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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

Contains fulltext : 240100.pdf (Publisher’s version ) (Open Access) Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.

Focal adhesion kinase (FAK) is a key regulator of growth factor receptor- and integrin-mediated signals, governing fundamental processes in normal and cancer cells through its kinase activity and scaffolding function. Increased FAK expression and activity occurs in primary and metastatic cancers of many tissue origins, and is often associated with poor clinical outcome, highlighting FAK as a potential determinant of tumor development and metastasis. Indeed, data from cell culture and animal models of cancer provide strong lines of evidence that FAK promotes malignancy by regulating tumorigenic and metastatic potential through highly-coordinated signaling networks that orchestrate a diverse range of cellular processes, such as cell survival, proliferation, migration, invasion, epithelial-mesenchymal transition, angiogenesis and regulation of cancer stem cell activities. Such an integral role in governing malignant characteristics indicates that FAK represents a potential target for cancer therapeutics. While pharmacologic targeting of FAK scaffold function is still at an early stage of development, a number of small molecule-based FAK tyrosine kinase inhibitors are currently undergoing pre-clinical and clinical testing. In particular, PF-00562271, VS-4718 and VS-6063 show promising clinical activities in patients with selected solid cancers. Clinical testing of rationally designed FAK-targeting agents with implementation of predictive response biomarkers, such as merlin deficiency for VS-4718 in mesothelioma, may help improve clinical outcome for cancer patients. In this article, we have reviewed the current knowledge regarding FAK signaling in human cancer, and recent developments in the generation and clinical application of FAK-targeting pharmacologic agents.

Introduction The extensive stromal deposition and remodelling of pancreatic ductal adenocarcinoma (PDAC) alters mechanical tumour-stroma integrations, promoting tumour development and metastatic spread. Few effective therapies mean that PDAC is predicted to be the second leading cause of cancer mortality by 2030. In highly metastatic mouse models of PDAC, we observed enhanced extracellular matrix (ECM) deposition and remodelling throughout disease progression. This was paralleled by an increased focal adhesion kinase (FAK) expression and activity, suggesting a role for FAK in the increased desmoplastic reaction that is typical of PDAC. Consequently, fine tuned manipulation of the dense stroma by streamlined FAK inhibition (FAKi) presents a novel opportunity for PDAC management and improved response to chemotherapy. Material and methods Intravital imaging of the FUCCI cell cycle reporter was used to dynamically monitor tumour cell response to combined FAKi and standard-of-care therapy with gemcitabine/Abraxane. This was overlaid with second harmonic generation (SHG) imaging of collagen fibres, to assess the efficacy of FAKi to disrupt the dense PDAC ECM. To complement our in vivo metastatic studies, we used sophisticated 3D in vitro models of invasion, anchorage-independent growth and shear-stress, in both primary and patient-derived PDAC cell lines. Results and discussions We systematically demonstrated that using FAKi to modulate ECM prior to standard-of-care therapy enhanced treatment efficacy whilst also reducing metastatic spread in vivo . Further analysis revealed that FAKi sensitised cells to shear stress, impairing metastatic colonisation and the establishment of fibrotic niches in the liver. Stratified patient samples revealed a subset of patients likely to respond to FAK priming regimes, where fine-tuned ECM manipulation prior to chemotherapy may offer a novel opportunity in metastatic PDAC. Conclusion This subtype-specific fine-tuned stromal manipulation may allow us to maximise gemcitabine/Abraxane therapy whilst reducing drug toxicity and potentially reducing metastatic spread in a preclinical setting.

Introduction The ‘Mutated in colorectal cancer’ (MCC) gene was originally discovered due to its linkage with the APC gene. MCC is silenced through gene promoter methylation in colorectal cancer (CRC) but its significance has been poorly understood. MCC promoter methylation occurs early in premalignant tumours and is associated with increased lymph node metastasis in advanced CRC. Here we determined the consequences of MCC knockdown on cell-cell adhesion and invasiveness of colon cancer cells and further characterised the gene methylation in patient tumours. Material and methods MCC promoter methylation was determined in a cohort of 365 CRC and compared with clinicopathological features, with research ethics approval by the Sydney Local Health District. MCC expression was stably knocked down in HCT116 colon cancer cells or induced in HCT15 cells using lentiviral vectors. The impact of MCC knockdown on invasiveness was determined using an organotypic assay and on cell-cell adhesion with dispase and transepithelial electrical resistance (TEER) assays. Protein complexes were characterised using Blue-Native PAGE, co-immunoprecipitation, immunofluorescence and Proximity Ligation Assay (PLA). Results and discussions MCC promoter methylation was found in 58% of cancers in the right colon, 28% in the left colon and 24% in the rectum. Methylation was associated with larger, poorly differentiated, circumferential or mucinous tumours, and higher T stage that indicates increased invasiveness. We established that the MCC protein interacts with the E-cadherin/beta-catenin complex in HCT116 and HCT15 cells. MCC knockdown in HCT116 cells caused a reduction in E-cadherin protein level, disrupted cell-cell adhesive strength and integrity, increased cell invasiveness and hepatocyte growth factor-induced cell scatter. MCC expression in HCT15 cells had the opposite effect in dispase, TEER and PLA assays. The invasive properties induced by MCC knockdown were abrogated by dasatinib, a candidate anti-invasive drug. Thus we have described a novel function of MCC in the regulation of E-cadherin mediated functions. Conclusion Our study advances current understanding of the protein-protein interactions that control cell-cell adhesion and suppress epithelial-mesenchymal-transition. Potential therapies exploiting our findings may include strengthening cellular junctions with dasatinib or other anti-invasive combination therapies.

Summary Here we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5β1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5β1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo., Highlights ► Activated integrins move from late endosomes/lysosomes to the plasma membrane ► Spatial segregation of pathways recycling active and inactive integrins ► CLIC3 regulates cell migration and invasion ► CLIC3 dictates whether Rab25 functions as cancer progression suppressor or promoter

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]

Introduction Pancreatic cancer remains a highly lethal cancer where response is limited by both intrinsic and acquired chemoresistance. Understanding resistance mechanisms may therefore lead to improved therapeutic strategies. We have recently defined specific molecular subgroups of pancreatic cancer associated with pre-clinical and clinical response to select tailored treatment strategies.1–3 Material and methods Using robust patient-derived xenografts (PDXs) of pancreatic cancer, here we generated novel in vivo models for the study of intrinsic and acquired chemoresistance mechanisms to clinically-used agents, gemcitabine, mitomycin C, and cisplatin. Here, we used whole genome sequencing (WGS) and microarray analysis to compare gemcitabine-resistant and gemcitabine-sensitive pancreatic tumours to identify relevant resistance mechanisms. Results and discussions Integrative analysis of WGS and microarray profiling of gemcitabine-resistant tumours revealed complex but potentially targetable resistance mechanisms, including increased DNA repair through activation of PARP1, MCM genes and RRM1, and changes within the tumour microenvironment. Importantly, acquired resistance to gemcitabine was effectively reversed by a novel PARP inhibitor, rucaparib, indicating that combination therapy involving this low toxicity agent may be useful in treating gemcitabine-resistant tumours defined by high genomic instability. Similarly, modulation of key components of the tumour microenvironment with fasudil, as recently achieved,2 provided another effective way of reversing gemcitabine resistance. Conclusion Significance our findings demonstrate the promise of patient-derived xenograft models for the study of in vivo mechanisms of chemotherapy resistance and efficacy testing of novel agents for the treatment of human pancreatic cancer. References Waddell N, et al. Nature (2015) 518(7540):495 Vennin C, et al. Science Translational Medicine (2017) pii: eaai8504 Chou A, et al. Gut (2017) pii: gutjnl-2017-315144 [epub ahead of print]

Introduction Pancreatic cancer (PC) has a 5 year survival of only 6%, and persists as the 4th most common cause of cancer-related death in Western societies. A more tailored treatment approach may be beneficial as the current standard-of-care therapies offer only a modest increase in overall patient survival patient overall. Recent large-scale genomic studies have revealed that the Src/JAK/STAT3 signalling pathway is deregulated in up to 35% of PC, and is yet to be systematically examined in this disease. Consequently, we hypothesised that targeting pancreatic tumours with activated JAK/STAT3 signalling with selective JAK1/JAK2 or JAK3 inhibitors and an Src inhibitor represents a promising novel therapeutic strategy for this disease. Material and methods We utilised well-annotated patient-derived cell-line models (ICGC), along with cell-lines generated from the aggressive the KPC mouse model. Using these pre-clinical models we assessed the in vitro efficacy of therapeutic strategies involving Src/JAK/STAT3 inhibition, using cell proliferation assays, 2D-drug synergy screens, and 3D organotypic invasion assays. Extracellular matrix integrity post-treatment was assessed using second-harmonic generation (SHG) imaging and picrosirius staining. To examine in vivo efficacy, we utilised a syngeneic KPC mouse model, and performed both orthotopic and subcutaneous studies. Results and discussions We show that selected JAK and Src-inhibitors inhibit cell proliferation in candidate PDCLs and KPC lines, characterised by activated Src/JAK/STAT3 signalling, with combination therapy being synergistic in the majority of these cell-lines. Cell invasion was significantly inhibited in organotypic matrices, and there was decreased collagen contractility, and reduced fibrillar collagen coverage. We also demonstrate the in vivo efficacy of these therapies, and show their ability to reduce regulatory T-cells, MDSCs and tumour-associated macrophages. Conclusion Our findings demonstrate the potential for tailored therapeutic strategies involving Src/JAK/STAT3 inhibition in PC, and suggest that therapeutic efficacy may be the result of targeting both tumour cells and the tumour microenvironment, as well as by overcoming tumour-induced immunosuppression.

Summary Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo , via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.

Background: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. Results: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. Conclusions: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.