Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a Kras G12D/p53 R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three‐dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK‐induced genes that facilitate extracellular matrix remodeling, with greatest fold‐changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13. MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three‐dimensional contexts. Treatment of Kras G12D/p53 R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor‐associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.
Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics. .
Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating Aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour suppressive mechanism.
The impermeable nature of the cell plasma membrane limits the therapeutic uses of many macromolecules and there is therefore a growing effort to circumvent this problem by designing strategies for targeted intracellular delivery. During the last decade several cell penetrating peptides, such as the HIV-1 tat peptide, have been shown to traverse the cell membrane, where integral protein transduction domains (PTDs) are responsible for their cellular uptake, and to reach the nucleus while retaining biological activity. It has since been discovered that PTDs can enable the cellular delivery of conjugated biomolecules and even nanoparticles, but nuclear delivery has remained problematic. This present study focuses on the development of water soluble, biocompatible gold nanoparticles of differing size functionalized with the HIV-1 tat PTD with the aim of producing nuclear targeting agents. The particles were subsequently tested in vitro with a human fibroblast cell line, with results demonstrating successful nanoparticle transfer across the plasma membrane, with 5 nm particles achieving nuclear entry while larger 30 nm particles are retained in the cytoplasm, suggesting entry is blocked via nuclear pores dimensions.
The bone marrow niche represents a specialized environment that regulates mesenchymal stem cell quiescence and self-renewal, yet fosters stem cell migration and differentiation upon demand. An in vitro model that embodies these features would open up the ability to perform detailed study of stem cell behavior. In this paper we present a simple bone marrow-like niche model, which comprises of nanomagnetically levitated stem cells cultured as multicellular spheroids within a type I collagen gel. The stem cells maintained are nestin positive and remain quiescent until regenerative demand is placed upon them. In response to coculture wounding, they migrate and appropriately differentiate upon engraftment. This tissue engineered regeneration-responsive bone marrow-like niche model will allow for greater understanding of stem cell response to injury and also facilitate as a testing platform for drug candidates in a multiwell plate format.
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