (mESCs) in tissue culture during exit from nave pluripotency. These results suggest that morphogenesis-associated apical actomyosin contractile activity may mechanically signal through the LINC complex to instruct appropriate chromatin remodeling at the apical nuclear surface which unlocks Sox1 for timely downstream transcriptional activation. We are currently delving more deeply into the cytoskeletal mechanism, aiming to determine which small GTPase and MRLC kinase directs apical constriction, and whether the barbed-end binding protein emerin plays a role in tethering actin to the NE.