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BackgroundGlioblastoma is the most aggressive type of brain cancer with high-levels of intra- and inter-tumour heterogeneity that contribute to its rapid growth and invasion within the brain. However, a spatial characterisation of gene signatures and the cell types expressing these in different tumour locations is still lacking.MethodsWe have used a deep convolutional neural network (DCNN) as a semantic segmentation model to segment seven different tumour regions including leading edge (LE), infiltrating tumour (IT), cellular tumour (CT), cellular tumour microvascular proliferation (CTmvp), cellular tumour pseudopalisading region around necrosis (CTpan), cellular tumour perinecrotic zones (CTpnz) and cellular tumour necrosis (CTne) in digitised glioblastoma histopathological slides from The Cancer Genome Atlas (TCGA). Correlation analysis between segmentation results from tumour images together with matched RNA expression data was performed to identify genetic signatures that are specific to different tumour regions.ResultsWe found that spatially resolved gene signatures were strongly correlated with survival in patients with defined genetic mutations. Further in silico cell ontology analysis along with single-cell RNA sequencing data from resected glioblastoma tissue samples showed that these tumour regions had different gene signatures, whose expression was driven by different cell types in the regional tumour microenvironment. Our results further pointed to a key role for interactions between microglia/pericytes/monocytes and tumour cells that occur in the IT and CTmvp regions, which may contribute to poor patient survival.ConclusionsThis work identified key histopathological features that correlate with patient survival and detected spatially associated genetic signatures that contribute to tumour-stroma interactions and which should be investigated as new targets in glioblastoma. The source codes and datasets used are available in GitHub:https://github.com/amin20/GBM_WSSM.

To fully investigate cellular responses to stimuli and perturbations within tissues, it is essential to replicate the complex molecular interactions within the local microenvironment of cellular niches. Here, the authors introduce Alginate-based tissue engineering (ALTEN), a biomimetic tissue platform that allows ex vivo analysis of explanted tissue biopsies. This method preserves the original characteristics of the source tissue's cellular milieu, allowing multiple and diverse cell types to be maintained over an extended period of time. As a result, ALTEN enables rapid and faithful characterization of perturbations across specific cell types within a tissue. Importantly, using single-cell genomics, this approach provides integrated cellular responses at the resolution of individual cells. ALTEN is a powerful tool for the analysis of cellular responses upon exposure to cytotoxic agents and immunomodulators. Additionally, ALTEN's scalability using automated microfluidic devices for tissue encapsulation and subsequent transport, to enable centralized high-throughput analysis of samples gathered by large-scale multicenter studies, is shown Refereed/Peer-reviewed

The 14-3-3 family of proteins have roles in regulating several key cellular processes. While their significant structural and functional homology had informed the idea that these proteins acted redundantly, it is now becoming clear that individual family members may have tissue and context specific functions, highlighting the need for a more nuanced understanding of these important proteins. Here, we demonstrate that mice deficient in 14-3-3ζ exhibit developmental defects of the mammary epithelium, associated with dysregulation of key transcription factors involved in the maintenance of mammary stem cell populations. We believe that this model will be prove useful for investigating the role of 14-3-3ζ in the maintenance of mammary stem cell populations and elucidating the transcriptional networks driving specification of the mammary epithelium.

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Forster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.

Description, Intravital imaging guides a personalized medicine approach to target mechanoreciprocity in pancreatic cancer., Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow–induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

The tissue microenvironment supports normal tissue function and regulates the behaviour of parenchymal cells. Tumour cell behaviour, on the other hand, diverges significantly from that of their normal counterparts, rendering the microenvironment hostile to tumour cells. To overcome this problem, tumours can co-opt and remodel the microenvironment to facilitate their growth and spread. This involves modifying both the biochemistry and the biophysics of the normal microenvironment to produce a tumour microenvironment. In this Cell Science at a Glance article and accompanying poster, we outline the key processes by which epithelial tumours influence the establishment of the tumour microenvironment. As the microenvironment is populated by genetically normal cells, we discuss how controlling the microenvironment is both a significant challenge and a key vulnerability for tumours. Finally, we review how new insights into tumour–microenvironment interactions has led to the current consensus on how these processes may be targeted as novel anti-cancer therapies.

Tumorigenesis involves a complex interplay between genetically modified cancer cells and their adjacent normal tissue, the stroma. We used an established breast cancer mouse model to investigate this inter-relationship. Conditional activation of Rho-associated protein kinase (ROCK) in a model of mammary tumorigenesis enhances tumor growth and progression by educating the stroma and enhancing the production and remodeling of the extracellular matrix. We used peptide matrixassisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify the proteomic changes occurring within tumors and their stroma in their regular spatial context. Peptides were ranked according to their ability to discriminate between the two groups, using a receiver operating characteristic tool. Peptides were identified by liquid chromatography tandem mass spectrometry, and protein expression was validated by quantitative immunofluorescence using an independent set of tumor samples. We have identified and validated four key proteins upregulated in ROCK-activated mammary tumors relative to those expressing kinase-dead ROCK, namely, collagen I, α-SMA, Rab14, and tubulin- β4. Rab14 and tubulin-β4 are expressed within tumor cells, whereas collagen I is localized within the stroma. α-SMA is predominantly localized within the stroma but is also expressed at higher levels in the epithelia of ROCK-activated tumors. High expression of COL1A, the gene encoding the pro-α 1 chain of collagen, correlates with cancer progression in two human breast cancer genomic data sets, and high expression of COL1A and ACTA2 (the gene encoding α-SMA) are associated with a low survival probability (COLIA, p = 0.00013; ACTA2, p = 0.0076) in estrogen receptor-negative breast cancer patients. To investigate whether ROCK-activated tumor cells cause stromal cancer-associated fibroblasts (CAFs) to upregulate expression of collagen I and α-SMA, we treated CAFs with medium conditioned by primary mammary tumor cells in which ROCK had been activated. This led to abundant production of both proteins in CAFs, clearly highlighting the inter-relationship between tumor cells and CAFs and identifying CAFs as the potential source of high levels of collagen 1 and α-SMA and associated enhancement of tissue stiffness. Our research emphasizes the capacity of MALDI-MSI to quantitatively assess tumor-stroma inter-relationships and to identify potential prognostic factors for cancer progression in human patients, using sophisticated mouse cancer models Refereed/Peer-reviewed

Reciprocal biochemical and biophysical interactions between tumor cells, stromal cells and the extracellular matrix (ECM) result in a unique tumor microenvironment that determines disease outcome. The cellular component of the tumor microenvironment contributes to tumor growth by providing nutrients, assisting in the infiltration of immune cells and regulating the production and remodeling of the ECM. The ECM is a noncellular component of the tumor microenvironment and provides both physical and biochemical support to the tumor cells. Rho–ROCK signaling is a key regulator of actomyosin contractility and regulates cell shape, cytoskeletal arrangement and thereby cellular functions such as cell proliferation, differentiation, motility and adhesion. Rho–ROCK signaling has been shown to promote cancer cell growth, migration and invasion. However, it is becoming clear that this pathway also regulates key tumor-promoting properties of the cellular and noncellular components of the tumor microenvironment. There is accumulating evidence that Rho–ROCK signaling enhances ECM stiffness, modifies ECM composition, increases the motility of tumor-associated fibroblasts and lymphocytes and promotes trans-endothelial migration of tumor-associated lymphocytes. In this review, we briefly discuss the current state of knowledge on the role of Rho–ROCK signaling in regulating the tumor microenvironment and the implications of this knowledge for therapy, potentially via the development of selective inhibitors of the components of this pathway to permit the tuning of signaling flux, including one example with demonstrated utility in pre-clinical models.

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

Glioblastoma is the most aggressive type of brain cancer with high-levels of intra- and inter-tumour heterogeneity that contribute to its rapid growth and invasion within the brain. Here, we have used a deep convolutional neural network (DCNN) as a semantic segmentation model to segment seven different tumour regions including leading-edge (LE), infiltrating tumour (IT), cellular tumour (CT), cellular tumour microvascular proliferation (CTmvp), cellular tumour pseudopalisading region around necrosis (CTpan), cellular tumour perinecrotic zones (CTpnz) and cellular tumour necrosis (CTne) in digitised glioblastoma histopathological slides from The Cancer Genome Atlas (TCGA). Analysis of segmentation results from tumour images together with matched RNA expression data identified genetic signatures that are specific to these different tumour regions. We found that spatially resolved gene signatures were strongly correlated with survival in patients with defined genetic mutations. Moreover, in silico cell ontology analysis and single-cell RNA sequencing data from resected glioblastoma tissue samples, showed that these tumour regions had different gene signatures, suggesting they are driven by different cell types in the tumour microenvironment. This points to a key role for interactions between microglia/pericytes/monocytes and tumour cells that occur in the IT and CTmvp regions, which may contribute to poor patient survival. Overall, this work identifies key histopathological features that are indicative of patient survival and detected spatially associated genetic signatures that mediate tumour-stroma interactions that should be investigated as new targets in glioblastoma. Citation Format: Amin Zadeh Shirazi, Mark D. McDonnell, Eric Fornaciari, Narjes Sadat Bagherian, Kaitlin G. Scheer, Michael S. Samuel, Mahdi Yaghoobi, Rebecca J. Ormsby, Santosh Poonnoose, Damon Tumes, Guillermo A. Gomez. A deep convolutional neural network for segmentation of whole-slide pathology images in glioblastoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-004.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a Kras G12D / p53 R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three‐dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK‐induced genes that facilitate extracellular matrix remodeling, with greatest fold‐changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13 . MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three‐dimensional contexts. Treatment of Kras G12D / p53 R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor‐associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM. Refereed/Peer-reviewed

The microenvironment encompasses all components of a tumor other than the cancer cells themselves. It is highly heterogenous, comprising a cellular component that includes immune cells, fibroblasts, adipocytes, and endothelial cells, and a non-cellular component, which is a meshwork of polymeric proteins and accessory molecules, termed the extracellular matrix (ECM). The ECM provides both a biochemical and biomechanical context within which cancer cells exist. Cancer progression is dependent on the ability of cancer cells to traverse the ECM barrier, access the circulation and establish distal metastases. Communication between cancer cells and the microenvironment is therefore an important aspect of tumor progression. Significant progress has been made in identifying the molecular mechanisms that enable cancer cells to subvert the immune component of the microenvironment to facilitate tumor growth and spread. While much less is known about how the tumor cells adapt to changes in the ECM nor indeed how they influence ECM structure and composition, the importance of the ECM to cancer progression is now well established. Plasticity refers to the ability of cancer cells to modify their physiological characteristics, permitting them to survive hostile microenvironments and resist therapy. Examples include the acquisition of stemness characteristics and the epithelial-mesenchymal and mesenchymal-epithelial transitions. There is emerging evidence that the biochemical and biomechanical properties of the ECM influence cancer cell plasticity and vice versa. Outstanding challenges for the field remain the identification of the cellular mechanisms by which cancer cells establish tumor-promoting ECM characteristics and delineating the key molecular mechanisms underlying ECM-induced cancer cell plasticity. Here we summarize the current state of understanding about the relationships between cancer cells and the main stromal cell types of the microenvironment that determine ECM characteristics, and the key molecular pathways that govern this threeway interaction to regulate cancer cell plasticity. We postulate that a comprehensive understanding of this dynamic system will be required to fully exploit opportunities for targeting the ECM regulators of cancer cell plasticity. 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 β common ([βc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use βc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the βc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the βc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease. Refereed/Peer-reviewed

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hedgehog ligand produced by neoplastic cells reprograms cancer-associated fibroblasts (CAFs) to provide a supportive niche for the acquisition of a chemo-resistant, cancer stem cell (CSC) phenotype via FGF5 expression and production of fibrillar collagen. Stromal treatment of patient-derived xenografts with smoothened inhibitors (SMOi) downregulates CSC markers expression and sensitizes tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. These studies identify Hedgehog signaling to CAFs as a novel mediator of CSC plasticity and an exciting new therapeutic target in TNBC., Stromal cell recruitment, activation and crosstalk with cancer cells is poorly understood. Here, the authors demonstrate that cancer cell-derived Hedgehog ligand triggers stromal remodeling that in turn induces a cancer-stem-cell like, drug-resistant phenotype of nearby cancer cells while treatment with smoothened inhibitors reverses these phenotypes.

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hh ligand produced by neoplastic cells reprogrammed cancer-associated fibroblast (CAF) gene expression, driving tumor growth and metastasis. Hh-activated CAFs upregulated expression of FGF5 and production of fibrillar collagen, leading to FGFR and FAK activation in adjacent neoplastic cells, which then acquired a stem-like, drug-resistant phenotype. Treatment with smoothened inhibitors (SMOi) reversed these phenotypes. Stromal treatment of TNBC patient-derived xenograft (PDX) models with SMOi downregulated the expression of cancer stem cell markers and sensitized tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. Markers of pathway activity correlated with response. These studies identify Hh signaling to CAFs as a novel mediator of cancer stem cell plasticity and an exciting new therapeutic target in TNBC.SIGNIFICANCECompared to other breast cancer subtypes, TNBCs are associated with significantly worse patient outcomes. Standard of care systemic treatment for patients with non-BRCA1/2 positive TNBC is cytotoxic chemotherapy. However, the failure of 70% of treated TNBCs to attain complete pathological response reflects the relative chemoresistance of these tumors. New therapeutic strategies are needed to improve patient survival and quality of life. Here, we provide new insights into the dynamic interactions between heterotypic cells within a tumor. Specifically, we establish the mechanisms by which CAFs define cancer cell phenotype and demonstrate that the bidirectional CAF-cancer cell crosstalk can be successfully targeted in mice and humans using anti-stromal therapy.

The epidermis is a highly regenerative tissue. YAP is a pivotal regulator of stem/progenitor cells in tissue regeneration, including in the epidermis. The molecular mechanisms downstream of YAP that activate epidermal cell proliferation remain largely unknown. We found that YAP and β-catenin co-localize in the nuclei of keratinocytes in the regenerating epidermis in vivo and in proliferating HaCaT keratinocytes in vitro. Inactivation of YAP in HaCaT keratinocytes resulted in reduced activated β-catenin and reduced keratinocyte numbers in vitro. In addition, we found that in the hyperplastic epidermis of YAP2-5SA-ΔC mice, the mutant YAP2-5SA-ΔC protein was predominantly localized in the keratinocyte nuclei and caused increased expression of activated nuclear β-catenin. Accordingly, β-catenin transcriptional activity was elevated in the skin of live YAP2-5SA-ΔC/TOPFLASH mice. Lastly, loss of β-catenin in basal keratinocytes of YAP2-5SA-ΔC/K14-creERT/CtnnB1–/– mice resulted in reduced proliferation of basal keratinocytes and a striking rescue of the hyperplastic abnormalities. Taken together, our work shows that YAP2-5SA-ΔC drives β-catenin activity to promote basal keratinocyte proliferation in the mouse skin in vivo. Our data shine new light on the etiology of regenerative dermatological disorders and other human diseases that display increased YAP and β-catenin activity. Refereed/Peer-reviewed

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Forster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer. Refereed/Peer-reviewed

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]

// Heidi A. Neubauer 1,2 , Duyen H. Pham 1,2 , Julia R. Zebol 1 , Paul A.B. Moretti 1 , Amanda L. Peterson 4 , Tamara M. Leclercq 1 , Huasheng Chan 1,2 , Jason A. Powell 1,3 , Melissa R. Pitman 1 , Michael S. Samuel 1,3 , Claudine S. Bonder 1,2,3 , Darren J. Creek 4 , Briony L. Gliddon 1 and Stuart M. Pitson 1,2,3 1 Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia, Australia 2 School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia 3 School of Medicine, University of Adelaide, Adelaide, South Australia, Australia 4 Monash Institute of Pharmaceutical Science, Monash University, Parkville, Victoria, Australia Correspondence to: Stuart M. Pitson , email: // Keywords : sphingosine kinase 2, neoplastic transformation, oncogenesis, proliferation, tumorigenesis Received : June 08, 2016 Accepted : August 25, 2016 Published : August 30, 2016 Abstract While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an anti-proliferative/pro-apoptotic function for SK2, while others indicate it has a pro-survival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo . These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.

International audience; The expression pattern of tight junction (TJ) proteins is frequently disrupted in epithelial tumors. In particular, isoform- and organ-specific alterations of claudins have been detected in human cancers, highlighting them as interesting tools for the prognosis or treatment of various carcinomas. However, the molecular mechanisms responsible for these alterations are seldom identified. Here, we analyzed the expression and localization of claudins 1, 4, and 7 in human bladder carcinoma. Claudin-4 expression was significantly altered in 26/39 tumors, contrasting with the rare modifications detected in the expression of claudins 1 and 7. Overexpression of claudin-4 in differentiated carcinomas was followed by a strong downregulation in invasive/high-grade tumors, and this expression pattern was associated to the 1-year survival of bladder tumor patients. A CpG island was identified within the coding sequence of the CLDN4 gene, and treatment with a methyl-transferase inhibitor restored expression of the protein in primary cultures prepared from high-grade human bladder tumors. In addition, claudin-4 expression correlated with its gene methylation profile in healthy and tumoral bladders from 20 patients, and downregulation of claudin-4 expression was detected in the urothelium of mice overexpressing DNA methyl transferase 3a (Dnmt3a). Delocalization of claudins 1 and 4 from TJs was observed in most human bladder tumors and in the bladder tumor cell line HT-1376. Although the CLDN4 gene was unmethylated in these cells, pharmacological inhibition of methyl transferases re-addressed the two proteins to TJs, resulting in an increase of cell polarization and transepithelial resistance. These biological effects were prevented by expression of claudin-4-specific siRNAs, demonstrating the important role played by claudin-4 in maintaining a functional regulation of homeostasis in urothelial cells. Results of this study indicate that the TJ barrier is disrupted from early stages of urothelial tumorigenesis. In addition, we identified hypermethylation as the mechanism leading to the alteration of claudin-4 expression, and maybe also localization, in bladder carcinoma.

Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing. Refereed/Peer-reviewed

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.

Plant α-1,4 glucanotransferases (disproportionating enzymes, or D-enzymes) transfer glucan chains among oligosaccharides with the concomitant release of glucose (Glc). Analysis of Chlamydomonas reinhardtii sta11-1 mutants revealed a correlation between a D-enzyme deficiency and specific alterations in amylopectin structure and starch biosynthesis, thereby suggesting previously unknown biosynthetic functions. This study characterized the biochemical activities of the α-1,4 glucanotransferase that is deficient in sta11-1 mutants. The enzyme exhibited the glucan transfer and Glc production activities that define D-enzymes. D-enzyme also transferred glucans among the outer chains of amylopectin (using the polysaccharide chains as both donor and acceptor) and from malto-oligosaccharides into the outer chains of either amylopectin or glycogen. In contrast to transfer among oligosaccharides, which occurs readily with maltotriose, transfer into polysaccharide required longer donor molecules. All three enzymatic activities, evolution of Glc from oligosaccharides, glucan transfer from oligosaccharides into polysaccharides, and transfer among polysaccharide outer chains, were evident in a single 62-kD band. Absence of all three activities co-segregated with thesta11-1 mutation, which is known to cause abnormal accumulation of oligosaccharides at the expense of starch. To explain these data we propose that D-enzymes function directly in building the amylopectin structure.

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

Pancreatic cancer is one of the leading causes of cancer death and despite advances in chemotherapeutic regimens the overall 5-year survival rate remains less than 5%. The actomyosin-regulating ROCK1 and ROCK2 kinases are downstream-targets of the Rho GTPase pathway. They contribute to various processes, such as cell adhesion, motility, proliferation, differentiation and survival, which then influence numerous stages of cancer growth and progression. Interestingly, exome sequencing of pancreatic cancer genomes revealed that 15% of pancreatic cancer patients carry an amplification of the ROCK1 gene (1). Moreover, we found significant increases in ROCK1 and ROCK2 RNA expression in pancreatic cancer datasets obtained using Oncomine. When we further determined the protein expression of ROCK2 in pancreatic ductal adenocarcinoma (PDAC) we found an up-regulation of ROCK2 during disease progression. Higher ROCK2 levels also correlated with less differentiated tumors. A characteristic of advanced stages of pancreatic cancer is a collagen and fibroblast enriched stroma. To analyze the effect of ROCK kinases on cancer cell invasion, we performed 3-dimensional organotypic assays. Our results demonstrate that increased ROCK signaling was sufficient to convert non-invasive PDAC cells into ones capable of invasion into organotypic collagen matrix. Using an RNA sequencing approach, we investigated the effect of ROCK activation on pancreatic cancer cell gene transcription. Differential expression analysis revealed an enrichment of gene sets that are involved in cell-matrix interaction, and we found a higher release of matrix metalloproteinases MMP10 and MMP13 upon ROCK activation. In addition, our organotypic studies revealed extensive tissue remodeling and an accumulation of cleaved collagen bundles at the sites of PDAC cell invasion. Furthermore, collagen degradation and cell invasion into organotypic matrices were significantly reduced by application of a broad-spectrum MMP inhibitor, confirming that ROCK-induced invasion is dependent on MMP activity. Interestingly, our studies also indicated that ROCK-driven invasion of PDAC cells into collagen matrix enabled cell growth. To study the function of ROCK kinase signaling in pancreatic cancer in vivo, we expressed conditionally active ROCK2 in a mouse model of PDAC. We found that an increase of ROCK activity in pancreatic cancer cells accelerated PDAC progression, which resulted in reduced survival. Contrary, the administration of a ROCK inhibitor during tumor progression had a beneficial effect on survival. In summary, our results suggest that targeting ROCK kinases should be considered for chemotherapy of invasive pancreatic cancer. (1) Biankin et al. Nature. 2012 Nov 15;491(7424):399-405 Citation Format: Nicola Rath, Shereen Kadir, Jennifer P. Morton, Andreia V. Pinho, Lena Helbig, Linda Julian, Ewan J. McGhee, Gabriela Kalna, Alexei Vazquez, Kurt I. Anderson, Ilse Rooman, Michael S. Samuel, Michael F. Olson. ROCK kinases drive invasive pancreatic tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C22.

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

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 .

In this issue of Cell Stem Cell, Chen et al. (2010) and Ohgushi et al. (2010) report that Rho-mediated actomyosin contractility underlies the proclivity of human embryonic stem cells to undergo apoptosis upon dissociation or during low-density propagation, a property that challenges their in vitro isolation and manipulation.

Genomic DNA fragments from Triticum tauschii (D-genome donor to wheat) carrying starch branching enzyme I (SBE I) type genes have been characterized. One fragment contains one complete gene and two partial genes in 16 kb of DNA. One of the partial genes is oriented in the opposite strand to the other two. The gene that is complete was sequenced. Its structure corresponds closely to that of rice in that exons 3–8 are retained at similar sizes and spacings. A cDNA closely corresponding to the complete gene was isolated and characterized; it codes for a putative protein that represents a novel type of SBE I, as it is shorter at the 3′ end than the forms reported so far in other plants. A second genomic fragment contains a different SBE I gene. There appear to be approximately 10 copies of SBE I type genes in wheat (approximately 5 in T. tauschii) and most of them have been assigned to group 7 chromosomes. In situ hybridization indicates that a major locus for the genes is located at the distal end of the short arm of chromosome 7D.Key words: starch, branching enzyme genes, wheat, Triticum tauschii, in situ hybridization.

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.

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