Ras promotes the accumulation of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Previous studies reported that acute Raf/MEK/ERK activation elevates p21 protein levels by increased transcription. However, we have found that p21 induction in Ras-transformed murine fibroblasts occurs principally by a post-translational mechanism. Chronic activation of the Raf/MEK/ERK pathway blocked proteasome-mediated p21 degradation, resulting in accumulation of p21 protein with an elevated half-life. The stabilization of p21 by Ras was accompanied by high levels of p21-associated cyclin D1 and, similarly to Ras, cyclin D1 was sufficient to inhibit the proteasome-mediated p21 degradation. Knock-down of cyclin D1 by RNA interference confirmed that Ras-induced p21 stabilization was dependent upon cyclin D1 expression. We show that p21 directly binds to the C8alpha subunit of the 20S proteasome complex and that by competing for binding, cyclin D1 inhibits p21 degradation by purified 20S complexes in vitro. Therefore, we propose that Ras stabilizes p21 by promoting the formation of p21-cyclin D1 complexes that prevent p21 association with, and subsequent degradation by, the 20S proteasome.

The RhoA and RhoC GTPases act via the ROCK1 and ROCK2 kinases to promote actomyosin contraction, resulting in directly induced changes in cytoskeleton structures and altered gene transcription via several possible indirect routes. Elevated activation of the Rho/ROCK pathway has been reported in several diseases and pathological conditions, including disorders of the central nervous system, cardiovascular dysfunctions and cancer. To determine how increased ROCK signalling affected gene expression in pancreatic ductal adenocarcinoma (PDAC) cells, we transduced mouse PDAC cell lines with retroviral constructs encoding fusion proteins that enable conditional activation of ROCK1 or ROCK2, and subsequently performed RNA sequencing (RNA-Seq) using the Illumina NextSeq 500 platform. We describe how gene expression datasets were generated and validated by comparing data obtained by RNA-Seq with RT-qPCR results. Activation of ROCK1 or ROCK2 signalling induced significant changes in gene expression that could be used to determine how actomyosin contractility influences gene transcription in pancreatic cancer.

Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development.

The 20 members of the Rho GTPase family are key regulators of a wide-variety of biological activities. In response to activation, they signal via downstream effector proteins to induce dynamic alterations in the organization of the actomyosin cytoskeleton. In this review, post-translational modifications, mechanisms of dysregulation identified in human pathological conditions, and the ways that Rho GTPases might be targeted for chemotherapy will be discussed.

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that functions at a constitutive level in cells, which may become further activated by stressors such as nutrient starvation or protein aggregation. Autophagy has multiple beneficial roles for maintaining normal cellular homeostasis and these roles are related to the implications of autophagy in disease mechanisms including neurodegeneration and cancer. We previously searched for novel autophagy regulators and identified Rho-kinase 1 (ROCK1) as a candidate. Here, we show that activated ROCK1 inhibits autophagy in human embryonic kidney 293 cells. Conversely, ROCK inhibitory compounds enhanced the autophagy response to amino acid starvation or rapamycin treatment. Inhibition of ROCK during the starvation period led to a more rapid response with the production of larger early autophagosomes that matured into enlarged late degradative autolysosomes. Despite the production of enlarged LC3-positive early autophagosomes, membrane precursors containing WD-repeat protein interacting with phosphoinositides 1 (WIPI1) and mammalian Atg9 were not affected by ROCK inhibition, suggesting that phagophore elongation had been unusually extended. However, the enlarged autophagosomes were enriched in ULK1 which was essential to allow progression of autophagy flux. Our results demonstrate a novel role for ROCK in the control of autophagosome size and degradative capacity.

The morphological changes during apoptosis help facilitate "immunologically silent" cell death. Caspase cleavage of the ROCK1 kinase results in its activation, which drives the forceful contraction of apoptotic cells. We previously showed that when ROCK1 was mutated to render it caspase-resistant, there was greater liver damage and neutrophil recruitment after treatment with the hepatotoxin diethylnitrosamine (DEN). We now show that acute DEN-induced liver damage induced higher levels of pro-inflammatory cytokines/chemokines, indicative of immunogenic cell death (ICD), in mice expressing non-cleavable ROCK1 (ROCK1nc). Hepatocellular carcinoma (HCC) tumours in ROCK1nc mice had more neutrophils and CD8+ T cells relative to mice expressing wild-type ROCK1, indicating that spontaneous tumour cell death also was more immunogenic. Since ICD induction has been proposed to be tumour-suppressive, the effects of two distinct ROCK inhibitors on HCC tumours was examined. Both fasudil and AT13148 significantly decreased tumour numbers, areas and volumes, but neither resulted in greater numbers of neutrophils or CD8+ T cells to be recruited. In the context of acute DEN-induced liver damage, AT13148 inhibited the recruitment of dendritic, natural killer and CD8+ T cells to livers. These observations indicate that there is an important role for ROCK1 cleavage to limit immunogenic cell death, which was not replicated by systemic ROCK inhibitor administration. As a result, concomitant administration of ROCK inhibitors with cancer therapeutics would be unlikely to result in therapeutic benefit by inducing ICD to increase anti-tumour immune responses.

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.This article has an associated First Person interview with the first author of the paper.

The Adenomatous Polyposis Coli (APC) gene is mutated in the majority of colorectal cancers (CRCs). Loss of APC leads to constitutively active WNT signaling, hyperproliferation, and tumorigenesis. Identification of pathways that facilitate tumorigenesis after APC loss is important for therapeutic development. Here, we show that RAC1 is a critical mediator of tumorigenesis after APC loss. We find that RAC1 is required for expansion of the LGR5 intestinal stem cell (ISC) signature, progenitor hyperproliferation, and transformation. Mechanistically, RAC1-driven ROS and NF-κB signaling mediate these processes. Together, these data highlight that ROS production and NF-κB activation triggered by RAC1 are critical events in CRC initiation.

Androgen receptor (AR) signalling and the PI3K pathway mediate survival signals in prostate cancer, and have been shown to regulate each other by reciprocal negative feedback, such that inhibition of one activates the other. Understanding the reciprocal regulation of these pathways is important for disease management as tumour cells can adapt and survive when either single pathway is inhibited pharmacologically. We recently carried out genome-wide exon-specific profiling of prostate cancer cells to identify novel androgen-regulated transcriptional events. Here we interrogated this dataset for novel androgen-regulated genes associated with the PI3K pathway. We find that the PI3K regulatory subunits PIK3R1 (p85α) and PIK3R3 (p55γ) are direct targets of the AR which are rapidly repressed by androgens in LNCaP cells. Further characterisation revealed that the PIK3CA p110α catalytic subunit is also indirectly regulated by androgens at the protein level. We show that PIK3R1 mRNA is significantly under-expressed in prostate cancer (PCa) tissue, and provide data to suggest a context-dependent regulatory mechanism whereby repression of the p85α protein by the AR results in destabilisation of the PI3K p110α catalytic subunit and downstream PI3K pathway inhibition that functionally affects the properties of prostate cancer cells.

Although chemotherapy for prostate cancer (PCa) can improve patient survival, some tumours are chemo-resistant. Tumour molecular profiles may help identify the mechanisms of drug action and identify potential prognostic biomarkers. We performed in vivo transcriptome profiling of pre- and post-treatment prostatic biopsies from patients with advanced hormone-naive prostate cancer treated with docetaxel chemotherapy and androgen deprivation therapy (ADT) with an aim to identify the mechanisms of drug action and identify prognostic biomarkers.

The contractile actin-myosin cytoskeleton provides much of the force required for numerous cellular activities such as motility, adhesion, cytokinesis and changes in morphology. Key elements that respond to various signal pathways are the myosin II regulatory light chains (MLC), which participate in actin-myosin contraction by modulating the ATPase activity and consequent contractile force generation mediated by myosin heavy chain heads. Considerable effort has focussed on the role of MLC kinases, and yet the contributions of the myotonic dystrophy-related Cdc42-binding kinases (MRCK) proteins in MLC phosphorylation and cytoskeleton regulation have not been well characterized. In contrast to the closely related ROCK1 and ROCK2 kinases that are regulated by the RhoA and RhoC GTPases, there is relatively little information about the CDC42-regulated MRCKα, MRCKβ and MRCKγ members of the AGC (PKA, PKG and PKC) kinase family. As well as differences in upstream activation pathways, MRCK and ROCK kinases apparently differ in the way that they spatially regulate MLC phosphorylation, which ultimately affects their influence on the organization and dynamics of the actin-myosin cytoskeleton. In this review, we will summarize the MRCK protein structures, expression patterns, small molecule inhibitors, biological functions and associations with human diseases such as cancer.

Wnt/β-catenin signal transduction directs intestinal stem cell (ISC) proliferation during homeostasis. Hyperactivation of Wnt signaling initiates colorectal cancer, which most frequently results from truncation of the tumor suppressor Adenomatous polyposis coli (APC). The β-catenin-TCF transcription complex activates both the physiological expression of Wnt target genes in the normal intestinal epithelium and their aberrantly increased expression in colorectal tumors. Whether mechanistic differences in the Wnt transcription machinery drive these distinct levels of target gene activation in physiological versus pathological states remains uncertain, but is relevant for the design of new therapeutic strategies. Here, using a Drosophila model, we demonstrate that two evolutionarily conserved transcription cofactors, Earthbound (Ebd) and Erect wing (Ewg), are essential for all major consequences of Apc1 inactivation in the intestine: the hyperactivation of Wnt target gene expression, excess number of ISCs, and hyperplasia of the epithelium. In contrast, only Ebd, but not Ewg, mediates the Wnt-dependent regulation of ISC proliferation during homeostasis. Therefore, in the adult intestine, Ebd acts independently of Ewg in physiological Wnt signaling, but cooperates with Ewg to induce the hyperactivation of Wnt target gene expression following Apc1 loss. These findings have relevance for human tumorigenesis, as Jerky (JRK/JH8), the human Ebd homolog, promotes Wnt pathway hyperactivation and is overexpressed in colorectal, breast, and ovarian cancers. Together, our findings reveal distinct requirements for Ebd and Ewg in physiological Wnt pathway activation versus oncogenic Wnt pathway hyperactivation following Apc1 loss. Such differentially utilized transcription cofactors may offer new opportunities for the selective targeting of Wnt-driven cancers.

The metastatic spread of cancer cells is a step-wise process that starts with dissociation from primary tumours and local invasion of adjacent tissues. The ability to invade local tissues is the product of several processes, including degradation of extracellular matrices (ECM) and movement of tumour cells through physically-restricting gaps. To identify properties contributing to tumour cells squeezing through narrow gaps, invasive MDA-MB-231 human breast cancer and MDA-MB-435 human melanoma cells were subjected to three successive rounds of selection using cell culture inserts with highly constraining 3 μm pores. For comparison purposes, flow cytometry was also employed to enrich for small diameter MDA-MB-231 cells. RNA-Sequencing (RNA-seq) using the Illumina NextSeq 500 platform was undertaken to characterize how gene expression differed between parental, invasive pore selected or small diameter cells. Gene expression results obtained by RNA-seq were validated by comparing with RT-qPCR. Transcriptomic data generated could be used to determine how alterations that enable cell passage through narrow spaces contribute to local invasion and metastasis.

Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

The Clostridium botulinum C3 exoenzyme has been an invaluable tool for the study of the biological functions of Rho GTPases. The C3 enzyme selectively catalyzes the ADP-ribosylation, and consequent inactivation, of RhoA, RhoB, and RhoC of the Rho GTPase protein family. Through the experimental use of C3, it has been possible to determine the contributions made by these signaling proteins to processes including the regulation of cell morphology, cell cycle progression, and gene transcription. Unlike bacterial toxins that have some means to attach to and/or enter cells, C3 does not have an element that facilitates efficient entry. As a result, numerous methods have been used to effectively deliver C3 into cells. One approach has been to engineer a recombinant C3 with an HIV TAT leader sequence that permits transduction of the protein across the plasma membrane. In this chapter, the purification and characterization of the recombinant TAT-C3 protein is described.

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

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 KrasG12D/p53R172H 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 KrasG12D/p53R172H 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.

Cutaneous squamous cell carcinomas (SCCs) are commonly diagnosed skin cancers that may progress to invasiveness in the absence of early intervention. Using a murine model of SCC, we have previously demonstrated that activation of the Rho-associated kinase (ROCK) signaling pathway promotes rapid progression of pre-neoplastic lesions to invasive SCC. Herein we demonstrate that in human cutaneous SCC, ROCK signaling is increasingly up-regulated with tumor progression in both tumor cells and cells of the tumor microenvironment and is accompanied by key tumor-promoting changes in the extracellular matrix protein composition. The mechanotransduction pathway mediated by integrin signaling through FAK, GSK3β, and the transcription coactivator β-catenin is also progressively activated in human cutaneous SCC. Our observations indicate that ROCK activation is a tumor promoter in human cutaneous SCC and acts via mechanotransduction of signals to β-catenin. Our experiments raise the possibility that inhibition of ROCK signaling could be a useful therapeutic approach to halt cutaneous SCC progression by reducing the signal flux through this pathway to physiologic levels, thereby normalizing the extracellular matrix composition.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.