Targeting ROCK activity to disrupt and prime pancreatic cancer for chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease; the identification of novel targets and development of effective treatment strategies are urgently needed to improve patient outcomes. Remodeling of the pancreatic stroma occurs during PDAC development, which drives disease progression and impairs responses to therapy. The actomyosin regulatory ROCK1 and ROCK2 kinases govern cell motility and contractility, and have been suggested to be potential targets for cancer therapy, particularly to reduce the metastatic spread of tumor cells. However, ROCK inhibitors are not currently used for cancer patient treatment, largely due to the overwhelming challenge faced in the development of anti-metastatic drugs, and a lack of clarity as to the cancer types most likely to benefit from ROCK inhibitor therapy. In 2 recent publications, we discovered that ROCK1 and ROCK2 expression were increased in PDAC, and that increased ROCK activity was associated with reduced survival and PDAC progression by enabling extracellular matrix (ECM) remodeling and invasive growth of pancreatic cancer cells. We also used intravital imaging to optimize ROCK inhibition using the pharmacological ROCK inhibitor fasudil (HA-1077), and demonstrated that short-term ROCK targeting, or ‘priming’, improved chemotherapy efficacy, disrupted cancer cell collective movement, and impaired metastasis. This body of work strongly indicates that the use of ROCK inhibitors in pancreatic cancer therapy as ‘priming’ agents warrants further consideration, and provides insights as to how transient mechanical manipulation, or fine-tuning the ECM, rather than chronic stromal ablation might be beneficial for improving chemotherapeutic efficacy in the treatment of this deadly disease

A Study Examining the Role of Rho Family GTPases in the Intracellular Targeting of Src Kinase During Cell Polarisation and Migration

The ability of a cell to polarise and move is governed by re-modelling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rad and Cdc42 during peripheral adhesion re-modelling that are required for directional migration. We show here that individual members of the Rho family, RhoA, Rac1 and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for adhesion re-modelling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rad-induced lamellipodia or Cdc42-induced filopodia. Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarisation towards migratory stimuli and reduced motility. Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localisation, and that RhoA, Rac1 and Cdc42 cannot co-ordinate cell structural changes and directed cell migration when Src is present at adhesion sites in an inactive form. Src's role, which involves phosphorylation of adhesion substrates including FAK, is to induce the necessary adhesion re-modelling downstream of Rac1 and Cdc42 during migratory cell responses

Rho-associated kinase signalling and the cancer microenvironment: novel biological implications and therapeutic opportunities

The Rho/ROCK pathway is involved in numerous pivotal cellular processes that have made it an area of intense study in cancer medicine, however, Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors are yet to make an appearance in the clinical cancer setting. Their performance as an anti-cancer therapy has been varied in pre-clinical studies, however, they have been shown to be effective vasodilators in the treatment of hypertension and post-ischaemic stroke vasospasm. This review addresses the various roles the Rho/ROCK pathway plays in angiogenesis, tumour vascular tone and reciprocal feedback from the tumour microenvironment and explores the potential utility of ROCK inhibitors as effective vascular normalising agents. ROCK inhibitors may potentially enhance the delivery and efficacy of chemotherapy agents and improve the effectiveness of radiotherapy. As such, repurposing of these agents as adjuncts to standard treatments may significantly improve outcomes for patients with cancer. A deeper understanding of the controlled and dynamic regulation of the key components of the Rho pathway may lead to effective use of the Rho/ROCK inhibitors in the clinical management of cancer

Monitoring the dynamics of Src activity in response to anti-invasive dasatinib treatment at a subcellular level using dual intravital imaging

Optimising response to tyrosine kinase inhibitors in cancer remains an extensive field of research. Intravital imaging is an emerging tool, which can be used in drug discovery to facilitate and fine-tune maximum drug response in live tumors. A greater understanding of intratumoural delivery and pharmacodynamics of a drug can be obtained by imaging drug target-specific fluorescence resonance energy transfer (FRET) biosensors in real time. Here, we outline our recent work using a Src-FRET biosensor as a readout of Src activity to gauge optimal tyrosine kinase inhibition in response to dasatinib treatment regimens in vivo. By simultaneously monitoring both the inhibition of Src using FRET imaging, and the modulation of the surrounding extracellular matrix using second harmonic generation (SHG) imaging, we were able to show enhanced drug penetrance and delivery to live pancreatic tumors. We discuss the implications of this dual intravital imaging approach in the context of altered tumor-stromal interactions, while summarising how this approach could be applied to assess other combination strategies or tyrosine kinase inhibitors in a preclinical setting

Genotype-based recall studies in complex cardiometabolic traits

In genotype-based recall (GBR) studies, people (or their biological samples) who carry genotypes of special interest for a given hypothesis test are recalled from a larger cohort (or biobank) for more detailed investigations. There are several GBR study designs that offer a range of powerful options to elucidate (1) genotype-phenotype associations (by increasing the efficiency of genetic association studies, thereby allowing bespoke phenotyping in relatively small cohorts), (2) the effects of environmental exposures (within the Mendelian randomization framework), and (3) gene-treatment interactions (within the setting of GBR interventional trials). In this review, we overview the literature on GBR studies as applied to cardiometabolic health outcomes. We also review the GBR approaches used to date and outline new methods and study designs that might enhance the utility of GBR-focused studies. Specifically, we highlight how GBR methods have the potential to augment randomized controlled trials, providing an alternative application for the now increasingly accepted Mendelian randomization methods usually applied to large-scale population-based data sets. Further to this, we consider how functional and basic science approaches alongside GBR designs offer intellectually intriguing and potentially powerful ways to explore the implications of alterations to specific (and potentially druggable) biological pathways

Inhibition of autophagy impairs tumor cell invasion in an organotypic model

Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis

Combating pancreatic cancer with PI3K pathway inhibitors in the era of personalised medicine

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly solid tumours. This is due to a generally late-stage diagnosis of a primarily treatment-refractory disease. Several large-scale sequencing and mass spectrometry approaches have identified key drivers of this disease and in doing so highlighted the vast heterogeneity of lower frequency mutations that make clinical trials of targeted agents in unselected patients increasingly futile. There is a clear need for improved biomarkers to guide effective targeted therapies, with biomarker-driven clinical trials for personalised medicine becoming increasingly common in several cancers. Interestingly, many of the aberrant signalling pathways in PDAC rely on downstream signal transduction through the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways, which has led to the development of several approaches to target these key regulators, primarily as combination therapies. The following review discusses the trend of PDAC therapy towards molecular subtyping for biomarker-driven personalised therapies, highlighting the key pathways under investigation and their relationship to the PI3K pathway

Morphogenesis of extra-embryonic tissues directs the remodelling of the mouse embryo at implantation

Abstract: Mammalian embryos change shape dramatically upon implantation. The cellular and molecular mechanism underlying this transition are largely unknown. Here, we show that this transition is directed by cross talk between the embryonic epiblast and the first extra-embryonic tissue, the trophectoderm. Specifically, we show via visualisation of a Cdx2-GFP reporter line and pharmacologically mediated loss and gain of function experiments that the epiblast provides FGF signal that results in differential fate acquisition in the multipotent trophectoderm leading to the formation of a tissue boundary within this tissue. The trophectoderm boundary becomes essential for expansion of the tissue into a multi-layered epithelium. Folding of this multi-layered trophectoderm induces spreading of the second extra-embryonic tissue, the primitive endoderm. Together, these events remodel the pre-implantation embryo into its post-implantation cylindrical shape. Our findings uncover how communication between embryonic and extra-embryonic tissues provides positional cues to drive shape changes in mammalian development during implantation