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It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time. Nobis et al. generated a RhoA-FRET biosensor mouse to characterize and quantify the spatiotemporal distribution of RhoA activity in native mammalian tissues in vivo during development and disease progression. They show that RhoA activity is tightly regulated during various normal biological processes and is co-opted in disease settings, such as invasive breast and pancreatic cancers. Refereed/Peer-reviewed

The ability to rapidly respond to applied force underpins cell/tissue homeostasis. This response is mediated by mechanotransduction pathways that regulate remodeling and tension of the actomyosin cytoskeleton to counterbalance external forces. Enhanced extracellular matrix tension hyper-activates mechanotransduction and characterizes diseased states such as cancer, but is also required for normal epidermal regeneration. While the impact of extracellular matrix tension on signaling and cell biology are well appreciated, that of acute compressive force is under-studied. We show here that acute compressive force applied to cells and tissues in a native 3-dimensional context elevates RHOA-GTP levels and increases regulatory myosin phosphorylation, actomyosin contractility and tension via ROCK. In consequence, cell proliferation was increased, as was the expression of regulators of epithelial-mesenchymal transition. Pharmacological inhibition of ROCK abrogated myosin phosphorylation, but not RHOA activation. Our results strongly suggest that acute compressive stress impairs cellular homeostasis in a RHO/ROCK-dependent manner, with implications for disease states such as cancer. Refereed/Peer-reviewed

// Zlatko Kopecki 1 , Gink N. Yang 1 , Jessica E. Jackson 1 , Elizabeth L. Melville 1 , Matthew P. Caley 2 , Dedee F. Murrell 3 , Ian A. Darby 4 , Edel A. O’Toole 2 , Michael S. Samuel 5 , Allison J. Cowin 1 1 Regenerative Medicine, Future Industries Institute, University of South Australia, Adelaide, South Australia, Australia 2 Centre for Cutaneous Research, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom 3 Department of Dermatology, St. George Hospital and University of New South Wales, Sydney, New South Wales, Australia 4 School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia 5 Centre for Cancer Biology, an alliance between SA Pathology and the University of South Australia, Australia Correspondence to: Zlatko Kopecki, e-mail: zlatko.kopecki@unisa.edu.au Keywords: Flightless, squamous cell carcinoma, cell invasion, skin cancer Received: April 20, 2015 Accepted: October 09, 2015 Published: October 19, 2015 ABSTRACT Flightless I (Flii) is an actin remodeling protein that affects cellular processes including adhesion, proliferation and migration. In order to determine the role of Flii during carcinogenesis, squamous cell carcinomas (SCCs) were induced in Flii heterozygous ( Flii +/− ), wild-type and Flii overexpressing ( Flii Tg/Tg ) mice by intradermal injection of 3-methylcholanthrene (MCA). Flii levels were further assessed in biopsies from human SCCs and the human SCC cell line (MET-1) was used to determine the effect of Flii on cellular invasion. Flii was highly expressed in human SCC biopsies particularly by the invading cells at the tumor edge. Flii Tg/Tg mice developed large, aggressive SCCs in response to MCA. In contrast Flii +/− mice had significantly smaller tumors that were less invasive. Intradermal injection of Flii neutralizing antibodies during SCC initiation and progression significantly reduced the size of the tumors and, in vitro , decreased cellular sphere formation and invasion. Analysis of the tumors from the Flii overexpressing mice showed reduced caspase I and annexin V expression suggesting Flii may negatively regulate apoptosis within these tumors. These studies therefore suggest that Flii enhances SCC tumor progression by decreasing apoptosis and enhancing tumor cell invasion. Targeting Flii may be a potential strategy for reducing the severity of SCCs.

The mechanical properties of the ECM strongly influence the behavior of all cell types within a given tissue. Increased matrix tension promotes epithelial cell proliferation by engaging mitogenic mechanotransduction signaling including the Salvador/Warts/Hippo, PI 3-kinase, Rho, Wnt and MAP kinase pathways. The Rho signaling pathways in particular are capable of increasing intra-cellular tension by elevating the production and contractility of the actomyosin cytoskeleton, which counteracts tension changes within the matrix in a process termed mechano-reciprocity. We have discovered that Rho-ROCK signaling increases the production of ECM through paracrine signaling between the epithelium and fibroblasts and also the remodeling of the ECM by regulating focal adhesion dynamics in fibroblasts. These two phenomena together cause increased ECM tension. Enhanced mechano-reciprocity results in ever-increasing intra- and extra-cellular tension in a vicious cycle that promotes cell proliferation and tumor progression. These insights reveal that inhibiting mechano-reciprocity, reducing ECM tension and targeting cancer-associated fibroblasts in a coordinated fashion has potential as cancer therapy. Refereed/Peer-reviewed

The chemokine receptor CXCR2 is a key mediator of neutrophil migration that also plays a role in tumor development. However, CXCR2 influences tumors through multiple mechanisms and might promote or inhibit tumor development depending on context. Here, we used several mouse models of spontaneous and inflammation- driven neoplasia to define indispensable roles for CXCR2 in benign and malignant tumors. CXCR2- activating chemokines were part of the secretome of cultured primary benign intestinal adenomas (ApcMin/+) and highly expressed by all tumors in all models. CXCR2 deficiency profoundly suppressed inflammation-driven tumorigenesis in skin and intestine as well as spontaneous adenocarcinoma formation in a model of invasive intestinal adenocarcinoma (AhCreER;Apcfl/+;Ptenfl/fl mice). Pepducin-mediated CXCR2 inhibition reduced tumorigenesis in ApcMin/+ mice. Ly6G+ neutrophils were the dominant source of CXCR2 in blood, and CXCR2 deficiency attenuated neutrophil recruitment. Moreover, systemic Ly6G+ cell depletion purged CXCR2-dependent tumor-associated leukocytes, suppressed established skin tumor growth and colitis-associated tumorigenesis, and reduced ApcMin/+ adenoma formation. CXCR2 is thus a potent protumorigenic chemokine receptor that directs recruitment of tumor-promoting leukocytes into tissues during tumor-inducing and tumor-driven inflammation. Similar leukocyte populations were also found in human intestinal adenomas, which suggests that CXCR2 antagonists may have therapeutic and prophylactic potential in the treatment of cancer. Refereed/Peer-reviewed

Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. beta-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters. [KEYWORDS: Actomyosin/ physiology, Animals, Biomechanics, Cell Proliferation, Cells, Cultured, Epidermis/ pathology, Humans, Hyperplasia, Mice, Papilloma/etiology, Signal Transduction, Skin Neoplasms/ etiology, beta Catenin/ physiology, rho-Associated Kinases/analysis/genetics/physiology]

The mode of activation of glycoprotein 130 kDa (gp130) and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III-like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. On expression in cells, L-gp130 stimulates ligand-independent signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase 1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the interleukin-3–dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Because gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells, which completely blocked their differentiation in the absence of cytokine stimulation and was consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of which is frequently observed in neoplastic diseases.

Aberrant activation of β-catenin is a common event in AML and is an independent predictor of poor prognosis. Although increased β-catenin signaling in AML has been associated with oncogenic translocation products and activating mutations in the FLT3R, the mechanisms that activate β-catenin in AML more broadly are still unclear. Here, we describe a novel link between IL-3 signaling and the regulation of β-catenin in myeloid transformation and AML. In a murine model of HoxB8 and IL-3 cooperation, we show that β-catenin protein levels are modulated by IL-3 and that Cre-induced deletion of β-catenin abolishes IL-3-dependent growth and colony formation. In IL-3-dependent leukemic TF-1.8 cells, we observed increased β-catenin protein levels and nuclear localization in response to IL-3, and this correlated with transcriptional induction of β-catenin target genes. Furthermore, IL-3 promoted β-catenin accumulation in a subset of AML patient samples, and gene-expression profiling of these cells revealed induction of WNT/β-catenin and TCF4 gene signatures in an IL-3-dependent manner. This study is the first to link β-catenin activation to IL-3 and suggests that targeting IL-3 signaling may be an effective approach for the inhibition of β-catenin activity in some patients with AML.

We describe a novel mutation in theChlamydomonas reinhardtii STA11 gene, which results in significantly reduced granular starch deposition and major modifications in amylopectin structure and granule shape. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides. The sta11-1mutation causes the absence of an α-1,4 glucanotransferase known as disproportionating enzyme (D-enzyme). D-enzyme activity was found to be correlated with the amount of wild-type allele doses in gene dosage experiments. All other enzymes involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, debranching enzymes, soluble and granule-bound starch synthases, branching enzymes, phosphorylases, α-glucosidases (maltases), and amylases, were unaffected by the mutation. These data indicate that the D-enzyme is required for normal starch granule biogenesis in the monocellular alga C. reinhardtii.

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants ofC. reinhardtii.

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. Refereed/Peer-reviewed

The Rho family of small GTP-binding proteins is comprised of 22 members, including the most well characterized members RhoA, Rac1 and Cdc42 (Jaffe and Hall 2005). The Rho family proteins share a high degree of homology with the Ras proto-oncogene, and indeed were first identified as a result of this similarity (Ras homologue). Activity of these proteins is dependent upon their nucleotide binding state; inactive when associated with GDP but active following exchange of GDP for GTP, which induces conformational changes that promote association/activation of downstream effector proteins. The GDP/GTP cycle is regulated by GAPs that accelerate GTP hydrolysis by providing a critical catalytic amino acid leading to a return to the inactive state (Bernards and Settleman 2005), and GEFs that promote guanine nucleotide exchange and consequent Rho activation (Rossman et al. 2005). The number of GAPs and GEFs far exceeds the number of Rho proteins, and the roles of individual GAPs and GEFs in specific cell types and biological processes is currently an intensively studied field. Although united by homology and function as regulators of the actin cytoskeleton, each of RhoA, Rac1 and Cdc42 has a distinct role in the organization of actin structures (Figure 1). RhoA is principally involved with the production of actin-myosin bundles and the generation of actomyosin contractile force. Rac1 contributes to the formation of actin meshworks that result in the emergence of large protrusive structures that lead to spreading or, if occurring in a polarized manner, will contribute to motility. Cdc42 promotes the formation of actin-rich filopodia. Together, coordinated programs of RhoA, Rac1 and Cdc42 activation/inactivation play prominent roles in processes such as endocytosis/exocytosis, adhesion and motility, which may subsequently impact upon proliferation and death/survival. Recent advances in the development of activation-state sensitive fluorescent probes have allowed temporal and spatial analysis of Rho protein activation, which has added significantly to our appreciation of Rho regulation and function (Hodgson et al. 2010). Much of the early research on Rho protein function relied upon over-expression of dominant-negative mutants that reduced affinity for GTP and constitutively-active mutants that reduced GTP hydrolysis; however, more refined analysis has become possible with the rise of RNAi and knockout methodologies (Heasman and Ridley 2008). The study of Rho family proteins has historically focused on their roles as molecular switches acting downstream of cell surface receptors to regulate the actin cytoskeleton (Jaffe and Hall 2005). Significant effort has gone into classifying signaling from Rho proteins into

The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms. Refereed/Peer-reviewed

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/β-catenin pathway, we challenged the allele combinations by genetically restricting intracellular β-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/β-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/β-catenin signaling capacity similar to that in the germline of the Apcmin mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apcmin mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/β-catenin pathway that regulate both physiological and pathophysiological conditions., Author Summary Germline or somatic mutations in genes are the underlying cause of many human diseases, most notably cancer. Interestingly though, even in situations where every cell of every tissue of an organism carries the same mutation (as is the case for germline mutations), some tissues are more susceptible to the development of disease over time than others. For example, in familial adenomatous polyposis (FAP), affected persons carry different germline mutations in the APC gene and are prone to developing cancers of the colon and the rectum—and, less frequently, cancers in other tissues such as stomach, liver, and bones. Here we utilize a panel of mutant mice with truncating or hypomorphic mutations in the Apc gene, resulting in different levels of activation of the Wnt/β-catenin pathway. Our results reveal that different pathophysiological outcomes depend on different permissive signaling thresholds in embryonic, intestinal, and liver tissues. Importantly, we demonstrate that reducing Wnt pathway activation by 50% is enough to prevent the manifestation of embryonic abnormalities and disease in the adult mouse. This raises the possibility of developing therapeutic strategies that modulate the activation levels of this pathway rather than trying to “repair” the mutation in the gene itself.

The Rho-associated protein kinases ROCK I and ROCK II are central effectors for the RhoA, B, and C small molecular weight GTP-binding proteins, and promote actin-filament stabilization as well as actin-myosin contractility. The effects of ROCK on actin filaments initiate changes in cytoskeletal structure and organization that are critical for many physiological and pathological processes. ROCK has been functionally associated with cell cycle control and proliferation, survival and apoptosis, as well as cell motility and migration. Interestingly, regulation of each of these biological process may be significantly altered in tumor cells, resulting in the promotion of cancer progression. Extensive clinical and preclinical investigations provide evidence supportive of a role for ROCK in multiple aspects of cancer, and pharmacological ROCK inhibitors have been developed as potential anticancer chemotherapeutic agents. This chapter reviews the structure and function of ROCK I and ROCK II, with a particular focus on their potential roles in cancer.

Background & Aims Aberrant DNA methylation is a common early event in neoplasia, but it is unclear how this relates to dysregulation of DNA (cytosine-5) methyltransferases (Dnmts). Here we use knock-in transgenic mice to investigate the consequences of intestinal epithelium-specific overexpression of de novo Dnmt3a. Methods A novel gene targeting strategy, based on the intestinal epithelium-specific, uniform expression of the A33 glycoprotein, is employed to restrict Dnmt3a overexpression in homozygous A33 Dnmt3a mutant mice. Results A33 Dnmt3a mice infrequently develop spontaneous intestinal polyps. However, when genetically challenged, tumor multiplicity in A33 Dnmt3a ; Apc Min compound mice is 3-fold higher than in Apc Min mice. Although we observe a requirement for spontaneous loss of heterozygosity of the adenomatous polyposis coli ( Apc ) gene to trigger tumorigenesis in Apc Min mice, lesions in A33 Dnmt3a ; Apc Min mice frequently retain the wild-type Apc allele. However, epithelia from normal mucosa and polyps of A33 Dnmt3a ; Apc Min mice show hypermethylation-mediated transcriptional silencing of the Wnt antagonists Sfrp5 , and to a lesser extent, Sfrp1 and increased nuclear β-catenin alongside activation of the Wnt-target gene Axin2/Conductin . Conversely, enforced Sfrp5 expression suppresses canonical Wnt-signaling more effectively in wild-type than in Apc Min cells. Conclusions Aberrant activation of the canonical Wnt pathway, either by mono-allelic Apc loss or transcriptional silencing of Sfrp5 is largely insufficient to promote polyposis, but epistatic interactions between these genetic and epigenetic events enables initiation and promotion of disease. This mechanism is likely to play a role in human colorectal cancer, because we also show that elevated DNMT3A expression coincides with repressed SFRP5 and enhanced AXIN2/CONDUCTIN expression in paired patient biopsies.

A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.

Analysis of barley shrunken grain mutants has identified lines with a novel high amylose starch phenotype. The causal mutation is located at the sex6 locus on chromosome 7H, suggesting the starch synthase IIa (ssIIa) gene as a candidate gene altered by the mutation. Consistent with this hypothesis, no evidence of SSIIa protein expression in either the starch granule or soluble fractions of the endosperm was found. Sequences of the starch synthase IIa gene, ssIIa, from three independent sex6 lines showed the presence of a stop codon preventing translation of the ssIIa transcript in each line. Perfect segregation of the starch phenotype with the presence of stop codons in the ssIIa gene was obtained, providing strong evidence for the lesion in the ssIIa gene being the causal mutation for the sex6 phenotype. The loss of SSIIa activity in barley leads to novel and informative phenotypes. First, a decrease in amylopectin synthesis to less than 20% of the wild-type levels indicates that SSIIa accounts for the majority of the amylopectin polymer elongation activity in barley. Secondly, in contrast to high amylose starches resulting from branching enzyme downregulation, the sex6 starches have a shortened amylopectin chain length distribution and a reduced gelatinisation temperature. Thirdly, the mutation leads to pleiotropic effects on other enzymes of the starch biosynthesis pathway, abolishing the binding of SSI, branching enzyme IIa and branching enzyme IIb to the starch granules of sex6 mutants, while not significantly altering their expression levels in the soluble fraction.

To study tumour progression mechanism in vivo, ROCK 2 signalling deregulation and co-operation with activated rasHa or PTEN loss was investigated in transgenic mouse skin carcinogenesis. Transgenic mice that expressed a 4-hydroxytamoxifen (4HT)-activated human ROCK 2-estrogen receptor fusion transgene from a keratin 14 promoter [K14.ROCKer] were crossed to mice expressing activated rasHa exclusively in epidermal transit amplifying keratinocytes [HK1.ras-ROCKer] or mice where cutaneous PTEN loss was achieved in basal layers, hair follicles and stem cell keratinocytes via topical RU486 treatment of bi-genic K14.creP/Δ5PTENflx genotypes [K14.Δ5PTEN-ROCKer]. Initial 4HT-treatments of K14.ROCKer mice [3/wk; 26 wks] induced epidermal and follicular hyperplasia but no papillomas; whilst treatment of HK1.ras line 1205 gave ear tag papillomas [10-12 wks] typically smaller than vehicle controls, but no malignant conversion. In contrast, 4-HT treated HK1.ras-Rocker papillomas [10-12 wks] exhibited areas of carcinoma in situ and well-differentiated squamous cell carcinoma [wdSCC]. With time [16-20wks] wdSCC areas increased, concomitant with loss of p53, increased p-AKT and altered differentiation marker expression; however histotypes remained wdSCC despite continued 4HT-induced Rock 2 activity; as confirmed by downstream MYPT-1 phosphorylation. Furthermore, papillomas regressed whenever 4-HT treated HK1.ras-Rocker mice lost ear tags, suggesting that continued promotion from wounding during papillomatogenesis was critical to achieving the appropriate [late-stage] papilloma context that facilitated Rock 2-mediated conversion. To test this idea, K14.ROCKer mice were crossed to HK1.ras line 1276, which is insensitive to wound promotion, and such bi-genic mice did not exhibit papillomas; while in reverse, demonstrating the need for initiation, classic TPA-promoted K14.ROCKer mice were also devoid of papillomas. In addition, as previously rasHa/PTENnull mice exhibited papillomas prone to conversion on acquisition of an additional oncogene [e.g. fos], K14.ROCKer mice were bred into a K14.cre.Δ5PTEN strain and bi-genic mice treated with Ru486 [3wks] and 4HT [>26wks]. Again, in the absence of rasHa no papillomas were observed and mice exhibited increased hyperplasia and altered differentiation consistent with disruption of epidermal physiology. These data suggest that Rock 2 deregulation plays major roles in malignant conversion and progression, and in cooperation with rasHa activation or PTEN loss, the mechanism requires additional promotion/initiation events during papillomatogenesis to create the context of late-stage papilloma where Rock 2 activities become causal; an idea being tested in tri-genic HK1.ras/Δ5PTEN-Rocker genotypes. Citation Format: Siti F. Masre, Michael S. Samuel, Michael F. Olson, David A. Greenhalgh. Inducible ROCK 2/rasHa cooperation requires wound promotion to achieve malignancy in transgenic mouse skin carcinogenesis, whereas inducible ROCK 2/PTEN loss fails to achieve benign papilloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 930. doi:10.1158/1538-7445.AM2014-930

Background Mast cells have gained notoriety based on their detrimental contributions to IgE-mediated allergic disorders. Although mast cells express the vitamin D receptor (VDR), it is not clear to what extent 1α,25-dihydroxyvitamin D 3 (1α,25[OH] 2 D 3 ) or its predominant inactive precursor metabolite in the circulation, 25-hydroxyvitamin D 3 (25OHD 3 ), can influence IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo . Objective We sought to assess whether the vitamin D 3 metabolites 25OHD 3 and 1α,25(OH) 2 D 3 can repress IgE-dependent mast cell activation through mast cell–25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) and mast cell–VDR activity. Methods We measured the extent of vitamin D 3 suppression of IgE-mediated mast cell degranulation and mediator production in vitro , as well as the vitamin D 3 –induced curtailment of PCA responses in WBB6F 1 - Kit W/W-v or C57BL/6J- Kit W-sh/W-sh mice engrafted with mast cells that did or did not express VDR or CYP27B1. Results Here we show that mouse and human mast cells can convert 25OHD 3 to 1α,25(OH) 2 D 3 through CYP27B1 activity and that both of these vitamin D 3 metabolites suppressed IgE-induced mast cell–derived proinflammatory and vasodilatory mediator production in a VDR-dependent manner in vitro . Furthermore, epicutaneously applied vitamin D 3 metabolites significantly reduced the magnitude of skin swelling associated with IgE-mediated PCA reactions in vivo ; a response that required functional mast cell–VDRs and mast cell–CYP27B1. Conclusion Taken together, our findings provide a mechanistic explanation for the anti-inflammatory effects of vitamin D 3 on mast cell function by demonstrating that mast cells can actively metabolize 25OHD 3 to dampen IgE-mediated mast cell activation in vitro and in vivo .

Three forms of starch branching enzyme (BE) from developing hexaploid wheat (Triticum aestivum) endosperm have been partially purified and characterized. Immunological cross-reactivities indicate that two forms (WBE-IAD, 88 kD, and WBE-IB, 87 kD) are related to the maize BE I class and that WBE-II (88 kD) is related to maize BE II. Comparison of the N-terminal sequences from WBE-IAD and WBE-II with maize and rice BEs confirms these relationships. Evidence is presented from the analysis of nullisomic-tetrasomic wheat lines demonstrating that WBE-IB is located on chromosome 7B and that the WBE-IAD fraction contains polypeptides that are encoded on chromosomes 7A and 7D. The wheat endosperm BE classes are differentially expressed during endosperm development. WBE-II is expressed at a constant level throughout mid and late endosperm development. In contrast, WBE-IAD and WBE-IB are preferentially expressed in late endosperm development. Differences are also observed in the kinetic characteristics of the enzymes. The WBE-I isoforms have a 2- to 5-fold higher affinity for amylose than does WBE-II, and the WBE-I isoforms are activated up to 5-fold by phosphorylated intermediates and inorganic phosphate, whereas WBE-II is activated only 50%. The potential implications of this activation of BE I for starch biosynthesis are discussed.

Wheat starch contains two classes of associated proteins: proteins which are embedded within the granule and loosely associated surface proteins. The characterisation of the major proteins that are embedded in the granule are described. Gel electrophoresis on the basis of size resolved these proteins into five bands of molecular weights 60, 75, 85, 100 and 105 kDa. These polypeptides were demonstrated to be within the granule by their resistance to proteinase K digestion when granules were ungelatinised. The N-terminal sequences of these polypeptides are reported. The most prominent polypeptide is the 60 kDa granule-bound starch synthase. The N-terminal sequence obtained from the 75 kDa polypeptide shows homology to rice soluble starch synthase. The 85 kDa band was resolved into at least two types of polypeptides, one of which reacted with polyclonal antiserum to the maize branching enzyme IIb. The 100 and 105 kDa polypeptides were located only in the granule and are related, on the basis of N-terminal sequence similarity and cross-reactivity to monoclonal antibodies. SDS-PAGE and monoclonal antibody cross-reactivity experiments suggest that the 100 and 105 kDa polypeptides are absent from starch granules from all other species examined, including other cereals. It is speculated that all the major granule proteins are involved in starch biosynthesis.

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechanotransduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano- reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3 zeta, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3 zeta(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3 zeta deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-33 zeta with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3 zeta, while cutaneous SCCs had reduced 14-3-3 zeta. These results reveal a novel 14-3-3 zeta-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities. Refereed/Peer-reviewed