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p190RhoGAP (p190), a Rho family GTPase-activating protein, regulates actin stress fiber dynamics via hydrolysis of Rho-GTP. Recent data suggest that p190 also regulates cell proliferation. To gain insights into the cellular process(es) affected by p190, we altered its levels by conditional or transient overexpression. Overexpression of p190 resulted in a multinucleated phenotype that was dependent on the GTPase-activating protein domain. Confocal immunofluorescence microscopy revealed that both endogenous and exogenous p190 localized to the newly forming and contracting cleavage furrow of dividing cells. However, overexpression of p190 resulted in abnormal positioning of the furrow specification site and unequal daughter cell partitioning, as well as faulty furrow contraction and multinucleation. Furthermore, levels of endogenous p190 protein were transiently decreased in late mitosis via an ubiquitin-mediated degradation process that required the NH2-terminal GTP-binding region of p190. These results suggest that a cell cycle–regulated reduction in endogenous p190 levels is linked to completion of cytokinesis and generation of viable cell progeny.

Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.

Background:The mechanisms of transcriptional regulation of TNFRSF10A and TNFRSF10B are not well described. Results: DDIT3 and KAT2A cooperatively up-regulated TNFRSF10A and TNFRSF10B. Conclusion: DDIT3 and KAT2A promote the transcription of TNFRSF10A and TNFRSF10B via the AP-1 and DDIT3 binding sites, respectively. Significance: Our findings offer new insights on the mechanisms of ER stress-mediated apoptosis.

BACKGROUND.The authors investigated whether coexpression and localization of E‐cadherin (E‐cad) and epidermal growth factor receptor (EGFR) had predictive and/or prognostic correlations with lymph node metastasis and/or survival in patients with squamous cell carcinoma of the head and neck (SCCHN).METHODS.Immunohistochemistry (IHC) of archival tissue was performed to measure expression of EGFR and E‐cad in surgical specimens of SCCHN (n = 143) that included primary tumors (PTs) with positive lymph nodes (Tu+Met) and their paired lymph node metastases (LnMet), PTs with negative lymph nodes (Tu−Met), and benign tissue biopsies as normal controls. IHC staining was quantified as a weighted index and as the ratio of membrane to cytoplasmic staining. Correlative expression between EGFR and E‐cad also was examined in SCCHN cell lines by immunoblotting and immunofluorescence analyses.RESULTS.Three distinct expression patterns of EGFR and E‐cad were observed. Membrane localization of E‐cad was significantly lower in the Tu+Met group than in the Tu−Met group (P = .01) and was associated inversely with lymph node status (P = .009). Wilcoxon analysis of the combined markers demonstrated that expression and/or membrane localization of EGFR and E‐cad were correlated with disease‐free survival and overall survival in patients with SCCHN. The study of SCCHN cell lines demonstrated that cells with positive but low EGFR expression and with negative E‐cad expression were relatively resistant to the EGFR tyrosine kinase inhibitor erlotinib.CONCLUSIONS.The current study suggested that examining not only the expression but also the localization of EGFR and E‐cad simultaneously may have clinical relevance in predicting lymph node metastasis, patient survival, and response to EGFR‐targeted therapy in patients with SCCHN. Cancer 2008. © 2008 American Cancer Society.

BackgroundParthenolide (PTL) is a sesquiterpene lactone which can induce apoptosis in cancer cells and eradicate cancer stem cells such as leukemia stem cells, prostate tumor-initiating cells and so on. However, the mechanism remains largely unclear.MethodsLung cancer cells were treated with parthenolide and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the cell survival was assayed by SRB and MTT assay. Cell cycle was evaluated by DNA flow cytometry analysis. TNFRSF10B, PMAIP1, ATF4 and DDIT3 genes were knocked down by siRNA technique. Apoptosis was evaluated by using Annexin V-FITC/PI staining and flow cytometry analysis.ResultsParthenolide (PTL) induces apoptosis and cell cycle arrest in human lung cancer cells. Moreover, PTL treatment in NSCLC cells increases expression of TNFRSF10B/DR5 and PMAIP1/NOXA. Silencing of TNFRSF10B or PMAIP1 or overexpression of CFLAR /c-FLIP (long form) could protect cells from PTL-induced apoptosis. Furthermore, PTL could increase the levels of endoplasmic reticulum stress hallmarks such as ERN1, HSPA5, p-EIF2A, ATF4 and DDIT3. Knockdown of ATF4 and DDIT3 abrogated PTL-induced apoptosis, which suggested that PTL induced apoptosis in NSCLC cells through activation of endoplasmic reticulum stress pathway. More importantly, we found that ATF4, DDIT3, TNFRSF10B and PMAIP1 were up-regulated more intensively, while CFLAR and MCL1 were down-regulated more dramatically by PTL in A549/shCDH1 cells than that in control cells, suggesting that PTL preferred to kill cancer stem cell-like cells by activating more intensive ER stress response in cancer stem cell-like cells.ConclusionWe showed that parthenolide not only triggered extrinsic apoptosis by up-regulating TNFRSF10B and down-regulating CFLAR, but also induced intrinsic apoptosis through increasing the expression of PMAIP1 and decreasing the level of MCL1 in NSCLC cells. In addition, parthenolide triggered stronger ER stress response in cancer stem cell-like cells which leads to its preference in apoptotic induction. In summary, PTL induces apoptosis in NSCLC cells by activating endoplasmic reticulum stress response.

Metastasis of squamous cell carcinoma of the head and neck (SCCHN) is a significant health-care problem worldwide. The 5-year survival rate is less than 50% for patients with lymph node metastases. Understanding the molecular basis of SCCHN metastasis would facilitate the development of new therapeutic approaches to the disease. To identify proteins that mediate SCCHN metastasis, we established a SCCHN xenograft mouse model and performed in vivo selection from a SCCHN cell line using the model. In the fourth round of in vivo selection, significant incidences of metastases in lymph nodes (7/10) and lungs (6/10) were achieved from a derived SCCHN cell line as compared with its parental cells, 1/5 in lymph nodes and 0/5 in lungs. Metastatic cell lines from lymph node metastases and parental cell lines from non-metastatic xenograft tumors were subjected to DNA microarray analysis using an Affymetrix gene chip HG-U133A, followed by data mining studies. The identified metastasis-related genes were further evaluated for their encoding protein products and the metastatic cells were examined by biological analyses. DNA microarray analysis highlighted molecular features of the metastatic SCCHN cells, including alteration of expression of cell-cell adhesion proteins, epithelial cell markers, apoptosis and cell cycle regulatory molecules. Further biological analyses of phenotypic alterations revealed that the metastatic cells gained epithelial-mesenchymal transition (EMT) features and were more resistant to anoikis, which are two of the important phenotypes for metastatic SCCHN.