Role of PINCH and Its Partner Tumor Suppressor Rsu-1 in Regulating Liver Size and Tumorigenesis

Particularly interesting new cysteine-histidine-rich protein (PINCH) protein is part of the ternary complex known as the IPP (integrin linked kinase (ILK)-PINCH-Parvin-α) complex. PINCH itself binds to ILK and to another protein known as Rsu-1 (Ras suppressor 1). We generated PINCH 1 and PINCH 2 Double knockout mice (referred as PINCH DKO mice). PINCH2 elimination was systemic whereas PINCH1 elimination was targeted to hepatocytes. The genetically modified mice were born normal. The mice were sacrificed at different ages after birth. Soon after birth, they developed abnormal hepatic histology characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells and increased deposition of extracellular matrix. After a sustained and prolonged proliferation of all epithelial components, proliferation subsided and final liver weight by the end of 30 weeks in livers with PINCH DKO deficient hepatocytes was 40% larger than the control mice. The livers of the PINCH DKO mice were also very stiff due to increased ECM deposition throughout the liver, with no observed nodularity. Mice developed liver cancer by one year. These mice regenerated normally when subjected to 70% partial hepatectomy and did not show any termination defect. Ras suppressor 1 (Rsu-1) protein, the binding partner of PINCH is frequently deleted in human liver cancers. Rsu-1 expression is dramatically decreased in PINCH DKO mouse livers. Increased expression of Rsu-1 suppressed cell proliferation and migration in HCC cell lines. These changes were brought about not by affecting activation of Ras (as its name suggests) but by suppression of Ras downstream signaling via RhoGTPase proteins. In conclusion, our studies suggest that removal of PINCH results in enlargement of liver and tumorigenesis. Decreased levels of Rsu-1, a partner for PINCH and a protein often deleted in human liver cancer, may play an important role in the development of the observed phenotype. © 2013 Donthamsetty et al

Characteristics of US-Based STEM Webcams \u3cem\u3eat a Glance\u3c/em\u3e

STEM organizations operate thousands of publicly available webcams, which have the potential to provide rich informal learning opportunities. To date, no research has analyzed the breadth of STEM webcams. In our study, we developed an inventory by performing internet searches for US-based STEM webcams and recorded operator name, organization type, webcam subject, etc. This inventory serves as the basis for a follow-up survey of webcam operators, which seeks to better understand the goals, outcomes, and investments of STEM webcam programs. We characterized nearly 1000 webcams that fit within our inclusion criteria. The majority of the cams fell within the life sciences or geosciences. Of the animal webcams, mammals (60%) and birds (23%) were strongly represented in contrast to reptiles (2%) and insects (4%). Within the mammal subjects, tigers, elephants, otters, and giraffes were most common (5-7% each). Within the 23 different bird webcam subjects, penguins (30%) and osprey (20%) were most common. Other studies have shown the over-representation of charismatic animals (e.g., elephants, giraffes, tigers, bears) in zoos, and our study suggests that webcams may amplify this phenomenon. Further, these preliminary results suggest that life sciences topics dominate STEM webcams, leaving potential for other visually engaging fields to participate

Neuroblastoma invasion strategies are regulated by the extracellular matrix

Neuroblastoma is a paediatric malignancy of the developing sympathetic nervous system. About half of the patients have metastatic disease at the time of diagnosis and a survival rate of less than 50%. Our understanding of the cellular processes promoting neuroblastoma metastases will be facilitated by the development of appropriate experimental models. In this study, we aimed to explore the invasion of neuroblastoma cells and organoids from patient-derived xenografts (PDXs) grown embedded in 3D extracellular matrix (ECM) hydrogels by time-lapse microscopy and quantitative image analysis. We found that the ECM composition influenced the growth, viability and local invasion of organoids. The ECM compositions induced distinct cell behaviours, with Matrigel being the preferred substratum for local organoid invasion. Organoid invasion was cell line- and PDX-dependent. We identified six distinct phenotypes in PDX-derived organoids. In contrast, NB cell lines were more phenotypically restricted in their invasion strategies, as organoids isolated from cell line-derived xenografts displayed a broader range of phenotypes compared to clonal cell line clusters. The addition of FBS and bFGF induced more aggressive cell behaviour and a broader range of phenotypes. In contrast, the repression of the prognostic neuroblastoma marker, MYCN, resulted in less aggressive cell behaviour. The combination of PDX organoids, real-time imaging and the novel 3D culture assays developed herein will enable rapid progress in elucidating the molecular mechanisms that control neuroblastoma invasion

Role of PINCH and Its Partner Tumor Suppressor Rsu-1 in Regulating Liver Size and Tumorigenesis

Particularly interesting new cysteine-histidine-rich protein (PINCH) protein is part of the ternary complex known as the IPP (integrin linked kinase (ILK)-PINCH-Parvin-a) complex. PINCH itself binds to ILK and to another protein known as Rsu-1 (Ras suppressor 1). We generated PINCH 1 and PINCH 2 Double knockout mice (referred as PINCH DKO mice). PINCH2 elimination was systemic whereas PINCH1 elimination was targeted to hepatocytes. The genetically modified mice were born normal. The mice were sacrificed at different ages after birth. Soon after birth, they developed abnormal hepatic histology characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells and increased deposition of extracellular matrix. After a sustained and prolonged proliferation of all epithelial components, proliferation subsided and final liver weight by the end of 30 weeks in livers with PINCH DKO deficient hepatocytes was 40% larger than the control mice. The livers of the PINCH DKO mice were also very stiff due to increased ECM deposition throughout the liver, with no observed nodularity. Mice developed liver cancer by one year. These mice regenerated normally when subjected to 70% partial hepatectomy and did not show any termination defect. Ras suppressor 1 (Rsu-1) protein, the binding partner of PINCH is frequently deleted in human liver cancers. Rsu-1 expression is dramatically decreased in PINCH DKO mouse livers. Increased expression of Rsu-1 suppressed cell proliferation and migration in HCC cell lines. These changes were brought about not by affecting activation of Ras (as its name suggests) but by suppression of Ras downstream signaling via RhoGTPase proteins. In conclusion, our studies suggest that removal of PINCH results in enlargement of liver and tumorigenesis. Decreased levels of Rsu-1, a partner for PINCH and a protein often deleted in human liver cancer, may play an important role in the development of the observed phenotype. © 2013 Donthamsetty et al

ECM changes in PINCH DKO mice.

<p>A) Photomicrograph of a liver of 5 week old PINCH DKO mouse showing increased stellate cell activation as evident by αSMA stain (shown by arrows). Similar photomicrograph from a Control mouse of the same age shows minimal activation of stellate cells B) Photomicrograph of a liver of a 30 week PINCH DKO mouse showing increased ECM deposition as evident by reticulin stain. All the figures are 100X. The insert are of 200X magnification.</p

Liver regeneration kinetics in PINCH DKO mice.

<p>C) PINCH DKO mice show no termination defect. At Day 14 after partial hepatectomy, the percentage body/liver weight of the control and PINCH DKO mice reaches 100% of the original.</p

Rsu-1 levels in HCC.

<p>A) Graphical representation of the number of HCC cases positive, negative or moderately positive for Rsu-1 in HCC tissue array (24 cases/48 cores). B) Protein levels of Rsu-1 in HCC cell lines compared to human hepatocytes (HH). Most of the HCC cell lines show decrease in Rsu-1 protein. C) Successful overexpression of Rsu-GFP (fusion protein) in Hep3B cell line. GFP tagged ORF clone of Homo sapiens Rsu-1 (#RG203334, Origene) was transfected into Hep3B cell line and analyzed for Rsu-1 48 h after transfection. Since it is a GFP fused protein, the MW of Rsu-1 is ~fifty-five kd instead of twenty-nine kd (MW of GFP is ~twenty-six kd). D) GFP-Rsu-1 fusion protein associates with PINCH inside the cell. Overexpression of GFP-Rsu-1 in Hep3B cell line leads to association of GFP-Rsu-1 with PINCH. GFP was immunoprecipitated 48 h after transfection. GFP precipitates were probed with either GFP or PINCH. Presence of PINCH in GFP precipitates shows association of GFP-Rsu-1with PINCH.</p

Components of the IPP complex are upregulated at the end of liver regeneration.

<p>Western blot of various components of IPP complex in rat hepatocyte pellet at various time points after partial hepatectomy. Hepatocytes were isolated by 2 step collagenase perfusion.</p

Quantitative assessment of hepatocyte proliferation and apoptosis in PINCH DKO mice.

<p>A) Number of Ki67 positive cells/field at different ages after birth. Each data point is the mean ± SE from two fields per slide from each animal in a total of at least 3 animals. B) Fold change in apoptosis (caspase3/7 activity) in the PINCH DKO cell lysates as compared to the controls at different ages. The numbers were derived as the ratio of caspace 3/7optical density between DKO and control mice. Each data point is the mean ± SE of at least 3 pairs of mice per time point. Comparison between two groups at the same time point is made by unpaired Student’s t test. The criterion for statistical significance is p ≤ 0.05. * indicates statistically significant difference.</p