p120-catenin phosphorylation status alters E-cadherin mediated cell adhesion and ability of tumor cells to metastasize.

p120-catenin is considered to be a tumor suppressor because it stabilizes E-cadherin levels at the cell surface. p120-catenin phosphorylation is increased in several types of cancer, but the role of phosphorylation in cancer is unknown. The phosphorylation state of p120-catenin is important in controlling E-cadherin homophilic binding strength which maintains epithelial junctions. Because decreased cell-cell adhesion is associated with increased cancer metastasis we hypothesize that p120-catenin phosphorylation at specific Serine and Threonine residues alters the E-cadherin binding strength between tumor cells and thereby affect the ability of tumor cells to leave the primary tumor and metastasize to distant sites. In this study we show that expression of the p120-catenin phosphorylation dead mutant, by converting six Serine and Threonine sites to Alanine, leads to enhanced E-cadherin adhesive binding strength in tumor cells. We observed a decrease in the ability of tumor cells expressing the p120-catenin phosphorylation mutant to migrate and invade using in-vitro models of cancer progression. Further, tumor cells expressing the phosphorylation mutant form of p120-catenin demonstrated a decrease in ability to metastasize to the lungs using an in-vivo orthotopic mammary fat pad injection model of breast cancer development and metastasis. This suggests that regulation of p120-catenin phosphorylation at the cell surface is important in mediating cell-adhesion, thereby impacting cancer progression and metastasis

Hepatocellular proliferation correlates with inflammatory cell and cytokine changes in a murine model of nonalchoholic fatty liver disease.

Nonalchoholic fatty liver disease (NAFLD) is a problem of increasing prevalence and clinical significance worldwide and is associated with increased risk of development of end stage liver disease and cirrhosis, and can be complicated by hepatocellular carcinoma (HCC). NAFLD is characterized by physical and molecular changes in the liver microenvironment which include an influx of inflammatory cell populations, fibrosis, changes in gene expression, and cytokine production. To better understand changes to the liver in the setting of steatosis, we used a murine model of diet induced hepatic steatosis and corroborated our results with human patient samples of NAFLD. Among the cellular changes, we identified a significant increase in hepatocellular proliferation in the setting of steatosis as compared to controls. Analysis of inflammatory cell populations revealed increased infiltration of CD11b positive myeloid and CD3 positive lymphocytic cell populations in steatotic livers compared to normal livers. Resident Kupffer cells of the liver comprise the largest percentage of these myeloid cells and appear to be responsible for important cytokine alterations impacting proliferation of cells in the liver microenvironment. Significant alterations in cytokine profiles in the plasma and liver tissue lysates from normal and steatotic mice were detected including leptin, CXCL1, CXCL2, and CXCL16 that were further shown to directly increase hepatocyte proliferation in vitro. This increased hepatocellular proliferation and turnover in the setting of steatosis may play important roles in the progression and complications of NAFLD

Changes in the lymphoid cell sub-populations in normal vs steatotic murine livers.

<p>Isolated CD45 positive cells from normal and steatotic livers were stained for lymphoid markers, subjected to flow cytometry and gated as a fraction of CD3e positive lymphocytic cells. CD3e⁺ cells were analyzed for changes in the total percentage of (A) CD4 positive helper T cells, (B) CD8 positive cytotoxic T cells, (C) CD25 positive regulatory T cells, and (D) CD62L negative activated T cells between normal and steatotic murine livers. Results only showed a significant increase in the overall CD25⁺ subpopulation of CD3e⁺ cells in the steatotic livers when compared to normal livers.</p

Cytokines effect hepatocyte proliferation in vitro.

<p>The effect of different concentrations of Leptin, CXCL1, CXCL2 and CXCL16 (100 ng/ml, 25 ng/ml and 1 ng/ml) on proliferation of (A) HepG2 and (B) HEPT cells determined by MTT assay. 10% FBS was used as a positive control. ** represents P<0.001 and *** P<0.0001 when compared to Serum Free conditions.</p

Changes in the myeloid cell sub-populations in normal vs steatotic murine livers.

<p>Isolated CD45 positive cells from normal and steatotic livers were stained for myeloid markers, subjected to flow cytometry and gated as a fraction of CD11b positive myeloid cells. (A) CD11b+ cells were analyzed for changes in the total percentage of F4/80 positive macrophages, (B) Gr1^hi expressing cells, (C) Ly6C positive Gr1 positive activated monocytes and (D) Ly6C negative Gr1 positive infiltrating neutrophils between normal and steatotic murine livers. Results show a significant decrease in the GR1hi and Ly6CpGr1p subpopulations and a significant increase in the Ly6CnGr1p subpopulation of steatotic versus normal livers, while there was not a significant difference the percentage of F4/80p subpopulation.</p

Quantification of inflammatory cell populations in human liver samples.

<p>Immunohistochemical staining was used to detect and quantify changes in inflammatory cell populations between normal and steatotic human liver samples. Positive immunoreactive staining (dark brown) was calculated as a percentage of total area for (A) CD45 positive inflammatory cells, (B) CD68 positive macrophages (C) CD3 positive T lymphocytes (D) CD8 positive cytotoxic T cells and (E) CD56 positive NK cells in frozen sections of normal and steatotic human livers. Results demonstrate a significant increase in the number of CD45 and CD3 positive cells in the steatotic livers when compared to normal liver samples. CD68 macrophages, CD8 cytotoxic T cells, and CD56 NK cells were not significantly altered between samples. Images are 20x.</p

Steatosis results in changes in the inflammatory cell populations in the murine liver.

<p>CD45 positive cells were isolated from normal and steatotic digested liver samples by immunomagnetic beads and then stained for the immune markers CD3 and CD11b before being subjected to Flow cytometric analysis. (A) Diagrams are representative scatter plot of flow cytometric analysis for overall CD45 positive cells in the livers. (B) CD45 positive cells were then gated for the percentage of CD3 positive T lymphocytes or CD11b positive myeloid cells. CD3+ and CD11b+ subpopulations were both increased in the steatotic livers versus the control livers.</p

Steatosis results in increased cellular proliferation in the liver.

<p>The percentage of total Ki67 positive cells and the percent of Ki67/HNF4α double positive hepatocytes are both increased in steatotic livers compared to normal livers of (A, B) murine and (C, D) human samples. Representative images of Ki67 (green), HNF4α (red) and DAPI (blue) immunofluoresence staining in livers of normal and steatotic (B) murine and (D) human liver sections showing increased number of Ki67 positive cells in steatotic livers. Images are taken at 40X and scale bars represent 50 microns.</p

Changes in cell sub-populations of B cells, Dendritic cells and Natural Killer cells in steatotic livers.

<p>Isolated CD45 positive cells from normal and steatotic livers were stained for markers for (A) B cells (CD19⁺), (B) dendritic cells (CD11c⁺, CD19^–) and (C) natural killers cells (NKp46⁺) before being subjected to flow cytometry and gated as a fraction of CD45⁺ cells to detect total percentage of subpopulations. Results demonstrate a significant decrease in B cells and a significant increase in total dendritic subpopulations in steatotic murine livers compared to normal livers. Percentage of the overall subpopulation of natural killer cells were not significantly different between groups.</p

Fold change in cytokine levels of high fat diet fed mice.

<p>Plasma, liver tissue lysates and conditioned media from CD11b⁺ magnetically isolated cells Cytokine arrays were used to detect changes in cytokine levels in the plasma, perfused liver tissue lysate (Liver Lysate), and from conditioned media of isolated CD45⁺CD11b⁺ subpopulations (CD11b⁺ Cell CM) between normal and steatotic samples. Cytokine values are presented as fold change with values greater than 1 representing increased levels and values less than 1 representing decreased levels in steatotic samples. (n = 3). Axl (Tyrosine protein kinase 7), CTACK (Cutaneous T-cell attracting chemokine, CCL27), CXCL16 (CXC chemokine ligand 16), IL-2 (Interleukin-2), IL-3 (Interleukin-3), IL-6 (Interleukin-6), IL-9 (Interleukin-9), IL-13 (Interleukin-13), IL-3 Rb (Interleukin-3 receptor beta, CD131), KC (CXC chemokine ligand 1), MCP-1 (Monocyte chemotactic protein-1, CCL2), MCP-5 (Monocyte chemotactic protein-5, CCL12), MIP-1γ (Macrophage inflammatory protein-1 gamma), MIP-2 (macrophage inflammatory protein -2, CXCL2), PF-4 (Platelet factor 4, CXCL4), RANTES (Regulated on activation normal T-cell expressed and secreted, CCL5), SCF (Stem cell factor), TNF (Tumor necrosis factor), VCAM-1 (Vascular cell adhesion molecule 1, CD106).</p