The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β

INTRODUCTION: Transforming growth factor-β (TGF-β) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-β-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-β. Recent findings suggest that therapeutics designed to antagonize TGF-β signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-β type II receptor antagonist that inhibits TGF-β binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-β, particularly its stimulation of mammary epithelial–mesenchymal transition (EMT), can be prevented by CystC. METHOD: Retroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Δ14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-β stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-β stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-β. RESULTS: CystC and Δ14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-β by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-β-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Δ14CystC also inhibited TGF-β signaling in two tumorigenic human breast cancer cell lines. CONCLUSION: Our findings show that TGF-β stimulation of initiating metastatic events, including decreased cell polarization, reduced cell–cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-β

Alterations in integrin expression modulates invasion of pancreatic cancer cells

Background Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. Methods In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. Results Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin β1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. Conclusion Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma

Solving the Puzzle of Metastasis: The Evolution of Cell Migration in Neoplasms

BACKGROUND: Metastasis represents one of the most clinically important transitions in neoplastic progression. The evolution of metastasis is a puzzle because a metastatic clone is at a disadvantage in competition for space and resources with non-metastatic clones in the primary tumor. Metastatic clones waste some of their reproductive potential on emigrating cells with little chance of establishing metastases. We suggest that resource heterogeneity within primary tumors selects for cell migration, and that cell emigration is a by-product of that selection. METHODS AND FINDINGS: We developed an agent-based model to simulate the evolution of neoplastic cell migration. We simulated the essential dynamics of neoangiogenesis and blood vessel occlusion that lead to resource heterogeneity in neoplasms. We observed the probability and speed of cell migration that evolves with changes in parameters that control the degree of spatial and temporal resource heterogeneity. Across a broad range of realistic parameter values, increasing degrees of spatial and temporal heterogeneity select for the evolution of increased cell migration and emigration. CONCLUSIONS: We showed that variability in resources within a neoplasm (e.g. oxygen and nutrients provided by angiogenesis) is sufficient to select for cells with high motility. These cells are also more likely to emigrate from the tumor, which is the first step in metastasis and the key to the puzzle of metastasis. Thus, we have identified a novel potential solution to the puzzle of metastasis

Evolutionary Dynamics of Intratumor Heterogeneity

Intraneoplastic diversity in human tumors is a widespread phenomenon of critical importance for tumor progression and the response to therapeutic intervention. Insights into the evolutionary events that control tumor heterogeneity would be a major breakthrough in our comprehension of cancer development and could lead to more effective prevention methods and therapies. In this paper, we design an evolutionary mathematical framework to study the dynamics of heterogeneity over time. We consider specific situations arising during tumorigenesis, such as the emergence of positively selected mutations (“drivers”) and the accumulation of neutral variation (“passengers”). We perform exact computer simulations of the emergence of diverse tumor cell clones over time, and derive analytical estimates for the extent of heterogeneity within a population of cancer cells. Our methods contribute to a quantitative understanding of tumor heterogeneity and the impact of heritable alterations on this tumor trait

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

<p>Abstract</p> <p>Background</p> <p>Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application.</p> <p>Results</p> <p>In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by <it>CaMV </it>35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells.</p> <p>Conclusions</p> <p>In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants.</p

Macrophages Homing to Metastatic Lymph Nodes Can Be Monitored with Ultrasensitive Ferromagnetic Iron-Oxide Nanocubes and a 1.5T Clinical MR Scanner

Background: Due to the ability of macrophages to specifically home to tumors, their potential use as a delivery vehicle for cancer therapeutics has been suggested. Tracking the delivery and engraftment of macrophages into human tumors with a 1.5T clinical MR scanner requires the development of sensitive contrast agents for cell labeling. Therefore, this study aimed to determine whether intravenously injected macrophages could target a primary tumor as well as metastatic LNs, and whether these cells could be detected in vivo by MRI. Methodology: Peritoneal macrophages were obtained from BALB/c nude mice. The viability, phagocytotic capacity and migratory activity of the macrophages were assessed. MR imaging was performed using a clinical 1.5 T MR scanner and we estimated the T2 * of the labeled macrophages. Metastatic lymph nodes were produced in BALB/c nude mice. We administrated 2610 6 macrophages labeled with 50 mg Fe/mL FIONs intravenously into the mice. In the 3D T2 * GRE MR images obtained one day after the injection of the labeled macrophages or FION solution, the percentages of pixels in the tumors or LNs below the minimum normalized SI (signal intensity) threshold were summated and reported as the black pixel count (%) for the FION hypointensity. Tumors in the main tumor model as well as the brachial, axillary and inguinal lymph nodes in the metastatic LN models were removed and stained. For all statistical analyses, single-group data were assessed using t test or the Mann-Whitney test. Repeated measurements analysis of variance (ANOVA) with Tukey–Krame

Day occupation is associated with psychopathology for adolescents and young adults with Down syndrome

Background: Young adults with Down syndrome experience increased rates of emotional and behavioural problems compared with the general population. Most adolescents with Down syndrome living in Western Australia participate in sheltered employment as their main day occupation. Relationship between day occupation and changes in behaviour has not been examined. Therefore, the aim of this research was to explore any relationship between post school day occupations and changes in the young person’s behaviour. Methods: The Down syndrome Needs Opinion Wishes database was used for case ascertainment of young adults aged 15 to 32 years with Down syndrome. Families of 118 young people in this population-based database completed questionnaires in 2004, 2009 and 2011. The questionnaires addressed both young person characteristics such as age, gender, presence of impairments, behaviour, functioning in activities of daily living, and family characteristics such as income and family functioning. Post-school day occupations in which the young people were participating included open and sheltered employment, training and day recreation programs. Change in behaviour of young adults who remained in the same post-school day occupation from 2009 to 2011 (n = 103) were examined in a linear regression model adjusting for confounding variables including age, gender, prior functioning and behaviour in 2004 and family income.Results: In comparison to those young adults attending open employment from 2009 to 2011, those attending day recreation programs were reported to experience worsening in behaviour both in the unadjusted (effect size -0.14, 95% CI -0.24, -0.05) and adjusted models (effect size -0.15, 95% CI -0.29, -0.01). Conclusions: We found that the behaviour of those participating in open employment improved compared to those attending other day occupations. Further examination of the direction of this association is required

Vandetanib (ZD6474), an inhibitor of VEGFR and EGFR signalling, as a novel molecular-targeted therapy against cholangiocarcinoma

Cholangiocarcinoma is an intractable cancer, with no effective therapy other than surgical resection. Elevated vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) expressions are associated with the progression of cholangiocarcinoma. We therefore examined whether inhibition of VEGFR and EGFR could be a potential therapeutic target for cholangiocarcinoma. Vandetanib (ZD6474, ZACTIMA), a VEGFR-2/EGFR inhibitor, was evaluated. Four human cholangiocarcinoma cell lines were molecularly characterised and investigated for their response to vandetanib. In vitro, two cell lines (OZ and HuCCT1), both of which harboured KRAS mutation, were refractory to vandetanib, one cell line (TGBC24TKB) was somewhat resistant, and another cell line (TKKK) was sensitive. The most sensitive cell line (TKKK) had EGFR amplification. Vandetanib significantly inhibited the growth of TKKK xenografts at doses ⩾12.5 mg kg−1 day−1 (P<0.05), but higher doses (50 mg kg−1 day−1, P<0.05) of vandetanib were required to inhibit the growth of OZ xenografts. Vandetanib (25 mg kg−1 day−1) also significantly (P=0.006) prolonged the time to metastasis in an intravenous model of TKKK metastasis. Inhibiting both VEGFR and EGFR signalling appears a promising therapeutic approach for cholangiocarcinoma. The absence of KRAS mutation and the presence of EGFR amplification may be potential predictive molecular marker of sensitivity to EGFR-targeted therapy in cholangiocarcinoma