Simulated microgravity inhibits cell focal adhesions leading to reduced melanoma cell proliferation and metastasis via FAK/RhoA-regulated mTORC1 and AMPK pathways

Abstract

Simulated microgravity (SMG) was reported to affect tumor cell proliferation and metastasis. However, the underlying mechanism is elusive. In this study, we demonstrate that clinostat-modelled SMG reduces BL6-10 melanoma cell proliferation, adhesion and invasiveness in vitro and decreases tumor lung metastasis in vivo. It down-regulates metastasis-related integrin α6β4, MMP9 and Met72 molecules. SMG significantly reduces formation of focal adhesions and activation of focal adhesion kinase (FAK) and Rho family proteins (RhoA, Rac1 and Cdc42) and of mTORC1 kinase, but activates AMPK and ULK1 kinases. We demonstrate that SMG inhibits NADH induction and glycolysis, but induces mitochondrial biogenesis. Interestingly, administration of a RhoA activator, the cytotoxic necrotizing factor-1 (CNF1) effectively converts SMG-triggered alterations and effects on mitochondria biogenesis or glycolysis. CNF1 also converts the SMG-altered cell proliferation and tumor metastasis. In contrast, mTORC inhibitor, rapamycin, produces opposite responses and mimics SMG-induced effects in cells at normal gravity. Taken together, our observations indicate that SMG inhibits focal adhesions, leading to inhibition of signaling FAK and RhoA, and the mTORC1 pathway, which results in activation of the AMPK pathway and reduced melanoma cell proliferation and metastasis. Overall, our findings shed a new light on effects of microgravity on cell biology and human health.

Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease

“…However, the data surrounding the response of the actin cytoskeleton to microgravity exposure are less clear. Many studies have reported that microgravity exposure had decreased expression of actin and actin-associated proteins, namely Arp2/3 and RhoA, subsequently resulting in the disorganization of the actin cytoskeleton (Carlsson et al, 2003;Higashibata et al, 2006;Corydon et al, 2016a,b;Louis et al, 2017;Tan et al, 2018). However, other studies have showed increased F-actin and stress fiber formation that accompanied the development of lamellipodia protrusions following exposure to microgravity (Gruener et al, 1994;Nassef et al, 2019).…”

“…Binding of integrins to matrix proteins promotes the bundling of F-actin at the cell-matrix adhesion and the subsequent maturation of both the focal adhesion and the actin stress fiber (Zaidel-Bar et al, 2003;Wolfenson et al, 2009Wolfenson et al, , 2011 to generate the tension required for cell adherence, migration, and tissue homeostasis. Exposure to microgravity reduces the formation, number, and total area of focal adhesions per cell (Guignandon et al, 2003;Tan et al, 2018) consequently affecting cellular adherence, migration capacity, and viability (Plett et al, 2004;Nabavi et al, 2011;Shi et al, 2015;Ahn et al, 2019;Dietz et al, 2019), albeit with contradictory results. Mechanical unloading has been shown to significantly reduce gene expression of a number of focal adhesion proteins, including FAK, DOCK1, and PTEN, while caveolin and p130Cas expression were shown to be increased (Grenon et al, 2013;Ratushnyy and Buravkova, 2017).…”

“…Mechanical unloading has been shown to significantly reduce gene expression of a number of focal adhesion proteins, including FAK, DOCK1, and PTEN, while caveolin and p130Cas expression were shown to be increased (Grenon et al, 2013;Ratushnyy and Buravkova, 2017). Thus, the activity of the downstream signaling pathways that govern the microgravity-induced cytoskeletal changes are significantly impaired and are at least in part due to the microgravitytriggered inhibition of FAK and/or RhoA signaling (Higashibata et al, 2006;Li et al, 2009;Tan et al, 2018). Furthermore, recent data suggest that changes to the cytoskeleton may also impact signaling via mechanically activated ion channels and contacts in response to both cell-generated (Nourse and Pathak, 2017;Ellefsen et al, 2019) and externally applied mechanical inputs (Bavi et al, 2019).…”

“…When applied to tumor cells microgravity has been found to impact tumor cell adhesion, proliferation, migration, and viability (Grimm et al, 2002;Plett et al, 2004;Infanger et al, 2006a;Shi et al, 2015;Tan et al, 2018), and to induce cell autophagy (Jeong et al, 2018). Changes in apoptotic rate were also observed in colorectal cancer cells (DLD-1) and a lymphoblast leukemic cell line (MOLT-4), accompanied by reduced transcription of the genes involved in colony formation, oncogenic progression, and metastatic potential (Vidyasekar et al, 2015).…”

Guanylyl Cyclase-cGMP Signaling Pathway in Melanocytes: Differential Effects of Altered Gravity in Non-Metastatic and Metastatic Cells

“…In addition, a reduction in the migration has also been reported for a human adenocarcinoma cell line [90] and human glioblastoma U87 cells during clinorotation (30 rpm) [91] as well as an impaired ability of fibroblasts to migrate in "a modeled microgravity conditions" using a RWV [92]. Moreover, it was reported that random positioning on a three-dimensional clinostat (with 30 • /s angular velocity) reduced BL6-10 melanoma cell proliferation, adhesion, and invasiveness in vitro and decreased tumor lung metastasis in vivo via FAK/RhoA-regulated mTORC1 and AMP-activated protein kinase pathways [93]. For the highly metastatic BL6-10 melanoma cells, it was further shown that RPM-exposure reduced the focal adhesions and altered the cytoskeleton and the nuclear positioning, leading to an enhanced cell apoptosis via suppression of the FAK/RoA-regulated mTORC1/NF-kB and ERK1/2 pathways [94].…”