Browsing by Author "Steele, Hannah E."

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    Fluid flow-induced activation of subcellular AMPK and its interaction with FAK and Src
    (Elsevier, 2020-01) Guo, Yunxia; Steele, Hannah E.; Li, Bai-Yan; Na, Sungsoo; Biomedical Engineering, School of Engineering and Technology
    AMP-activated protein kinase (AMPK) is a metabolic energy sensor that plays a critical role in cancer cell survival and growth. While the physical microenvironment is believed to influence tumor growth and progression, its role in AMPK regulation remains largely unknown. In the present study, we evaluated AMPK response to mechanical forces and its interaction with other mechano-responsive signaling proteins, FAK and Src. Using genetically encoded biosensors that can detect AMPK activities at different subcellular locations (cytosol, plasma membrane, nucleus, mitochondria, and Golgi apparatus), we observed that AMPK responds to shear stress in a subcellular location-dependent manner in breast cancer cells (MDA-MB-231). While normal epithelial cells (MCF-10A) also similarly responded to shear stress, they are less sensitive to shear stress compared to MDA-MB-231 cells. Inhibition of FAK and Src significantly decreased the basal activity level of AMPK at all five subcellular locations in MDA-MB-231 cells and selectively blocked shear stress-induced AMPK activation. Moreover, testing with cytoskeletal drugs revealed that myosin II might be the critical mediator of shear stress-induced AMPK activation in MDA-MB-231 cells. These findings suggest that breast cancer cells and normal epithelial cells may have different mechanosensitivity in AMPK signaling and that FAK and Src as well as the myosin II-dependent signaling pathway are involved in subcellular AMPK mechanotransduction in breast cancer cells.
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    Mechanotransduction of mitochondrial AMPK and its distinct role in flow-induced breast cancer cell migration
    (Elsevier, 2019-06) Steele, Hannah E.; Guo, Yunxia; Li, Bai-Yan; Na, Sungsoo; Biomedical Engineering, School of Engineering and Technology
    The biophysical microenvironment of the tumor site has significant impact on breast cancer progression and metastasis. The importance of altered mechanotransduction in cancerous tissue has been documented, yet its role in the regulation of cellular metabolism and the potential link between cellular energy and cell migration remain poorly understood. In this study, we investigated the role of mechanotransduction in AMP-activated protein kinase (AMPK) activation in breast cancer cells in response to interstitial fluid flow (IFF). Additionally, we explored the involvement of AMPK in breast cancer cell migration. IFF was applied to the 3D cell-matrix construct. The subcellular signaling activity of Src, FAK, and AMPK was visualized in real-time using fluorescent resonance energy transfer (FRET). We observed that breast cancer cells (MDA-MB-231) are more sensitive to IFF than normal epithelial cells (MCF-10A). AMPK was activated at the mitochondria of MDA-MB-231 cells by IFF, but not in other subcellular compartments (i.e., cytosol, plasma membrane, and nucleus). The inhibition of FAK or Src abolished flow-induced AMPK activation in the mitochondria of MDA-MB-231 cells. We also observed that global AMPK activation reduced MDA-MB-231 cell migration. Interestingly, specific AMPK inhibition in the mitochondria reduced cell migration and blocked flow-induced cell migration. Our results suggest the linkage of FAK/Src and mitochondria-specific AMPK in mechanotransduction and the differential role of AMPK in breast cancer cell migration depending on its subcellular compartment-specific activation.
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    Mechanotransduction of subcellular AMPK and its role in breast cancer cell migration
    (2018-04) Steele, Hannah E.; Na, Sungsoo
    The biophysical microenvironment of the tumor site has significant impact on breast cancer progression and metastasis. The importance of altered mechanotransduction in cancerous tissue through the integrin-mediated signaling axis has been documented, yet its role in the regulation of cellular metabolism and the potential link between cellular energy and cell migration remain poorly understood. In this study, we investigated the role of mechanotransduction (via Src and FAK) in AMP-activated protein kinase (AMPK) activation in breast cancer cells in response to interstitial fluid flow. Additionally, we explored the involvement of AMPK in breast cancer cell migration. An in-vitro three-dimensional (3D) cell culture model utilizing collagen-Matrigel matrices was used. Interstitial fluid flow was applied to the 3D cell-matrix construct inside a flow chamber. The sub-cellular signaling activity of Src, FAK, and AMPK was visualized in real-time using fluorescent resonance energy transfer (FRET). We observed that breast cancer cells (MDA-MB-231) are more sensitive to interstitial fluid flow than normal epithelial cells (MCF-10A) in the regulation of FAK and Src. AMPK was activated in the mitochondria of MDA-MB-231 cells by interstitial fluid flow, but not in other subcellular domains (i.e., cytosol, plasma membrane, and nucleus). Subcellular AMPK in MCF-10A cells did not respond to interstitial fluid flow. The inhibition of FAK or Src abolished flow-induced AMPK activation in the mitochondria of MDA-MB-231 cells. We also observed that global AMPK activation reduced MDA-MB-231 cell migration. Interestingly, specific AMPK inhibition in the mitochondria reduced cell migration and blocked interstitial fluid flow-induced cell migration. Our results suggest the linkage of FAK/Src and mitochondria-specific AMPK in mechanotransduction and the dual role of AMPK in breast cancer cell migration depending on its subcellular activation. Therefore, subcellular AMPK activation may play an important and distinct role in cancer invasion and progression.
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    Subcellular domain-dependent molecular hierarchy of SFK and FAK in mechanotransduction and cytokine signaling
    (Springer NPG, 2017-08-22) Wan, Qiaoqiao; TruongVo, ThucNhi; Steele, Hannah E.; Ozcelikkale, Altug; Han, Bumsoo; Wang, Yingxiao; Oh, Junghwan; Yokota, Hiroki; Na, Sungsoo; Biomedical Engineering, School of Engineering and Technology
    Focal adhesion kinase (FAK) and Src family kinases (SFK) are known to play critical roles in mechanotransduction and other crucial cell functions. Recent reports indicate that they reside in different microdomains of the plasma membrane. However, little is known about their subcellular domain-dependent roles and responses to extracellular stimuli. Here, we employed fluorescence resonance energy transfer (FRET)-based biosensors in conjunction with collagen-coupled agarose gels to detect subcellular activities of SFK and FAK in three-dimensional (3D) settings. We observed that SFK and FAK in the lipid rafts and nonrafts are differently regulated by fluid flow and pro-inflammatory cytokines. Inhibition of FAK in the lipid rafts blocked SFK response to fluid flow, while inhibition of SFK in the non-rafts blocked FAK activation by the cytokines. Ex-vivo FRET imaging of mouse cartilage explants showed that intermediate level of interstitial fluid flow selectively decreased cytokine-induced SFK/FAK activation. These findings suggest that SFK and FAK exert distinctive molecular hierarchy depending on their subcellular location and extracellular stimuli.