Irina Jahin & Thomas A Phillips: Extracellular matrix stiffness activates mechanosensitive signals but limits breast cancer cell spheroid proliferation and invasion
Extracellular matrix stiffness increases as tumors develop and spread, with stiffer environments thought to correlate with poorer disease prognosis. Changes in extracellular stiffness and other physical characteristics are sensed by integrins which integrate these extracellular cues to intracellular signaling, resulting in modulation of proliferation and invasion. However, the co-ordination of mechano-sensitive signaling with functional changes to groups of tumor cells within 3-dimensional environments remains poorly understood.
Here we provide evidence that increasing the stiffness of collagen scaffolds results in increased activation of ERK1/2 and YAP in human breast cancer cell spheroids. We also show that ERK1/2 acts upstream of YAP activation in this context. We further demonstrate that YAP, matrix metalloproteinases and actomyosin contractility are required for collagen remodeling, proliferation and invasion in lower stiffness scaffolds.
However, the increased activation of these proteins in higher stiffness 3-dimensional collagen gels is correlated with reduced proliferation and reduced invasion of cancer cell spheroids. Our data collectively provide evidence that higher stiffness 3-dimensional environments induce mechano-signaling but contrary to evidence from 2-dimensional studies, this is not sufficient to promote pro-tumorigenic effects in breast cancer cell spheroids.
Solid tumors exist within complex three-dimensional (3D) extracellular matrices (ECM) comprised of a complex network of collagens with other constituent macromolecules such as fibronectin, hyaluronic acid and proteoglycans (Mohan et al., 2020). Cells sense biophysical properties of the surrounding ECM through integrins and other surface receptors driving downstream signaling pathways and transcription (Cooper and Giancotti, 2019). These mechano-sensitive signaling pathways control a range of functional outcomes including mitotic signaling and cytoskeletal re-organization to promote invasion through the surrounding matrix (Yamada and Sixt, 2019). Increased ECM stiffness can occur during tumor progression (Handorf et al., 2015), with in vitro data supporting roles for these changes in control of cancer cell motility and proliferation (Wullkopf et al., 2018). The pathogenic role of increasing stiffness within the tumor microenvironment has been particularly well documented in breast cancer progression, with higher ECM stiffness correlating with poor prognosis and drug resistance (Provenzano et al., 2008; Levental et al., 2009; Boyd et al., 2014; Acerbi et al., 2015; Lindstrom et al., 2015; Joyce et al., 2018).
Several key mechanosensitive signaling pathways have been identified as potential regulators of cancer cell responses to increasing ECM stiffness. The transcription factor YAP is perhaps the best studied of these pathways, with its activation triggered by extracellular stiffness leading to downstream transcriptional regulation. YAP (and its paralog TAZ) are central, overlapping, but distinct components of the Hippo signaling pathway that controls cell fate, cell growth, organ size, and morphogenesis (Davis and Tapon, 2019). The core pathway functions with MST1/2 and binding partner SAV1 activating LATS1/2 kinases and their scaffold MOB1A/B. Activated LATS kinase then phosphorylates and inactivates YAP/TAZ. Phosphorylated YAP/TAZ is sequestered in the cytoplasm through interaction with 14-3-3 proteins or is subsequently ubiquitinated and degraded by the proteasome (Cai et al., 2021). YAP/TAZ activity promotes interaction with TEAD transcription factors in the nucleus to induce expression of a plethora of downstream targets which promote proliferation, cell survival and ECM remodeling (Davis and Tapon, 2019). Biomechanical regulation of YAP activity by cell-cell junctions, focal adhesions and by the actin cytoskeleton have all been suggested to play roles in tumourigenesis (Moreno-Vicente et al., 2019), but contributions of YAP activity to breast cancer progression remain controversial (Lee et al., 2019; Fresques and LaBarge, 2020; Qin et al., 2020).
Emerging evidence also suggests that the MAP kinase (MAPK) signaling pathways are important mechano-sensitive proteins that may play a role in stiffness sensing in cancer. Oncogenic mutations to MAPK signaling components have been well studied as contributing to uncontrolled growth and tumorigenesis (Braicu et al., 2019). ERK1/2 is one family member that typically integrates growth factors and mitogens to induce cell growth and differentiation (McKay and Morrison, 2007). The ERK signaling cascade is triggered by the small GTPase Ras, which is activated in response to mitogen stimulations of receptor tyrosine kinases at the plasma membrane. Once phosphorylated, ERK enters the nucleus and alters gene expression by phosphorylating transcription factors such as Elk1, Ets-2 and c-Myc in addition to cytoplasmic substrates (Unal et al., 2017). ERK has been suggested to be mechano-responsive downstream of stiffness-induced receptor tyrosine kinase activation (Hwang et al., 2015; Zhang et al., 2017; Yang et al., 2018; Farahani et al., 2021). Moreover, MEK inhibitors have been suggested to specifically block stiffness induced drug resistance in breast cancer cells (Schwartz et al., 2017). Interestingly, several lines of evidence suggest that YAP and ERK may be co-regulated by extracellular stiffness in cancer. A reduction in the YAP target Cyr61 leads to reduced ERK phosphorylation and breast cancer cell invasion (Hellinger et al., 2019). ERK has also been shown to be upstream of YAP in breast cancer cell lines subjected to low shear stress (Qin et al., 2019). Conversely, ERK phosphorylation is reduced following YAP knockdown in colon cancer cell lines (Liu et al., 2019) suggesting a complex regulatory axis may be in play. Evidence for ERK/YAP synergy can also be found in ovarian cancer (Shi et al., 2020) and melanoma (Yu et al., 2015) cell lines.