Breast tumours have a tendency to be highly desmoplastic with high collagen content. This work explores ROCK1 activity, regulation and cell contractility function during cell migration in high-density (HD) matrices. Live-cell imaging showed that tumour cells navigated through HD matrices by contraction of the cell body. Treatment with inhibitors demonstrated a role for ROCK1 and MMPs in cell migration. There was increased expression of invasive genes in HD compared to LD matrices including ROCK1, whereby both its expression and activity were significantly upregulated in denser matrices. This effect of the microenvironment on ROCK1 was sensitive to treatment with a HDAC inhibitor, MS-275, which upregulated Notch1 that in turn, suppressed ROCK1. This was shown by downregulation of Notch1 using siRNA knockdown and DAPT, which abrogated the inhibition of ROCK1 by MS-275.
Dense breast tissue shows increased stromal collagen and analyses of tumour material indicate that cancerous breast tissues are stiffer than healthy tissue. Stiffness or resistance to deformation measured from Young’s modulus of collagen matrices is dependent on the number of fibrillar cross-links and higher fibre densities[19, 54, 55]. Stiffer matrices promote invasion by increasing the numbers of active invadopodia and increase cell proliferation by elevating Rho-GTPase activity and cell adhesion. Tumour cells in turn, remodel the extracellular matrix for example, by realigning randomly organised collagen fibres into a radial configuration to help facilitate local invasion[14, 15, 58]. Tumour cells are also known to use protease to cleave ECM components and together with other mechanisms to contract and reorganise the collagen matrix to provide space required for cell migration. It is conceivable that matrix reorganisation via pushing of protrusions, contraction of the cell body and local matrix proteolysis serve to reduce matrix stiffness and facilitate cell migration. This study showed that levels of ROCK transcript, protein and protein activity were significantly upregulated in stiff matrices coincident with the observation of cell body contractility utilised for migration. Unlike other biological programs such as proliferation and differentiation where cells are committed to specific pathways, cells can switch between regulatory pathways and migration modes for invasion. Protrusion-, contractility- or protease-led mechanisms are interchangeably utilised by tumour cells. These are dependent on environmental conditions and cell proclivities related to genetic make-up governing polarity, adhesion and cytoskeletal functions. Variations in these factors lead to a number of permutations in the migration mode of tumour cells. For example, blockade of MMPs causes mesenchymal tumour cells to switch to cell contractility for migration similar to amoeboid cells in LD matrices[15, 22, 57, 60].
In HD matrix, ROCK inhibition had no effect on migration even though live DIC microscopy showed evidence of cell contractility. It is possible that in the absence of ROCK, protease-led migration might compensatory. Indeed, inhibiting both MMPs and ROCK, cooperatively/synergistically reduced migration levels, albeit at the lower end of the GM6001 concentration (12.5 μm) used. This suggests that at a critical level of MMPs, ROCK is required for efficient cell migration. At higher GM6001 concentrations (above 12.5 μm), addition of ROCK inhibitors has no further effects suggesting that ROCK can no longer compensate for migration. Here, we are able to glimpse into how tumour cells are inherently plastic where cells can swap between migration modes utilising ROCK1 and/or MMPs. Residual migration (when ROCK1 and proteolysis are inhibited) suggests that a third pathway is utilised, possibly one that controls protrusion-led migration. Indeed, we observe that tumour cells migrate into dense matrices utilising enlarged protrusions that interacts with collagen fibrils to gain traction ( Additional file1: Movie S1).
Epigenetics have been shown to play a role in regulating ROCK1 expression as a function of cell adhesion, an environmental cue. Cells in suspension expressed more ROCK1 compared with adherent cells and the use of an HDAC inhibitor further increased the expression of ROCK1 in suspension cultures. The function of ROCK1 was to generate cell contractility that blocked adhesion in the cells in suspension. Here we explored whether epigenetics might also play a part in the regulation of ROCK1 when cells experience microenvironmental differences in matrix stiffness. ROCK1 expression and activity was significantly upregulated in the highly elastic HD matrix compared to LD matrix. Blocking HDAC function using MS-275 downregulated ROCK1 and this could result from either direct or indirect effects of the drug.
HDACs deacetylate histones reducing accessibility of DNA to the transcription machinery resulting in inactive chromatin. Furthermore, histone deacetylation can also lead to methylation-dependent transcriptional activation[61, 62]. There are two possibilities as to how HDACs might increase ROCK1 transcript in HD matrix. This might occur directly through HDAC promotion of histone methylation at H3K4Me and activation of ROCK1 gene transcription (as opposed to downregulating genes at other methylated sites such as H3K9Me.). The alternative hypothesis is that in HD matrix, HDAC suppresses an inhibitor of ROCK1 so that addition of MS-275 abrogates this suppression, leading to the downregulation of ROCK1. To test this, we used cycloheximide (CHX) to block protein translation and showed that CHX prevented the downregulation of ROCK1 transcript and protein activity in the presence of MS-275. This suggests that the effect of MS-275 on ROCK1 is indirect and it is dependent on another protein(s) upregulated by MS-275.
ROCK activity is regulated by Rho GTPase, which frees the kinase region from the autoinhibitory carboxy-terminal region of ROCK1 and it is also activated autonomously from Rho. ROCK phosphorylates substrates that function in the assembly of actin filaments and in cell contractility including ezrin-radixin-moesin (ERM) proteins and MLC. Phosphorylation of the MLC of myosin II activates myosin ATPase and consequently promotes cell contractility. Furthermore, ROCK also phosphorylates the myosin-binding subunit (MYPT1) of myosin light–chain phosphatase (MLCP), a negative regulator of MLC, resulting in enhanced contractility. We show that using blebbistatin to block myosin II, downstream of ROCK, has no effect on cell migration ( Additional file4: Figure S3).
Apart from self-regulation at the protein level, ROCK can be controlled at the transcript level. In keratinocytes, p53 positively regulates Notch1 and both these factors inhibit ROCK1/2. Notch is a type I transmembrane receptor with a key role in cell fate determination and the differentiation of cells during development. Inhibition of Notch increases tumour formation by primary human keratinocytes expressing oncogenic Ras, suggesting a tumour suppressor role for Notch. Blockade of Notch also suppressed differentiation and increased stem cell populations. The binding of cognate ligands to the Notch receptor is followed by proteolytic cleavage of Notch, releasing its intracellular active domain. Notch translocates to the nucleus and interacts with DNA-binding proteins such as CSL, converting it from a transcriptional repressor to an activator[67, 68]. Notch also binds Mastermind-like 1 (MAML1) to further elevate CSL-regulated transcriptional activation. The Notch/CSL/MAML pathway targets the HES and HERP families of basic helix-loop-helix (bHLH) transcriptional repressors. Conserved HES-binding sites in turn, can be found in the promoter regions of ROCK2 and MRCKα genes, the effectors of RhoA and CDC42, respectively. Notch promotes the repressor function of HES1 leading to the downregulation of ROCK2 and MRCKα gene expression. Furthermore, use of DAPT increased the expression of ROCK1 and 2, supporting the idea that Notch1 normally controls these genes in keratinocytes to prevent tumour progression. The transcript levels of Notch have been shown to be upregulated in mouse embryos treated with trichostatin A, a potent HDAC-inhibitor. Therefore, there is evidence to suggest that Notch1 not only negatively regulates ROCK1 at the promoter level but that HDAC inhibitors upregulate Notch1 gene expression.
In HD matrix, we find that Notch1 but not p53 was upregulated by MS-275 and the increase in Notch1 levels was independent of CHX. When Notch1 activation was blocked using a γ-secretase inhibitor, DAPT, or when Notch1 levels were reduced by pooled siRNA transfection, the effect of MS-275 on ROCK1 activity was abrogated. The data suggest that MS-275 directly upregulates Notch1, which in turn blocks ROCK1 expression perhaps via repressor activities on the ROCK1 promoter.