Down-Regulation of GEP100 Causes Increase in E-Cadherin Levels and Inhibits Pancreatic Cancer Cell Invasion

AIMS: Invasion and metastasis are major reasons for pancreatic cancer death and identifying signaling molecules that are specifically used in tumor invasion is of great significance. The purpose of this study was to elucidate the role of GEP100 in pancreatic cancer cell invasion and metastasis and the corresponding molecular mechanism. METHODS: Stable cell lines with GEP100 knocked-down were established by transfecting GEP100 shRNA vector into PaTu8988 cells and selected by puromycin. qRT-PCR and Western blot were performed to detect gene expression. Matrigel-invasion assay was used to detect cancer cell invasion in vitro. Liver metastasis in vivo was determined by splenic injection of indicated cell lines followed by spleen resection. Immunofluorescence study was used to detect the intracellular localization of E-cadherin. RESULTS: We found that the expression level of GEP100 protein was closely related to the invasive ability of a panel of 6 different human pancreatic cancer cell lines. Down-regulation of GEP100 in PaTu8988 cells significantly decreased invasive activity by Matrigel invasion assay, without affecting migration, invasion and viability. The inhibited invasive activity was rescued by over-expression of GEP100 cDNA. In vivo study showed that liver metastasis was significantly decreased in the PaTu8988 cells with GEP100 stably knocked-down. In addition, an epithelial-like morphological change, mimicking a mesenchymal to epithelial transition (MET) was induced by GEP100 down-regulation. The expression of E-cadherin protein was increased 2-3 folds accompanied by its redistribution to the cell-cell contacts, while no obvious changes were observed for E-cadherin mRNA. Unexpectedly, the mRNA of Slug was increased by GEP100 knock-down. CONCLUSION: These findings provided important evidence that GEP100 plays a significant role in pancreatic cancer invasion through regulating the expression of E-cadherin and the process of MET, indicating the possibility of it becoming a potential therapeutic target against pancreatic cancer

Statins may have double-edged effects in patients with lung adenocarcinoma after lung resection

Shigeto Nishikawa,1 Toshi Menju,1 Koji Takahashi,1 Ryo Miyata,1 Toyofumi Fengshi Chen-Yoshikawa,1 Makoto Sonobe,1 Akihiko Yoshizawa,2 Hisataka Sabe,3 Tosiya Sato,4 Hiroshi Date11Department of Thoracic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 2Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto, Japan; 3Department of Molecular Biology, Faculty of Medicine, Hokkaido University, Sapporo, Japan; 4Department of Biostatistics, Kyoto University School of Public Health, Kyoto, JapanPurpose: The epithelial to mesenchymal transition (EMT) is pivotal for driving metastasis and recurrence in lung cancer. Some in vitro reports have shown that statins suppress EMT by inactivating mutant p53 functions. Several clinical trials of conventional treatments with statins have been performed, but the effect of these drugs on prognosis is still uncertain. The purpose of this study is to examine the impact of statins on EMT and the prognosis of patients with lung adenocarcinoma.Materials and methods: Morphological changes were evaluated and EMT markers (E-cadherin, vimentin) were analyzed by Western blotting in p53-overexpressing H1650 and mutant p53-harboring H1975 lung adenocarcinoma cells, with and without simvastatin administration. The invasive ability of these cells was analyzed in a Matrigel chemoinvasion assay. A total of 250 lung adenocarcinoma specimens were also collected from patients who underwent surgery in our institute. EMT markers in these tumor specimens were evaluated by immunostaining and p53 mutation status was determined by direct sequencing. Associations among EMT status, p53 mutation status, and statin use were evaluated, and prognosis was analyzed using a marginal structural model.Results: Mutant p53 induced EMT and increased the invasive ability of H1650 cells. Simvastatin restored the epithelial phenotype and decreased the invasive ability of both H1650 and H1975 cells. Statin administration was associated with inactivation of EMT only in patients with mutant p53, which was consistent with the in vitro results. Moreover, in patients with mutant p53, statin users had significantly better survival than non-statin users. In contrast, statins significantly worsened the prognosis of patients with wild type p53 (HR 2.10, 95% CI 1.14–3.85).Conclusion: Statins suppress EMT and change the prognosis of patients with lung adenocarcinoma in a p53 mutation-dependent manner.Keywords: p53, epithelial to mesenchymal transition, statin, survival analysis, non-small cell lung cance

Monitoring chronic lymphocytic leukemia progression by whole genome sequencing reveals heterogeneous clonal evolution patterns.

Chronic lymphocytic leukemia is characterized by relapse after treatment and chemotherapy resistance. Similarly, in other malignancies leukemia cells accumulate mutations during growth, forming heterogeneous cell populations that are subject to Darwinian selection and may respond differentially to treatment. There is therefore a clinical need to monitor changes in the subclonal composition of cancers during disease progression. Here, we use whole-genome sequencing to track subclonal heterogeneity in 3 chronic lymphocytic leukemia patients subjected to repeated cycles of therapy. We reveal different somatic mutation profiles in each patient and use these to establish probable hierarchical patterns of subclonal evolution, to identify subclones that decline or expand over time, and to detect founder mutations. We show that clonal evolution patterns are heterogeneous in individual patients. We conclude that genome sequencing is a powerful and sensitive approach to monitor disease progression repeatedly at the molecular level. If applied to future clinical trials, this approach might eventually influence treatment strategies as a tool to individualize and direct cancer treatment

Heterogeneous expression of apolipoprotein-E by human macrophages

Apolipoprotein-E (apoE) is expressed at high levels by macrophages. In addition to its role in lipid transport, macrophage-derived apoE plays an important role in immunoregulation. Previous studies have identified macrophage subpopulations that differ substantially in their ability to synthesize specific cytokines and enzymes, however, potential heterogeneous macrophage apoE expression has not been studied. Here we examined apoE expression in human THP-1 macrophages and monocyte-derived macrophages (MDM). Using immunocytochemistry and flow cytometry methods we reveal a striking heterogeneity in macrophage apoE expression in both cell types. In phorbol-ester-differentiated THP-1 macrophages, 5% of the cells over-expressed apoE at levels more than 50-fold higher than the rest of the population. ApoE over-expressing THP-1 macrophages contained condensed/fragmented nuclei and increased levels of activated caspase-3 indicating induction of apoptosis. In MDM, 3–5% of the cells also highly over-expressed apoE, up to 50-fold higher than the rest of the population; however, this was not associated with obvious nuclear alterations. The apoE over-expressing MDM were larger, more granular, and more autofluorescent than the majority of cells and they contained numerous vesicle-like structures that appeared to be coated by apoE. Flow cytometry experiments indicated that the apoE over-expressing subpopulation of MDM were positive for CD14, CD11b/Mac-1 and CD68. These observations suggest that specific macrophage subpopulations may be important for apoE-mediated immunoregulation and clearly indicate that subpopulation heterogeneity should be taken into account when investigating macrophage apoE expression

Lysophosphatidic acid activates Arf6 to promote the mesenchymal malignancy of renal cancer

Acquisition of mesenchymal properties by cancer cells is critical for their malignant behaviour, but regulators of the mesenchymal molecular machinery and how it is activated remain elusive. Here we show that clear cell renal cell carcinomas (ccRCCs) frequently utilize the Arf6-based mesenchymal pathway to promote invasion and metastasis, similar to breast cancers. In breast cancer cells, ligand-activated receptor tyrosine kinases employ GEP100 to activate Arf6, which then recruits AMAP1; and AMAP1 then binds to the mesenchymal-specific protein EPB41L5, which promotes epithelial-mesenchymal transition and focal adhesion dynamics. In renal cancer cells, lysophosphatidic acid (LPA) activates Arf6 via its G-protein-coupled receptors, in which GTP-G alpha 12 binds to EFA6. The Arf6-based pathway may also contribute to drug resistance. Our results identify a specific mesenchymal molecular machinery of primary ccRCCs, which is triggered by a product of autotaxin and it is associated with poor outcome of patients