The CDC42 effector protein MRCKß autophosphorylates on Threonine 1108.

The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the Myotonic dystrophy related CDC42-binding protein kinases (MRCK) α and ß. We previously identified Serine 1003 of MRCKα as a site of autophosphorylation, and showed that a phosphorylation-sensitive antibody raised against this site could be used as a surrogate indicator of kinase activity. In this study, a kinase-dead version of MRCKß was established by mutation of the conserved Lysine 105 to Methionine (K105M), which was then used for mass spectrometry analysis to identify phosphorylation events that occurred in catalytically-competent MRCKß but not in the kinase-dead form. A total of ten phosphorylations were identified on wild-type MRCKß, of which the previously undescribed Threonine 1108 (Thr1108) was not found on kinase-dead MRCKß K105M, consistent with this being due to autophosphorylation. Mutation of Thr1108 to non-phosphorylatable Alanine (T1108A) or phosphomimetic Glutamate (T1108E) did not affect the ability of MRCKß to phosphorylate recombinant myosin light chain in vitro, or observably alter the subcellular localization of green fluorescent protein (GFP)-tagged MRCKß expressed in MDA MB 231 human breast cancer cells. Although phosphorylation of Thr1108 did not appear to contribute to MRCKß function or regulation, the identification of this phosphorylation does make it possible to characterize whether this site could be used as a surrogate biomarker of kinase activity and inhibitor efficacy as we previously demonstrated for Ser 1003 in MRCKα.

Rho Kinase Inhibition by AT13148 Blocks Pancreatic Ductal Adenocarcinoma Invasion and Tumor Growth.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321-36. ©2018 AACR.

Discovery of Potent and Selective MRCK Inhibitors with Therapeutic Effect on Skin Cancer.

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKß contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy.Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR.

A novel small-molecule MRCK inhibitor blocks cancer cell invasion.

BACKGROUND: The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKß regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK. RESULTS: Screening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKß kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKß over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 µM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation. CONCLUSIONS: BDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.

The actin-myosin regulatory MRCK kinases: regulation, biological functions and associations with human cancer.

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, MRCKß 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.

In vivo maturation of functional renal organoids formed from embryonic cell suspensions.

The shortage of transplantable organs provides an impetus to develop tissue-engineered alternatives. Producing tissues similar to immature kidneys from simple suspensions of fully dissociated embryonic renal cells is possible in vitro, but glomeruli do not form in the avascular environment. Here, we constructed renal organoids from single-cell suspensions derived from E11.5 kidneys and then implanted these organoids below the kidney capsule of a living rat host. This implantation resulted in further maturation of kidney tissue, formation of vascularized glomeruli with fully differentiated capillary walls, including the slit diaphragm, and appearance of erythropoietin-producing cells. The implanted tissue exhibited physiologic functions, including tubular reabsorption of macromolecules, that gained access to the tubular lumen on glomerular filtration. The ability to generate vascularized nephrons from single-cell suspensions marks a significant step to the long-term goal of replacing renal function by a tissue-engineered kidney.

Dissociation of embryonic kidney followed by re-aggregation as a method for chimeric analysis.

This chapter presents three methods for re-constructing mouse foetal kidney tissue from simple suspensions of cells. These techniques are very useful for a number of purposes: (1) they allow the production of fine-grained chimaeras in which cell autonomy of mutations can be tested, (2) they provide an environment that allows the renal differentiation potential of stem cells to be assessed, and (3) they are an excellent system in which to study the mechanisms of self-organization. Each of the methods described here begins with disaggregation of embryonic mouse kidneys, followed by re-aggregation and culture; the main differences are in the culture methods, each of which has advantages for particular purposes.

siRNA-mediated RNA interference in embryonic kidney organ culture.

In principle, treatment of embryonic kidneys growing in organ culture with short interfering RNA (siRNA) offers a powerful means of investigating molecular function quickly and cheaply. Experiments using this approach have yielded significant new data, but they have also highlighted important limitations. Here, we briefly describe the published successes and limitations and present detailed instructions for two methods of siRNA treatment. The first method applies siRNA to intact cultured kidneys; this method is the quicker and easier of the two, but it is the one most affected by problems of siRNA uptake by certain renal tissues. The second method reduces kidney rudiments to a suspension of single cells, applies siRNA at that stage, when the cells are highly accessible, and then reaggregates the kidney; this method is more time-consuming but suffers less from problems of limited uptake. As well as proving instructions for the methods, we provide a brief discussion of necessary controls.

Evaluation of methods for one-dimensional spatial analysis of two-dimensional patterns in mouse chimaeras.

The relative extent of cell mixing in tissues of mouse chimaeras or mosaics can be studied by comparing the distributions of the two cell populations in the tissues. However, the mean patch size is misleading because it is affected by both the extent of cell mixing and the relative contributions of the two cell populations. Previous work suggested that effects attributable to differences in tissue composition among chimaeras can be factored out either by correcting the mean patch size or by using the median patch size for the minority cell population and restricting the analysis to grossly unbalanced chimaeras. In the present study, computer simulations of two-dimensional mosaic arrays of black and white squares (representing cells) were used to simulate chimaeric tissues. Random arrays simulated tissues with extensive cell mixing, arrays of cell clumps (representing coherent clones) simulated less mixed tissues, and striped arrays simulated tissues with elongated but fragmented descendent clones. The computer simulations predicted that (i) the median patch length (minority cell population) and the corrected mean patch length would both distinguish between random and clumped patterns and (ii) differences in the variation of the composition of two perpendicular series of one-dimensional transects would distinguished between stripes and randomly orientated patches. Both predictions were confirmed by analysis of histological sections of the retinal pigment epithelium from fetal and adult mouse chimaeras. This study demonstrates that two types of non-random two-dimensional variegated patterns (clumps and stripes) can be identified in chimaeras without two-dimensional reconstruction of serial sections.

An improved kidney dissociation and reaggregation culture system results in nephrons arranged organotypically around a single collecting duct system.

Methods for constructing engineered "tissues" from simple suspensions of cells are valuable for investigations into basic developmental biology and for tissue engineering. We recently published a method for producing embryonic renal tissues from suspensions of embryonic mouse renal cells. This method reproduced the anatomies and differentiation states of nephrons and stroma very well; it had the limitation, however, that what would, in normal development, be a single, highly branched collecting duct tree leading to a ureter developed, in the engineered system, as a multitude of very small collecting duct trees. These were isolated from each other and therefore would not be effective for draining urine to a common exit, were the tissue to be supplied with blood and physiologically active. Here, we report an improvement on the original method; it results in the formation of nephrons arranged around one single collecting duct tree as would happen in a normal kidney.

Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR.

Human amniotic fluid stem cells (hAFSCs) can be grown in large quantities, have a low risk for tumour development and harbour a high differentiation potential. They are a very promising new fetal stem cell type for cell-based therapy approaches and for studying differentiation processes without raising the ethical concerns associated with embryonic stem cells. Recently, a protocol for studies on renal development has been established in which murine embryonic kidneys are dissociated into single-cell suspension and then reaggregated to form organotypic renal structures. Using this approach, we formed chimeric renal structures via mixing murine embryonic kidney cells with monoclonal hAFSCs. We demonstrate here that hAFSCs harbour the potential to contribute to renal tissue formation accompanied by induction of specific renal marker expression. As part of the two kinase complexes mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signalling pathway, which is involved in the regulation of differentiation and in the development of a wide variety of human genetic diseases many with characteristic kidney symptoms. Modulating endogenous mTOR activity via specific siRNA approaches revealed that contribution of hAFSCs to renal tissue formation is regulated by mTORC1 and mTORC2. These findings (i) demonstrate renal differentiation potential of hAFSCs, (ii) prove chimeric cultures of mixtures of murine embryonic kidney cells and hAFSCs to be a powerful tool to study the effects of gene knockdowns for renal structure formation and (iii) provide new insights into the role of the mTOR pathway for renal development.

A novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization.

Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1-3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium-around 85 microliters-using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle.

Introducing the scanning air puff tonometer for biological studies.

It is getting increasingly evident that physical properties such as elastoviscoplastic properties of living materials are quite important for the process of tissue development, including regulation of genetic pathways. Measuring such properties in vivo is a complicated and challenging task. In this paper, we present an instrument, a scanning air puff tonometer, which is able to map point by point the viscoelastic properties of flat or gently curved soft materials. This instrument is an improved version of the air puff tonometer used by optometrists, with important modifications. The instrument allows one to obtain a direct insight into gradients of material properties in vivo. The instrument capabilities are demonstrated on substances with known elastoviscoplastic properties and several biological objects. On the basis of the results obtained, the role of the gradients of elastoviscoplastic properties is outlined for the process of angiogenesis, limb development, bacterial colonies expansion, etc. which is important for bridging the gaps in the theory of the tissue development and highlighting new possibilities for tissue engineering, based on a clarification of the role of physical features in developing biological material.

Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues.

Here we describe a novel method in which embryonic kidneys are dissociated into single-cell suspensions and then reaggregated to form organotypic renal structures. Kidney cell reaggregates were transiently cultured with small-molecule Rho kinase inhibitors, which caused ureteric bud structures to form and induced formation of nephrons. These structures displayed normal morphology, expressed appropriate differentiation markers, and were connected at their distal ends to the ureteric buds, thus forming artificial tissues very similar to those found in normal embryonic kidneys. Using this culture method, it was straightforward to make fine-grained chimeras by mixing different cell types or by mixing cells transfected with different constructs before reaggregation. Chimeric renal cultures were formed using mixtures of unmarked normal host embryonic kidney cells and CellTracker-marked WT1 siRNA-carrying cells to test the hypothesis that WT1 is important to a cell's ability to contribute to nephron formation. We found a significant reduction in the ability of WT1 knockdown cells to contribute to nephron formation. This dissociation and reaggregation procedure can also be applied to embryonic lungs and to form coarse-grained hybrid tissues from mixtures of lung and kidney cells. Overall, our protocol allows very simple mixing of cells from different sources or cells subjected to different pretreatments to make fine-grained, highly dispersed chimera tissues.

During vertebrate development, arteries exert a morphological control over the venous pattern through physical factors.

The adult vasculature is comprised of three distinct compartments: the arteries, which carry blood away from the heart and display a divergent flow pattern; the capillaries, where oxygen and nutrient delivery from blood to tissues, as well as metabolic waste removal, occurs; and the veins, which carry blood back to the heart and are characterized by a convergent flow pattern. These compartments are organized in series as regard to flow, which proceeds from the upstream arteries to the downstream veins through the capillaries. However, the spatial organization is more complex, as veins may often be found paralleling the arteries. The factors that control the morphogenesis of this hierarchically branched vascular network are not well characterized. Here, we explain how arteries exert a morphological control on the venous pattern. Indeed, during vertebrate development, the following transition may be observed in the spatial organization of the vascular system: veins first develop in series with the arteries, the arterial and venous territories being clearly distinct in space (cis-cis configuration). But after some time, new veins grow parallel to the existing arteries, and the arterial and venous territories become overlapped, with extensive and complex intercalation and interdigitation. Using physical arguments, backed up by experimental evidence (biological data from the literature and in situ optical and mechanical measurements of the chick embryo yolk-sac and midbrain developing vasculatures), we explain how such a transition is possible and why it may be expected with generality, as organisms grow. The origin of this transition lies in the remodeling of the capillary tissue in the vicinity of the growing arteries. This remodeling lays down a prepattern for further venous growth, parallel to the existing arterial pattern. Accounting for the influence of tissue growth, we show that this prepatterned path becomes favored as the body extends. As a consequence, a second flow route with veins paralleling the arteries (cis-trans configuration) emerges when the tissue extends. Between the cis-cis and cis-trans configurations, all configurations are in principle possible, and self-organization of the vessels contributes to determining their exact pattern. However, the global aspect depends on the size at which the growth stops and on the growth rate.

Tracheal occlusion increases the rate of epithelial branching of embryonic mouse lung via the FGF10-FGFR2b-Sprouty2 pathway.

Tracheal occlusion during lung development accelerates growth in response to increased intraluminal pressure. In order to investigate the role of internal pressure on murine early lung development, we cauterized the tip of the trachea, to occlude it, and thus to increase internal pressure. This method allowed us to evaluate the effect of tracheal occlusion on the first few branch generations and on gene expression. We observed that the elevation of internal pressure induced more than a doubling in branching, associated with increased proliferation, while branch elongation speed increased 3-fold. Analysis by RT-PCR showed that Fgf10, Vegf, Sprouty2 and Shh mRNA expressions were affected by the change of intraluminal pressure after 48h of culture, suggesting mechanotransduction via internal pressure of these key developmental genes. Tracheal occlusion did not increase the number of branches of Fgfr2b-/- mice lungs nor of wild type lungs cultured with Fgfr2b antisense RNA. Tracheal occlusion of Fgf10(LacZ/-) hypomorphic lungs led to the formation of fewer branches than in wild type. We conclude that internal pressure regulates the FGF10-FGFR2b-Sprouty2 pathway and thus the speed of the branching process. Therefore pressure levels, fixed both by epithelial secretion and boundary conditions, can control or modulate the branching process via FGF10-FGFR2b-Sprouty2.

The Textural Aspects of Vessel Formation during Embryo Development and Their Relation to Gastrulation Movements.

We have investigated the microscopic physical inhomogeneity ("texture") of the avian embryo in vivo by shadowgraph. This noninvasive technique allows one to correlate the shape of blood vessels to the physical, micro-structural, pattern that exists in the embryo prior to vessel appearance. Before any vessel forms, vascular paths are present and are prepatterned, by fields of cellular orientations and lumen anisotropies. We find the origin of this prepattern in the movements of the embryo during gastrulation, and the related deformation and force field, which establish both the animal and vascular pattern.

Molecular mechanisms of early lung specification and branching morphogenesis.

The "hard wiring" encoded within the genome that determines the emergence of the laryngotracheal groove and subsequently early lung branching morphogenesis is mediated by finely regulated, interactive growth factor signaling mechanisms that determine the automaticity of branching, interbranch length, stereotypy of branching, left-right asymmetry, and finally gas diffusion surface area. The extracellular matrix is an important regulator as well as a target for growth factor signaling in lung branching morphogenesis and alveolarization. Coordination not only of epithelial but also endothelial branching morphogenesis determines bronchial branching and the eventual alveolar-capillary interface. Improved prospects for lung protection, repair, regeneration, and engineering will depend on more detailed understanding of these processes. Herein, we concisely review the functionally integrated morphogenetic signaling network comprising the critical bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, transforming growth factor-beta, vascular endothelial growth factor, and Wnt signaling pathways that specify and drive early embryonic lung morphogenesis.