SNAI1-Mediated Epithelial-Mesenchymal Transition Confers Chemoresistance and Cellular Plasticity by Regulating Genes Involved in Cell Death and Stem Cell Maintenance

<div><p>Tumor cells at the tumor margin lose epithelial properties and acquire features of mesenchymal cells, a process called epithelial-to-mesenchymal transition (EMT). Recently, features of EMT were shown to be linked to cells with tumor-founding capability, so-called cancer stem cells (CSCs). Inducers of the EMT include several transcription factors, such as Snail (SNAI1) and Slug (SNAI2), as well as the secreted transforming growth factor (TGFß). In the present study, we found that EMT induction in MCF10A cells by stably expressing SNAI1 contributed to drug resistance and acquisition of stem/progenitor-like character as shown by increased cell population for surface marker CD44⁺/CD24⁻ and mammosphere forming capacity. Using a microarray approach, we demonstrate that SNAI1 overexpression results in a dramatic change in signaling pathways involved in the regulation of cell death and stem cell maintenance. We showed that NF-κB/MAPK signaling pathways are highly activated in MCF10A-SNAI1 cells by IL1ß stimulation, leading to the robust induction in <i>IL6</i> and <i>IL8</i>. Furthermore, MCF10A-SNAI1 cells showed enhanced TCF/ß-catenin activity responding to the exogenous Wnt3a treatment. However, EMT-induced stem/progenitor cell activation process is tightly regulated in non-transformed MCF10A cells, as <i>WNT5A</i> and <i>TGFB2</i> are strongly upregulated in MCF10A-SNAI1 cells antagonizing canonical Wnt pathway. In summary, our data provide new molecular findings how EMT contributes to the enhanced chemoresistance and the acquisition of stem/progenitor-like character by regulating signaling pathways.</p></div

SNAI1 overexpression in MCF10A cells induced a dramatic change in gene expression profile.

<p>(A) The microarray discovered 966 genes differentially expressed in MCF10A and MCF10A-SNAI1 cells (fold change >2, <i>p</i>-value <0.01). 966 genes were subjected to GO analysis using Gene Spring GX program to gain insights on biological functions of the genes. Majority of differentially regulated genes were shown to be involved in signaling. FDR-corrected p-values were displayed and all significantly enriched processes were highlighted by bold characters. (B) Using the functional annotation clustering tool available in DAVID bioinformatics database, genes were classified into functionally related gene groups. Selected sets of genes under the categories like signaling and response were displayed. (C) In the heat map, genes involved in GO term, regulation of cell death were shown. Many of them were revealed to be associated with signaling pathways like NF-κB (red arrow), Wnt (yellow arrow), TGFß (blue arrow), and Notch/STAT (green arrow).</p

EMT generates cells with increased resistance to cell death.

<p>(A) MCF10A cells stably expressing SNAI1 (MCF10A-SNAI1) induced an EMT as shown by fibroblast-like appearance. Bar = 10 µm. Western blot analysis showed down-regulation of the epithelial marker, E-cadherin (CDH1) in SNAI1-overexpressing cells. (B) MCF10A-SNAI1 cells were more resistant to camptothecin (CPT) - and doxorubicin-induced cell death, as determined by MTT analysis. (C) MCF10A-SNAI1 cells were more resistant to the CPT-induced apoptosis as revealed by Hoechst staining and immunofluorescence using anti-active caspase-3 antibody. Positive cells for Hoechst or active caspase-3 staining were counted in the table. Bar = 100 µm. (D) Using Annexin V apoptosis detection kit, apoptotic cells were quantified upon CPT treatment (24 h). Viable cells are Annexin V-PE and 7-AAD-PerCP negative, and cells that are in early apoptosis are Annexin V-PE positive and 7-AAD-PerCP negative. Cells that are in late apoptosis or already dead are both Annexin V-PE and 7-AAD-PerCP positive. (E) To investigate the cellular response to CPT-induced DNA damage, western blot analysis was performed using antibody against γ-H2A.X, which is an indicator for DNA repair response. No difference in the accumulation of γ-H2A.X was observed in two cell lines for the indicated times, whereas MCF10A-SNAI1 cells showed higher AKT activation 6, 18 and 24 h after CPT treatment. (F) Serum-depletion for 10–15 days induced more cell death in MCF10A cells than MCF10A-SNAI1 cells as determined by a trypan blue assay. *<i>P</i><0.05. (G) Hoechst positive cells upon serum-depletion were counted in three independent fields under a fluorescence microscopy. Bar = 100 µm.</p

Differential gene expression pattern in NF-κB signaling and significant responses to IL1ß in MCF10A-SNAI1 cells.

<p>(A) Quantitative real-time PCR analysis confirmed the differential expression of genes revealed by microarray. (B) Treatment of LPS, IL1α, ILβ or combination of IL1α and ILβ increased <i>IL6</i> and <i>IL8</i> mRNA level in MCF10A-SNAI1 cells but not in MCF10A cells as analyzed by qRT-PCR. (C) WB analysis demonstrated that IL1β incubation leads to the activation of NF-κB, p38, ERK1/2 and AKT in MCF10A-SNAI1 cells but not in MCF10A cells. Constantly activated STAT3 was detected in MCF10A-SNAI1 cells regardless of IL1β addition. (D) Prolonged incubation of MCF10A-SNAI1 cells with IL1ß augmented the mammosphere formation ability.</p

EMT generates CSC-like but not tumorigenic cells.

<p>(A) Cell population for surface marker CD44⁺/CD24⁻ which is associated with breast cancer stem/progenitor cell features was increased in MCF10A-SNAI1 cells. MDA-MB-231 cells and MCF7 cells were used as positive controls for CD44⁺ and CD24⁻, respectively. (B) MCF10A-SNAI1 cells showed increased mammosphere formation, while MCF10A cells underwent apoptotic death as shown by arrows. Subpopulation of MCF10A-SNAI1 cells (MCF10A-SNAI1-R) which survived the 5 nM doxorubicin-induced cellular toxicity showed no increased mammosphere forming ability. MDA-MB-231 cells were used as a positive control. Only mammospheres larger than 60 µm were counted. Bar = 100 µm. *<i>P</i><0.05. (C) <i>In vitro</i> soft agar colony formation assay revealed that MCF10A-SNAI1 cells are non-transformed cells. MDA-MB-231 cells were used as a positive control. Bar = 100 µm.</p

Image_1_CDK7 is a prognostic biomarker for non-small cell lung cancer.pdf

AimNon-small cell lung cancer (NSCLC) remains the leading cause of cancer-related death globally despite promising progress of personalized therapy approaches. Cyclin-dependent kinase 7 (CDK7) is a kinase involved in transcription, overexpressed in a broad spectrum of cancer types and found to be associated with an unfavourable prognosis. In this study, we aimed to investigate the protein expression of CDK7 in a large cohort of NSCLC incorporating adenocarcinomas (adNSCLC) and squamous cell carcinomas (sqNSCLC) and to correlate its expression with clinicopathological data.MethodsWe performed immunohistochemical staining of CDK7 on our cohort of NSCLC including 258 adNSCLC and 101 sqNSCLC and measured protein expression via a semi-automated read out. According to the median value of CDK7 the cohort was stratified in a CDK7 high and low expressing group, respectively, and results were correlated with clinico-pathological data.ResultsCDK7 was significantly higher expressed in sqNSCLC than in adNSCLC. In the group of sqNSCLC, CDK7 expression was significantly higher in sqNSCLC with lymph node metastases than in sqNSCLC with N0 stage. We found a significantly worse overall survival and disease-free survival for patients with CDK7 high expressing NSCLC.ConclusionSince a high CDK7 expression seems to be linked with a poor prognosis it might serve as a promising novel prognostic biomarker and its assessment could be implied in future routine diagnostic workup of NSCLC samples. Considering that CDK7 inhibitors are currently tested in several trials for advanced solid malignancies, it may also be a new target for future anti-cancer therapy.</p

ATZ11 Recognizes Not Only Z-α₁-Antitrypsin-Polymers and Complexed Forms of Non-Z-α₁-Antitrypsin but Also the von Willebrand Factor

<div><p>Aims</p><p>The ATZ11 antibody has been well established for the identification of α₁-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue.</p><p>Methods and Results</p><p>Ultrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens.</p><p>ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes.</p><p>Conclusions</p><p>ATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes.</p></div

Recombinant von Willebrand Factor Expression in four Transfection Experiments in HEK293 cell lines.

<p>rVFW – recombinant von Willebrand factor.</p

Confocal imaging of VWF-transfected HEK293 cells: Pseudo-Weibel-Palade-Body (pseudo-WPB) granules formed after transfection of HEK293 cells using recombinant wild-type VWF (rVWF-WT) constructs: (A, B) Pseudo-WPB granules are shown in green (anti-VWF staining).

<p>(C) The same intracellular structures are stained with ATZ11 (red). (D) Small dot-like signals of less than .25 µm were found in very few HEK293 cells stained with anti-AAT (arrow). (E) Merged images of anti-VWF and ATZ11 stains highlight the co-localization of the antibody-binding sites. At a single cell level, small dot-like positive signals were found in the ATZ11 reaction, which were not co-localized with VWF staining (arrow). (F) Merged images of anti-VWF staining and anti-AAT signals demonstrated that the dot-like anti-AAT positive signals were not associated with pseudo-WPBs (arrow). Scale bar = 10 µm.</p

Protein-electrophoretic studies on VWF-transfected HEK293 cells and human serum samples:

<p>(A) <b>SDS-PAGE electrophoresis and subsequent western blotting (WB) and visualization using anti-VWF: (lane 1) cell lysates of recombinant wild-type VWF (rVWF-WT)-transfected HEK293 cells, (lane 2) mock-transfected HEK293 cells.</b> (B) SDS-PAGE electrophoresis and subsequent WB and visualization using ATZ11: (lane 1) of cell lysates of rVWF-WT-transfected HEK293 cells and (lane 2) mock-transfected HEK293 cells. A congruent single band of 225 kDa was detected in the VWF-transfected HEK293 cells using both anti-VWF (A) and ATZ11 (B). (C) SDS-PAGE electrophoresis and subsequent WB of human serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with anti-VWF. (D) SDS-PAGE electrophoresis and subsequent WB of serum samples of a non-Z healthy individual (lanes 1–2) and of VWF-deficient patients (lanes 3–5) stained with ATZ11. (E) Native PAGE electrophoresis and subsequent WB of a recombinant VWF (lanes 1–2) and serum samples of a non-Z healthy individual (lanes 3–5) stained with the anti-VWF antibody.</p