Regional engagement and spatial modelling for natural resource management planning.

Changing unsustainable natural resource use in agricultural landscapes is a complex social-ecological challenge that cannot be addressed through traditional reductionist science. More holistic and inclusive (or transdisciplinary) processes are needed. This paper describes a transdisciplinary project for natural resource management planning in two regions (Eyre Peninsula and South Australian Murray-Darling Basin) of southern Australia. With regional staff, we reviewed previous planning to gain an understanding of the processes used and to identify possible improvement in plan development and its operation. We then used an envisioning process to develop a value-rich narrative of regional aspirations to assist stakeholder engagement and inform the development of a land use management option assessment tool called the landscape futures analysis tool (LFAT). Finally, we undertook an assessment of the effectiveness of the process through semi-structured stakeholder interviews. The planning process review highlighted the opinion that the regional plans were not well informed by available science, that they lacked flexibility, and were only intermittently used after publication. The envisioning process identified shared values-generally described as a trust, language that is easily understood, wise use of resources, collaboration and inclusiveness. LFAT was designed to bring the best available science together in a form that would have use in planning, during community consultation and in assessing regional management operations. The LFAT provided spatially detailed but simple models of agricultural yields and incomes, plant biodiversity, weed distribution, and carbon sequestration associated with future combinations of climate, commodity and carbon prices, and costs of production. Stakeholders were impressed by the presentation and demonstration results of the software. While there was anecdotal evidence that the project provided learning opportunities and increased understanding of potential land use change associated with management options under global change, the direct evidence of influence in the updated regional plan was limited. This project had elements required for success in transdisciplinary research, but penetration seems limited. Contributing factors appear to be a complexity of climate effects with economic uncertainty, lack of having the project embedded in the plan revision process, limited continuity and capacity of end users and limited after project support and promotion. Strategies are required to minimise the controlling influence that these limitations can have.

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.

We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment.

CXCR4 drives the metastatic phenotype in breast cancer through induction of CXCR2 and activation of MEK and PI3K pathways.

Aberrant expression of CXCR4 in human breast cancer correlates with metastasis to tissues secreting CXCL12. To understand the mechanism by which CXCR4 mediates breast cancer metastasis, MCF-7 breast carcinoma cells were transduced to express wild-type CXCR4 (CXCR4WT) or constitutively active CXCR4 (CXCR4ΔCTD) and analyzed in two-dimensional (2D) cultures, three-dimensional reconstituted basement membrane (3D rBM) cultures, and mice using intravital imaging. Two-dimensional cultures of MCF-7 CXCR4ΔCTD cells, but not CXCR4WT, exhibited an epithelial-to-mesenchymal transition (EMT) characterized by up-regulation of zinc finger E box-binding homeobox 1, loss of E-cadherin, up-regulation of cadherin 11, p120 isoform switching, activation of extracellular signal-regulated kinase 1/2, and matrix metalloproteinase-2. In contrast to the 2D environment, MCF-7 CXCR4WT cells cultured in 3D rBM exhibited an EMT phenotype, accompanied by expression of CXCR2, CXCR7, CXCL1, CXCL8, CCL2, interleukin-6, and granulocyte-macrophage colony stimulating factor. Dual inhibition of CXCR2 with CXCR4, or inhibition of either receptor with inhibitors of mitogen-activated protein kinase 1 or phosphatidylinositol 3-kinase, reversed the aggressive phenotype of MCF-7 CXCR4-expressing or MDA-MB-231 cells in 3D rBM. Intravital imaging of CXCR4-expressing MCF-7 cells revealed that tumor cells migrate toward blood vessels and metastasize to lymph nodes. Thus CXCR4 can drive EMT along with an up-regulation of chemokine receptors and cytokines important in cell migration, lymphatic invasion, and tumor metastasis.

Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer.

A fluorescence-based, high-resolution imaging approach was used to visualize longitudinally the cellular events unfolding during T cell-mediated tumor destruction. The dynamic interplay of T cells, cancer cells, cancer antigen loss variants, and stromal cells-all color-coded in vivo-was analyzed in established, solid tumors that had developed behind windows implanted on the backs of mice. Events could be followed repeatedly within precisely the same tumor region-before, during and after adoptive T cell therapy-thereby enabling for the first time a longitudinal in vivo evaluation of protracted events, an analysis not possible with terminal imaging of surgically exposed tumors. T cell infiltration, stromal interactions, and vessel destruction, as well as the functional consequences thereof, including the elimination of cancer cells and cancer cell variants were studied. Minimal perivascular T cell infiltrates initiated vascular destruction inside the tumor mass eventually leading to macroscopic central tumor necrosis. Prolonged engagement of T cells with tumor antigen-crosspresenting stromal cells correlated with high IFNγ cytokine release and bystander elimination of antigen-negative cancer cells. The high-resolution, longitudinal, in vivo imaging approach described here will help to further a better mechanistic understanding of tumor eradication by T cells and other anti-cancer therapies.

Elevation of receptor tyrosine kinase EphA2 mediates resistance to trastuzumab therapy.

One arising challenge in the treatment of breast cancer is the development of therapeutic resistance to trastuzumab, an antibody targeting the human epidermal growth factor receptor-2 (HER2), which is frequently amplified in breast cancers. In this study, we provide evidence that elevated level of the receptor tyrosine kinase Eph receptor A2 (EphA2) is an important contributor to trastuzumab resistance. In a screen of a large cohort of human breast cancers, we found that EphA2 overexpression correlated with a decrease in disease-free and overall survival of HER2-overexpressing patients. Trastuzumab-resistant cell lines overexpressed EphA2, whereas inhibiting EphA2 restored sensitivity to trastuzumab treatment in vivo. Notably, trastuzumab treatment could promote EphA2 phosphorylation by activating Src kinase, leading in turn to an amplification of phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signaling in resistant cells. Our findings offer mechanistic insights into the basis for trastuzumab resistance and rationalize strategies to target EphA2 as a tactic to reverse trastuzumab resistance.

Establishment and quantitative imaging of a 3D lung organotypic model of mammary tumor outgrowth.

The lung is the second most common site of metastatic spread in breast cancer and experimental evidence has been provided in many systems for the importance of an organ-specific microenvironment in the development of metastasis. To better understand the interaction between tumor and host cells in this important secondary site, we have developed a 3D in vitro organotypic model of breast tumor metastatic growth in the lung. In our model, cells isolated from mouse lungs are placed in a collagen sponge to serve as a scaffold and co-cultured with a green fluorescent protein-labeled polyoma virus middle T antigen (PyVT) mammary tumor cell line. Analysis of the co-culture system was performed using flow cytometry to determine the relative constitution of the co-cultures over time. This analysis determined that the cultures consisted of viable lung and breast cancer cells over a 5-day period. Confocal microscopy was then used to perform live cell imaging of the co-cultures over time. Our studies determined that host lung cells influence the ability of tumor cells to grow, as the presence of lung parenchyma positively affected the proliferation of the mammary tumor cells in culture. In summary, we have developed a novel in vitro model of breast tumor cells in a common metastatic site that can be used to study tumor/host interactions in an important microenvironment.

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5.

Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (Grl), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in grl cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression.

C/EBPbeta-2 confers EGF-independent growth and disrupts the normal acinar architecture of human mammary epithelial cells.

BACKGROUND: The transcription factor, C/EBPbeta, is a key regulator of growth and differentiation in the mammary gland. There are three different protein isoforms of C/EBPbeta. C/EBPbeta-1 and -2 are transactivators, and differ by only 23 N-terminal amino acids present in beta-1 only. C/EBPbeta-3 (LIP) lacks the transactivation domain and represses transcription. Elevated C/EBPbeta-2 expression causes MCF10A normal human mammary epithelial cells to become transformed, undergo an epithelial to mesenchymal transition (EMT), and acquire an invasive phenotype. C/EBPbeta is a downstream transcriptional target of Ras signaling pathways and is required for Ras transformation of some cell types. Ras signaling pathways are activated in mammary epithelial cells by the ErbB receptor tyrosine kinase family. Therefore, we considered whether elevated C/EBPbeta-2 expression would resemble ErbB RTK activation in MCF10A cells. RESULTS: We show that elevated C/EBPbeta-2 expression confers EGF-independent growth in MCF10A mammary epithelial cells. However, MCF10A cells expressing C/EBPbeta-3 are not EGF-independent, and high C/EBPbeta-3 or LIP expression is incompatible with growth. C/EBPbeta-2 overexpression disrupts the normal acinar architecture of MCF10A cells in basement membrane cultures and induces complex multiacinar structures with filled lumen, similar to the consequences of aberrant ErbB2 activation. CONCLUSION: Given the ability of C/EBPbeta-2 to confer EGF-independent growth to mammary epithelial cells as well as its capability for disrupting normal epithelial architecture and causing EMT, it is worth considering whether inhibitors which target ErbB family signaling pathways could be less effective in mammary epithelial cells with elevated nuclear C/EBPbeta-2 expression.

A structural requirement for processing the cardiac K+ channel KCNQ1.

Normal membrane protein function requires trafficking from the endoplasmic reticulum. Here, we studied processing of the KCNQ1 channel mutated in LQT1, the commonest form of the long QT syndrome. Serial C terminus truncations identified a small region (amino acids (aa) 610-620) required for normal cell surface expression. Non-trafficked truncations assembled as tetramers but were nevertheless retained in the endoplasmic reticulum. Further mutagenesis did not identify specific residues mediating channel processing; cell surface expression was preserved with the mutation of known trafficking motifs in the channel and with alanine scanning across aa 610-620. Structural prediction algorithms place aa 610-620 at the C-terminal end of an alpha-helix (aa 586-618) that includes a leucine zipper and is part of a coiled coil. Mutants disrupting the leucine zipper but preserving the predicted coiled coil reached the cell surface, whereas those disrupting the coil did not. These data suggest that specific protein-protein interactions are required for normal channel processing. Further biochemical studies ruled out three candidate proteins, namely KCNE1, yotiao, and KCNQ1 itself, as effectors of this coiled coil-mediated trafficking. Four LQT1 mutations within this helix generated little or no current and were not expressed on the cell surface, whereas LQT1 mutations in adjacent residues, which produce a milder clinical phenotype, generate only slightly reduced current and are expressed on the cell surface. These data suggest that mutations within this domain cause human disease by interfering with normal channel processing. More generally, we have identified a domain whose structural integrity is required for normal surface expression of the KCNQ1 channel.

An adenoviral vector containing an arg-gly-asp (RGD) motif in the fiber knob enhances protein product levels from transgenes refractory to expression.

Genetic manipulation of the adenovirus type 5 represents one strategy to modify viral transduction properties in vitro and in vivo. In the majority of studies to date, reporter gene activity has been monitored to assess transduction efficiency. BRCA1 is a gene whose protein product is clinically important, biologically toxic, difficult to overexpress, and difficult to detect as an untagged protein species. Thus, it represents an attractive candidate from which to evaluate the efficacy of a gene delivery system. In the present study, transgene expression was assessed employing otherwise isogenic viruses, which differed only in the presence or absence of an RGD integrin-binding motif in the HI loop of the Ad fiber knob. We utilized a combination of BRCA1 expression level comparisons among several human BRCA1/mutant BRCA1/murine Brca1 constructs and reporter gene activity following transduction of a panel of human breast and ovarian tumor cell lines representative of both sporadic and hereditary cases. A general overall concordance in efficiency was observed, whether the biological readout measured was reporter gene activity or steady-state level of ectopic BRCA1 protein produced. Importantly, the expression of full-length wild-type BRCA1 protein, clinically relevant mutant BRCA1 proteins or murine Brca1 was superior when the gene was delivered via the RGD-modified Ad. The ectopic BRCA1 stabilized endogenous BARD1 and this functional effect was evident at lower input viral doses when BRCA1 was delivered via the RGD-modified Ad. Quantitative, noninvasive, real-time image analysis of reporter gene function in nude mice harboring human ovarian tumor xenographs demonstrated a similar enhancement of expression in vivo by the RGD fiber modification, with low levels of transduction of normal mouse mesothelium. These results provide additional evidence supporting the concept that rational modification of viral vectors can result in the delivery of functionally active therapeutic proteins such as BRCA1 that present with technical difficulties with regard to their expression.