The Role of Mechanotransduction in Contact Inhibition of Locomotion and Proliferation

Abstract

Contact inhibition (CI) represents a crucial tumor-suppressive mechanism responsible for controlling the unbridled growth of cells, thus preventing the formation of cancerous tissues. CI can be further categorized into two distinct yet interrelated components: CI of locomotion (CIL) and CI of proliferation (CIP). These two components of CI have historically been viewed as separate processes, but emerging research suggests that they may be regulated by both distinct and shared pathways. Specifically, recent studies have indicated that both CIP and CIL utilize mechanotransduction pathways, a process that involves cells sensing and responding to mechanical forces. This review article describes the role of mechanotransduction in CI, shedding light on how mechanical forces regulate CIL and CIP. Emphasis is placed on filamin A (FLNA)-mediated mechanotransduction, elucidating how FLNA senses mechanical forces and translates them into crucial biochemical signals that regulate cell locomotion and proliferation. In addition to FLNA, trans-acting factors (TAFs), which are proteins or regulatory RNAs capable of directly or indirectly binding to specific DNA sequences in distant genes to regulate gene expression, emerge as sensitive players in both the mechanotransduction and signaling pathways of CI. This article presents methods for identifying these TAF proteins and profiling the associated changes in chromatin structure, offering valuable insights into CI and other biological functions mediated by mechanotransduction. Finally, it addresses unanswered research questions in these fields and delineates their possible future directions.

Keywords: WW domain-containing transcription regulator protein 1 (TAZ); Yes-associated protein (YAP1); contact inhibition; contact inhibition of locomotion; contact inhibition of proliferation; filamin; gene expression; mechanical force; mechanotransduction; proteomics; ubiquitin-conjugating enzyme E2 A and B (UBE2A/B).

Figure 5

Regulation of chromatin structure and stretching of NPC in CI and mechanotransduction. (A) The physical connection between chromatin and the ECM may lead to the opening of chromatin regions where transcription-associated factors (TAFs) can bind for gene expression. This process could be regulated by the transmission of mechanical force through transmembrane proteins, the cytoskeleton, the LINC complex, and nuclear lamins. Additionally, the movement of TAFs could be influenced by the mechanical stretching of NPC. The SUN domains of SUN proteins directly interact with the KASH domains of nesprin proteins. Nuclear lamin-associated membrane proteins include emerin, LAP1/2, LBR, and MAN1 (inner nuclear membrane protein Man1). PTM: post-translational modification, HP1: heterochromatin protein 1. Numerous additional proteins have been detected in the nuclear envelope using proteomic methods, yet these findings require further confirmation [170]. (B) TAFs (red) bind to open chromatin to regulate gene expression. These binding loci of TAFs are distinct from the gene body, where modifications like histone H3 lysine-79 dimethylation (H3K79me2, blue) are present. Furthermore, histone H3 lysine-4 trimethylation (H3K4me3) modifications (promoter, magenta) are found in broader regions upstream of the transcription start site (TSS). TES, transcription end site. In various biological processes, such as CI and other mechanotransduction-mediated biological processes, gene regulation involves changes in chromatin structure due to epigenetic modifications. The DSP-MNase-proteogenomics method identifies TAFs and their binding loci [171]. The heatmaps represent the read coverage or signal intensities across genomic regions and DNA samples extracted from open chromatin using MNase (see Figure 6 for details). Heatmaps identify patterns of enrichment or depletion in specific regions of open chromatin and can provide insights into the regulatory landscape of the genome. Heatmap analysis of DNA fragments extracted from low- and high-density cells revealed that TAFs bind to accessible regions of chromatin, which are situated near the upstream of TSS, and displace histone H3 in the process. As expected, H3K4me3 and H3K79me2 modifications are enriched in the promoter region and gene body, respectively.

Figure 1

Contact inhibition of locomotion and proliferation in normal and cancer cells. (A) Top view: When cells contact each other, the cells change the direction of movement (CIL). At high density, cells stop proliferation (CIP). (B) Side view: When normal cells reach the monolayer, the cells stop movement and growth; thereby, both CIL and CIP are on. In cancer cells, metastatic cells migrate (CIL is off) and settle at a target organ (CIL is on). Some cancer cells leave the primary site and move (CIL is off). Cancer cells can grow even at high density (CIP is off).

Figure 2

Signaling pathways in CIL and CIP. (A) Signaling pathways of CIL. In type I CIL, cell protrusion is inhibited through transmembrane proteins that regulate the balance of RhoA and Rac activities. Wnt-planar cell polarity (PCP) pathway, junctional adhesion molecules (JAMs). In type II CIL, inhibition of protrusion may be regulated by (a) force generated by collision and alteration of membrane tension, and/or (b) loss of adhesion to a colliding cell. (B) Signaling pathways of CIP. At low cell density, TAFs (e.g., transcriptional coactivator YAP1, with the transcription factor represented in magenta and the repressor in cyan) bound to chromatin either induce or suppress gene expression for proliferation, respectively. Both biochemical signals and mechanical forces control the activation of genes. At high density, transmembrane proteins, membrane tension, and the relaxation of myosin contraction promote the translocation and dissociation/association of TAFs to chromatin, leading to growth arrest. Some TAFs may bind to chromatin only in high-density cells, although such TAFs have not yet been identified. Receptor tyrosine kinase (RTK).

Figure 3

Molecules that change conformation by mechanical forces.

Figure 4

Mechanotransduction in animal cells.

Figure 6

Methods to identify mechanosenstive TAFs. (A) DSP-MNase-proteogenomics [171]. DNA-TAF complex is stabilized with a reversible cross-linker, dithiobis(succinimidyl propionate) (DSP). After washing out the majority of proteins, the DNA-TAF complex is cleaved off using micrococcal nuclease (MNase) and separated from cross-linked chromatin by centrifugation. After de-cross-linking the cleaved DNA-TAF complex, TFA proteins can be identified by conventional or stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. DNA fragments can be sequenced by NGS. (B) DNase I hypersensitive sites (DHSs) proteomics [87]. After washing out soluble cytoplasmic proteins using hypotonic buffer, loose and open chromatin is cleaved off using DNase I and fractionated. Proteomics using SILAC quantitatively identify proteins that increased or decreased after stimulation. Since no crosslinking and extensive washing are involved, proteins that transiently associate with chromatin can be detected. Light blue indicates nucleosome. Red (strong interaction) and purple (transient interaction) indicate TAFs. Green indicates enzyme. Magenta indicates PTM of histone. Orange indicates DSP crosslinker.

Figure 7

FLNA-mediated mechanotransduction. (A) The structure of FLNA includes paired domains that may undergo unfolding in response to mechanical forces [99,203]. (B) Mechanical force induces conformational changes in the FLNA molecule, exposing cryptic binding sites and altering the geometry of FLNA subunits (e.g., A: FilGAP, B: integrins). (C) FLNA and F-actin are highly concentrated at the leading edge of the cell. 100 × 100 μm. (D) The model proposes that cell protraction at the leading edge is regulated by the force-dependent interaction between FLNA and FilGAP. The yellow-highlighted “P” indicates phosphorylation (activated FilGAP). For further details, refer to the text.

Figure 8

Hippo-dependent and -independent pathways for cell proliferation. The pink-colored nucleus indicates the localization of YAP1/TAZ in the nucleus, suggesting a cell growth state. Nevertheless, it has been reported that YAP1 nuclear localization alone is insufficient as an indicator of YAP1 activity (see Section 3). (A,B) The Hippo pathway can be turned off or inhibited by several mechanisms and factors, such as alterations in cell polarity, growth factors, oncogenic signaling, nutrient availability and metabolic signals. When the Hippo pathway is turned off, the core kinases of the pathway, particularly LATS1/2, become inactive. This leads to reduced or no phosphorylation of YAP1. Non-phosphorylated YAP1 then translocates to the nucleus and acts as a transcriptional co-activator. It binds to various transcription factors, most notably those in the TEAD family. This interaction triggers the activation of genes that promote cell proliferation and survival (resistance to apoptosis), as well as other genes involved in processes such as stem cell activation. The S127A mutant of YAP1 is resistant to phosphorylation and is retained within the nucleus [230]. The Hippo pathway is activated by various extracellular and intracellular signals, including G-protein-coupled receptor signaling, as well as signals downstream of cellular metabolism and CIP. These signals can influence the activity of the pathway’s core components, which include mammalian STE20-like protein kinases (MST1/2) and LATS1/2. SAV1, MOB1, and other factors also promote the kinase activity of their binding partners. Once activated, LATS1/2 phosphorylate YAP1/TAZ. When phosphorylated, YAP1/TAZ are retained in the cytoplasm and become subject to degradation. Normally, the Hippo pathway restricts growth by suppressing YAP1. However, oncogenes such as Ras and Src can either inhibit the Hippo pathway or directly activate YAP1, thus promoting uncontrolled cell growth and contributing to cancer development. (C,D) Hypothesis: Cells can proliferate through various growth signals even in the absence of the Hippo pathway or YAP1 (this can occur even when the Hippo pathway is active), potentially leading to uncontrolled growth. (E,F) Mechanical forces can influence YAP1 translocation into the nucleus independently of the Hippo pathway. YAP1 remains in the cytosol on soft substrates, even when its phosphorylation is inhibited, indicating that dephosphorylation alone does not ensure its nuclear entry. The stretching of NPC through mechanical forces and substrate stiffness, mediated by the LINC complex and actin cytoskeleton, plays a key role in YAP1’s nuclear import, which varies with substrate rigidity [62]. Nevertheless, it has been observed that the opening of the NPC alone is insufficient for the nuclear entry of YAP1 (see Section 3). (G,H) The binding of SAV1 to FLNA under tension retains MST1/2 and LATS1/2 in the cytoplasm, thereby endorsing the activity of YAP1 in the nucleus. In relaxed cells, SAV1 is released from FLNA to induce apoptosis by an unknown mechanism. Some of the released SAV1 might translocate to the nucleus, presumably with MST1/2 and LATS1/2. Phosphorylation of YAP1 might take place in the cytosol if non-phospho-YAP1 can exit the nucleus [225].