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Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin β1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin β1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin β1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch. [Cancer Res 2008;68(15):6241–50]

Supplementary Video 1 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Supplementary Methods from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Supplementary Video 2 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Supplementary Figure Legends 1-5 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Supplementary Figures 1-5 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Loch Drunkie is retained by two earth dams built in 1859. The outlet pipe is a 24 in dia. cast iron pipe through the highest part of the east dam with downstream control only. Although no record drawings exist, it is considered that the pipe is laid directly within the foundation or embankment fill and the presence of any concrete surround is unlikely. The pipe is over 160 years old and, due to the valve arrangement, is always subject to internal pressure from the reservoir. The east dam was inaccessible with no vehicular access to within 500 m. In the 2014 Statutory Report, a recommendation in the interest of safety was made to ascertain the condition of the outlet pipe and carry out any works deemed to be required to reduce any potential threats posed to the safety of the dam. This paper describes the series of investigations and subsequent pipe lining works that were carried out at the reservoir. Due to concerns regarding the integrity of the pipe, all investigations and pipe lining works had to be carried out with the reservoir at top water level to ensure the internal pressure to the pipe was maintained throughout.

In December 2020, the British Dam Society (BDS) conducted a year-end survey of both its individual and corporate members. The survey asked that members rate how much they agreed with a series of statements and were given the opportunity to submit additional comments. The survey contained 14 questions for individual members and three questions for corporate members. This paper presents the results of the survey along with feedback from the BDS Committee.

Oncogenic RAS induces perinuclear translocation of the oncogenic kinases ERK1/2 and CK2 and the MAPK scaffold KSR1, forming endosomal signaling hubs termed perinuclear signaling centers (PSCs). PSCs are found in nearly all cancer cell lines and tumor tissues, suggesting that compartmentalization of oncogenic kinases drives tumorigenesis. We show here that the endosomal adaptor, TOLLIP, tethers RAB11A+ signaling endosomes containing CK2 and KSR1, but not ERK, to the perinuclear ER. TOLLIP binds to the KSR1 CA5 pseudo-kinase domain through a conserved region predicted to form a β-hairpin, thus anchoring PSCs to perinuclear endosomes. TOLLIP is perinuclear in cancer cells and KRasG12D-driven mouse tumors but is pan-cytoplasmic in non-transformed cells, correlating with the presence of PSCs. TOLLIP is required for proliferation/survival of KRAS mutant tumor cells but not HRAS, NRAS and BRAF cancers or non-transformed cells. KRasG12D-induced lung lesions in TollipKO/KOmice showed reduced numbers of carcinomatous lesions, implicating TOLLIP in progression to malignant cancer. Phosphoproteomics studies revealed that perinuclear CK2 phosphorylates specific substrates, particularly proteins involved in ribosome biogenesis and translation such as RIOK1 and eIF4B. In summary, our findings identify TOLLIP as a key signaling adaptor in KRAS tumors whose inhibition is a potential vulnerability of these cancers.

C/EBPβ is a potent regulator of RAS-induced senescence (RIS) and the SASP. C/EBPβ is post- translationally activated in RIS cells by the effector kinases ERK1/2 and CK2, but in tumor cells activation is suppressed by the CEBPB 3’UTR. 3′UTR regulation of protein activity (UPA) requires a G/U-rich element (GRE) and its cognate binding protein, HuR. These components segregate CEBPB transcripts away from a perinuclear compartment harboring ERK1/2 and CK2, restricting C/EBPβ from its activating kinases. We report here that the mRNA decay proteins UPF1 and Staufen1/2 are essential UPA factors enriched within the perinuclear cytoplasm. STAU1/2 colocalize with CK2 on perinuclear signaling endosomes where they promote localized CEBPB mRNA decay. UPF1 or STAU1/2 depletion in tumor cells increased CEBPB transcripts adjacent to CK2 foci, coinciding with C/EBPβ activation and senescence. The GRE and an adjacent STAU binding site act to suppress C/EBPβ-mediated senescence, while a separate 3’UTR region inhibits SASP induction. Accordingly, KrasG12D-driven lung tumors in mice carrying a Cebpb GRE deletion rarely progressed to malignant adenocarcinomas. Our findings identify kinase-proximal mRNA decay as a novel mechanism to inhibit C/EBPβ activating modifications in tumor cells to facilitate senescence bypass.

Breast cancer is the second leading cause of cancer death among U.S. women although the mortality is on a steadily decreasing trend since early 90s. We previously demonstrated a strong correlation between elevated NOS2 and COX2 expressions suggesting these genes can serve as markers of poor prognosis in estrogen receptor negative (ER-) breast cancer. In other words, NOS2 and COX2 is generally associated with the immune response. Induction of NOS2 and COX2 has also been shown to orchestrate multiple pro-oncogenic pathways that may contribute to the immunosuppressive nature of cancer cells. To better understand the interactions between cytokines and breast cancer cells and develop effective therapeutics for breast cancer, we examined the effects of selected cytokines on MDA-MB-231, a human (ER-) breast adenocarcinoma cell line. We found that in combination with cytokines, such as IFNG plus TNF, IL1B, or IL17, significantly induced NOS2 and COX2. IFNG plus TNF or IL1B most strongly induced NOS2 and COX2 where IFNG or IL17 to a lesser extent. It indicates that expressions of NOS2 and COX2 required IFNG but at a low dose in presence of other cytokines. Single-cell RNAseq analysis shows that cells treated with IFNG plus TNF or IL1B clustered with those expressed NOS2 and COX2 as revealed in tSNE plots. Expression of other factors such as IL8 and IL6 (suggested markers for prognosis of breast cancer) or PDL1 and IDO1 (suggested cancer therapeutic targets) also co-clustered with NOS2 and COX2 expressions. In addition, we found increased IL1A and IL1B expressions provide fortification to NOS2 and COX2 expressions. Taken together, our current findings indicate the interactions between breast cancer cells and cytokines. We believe further spatial transcriptomic and proteomic studies may be beneficial for clarifying the complex networks of the immunosuppressive tumor microenvironment. Citation Format: Yuk Sing Robert Cheng, Lisa Ridnour, Veena Somasundaram, Dana Bhattacharyya, Christopher McGinity, Daniel McVicar, Stephen Anderson, Stephen Lockett, David Wink. Interferon gamma induces NOS2 and COX2 expression in estrogen receptor negative breast cancer leading to immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3804.

At Talla Reservoir the main scour outlet was impracticable to operate due to its arrangement; discharge would impinge at high velocity against the adjacent supply pipe stack, which carries a substantial part of the water supply to Edinburgh. The scour capacity available without the use of the main scour arrangement was deemed inadequate. Works were carried out to amend the arrangement of the scour pipe by installing specially manufactured steel bends and tapers to discharge reservoir water through a diffuser pipe located downstream of the existing supply stack. This paper describes the design, installation and commissioning of the works including the principal considerations and challenges that were overcome during the design process.

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in

Children and adolescents with chronic memory impairment may develop coping strategies that enable functioning, yet these often remain undetectable using traditional psychometric measures. Personalized intervention studies that promote the use of such strategies designed specifically for use by this young cohort are scarce.To investigate the effect of a novel virtual reality peer-delivered memory intervention on the everyday functioning and well-being of SE, a 17-year-old female with a history of chronic verbal memory issues, impaired autobiographical event recall and elevated mood symptoms.A single-case ABA experimental design study was used to assess change.Following initial baseline assessment using objective neuropsychological and subjective functional questionnaires and intervention training, case SE used the intervention daily for 3 weeks before repeating key outcome measures.Using non-overlap of all pairs and qualitative feedback analysis, the results revealed a significant increase in event recall and self-reported positive changes to levels of everyday functioning.Supporting autobiographical event recall and prospective memory via a virtual peer-delivered intervention may lead to reduction in cognitive load, and benefit overall well-being and everyday functioning.

Actin fibers (F-actin) control the shape and internal organization of cells, and generate force. It has been long appreciated that these functions are tightly coupled, and in some cases drive cell behavior and cell fate. The distribution and dynamics of F-actin is different in cancer versus normal cells and in response to small molecules, including actin-targeting natural products and anticancer drugs. Therefore, quantifying actin structural changes from high resolution fluorescence micrographs is necessary for further understanding actin cytoskeleton dynamics and phenotypic consequences of drug interactions on cells. We applied an artificial neural network algorithm, which used image intensity and anisotropy measurements, to quantitatively classify F-actin subcellular features into actin along the edges of cells, actin at the protrusions of cells, internal fibers and punctate signals. The algorithm measured significant increase in F-actin at cell edges with concomitant decrease in internal punctate actin in astrocytoma cells lacking functional neurofibromin and p53 when treated with three structurally-distinct anticancer small molecules: OSW1, Schweinfurthin A (SA) and a synthetic marine compound 23′-dehydroxycephalostatin 1. Distinctly different changes were measured in cells treated with the actin inhibitor cytochalasin B. These measurements support published reports that SA acts on F-actin in NF1−/− neurofibromin deficient cancer cells through changes in Rho signaling. Quantitative pattern analysis of cells has wide applications for understanding mechanisms of small molecules, because many anticancer drugs directly or indirectly target cytoskeletal proteins. Furthermore, quantitative information about the actin cytoskeleton may make it possible to further understand cell fate decisions using mathematically testable models. Published 2014 Wiley Periodicals Inc.†